Supplemental Digital Content is available in the text. If the benefits of newborn hearing screening and early intervention are to be fully realized, there is a need to understand the challenges of hearing aid management in infants. The aim was to investigate longitudinal changes in hearing aid use and hearing aid management challenges in very young infants.Eighty-one primarily female (99%) caregivers of infant hearing aid users completed a questionnaire about hearing aid management experiences, first when their infants were 3 to 7 months old (1 to 6 months after hearing aid fitting) and again at 7 to 21 months of age. Hearing aid data logging was compared with caregiver reports of daily use for 66 infants.The main hearing aid management challenges reported by caregivers were performing listening checks and troubleshooting. These challenges reduced over the approximately 5-month time period but remained a problem with around a quarter of respondents still not confident or unsure about troubleshooting, and around a third not performing a daily listening check. Mean daily hearing aid use, obtained from data logging, declined significantly over time from 6.6 to 5.3 hours. Further analysis revealed reduced hearing aid use was primarily among infants with profound losses (n = 11). Caregivers overestimated daily hours of use at both time points. Caregivers reported difficulty with the infants pulling out their hearing aids, especially at the later time point.The findings from this relatively large sample of caregivers of young infants, assessed at two time points, revealed significant challenges in hearing aid management, including highly variable daily hearing aid use. Interventions that use behavior change techniques may be needed to ensure intentions are consistently turned into successful actions, if the benefits of newborn hearing screening and early intervention are to be fully realized. The importance of early and effective intervention for children with hearing loss is well established. In England, a national newborn hearing screen has been in operation since 2006. Since the introduction of the screening program, the median age of first hearing aid fitting has reduced from around 26 months to just 2.7 months . Early iThe benefits of early intervention and consistent hearing aid use are well established e.g., . HoweverCaregiver questionnaires have been used to investigate the challenges faced by caregivers of children and infant hearing aid users. Caregivers may lack the required knowledge for good hearing aid management, or may struggle to put this knowledge into practice. In a survey of 349 US audiologists, nearly half (44%) reported they did not routinely teach caregivers all aspects of hearing aid maintenance  and 38% did not routinely show caregivers how to teach other caregivers about hearing aid management . Even whCaregivers may be highly motivated to ensure their babies are wearing functioning hearing aids, but may require specific strategies to develop capabilities and provide opportunities for good hearing aid management and maintenance skills. Behavior change approaches based on the Capability-Opportunity-Motivation-Behavior (COM-B) model have been shown to be potentially useful in adult audiology . The COMThe children in Muñoz et al.’s (2015) study had a mean age of 22 months, and other questionnaire studies have focused on children aged up to 5 to 6 years . If the Caregivers of 103 infants with a hearing loss ranging from mild to profound were recruited from 53 NHS audiology departments across the United Kingdom, between June 2016 and February 2019. The families were recruited as part of a larger longitudinal study investigating the feasibility of using aided evoked potential measures to assess infant hearing. Families received an information pack from their audiologist or teacher of the deaf, and contacted the researchers directly if they were interested in taking part. Ethical approval was obtained from the North West National Research Ethics Service Ethics Committee (reference 172044), and all caregivers gave written informed consent to participate. Caregivers completed a hearing aid management questionnaire and data logging was recorded at two time points, which were opportunistic to coincide with participation in the wider study.Of 103 participants, 17 were excluded from questionnaire analysis due to not fully completing the questionnaire at both time points , and five were excluded due to a different caregiver completing the questionnaire at each time point, leaving a total of 81 participants. Eighty of these 81 respondents were female primary caregivers. These 81 infants had a median age of 5.1 months (range 3.4 to 7.3) at the first time point and median age 10.2 months (range 7.4 to 21.4) at the second time point. For premature infants, all ages are reported corrected. The median age at hearing aid fitting was 1.8 months (range 0.6 to 5.0 months), and the median duration of hearing aid use at the initial time point was 3.4 months (range 0.8 to 6.3 months).Thirty-three of the 103 infants were excluded from data logging analysis due to data logging being unavailable in at least one of the two sessions. A further three were excluded because the data logging appeared unreliable  and a further one was excluded due to not having provided a subjective response about hearing aid use. One case was included in the analysis with an average daily usage of 16.9 hours as the caregiver indicated that hearing aids were worn all day, including for naps. This left 66 cases for data logging analysis (median age 5.1 months (range 3.0 to 7.5) and 10.1 months (range 7.4 to 21.4) at the two time points). Twelve participants included in data logging analysis were not included in the questionnaire analysis.http://links.lww.com/EANDH/A738), whereby questions were removed if they were not relevant to the aims of the study. For example, questions on affordability of batteries and ear moulds were removed as these are free at the point of delivery in the UK National Health Service. The resulting questionnaire covered three main areas: (1) Information provided by the audiology clinic ; (2) hearing aid management skills ; and (3) factors influencing hearing aid use . Likert-scale questionnaire responses were given a numeric value from 1 (strongly agree) to 5 (strongly disagree). There were four additional free-text questions for caregivers to provide further details about the three main areas and any other comments they had on their experience of their child’s hearing aid use. Caregivers were also asked how often their child wore their hearing aids: all waking hours, most of the day (over 5 hours), some of the day (1 to 5 hours), or hardly at all (less than 1 hour).An adapted version of Muñoz et al.’s (2015) PHAMI was used , corrections were made for multiple comparisons based on the number of questions within each category of questions .Where paired-samples For the four free-text questions a qualitative content analysis  was initt-test. To compare data logging with caregiver reported use, data logging values were converted into categorical data  and compared with the equivalent categories used in the caregiver-reported data. The categorical data were assigned a numerical value (1 to 3), and agreement between categorical scores for subjective (caregiver) and objective (data logging) values was determined using paired-samples t-tests for the n = 66 participants with both data logging and caregiver report available.Differences in data logging between the two time points were compared using a paired-samples t(79) = 1.96, p = 0.054). Figure Figure The response categories emerging from the question “What other information would have been helpful?” are summarized in Table t(79) = 2.87, p = 0.005). Figures t(79) = 5.51, p < 0.001). Figure t(76) = 4.06, p < 0.001). Figure t(67) = 5.08, p < 0.001). Figure t(74) = 4.45, p < 0.001). Figures Figure Response categories emerging from the question: “What other support would have been helpful in learning hearing aid management?” are summarized in Table t(62) = 2.73, p = 0.008). Figure t(79) = 3.01, p = 0.004). Figure t(62) = 3.40, p = 0.001). Figure Figure Table A few new categories emerged from the final prompt: “Any other comments about my experiences with my child’s hearing aids.” A large number of caregivers reported positive overall experiences with audiology services and other professionals , and positive experiences of using the hearing aids (“I am grateful for the hearing aids to give my child better hearing to help with development”). Minor categories included: problems attending audiology appointments  and difficulties with public perception/awareness of hearing loss .t(65) = 3.14, p = 0.003). There was a statistically significant correlation between reduction in daily hours’ hearing aid use and four-frequency average hearing threshold, with greater reduction in use being associated with greater degrees of hearing loss . Further analysis revealed that for the n = 11 infants with profound loss (better ear four-frequency average hearing threshold > 95 dB HL), mean daily use reduced significantly from 7.7 hours at the early time point to 4.2 hours at the later time point (t(10) = 4.90, p < 0.001). This remained significant excluding two infants with likely progressive loss between the two time points (t(8) = 3.94, p = 0.004). For the remaining n = 55 infants without profound loss, there was a trend for a decline in use from 6.4 to 5.6 hours, but this did not reach statistical significance (t(54) = 1.87, p = 0.067). For cases where bilateral data logging was available (n = 61) the median absolute difference between the left and right data logging was 0.3 hours at the early time point and 0.4 hours at the later time point, indicating largely bilateral hearing aid use for these infants.Figure Figure t-tests  at the two time points revealed that at both time points caregivers reported significantly greater daily use than was revealed by data logging (3 to 7 months: t(65) = 4.09, p < 0.001; 7 to 21 months: t(65) = 4.06, p < 0.001).Paired-samples r = −0.200, p = 0.147; early time point/factors impacting hearing aid use r = 0.115, p = 0.406; late time point/hearing aid management skills r = −0.090, p = 0.516; late time point/factors impacting hearing aid use r = −0.244, p = 0.075).To investigate whether self-reported hearing aid management skills or factors affecting use (sections 2 and 3 of the questionnaire) were negatively associated with objectively measured hearing aid use, scores from these sections of the questionnaire were summed and correlated with hearing aid data logging, for the 54 participants with complete data logging and questionnaire data. No significant correlations were found . This suggests that such practical demonstrations are helpful to convey this information. Caregivers also wanted to know about their infant’s future. Whilst the ability to predict longer-term outcomes is limited, hearing/educational professionals could perhaps provide more information about educational support services in the early months after diagnosis. Caregivers generally reported feeling confident with most aspects of hearing aid management including changing hearing aid batteries (98 to 100%), inserting earmolds (98 to 99%), and cleaning earmolds (88 to 98%). Confidence knowing when new earmolds were needed increased over time, with 82% agreeing they were confident at the early time point and 94% agreeing they were confident at the later time point. As the caregivers taking part in this study are likely to be highly motivated, it is possible that these results overestimate caregivers’ success with hearing aid management; other caregivers may struggle more with hearing aid management tasks. In general, caregivers who are struggling more with their child’s hearing loss and hearing instruments may be less motivated to participate in research.There are some areas in which caregivers reported lower confidence when the child was younger and, although there was some improvement over time, these remained a challenge. An example of this was daily listening checks using a stethoscope. At the early time point, around a third of caregivers did not agree that they knew how to do a listening check or how to teach others to do a listening check and less than half carried out the check daily. Caregivers felt more confident about listening checks when their infant was older. Listening checks have previously been reported by caregivers as something they do not feel confident doing , 2019. TAnother area that caregivers were unsure about at both time points was troubleshooting, with over half of caregivers answering “unsure” or disagreeing that they had the skills when their infant was 3 to 7 months. While significantly more caregivers reported they knew how to do this when their babies were 7 to 21 months, over a quarter still reported that they were unsure or disagreed. This is consistent with Two primary areas that caregivers felt were affecting their infant’s hearing aid use, across both age groups, were frequent acoustic feedback and the hearing aids not working correctly. Although a quarter of caregivers reported that hearing aids not working correctly did affect hearing aid use, we do not know to what extent they felt it affected use . Faulty hearing aids are something that can be easily addressed if caregivers have appropriate skills, clear information, and access to services. These findings suggest that caregivers need better support to be able to troubleshoot the aids themselves, carry out basic maintenance, and know how and when to ask for support to get replacement aids or have the problem corrected professionally.Feedback is a slightly trickier issue, especially when the infant is very young and growing rapidly out of their earmolds. When asked in a free-text question what other issues affect hearing aid use, poorly fitting moulds, and the time it takes for new moulds to arrive were cited as problems by a number of caregivers. Feedback suppression features on the hearing aids should help to reduce the amount of feedback whilst enabling the hearing aids to continue giving a high level of gain, however this feature can distort sound, so regularly replacing ear moulds to reduce feedback is important  particulAnother two areas that many caregivers agreed affected their infant’s hearing aid use relate more to family life than to the hearing aids themselves. These included distractions and needs of other children, and difficulty getting into a set routine. When the infants were younger, one-fifth of caregivers reported that the needs of other children affected hearing aid use, and this increased to nearly one third when the infants were 7 to 21 months. Conversely, when the infant was older, and the families had more experience with hearing aids and hearing loss, fewer caregivers (16%) agreed that getting into a routine affected hearing aid use compared to when the infant was younger (31%). These are difficult areas for hearing professionals to address, as family life and the needs of other children will always continue after diagnosis of a hearing loss. Teachers of the Deaf and other early intervention professionals are better placed than audiologists to offer support in these areas as they have more involvement with the family in their home. The process by which Teachers of the Deaf could provide this support and how hearing aid management fits in with caregivers’ existing routines is a topic for future research.When asked in a free-text question what other reasons make their child’s hearing aid use difficult, a great many caregivers mentioned their child removing their hearing aids. This is despite the majority of caregivers agreeing or strongly agreeing that they knew how to keep the hearing aids on their child. More caregivers mentioned this when their infant was 7 to 21 months compared to 3 to 7 months, although this was by far the most cited difficulty at both time points. This is in keeping with previous studies, such as There was a wide range of daily hearing aid use in both age groups (0 to 16.9 and 0 to 13.5 hours), but hearing aid use was generally low compared with the number of hours an infant is expected to be awake. This is in line with previous research showing that most caregivers struggle to achieve consistent hearing aid use during all waking hours until their children are around 28.5 months, largely due to lower use in situations where infants cannot be closely monitored, for example during car journeys . Data loAn alternative way to record hearing aid usage is by percentage of waking hours, which may be a better way of recording longitudinal changes, as it accounts for developmental changes in sleeping patterns. This is the approach used in the PEACH  questionnaire , which aCaregiver reported hearing aid use was significantly higher than that recorded from data logging at both time points. This is in keeping with other research which has similarly found that caregivers overestimate hearing aid use and indicates caregiver self-report should not be solely relied upon ; 2015. ONo correlation was found between hours of daily hearing aid use and self-reported hearing aid management skills or factors having a negative impact on hearing aid use. To address the areas of difficulty with hearing aid management and factors affecting hearing aid use identified in this study, a more targeted behavior change approach may be useful. Having a course/class and being pointed towards other sources of information was suggested by the caregivers when asked what else might be useful in learning hearing aid management . Recent survey data, since the COVID-19 pandemic, has shown keenness from pediatric audiologists and caregivers to engage in, and expand the scope of, remote care practice . There iThe COM-B behavior change model may be particularly useful for this type of intervention. In this model, “capability,” “opportunity,” and “motivation” are the key factors that need to be influenced in order to change behavior . The behOne limitation of the study is that predominantly (99%) female caregivers were sampled. It would be valuable to determine and compare the experiences of both female and male caregivers of infants from a very young age. Future research on behavior change techniques would need to consider the experiences of all caregivers, potentially including extended family in multi-generational homes . The timThis study identified challenges with hearing aid management for caregivers of young infants, both soon after hearing aid fitting, and after some months of hearing aid use. Caregivers reported difficulty with both troubleshooting aids and infants pulling aids out, both of which can impact daily hearing aid use. By better understanding the challenges facing caregivers, hearing professionals can improve the support they can offer. Caregivers were generally positive regarding information provided at the time of hearing aid fitting, but ongoing challenges in hearing aid management and the reduction over time in daily hours of hearing aid use highlight the need for a new approach to ensure that the benefits of early identification of hearing loss are fully realized.The authors are grateful to all the families who took part and all the audiologists and teachers of the deaf around the UK who helped to recruit families to the study. Thanks to Jo Brooks and Rhiannon Morgan for contributing to data collection and to Michael Stone for support on the project. Thanks to Karen Muñoz for permission to use and reproduce sections of the PHMI questionnaire.


To explore whether patients with unilateral idiopathic sudden sensorineural hearing loss (uISSNHL) have normal speech in noise (SIN) perception under different masking conditions after complete recovery of pure tone audiometry. Eight completely recovered uISSNHL patients were enrolled in ISSNHL group, while 8 normal-hearing adults matched with age, gender, and education experience were selected as the control group. Each group was tested SIN under four masking conditions, including noise and speech maskings with and without spatial separation cues. For both ISSNHL and control groups a two-way ANOVA showed a statistically significant effect of masking type (p = 0.007 vs p = 0.012). A significant effect of perceived spatial separation (p < 0.001 vs p < 0.001). A significant interaction between masking type and perceived spatial separation was found (p < 0.001 vs p < 0.001). A paired sample T-test showed that the SIN perception of the control group was statistically significant lower than that of ISSNHL patients only under speech masking without spatial separation cues (p = 0.011). There were still abnormalities in the auditory center shortly after complete recovery in the ISSNHL group (within 2 weeks). However, the auditory periphery and higher-level ability to use spatial cues was normal. The pathogenesis of ISSNHL is still unknown, although a number of hypotheses have been proposed, including viral infection, vascular compromise, chronic inflammation, immunological diseases3, cochlear membrane rupture4, inner ear cell stress reaction5, hemorrhage of inner ear6, migraine7. Fortunately, about one-third of ISSNHL obtains complete recovery of hearing either spontaneously or after appropriate interventions8.Idiopathic sudden sensorineural hearing loss (ISSNHL) is defined as at least 30 dB sensorineural hearing loss in three sequential frequencies within 3 days with no identifiable cause9. Clinically, among patients with uISSNHL who have recovered completely (ISSNHL group) based on pure tone audiometry after treatment, some still complain of difficulty in speech perception in daily life. Previous studies on temporary hearing loss caused by noise exposure have found that despite the normal hair cell populations, temporary threshold shift caused by noise exposure can result in a permanent loss of low-spontaneous-rate auditory nerve fibers and reduction of auditory brainstem response wave-I amplitudes may affect SIN10. Although there have been some studies on the SIN ability of patients after sudden deafness10, to our knowledge, there have been no previous studies taking into account different mask conditions in the uISSNHL patients following complete recovery of pure tone hearing threshold.At present, the standard of hearing recovery of ISSNHL is mainly based on pure tone audiometry, however, the result of pure tone audiometry and speech recognition in quiet environment cannot fully reflect the auditory ability in daily life, which is full of reverberation and background noise11. Two types of maskers resulted in difficulties of SIN perception: noise masking and speech masking14. Noise masking is the difficulty when the auditory peripheral representation of the target sound is overwhelmed by competing sounds (when the masker are noise sounds and target is speech sound), originating primarily in the auditory periphery; speech masking refers to difficulty in the higher-order selective processing of audible target sounds due to perceptual/sensory similarities between the target and the masker (when both the target and masker are speech sounds), which was originated from the auditory central level. The role of higher-level cognitive abilities is important in speech recognition, the SIN perception performance is better when there is perceived spatial separation between targets and maskers. For example, the threshold of signal-to-noise ratios in speech perception is about 4–9 dB lower because of subjective 90-degree spatial separation 13. These results proved that SRM is one of the higher-level cognitive functions to improve speech intelligibility.SIN perception is a complicated multifaceted process. It is established that peripheral hearing loss, declined central auditory processing and impaired cognitive abilities are critical predictors to the deficit in understanding SINThe present study is to investigate whether pure tone audiometry completely recovered uISSNHL patients have normal behavior in speech perception under noise masking and speech masking with or without using a perceived spatial separation paradigm, including noise and speech maskings with (noise masking’s separation condition and speech masking’s separation condition) or without spatial separation cues (noise masking’s colocation condition and speech masking’s colocation condition).15; (4) Patients with middle ear disease, retro cochlear disorders, and congenital deafness had been excluded. The clinical characteristics of the ISSNHL group are shown in Table Eight uISSNHL patients  met the following criteria were enrolled in the present study as the ISSNHL complete recovery (ISSNHL) group: 1) 18–65 years old; (2) acute unilateral SSNHL (≥ 30 dB) in three sequential frequencies of 0.25, 0.5, 1, 2, 3, 4, and 8 kHz compared to the healthy ear; (3) After treatment according to the Chinese sudden deafness guideline , 0.25, 0.5, 1, 2, 3, 4, and 8 kHz hearing defined as final hearing level better than 25 dB  or reached healthy side hearing level (2/8) 18–65 yeAudiometric measurements were obtained following the procedure recommended by the Chinese Acoustics Standardization Committee, using a Astera  Unity PC audiometer transducer in a sound-attenuating booth. Hearing thresholds had been obtained for air and bone conduction at 0.25, 0.5, 1, 2, 3, 4, and 8 kHz Separately. Pure-tone audiometry had been performed every 5–7 days. The patients participated the study within two weeks when their pure-tone audiometry recovered back to normal hearing (≤ 25 dB) or reached healthy side hearing level.16 were used as target ones which were translated from English nonsense sentences developed by Helfer19. For instance, the English translation of a Chinese nonsense sentence “一些条令已经翻译我的大衣” is “Some rules had translated my coat” 17. Target sentences with a naturally stable rate were spoken by a young female talker  at an average rate of 5.4 syllables/s (with the standard deviation of 0.7 syllables/s), while the duration of a sentence was about 2–3 s. The noise masker was a stream of steady-state speech-spectrum noise, whose spectrum was representative of the average spectrum of target sentences. The speech masker was a 47-s loop of digitally combined continuous recordings for Chinese nonsense sentences, spoken by two different young female talkers .A set of special Chinese nonsense sentencesPatients from the ISSNHL group and control group were tested bilateral  SIN perception of various signal-to-noise ratios (SNRs) under speech and noise masking conditions. Each participant has to complete four consequent levels of SNRs . In addition, the benefits of spatial separation cues under these two types of masking were analyzed. Namely, the experiment had four within-subject factors: masker type , the side from which participants perceived target , perceived spatial separation  and SNRs . Besides, there was a between-subject factor: group . Fifteen target sentences were used in each condition, and 480 trials totally were used for each participant.This cohort research was approved by the Peking University People’s Hospital Research Ethics Committee  (2021PHB147-001). Written informed consent for participating this experiment was received from all participants (including control group as well). And all methods were performed in accordance with the relevant guidelines and regulations.A logistic psychometric function (Eq. ) was fitThe release amount of spatial unmasking was calculated as the difference in the threshold between separation and colocation, for the noise masker and speech masker separately. Larger spatial release amount indicates larger cognitive benefit from spatial attention and better binaural auditory processing to the target in perceiving SIN.SPSS software  was used for statistical analysis. Categorical variables were analyzed by the chi-squared test. Binary logistic regression models were further to explore the results of univariate analyses. In all analyses, p < 0.05 was considered as statistical significance.8 uISSNH patients’ clinical and epidemiological characteristics are shown in Table In view of the situation of unilateral hearing loss in ISSNHL patients, there were two kinds of perceived target speech locations, left and right. This within-subject factor can help us to pursue whether the SIN perception ability of the uISSNHL patients has lateral bias. The mean SINs threshold (μ) of two groups under each condition was calculated as the Fig. For ISSNHL patients, a two-way ANOVA showed a statistically significant effect of masking type , a significant effect of perceived spatial separation , a statistically significant interaction between masking type and perceived spatial separation  shown in Fig. For control group, a repeated measures analysis of variance ANOVA showed a significant main effect of masking type , a statistically significant effect of perceived spatial separation , a significant interaction between masking type and perceived spatial separation  as show in Fig. Figure Besides, a paired sample T test showed that there were no significant differences of SRM between ISSNHL patients and control under noise masking  or speech masking . Moreover, the effect of spatial release from speech masking was statistically significantly higher than that from noise masking for ISSNHL patients , just as well as control group . These results indicate that ISSNHL patients could benefit from spatial separation cues, especially under speech masking, and their ability of spatial release was the same as their normal hearing peers  loss on hearing have been well described in several species across a number of studies, as it can result in increased hearing thresholds of associated frequencies and decline of frequency selectivity. In contrast, damage to inner hair cells (IHCs) and auditory nerve fibers are difficult to be detected, for they can only affect the threshold sensitivity when exceeding 80% are damaged. Fortunately, the SIN perception tests are sensitive to moderate to severe IHCs’ damage, so in this study, it was used to test the function of IHCs21. In this study, there was no statistically significant difference in SIN perception between these two groups under noise masking, suggesting that the hair cell function of the ISSNHL group was similar with that of the control group. Although some studies have shown that there may be hidden loss of hair cells that cannot be recognized by psychoacoustic tasks22, in this study, it is illustrated that the inner ear function of the ISSNHL group was equivalent to that of the control group, which suggested that the function of auditory periphery had recovered completely.Noise masking which is interference produced in the spectrum of noise, originating primarily in the auditory periphery. The cochlea is still considered to be the most probable lesion site of sudden deafness23 found that a measure named QuickSIN (Quick Speech in Noise test) that syntactically correct sentences with low semantic cues, is more sensitive to performance difference between normal hearing and hearing impaired groups than the BKB-SIN  and Hearing In Noise Test (HINT), which use meaningful sentences. Thus, as a more sensitive measurement, the nonsense sentences in Mandarin Chinese which is described in detail in part method were used in current SIN perception research.In previous research, sentences with no context or sense have been widely used to investigate different groups of people’s speech perception. Wilson et al.13. The damage of sensory system can ultimately lead to changes in neural representation via injury-induced plasticity. In the auditory system, restricted damage to the peripheral hearing can lead to robust plastic changes in the auditory cortex. A large number of studies have described the pattern and time course of plasticity in various cortical and brainstem regions caused by unilateral deafness. That is, even if there is no permanent change in the peripheral hearing threshold, the neural activity of the auditory cortex can be changed26. The neurophysiological evidence of functional reorganization of the central auditory pathway in patients with acute uISSNHL can be found in the very early central processing of transient sounds arriving at the auditory cortex20. The results of this study also suggested that although pure tone audiometry and peripheral auditory function completely recovered, the changes of central auditory function caused by the sudden deafness could not be fully restored in a short period (2 weeks).It has been documented that when the target signal and the masking signal are both speech signals, because the speech masking signal can also activate the speech system related to speech recognition and comprehension, it will interfere with the recognition of the target signal. It is suggested that speech masking starts from the auditory central level13. The performance of speech perception in different types of masking and the ability that separating target signal from masking by using spatial separation cues reflect the central auditory processing and cognitive function. After unilateral hearing loss, learning-induced changes in adult tonotopic map plasticity are traditionally thought to require the precisely-timed involvement of neuromodulators recruited by arousal or attention. In this study, there was no difference in the ability of using spatial cues between the two groups, namely there was no difference in the higher cognitive function.In the process of speech masking, the cognitive processing systems need to use some certain clues, including the visual information such as lip shape and body posture, familiarity with the spatial position and the contents of the speech, and other related information like voice are all related to this cognitive process. It has been well documented that spatially separating the target signal from the masker can improve the recognition of the target signalFirst, longitudinal follow-up can determine whether the central changes of these patients can be improved and whether intervention is needed, which is of greater clinical importance. Secondly, further electrophysiological studies, such as frequency following response (FFR) or auditory brainstem response(ABR), can be carried out on the patients in the follow-up to locate the lesion site of central auditory system. Finally, although there are some deficiencies, the preliminary exploration of this paper confirmed that patients who have complete recovery of pure tone audiometry after uISSNHL do have difficulties in SIN perception, which is worthy of attention, and also provides scientific basis for the treatment of unilateral sudden deafness. In addition, our study sample is relatively small and further study is needed in the future.This study is to explore the SIN perception among uISSNHL patients after complete recovery of pure tone audiometry under different types of masking conditions. The results of this study indicated that with peripheral auditory function completely recovery, there were still some abnormalities in the function of central auditory system, resulting in the impairment of speech perception under speech masking. We speculated that it may be related to the central compensation induced by unilateral sudden deafness, and further follow-up is needed. However, the higher cognitive function of using spatial cues of the ISSNHL group was the normal as the control group.


Invasive species cause enormous costs of over $120 billion to the U.S. economy. Among biological invasions, the invasion by pheretimoid earthworms has gone relatively unnoticed and their invasion imposes yet unknown damage on USA agriculture and horticulture. The main dispersal is with horticultural goods such as plant material and composts. Pheretimoids affect commercially important hardwood forest. With no chemical agents currently certified for earthworm control nor any best horticultural practices, slowing the invasion is difficult.B. bassiana (BotaniGard®) to kill pheretimoid earthworms under greenhouse conditions. Four treatments of B. bassiana were applied: The commercial product as per label, re-cultured commercial B. bassiana, 15 g and 25 g millet grains mycotized with recultured product. In all, three bioassays were conducted in 2 consecutive years with two batches of BotaniGard®.In this study we measured the efficacy of a commercial entomopathogenic fungal isolate of B. bassiana treatments with re-cultured product resulted in greater than 70% mortality within 4 weeks. Mortality was less than 60% when BotaniGard® was used as prescribed by the label. When using 1-year old spores (refrigerated at 4 °C), mortality rates for B. bassiana treatments were less than 20% and not significantly different from the controls. However, B. bassiana still affected the earthworms by slowing their development from juvenile to adult stage.With fresh batches, all B. bassiana was effective against pheretimoid earthworms. Overall, mycotized millet grains did not significantly increase mortality over the re-cultured, directly applied B. bassiana spores. More experimentation is needed to find the mode of action of the re-cultured B. bassiana before investigating ways to improve the efficacy of B. bassiana when applied as prescribed on the label. The damage and economic losses in agriculture and forestry associated with biological invasions in the United State are enormous . Even wiAlthough there are several native North American earthworms’ species in northern North America , most inAmynthas agrestis, A. tokioensis and Metaphire hilgendorfi  Vuillemin in wettable powder formulation has been widely used for the control of many insect pests afflicting horticultural crops in North America. To test the efficacy of B. bassiana on pheretimoid earthworms, three greenhouse assays were conducted with both spore suspensions and mycotized millet grains.With some US states regulating pheretimoids in horticulture, environmentally friendly, easy to use methods that are acceptable in horticulture, must be found. Three co-occurring f litter . The epif litter . SeveralJuvenile pheretimoids were collected at the University of Vermont’s Centennial Woods (CW) Natural Area  in June of 2018 and May and July 2019. At these times pheretimoids are still in their juvenile stage and cannot be identified to species by morphology. In July 2019, the earthworms were fully extended but still had not developed a clitellum. At this site the earthworm community is exclusively pheretimoids , they wePinus strobus Linnaeus), hemlock (Tsuga canadensis (L.) Carriere), and sugar maple (Acer saccharum Marsh). The understory at the site was dominated by jewel weed (Impatiens capensis Meerb.). The likely local origin of the earthworms is a housing development with ornamental and vegetable gardens some 250 m up-slope from the study site where the presence of A. agrestis, A. tokioensis and M. hilgendorfi was confirmed. Collections from CW for another project indicated that A. agrestis was dominant there accounting for 95% of specimens  . The canpecimens . In 2018B. bassiana from the commercial product  on Potato Dextrose Agar  in the dark at 23 ± 2 °C. Liquid culture inoculate was prepared, according to a protocol developed by 8 conidia mL−1 in a 250 mL flask) of B. bassiana from the PDA to one-quarter-strength Sabouraud dextrose broth . Flasks were held on a rotary shaker (150 rpm) at 24 ± 2 °C for 3 days. The treatment strength of 108 conidia per mL was obtained by serial dilution.We cultured pure Panicum miliaceum L.) grains  using a method modified from B. bassiana (−1). All bags were flattened in a tray and autoclaved at 121 °C for 40 min and cooled to ambient temperature. Each bag of autoclaved grains was inoculated with a homogenized mixture of 5 mL of liquid culture broth and held for 14 days at 23 ± 2 °C and a 16:8 h L:D photoperiod. After 10 days when millet grains were entirely covered by B. bassiana, the bags were opened at ambient temperature until a moisture content of <5% was reached.We mycotized organic whole millet  were inoculated with 0.1 mL of the conidial suspension and held in the dark at 25 °C for 24 h. Conidia with germination tubes longer than their width were considered germinated by inspecting 400 conidia per Petri dish . ConidiaBeauvaria bassiana (BotaniGard®) on juveniles of Amynthas spp. The first was conducted in the spring of 2018, the second and third in the spring and summer of 2019, respectively. The assays in the spring of 2018 and 2019 were conducted with the same batch of a BotaniGard® obtained in April 2018. These two assays differed only in the age of the BotaniGard®. For both of these assays fresh cultures were prepared as described above and the age of the juveniles was comparable. For assay 3 in the summer 2019, fresh product was purchased 4 weeks before the trial and, in this aspect, it resembled the first bioassay. However, the age of the juveniles was more mature in the summer 2019 assay. The bioassays were distributed over 2 years because these earthworms are annual species and develop fast so that similar life stages had to be collected over 2 years.Three mesocosm bioassays were conducted to test the effect of the entomopathogenic fungus B. bassiana cultured from the commercial product, 15 g and 25 g of mycotized millet (® suspension prepared according to the label (1 × 1012 spores/mL). Treatments and controls were each replicated 15 times. The suspensions were applied directly to the earthworms before adding them to the pots. Millet was added to the growing medium prior to the earthworms.The treatments used in all trials included two controls  and the following three treatments: suspension of pure d millet . To have50.Experimental units were prepared using 1-gallon pots lined with zippered, fine-meshed laundry bags 11 × 15 inch  to prevent worms from escaping. To each bag were added 1,000 g of organic potting mix  and seven non-clitellated (juveniles) pheretimoid earthworms. The pots were then arranged on a growing bench at the center of a greenhouse. They were watered twice a week until the pots leaked leachate. In 2018, the earthworms were enumerated at the end of a 5-week period. In the summer of 2019, earthworms were counted every 3 to 4 days during the experimental period in order to calculate LTPrior to analysis we used the arcsine-square-root transformation [x′ = asin(sqrt(x))] to normalize mortality data, followed by the Kolmogorov-Smirnov test (KS test) to confirm normality. The arcsin square root transformed mortality data sets for each treatment were normal at the 0.05 level. In all trials, ANOVA was used to test whether there were any differences in mortality data among treatments at the end of the experiments. Tukey’s Honestly Significant Difference (HSD) test was used to test whether the treatments were significantly different from the appropriate control. While statistics was done on arcsine-square-root transformed data, in 50 and LT99:In addition, Abbott’s formula was applied to calculate mortality rates corrected for the control response  to calcuMcorr is the corrected mortality, <TM> is the average observed treatment mortality, and <cM> is the average observed mortality in the control.where Mcorr, Var (Mcorr) was calculated following The variance of n is the number of replicates, Var (TM) and Var (cM) are the variances of the treatment and control mortalities.where 50 and LT99 assuming a logit function. These were estimated using R package Ecotox  for the 15 g mycotized millet and (P < 0.0001) for the 25 g mycotized milled). Likewise, the recultured B. bassiana treatment differed significantly from the control (P < 0.0001). The fungal treatments showed mortalities between 72 ± 6% and 78 ± 6%. Compared with the controls that varied between 22 ± 6% 12 ± 4% and . The comparison between water and BotaniGard® was also not significant (P = 0.939). The comparison between B. bassiana and water was also significant (P = 0.036). Comparison between millet 25 g and mycotized millet 25 g was significant (P < 0.0001). The recultured B. bassiana suspension added directly to the earthworms prior to placing them in the soil had the highest mortality (96% ± 3%) which was significantly different from all other fungal treatments (P < 0.043)  and the untreated control (71% ± 7%).Assay 3 obtained similar results to assay 1 . Compari< 0.043) . Lowest There were no significant differences in mortality in assay 2 between controls and treatments in which 1 year old product was used . MortaliF = 34, P > 0.001). There were significant differences between 15 g mycotized millet (4% ± 8%) and the 15 g millet control (40% ± 29%), and between 25 g mycotized millet (50% ± 30%) and the control with 25 g millet (73% ± 24%). The more millet, the greater the advance from juvenile to adult. And, more earthworms advanced to adult in the control treatments than the fungal treatments.While there was no difference in mortality found for treatments in assay 2 , there wIn assay 1 only earthworms in the control with 25 g millet reached maturity (87% ± 35%).F = 53, P > 0.001). Controls with 15 g and 25 g millet, not mycotized, had the largest number of adults: 75% ± 16% and 76% ± 13% respectively. In contrast, in treatments with B. bassiana occurrence of adults were lower: 2% ± 6% for recultured B. bassiana, 15 g mycotized millet 33% ± 27% for 15 g of mycotized millet, and 26% ± 28%, for the 25 g mycotized millet treatment. The untreated control scored a similar development rate to the fungal treatments (20% ± 21%).In the third assay, in which juveniles were further along in their development, adults were observed in all treatments but with very significant differences among treatments . In contrast, lethal times were longer for BotaniGard® (30.2 and 76.1 days). The more mycotized millet is added, the shorter the lethal times.LTE. foetida caused by fungi, specifically Aspergillus spp., Penicillium spp. and Trichoderma spp.  than pre-adult juveniles collected in late August (assay 3). Untreated millet accelerated maturation of these earthworms. This may be of interest to those who want to grow experimental populations of pheretimoids.The life stage of the earthworms might have also influenced the outcome of this study. Pheretimoid juveniles collected in June were more susceptible to both 50 values in this experiment with earthworms were longer than observed in insect larvae subjected to this control agent. The Pheretimoid earthworm LT50 was between 13 and 22 days. 50 values between 4 and 14 days for crucifer pests with the longer LT50 value associated with conidial concentrations two order of magnitudes lower than ours. 50 for different aphid species of around 3 days. LT90 values in this experiment were about twice as long as reported by B. bassiana . It is possible that the innate immune response of earthworms can resist the fungus and slow the infection (In this experiment the commercial formulation of nfection . The celnfection . Earthwonfection  that mayB. bassiana saprophytic nature, millet provides a good substrate for it to stay viable for longer in soil (Mycotized millet was added to the soil was as effective as the recultured suspension sprayed directly on the earthworms. Mycotized millet has two potential functions that make it effective. Because of  in soil . It also50? Are there other pathogens that can kill these invasive forest pests faster?Immediate questions for future research arising from this study are: Why does the recultured formulation perform better than the commercial product from which it was derived? What causes the long LTB. bassiana might affect the viability of cocoons. These are not only reproductive, but also survival structures and may afford the embryos some protection from the fungus. The one study that evaluated the effect of entomopathogenic fungi on cocoons was not conclusive (This study did not address how nclusive  and moreB. bassiana can be considered as a potential biocontrol agent to prevent the spread of invasive pheretimoids from greenhouses and nurseries into forest ecosystems. However, its mode of action causing the morbidity of earthworms is not clear and further studies are needed.Our results suggest that using 10.7717/peerj.11101/supp-1Supplemental Information 1Bioassay 1, 2, and 3 results.Click here for additional data file.


Perrault syndrome (PRLTS) is a rare autosomal recessive disorder characterised by ovarian failure in females and sensorineural hearing loss (SNHL) in both genders. In the present paper we describe a child affected by PRLTS3, due to CLPP homozygous mutations, presenting auditory neuropathy spectrum disorder (ANSD) with bilateral progressive SNHL. This is the first case reported in the literature of an ANSD in PRLTS3. CLPP is a nuclear encoded mitochondrial protease directed at the mitochondrial matrix. It is encoded on chromosome 19. This protease participates in mitochondrial protein quality control by degrading misfolded or damaged proteins, thus maintaining the normal metabolic function of the cell. In PRLTS3, the peptidase activity of CLPP is suppressed. Neurological impairments involved in PRLTS3 suggest that the pathogenic mutations in CLPP might trigger a mitochondrial dysfunction. A comprehensive description of the clinical and audiological presentation, as well as the issues related to cochlear implant (CI) procedure and the results, are addressed and discussed. A brief review of the literature on this topic is also provided. Perrault syndrome (PRLTS) is a rare autosomal recessive disorder characterised by ovarian failure in females and sensorineural hearing loss (SNHL) in both genders . The iniThe genes involved in PRLTS expression are HSD17B4, HARS2, CLPP, LARS2, TWNK and ERAL1, defining, respectively, the subtypes PRLTS1 (OMIM# 233400), PRLTS2 OMIM# 614926), PRLTS3, PRLTS4 OMIM# 615300), PRLTS5 (OMIM# 616138) and PRLTS6 (OMIM# 617565) [300, PRLT14926, PRIn PRLTS, SNHL is generally reported to be due to a cochlear dysfunction, and only three cases due to a neural deficit (auditory neuropathy) are reported in the scientific literature, two cases with PRTLS5, associated with TWNK mutation  and a fuIn the present paper we describe a child affected by PRLTS3, due to CLPP homozygous mutations, presenting auditory neuropathy spectrum disorder (ANSD) with bilateral progressive SNHL. This is the first case reported in the literature of an ANSD in PRLTS3. A comprehensive description of the clinical and audiological presentation, as well as the issues related to cochlear implant (CI) procedure and the results, are addressed and discussed. A brief review of the literature on this topic is also provided.The case presentation is compliant with the SCARE guidelines .A female infant from consanguineous Senegalese parents was born preterm  by caesarean section due to alteration of cardiotocography tracking. During the pregnancy a severe intrauterine growth restriction occurred from the second trimester. Body weight at birth was 1350 g, and APGAR score , Pulse (heart rate), Grimace (reflex irritability), Activity (muscle tone), and Respiration of a newborn) was 2 in the first minute of life and which increased at 7 after 10 min. The child underwent cardiopulmonary resuscitation manoeuvres and tracheal intubation and was admitted to the neonatal intensive care unit for ventilation support. After 7 weeks, given the improvement of her general condition, the baby was discharged from the hospital. While hospitalized, the baby underwent newborn hearing screening, according to the protocol of the Tuscan region  and resuGiven a retardation of her neuromotor and linguistic development, the baby was entrusted to the infantile neuropsychiatric service at 24 months of age. During this period of hospitalization, the infant underwent brain magnetic resonance, genetic testing and an audiologic revaluation. She presented global developmental delay involving the motor, cognitive and linguistic areas. However, the neurological examination did not reveal any specific neurological signs.Brain magnetic resonance imaging (MRI) showed leukoencephalopathy and suggested a diagnostic hypothesis of mitochondrial disease.Muscle biopsy and genetic tests showed signs suggestive of mitochondrial pathology due to a homozygous mutation of CLPP gene (mut. Homozygosity C.425C> T/p.Pro142Leu), defining a picture of PRLST3. Both the parents were heterozygous for the same mutation.The audiologic assessment performed at 24 months revealed severe-to-profound hearing loss, with a free field threshold at behavioural audiometry around 95 dB (pure tone audiometry between 0.5 and 1-2-4KHz), with no sound detection at Ling six sound testing. Furthermore, the TEOAE were bilaterally present, while auditory brainstem responses (ABR) were absent, even at the higher level of stimulation. Tympanometry was bilaterally normal and stapedial reflexes bilaterally absent even at the higher stimulation levels. The audiologic picture was that of an ANSD.The child was promptly fitted with high power hearing aids bilaterally. After 4 months of traditional hearing aid fitting and speech therapy, with very limited benefit in terms of sounds and speech perception and language development, a CI was proposed. A CT scan of the petrous bone showed normal middle and inner ear anatomy see  and a fuAt 29 months she presented motor regression with loss of walking, which she had acquired around the age of 25 months. She did not have any infectious and traumatic episodes. At the neurological examinations she manifested fluctuating tone with rigidity on tibio-tarsal joint and hyperactive patellar and Achille’s reflex. A brain magnetic resonance was repeated, which evidenced a worsening of the existing lesions . The dev® CI612 device with a perimodiolar electrode was used. The surgery was uneventful through a standard facial recess approach to the cochlea [A NucleusOne month after surgery the child underwent the first CI fitting session and continued to attend psychomotor and speech rehabilitation. The speech therapy sessions were only for 1 h a week, due to difficulties the parents had in reaching the hospital. She also underwent periodic CI fitting sessions and audiological follow-up at our institute.One year after implantation, the child uses the implant for about 7 h per day . Free field threshold with CI is about 30 dB (pure tone audiometry between 0.5 and 1-2-4KHz). The child is able to detect voice, every Ling test phoneme and environmental sounds, and she produces various vocalizations and a few onomatopoeias.We reported a case of ANSD in PRLTS3, due to CLPP homozygous mutation, diagnosed in a 24-month-old girl, who was submitted to CI at the age of 40 months.Mutations in CLPP are related with PRLTS3, which presents with progressive hearing loss, female infertility, microcephaly, epilepsy, and growth and mental retardation ,13,14,15CLPP is a nuclear encoded mitochondrial protease directed at the mitochondrial matrix. It is encoded on chromosome 19 . This prIn patients with PRLTS, the SNHL is generally bilateral, symmetric, early onset and progressive, very often resulting in a severe-to-profound deficit ,18,19,20Generally, the hearing loss associated to PRLSTS is reportedly due to a cochlear damage, and very few cases of ANSD have been previously reported. In one case, it was related with biallelic mutation of TWNK gene  and in aAs far as we know, to date, the CLPP mutation detected in the patient herein reported has never been related to ANSD.It should be observed that, even if ANSD has been found in just two papers ,8, most It is important to point out that a few other articles also describe unproportionally poor speech discrimination in relation to the hearing deficit degree, in the reported cases ,23, perhAs far as we know, no cases of ANSD in PRLTS submitted to CI have been reported in literature. In the literature, only six patients with PRLTS and severe-to-profound hearing loss submitted to CI have been reported ,19,21,22For what specifically concerns PRLTS3, in the previous scientific literature 20 cases have been reported. Analysing the audiologic assessment of those cases, in 12 cases the grade of SNHL was profound , in fourIn the case herein reported, a comprehensive audiological assessment was completed at the age of 24 months, defining a picture of ANSD; furthermore, the hearing loss had a delayed onset, since at birth the newborn hearing screening was bilaterally pass both for TEOAE and AABR . The aidSome issues related to CI concerning the present case have to be addressed. The first is how a CI can manage to restore hearing in the ANSD and what the intended outcome is. Previous studies showed that individuals with ANSD in which the cochlear sensory system and the synapse are affected, which are bypassed by the CI, have optimal post-implantation outcomes. Besides, also some patients with ANSD due to neural damage benefit from implantation because the stimulus provided by the implant is synchronous and regular, but others show poor performance with CI because of the poor neural transmission of the electrical signal ,27. ANSDFurthermore, in the present case, the results after implantation may be affected both by the neuromotor delay and by the delay in intervention, to the extent that it was only partially pre-operatively predictable ,30.The second concern regards the need to submit the patient to serial brain MRI study in the future for the evaluation of the neuroradiologic picture. It is known that, even if the last generation of CI equipped with diametrically bipolar magnets allow pain-free MRI scans even at 3 T and without a headband , the magThe choice of the brand of the implant was due to the possibility to modify many stimulation parameters during intraoperative and post-operative neural response telemetry testing and to the possibility to manually adjust the pulse width and other stimulation parameters during the fitting of the implant, which can be useful in the case of a poor nerve.In the reported case, we demonstrated good neural stimulation by the implant: we recorded stable and consistent compound action potentials with neural response telemetry test on apical, medial and basal electrodes, both intraoperatively and post-operatively during the fitting sessions. We conducted a standard CI fitting procedure, and it was not necessary to use high stimulation parameters, such as high stimulation levels or larger pulse-width.Since the implant was activated, the patient has been undergoing speech and language and neurodevelopmental disorder rehabilitative treatment once a week for about 12 months. The datalogging of the device showed a constant use of the processor. The sound and speech perception increased significatively, but despite perceptive benefits, to date, speech abilities lag beyond those expected. However, it must be considered that both the delay in intervention and the relevant neurological involvement of the child undoubtfully play a role in limiting the results, even if an effective audiological input is provided.To our knowledge this is the first case of ANSD related to PRLTS and CLPP mutation reported in the literature and the first report of the results after CI procedure. Our report adds evidence that CI may be a viable option for cases with ANSD that show unsatisfactory results with traditional hearing aids and particularly in cases affected by PRLTS5.The need for a comprehensive audiological assessment in paediatric cases with hearing loss is highlighted to completely define the clinical picture, especially in the case of a genetic and syndromic disease; this is important for the global management of the child and also for the audiological and rehabilitative management. Rehabilitative options, including the possibility of a CI, must be evaluated with great attention and on a case-by-case basis, with regard to pre-operative evaluation issues, to issues concerning the choice of the right device, the device fitting and the post-operative rehabilitation and follow-up. In the case herein reported, the presence of a syndromic disease with neuropsychiatric disabilities adds critical aspects to deal with in every phase of the procedure with a multidisciplinary approach.


These results highlight that our method has sufficient ability to distinguish facial grooming and body grooming in mice.Grooming is a common behavior for animals to care for their fur, maintain hygiene, and regulate body temperature. Since various factors, including stressors and genetic mutations, affect grooming quantitatively and qualitatively, the assessment of grooming is important to understand the status of experimental animals. However, current grooming detection methods are time-consuming, laborious, and require specialized equipment. In addition, they generally cannot discriminate grooming microstructures such as face washing and body licking. In this study, we aimed to develop an automated grooming detection method that can distinguish facial grooming from body grooming by image analysis using artificial intelligence. Mouse behavior was recorded using a standard hand camera. We carefully observed videos and labeled each time point as facial grooming, body grooming, and not grooming. We constructed a three-dimensional convolutional neural network (3D-CNN) and trained it using the labeled images. Since the output of the trained 3D-CNN included unlikely short grooming bouts and interruptions, we set posterior filters to remove them. The performance of the trained 3D-CNN and filters was evaluated using a first-look dataset that was not used for training. The sensitivity of facial and body grooming detection reached 81.3% and 91.9%, respectively. The positive predictive rates of facial and body grooming detection were 83.5% and 88.5%, respectively. The number of grooming bouts predicted by our method was highly correlated with human observations (face:  Experimental animals exhibit various behaviors, such as ambulation, immobility, rearing, scratching, and grooming. Since behavior reflects the mental and physical condition of an animal, we can estimate it by observing its behavior. Grooming is one of the common behaviors to care for fur, maintain hygiene, and regulate body temperature in experimental animals such as mice, rats, and others . GroominThere are several methods for assessing the grooming behavior of mice and rats. Human observation is a traditional and standard way to detect grooming. Although human observation does not require any specific equipment, it is labor-intensive and time-consuming. Additionally, the results vary between observers. Several automated methods have been developed to detect grooming in rodents. For example, de facto standard method for image recognition. CNN-based algorithms have also been developed in biology. For example, The development of deep neural network algorithms is noteworthy. Since In this study, we aimed to establish a novel automated method to detect facial and body grooming in mice. Here, we showed that 3D-CNN can accurately classify facial and body grooming.n = 9) were treated with lysophosphatidic acid . Mice were then placed in a black square arena and their behavior was recorded by the hand camera  set at a height of 150 cm above the arena. Recording conditions were as follows: frame rate, 60 Hz; resolution, 1,920 × 1,080 pixels, 24-bit color. We finally obtained 30 9-min video files and used them for grooming detection. We split the whole 30 videos into training (1–23), validation (24–25), and test (26–30) datasets. Detailed information is shown in In this study, we used the videos recorded in the previous study to establish an automated scratching detection method . Here, wt ± 10, t ± 20, t ± 30, or t ± 40 for each time point t to use them for an input , licking or washing of paws, body, tail, and genital (body grooming). These thresholds were decided empirically during annotation processes. We carefully watched the videos and labeled each frame as “not grooming”: 0, “facial grooming”: 1, and “body grooming”: 2 . The labges at t . We noteThe architecture and hyper-parameters of the convolutional recurrent neural network (CRNN) were almost similar to our previous study  Kobayas. Brieflyt, w, h) =  where t is temporal, w is width, and h is height axis) followed by 3D-max pooling layer [pooling size:  =  or ]. Output feature extracted by 3D-CNN block was flattened and input into Fc blocks. Fifty percent of units were randomly dropped out after each 3D-max pooling layer and between the first and second Fc layers. The activation function of the last layer was softmax and that of all the other layers was ReLU. Detailed information of layers is summarized in We constructed 3D-CNN having two blocks: 3D-CNN block and Fc block. We referred to the C3D model  in const–4 and 3 × 10–5 learning rates for CRNN training and 3D-CNN training, respectively. A categorical cross-entropy loss function was used for both trainings.Twenty-three videos (1–23) were used for the training of neural networks. We randomly reduced the number of grouped images labeled as not grooming. For one epoch, 1,000 of not grooming, 200 of facial grooming, and 400 of body grooming grouped images were randomly picked allowing duplicates (batch size was 8). Grouped images were randomly rotated by multiples of 20° and flipped for data augmentation. We used AMSGrad optimizer with 1 × 10The last layer of neural networks outputs three decimal values from 0 to 1 for each grouped image. These values were interpreted as the probability that the input grouped image belongs to three categories . The category having the largest probability was adopted as the prediction of neural networks.Trainings and predictions of neural networks were conducted on AI-COMPLIANT ADVANCED COMPUTER SYSTEM at the information initiative center, Hokkaido University, Sapporo, Japan, which was equipped with Intel Xeon Gold 6230 (2.1 GB) and NVIDIA Tesla V100 GPU (32 GB). Trainings and Predictions were conducted using the TensorFlow library (version 2.2.0) in Python.n = 9) and obtained 30 9-min video files  sporadic misprediction, (ii) unlikely interruption, and (iii) unlikely transition . SporadiIn the raw prediction data, the number of grooming bouts was overestimated owing to these improbable predictions . Filter r = 0.93; body: r = 0.98).We then evaluated the performance of 3D-CNN and posterior filters for the test dataset, which has not been used up to here. After all filters were applied, the sensitivity and PPR of mouse body grooming were 91.9% and 88.5%, respectively, and those of facial grooming were 81.3% and 83.5%, respectively . We alsoWe investigated the frame-by-frame differences between post-filtered predictions and human observations. Discrepant frames between them  were classified into three mutually exclusive error types: (i) boundary errors, (ii) oversights, and (iii) false detections. Boundary errors were defined as differences in the start and/or end of each grooming bout. Oversights were defined as bouts that were labeled as grooming by humans, although they were predicted as not grooming. False detections were defined as bouts that were incorrectly predicted as grooming. We found that boundary errors, oversights, and false detections accounted for 58.6%, 12.1%, and 29.3% of all discrepant frames, respectively .We also evaluated the types of behavior mice exhibited in oversights and false detection frames. We revealed that fine movements around the face were often overlooked .Since it is impossible to directly determine the mental and physical conditions of experimental animals, many researchers have paid attention to their behavior. We focused on grooming and successfully established a method to detect facial and body grooming in mice using 3D-CNN. Detailed evaluation of grooming has been focused on because various factors, including stressors, drug treatment, and genetic mutation, affect grooming frequency and pattern . In partAn automated grooming detection system is required as human observation is labor-intensive and has low throughput. An automated system is essential to be convenient and to have sufficient performance compared with human observations. In this study, we developed a novel automated grooming classifier composed of a commercially available hand camera, normal home cage, and GPU-mounted computer. This simplicity is superior to existing methods, which often require special equipment. More importantly, our system achieved sufficient performance in distinguishing facial and body grooming after the application of filters . Analyside facto standard method of image recognition in many research fields, including animal ethology. Recently, to treat time-series data effectively, we and others combined 2D-CNN and RNN and succeeded in detecting animal behavior, including scratching in mice -CNN has become the  in mice  and dail in mice . We also in mice . Here, wThis study has the following limitations. First, our 3D-CNN was not suitable for detecting fine movements . It is sIn conclusion, we developed a 3D-CNN-based grooming detection method that can automatically distinguish facial and body grooming.The raw data supporting the conclusions of this article will be made available by the corresponding author, without undue reservation.All experiments were reviewed and approved by the institutional Animal Care and Use Committee at the University of Tokyo (P19–079). Animal care and treatments were performed in accordance with the guidelines outlined within the Guide to Animal Use and Care of the University of Tokyo.NS, KK, MY, and TM contributed to the conception and design of the study. NS, KK, TY, and SM performed the experiments. NS and KK analyzed the data. NS wrote the first draft of the manuscript. NS, KK, and TM revised the manuscript. All authors read 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.


Vision loss is among the characteristic symptoms of neuronal ceroid lipofuscinosis (NCL), a fatal neurodegenerative lysosomal storage disorder. Here, we performed an in-depth analysis of retinal degeneration at the molecular and cellular levels in mice lacking the lysosomal aspartyl protease cathepsin D, an animal model of congenital CLN10 disease. We observed an early-onset accumulation of storage material as indicated by elevated levels of saposin D and subunit C of the mitochondrial ATP synthase. The accumulation of storage material was accompanied by reactive astrogliosis and microgliosis, elevated expression of the autophagy marker sequestosome 1/p62 and a dysregulated expression of several lysosomal proteins. The number of cone photoreceptor cells was reduced as early as at postnatal day 5. At the end stage of the disease, the outer nuclear layer was almost atrophied, and all cones were lost. A significant loss of rod and cone bipolar cells, amacrine cells and ganglion cells was found at advanced stages of the disease. Results demonstrate that cathepsin D deficiency results in an early-onset and rapidly progressing retinal dystrophy that involves all retinal cell types. Data of the present study will serve as a reference for studies aimed at developing treatments for retinal degeneration in CLN10 disease. Neuronal ceroid lipofuscinosis (NCL) comprises a group of mainly recessively inherited neurodegenerative lysosomal storage diseases usually starting in childhood ,2,3. Whihttps://www.ucl.ac.uk/ncl-disease/mutation-and-patient-database/mutation-and-patient-datasheets-human-ncl-genes; accessed on 21 March 2021) in 13 different genes have been shown to cause different NCL forms (CLN1-8 and CLN10-CLN14) [Originally classified mainly by the age at disease onset and ultrastructural characteristics of the storage material, NCLs are now grouped according to the affected gene ,6,7. To 0-CLN14) . The maj0-CLN14) .CTSD gene [CTSD gene cause congenital NCL, the most severe NCL variant with prenatal disease onset and death within days or weeks after birth. Affected patients present with a severe phenotype, including skull and brain deformation, microcephaly, seizures and respiratory insufficiency [CTSD gene resulting in decreased CTSD enzymatic activity, in comparison, present with an infantile or juvenile disease onset and a milder and more slowly progressing phenotype [CLN10 disease is caused by mutations in the gene encoding the lysosomal enzyme cathepsin D (CTSD), a ubiquitously expressed aspartyl protease ,13. To dTSD gene . In addiTSD gene ,16. Lossficiency ,17,18,19henotype ,21,22. Ahenotype ,21.Ctsd gene display many phenotypic similarities to affected patients [Ctsd ko) mice are born viable and initially develop normally [Ctsd ko mice display a progressive regression of visceral organs. In the brain, pronounced neurodegeneration becomes apparent during the third postnatal week and is accompanied by seizures, ataxia and bradykinesia. Reactive astrogliosis and microgliosis and progressive neuronal loss in different brain regions are accompanied by an accumulation of storage material with the ultrastructure of GRODs and fingerprint profiles [Ctsd ko mouse recapitulates many of the characteristic pathological features of congenital CLN10 disease.Naturally occurring and transgenic animal models carrying mutations in the patients ,24. Simipatients ,26,27,28normally . Howevernormally ,30. At 2profiles ,31,32,33profiles . TogetheCtsd ko mice [Ctsd ko mice, and quantified the thinning of different retinal layers, the progressive loss of various retinal cell types and the dysregulation of various lysosomal proteins. Results will serve as a reference for preclinical studies aimed at evaluating the efficacy of therapeutic strategies for the treatment of retinal degeneration in CLN10 disease.Progressive visual impairment as a result of retinal degeneration is among the characteristic clinical symptoms of most NCLs ,34. Opht ko mice ,36. RetiCtsd ko) mice [Cathepsin D knockout (ko) mice  on a C57ko) mice ,29. We, Ctsd ko mice have a life expectancy of only around 26 days [Ctsd ko and age-matched wild-type or heterozygous mice of both sexes. Eyes were enucleated and fixed overnight in 4% paraformaldehyde  in phosphate buffered saline . After cryoprotection with an ascending series of sucrose, eyes were frozen in Tissue-Tek  and serially sectioned with a cryostat  at a thickness of 25 µm, blocked in PBS (pH 7.4) containing 0.1% bovine serum albumin (BSA) and 0.3% Triton X-100 , incubated with primary antibodies  and ZEN2.3 software. The thickness of the entire retina, the outer retina ) and the inner nuclear layer was measured at 18 equidistant positions between the nasal and temporal retina periphery using Fiji Image J 1.51 s software .Rows of photoreceptor cell nuclei were counted in both the temporal and nasal retina close to the optic disc in sections stained with DAPI and antibodies to recoverin. BRN-3A-positive ganglion cells and calbindin-positive horizontal cells with a clearly visible nucleus were counted in entire retina sections, and their numbers per 1000 µm retina length were calculated. The density of ganglion cells in retinal flatmounts was determined as described in ,38. NumbCtsd ko mice display an early-onset and rapidly progressing phenotype characterized by lymphopenia, intestinal necrosis, loss of body weight, brain atrophy, motor impairment and retinal degeneration, and die around P26 [ound P26 ,29,36. TCtsd ko retina at P5 and P10  and the outer nuclear layer , but not the inner nuclear layer . Interactions between “genotype and age” were significant for all thickness measurements ; outer nuclear layer:  = 94.17, p < 0.001); inner nuclear layer: . The Bonferroni post hoc test revealed no significant differences in the thickness of the entire retina (p < 0.001), P20 (p < 0.001) and P25 (p < 0.001), as indicated by the Bonferroni post hoc test . Similar numbers of IBA1-positive cells were found in the retinas of older Ctsd ko mice with the only exception of the outer retina, where the density of IBA1-positive cells decreased significantly between P20 and P25  antibodies. In the inner retina of 5-day-old control retinas Aa, the d retinas Ab. Quant retinas B. At P15Ctsd ko retinas, a few Müller cell processes were GFAP-positive, and GFAP expression in retinal astrocytes was elevated compared to the control retinas  in the control retinas was confined to retinal astrocytes at all ages analysed Am,Ao,Aq. retinas . In oldeCtsd ko retinas, we performed immunostainings with antibodies to SCMAS and saposin D  and lysosomal-associated membrane protein 2 LAMP2; . ElevatePE cells . The exp compare m,n. As t compare p,r and t compare . Age-mat compare o,q.Ctsd ko retinas, sequestosome 1/p62 (SQSTM1/p62)-positive punctae were mainly confined to the outer nuclear layer  cones/250 µm retina length in mutant retinas compared to 42.4 ± 3.4 cones/250 µm in age-matched control retinas  horizontal cells/mm retina length compared to 12.0 ± 0.5 horizontal cells/mm in control retinas. However, at P25, the density of horizontal cells was significantly reduced in the mutant  when compared with the control mice .Significant thinning of the inner nuclear layer of mutant mice Bc additiCtsd ko retinas (37.6 ± 1.1 (mean ± SEM) RGCs/mm retina length) when compared with the control retinas  survival using anti-BRN-3A antibodies. At P10 Aa,Ab andCtsd ko mice [Ctsd ko mice to obtain insights into the pathological alterations associated with CTSD deficiency. Specifically, we studied the molecular changes associated with retinal degeneration in this mutant mouse, identified the cell types affected in the Ctsd ko retina and quantified their degeneration during the course of the disease. A retinal pathology was already evident in young postnatal Ctsd ko mice, as indicated by an accumulation of storage material and a significant loss of cone photoreceptor cells. Reactive astrogliosis, reactive microgliosis and degeneration of rod photoreceptor cells became evident slightly later, while a significant loss of retinal interneurons and ganglion cells was observed at the end stage of the disease.CLN10 disease, the most severe NCL form, is caused by dysfunctions of the lysosomal aspartyl protease CTSD ,20. Ctsd ko mice  faithful ko mice ,36. In tCtsd ko retina, we found a few CD68-positive microglia/macrophages as early as at P5, and elevated levels of GFAP expression in retinal astrocytes and some Müller cells at P10. In older animals and coincident with the massive loss of photoreceptor cells, the density of microglial cells increased dramatically when compared with control mice, in line with a previous report [Ctsd ko mice have implicated activated microglia in the progression of neurodegeneration through the production of neurotoxic levels of nitric oxide. In support of this view, multiple intraperitoneal injections of nitric oxide inhibitors were shown to result in the significant attenuation of neuron loss in the thalamus and the inner retina of the mutant mouse [Neuroinflammation characterized by reactive microgliosis and reactive astrogliosis closely accompanies or even precedes neurodegeneration in different NCLs, and has been implicated in the progression of the neuropathology ,42. In ts report . Microglnt mouse ,36,43. Ant mouse ,46,47,48Ctsd ko retina. The prevalence of two of the major components of lysosomal storage material, SCMAS and the sphingolipid activator proteins saposin A and D, differs between different NCL forms [Ctsd ko retina, we detected elevated levels of SCMAS and saposin D as early as at P5. At this age, the accumulation of both proteins was mainly confined to the inner retina, from which they spread to the outer retina later during disease progression. The loss of retinal neurons, in comparison, was first apparent in the outer nuclear layer, subsequently in the inner nuclear layer and finally in the ganglion cell layer (see below). Thus, we observed no strict spatio-temporal correlation between the accumulation of storage material and neurodegeneration, similar to findings in the brain of a CLN6 sheep model [Ctsd ko mice resulted in an attenuation of the neurological deficits and a pronounced extension of the mutant’s lifespan, despite the presence of high amounts of storage material in the brain [Ctsd ko retina without, however, attenuating retinal degeneration in the treated animals [The accumulation of storage material is a hallmark of all NCL forms, and was among the earliest pathological alterations observed in the developing CL forms ,13,34,49ep model ,51. Resuhe brain . Moreove animals .Ctsd ko retinas, as indicated by the increased expression levels of LAMP1, LAMP2 and the lysosomal cysteine protease CTSX/P/Z. The elevated expression of lysosomal proteins followed a spatiotemporal pattern closely resembling that of storage material accumulation, suggesting a link to lysosomal stress and activation of the transcription factor EB (TFEB) [Similar to our observations in the dystrophic retinas of CLN1, CLN6 and CLN7 mouse models ,55,56, wB (TFEB) ,58,59. IB (TFEB) ,61,62, iB (TFEB) ,63. AccoB (TFEB) ,65. SQSTB (TFEB) .Ctsd ko retina starting at P15. Retina thinning was mainly the result of a rapidly progressing atrophy of the outer nuclear layer, in agreement with another report [Morphometric analyses revealed a progressive thinning of the r report . HoweverCtsd ko retinas as early as at P5, slightly before a pronounced reactive microgliosis or astrogliosis became evident. In the mouse retina, cones are generated prenatally [Ctsd ko retina start to degenerate shortly after they are born. However, it is also possible that the reduced number of cones is the result of a developmental defect. In older mutants, the loss of cones progressed rapidly, with no cones remaining at P25. Cones constitute only 3% of the total photoreceptor population [Ctsd ko mouse suffers from a cone-rod dystrophy. Early-onset photoreceptor degeneration has also been observed in mouse models of other NCLs, such as CLN4 [Of note, we found a significantly reduced density of cone photoreceptors in enatally ,68, suggpulation . The maj as CLN4 , CLN5 [7 as CLN4 , CLN6 [4 as CLN4 ,54,71, C as CLN4  and CLN8 as CLN4 ,73,74, i as CLN4 ,79,80,81 as CLN4 . Disrupt as CLN4 ,85,86,87Ctsd ko mice was a significant thinning of the inner nuclear layer, although it was not as pronounced as that observed for the outer nuclear layer. At the cellular level, the thinning of this layer was mainly the result of a progressive degeneration of rod and cone bipolar cells. The loss of rod and bipolar cells was moderate at P20 , but pronounced at P25 . At the end stage of the disease, we additionally observed a significantly reduced number of horizontal cells. The degeneration of retinal interneurons has also been observed in animal models of other NCL forms. In fact, in some NCL forms, the retinal pathology has been reported to start mainly in the inner retina with the outer retina being only mildly affected. In a canine CLN2 model and in murine CLN1 and CLN3 models, for example, the retinal phenotype is characterized by a thinning of the inner nuclear layer and a relative preservation of the outer nuclear layer, a significant neurodegeneration in the inner nuclear layer and/or reduced b/a wave ratios in electroretinogram recordings [nclfCln6 mouse is caused by defects in bipolar cells, which, however, start to degenerate only after a significant number of photoreceptors is lost [Ctsd ko retina at P20, which increased to ~20% at P25. The accumulation of high amounts of storage material in these large projection neurons already at initial stages of the disease, but the onset of RGC degeneration at relatively late stages of the retinal pathology, has also been observed in animal models of other NCL forms [Another prominent feature of the retinal pathology of cordings ,89,90,91 is lost . FinallyCL forms ,56,93,94CL forms ,95,96. OCL forms ,98. TogeCtsd ko mouse, despite the rapid disease progression using an adeno-associated virus (AAV) vector-mediated gene transfer to the brain and/or the viscera [CTSD gene that impair, but not completely abolish, the enzymatic activity of CTSD present with an infantile or juvenile disease onset and progressive retinal degeneration [Results from preclinical studies suggest that enzyme replacement strategies represent promising treatment options for NCLs caused by the dysfunction of soluble lysosomal enzymes, such as palmitoyl-protein thioesterase 1 (PPT1) in CLN1 disease, tripeptidyl peptidase 1 (TPP1) in CLN2 disease or CTSD in CLN10 disease ,101,102. viscera ,104. Fur viscera . In addi viscera . Resultsneration ,21,35.


Overall, the prevalence of hearing loss was 58.1%, 61.4%, and 85.0% in the no DR, NPDR, and PDR groups, respectively. After adjusting for confounding factors, the logistic regression model showed that there was no significant association between the prevalence of DR and hearing loss in the overall sample. However, the presence of PDR  was significantly associated with hearing loss in the middle-aged group. Middle-aged people with diabetes may have an association between DR severity and hearing loss. The potential role of microvascular diseases in the development of hearing loss, especially in middle-aged patients, could be considered.We investigated the association between the severity of diabetic retinopathy (DR) and hearing loss based on vascular etiology. We used data from the Korean National Health and Nutrition Survey 2010–2012. Adults aged >40 years with diabetes were enrolled. Demographic, socioeconomic, general medical, noise exposure and biochemical data were used. Participants were classified into three groups: diabetes without DR, non-proliferative DR (NPDR), and proliferative DR (PDR); participants were also divided into two groups (middle age (40 ≤ age < 65 years) vs. old age (age ≥ 65 years)). The association between hearing loss and DR was determined using logistic regression analysis. A total of 1045 participants ( Sensorineural hearing loss is one of the most common sensory abnormalities in elderly people, and its pathophysiology remains unknown. Old age, male sex, history of noise exposure, and vascular risk factors (diabetes mellitus (DM), hypertension, and dyslipidemia) are known risk factors ,2. AmongThe retina is a tissue in the eye through which the vascular status can be easily observed directly from outside the body in a non-invasive manner. Due to this characteristic, many studies have investigated the relationship between systemic disease states and retinal vascular conditions ,5,6,7. TThe retina and inner ear are known to be regulated by some of the same genes , and sysThe data for this study were obtained from the fifth Korea National Health and Nutrition Examination Survey (KNHANES V), conducted from 2010 to 2012 . The KNHThe KNHANES consists of three parts: a health interview survey, a health examination survey including ophthalmologic and otologic examinations, and a nutritional survey. The health interview survey was administered to all study participants, and ophthalmic and otologic examinations were performed simultaneously in the same mobile examination unit. The KNHANES was approved by the KCDC Institutional Review Board, and the survey adhered to the tenets of the Declaration of Helsinki; all participants provided written informed consent.We included participants with DM aged 40 years and older in the analysis. DM was defined as a previous diagnosis of diabetes made by a physician, current treatment with insulin or oral hypoglycemic agents, and/or a fasting blood glucose level of ≥126 mg/dL. Participants were excluded from our analyses if they did not have gradable fundus photographs due to poor image quality or missing examination or interview data.A digital non-mydriatic fundus camera  and a Nikon D-80 digital camera  were used to obtain the fundus images. Seven standard field photographs were obtained for each eye of the participant after pharmacological pupil dilatation, as per the Early Treatment for Diabetic Retinopathy Study protocol, from 2010 to 2011 . In contDR was identified based on the presence of any characteristic lesion as per the Early Treatment for Diabetic Retinopathy Study severity scale: microaneurysms, hemorrhages, hard exudates, cotton wool spots, intraretinal microvascular abnormalities, venous beading, and new retinal vessels . The preThe pure-tone air-conduction threshold was measured in a sound-treated booth using an automatic audiometer . Trained otolaryngologists collected data independently for each ear at six frequencies: 0.5, 1.0, 2.0, 3.0, 4.0, and 6.0 kHz. All audiometric tests were performed under the supervision of an otolaryngologist. Hearing loss was defined as an average pure-tone air-conduction hearing thresholds in the better ear worse than 25 decibel hearing level (dBHL) at 0.5, 1, 2, and 4 kHz, according to the World Health Organization (WHO) definition. We classified hearing impairment into three categories based on severity: mild, moderate, and severe. Mild hearing impairment was defined as an unaided pure-tone hearing threshold level for the better ear of 25–39 dBHL and average hearing threshold levels at 0.5, 1, 2, and 4 kHz. Moderate hearing impairment was defined as an unaided pure-tone hearing threshold level for the better ear of 40–69 dBHL and average hearing threshold levels at frequencies of 0.5, 1, 2, and 4 kHz. Severe hearing impairment was defined as an unaided pure-tone hearing threshold level for the better ear of 70 dBHL or greater and average hearing threshold levels at frequencies of 0.5, 1, 2, and 4 kHz.In this study, we have included several related factors that may affect hearing loss in patients with DM, as suggested in previous studies ,22,23,24Participants with diabetes were divided into three groups: diabetes without DR, diabetes with NPDR, and diabetes with PDR; all participants were divided into two groups based on an age cutoff of 65 years: middle-aged group (over 40 years old and under 65 years old) and old-age group (over 65 years old).p-value of 0.05.The KNHANES sampling data were weighted by statisticians and statistical analysis was performed according to the statistical guidelines of the Korean Center for Disease Control and Prevention, an institution supervising the KNHANES. Demographic data and differences between the groups were analyzed using the Pearson’s chi-square test for categorical variables and an independent-sample t-test for continuous variables. The association between hearing loss and DR was determined using logistic regression analysis. Univariate logistic regression analyses were performed for analyzing the association between factors and hearing loss in all participants according to age group. Multivariate logistic regression analyses were performed for analyzing the association between hearing loss and DR after adjusting for all potential confounding factors. Complex sample analyses considering weights, stratified variables, and cluster variables were performed using SPSS software . All data are presented as the mean ± standard error, and statistical significance was set at a Of the 23,353 participants who underwent ophthalmic and otologic examinations, we excluded 21,572 participants because they were diabetes-free and aged <40 years. Of the remaining 1781 participants, 624 were excluded from the analysis because they did not have gradable fundus photographs due to poor image quality or were missing interviews, or laboratory data, and 112 participants with conductive hearing loss based on the otoendoscopic examination were excluded .p < 0.001). Female sex was less common in participants with hearing loss than in those without hearing loss . Participants with hearing loss were more likely to have a lower education level  and household income  than those without hearing loss. The prevalence of smoking was greater in participants with hearing loss than in those without hearing loss , while a higher body mass index (BMI) was observed in participants without hearing loss than in those with hearing loss . Participants with hearing loss were more likely to have a history of noise exposure than those without hearing loss . Hypertension , chronic kidney disease (CKD) , and stroke history  were more frequently observed among participants with hearing loss than among those without hearing loss. Regarding laboratory data, the fasting blood sugar level (140.3 ± 2.2 vs. 149.4 ± 3.7 mg/dL), serum total cholesterol level (184.0 ± 2.1 vs. 192.0 ± 3.2 mg/dL), serum creatinine level (0.9 ± 0.0 vs. 0.8 ± 0.0 mg/dL), and estimated glomerular filtration rate  were significantly lower in participants with hearing loss than in those without hearing loss. There was no significant difference in the presence of DR (10.2% ± 1.5% vs. 7.5% ± 1.8%) between the two groups (p = 0.277). The proportions also did not differ according to the severity of DR between the two groups.A total of 1045 participants were eligible for this study (411 participants in the middle-aged group and 634 participants in the old-aged group). p < 0.001) and higher household income  than the excluded group.Comparing the demographic data of the exclusion and inclusion groups shows differences in age and household income; the group included in the study had a lower mean age  and in all participants . The proportions of participants with hearing loss among participants with DR are shown in According to multivariate logistic regression analysis, adjusting for all potential confounding factors, DR was not significantly associated with hearing loss in the entire group . However, in the middle-aged group, hearing loss was more common in participants with PDR than in those without DR  .In this population-based, cross-sectional study, we assessed all risk factors for hearing loss in participants with DM that had shown significance in previously published population-based studies. There was no association between overall DR and sensorineural hearing loss for both age groups combined. However, after adjusting for potential associated factors, DR severity was significantly associated with hearing loss. In particular, the group with PDR had a 7.74 times higher risk of hearing loss than the group without DR in the middle-aged group.The relationship between DR and hearing loss has long been studied. Jorgensen and Bush  reportedThe Blue Mountains Eye Study was a population-based study of hearing loss , in whicOoley et al.  classifi2 vs. 29.99 ± 4.22 kg/m2), and HbA1c levels (8.61 ± 2.02% vs. 8.38 ± 1.67%) did not show significant differences between the hearing loss and non-hearing loss groups. However, in our study, fasting blood glucose levels and BMI of participants with hearing loss were lower than those of participants without hearing loss.In a previous study  comparinHearing loss in patients with DM is caused by chronic hyperglycemia causing vascular defects in the stria vascularis and vestibulocochlear nerves ,3. HyperInterestingly, in this study, the middle-aged group with PDR but not the old-age group with PDR was associated with hearing loss. Hearing loss is closely related to the aging process. We assume that diabetic vasculopathy has a greater influence on hearing loss than the aging process in the middle-aged group, while both aging and diabetic vascular changes simultaneously cause hearing loss in the older age group. The loss of a significant association between PDR and hearing loss in patients aged >65 years may be due to a potent aging factor that dilutes the effect of DR on hearing function.This study had several limitations. First, it was impossible to define a causal relationship between risk factors and hearing loss because this was a cross-sectional study. A prospective longitudinal cohort study could help identify causal risk factors. Second, DR evaluation using fundus photos can underestimate the prevalence of DR because of the narrow viewing angle. Ultrawide fundus photography-based epidemiologic studies can provide more informative data in the future. Third, although risk factors were selected based on existing studies, there may have been factors that were not considered, leading to unexpected biases. Fourth, we did not divide the hearing loss group according to the frequency of hearing state. Fifth, the number of NPDR and PDR subjects was small. Ophthalmology and otolaryngology examinations were conducted simultaneously in the KNHANES for only three years since 2010. In addition, we excluded many subjects from the study if any of the health interview surveys, laboratory data, and fundus photography had problems. As a result, however, there were fewer subjects with DR in our study, showing a significant association with DR severity and hearing loss in middle-aged patients; however, additional studies are needed to prove a clearer association. Sixth, in this study, 35% (624 out of 1781) of the subjects were excluded because of incomplete data in interviews or laboratory findings and poor quality of fundus photography. The demographic data of the exclusion and inclusion groups show that the group included in the study had a lower mean age and higher household income than the excluded group. There may be socioeconomic factors influencing the selection of subjects, hence, care should be taken when interpreting the results. Nevertheless, this study had strengths, in that it used a large sample size. The KNHANES is a government-led survey conducted using a standardized process, and the data are representative of the South Korean population.The retina is the only tissue that can be evaluated to directly observe the state of the internal blood vessels in the body. Retinopathy might reflect vascular pathologies in other organs. Therefore, studies have been conducted to evaluate systemic vascular conditions based on retinal vasculature status. To our knowledge, this is the first population-based study to investigate the association between DR severity and hearing loss in an Asian population. In our study, no association was found between the presence of DR and hearing loss in patients with diabetes at all ages. However, middle-aged people with diabetes may have an association between DR severity and hearing loss. The relationship between DR severity and hearing loss remains controversial; therefore, more evidence is needed to support this association. Further well-designed and longitudinal cohort studies on the relationship between DR severity and hearing loss will provide a more precise evidence.