Hearing loss and cognitive impairments are both highly prevalent neurological complications for older adults. While there is growing evidence to suggest that these two conditions are interrelated, little research has been conducted that directly examines the progression and developmental trajectories of these complications contemporaneously. The aim of the study is to identify the distinct trajectory profiles for hearing loss and cognitive function in an older population over a 10-year period. Through dual trajectory modeling, the interrelationship, co-occurring movements, and overlaps between these two complications were examined. We also investigated the influence of hearing aid ownership on cognitive function trajectories. We utilized longitudinal data from 1,445 participants in the Blue Mountains Hearing Study (aged 55+ years) involving repeated measures from a population-based survey with audiometric hearing assessments. Cognitive function was assessed using the Mini-Mental State Examination (MMSE). The group-based trajectory modeling (GBTM) identified three trajectory profiles for both hearing loss and cognitive function in two older age groups (55–69 years and 70+ years). The outputs from the dual trajectories models showed the conditional probability for “no hearing loss” trajectories to be around 90% more likely to have “high-normal” cognitive function, demonstrating co-occurring overlap. In contrast, for “moderate to severe hearing loss” trajectories, the conditional probability drops to 65% and 79% for the 55–69 age group and 70+ age group respectively. There was also an increasing probability for “cognitive decline” conditional on the severity of hearing loss with 6.7%, 7.5%, and 8.7% for no hearing loss, mild hearing loss, and moderate to severe hearing loss trajectory groups. While we did not find any statistically significant difference in the influence of hearing aid use in the cognitive function trajectories, there was a consistent greater representation of non-hearing aid users in the trajectories with poorer cognitive function. This study found GBTM to identify trajectories that were in agreement with our current understanding of hearing loss and cognitive impairment in older adults. This study also adds to the existing evidence-base as dual trajectories demonstrated co-occurrence in developmental changes in these two common neurological complications for the older population. Growth in the older population is increasing worldwide mostly due to the rise in life expectancy. It has been estimated that by 2050 the number of persons aged 65 years or over will double to 1.5 billion , a greater hearing loss was found to be associated with poorer cognitive function with lower scores on measured mental status , memory , and executive function , with 5- and 10-year follow-up to 2009  calibrated to Australian standards. Stimuli were presented through supra-aural headphones using standard TDH-39 earphones. Audiometric thresholds for air-conduction stimuli in both ears were established for frequencies at 0.25–8.0 kHz. For a measure of hearing loss, we used the conventional hearing status in accordance with the World Health Organization (WHO), using averaged pure-tone audiometric hearing thresholds at 0.5, 1, 2, and 4 kHz. A four-frequency pure tone average PTA (dB HL) greater than 25 dB HL in the better hearing ear was used to define hearing loss. This defines hearing loss as bilateral. Hearing loss was defined as mild at 25–45 dB, and moderate to severe at greater than 45 dB , often used as a check for cognitive impairment  was used. GBTM is an exploratory technique used for isolating developmental trajectories within a population with the goal of identifying subgroups that follow distinct trajectories over time Nagin, . It was To examine the co-occurring relationship between hearing loss and cognitive function trajectories we used the extension of the GBTM, the dual trajectory analysis  = 46.13, p < 0.001). There were also significant sex differences between the three trajectories , with a larger proportion of females in the no hearing loss trajectory and a smaller proportion in the moderate hearing loss trajectory. The three classes identified from GBTM for hearing loss were labeled as no hearing loss, mild hearing loss, and moderate to severe hearing loss. This was named based upon their baseline hearing loss status since hearing loss is considered as a status from a specific timepoint rather than over a period of time. The mean (95% CI) of any hearing loss for the three trajectories were 14.8 (14.3–15.3), 30.2 (29.6–30.9), and 57.6 (55.9–59.3) dB HL. Hearing loss was found to increase over time in all three trajectories over the 10-year period with an average rate of change in the three trajectories being 1 dB hearing loss per year. For the 55–65 age group, 62.8% belonged to the trajectory with no hearing loss, 32.0% belonged to a trajectory with mild hearing loss, and only 5.2% of the group in the moderate to severe hearing loss trajectory. There were three cognitive function trajectories labeled as a high-normal cognitive function, low-normal cognitive function, and declining cognitive function. The high-normal cognitive trajectory stayed within the normal MMSE range for the 10-year period. The low-normal cognitive trajectory showed a decline towards mild cognitive impairment by 10 years, whereas the declining cognitive function trajectory showed cognitive impairment at both 5- and 10-year follow-ups  = 3.11, p = 0.045) between the trajectories. Similar to the hearing loss trajectories there were differences in the proportion of males and females for the cognitive function trajectories . There was a smaller proportion of females in the low-normal and decline trajectories. The rate of change for the declining trajectory is at 1.1 MMSE points per year. The mean (95% CI) MMSE scores for these three trajectories were 28.9 (28.8–29.0), 26.1 (25.9–26.3), and 21.0 (20.2–21.8). For this age group, 86.9% of this cohort belonged to a stable high-normal cognitive function trajectory, 11.7% in the stable low-normal cognitive function trajectory, and only 1.4% belonged to a trajectory with declining cognitive function.Fit indices determined that three trajectories were the best solution for both hearing loss and cognitive function  = 22.95, p < 0.001), with increasing age associated with greater hearing loss. However, there were no significant differences in the proportion of males and females for these trajectories. The rates of hearing loss for all three trajectories were also increasing over the 10-year period, an average rate of change in the three trajectories of 1.2 dB hearing loss per year. The mean (95% CI) bilateral hearing loss for the three trajectories were 21.3 (20.5–22.1), 38.1 (37.4–38.9), and 56.6 (55.0–58.3) dB HL. The three trajectories identified from GBTM for this age group had 42.7% belonging to the trajectory with no hearing loss, 45.4% belonged to a trajectory with mild hearing loss, and 11.9% of the group in the moderate to severe hearing loss trajectory. Similarly, there were three cognitive function trajectories labeled the same as the 55–69 age group. There were no significant differences in the proportion of males and females or in the ages of the three cognitive function trajectories. The mean (95% CI) MMSE scores for these three trajectories were 28.2 (28.1–28.4), 25.5 (24.8–26.2), and 21.6 (20.8–22.3). For this age group, 87.9% of this cohort belonged to the high-normal cognitive function trajectory, 7.3% in the low-normal cognitive function trajectory, and only 4.9% belonged to a trajectory with declining cognitive function. Once again, the rate of decline in the declining trajectory was also at 1.1 MMSE score per year.n = 491) probability for those in the no hearing loss to also belong to the high-normal cognitive function trajectory. From the linkage probabilities, there is evidence for large overlap or co-movement between no hearing loss and mild hearing loss 83.4% (n = 219) and high-normal cognitive function. For no hearing loss there was an 8.4% (n = 44) probability of belonging to the low-normal trajectory and 14.6% (n = 40) probability for mild hearing loss. In contrast, those that belong to the moderate to severe hearing loss trajectory had only a 65.2% (n = 28) chance of belonging to the high-normal cognitive function trajectory with the remaining 34.8% (n = 15) probability of belonging to the trajectory with lower  cognitive function trajectory. The cognitive decline group was small and as mentioned earlier only accounted for 1.4% of the whole group. Those in the mild hearing loss trajectory had a 2% (n = 5) probability of belonging to this trajectory and those in the no hearing loss trajectory had a 0.9% (n = 5) chance of belonging to this trajectory.To examine the contemporaneous development of hearing loss and cognitive function dual-GBTMs were examined. The conditional probabilities of co-occurring developmental trajectories for hearing loss and cognitive function for the 55–69 age group are shown in n = 225). There was a larger probability for mild hearing loss trajectory to also belong to the high-normal cognitive function trajectory compared with those in the younger 55–69 age group at 89.2% (n = 248). There were smaller probabilities of belonging to low-normal cognitive trajectories compared to the younger age group at 4.8% (n = 12) for no hearing loss, 3.3% (n = 8) for mild hearing loss, and 12.4% (n = 8) for moderate hearing loss. There was an increasing conditional probability between levels of hearing loss trajectories and the cognitive decline trajectory with 6.7% (n = 20), 7.5% (n = 20), and 8.7% (n = 6) for no hearing loss, mild hearing loss, and moderate to severe hearing loss respectively.For the 70+ group, the conditional probabilities are shown in PTA(dB HL) = 1.14, showing acceptable matching. There was no significant difference for age  = 1.65, p = 0.2) or sex , however, the hearing loss group remains significantly more severe in hearing loss from PTA = 8.43, p = 0.004) between the two subgroups. We tested for differences in the proportion of hearing aid users and non-hearing aid users for the three cognitive function trajectories While there was no significant difference in proportion between the two groups in the three cognitive function trajectories , there was a greater percentage of hearing aid users in the high-normal cognitive function trajectory with 91.7% (n = 133) compared with 84.1% (n = 122). Fewer hearing aid users were in poorer cognitive function trajectories such as the low-normal trajectory [2.1% (n = 3) compared with 4.8% (n = 7)] and cognitive decline trajectory [6.2% (n = 9) compared with 11.0% (n = 16)]. Despite the higher severity of hearing loss in the hearing-aid users’ group, there were modest protective effects found from hearing aid use on cognitive functions.The influence of hearing aid use on the cognitive function trajectories was also examined. We utilized a subgroup of hearing aid users and a matched comparison group from propensity score matching. For distribution of the propensity scores for the unmatched and matched subgroups see This study examined the developmental pathways of two interrelated complications prevalent in the older population—hearing loss and cognitive impairment. We identified through the GBTM approach, three trajectory profiles with frequency distributions for both hearing loss and cognitive function in two older age groups (55–69 years and 70+ years). For hearing loss profiles, the trajectories were, no-hearing loss (62.8% and 42.7%), mild hearing loss (32.0% and 45.4%), and moderate to severe hearing loss (5.2% and 11.9%) for the two age groups, respectively. For the cognitive function profiles, the trajectories were, high-normal (86.9% and 87.9%), low-normal (11.7% and 7.3%), and declining cognitive function (1.4% and 4.9%), for the two age groups, respectively. Further, the findings have extended our understanding of hearing loss and cognitive impairment by examining the two complications as they co-occur. Dual-GBTM allowed for a comprehensive understanding of the underlying relationship between these two outcomes by examining the dynamic linkage between the concurrent trajectories (Xie et al., Despite the known cognitive function differences that occur at 70 years and older (Harada et al., Surprisingly, the profile for high-normal cognitive function for the two age groups was very similar at 86.9% and 87.9%. This may be a limitation of the MMSE, whereby ceiling effects are common especially in normal or predementia stages (Trzepacz et al., Further, the results from this study also demonstrated that the different hearing loss profiles have an impact on the contemporaneous development of cognitive impairment. Dual trajectories were applied to examine the co-occurring pathways and showed that up to 90% probability of belonging in the high-normal cognitive function trajectory was conditional on belonging to the no hearing loss trajectory, irrespective of age group. The linkage probabilities show a high level of temporal correspondence between no hearing loss and normal cognitive function. Having a higher severity of hearing loss reduces the probability of belonging to a high-normal cognitive trajectory to 65–79%. This result strongly supports maintaining optimal normal hearing function for normal cognitive function. There was a relationship between severity of hearing loss and cognitive decline. Those belonging to the no hearing loss trajectory had a 6.5 probability of being in the cognitive declined trajectory and this increased with severity of hearing loss to 7.5% probability for mild hearing loss and 8.7% probability for moderate to severe hearing loss. These results demonstrate a steady decline in cognitive function for those belonging to increasing hearing loss severity trajectories. Similarly, compared to normal hearing, another research has found a two-, three- and five-fold increase in risk for dementia with mild, moderate, and severe hearing impairment (Lin et al., It is of concern that hearing loss, although highly prevalent in older adults and prospectively associated with incident dementia, often remains untreated (Popelka et al., The strengths of this study include utilizing a modeling technique that allows for the identification of distinct heterogenous patterns within a large representative longitudinal dataset. In addition, we were able to explore developmental changes in these two common neurological complications for the older population as they evolve contemporaneously, showing their relationship and co-occurrence of movement with each other. As to limitations, we examined the trajectories as they were occurring without examining potential unmeasured confounders. We have tried to limit the confounding influence of demographic confounders such as age through stratification into subgroups and have also examined effects of sex on these trajectories, however, other potential unmeasured confounders, such as noise exposure (Huang et al., This study utilized a representative population-based sample of older adults in the Australian community. It identified trajectories in proportions that were in agreement with our current understanding of hearing loss and cognitive impairment in the older population worldwide, showing support for the generalizability of the trajectories. With the growing evidence that these two conditions are interrelated, this study directly examined the progression and developmental trajectories of these complications contemporaneously. This adds to the existing evidence-base as dual trajectories demonstrated co-occurrence in developmental changes in these two common neurological complications for the older population. Results demonstrate a high probability for normal cognitive function with optimal/normal hearing. Examining both hearing loss and cognitive function longitudinally allowed the heterogeneity of both complications to be addressed together.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by The University of Sydney. The patients/participants provided their written informed consent to participate in this study.YT, DT, and BG conceived the study. YT, DT, BG, CL, CM, and PM developed the study design. CL conducted the data wrangling. YT drafted the article and conducted the analysis. YT, DT, BG, and CL made important contributions to the interpretation of data. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Although the two groups felt capable and reported similar opportunities to use hearing protection, never-performers lacked automatic motivation (p < 0.001) and reflective motivation (p < 0.001) compared to ever-performers. For the first time, the present study identifies potential groups at whom hearing protection interventions might be targeted and what those interventions may contain. Further work is required to develop interventions targeted at older people, women and those in non-manual occupations. Lack of motivation is a key concern, and further work that uses specific theoretical frameworks, such as the PRIME  theory of motivation, may shed light on the kinds of interventions that are needed to boost hearing protection use effectively.Interventions to increase hearing protection behaviours within noisy recreational settings are limited by the lack of an underpinning evidence base. The aim of the present study was to identify targets for interventions in a population exposed to recreational noise, including those who had used hearing protection (ever-performers) versus those who had not (never-performers). A cross-sectional survey was administered to 185 UK adults who had been involved in noisy recreational activities. Participants had an average age of 36.79 years; the majority were women (68.1%), from a white ethnic background (87.6%), and with non-manual occupations (75.7%). Using Chi-square, MANOVA and ANOVA, we looked for differences in sociodemographic variables and variables from the capabilities, opportunities and motivations model of behaviour change (COM-B) between ever- and never-performers. Ever-performers were more likely to be younger ( Hearing loss is ranked third in total years lived with a disability, ahead of both diabetes and asthma . ExposurLoughran et al.’s  systematThus, a first step in developing hearing protection interventions is to understand what needs to change in terms of people’s capabilities, opportunities and/or motivations. In order to address this question, for the first time, we proactively recruited a group of people who had been exposed to excessive recreational noise over their lifetimes and examined what discriminated those who did use hearing protection (ever-performers) from those who did not (never-performers). Due to ever-performers having already taken the steps to perform hearing protection behaviour, we can hypothesise that they will have significantly greater capabilities, opportunities and motivations than never-performers. It is also important to establish whether any sociodemographic variables  differ within these naturally occurring hearing protection groups in order to help identify groups to target in future interventions. Based upon recent epidemiological data from the UK , we can Ethical approval was granted by the Division of Psychology and Mental Health, University of Manchester, June 2018. Reference: 2018-3556-6133.A cross-sectional survey was made available from June 2018 until June 2019 to any UK-based individual aged 18–69 years. The final dataset included anyone able to confirm their involvement in at least one noisy recreational activity , defined by vocal exertion in presence of background noise >85 dBA ,12). Rec. Rec12])Those included had the option to say whether they had performed any hearing protection behaviours  while taking part in noisy recreational activities, or not (never-performers). Originally, the participants were asked, “Do you use hearing protection when you do this activity?” , to whicSociodemographic measures included age, gender, ethnicity and socioeconomic status. According to the UK Office of National Statistics, ethnicity was divided into White versus Black, Asian, or Minority Ethic (BAME), and socioeconomic status was split into manual versus non-manual work .Psychological capability captured if the person felt they had the knowledge and skills to use hearing protection, “I have the knowledge and skills, and I have the ability to remember, pay attention and make decisions to use hearing protection”. Physical capability explored if they felt they had the physical skills and strength to perform the behaviour, “I have enough physical stamina and I have sufficient physical skills to use hearing protection”. Physical opportunity assessed people’s perceptions of having the necessary resources and reminders to use hearing protection, “I have sufficient time, the necessary resources, and the reminders to use hearing protection”. Social opportunity considered if they felt they had adequate social support, “I have the necessary support from people  to use hearing protection”. Reflective motivation evaluated if the person had the desire and wanted to use hearing protection, “I have the desire to and I want to use hearing protection”. Automatic motivation assessed if the behaviour was performed without realisation, “Using hearing protection is something I do before I realise I’m doing it”.Keyworth et al.’s  validateStatistical analyses were conducted using SPSS version 23. Chi-squared and univariate analyses of variance (ANOVAs) were used to explore differences between the current sample of adults exposed to recreational noise and a UK population study for representativeness [n = 126), with a white ethnic background , a higher education , non-manual occupation , and with an average age of 36.79 years (SD = 15.328). Compared with a nationally representative sample [2  = 0.94, p = 0.333) by adults exposed to recreational noise. However, they were younger  = 3287.007, p < 0.050), more likely to be women  = 21.82, p < 0.050), more likely to report a white ethnic background  = 5.85, p < 0.050) and more likely to be in a non-manual occupation  = 108.10, p < 0.050).Of the 185 participants recruited, most were women  exceeded ever-performers , and with only 4.3% (n = 8) “always” using hearing protection, 2.7% (n = 5) “often”, 8.6% (n = 16) “sometimes”, and 8.1% (n = 15) “seldom”, there is still work to be done to promote hearing protective behaviours. Consistent with our hypothesis, comparisons revealed that ever-performers were younger  = 5.348, p < 0.050), and more likely to be men  = 6.19, p < 0.050) and in a manual occupation  = 5.69, p < 0.050). No differences between hearing protection groups were found for ethnicity  = 0.055, p = 0.814), nor education  = 2.27, p = 0.132)  see .p < 0.001). ANOVAs revealed significant differences between groups for reflective motivation  = 22.081, p < 0.001), and automatic motivation  = 18.667, p < 0.001)  = 5.223, 001) see . The resp = 0.074) and physical  = 2.777, p = 0.097) opportunities. However, although results indicate similar opportunities, with scores close to the midpoint it would suggest that there is quite a large scope available to improve physical and social opportunities for both. There were also no differences found for psychological  = 0.014, p = 0.907) and physical  = 0.220, p = 0.639) capabilities, and it appears that both groups feel they have sufficient capabilities.No statistically significant differences were found between groups in terms of their social  = 3.228, The major finding of this study is that, with similar rates of capability  and opportunity  recorded across both groups, it is ever-performers’ greater reflective  and automatic motivation  driving their use of hearing protection. Use of the COM-B model has highlighted never-performers’ lack of reflective and automatic motivation as a concern, and potential targets of the content addressing what needs to change to increase use, and uptake of hearing protection. In order to address this gap, further research is required to pinpoint what is energising these motivational differences, and implementation of theoretical frameworks such as the PRIME  theory of motivation  could aiComparable with Armitage et al.’s  researchOther potential avenues for change may have presented themselves through the low scoring recorded for physical and social opportunities. In order to address these gaps, further explorative research is required, potentially achieved through qualitative fact-finding informed by the COM-B model itself, similar to that seen within the fields of dietary patterns ,24 and cFirst, this study is the first of its kind to assess the capabilities, opportunities and motivations of ever- and never-performers of hearing protection during noisy recreational activities, highlighting potential targets to drive improved behaviour, and although these measures were self-reported, the tool is known to be reliable and valid . Second,First, although the present sample was representative in terms of the numbers of UK adults known to use hearing protection recreationally , it was Interventions addressing reflective and automatic motivation, and targeting older people, women, and those in non-manual occupations, may have the potential to increase hearing protection behaviour during noisy recreational settings. Further research is required to explore the motivational differences between ever- and never-performers using theoretical frameworks, such as the PRIME theory of motivation, with the prospect of developing an intervention. Additionally, it would be valuable to replicate the findings in a larger more representative sample.


This case report presents bilateral hearing loss after vestibular‐evoked myogenic potential (VEMP) testing. The loud acoustic stimulus used in VEMP testing can cause noise‐induced hearing loss in some cases with high sensitivity to noise exposure or by exceeding individual cochlear safety thresholds. This case report presents bilateral hearing loss after vestibular‐evoked myogenic potential (VEMP) testing. The loud acoustic stimulus used in VEMP testing can cause noise‐induced hearing loss in some cases with high sensitivity to noise exposure or by exceeding individual cochlear safety thresholds. However, the acoustic stimulus used in VEMP testing is a loud sound of around 120–130 dBSPL, and the safety evaluation of the sound pressure level is still under investigation.2A 78‐year‐old man with a history of lumbar spinal canal stenosis and chronic pain after lumbar spine surgery was referred to our hospital for vestibular evaluation of floating dizziness and wobbling throughout the day and a tendency to waver to the right during walking for the previous one month. He also had non‐pulsatile mild right occipital pain and stiffness in his shoulders. His medication included ketoprofen patch, fentanyl patch, fexofenadine, tramadol hydrochloride, mirogabalin besylate, mecobalamin, and limaprost alfadex, which had been newly prescribed 5 days before the vestibular examination. In the vestibular assessment, there was no gaze‐evoked nystagmus or positional nystagmus. Pure tone audiometry (PTA) showed age‐appropriate thresholds. The total trajectory lengths of posturography for 60 s were 254.17 cm (eyes open) and 339.82 cm (eyes closed), and the gravitational chart was teardrop‐shaped, suggesting a psychogenic vertigo. Videonystagmography (VNG) revealed no gaze‐evoked nystagmus, normal smooth pursuit, and good optokinetic nystagmus. The maximum slow phase velocities recorded in the ice water caloric test were 36 degrees/second on the right and 38 degrees/second on the left. The results of cervical VEMP (cVEMP) and ocular VEMP (oVEMP) testing were all normal, except for absent oVEMPs with stimulation at 1kHz on the left. The Dizziness Handicap Inventory (DHI) score was 18, the hospital anxiety and depression scale (HADS) scores were 10 (A) and 16 (D), and the Geriatric Depression Scale (GDS) score was 13. There was no evident peripheral or central vestibular dysfunction during the examination, and the patient was diagnosed with psychogenic vertigo.While testing the right ear for cVEMPs with a 500 Hz stimulation, the patient suddenly stopped responding to the examiner's instructions and complained of sudden bilateral hearing loss. The examination was immediately stopped, and the physician checked both ears, but found no abnormalities. After a 5‐min break, the examination of cVEMPs and oVEMPs was continued with the patient's consent. After all tests were completed, he still complained of difficulty hearing, and pure tone audiometry showed threshold increases of about 30 dB in both ears. The results of distortion product otoacoustic emissions (DPOAE) showed no responses bilaterally. Subsequently, he was treated with oral steroid therapy and bilateral intratympanic dexamethasone, but there was no recovery in hearing thresholds after 1 month  of National Institutes of Occupational Safety and Health (NIOSH) was 89.1 dBA (left) and 89.5 dBA (right), which slightly exceeded the recommendations by 4.1 dB and 4.5 dB, respectively. In the past, only one case of bilateral hearing loss after VEMP testing has been reported, in which the LAeq,8hr equivalents of NIOSH were exceeded by 6 dB and 7 dB.Aeq,8hrThere are three possible causes of bilateral hearing loss in this case. First is bilateral sudden hearing loss. Although sudden deafness usually occurs on one side only, there have been several reports of bilateral sudden deafness in the past. The reported time of onset of these cases varied from morning to noon and night, allowing the possibility that bilateral sudden deafness occurred coincidentally during the examination. The second possible cause is an enlarged vestibular aqueduct (EVA), which can cause hearing loss as a result of loud sounds. We could not evaluate the presence of EVA in our patient, however, because he did not agree to undergo MRI or CT scan. Because the onset of hearing loss with EVA generally ranges from around 10 years old up to patients in their 20s,4Vestibular evoked myogenic potential sound stimulation can cause noise‐induced hearing loss in some cases with high sensitivity to noise exposure or by exceeding individual cochlear safety thresholds. It is necessary for healthcare providers to understand the risk of hearing loss with VEMP sound stimulation and to explain this to patients in advance. To prevent acoustic trauma, it is necessary to have the patient listen to the stimulus sound before starting the test, to make the patient take a break during the test, and to reduce the number of stimuli and stimulus sound pressure levels. A hearing threshold check after VEMP testing should be considered to make sure there are no hearing problems.The authors have no conflicts of interest to disclose.All authors contributed to the writing of the manuscript. S. A. and T. K. equally contributed to the study. All authors conceived this study and contributed to the writing of the manuscript.This study was approved by the regional ethical standards committee in the Faculty of Medicine at the University of Tokyo (2487‐(13)).The authors have confirmed during submission that patient consent has been signed and collected in accordance with the journal’s patient consent policy.


HMOX1, ANGPTL4, VEGFA, MMP-2, MMP-9, and upregulated inflammatory genes p65, IL-6, IL-1β, and TNF-α in vitro and in vivo. Further investigation showed that overexpression or activation (rosiglitazone) alleviated cell viability, migration, angiogenesis inhibition, and inflammatory response caused by TCS, while knockdown or inhibition (GW9662) of PPARγ had the opposite effect. Moreover, TCS caused placenta dysfunction characterized by the significant decrease in weight and size of the placenta and fetus, while PPARγ agonist rosiglitazone alleviated this damage in mice. Taken together, our results illustrated that TCS-induced placenta dysfunction, which was mediated by the PPARγ pathway. Our findings reveal that activation of PPARγ might be a promising strategy against the adverse effects of TCS exposure on the placenta and fetus.Exposure to the antibacterial agent triclosan (TCS) is associated with abnormal placenta growth and fetal development during pregnancy. Peroxisome proliferator-activated receptor γ (PPARγ) is crucial in placenta development. However, the mechanism of PPARγ in placenta injury induced by TCS remains unknown. Herein, we demonstrated that PPARγ worked as a protector against TCS-induced toxicity. TCS inhibited cell viability, migration, and angiogenesis dose-dependently in HTR-8/SVneo and JEG-3 cells. Furthermore, TCS downregulated expression of PPARγ and its downstream viability, migration, angiogenesis-related genes  Triclosan (TCS) is a synthetic broad-spectrum antimicrobial and exposure mainly occurs in dermal application  and oral use of consumer products  ,2,3. TCSThe nuclear receptors, peroxisome proliferator-activated receptors (PPARs) are activated by binding natural ligand-inducible transcription factors such as glitazones ,12. PPARThe effect of prenatal TCS exposure on placental toxicity and its role in fetal growth and development are still unclear. Whether the effect of prenatal TCS exposure on placental toxicity is associated with PPARγ remains to be revealed. Our research aims to clarify the potential character of prenatal TCS exposure on placenta function and its potential mechanism of PPARγ in the process of placental exposure to TCS.PPARγ vector and si-PPARγ were purchased from GenePharma . Moreover, the rosiglitazone (a specific PPARγ agonist) and GW9662 (a specific PPARγ antagonist) were obtained from MedChemExpress .TCS  was obtained from Sigma–Aldrich . DMEM/F12 medium and MEM medium were purchased from KeyGEN BioTECH . RPMI 1640 medium was purchased from GIBCO . Dimethyl sulfoxide (DMSO), fetal bovine serum (FBS), the Cell Counting Kit-8 (CCK-8), and corn oil used was obtained from Vicmed  and Macklin , respectively, and were analytical grades. The pcDNA-2 humidified atmosphere at 37 °C. Based on the manufacturer’s instructions, these two cell lines were transfected with small interfering RNA (siRNA) , the control siRNA , pcDNA-PPARγ (2 µg), and pcDNA 3.1 (2 µg) targeting PPARγ by Lipofectamine 2000 . The medium was replaced after 4 h of cell transfection. After cell transfection for 24 h, they were incubated with triclosan for 24 h. For PPARγ activation or deactivation, cells were treated with rosiglitazone or GW9662 with 10 µM for 24 h and pre-treated for 0.5 h before incubating with TCS.Thanks to Dr. Xinru Wang from Nanjing Medical University  for sending HTR-8/SVneo and JEG-3 cells as a gift to us. HTR-8/SVneo and JEG-3 cells were seeded in DMEM/F12 and MEM medium, respectively, and supplemented with 10% FBS in a 5% COn = 8 per group). All pregnant mice were orally managed with 0, 10, 50, and 100 mg/kg/day TCS and from GD 7.5 to GD 17.5, while control pregnant mice accepted the same volume of corn oil. Firstly, rosiglitazone was dissolved in DMSO, and the ratio of DMSO to corn oil was 1:1000, and pregnant mice were orally administered with rosiglitazone (20 mg/kg/day) together with TCS (100 mg/kg/day). Mice placentas were isolated, put into liquid nitrogen to be quickly frozen and stored at −80 °C. According to manufacturers of Xuzhou Medical University Animal Ethics Committee, the entire experiment was performed in a Specified Pathogen-Free (SPF) environment .Institute of Cancer Research (ICR) mice, at 10 weeks old and weighing about 30 g, were obtained under a protocol approved by Xuzhou Medical University Animal Center. On day GD 7.5, after the vaginal plug (GD 0.5) was observed in pregnant mice, they were randomly separated into five groups (3 cells/well) were seeded into sterile plates of 96 wells in complete medium . Cells were incubated and cultured with TCS of different concentrations for 24 h. An amount of 10 μL CCK-8  solution was added to each well and incubated for 0.5–4 h at 37 °C in a 5% CO2 atmosphere. The plate was detected at 450 nm on a microplate reader Spark .HTR-8/SVneo or JEG-3 cells , and the migration distance was measured by Image J analysis software . The whole wound closure distance was calculated and the distance of newly covered cells was measured at 0 and 24 h to evaluate migration.3/mL HUVECs were suspended in a conditioned medium .Approximately 50 µL Matrigel (10 mg/mL)  was decked into 96-well plates and allowed to completely solidify at 37 °C for 1 h to form a gel. Approximately 5 × 10−ΔΔCT), and the relative levels were normalized to the level of GAPDH. The primers were designed by Sangon Biotech , and primer sequences were listed in Placental tissues, trophoblast cells, and Trizol  were used to isolate total RNA. RNA concentration was detected by Nanodrop 2000 . The reverse transcription was conducted using SYBR Green qPCR SuperMix and 500 ng of the total RNA was reverse transcribed to cDNA. The quantitative RT-PCR was carried out using SYBR Green Master Mix on a 7500 fast real-time PCR System  according to the manufacturer’s instructions. The mRNA levels were analyzed using the comparative cycle threshold method  and phosphatase inhibitors . Proteins were separated with 10% SDS-PAGE gels and transferred to PVDF membranes . Membranes were blocked with 5% non-fat milk for 1h and then incubated with primary antibodies of PPARγ, ANGPTL4, MMP-2, IL-1β, GAPDH, and tubulin β  overnight at 4 °C. After being washed five times for five minutes, the membranes were incubated with the corresponding secondary antibodies for 1h at room temperature. The protein bands were visualized with an Enhanced Chemiluminescence (ECL) detection kit . The protein bands were analyzed using Image Lab Software .t-test or one-way ANOVA was implemented using the SPSS 19.0 . p-value < 0.05 and p-value < 0.01 were considered a statistically significant difference and higher significance, respectively.The above assays were carried out three independent times and all data were carried out by GraphPad Prism 8.3 software , and data were exhibited as the mean ± SEM. The comparison of the data was subjected to a one-way analysis of variance among each independent group. The PPARγ mRNA expression levels in those two cell lines especially HTR-8/SVneo cells  or knockdown and inhibition (GW9662), and then exposed to TCS. Rosiglitazone and GW9662 with the concentration of 10 μM were not toxic in HTR-8/SVneo and JEG-3 cells . The eff-3 cells C,D. In cll lines C,E. In ate trend .The wound healing assay confirmed that the migration ability of HTR-8/SVneo and JEG-3 cells were inhibited after concentration dependent on exposure to TCS A. To assAn angiogenesis assay was implemented to describe the influence of TCS on tube branch length. The HTR8/SVneo and JEG-3 cell lines were treated with different concentrations of TCS. TCS showed significantly decreased tube branch length compared to the control (DMSO) at 30 μM or 40 μM in the two cell lines, respectively A. The imHMOX1, ANGPTL4, VEGFA, MMP-2, and MMP-9 genes were determined as the target genes of PPARγ and play considerable roles in cell viability, angiogenesis, and migration [HMOX1, ANGPTL4, VEGFA, MMP-2, and MMP-9 mRNA were significantly decreased in the TCS-exposed group  and Heme oxygenase 1 (HO-1) are key modules of the angiogenesis process [ANGPTL4) is a secretory glycoprotein member of the angiopoietin family, which participates in the regulation of migration and angiogenesis [ANGPTL4 in placental development and angiogenesis, mediating the survival, proliferation, migration, and invasion of HTR8/SVneo cells [HMOX1, ANGPTL4, and VEGFA expression caused by TCS. MMPs have been demonstrated to mediate the migration and invasion of trophoblast cells, and MMP-2 and MMP-9 expression levels were decreased in preeclamptic placental tissues [MMP-2 and MMP-9 expression. In this study, cell growth, migration, and angiogenesis inhibition by TCS was partly ameliorated by PPARγ elevation or activation. These findings prove that TCS affects cell viability, migration, and angiogenesis through the PPARγ pathway.Vascular endothelial growth factors A ( process . Angiopoogenesis ,38. Furteo cells ,40. Our  tissues ,42. SimiTNF-a and IL-6 expression in HUVEC [TNF-α and IL-6, which were linked to preterm labor, miscarriage, and preeclampsia [IL-6, IL-8, and TNF-α, in human gestational tissue [Pregnancy is a process of dynamic inflammatory phases, and the inflammatory state is present almost throughout pregnancy and towards the end . Activatin HUVEC . Interesclampsia . Moreovel tissue ,47. Furtl tissue . Similarl tissue . Previoul tissue . The PPAl tissue ,52. Our si-PPARγ aggravated the toxicity. Similarly, animal studies also indicated that rosiglitazone mitigated the decrease of placental diameter and fetal weight caused by TCS. In addition, curcumin, a natural compound and a modulator of PPARγ, has been known to have beneficial effects on pregnancy outcomes [All the results in this study illustrated that TCS caused significant abnormal functions of HTR-8/SVneo and JEG-3 cells, including viability, migration, angiogenesis, and inflammation response. In addition, treatment with rosiglitazone or overexpression of PPARγ almost completely prevented the abnormal cell function changes in vitro. On the other hand, treatment with GW9662 or outcomes ,54. It w


The ternary complex (KDB) was further subjected for in vivo pharmacokinetic studies in rats and a significant improvement in the bioavailability (2.17 fold) was observed in comparison with pure ADL. Therefore, it can be concluded that the solubilization and bioavailability of ADL can be remarkably increased by ADL/β-CD complexation in the presence of a third component, poloxamer 188.In the current study, the effect of poloxamer 188 on the complexation efficiency and dissolution of arbidol hydrochloride (ADL), a broad-spectrum antiviral agent, with β-cyclodextrin (β-CD) was investigated. Phase solubility studies confirmed a stoichiometry of a 1:1 ratio for both ADL:β-CD and ADL/β-CD with a 1% poloxamer 188 system with an AL type of phase solubility curve. The stability constants (K1:1) calculated from the AL type diagram were 550 M-1 and 2134 M-1 for AD:β-CD and ADL/β-CD with 1% poloxamer 188, respectively. The binary ADL/β-CD and ternary ADL/β-CD with 1% poloxamer 188 complexes were prepared by kneading and a solvent evaporation method and were characterized by aqueous solubility, FTIR, PXRD, DSC and SEM in vitro studies. The solubility (13.1 fold) and release of ADL were markedly improved in kneaded ternary ADL/β-CD with 1% poloxamer 188 (KDB). The binding affinity of ADL and β-CD was confirmed by  Arbidol hydrochloride (ADL), also known as umifenovir, is a Russian-made potent broad-spectrum antiviral agent. ADL is used for prophylaxis as well as the treatment of human pulmonary diseases caused by influenza A and B viruses, hepatitis C and other human pathogenic respiratory viruses ,2,3. SinBeing indole derivative, ADL is hydrophobic in nature and is poorly soluble in aqueous media, which results in poor bioavailability in humans (40%) and rats 18.8%) 9]. SeverOwing to their non-toxic nature and complexation ability, a cyclodextrins (CDs)-based drug delivery system is commonly employed as a promising platform to increase drug solubility and stability, which enhances the drug release profile . In thisPoloxamer 188 is an FDA (Food and Drug Administration) approved non-ionic block polymer composed of two hydrophilic side chains attached to a hydrophobic core and is frequently used for the solubility enhancement of poorly soluble drugs due to its excellent surfactant property ,20. Here−1 and 2134 M−1, respectively. The complexation efficiency (CE) was considered as more reliable data for complexation. The CE of ADL/β-CD and ADL/β-CD with 1% poloxamer 188 systems were found to be 0.0064 and 0.0250, respectively. It was revealed from the data that the addition of 1% poloxamer 188 to β-CD enhanced the interaction of ADL with β-CD by increasing the stability constant.The phase solubility diagrams of binary ADL/β-CD and ternary ADL/β-CD with 1% poloxamer 188 systems showed linear AL type solubility curves of ADL against an increasing concentration of β-CD and β-CD with 1% poloxamer 188 . AccordiThe results of the aqueous solubility of pure ADL and the PM and their binary and ternary complexes are shown in The results of the DSC curve are presented in −1 for O-H stretching vibrations, 3100 cm−1 for aromatic C-H stretching vibrations and 1680 cm−1 for C=H stretching vibrations, confirming the purity of the ADL drug [−1 for O-H stretching vibrations and 2934 cm−1 for C-H stretching vibrations and other peaks at 1657 cm−1, 1278 cm−1, 1042 cm−1 and 863 cm−1 assigned for the presence of H-OH stretching, C-O stretching, C-O-C stretching for the glucose unit and C-O-C stretching vibrations for the ring β-CD. Compared with pure ADL, the binary ADL/β-CD and ternary ADL/β-CD with 1% poloxamer 188 complexes showed the absence of or diminished sharp ADL peaks; other sharp peaks belonging to β-CD could be seen in the spectra of the complexes. However, the ternary ADL/β-CD with 1% poloxamer 188 complex prepared by the kneading technique showed almost a complete disappearance of typical peaks of ADL. Therefore, these results confirmed the complexation of ADL inside the cavity of β-CD.The results of the FTIR spectra of ADL and its inclusion complexes  are shown in ADL drug . The FTIThe PXRD pattern of ADL, the physical mixture (PM) and the binary and ternary complexes are shown in A comparative release profile of pure ADL, the PM and their binary  and ternary  complexes measured at different time intervals  are shown in The SEM images of the PM and KDB are shown in 1H NMR studies. A significant upfield chemical shift was observed when ADL was complexed with β-CD (KDA) and β-CD with 1% poloxamer 188 (KDB). The H3 and H5 (present in the inner surface of β-CD) showed a maximum CIS in both the KDA and KDB complexes. The higher CIS at H3 (−0.030 and −0.041) and H5 (−0.020 and −0.064) signals were observed in the KDA and KDB complexes, respectively, which suggested the inclusion of ADL inside the cavity of β-CD [The formation of the inclusion complexes of ADL into the β-CD cavity with and without 1% poloxamer 188 was evidenced by chemical shift changes detected in  of β-CD ,25. The  of β-CD . p < 0.05), AUC0-T and AUC0-∞ (p < 0.01) were significantly higher in the case of the ADL/β-CD with 1% poloxamer 188 formulation (KDB) compared with the normal suspension of ADL. Therefore, the relative bioavailability was 2.17 fold higher with the optimized ternary complex (KDB). However, the Kel and T1/2 (h) values were not statistically different between these two formulations, indicating that the bioavailability enhancement was due to the rapid absorption of the novel formulation. A good relation between the in vitro release profile and the apparent absorption in the case of the KDB formulation suggested that the rate determining process was the in vivo dissolution of ADL. The MRT value of the optimized ternary complex (KDB) was higher compared with the normal suspension indicating a higher retention of the novel formulation in the body.The pharmacokinetic results of a normal ADL suspension and the optimized ternary complex (KDB) are presented in ® F-68 (non-proprietary name: poloxamer 188) were obtained from Sigma–Aldrich Pvt. Ltd. . All reagents were of analytical reagent grade and were used without further purification. Deionized water was used for the preparation of aqueous samples. ADL was purchased from the Beijing Mesochem Technology Co. Ltd. . β-CD and PluronicKs) using the following equation:oS was the intrinsic solubility of arbidol in water and Slope was the slope of the phase solubility diagram.Phase solubility studies of ADL/β-CD and ADL/β-CD with 1% poloxamer 188 were used to evaluate the complexation . In thisCE) was the ratio of the complex formed to free the β-CD concentration, which was an excellent method of calculating the solubilizing effect of β-CD. The CE was estimated using the following formula.The complexation efficiency (The inclusion complex of ADL/β-CD (1:1) was prepared by mixing in a clean glass mortar and pestle.v/v). The mixture was continuously kneaded for 30 min until a thick paste was formed, which was evaporated at 50 °C for 4 h. The solid powder was then crushed, passed through a sieve and stored in airtight containers. The same procedure was followed to prepare the ternary complex of ADL /β-CD as described above but 1% poloxamer 188 was kneaded with the ADL /β-CD complex (The inclusion complexes containing ADL/β-CD (1:1) were established in preliminary phase solubility studies. The inclusion complexes of ADL/β-CD and ADL/β-CD with 1% poloxamer 188 in a 1:1 molar ratio were prepared by utilizing kneading. The binary complex of ADL/β-CD in a molar ratio (1:1) was prepared by mixing one mole of ADL and one mole of β-CD in a mortar followed by the addition of about 6 mL of a water-ethanol solution . The obtained solution was then dried on a rota evaporator at 50 rpm for 4 h (Buchi Rotavapor R-215). The same procedure was followed to prepare the ternary complex of ADL/β-CD but 1% poloxamer 188 was dissolved with the ADL/β-CD complex .Excess amounts of pure ADL, the PM and their binary and ternary complexes were kept in conical flasks containing 10 mL of distilled water and stirred on a thermostatic mechanical shaker  at 25 °C for 72 h . All samThe DSC thermal curves of the prepared complexes were recorded using a DSC instrument . The samples (5 mg) were crimped in aluminum pans by applying pressure and the pan was placed in the sample holder of the instrument and heated at a temperature of 50–200 °C at a heating rate of 10 °C/min. The DSC instrument was purged with nitrogen gas during the analysis ,31.The FTIR spectra of pure ADL, the PM and their binary and ternary complexes prepared by the KD and SE methods were recorded using an FTIR spectrometer . The samples were pressed into transparent pellets by diluting with crystalline potassium bromide. The FTIR spectra were interpreted using spectra manager software ,31.The PXRD pattern of pure ADL, the PM and their binary  and ternary  complexes were recorded by an Ultima IV diffractometer  in the range of 0–80° (2θ) at a scan rate of 0.5°/min. The recorded PXRD patterns of each sample were evaluated for inclusion complexation .In vitro dissolution studies were performed to compare the release profile of the physical mixture (PM) with the binary kneaded (KDA), solvent evaporated (SEA), ternary kneaded (KDB) and solvent evaporated (SEB) inclusion complexes with pure ADL. The pure ADL and complexes  were filled into hard gelatin capsules and each filled capsule was dipped into a basket containing 900 mL of 0.1N HCl (pH 1.2). The stirring speed was set at 50 rpm at a temperature of 37 ± 1 °C. The samples were withdrawn at different time intervals and analyzed by UV at 258 nm .The surface morphology of the physical mixture (PM) and the optimized kneaded ternary inclusion complex (KDB) were studied under the scanning electron microscopy (SEM) . The samples were vortexed for 5 min after a suitable dilution with methanol. One drop of solution was spread onto a slide and kept for drying before the slide was examined for morphology .1HNMR spectra were recorded using a DMSO-d6 solvent and an UltraShield Plus 500 MHz Bruker spectrometer operating at 500 MHz for proton atoms at the Prince Sattam Bin Abdulaziz University and 2D NMR (ROSEY) spectra were acquired using the Bruker library. Briefly, the pure ADL drug, β-CD, the binary kneaded (KDA) and ternary kneaded (KDB) complexes were dissolved in DMSO-d6 and filtered in a 5 mm glass capillary tube and then the 1HNMR spectra were recorded. The chemical shift values were measured in δ (ppm). The complexation induced shifts (CIS = δcomplexed β-CD = δfree β-CD) were also calculated to discover the inclusion of ADL into the β-CD cavity.The quantitative analysis of ADL was performed on an Acquity UPLC system connected with a triple quadruple (TQD) detector  using ibrutinib as an internal standard (IS). The electrospray ionization in a positive mode was used for the sample ionization in a multiple reaction monitoring (MRM) mode. The precursor to the production ion transition of 477.05 > 279.02 and 441.16 > 84.4 were used in the MRM mode for the detection of ADL and the IS. The optimized mass spectrometry conditions were capillary voltage (0.5 kV), source temperature (150 °C), desolvation temperature (350 °C), desolvation gas (nitrogen) flow rate (600 L/h) and collision gas (argon) flow rate (0.13 mL/min). The cone voltage and collision energy of ADL and the IS were optimized to 30 and 48 V and 36 and 40 eV, respectively .v/v) was used at a flow rate of 0.3 mL/min. The total run time of the analysis was 2.5 min only. The calibration curves were linear between the concentration ranges of 1.32–625 ng/mL with a lower limit of quantification (LOQ) of 1.32 ng/mL. All of the validation parameters were within the acceptable limits as per the guidelines for a bioanalytical method validation.The chromatographic separation was performed on an Acquity UPLC BEH column . The mobile phase comprising of acetonitrile and 15 mM ammonium acetate in a composition ratio of 80:20  was then spiked into each tube except the blank samples and vortex-mixed properly. One mL of ethyl acetate was transferred to each tube followed by cold centrifugation at 4500× n = 6 each); one group received a normal suspension of pure ADL prepared in 0.5% w/v carboxyl methyl cellulose and the other group received the optimized ternary complex (KDB) (20 mg/Kg). The blood samples (0.5 mL) were collected in a pre-heparinized tube at different time intervals . All blood samples were centrifuged at 4500× g for 5 min and plasma was harvested and placed in a deep freezer maintained at 80 ± 10 °C until further UPLC-MS/MS analysis.The pharmacokinetic study of the newly developed and the optimized ternary complex (KDB) was compared with the normal suspension of ADL in male wistar albino rats. The experimental protocol was reviewed and approved by the Research Ethics Committee  and all applicable international guidelines including the Animal Care and Use Committee of the Prince Sattam Bin Abdulaziz University were followed. The twelve rats (weight 180–210 gm) were received from the Animal Care Centre, College of Pharmacy, Prince Sattam Bin Abdulaziz University, Alkharj, Saudi Arabia and were kept under the recommended conditions with access to food and water ad libitum. Before the experiment, all animals were allowed to acclimatize to the normal conditions for one week. The rats were divided into two groups  and the results were expressed as a mean ± standard deviation (SD). A non-compartmental pharmacokinetic model was used to calculate the maximum concentration (Cmax) and the time to reach the maximum concentration (Tmax), the AUC from 0 to t (AUC0–12) and 0–inf (AUC0-∞), the elimination rate constant (kz), the half-life (T½) and the mean residence time (MRT). An unpaired t-test was used to compare the results between the normal ADL suspension and ADL/β-CD with 1% poloxamer 188 considering In the present study, the complexation efficiency of ADL with β-CD was investigated in the presence and absence of poloxamer 188. Both the binary ADL/β-CD and the ternary ADL/β-CD with 1% poloxamer 188 complexes were prepared by kneading and solvent evaporation methods and were further characterized by aqueous solubility, FTIR, PXRD, DSC, SEM, 1HNMR and in vitro studies. The solubility and release of ADL was remarkably improved (13.1 fold) in the kneaded ternary complex ADL/β-CD with 1% poloxamer 188 (KDB). The pharmacokinetic study revealed that the ADL formulation composed of KDB showed a significant improvement in relative bioavailability (2.71 fold) in comparison with the pure ADL suspension. Thus, the above study demonstrated that the solubilization and bioavailability of ADL can be improved by the formation of an ADL/β-CD complexation in the presence of a third component, which is poloxamer 188.


LOXHD1, a gene that is known to be essential for cochlear hair cell function and associated with nonsyndromic hearing loss in humans and mice. The canine LOXHD1 variant was specific to the Rottweiler breed in our study cohorts of pure-bred dogs. However, it also was present in some mixed-breed dogs, of which the majority showed Rottweiler ancestry. Low allele frequencies in these populations, 2.6% and 0.04%, indicate a rare variant. To summarize, our study describes the first genetic variant for canine nonsyndromic hearing loss, which is clinically and genetically similar to human LOXHD1-related hearing disorder, and therefore, provides a new large animal model for hearing loss. Equally important, the affected breed will benefit from a genetic test to eradicate this LOXHD1-related hearing disorder from the population.Hearing loss is a common sensory deficit in both humans and dogs. In canines, the genetic basis is largely unknown, as genetic variants have only been identified for a syndromic form of hearing impairment. We observed a congenital or early-onset sensorineural hearing loss in a Rottweiler litter. Assuming an autosomal recessive inheritance, we used a combined approach of homozygosity mapping and genome sequencing to dissect the genetic background of the disorder. We identified a fully segregating missense variant in The online version contains supplementary material available at 10.1007/s00439-021-02286-z. Despite the accumulating knowledge, a large number of patients remain without a molecular diagnosis. Thus, there is a need to better understand the molecular underpinnings of hearing loss and the associated genes and variants in cochlear biology and disease pathophysiology.Hearing loss (HL) is the most common sensory impairment and consequently constitutes a major medical issue, affecting 1–3/1000 newborns and becoming more prevalent by age (Morton and Nance MITF)  and tyrosine phosphatase, receptor type Q (PTPRQ) . DNA concentration was determined either with NanoDrop ND-1000 UV/Vis Spectrophotometer or Qubit 3.0 Fluorometer (Thermo Fisher Scientific Inc.). Sample collection was ethically approved by the Animal Ethics Committee of State Provincial Office of Southern Finland (ESAVI/343/04.10. 07/2016 and ESAVI/25696/2020).–8. One of the cases was discarded due to a poor call rate. As recommended by Meyermans et al. . Pre-analytical QC was conducted using PLINK  was also performed with PLINK 1.9  was carried out for one affected dog of the initially identified Rottweiler litter. The exome library was prepared with 140702_canFam3_exomeplus_BB_EZ_HX1 kit with a capture size of 152 Mb from the Roche NimbleGen SeqCap EZ target enrichment design  and small insertions and deletions (indels) was done using the HaplotypeCaller in gVCF mode (v3.7-0), which is combined with gVCFs from our cohort with combineGVCFs and joint genotyping was done by GenotypeGVCFs in Genome Analysis Tool Kit GATK version 4.1 . The products were directly sequenced using the PCR primers on an ABI 3730 capillary sequencer (Life Technologies) after treatment with exonuclease I (New England Biolabs) and rapid alkaline phosphatase (Roche Diagnostics). The Sanger sequence data were analyzed using either Sequencher 5.4 (GeneCodes) or Unipro UGENE v1.32.0  and a reverse primer (5′–TAGTTGCCTGACACCCTGAG–3′) flanking the A sample of 28,116 dogs, including 374 different breeds (Online Resource 7), was submitted for commercial genetic testing at Genoscoper Laboratories  during 2017–2020. Another study sample of 771,864 mixed-breed dogs was screened using Wisdom Panel™  genetic testing, including breed detection assessment, during 2019–2021.n = 2), 5, or 19 months of age, and no auditory response was detected in any of them. However, owners’ observations suggested that the puppies had already been affected by hearing impairment at a few weeks of age. No other clinical signs were observed.Sensorineural bilateral deafness was diagnosed in four Rottweiler siblings  in a litter of ten puppies using brainstem auditory evoked response (BAER) testing. BAER testing was performed either at 4 , a gene that is known to cause hearing loss in humans and mice  was predicted to cause a glycine-to-serine substitution. We assessed the pathogenicity of these variants in silico using two protein prediction tools, PROVEAN and PolyPhen-2. First, PROVEAN predicted both the LOXHD1 and MROH8 substitutions as “deleterious” with a score of − 4.517 and − 3.336, respectively. Similarly, PolyPhen-2 predicted the LOXHD1 variant as “probably damaging” with a HumVar score of 0.992 and the MROH8 variant as “possibly damaging” with a score of 0.550. As MROH8 has been associated with red blood cell volume, body mass index, telomere length and hippocampal atrophy in humans  in the fourteenth PLAT domain of the canine LOXHD1 protein . Of the seven case-specific variants that resided in ROH, two exonic variants were considered for further analyses. First, a G>C missense variant at chr7:44,806,821 in lipoxygenase homology domains 1 . In these species, the glycine residue and several flanking amino acids are fully conserved.LOXHD1 variant, we genotyped it in a cohort of 585 Rottweilers, including the four affected siblings and 581 unaffected dogs. We observed complete segregation of the variant with hearing loss, as all four affected dogs were homozygous for the variant. The unaffected dogs were either heterozygous (n = 33) or wild-type (n = 548). The six unaffected littermates of the probands were either wild-type or heterozygous for the variant. The allele frequency in the population, excluding the affected family, was 2.6% and carrier frequency 5.3%.To validate the LOXHD1 variant to explore its distribution across breeds. All 28,116 tested dogs representing 374 breeds, breed varieties or designer dog mixes were found homozygous for the wild-type allele (Online Resource 7). Finally, the variant was also screened in a larger study sample of 771,864 dogs submitted to genetic testing, including breed detection assessment. A variant carrier frequency of 0.08% and allele frequency of 0.04% were observed in this dataset. Interestingly, six dogs were found homozygous for the LOXHD1 variant. We were able to contact the owners of 4/6 of the homozygous dogs and the owners reported profound hearing loss or deafness in all of them. One of the deaf dogs did not show any immediate Rottweiler ancestry, while one was a purebred Rottweiler and two were mixed-breed with Rottweiler ancestry. Altogether, of the dogs carrying at least one copy of the deafness candidate variant, 63.4% showed evidence of Rottweiler ancestry in their immediate three-generation pedigree going back to great-grandparents, providing further support for a link between this specific breed background and the presence of the variant.An additional sample of dogs submitted for commercial genetic testing was screened for the LOXHD1 variants in humans have been suggested to contribute to autosomal dominant late-onset Fuchs endothelial corneal dystrophy (FECD). For this reason, we assessed the eye examinations performed as a part of the breeding programs and regular health check-ups of the Rottweilers genotyped either homozygous or heterozygous for the LOXHD1 variant. Two of the homozygous deaf dogs had been eye examined healthy, one at 2 years and the other at 8 years old. In addition, eye examination reports were available for 22/33 heterozygous dogs, examined between 1 and 7 years and 4 months old. Three heterozygous dogs were diagnosed with different forms of cataract, which is a relatively common eye disease in Rottweilers. No signs of corneal dystrophy were reported in any of the dogs.According to a study by Riazuddin et al. , heterozLOXHD1 associated with an autosomal recessive congenital nonsyndromic hearing loss in Rottweilers. The variant is rare, yet we confirmed it to be fully segregating with the disease in the breed. This is the first genetic defect identified for NSHL in dogs.We describe here a missense variant in LOXHD1 variant had either congenital or early-onset hearing loss. The owners’ reports suggest that the puppies had at least some hearing impairment already at a few weeks of age. However, the confirmed diagnosis by BAER was acquired later and by that time, the hearing loss was total. Therefore, it is likely that the hearing impairment was congenital and progressed to deafness in a few months, although this remains unconfirmed. The hearing loss in Rottweilers can be defined as nonsyndromic as the affected dogs showed no other consistent clinical features.The dogs with the identified samba mouse line generated in an ethylnitrosourea (ENU) mutagenesis screen, where a Loxhd1 missense variant leads to hearing loss  domains are likely involved in targeting the protein to the plasma membrane    and a p.(Y1541*) variant, had been diagnosed with severe congenital hearing loss at the age of two years  has been observed in one study, suggesting that LOXHD1 could be relevant to the FECD pathogenesis Supplementary file2 (XLSX 10 kb)Supplementary file3 (XLSX 15 kb)Supplementary file4 (XLSX 18 kb)Supplementary file5 (PDF 132 kb)Supplementary file6 (XLSX 14 kb)Supplementary file7 (XLSX 19 kb)Below is the link to the electronic supplementary material.


Despite the increasing prevalence of hearing loss, the cost and psychological barriers to the use of hearing aids may prevent their use in individuals with hearing loss. Patients with hearing loss can benefit from smartphone-based hearing aid apps (SHAAs), which are smartphone apps that use a mobile device as a sound amplifier.The aim of this study is to determine how ear, nose, and throat outpatients perceive SHAAs, analyze the factors that affect their perceptions, and estimate the costs of an annual subscription to an app through a self-administered questionnaire survey of smartphone users and hearing specialists.This study used a cross-sectional, multicenter survey of both ear, nose, and throat outpatients and hearing specialists. The questionnaire was designed to collect personal information about the respondents and their responses to 18 questions concerning SHAAs in five domains: knowledge, needs, cost, expectations, and information. Perception questions were rated on a scale of 1 (strongly disagree) to 5 (strongly agree). Questions about the expected cost of SHAAs were included in the questionnaire distributed to hearing experts.P<.001), and an increased level of hearing impairment resulted in significantly higher points in the needs category (P<.001). Patients expected the cost of an annual app subscription to an SHAA to be approximately US $86, and the predicted cost was associated with economic status (P=.02) and was higher than the prices expected by hearing specialists (P<.001).Among the 219 smartphone users and 42 hearing specialists, only 8 (3.7%) respondents recognized SHAAs, whereas 18% (47/261) of respondents reported considering the use of an assistive device to improve their hearing capacity. The average perception score was 2.81 (SD 1.22). Among the factors that shaped perceptions of SHAAs, the needs category received the lowest scores , whereas the cost category received the highest scores . Age was correlated with the information domain (Outpatients expected SHAAs to cost more than hearing specialists. However, the perception of the SHAA was relatively low. In this regard, enhanced awareness is required to popularize SHAAs. Hearing loss is one of the most common health care problems worldwide. When the World Health Organization started reporting hearing loss in 1985, the number of people with moderate-to-profound hearing impairment was estimated to be 42 million. Furthermore, the number of people with disabling hearing loss reached 466 million in 2018 and is projected to reach approximately 630 million by 2030 [Nevertheless, HA adoption rates are extremely low. Globally, only 17% of those who need appropriate hearing rehabilitation use HAs . In addiSHAAs were originally developed to mimic conventional HA devices. SHAAs refer only to the software installed on a mobile device for hearing support, which is different from the traditional HA hardware–software complex. SHAAs require wired or Bluetooth headsets or headphones instead of hardware resources. Many free or low-price HA apps are available on the web. Although they enhance hearing capabilities through sound amplification, SHAAs were previously far less sophisticated because they could not exactly fit an individual’s prescribed target gain as could HAs fitted using real-ear measurement . Some SHEasy accessibility is a notable advantage of SHAAs. Users can simply download the app on their smartphones and prepare headsets or headphones for use. SHAAs may particularly help overcome psychological resistance to the use of HAs. Trials with SHAAs showed a reduction in the degree of anxiety and personal distress and increased self-esteem. In addition, reduced stigma or body image of HA users can be expected because of the growing number of individuals who wear headphones with their smartphones . MaidmenThe mobile app market is rapidly growing. As of 2019, about 61% of the global population was able to access the internet from mobile devices, and this number is projected to increase to approximately 79% by 2025 . FurtherWe performed a multicenter survey of 5 general hospital outpatients who use smartphones and hearing specialists, including otology specialists, audiologists, and HA researchers. Before gaining access to a questionnaire, the potential participants were informed about the survey, and those who agreed to participate were asked to fill out the questionnaire under the direction of a health care provider.This study was carried out in accordance with the Declaration of Helsinki on biomedical research for human participants, and the study protocol was approved by the institutional review board of each participating hospital .F=28.5, P<.001 [hearing aids was replaced with smartphone-based hearing aid apps. A total of 18 questions in the questionnaire were reviewed by 42 hearing rehabilitation specialists who participated in the opinion survey, and Cronbach α for each question was recalculated  sociodemographic characteristics, including age, gender, residence, educational background, economic status, and occupation; (2) clinical characteristics, including the recognition of hearing loss and inconvenience level, the presence of tinnitus and inconvenience level, previous experience with PSAPs or SHAAs by the respondent or their family member, respondent’s willingness to use PSAPs or SHAAs, and expected cost of the app; and (3) perception status. In the clinical characteristics section, respondents with hearing loss or tinnitus were asked to assess the degree of their symptoms using a visual analogue scale (VAS). In the perception status section, they were asked to rate 18 questions in 5 categories on a scale from 1 (strongly disagree) to 5 (strongly agree); lower scores indicate poorer awareness. The questions were grouped into 5 categories by similar objectives, which were reviewed by the hearing specialists, allowing the analysis to be simpler and clearer. Questions 1-4 were grouped in the knowledge category, which aimed to evaluate whether respondents were aware of SHAAs as hearing rehabilitation options and how they differed from conventional HAs. Questions 5-6 in the needs category were designed to evaluate whether respondents thought that SHAAs were necessary for hearing discomfort. Questions 7-9 in the cost category were used to identify the influence of price on the decision to purchase, and questions 10-13 were used to evaluate respondents’ expectations regarding the ability of SHAAs to improve hearing capabilities. Finally, questions 14-18 in the information category attempted to determine whether participants had accurate information about how to use SHAAs .The opinion survey for hearing specialists contained questions to determine demographic information such as employment history, educational background, and professional experience (length of career) as well as the expected annual subscription rate for an SHAA and the main selection criteria for HA devices .Korean Won was used as the standard currency in the questionnaire and was converted into US $ in this report (US $1=₩1082.50).P values <.05 were considered statistically significant. The 2-sample 2-tailed t test was used to compare the expected costs between the hearing specialists and potential users. All statistical analyses were performed using R version 3.6.0 .Age, gender, education background, and economic status data were treated as categorical variables. Reference variables were 20-39 years for age, male for gender, middle school graduate for educational background, and 1 for economic status. VAS score of hearing loss and VAS score of tinnitus were regarded as continuous variables. Linear regression models with the perception level and the expected annual subscription rate as response variables were applied. Robust variance estimation was used for SEs and CIs. Age, gender, educational background, economic status, VAS score of hearing loss, and VAS score of tinnitus were used as explanatory variables in the regression models. Bonferroni-corrected A total of 98.6% (219/222) of respondents’ answers were analyzed after the survey responses of 3 participants with a survey completion rate <50% were excluded.The clinical characteristics of the participants are presented in When asked about SHAAs, 21.5% (47/219) of respondents stated that they had considered using an assistive device for hearing loss, but only 3.7% (8/219) respondents knew the difference between traditional HAs and SHAAs. Only 0.9% (2/219) of respondents had experience with an SHAA. However, 26.5% (58/219) of respondents expressed a willingness to use an SHAA in the future.A total of 42 responses were received from the hearing specialist group, which comprised 29 (69%) otologists and 13 (31%) audiologists. The average number of years working in this profession was 11.83 (SD 7.81) years. A total of 40% (17/42) of respondents had a bachelor’s degree, followed by 33% (14/42) of respondents with a master’s degree and 26% (11/42) of respondents with a doctorate degree.The overall score of awareness of SHAAs of the 219 respondents was 2.81 (SD 1.21). Among the 5 categories, the needs category received the lowest score of 2.02 (SD 1.42), whereas the cost category ranked first with a score of 3.29 SD 1.14; .In the opinion survey of hearing specialists, the main consideration factor for recommending an SHAA was basic performance , followed by price  and additional functions . In addition, noise reduction and the number of channels were mentioned by 1 respondent each.P=.05) in this age group. Meanwhile, in the ≥60-year group, there was a remarkable positive correlation between the total SHAA perception score and age in comparison with the 20-39–year group (P=.002), and there were also strong associations between the information and perception scores among the 5 categories . The presence of hearing loss was strongly associated with the needs category (P<.001), but there were no significant associations with the other categories. The degree of hearing loss indicated by the VAS score was closely related to the total scores (P=.001) and needs . The VAS score for tinnitus did have significant associations with SHAA perception , whereas it was not significantly associated with other categories . Analyzed by age group, the average expected prices were US $97.37 (95% CI 75.10-119.54), US $78.98 (95% CI 63.46-94.41), and US $86.47 (95% CI 62.91-109.93) in the 20-39–year group, the 40-59–year group, and in the ≥60-year group, respectively . The exptegories .The experts’ average expected cost for an annual subscription to a premium version app was US $32.48 (95% CI 17.81-47.24), and 33% (14/42) of respondents answered that the app should be available at no cost. As for an entry-version app, the expected cost for an annual subscription was US $9.69 (95% CI 2.68-16.70) on average, and 71% (30/42) of respondents expected this app to be provided free of charge.P<.001). A total of 45% (19/42) of respondents among the potential users were not willing to pay for the app, which was much lower than the percentage of the hearing specialist group who thought the SHAA should be provided for free (P<.001).The average cost for an annual subscription expected by potential users was markedly higher than that expected by hearing specialists  of respondents were aware of the differences between SHAAs and conventional HAs. In addition, only 0.9% (2/219) of the respondents had experience using an SHAA. These results indicated an extremely low level of perception regarding SHAAs. However, it is noteworthy that 26% (57/219) of the respondents stated their intention to consider using SHAAs after they obtained information about SHAAs during the survey. This suggests that increased awareness of SHAAs may lead to their use by more individuals with hearing loss.As the perception of SHAAs was more meaningful to people with hearing problems than to the general population, the survey was conducted for ear, nose, and throat outpatients and resulted in a relatively high proportion of participants with hearing loss or tinnitus. There was also a strong association between the perception of SHAAs and the age and degree of hearing loss. The amount of information increased with age, whereas gender showed no relationship with the information. Although the degree of hearing loss influenced the purchase of an SHAA, the level of tinnitus was not related to the perception of SHAAs. These findings suggest that the demand for hearing rehabilitation devices increases with age and the development of hearing loss, indicating the necessity of providing further relevant information to elderly individuals with hearing impairment.The expected cost was associated with the economic status. The prices that respondents were willing to pay for SHAA were relatively high in the 20-39–year group and the >60-year group. This is perhaps because the younger generation group would like to improve their own or their parents’ hearing capacity, and the older adult group faces more inconvenience from hearing loss. In addition, price was regarded as one of the most crucial factors determining whether or not to purchase an SHAA. Respondents anticipated more advanced features with an increase in price. The average expected cost for an annual subscription to an SHAA was higher than that expected by the hearing specialists. We assumed that those with hearing impairment were willing to pay a higher price than expected by specialists because of the effect of hearing loss on their quality of life. Furthermore, the expected cost was higher than the actual price of Petralex , one of the most expensive SHAAs on the market . It is nSmartphone-based mobile health is widely used for diagnostics and therapy  and alsogateway products to more sophisticated devices, such as conventional HAs [SHAAs have several advantages. First, SHAAs range in price from free to US $70, and are therefore cheaper than conventional HAs overall. SHAAs are therefore likely to substitute for traditional HAs . Second,onal HAs .South Korea’s gross domestic product per capita is US $32,310, ranking South Korea 28th across the globe . In partHowever, it should be noted that the effectiveness of SHAAs has not been fully proven. Amlani et al  recommenPrevious studies have demonstrated that SHAAs can improve hearing performance in patients with and without hearing loss ,40. MostOne strength of this study is that it is the first study to measure the perception of SHAAs. These findings are expected to pave the way for more surveys regarding awareness of other hearing rehabilitation devices. In addition, because of the multicenter nature of this study, our findings are generalizable to a broad population. According to a survey conducted by the government, the urban population of South Korea is approximately 90% of the total population, which is similar to the population distribution in our results  .One limitation of this study is that because no standardized questionnaires are available to evaluate perceptions of hearing rehabilitation devices, we modified the questionnaire of a preceding study that investigated awareness about HAs . As thisIn addition, the participants in this study were younger than those of known typical HA seekers . In addiFurthermore, our findings do not provide insight into the efficacy of SHAAs in remediating hearing loss; our focus was primarily on the perception of SHAAs. Thus, clinical validation of the effectiveness of the SHAA is required .SHAAs are an alternative hearing rehabilitation option for smartphone users with hearing loss who have no access to appropriate hearing rehabilitation devices because of their high costs. However, the perception of SHAAs was very low. Age and degree of hearing loss were correlated with perception scores. Potential users estimated the cost of an SHAA as approximately US $86 for a 1-year subscription. Those with hearing loss and requiring hearing rehabilitation were willing to pay a higher price than what the hearing specialists expected the price to be. In addition, a higher economic status was associated with an increased willingness to pay higher prices. Considering that a large portion of respondents showed interest in SHAA after obtaining information from the survey, enhancement of perception of SHAAs is likely crucial to expand their market base.


The use of illegal drugs may be a risk factor of hearing loss. However, very few studies with large sample size have investigated the relationship between illegal drug use and hearing loss. Therefore, to evaluate the association between illegal drug use and hearing loss, this cross-sectional population-based study collected data from the US National Health and Nutrition Examination Survey 2011. The study included 1772 participants aged 20 to 59 years who underwent the Drug Use Questionnaire and Audiometry Examination. Of the 1772 participants in this study, 865 were men (48.8%) and 497 were illegal drug users. The mean (SD) age of the patients was 40.0 (11.4) years. After considering age, sex, and comorbidities, the participants who used illegal drugs were found to have higher risks of high-frequency hearing loss (adjusted odds ratio (OR), 1.69; 95% confidence interval (CI), 1.35–2.10) and overall hearing loss  as compared with the nonusers. In the second analysis, the participants who used ≥ 2 types of illegal drugs were associated with higher risks of high-frequency hearing loss  and overall hearing loss . In the third analysis, cocaine use was associated with increased risks of high-frequency hearing loss  and overall hearing loss . The adjusted OR for overall hearing loss in the methamphetamine users was 1.54  as compared with that in the nonusers. This study shows that illegal drug users might have a higher risk of overall hearing loss than nonusers. In addition, the analysis results demonstrated that the more kinds of illegal drugs used, the higher the risk of hearing loss. Further experimental and longitudinal research studies are required to confirm the causal relationship between illegal drug use and hearing loss. Hearing loss is a common chronic disorder and affects approximately 466 million people worldwide ,2. In adIllegal drug use is recognized as a major public concern and may contribute to health and social problems ,11,12. IHowever, although illegal drugs and hearing loss are all major public concerns, to date, the existing population-based research studies with large sample sizes that investigated the association between the use of illegal drugs and hearing loss are still insufficient. Owing to the ethical issue and difficulties in recruiting participants, most previous studies were only basic research and case reports. Therefore, this study aimed to use large-scale data from the US National Health and Nutrition Examination Survey (NHANES) to determine the actual association between illegal drug use and hearing loss.https://www.cdc.gov/nchs/nhanes/index.htm) (accessed on 12 November 2021) for data users and researchers throughout the world. As the publicly available database did not include the participants’ identifying information, our study was exempt from a full review by the institutional review board.The data used in this cross-sectional study were sourced from the US NHANES. NHANES has been a continuous program of the National Center for Health Statistics (NCHS) for health and nutrition measurements since 1999. The database includes interview and physical examination data. The sample of approximately 5000 persons from counties across the United States that are included in the yearly NHANES investigation is nationally representative. The NHANES research protocols were approved by the research ethics review board of the NCHS, and all the participants provided their written informed consent to the NCHS. These survey data are free, publicly available, and can be directly downloaded on the NHANES website (n = 4500). All missing data in the questionnaires and examinations were excluded (n = 1666). We further excluded respondents who only used cannabis as indicated in the Drug Use Questionnaire (n = 1062) because cannabis is legal in several counties. Finally, 497 participants were defined as illegal drug users for inclusion as the exposed group, and 1275 participants were defined as nonusers for inclusion as the control group. Overall, 1772 participants were included in the final analyses. Illegal drug users were identified by using the following questions from the Drug Use Questionnaire: “Ever use any form of cocaine?” “Ever used heroin?,” and “Ever used methamphetamine?”. Participants who responded “yes” to any of the three previous questions were identified as illegal drug users in this study. Flow diagram for the selection of sampled patients was displayed in This study first included respondents from ages 20 to 69 years who had received both the Drug Use Questionnaire and Audiometry Examination during the period of 2011–2012 . We compared the exposed and control groups in terms of demographic characteristics and comorbidities by using the chi-square test, independent t test, mean, and standard deviation. We further used logistic regression models to evaluate the association between the two groups and calculated unadjusted and adjusted odds ratios (ORs) and 95% confidence intervals (CIs). CI was defined as a range of values in which the true parameter was expected to fall. Additionally, age, gender, race, hypertension, ear infection, and diabetes were all adjusted in the logistic regression models. In all the statistical analyses, p = 0.012), sex (p < 0.001), race (p < 0.001), hypertension (p < 0.001), and ear infection (p < 0.001), but not in terms of the incidence of diabetes (p = 0.954). This study further classified the illegal drug users into two groups as follows: (1) those who were using 1 type of illegal drug and (2) those who were using ≥ 2 types of illegal drugs. The relevant findings presented significant differences between the groups  in terms of age (p < 0.001), sex (p < 0.001), race (p < 0.001), hypertension (p < 0.001), and ear infection (p < 0.001), but not in terms of the incidence of diabetes (p = 0.917). We considered these factors in the following regression models.This cross-sectional study consisted of 1772 people aged ≥ 20 years. The mean age of all the participants was 40.0 ± 11.4 years. p = 0.012), sex (p < 0.001), race (p < 0.001), hypertension (p < 0.001), and ear infection (p < 0.001), but not in terms of the incidence of diabetes (p = 0.954). This study further classified the illegal drug users into two groups as follows: (1) those who were using 1 type of illegal drug and (2) those who were using ≥ 2 types of illegal drugs. The relevant findings presented significant differences between the groups  in terms of age (p < 0.001), sex (p < 0.001), race (p < 0.001), hypertension (p < 0.001), and ear infection (p < 0.001), but not in terms of the incidence of diabetes (p = 0.917). We considered these factors in the following regression models.This cross-sectional study consisted of 1772 people aged ≥ 20 years. The mean age of all the participants was 40.0 ± 11.4 years. This study used the US NHANES data and found that illegal drug users had higher odds of developing overall and high-frequency hearing loss than nonusers even after adjustment for their demographic characteristics and comorbidities. This study further demonstrated that the more kinds of illegal drugs used, the higher the risk of hearing loss. In addition, the relevant findings include the possible association of cocaine use with high-frequency and overall hearing loss. Methamphetamine use may also be related with overall hearing loss after considering confounders.To date, only a few population-based studies with large sample sizes have investigated the relationship between illegal drug use and hearing loss. Most previous human studies only attempted to demonstrate the relationship between alcohol and cigarette use and hearing loss ,25,26,27Although the exact mechanisms of hearing impairment and the association between the use of illegal drugs and hearing loss are hypothetical and unclear, there are two probable reasons regarding hearing loss and its types. First, it is plausible that neurotransmitters may play roles in the association between illegal drug use and hearing. To date, many studies have found that neurotransmitters may affect audition ,17,31. ASecond, the types of hearing loss are related to lesions of different cochlear sites. We found that the type of hearing loss more frequently associated with illegal drug use was high-frequency hearing loss. In this study, the participants who used illegal drugs had a higher risk of high-frequency hearing loss than the nonusers . A higher risk of high-frequency hearing loss was found in both the users of 1 type of illegal drug  and users of ≥2 types of illegal drugs  than in the nonusers. Cocaine use was also observed to be associated with high-frequency hearing loss. The pure tone audiograms of the previous case reports also showed similar trends ,28. For This study has some limitations. First, the study focused on illegal drugs, including cocaine, heroin, and methamphetamine. As this study used questionnaires to identify illegal drug users, the number of illegal drug users might have been underestimated. Second, the self-reported questionnaire may cause information bias. In addition, recall bias may be caused by the participants’ incorrect recall of using illegal drugs. Third, owing to the cross-sectional design of the study, we could not determine the causal relationship between hearing loss and the use of illegal drugs.In conclusion, this study demonstrated the association between the use of illegal drugs and hearing loss, including high-frequency and overall hearing loss. In addition, we found that the risk of overall hearing loss for the users of ≥2 types of illegal drugs was high. Our study also revealed that cocaine and methamphetamine users had higher risks of hearing loss than the nonusers. The relevant information could provide some evidence for policy makers and medical professionals. However, further experimental and longitudinal research studies are required to confirm the causal relationship between illegal drug use and hearing loss.