The aging population is prone to hearing loss, which has several adverse effects on quality of life, including difficulty following conversations in noisy environments. Personal Sound Amplification Products (PSAPs) are a less expensive, over-the-counter alternative to traditional, more expensive hearing aids. Although some studies have shown that PSAPs can mitigate hearing loss, the literature generally only addresses group differences without considering interindividual variability. This study aimed to 1) determine how PSAPs affect listening effort and speech perception in noise and 2) measure interindividual variability and identify contributing demographic and health factors.We used a cross-over design in which all participants were assigned to each condition.Twenty-eight adults aged 60 to 87 years with normal hearing and mild hearing loss fulfilled the study requirements.In one session, speech-in-noise perception tasks were performed without PSAPs, and in the other, the tasks were performed with bilateral PSAPs. The two sessions were separated by one week, and the order of the sessions was balanced across participants.In both sessions, participants performed the Quick speech-in-noise test and a word discrimination task in noise, in which their self-reported listening effort was measured.PSAPs use improved speech perception in noise in both tasks and reduced listening effort. There was considerable variability between individuals, with approximately 60–70% of participants showing benefit. Age, hearing and cognitive status were significant predictors of the benefits.Not all individuals may benefit from the effect of PSAPs to the same extent at their first use, and this depends on specific health and demographic factors, particularly age, hearing, and cognitive status. These results underscore the importance of demographic and health factors in assessing the benefits of hearing amplification in older adults.NCT05076045.ClinicalTrials.gov,  Hearing loss is highly prevalent among older adults. According to the World Health Organization, one-third of people over the age of 65 have debilitating hearing loss worldwide . HearingThe traditional method of treating mild to moderate hearing loss is through hearing aids, which are sound amplification devices adjusted by audiologists to the level and specificity of the person’s hearing loss. Chien and Lin  estimateMarket research and surveys have listed the cost of hearing aids as the main reason older adults do not seek help for their hearing loss , 13. AccIn recent years, there has been an upsurge in the popularity of personal sound amplification products (PSAPs) and over-the-counter (OTC) hearing aids as affordable alternatives to traditional hearing aids. Both types of devices are readily available and may be bought by customers without the help of a healthcare professional. There are, however, differences between PSAPs and OTC hearing aids. OTC hearing aids are medical devices that must comply with Food and Drug Administration (FDA) regulations and are used to treat mild to moderate hearing loss in people 18 years of age and older. The FDA established this new category of hearing aids to improve consumer access to hearing aids by enabling their purchase over-the-counter, without a prescription . PSAPs aAlthough PSAPs are gaining popularity, their appreciation and benefits on hearing ability are still equivocal. In a study of customer opinions, the authors identified many customers’ negative impressions across all types of PSAPs studied, with more positive comments expressed about high and medium-cost products than about low-cost products . This suThis study aimed to recruit older adults with different hearing profiles to determine whether using PSAPs is associated with improved speech perception in noisy environments compared with an unaided condition. None of the participants had been diagnosed with hearing loss, but they were keen to explore the potential benefits of PSAPs for their hearing needs. PSAPs are primarily designed for people who may have difficulty hearing in specific situations, even if their hearing is normal for their age. By including people with different hearing sensitivity as measured with pure tone audiometry, the study aimed to examine the effectiveness of PSAPs in improving the hearing experience of people who may have difficulties in certain listening situations, even if they have not been clinically diagnosed with hearing loss. We hypothesized that using PSAPs improves speech perception in noise, as measured by task performance and self-reported listening effort. This improvement would be predicted by hearing and cognitive status.n = 2) and medical (n = 2) reasons, resulting in a sample size of 28 participants . This sample size was determined based on previous research examining the effects of PSAPs. Previous studies did not report effect sizes or descriptive statistics between groups, making it difficult to conduct an accurate power analysis.Recruitment took place from March 2022 to September 2022. Participants were recruited from Baycrest’s participant database and through advertisements and word of mouth. The recruitment process of the participants is summarized in Participants ranged in age from 60 to 87 years . They all completed a phone interview to verify eligibility. The following criteria were required for inclusion: being right-handed, 60 years of age or older, and having English as a first language (or having learnt it before age five). The exclusion criteria included language impairment, significant medical or neurocognitive conditions (such as dementia) or interventions that might affect cognitive function, untreated visual impairment, tinnitus or otologic disorders, known cerebrovascular disease, past or present experience with any hearing device, and contraindications to the PSAPs as described in the user manual.This study was approved by the Baycrest Research Ethics Board (project #21–32) and is registered at ClinicalTrials.gov (NCT03008174). All participants gave written informed consent before the start of the experiment and received financial compensation.The CONSORT checklist and the study protocol are provided in All participants attended two sessions of approximately three hours each, with at least one week between them. At the start of the first session, participants completed several assessments, including the Montreal Cognitive Assessment (MoCA) , Speech,A randomized cross-over design was employed. The tasks were carried out in two sessions, one without PSAPs and the other with bilateral PSAPs. The order of the sessions was counterbalanced between participants using the block randomization method with blocks of three by the experimenters . The order of sessions was determined by the recruitment order . As a result, the order was made regardless of the participant’s health. Thirteen participants performed the tasks without PSAPs first, while 15 participants completed the tasks with PSAPs first. All procedures took place at Baycrest’s Rotman Research Institute in a double-walled soundproof room. Participants were given breaks during testing if necessary. The study design remained unchanged throughout the experiment.Pure-tone thresholds in decibel (dB) hearing level (HL) were measured with a calibrated clinical audiometer . The following frequencies: 0.25, 0.5, 1, 2, 3, 4, 6, and 8 kHz were assessed in each ear separately. None of the participants had been diagnosed with hearing loss by an audiologist. Audiograms are provided in Line graphs present an overview of participants’ hearing thresholds for the right ear (right panel) and the left ear (left panel). Each light gray line represents one participant. The black line represents the average thresholds of all participants.Participants were fitted with bilateral PSAPs . We used this device because it is one of the most studied PSAPs in the literature, making it easier to integrate our results with the results of previous studies. It is a high-end behind-the-ear sound amplifier with a 16-channel compressor, 16-channel noise reduction, feedback suppression, and a dual-directional microphone that is more sensitive to sounds from the front and less sensitive to sounds from the back. The price is approximately USD300 dollars per device, which is still significantly lower than traditional hearing aids.The participants fitted the bilateral PSAPs themselves. They first selected the most appropriate ear tips from the three sizes  according to their preferences and comfort level. They then took a hearing test in the PSAPs. The PSAPs were connected through Bluetooth to a mobile application on an iPhone  called Customizer by Sound World Solutions. Participants pressed a button when they heard a tone in one of the devices. This test is similar to a traditional pure-tone audiometry test in which pure tones of different frequencies are played in each ear separately. Instructions on how to conduct the test were provided in the iPhone app. No guidance was given to participants during the test, as these devices are intended to be fitted at home without the help of a healthcare professional. Although participants received no guidance, they were supervised during the fitting. The majority of participants were able to fit the devices themselves. However, on a few occasions, participants received assistance when encountering difficulties. Occasional guidance was provided to ensure that we were measuring the effectiveness of the devices rather than the participants’ ability to fit them. After the hearing test, the iPhone app automatically sets the gain prescription (amplification) according to the in situ audiometric assessment results using the NAL-NL2 prescription . The resIt should be noted that we did not use objective methods to test the PSAPs fitting process. In other words, we did not assess the consistency of audibility or the extent to which the amplification matched the NAL-NL2 targets. Instead, the study relied on participants’ subjective feedback to confirm the effectiveness of PSAPs. Indeed, after the fitting, participants were asked if they heard a change in amplification, and the experimenter changed the amplification level to confirm that the devices were working properly. Finally, the devices were put into "restaurant mode" to activate the dual directionality of the microphone and were set to the amplification level determined by the app. These options were retained for each task.This study included three primary outcome measures and one secondary outcome measure. The three primary outcome measures included 1) accuracy on the Quick Speech-In-Noise (QuickSIN) , which mSpeech comprehension in noise was measured using the QuickSIN test . The Quihttps://freesound.org/people/mefrancis13/sounds/210611/). The audio file for the noise was segmented into 5-second segments that were played during the entire trial.Phonological AX discrimination in noise was measured using a task similar to that developed by Tremblay and colleagues –37. It iThree different signal-to-noise ratios , calculated as the ratio of pressure signal to pressure noise, were utilized in the study. The audio files for the words were edited using PRAAT script to normalize root-mean-square (RMS) intensity at 70 dB sound pressure level (SPL). The audio files for noise were edited to normalize the RMS intensity to 67, 70, or 73 dB SPL. Thus, word intensity was constant, while noise intensity varied across trials to mimic a real-world situation. The sound level was adjusted at 70 dB SPL for all participants.To utilize the dual-microphone directionality feature of the PSAPs, words were presented simultaneously through two speakers  placed in the front corners, while background noise was played through two speakers in the back corners of the soundproof room. This configuration was used to create a complex immersive auditory experience. All speakers were located approximately 1.5 meters from the participant’s head, with a height of 1.2 meters and an angle of 45°. To ensure that the participant remained seated and looked straight ahead without turning slightly toward the speakers, participants were instructed to focus on a fixation cross on a computer screen throughout the task. Video monitoring was used to ensure that participants complied with task instructions.https://doi.org/10.5683/SP3/HTMDLI/.Two versions of the same task were created, one for each testing session. In both versions, the material was the same, except that the order of the words in each pair was reversed, and the pairs were presented in different SNRs. In both sessions, the experiment began with a practice block of 16 trials. The main task was divided into three blocks of 10 minutes each, with a short break between blocks. A total of 306 pairs were presented in both sessions, including 102 pairs in each SNR condition. In each trial, the babble noise began to play for 1000 milliseconds (ms), and then the two words were presented with an inter-stimulus interval of 300 ms. After the words were presented, participants were asked to determine whether the words were the same or different using a response box . A white fixation cross centred on a dark gray background was presented during the words, followed by a green question mark (?) to instruct participants to respond. The inter-trial interval was 1000 ms. Participants had a maximum of 3 s to respond. All stimuli and experiment files are available at Participants used a seven-point Likert scale to rate the listening effort required to complete each of the three blocks of the word discrimination task. The scale used was the one adopted by Johnson et al. . The speDemographic factors included age and biological sex. Health factors included cognitive functioning, depression, anxiety, self-rated health, and subjective measure of hearing. Cognitive functioning was measured using the Montreal Cognitive Assessment Hearing Impairment (MoCA-HI) . Depresshttps://doi.org/10.5683/SP3/HTMDLI. Data were analyzed using R version 4.1.1 , 95% CI = ) than with , z = 3.98, p < 0.001.The probability of reporting each score for each session  is shown in For each behavioural measure that showed differences between sessions , we performed an additional LME model to identify predictors of the difference.For QuickSIN, a generalized LME model was used. Because the scores were positively skewed with a continuous positive range, the model was fitted using a gamma distribution and a log link function. The model revealed significant interactions between Session and Age, Session and Better ear PTA4, and Session and Speech SSQ . Age wasFor the word discrimination task, an LME model revealed a significant interaction between Session and the MoCA-HI, with no other significant interactions . CognitiFor the listening effort score, an LME model revealed no significant interaction between Session and the four factors .Personal sound amplification products (PSAPs) and over-the-counter (OTC) hearing aids are becoming a new alternative for individuals with hearing loss. Although a growing body o advantages of using these devices at the group level, there has been limited focus on individual differences. Therefore, the full extent of their benefits remains unknown. To clarify the interindividual variability in benefits, the present study used a cross-over design to assess the beneficial effects of PSAPs on speech perception in noise and self-reported listening effort in two counterbalanced sessions. Consistent with previous research, this study supports that PSAPs use is associated with speech-in-noise benefits in terms of performance and listening effort. However, the benefits are small and only observed for some individuals.In this study, we focused on speech perception in noise because difficulty following a conversation in a noisy environment is one of the most common complaints of older adults with and without hearing loss. Two different tasks were used to measure speech-in-noise perception. The first was the QuickSIN , a cliniIn the present study, PSAPs use was associated with reduced self-reported listening effort. According to the information degradation hypothesis , listeniAnother factor that could explain the gain in speech-in-noise perception is that the PSAPs currently on the market might not be effective enough to provide clinically relevant benefits, as they do not all have the same features as conventional hearing aids. This would be consistent with a previous study that examined the impact of five different PSAPs and hearing aids on listening ability . Their rThere are important conceptual differences to consider when comparing traditional hearing aids, OTC hearing aids and PSAPs. These differences relate to personalization, effectiveness, safety and affordability. Traditional hearing aids are medical devices that must be prescribed, are tailored to the specific needs of the user, and are regulated by the FDA to ensure their effectiveness and safety. Similar features are found in OTC hearing aids, but they do not require a prescription and can be adjusted by the users themselves, making them more widely available and less expensive. On the other hand, PSAPs are not subject to the same regulations as medical devices and may have fewer and less advanced features, which could reduce their effectiveness. While conventional hearing aids should remain the preferred choice for hearing care, the results of this study suggest that PSAPs may be a viable alternative for those who cannot afford both conventional or OTC hearing aids, as they appear to offer significant benefits compared to an unaided condition. The results of this study come in the context that conventional hearing aids are rarely reimbursed in full or even in part in most countries. If conventional devices were reimbursed, they would be a safe and effective option for treating hearing loss. In this case, PSAPs may still be an attractive option in some cases, such as for very mild hearing loss or when a person needs amplification under certain conditions. In line with this idea, the current study shows that individuals with diverse hearing profiles, even without any clinical diagnosis of hearing loss, can experience benefits from PSAPs, providing tangible evidence that these devices can help people who want to improve their auditory communication skills.Future studies are, however, needed to compare different PSAP brands on speech perception and listening ability. However, this may be challenging as the market will likely grow in the coming years, making it challenging to compare the different types of PSAPs. Future studies should examine which parameters and settings are most beneficial on PSAPs, thus providing guidelines for those who opt for this option instead of conventional or OTC hearing aids. Here, we used high-end PSAPs with noise cancellation features, making our results only generalizable for this device and other devices with the same characteristics.Notwithstanding, it is encouraging that most participants did better with PSAPs than without, even for a short period of use. There are no longitudinal studies to date exploring the effects of these devices on measures of speech perception and auditory processing. The next step would be to conduct longitudinal studies to explore the benefits of these devices on speech perception and compare these benefits to those of hearing aids.Finally, another factor that may contribute to the interindividual variability we examined in this study is the impact of demographic and health factors. Age-related hearing loss is a complex condition that does not affect all individuals equally. Some people with specific characteristics are more likely to have hearing loss, or report hearing loss than others. For example, in a recent study conducted by our group, we found significant differences in speech-in-noise ability between males and females, with males showing a higher loss than females . We alsoStepwise regression analyses were used to determine whether interindividual differences in performance with and without PSAP could be explained by age, hearing thresholds (Better ear PTA4), self-reported listening ability  and cognitive functioning (MoCA-HI). We found that the benefits of PSAPs can be explained by some demographic and health factors, at least for the PSAPs we employed in the current study and others with comparable features. Although these factors do not necessarily explain who will benefit most from these devices in the long term, they highlight important factors that future studies should consider when evaluating the impact of these devices on hearing and listening.First, we observed that people who met the criterion for hearing loss, who were younger older adults and reported having good speech-listening skills, had higher benefits on the QuickSIN with the devices. The effect of hearing thresholds is not surprising because few studies show that hearing thresholds are a significant determinant of the impact of PSAPs on listening ability. The devices seem beneficial for people with mild-to-moderate hearing loss but not for severe hearing loss , 22. TheAnother interesting result is that individuals with higher scores on the MoCA-HI benefitted more from the devices than those with lower scores. Here, all participants’ MoCA-HI scores were within a range considered cognitively healthy, showing that even normal cognitive score variability can lead to changes in PSAPs effects. The difference in PSAPs uptake as a function of cognitive status suggests that individuals with lower cognitive scores may be more overwhelmed by amplification than those with higher cognitive functioning. For instance, individuals with lower cognitive functioning may have fewer cognitive resources to adjust for the effect of amplification. This would be consistent with the literature on conventional hearing aids showing that speech intelligibility in noise appears worse for listeners with low working memory capacity than for those with high capacity . It has Finally, for listening effort, no single factor significantly explains the variability in the benefits. Looking at the individual data, we observe that almost all participants reported lower listening effort with the devices than without the devices. This suggests that one mechanism by which devices can help improve listening ability is by reducing listening effort. However, participants may also report less listening with the device because they believe it should help them hear better. Future studies should consider supplementing subjective rating of listening effort with more objective metrics such as pupil dilatation e.g., , 49.In the present study, we focused on a single brand of high-end PSAPs. This choice limits the generalization of our results to only PSAPs similar to the one used in our study. Several brands of PSAPs are on the market, ranging from a few dozen dollars to a few hundred dollars, with different features. The OTC market (OTC hearing aids and PSAPS) rapidly expands, providing many options when purchasing OTC amplifiers. It is urgent to conduct studies comparing other brands of devices, focusing on the features that best maximize the benefits, especially in adverse listening conditions. Noise suppression and directional microphones could be an essential feature, as difficulty listening to speech-in-noise is a frequently reported complaint in aging, even in older adults with normal hearing. We also did not compare the benefits of PSAPs with conventional hearing aids. PSAPs may provide significantly more benefits than the unaided condition but substantially less than traditional hearing aids, which is consistent with the results of a previous study. If this is the case, it would suggest that conventional hearing aids should be preferred over PSAPs for treating hearing loss. It is also possible that PSAPs and conventional hearing aids offer similar benefits, consistent with the meta-analysis mentioned above, indicating that PSAPs may be as effective as conventional hearing aids in treating hearing loss. Although this comparison is critical to understand the extent of the benefits of PSAPs, it is difficult to do given the many different types of hearing aids and PSAPs. Further studies should compare PSAPs and hearing aids with similar characteristics.The lack of objective device output verification is another limitation of the study, as it raises questions about the reliability and accuracy of the data and may be related to the large individual differences in benefit from these devices. The study did not quantify or account for variations in the output of the devices in the ear canal or how well they were coupled to the ear. This factor could be an important predictor of interindividual variability in benefit and was not considered in the current study’s design. Nevertheless, we have effectively overcome this issue by verifying the device’s functionality with self-reported measures, ensuring a minimum level of amplification that participants found noticeable. This study is encouraging because it demonstrates that the participants were able to benefit from self-fitting the devices with very little guidance.Another limitation of this study is the placement of speakers in corners instead of using a single forward-facing speaker, which may not fully reproduce the ecological nature of face-to-face conversations. In addition, the use of babble noise, while providing some background interference, may not fully reflect the complexities of natural listening environments. The use of a multi-speaker background noise, which more closely resembles the acoustic conditions of group conversations, might have been more appropriate for improving the ecological validity of the task.Finally, our sample size is relatively small, which may have reduced the statistical power of our results. We relied on sample sizes from previous studies to determine our sample size because we could not perform a power analysis based on the information provided in those studies. To address this literature problem, we systematically reported the effect sizes of our analyses. This will allow future studies to calculate their sample sizes more accurately based on the effect sizes reported in the current study.In an environment where OTC devices, including PSAPs, are becoming more widespread and readily available, our results are important for three reasons. First, we showed that PSAPs can help people with different hearing profiles, particularly those with higher hearing thresholds, to improve their listening ability compared with an unaided situation. Second, we demonstrated that adaptation to the devices is rapid and that benefits are apparent only after a few hours of use. Longer use is likely to yield greater benefits. Finally, we observed that not all users initially experience the same level of benefit, which varies with age, hearing status, and cognition. This reveals the importance of considering demographic and health factors to fully understand the benefits of hearing amplification. Previous studies have not included demographic and health factors in the analyses. Therefore, this may explain why some studies have favoured PSAPs over conventional hearing aids, and others have favoured conventional hearing aids over PSAPs. However, it needs to be clarified whether these factors only contribute to rapid acclimation to the device or whether they also indicate which users will experience long-term benefits.S1 File(DOCX)Click here for additional data file.S2 File(DOCX)Click here for additional data file. 22 Mar 2023 <div> PONE-D-23-03040 </div>  <div> Interindividual variability in the benefits of over-the-counter hearing devices on speech perception in noise: a randomized cross-over clinical trial </div>  <div> PLOS ONEDear Dr. Perron, </div>  <div>   </div>  <div> As you can see from the reports included below, your manuscript has been assessed by three reviewers. They appreciated the timeliness of your research question, but they also raised several significant concerns that need your attention. For example, they noted overlapping concerns regarding the methodological reporting and the  statistical analysis of your data, which impact on the overall strength of your conclusions. Given the nature of the concerns raised, your revised manuscript will likely be sent to the original reviewers for re-assessment.      </div>  <div> Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.Please submit your revised manuscript by May 06 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at  </div> A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.Please include the following items when submitting your revised manuscript:If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: We look forward to receiving your revised manuscript.Kind regards,Dario Ummarino, PhDSenior EditorPLOS ONEJournal Requirements:When submitting your revision, we need you to address these additional requirements.1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf2. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide.[Note: HTML markup is below. Please do not edit.]Reviewers' comments:Reviewer's Responses to Questions <font color="black"> Comments to the Author1. Is the manuscript technically sound, and do the data support the conclusions? </font> The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: PartlyReviewer #2: PartlyReviewer #3: Partly********** <font color="black"> 2. Has the statistical analysis been performed appropriately and rigorously?  </font> Reviewer #1: YesReviewer #2: NoReviewer #3: No********** <font color="black"> 3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. </font> The Reviewer #1: YesReviewer #2: YesReviewer #3: No********** <font color="black"> 4. Is the manuscript presented in an intelligible fashion and written in standard English? </font> PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: YesReviewer #3: Yes********** <font color="black"> 5. Review Comments to the Author </font> Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: Thank you for the opportunity to review this manuscript. The manuscript is well written and the research topic is timely. Please see my comment below for your consideration.Major Concerns:1. The introduction details quite nicely the impact hearing loss can have on individuals and the role hearing aids play in mitigating these issues. However, 13 of the 28 participants had normal hearing. It is unclear to this reviewer why participants with normal hearing were included given the rationale for the study related to mitigating hearing loss with low cost PSAPS. I suggest removing the normal hearing participants as I have difficulty understanding the interest in studying this group. If they are to be included, consider adding Group as a between subject variable in the statistical analyses to determine if the effects—although small—are different for normal versus impaired groups. By including the normal hearing participants in the current analysis, I suspect that the statistical power has been increased to the point where these small effects reach statistical significance.2. Many studies investigating PSAPs and OTCs note that these devices are self-fitted by the individual, yet they typically involve assistance from the research team. This is a flaw of the current study as well. Page 11, lines 215-227 detail the fitting process of the device. Lines 220-221 suggest the participants were not guided or assisted and this fitting was typical of anyone that would use the device. The lines above suggest otherwise as these participants were fitted in a double-walled sound booth, earmolds were selected by the researchers, instructions and examples regarding the fitting were provided, additional assistance was provided if requested. It is unclear why the researchers did not provide the device and instructions as any person that purchased these devices on-line would receive, have them do an actual self-fit, then do the testing.Other Concerns:3. Page 8, participants, please include a power analysis to determine the appropriate sample size.4. Figure 1 is not necessary.5. Page 10, line 204-205, the rationale for using this device stated here is vague. What is this based on—‘good representation’? I suggest deleting this sentence. The sentence that follows makes a stronger rationale for selecting this device.6. Page 11, lines 208-209, is this $300 cost per device or for 2 devices? Also, I searched this device on-line and it appears to be a BTE and not an in-ear device.7. Page 11, line 210-211, consider changing this description to ‘dual directional microphone that is more sensitive to sounds from the front and less sensitive to sounds from the back.’8. Page 11, line 224, was the NAL-NL2 fitting used based on the app? Is this the only option available or was this an option selected by the researchers for the participants?9. Page 12, line 251, McArdle & Wilson (2006) determined the QuickSIN lists were not equivalent in difficulty for listeners with hearing loss and suggested lists 4, 5, 13 & 16 should not be used. List 4 was used in the current study.10. Page 13, line 261, the example for identical is /tap/-/bat/ but these words are different.11. Page 13, line 275, were the words presented through 2 speakers simultaneously? Why would you present the words from 2 speakers at +/-45 degrees as this isn’t a realistic situation.12. Page 14, lines 290-293, why did you change from referring to the stimuli as CVC words to ‘syllables’?13. Page 15, line 311, is Field a typo?14. Page 20, line 398, why is there no result reported for the interaction of SNR x session?15. Page 23, lines 478-479, see comment #1 in this review.16. Page 24, lines 503-505, I am not sure the data support this statement, particularly the notion this could reduce the negative impact on cognitive performance. Also, a picky thought, but hearing aids do not restore hearing. Same on page 28, line 593.17. Lastly, the manuscript is lengthy and has quite a bit of redundancies throughout. I get frustrated when a reviewer makes a comment that doesn't have a direct solution, so I realize this isn’t helpful. Just consider.Reviewer #2: The aim of the study is to determine how Personal Sound Amplification Products (PSAPs) affect listening effort and speech perception in noise and measure interindividual variability and identify contributing demographic and health factors.This is quite an interesting study, however, the manuscript requires improvement.CommentsThe following are to be provided/stated/described:Line 154, the word abbreviation sdLine 161, exclusion criteriaLine 168, the conditionLine 172, the second session.Line 181, allocation concealment.Line 179-182, information to be incorporated into Figure 1.The study design not very clear and requires more description and with proper flow.Line 208, $300 US to be written as USD300Line 269, ‘ were used .’ to be rewritten.Line 311, typographical  hearing aids in the United States. However, there were several significant limitations that would need to be addressed before the paper would be suitable for publication.One major concern is the conceptual differences between the PSAP devices tested here and over-the-counter hearing aids. The title and various other parts of the paper describe PSAP and OTC devices interchangeably, but a more careful description of how the devices used in this study compare to OTC devices would be helpful. There is some overlap between these terms, but they are also distinctions that should be made so that readers understand the type of device that was evaluated and how that might compare to the more general category of OTC devices. Referencing the US FDA Final Rule on OTC definitions might help to provide some additional detail on how to classify this device using terminology that has recently become familiar to readers.The description of the process of fitting the devices was confusing and replicating this study based on the details provided in the method would be challenging without further elaboration. Starting on line 215, the following questions should be addressed:-The is reference to an earmold in the fitting. An earmold is traditionally a custom mold fitted to the specific patient’s ear by taking an impression. A clear description of the device coupling to the ear should be made in terms of whether this was a custom fitting or if the fitting was non-custom using different sized domes or ear tips.-It says that the patients self-fitted the device, but in one sentence (Line 218) instructions were given and in the next sentence (Line 220), it says that fitting assistance was not provided to simulate self-fitting at home. This should be clarified as it might impact how these results relate to current OTC devices that do not include professional guidance or instructions.-The fitting procedure describes a hearing test, assumably through the device (in situ), but that is not clear.-The device based the fitting on the NAL-NL2 prescriptive formulae, but in the previous paragraph the device is described as having three pre-sets. Thus, it is unclear whether the fitting was customized based on the hearing test or if the device selects one of the presets closest to NAL-NL2 based on the hearing test. This is important because fitting to NAL-NL2 targets using only three presets for the range of hearing losses included in the sample would be difficult.-Was the fitting verified in any way using probe microphone or electroacoustic measures to determine whether the devices provided consistent audibility or how closely they matched the NAL-NL2 targets?The lack of verification of the output of the devices is a major limitation of the experimental design. The large individual differences in benefit from these devices could be related to variation in the output of the devices in the ear canal and variation in how well they were coupled to the ear. It is true that real-ear verification or coupler measures would not be available if these devices were self-fitted at home as PSAPs are, but the same argument could be made for all of the other outcomes in the experiment, and the lack of consideration for how these devices might have restored audibility for these participants is a significant limitation in that a key factor that could have determined the outcome was omitted from the design.The use of a difference score in the statistical analyses, rather than creating a repeated-measures fixed effect for aided condition is problematic as the difference score ignores the variance of the unaided and aided conditions. The linear mixed effects models used here with random effects for each subject can account for the correlation between repeated measures within subject quite well, so it is unclear why the repeated measure was reduced to a difference score instead.The stepwise regression approach based on AIC is concerning because the model comparisons and AIC criteria are not described. One can assume the mode with the best AIC was reported, but it is unclear how much better the final models were than comparator models for both speech recognition and listening effort. Much more detail about the statistical approach would be needed to interpret these models.It is unclear whether the design has adequate statistical power to assess the number of predictors that were included in the model and whether the lack of power influenced the stepwise regression outcome.The discussion includes overly general statements about potential benefits from PSAP and OTC devices that should be heavily qualified. This is an example where a clearer description of how these PSAP devices fit within the category of OTC devices would be helpful, as it could help readers to understand how the results with these devices might generalize to the broader landscape of OTC hearing aids.A minor comment on the Introduction. The need for OTC devices in the introduction is heavily based on the cost of current prescription hearing aid being too high. It is worth acknowledging several studies that have shown that hearing aid adoption and hearing aid use are only marginally better in countries where prescription hearing aids are provided for free as part of government healthcare programs or at a significantly reduced cost. This should be acknowledged, and the revised discussion should include some consideration for how the benefit of OTC devices might compare to the benefit of prescription devices, if the latter were available at low or no cost.********** <font color="black"> 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. </font> Reviewer #1: NoReviewer #2: NoReviewer #3: No**********https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool,  21 Apr 2023Responses to reviewers are attached.AttachmentResponse to reviewers.docxSubmitted filename: Click here for additional data file. 13 Jun 2023 <div> PONE-D-23-03040R1 </div>  <div> Interindividual variability in the benefits of over-the-counter hearing devices on speech perception in noise: a randomized cross-over clinical trial </div>  <div> PLOS ONEDear Dr. Perron, </div>  <div>   </div>  <div> After further  consideration, therefore, whilst we feel that your manuscript has merit, it does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that gives further consideration to the points raised during the review process.Thank you for submitting your revised manuscript to PLOS ONE and for your attention to the previous comments made by the reviewers. As you will see below, they still have some concerns. In particular the extent of assistance given to the participants, and the inclusion of those with normal hearing in the analysis. You do not seem to adequately address these, nor highlight them in the limitations section.plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.Please submit your revised manuscript by Jul 28 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at  </div> A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. <div> If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.Please include the following items when submitting your revised manuscript:https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: We look forward to receiving your revised manuscript.Kind regards,Antony BayerAcademic EditorPLOS ONEJournal Requirements:Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.[Note: HTML markup is below. Please do not edit.]Reviewers' comments:Reviewer's Responses to Questions <font color="black"> Comments to the Author </font> 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.Reviewer #1: (No Response)Reviewer #2: All comments have been addressedReviewer #3: All comments have been addressed**********  <font color="black"> 2. Is the manuscript technically sound, and do the data support the conclusions? </font> The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: PartlyReviewer #2: PartlyReviewer #3: Yes**********  <font color="black"> 3. Has the statistical analysis been performed appropriately and rigorously?  </font> Reviewer #1: YesReviewer #2: NoReviewer #3: Yes**********  <font color="black"> 4. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. </font> The Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********  <font color="black"> 5. Is the manuscript presented in an intelligible fashion and written in standard English? </font> PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********  <font color="black"> 6. Review Comments to the Author </font> Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: Thank you for the opportunity to review this revised manuscript. The authors have not satisfactorily addressed the following concerns.Major Concerns:1. The authors added justification for including normal hearing participants in the study design. The justification is “PSAPs are designed for people that have difficulty hearing in certain situations, despite having normal hearing for their age.” They further noted “We focused on speech perception in noise because it is challenging for both older adults with and without hearing loss.” However, there is no information regarding the difficulties encountered by the normal hearing participants in the study. These were not normal hearing people with difficulties in noise based on the inclusion criteria. That being said, the normal hearing participants could have been evaluated separately from the hearing-impaired participants, as suggested, yet the authors did not do so. The effects observed were already very small. Including the normal hearing participants in the current analysis has increased the statistical power to the point where these small effects reach statistical significance. The authors suggest that using PTA as a continuous variable addressed my concern; however, the results showed session was related to PTA—further suggesting normal hearing participants aren’t candidates for these devices. Moreover, throughout the discussion, the authors seem to go back and forth between the findings being small and not clinically significant to PSAPs being a viable alternative to hearing aids.2. As noted in the previous review, many studies investigating PSAPs and OTCs note that these devices are self-fitted by the individual, yet they typically involve assistance from the research team. This remains a flaw of the current study as well. In the revision, the authors note “No guidance (except if necessary) was necessary to participants during the test, as these devices are intended to be fitted at home without assistance of a healthcare professional.” How often was assistance necessary and to what extent? Again, it is unclear why the researchers did not provide the device and instructions as any person that purchased these devices on-line would receive, have them do an actual self-fit, then do the testing.3. The test setup playing the target speech signal from 2 speakers at 45 degrees simultaneously is not realistic. The design does not, in any way, “mimic a complex and immersive listening environment, similar to those typically experienced by people with hearing loss, such as in group conversations or crowded restaurants, where sounds can emanate from all directions.” The use of a multi-talker babble, such as the one used in the QuickSin, would have been more appropriate.4. In the discussion, line 543-546, again the authors are making claims about the value of PSAPs for normal hearing listeners that the data do not support as presented. The normal hearing data are not evaluated alone. The same, unfounded claim is made in the conclusions.Reviewer #2: Minor comments:Line 409, typo numerator.Line 445 – 462, the results are to be presented in table form.For the reduction of predictors in the analysis, the model fit is to be discussed.Reviewer #3: The revision substantially addressed my comments from the original manuscript. I have two minor comments that should be easy to resolve.-The fitting procedure has been substantially clarified in the revision. However, the method section now refers to the "NAL-NL2 procedure" when describing how gain for the devices is determined. There really is not an NAL-NL2 procedure, and if there were, if would suggest that the output of the devices was verified using traditional probe microphone measures. To avoid any confusion, I would state that the software set the gain prescription based on the results of the in-situ audiometric evaluation using the NAL-NL2 prescription.-The term "over-the-counter devices" in the title should be changed to " a personal sound amplification product" since a single PSAP device was used, unless this device is classified as an OTC under the FDA definition and regulations.**********  <font color="black"> 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. </font> Reviewer #1: NoReviewer #2: NoReviewer #3: No**********https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool,  20 Jun 2023The response to the reviewers is attached.AttachmentResponse_to_reviewer_R2.docxSubmitted filename: Click here for additional data file. 28 Jun 2023Interindividual variability in the benefits of personal sound amplification products on speech perception in noise: a randomized cross-over clinical trialPONE-D-23-03040R2Dear Dr. Perron,Thank you for your revised manuscript and for your further attention to the reviewers' comments. We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Antony BayerAcademic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments: 11 Jul 2023PONE-D-23-03040R2 Interindividual variability in the benefits of personal sound amplification products on speech perception in noise: a randomized cross-over clinical trial Dear Dr. Perron:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staffon behalf ofProfessor Antony Bayer Academic EditorPLOS ONE


Maintaining static balance is a process coordinated by central integration of visual, vestibular and somatosensory information. Whether or not hearing and spatial acoustic information contributes to the maintenance of static postural balance is unclear.A prospective observational pilot study was performed. Twenty-five normal hearing adults  underwent a computerized dynamic posturography test battery including the Sensory Organization Test (SOT), the Motor Control Test (MCT), and the Adaptation Test (ADT). The balance tests were performed two times, in a randomized sequence without or with acute hearing loss. Earplugs (sound insulation 37 dB) or headphones with white noise (sound volume 75 dB) induced the conductive hearing loss. Hence, all participants passed through four sequences of the balance test battery. A repeated-measures analysis of variance (ANOVA) was used to analyze the results.p > 0.05). The ANOVA showed no longer latencies with simulated hearing loss compared to no hearing loss in both experiments with one exception: the reaction of the right foot during large forward translation was longer with hearing loss than without hearing loss in both experiments (p = 0.025).The ANOVA revealed no difference for any SOT and ADT subtest without hearing loss and simulated hearing loss (either earplugs or headphones; all Overall, a simulated acute conductive bilateral moderate or severe hearing loss did not disturb the static balance function in normal hearing younger adults in this first small pilot study.The online version contains supplementary material available at 10.1007/s00405-023-07942-w. The continuous maintenance of static balance in upright standing is a complex process. The position of the human body relative to gravity and spatial surroundings is mainly sensed by central integration of visual, vestibular and somatosensory inputs . The rolIn this regard, it is of interest to see if sudden acute hearing loss in younger patients affects postural stability. In a recent study by Horowitz et al., all but one static balance tests remained unchanged in young normal-hearing persons before and after the ears were plugged with earplugs . SurprisThis prospective observational pilot study was carried out by the Department of Otorhinolaryngology, Jena University Hospital, Jena, Germany. Approval for the study was obtained through the local ethics committee (No. 2021–2248-BO) and written informed consent was obtained from all study participants. The study included two experiments using two different methods to simulate the acute hearing loss in 25 healthy subjects. Earplugs were used in the first experiment and headphones with white noise in the second experiment. All participants performed the static balance test without and with simulated hearing loss. To rule out a learning effect, the sequence, without and with simulated hearing loss, was randomized in each participant in both experiments [Only healthy, normal hearing subject with normal balance function were included. The participants received a pure tone audiogram . The bone conduction hearing threshold at 0.5, 1, 2, and 4 kHz had to be below 20 dB. Unterberger stepping test and video head impulse test  had to be normal. Persons with a history of an ear or balance disease were excluded.Standard earplugs were used  in both ears. The sound insulation value of these plugs is 37 dB. An example of a pure tone audiogram of a subject wearing the earplugs is shown in Supplement Fig. 1.In this setting, the simulation was performed using headphones  and white noise with a sound volume of 75 dB. An example of a pure tone audiogram and a speech audiogram of a subject wearing the headphones with white noise is shown in Supplement Fig. A computerized dynamic posturography system  was used. The SMART EquiTest uses a computerized dual force plate system that allows real-time movement and center of gravity (COG) estimations based on pressure changes on the platform related to the participant’s height. The force sampling frequency was set at 100 Hz. The test battery consisted of the Sensory Organization Test (SOT), the Motor Control Test (MCT) and the Adaptation Test (ADT) protocols.The SOT allows a separated or combined manipulation of visual, vestibular, and somatosensory feedback of the static balance system using six test conditions with eyes open/closed, fixed/moving surround, fixed/moving platform . The subThe MCT measures the subject’s automatic postural reaction as a reaction to support unexpected surface translations in a standardized manner : particiThe translations were scaled according to the person’s height. The durations were the same for everyone. Each translation started randomly after 1.5–2.5 s to prevent a foreseeability of the translation. As outcome measure of the MCT, the latency scores from the MCT were determined by calculation of the elapsed time in ms between onset of support surface translation until the point when the person actively resisted the induced sway.The ADT measures motor reactions following abrupt platform rotations to the direction of toes up and down, with an amplitude of 8º and duration of 400 ms . Particip values of 0.05 or less were considered significant.Participants’ characteristics and outcome variables were analyzed with IBM SPSS statistics software (Version 23.0) for medical statistics. Data are presented as mean ± standard deviation (SD) if not otherwise indicated. Explorative statistics were used as no sufficient data on the effects of a simulated hearing loss on static balance were available. SOT, MCT, and ADT outcomes were compared using repeated-measures analysis of variance (ANOVA). The results 1) without and with simulated hearing loss, 2) between experiment 1 and 2, and 3) also interactions were compared. Seventeen female and eight male participants were examined. The mean age was 22.4 ± 2.2 years (range 19–31). Mean body height was 1.7 ± 0.1 m (1.6–2.0). Mean body weight was 66.4 ± 12.5 kg (range 49–110). This resulted in a BMI of 22.0 ± 2.6 (range 19.1–31.5).p > 0.05; Table p = 0.168) were significantly better in experiment 2 than in experiment 1 . There was no significant interaction .The highest (best) values were achieved in condition 1 . There was no significant interaction .The results of the MCT are also presented in Table p = 0.003; toe up 3: p = 0.001; toe up 4: p = 0.041; toe up 5: p = 0.02; toe up mean: p = 0.001; toe down 1: p = 0.003; toe down 2: p = 0.031; toe down 4: p = 0.030; toe down 5: p = 0.027; toe down mean: p = 0.003). There was no significant interaction .The results of the ADT are also presented in Table Studies on the associations between acute or chronic hearing acuity and postural balance as well as between hearing acuity and falls are scarce and the results have been contradictory . Some stp = 0.0495), in a soundproof room with open eyes on foam (p = 0.0164), or with closed eyes on foam (p = 0.0495), wearing ear defenders resulted on more postural sway. The p values are even marginal in two of these three setting. In all other four conditions, the defenders had no significant impact . Hence, also the work of Kanegaonkar et al. is more confirming than contradicting the present results. From a practical point of view, it can be concluded that acute hearing loss (for instance by a noise trauma at work) should not affect static balance. This could be important for occupations with high demands on static postural stability.To analyze the role of normal hearing on static balance in people with normal vestibular function, experiments like the present study simulating acute hearing loss not allowing any adaptation to the acute impairment are important. Such studies are even scarcer than studies with people with chronic hearing loss. Horowitz et al. used also earplugs (like in experiment 1 in the present study) to induce acute bilateral hearing loss in 20 normal hearing young adults . They alThe present study was limited to normal hearing 19–31-year-old adults. The sample size was small. Hence, the results should be interpreted with caution. As a next step, we want to confirm the results in a larger cohort of young adults. Second, we want to repeat the study with older adults allowing the normal spectrum of hearing loss in such an older cohort. Furthermore, our focus is on static balance. That does not simply mean that acute bilateral moderate or severe hearing loss also has no influence on dynamic balance. When older adults (65–86 years) with no diagnosed hearing loss walk on a treadmill, ear plugging results in longer step length and step time . HoweverA large test battery consisting of the SOT, MCT, and ADT has shown that normal hearing adults between 19 and 31 years of age principally show an unaltered normal static balance during a simulated acute conductive moderate or severe bilateral hearing loss, at least in this first small pilot study. This suggest that hearing or spatial information does not play an important role to support static balance even during postural challenges in young adults.Supplementary file1 (PPTX 121 KB)Below is the link to the electronic supplementary material.


Atp2b2, an outer hair cell gene, cause dominant hearing loss in humans. Using a mouse model Atp2b2Obl/+, with a dominant hearing loss mutation (Oblivion), we show that liposome-mediated in vivo delivery of CRISPR-Cas9 ribonucleoprotein complexes leads to specific editing of the Obl allele. Large deletions encompassing the Obl locus and indels were identified as the result of editing. In vivo genome editing promotes outer hair cell survival and restores their function, leading to hearing recovery. We further show that in a double-dominant mutant mouse model, in which the Tmc1 Beethoven mutation and the Atp2b2 Oblivion mutation cause digenic genetic hearing loss, Cas9/sgRNA delivery targeting both mutations leads to partial hearing recovery. These findings suggest that liposome-RNP delivery can be used as a strategy to recover hearing with dominant mutations in OHC genes and with digenic mutations in the auditory hair cells, potentially expanding therapeutics of gene editing to treat hearing loss.Mutations in  Liposome-mediated gene editing was used to abolish a mutation in gene Atp2b2 and recover hearing in a mouse model of dominant deafness. Editing was also used to target two mutations to recover hearing. The study detected large deletions due to editing. The most common type is sensorineural hearing loss (SNHL) of which pathogenesis occurs in the inner ear, the sensory organ or the auditory nerve. Hereditary types of hearing loss account for more than 50% of all congenital sensorineural hearing loss cases and are caused by genetic mutations1. To date, over 120 genetic loci have been linked to hereditary hearing loss (http://hereditaryhearingloss.org/), but there are currently no biological treatments for any form of hereditary deafness3. In the last two decades, the use of gene therapy strategies as treatment for inner ear dysfunction has emerged as a powerful therapeutic approach in deaf mouse models. Gene replacement using adeno-associated viral (AAV) vectors, gene silencing using either antisense oligonucleotides or AAV delivered microRNAs are strategies that have shown promising results in improving hearing in mouse models of deafness8. However, these approaches have limitations including the size of genes to be inserted into an AAV, inefficient targeting dominant mutations, and potential long term safety concerns of viral vectors.Deafness affects about 466 million people, including 34 million children, constituting 6% of the global population (13. We and others have applied the CRISPR-based gene editing agents to recover hearing in mouse models of human genetic deafness. By lipid and AAV mediated delivery of Streptococcus pyogenes Cas9 (SpCas9) ribonucleoprotein (RNP) complexes, we and others have improved hearing in a mouse model of dominant hearing loss of hair cell origin, the Beethoven (Bth) mouse, by targeting the mutant allele of the transmembrane channel-like 1 (Tmc1)15. Recently, the new generation of base editors were used to repair a recessive mutation on the Tmc1 gene, which transiently improved hearing in the Baringo mice16. The editing treatment for hearing loss, however, has been limited to inner hair cells (IHC) due to the primary role of Tmc1. The auditory function requires functional inner and outer hair cells (OHC). Besides, multiple types of genetic hearing loss are associated with mutations in OHCs17. In our editing treatment of the Beethoven mutant mice for hearing recover, we detected deterioration in OHC function shown by distortion product of acoustic emissions (DPOAE) in the treated inner ears14, raising the question if our liposome-mediated delivery and editing is suitable to target OHC mutations to recover hearing. Thus, it is necessary to address the limited efficiency of gene editing therapies in targeting and functionally rescuing the OHC.The advent of genome editing has made it possible to treat genetic diseases by the modifications at genomic DNA level to disrupt or correct the mutations. Furthermore, the recent discovery and development of CRISPR-associated protein 9 (Cas9)-based genome editing has facilitated the effective targeted gene disruption or repair of virtually any sequence of interest in the genome, and has been used to treat hereditary diseases in mouse modelsAtp2b2Obl/+ mouse), which carries a mutation Oblivion (Obl) in the second isoform of the plasma membrane Ca2+-ATPase (Atp2b2) gene that impairs the calcium pumping ability of the PMCA2 protein18. The PMCA2 calcium pump is produced at high level in the stereocilia bundle of the OHC19. Mutations of PMCA2 Ca2+ pump of the stereocilia cause deafness and loss of balance in both mice and humans21. Obl heterozygous mice show progressive hearing loss starting at P20 and progressive degeneration of the sensory hair cells. Obl homozygous mutants are completely deaf from birth and show a severe degeneration of the organ of Corti. In this work, we apply our previously described CRISPR-Cas9 lipid-mediated delivery paradigm to the Oblivion mouse model to treat hearing loss of the OHC origin. We first selected and evaluated the ability and specificity of different sgRNAs in disrupting the Obl mutation in vitro. Then, we performed lipid-mediated delivery of Cas9 and sgRNA ribonucleoprotein complexes into the inner ear of postnatal heterozygous Atp2b2Obl/+ mouse to recover hearing.To evaluate liposome delivery of gene editing complex targeting the OHCs, we selected a dominant deafness mouse model  mutation and a nearby NGG protospacer adjacent motif (PAM) sequence required by SpCas9. The three candidate sgRNAs  place the Obl mutation at the position of 18, 12, and 20, respectively, of the protospacer, counting the PAM as positions 21-23  efficiency at the on-target by next-generation sequencing (NGS) and detected indels (1.24–2.67%) in both Atp2b2Obl/+ and Atp2b2Obl/Obl mouse primary fibroblasts  and small (with large deletions) DNA fragments . In injected inner ears, an Obl:WT ratio percentage of 46.18 ± 2.55: 53.25 ± 2.27 (mean ± sd) was detected. There was 6.4% shift from the Obl towards the WT allele in hair cells after injection. We calculated that in injected ears, the editing efficiency on the Obl is ~21% 31. Previous studies have shown that the Atp2b2Obl/+ mice exhibited progressive base to apex degeneration of hair cells, with OHC more affected than inner hair cells (IHC)21. To evaluate the effect of the Cas9:Atp2b2-mut1 sgRNA complex to target the Obl allele in the OHCs in vivo, we complexed the Cas9:Atp2b2-mut1 sgRNA with Lipofectamine 2000 and injected the mixture into the scala media of neonatal (P0-2) Atp2b2Obl/+ mice by cochleostomy, with the mouse inner ears harvested at 2 months of age. We used an anti-Parvalbumin antibody to label all hair cells and an anti-PMCA2 antibody to visualize the distribution of PMCA2, the protein encoded by the Atp2b2 gene, in the OHC. In uninjected Atp2b2Obl/+ inner ear, there was a severe loss of HCs. In the high-frequency region (45.25 kHz), virtually all OHCs (PMCA2+) were absent with some missing IHC, whereas in other frequency regions, partial loss of OHCs was evident 21. The lack of OHC function results in profound hearing loss in the Atp2b2Obl/+ mice21.The goal of our study is to use editing to target the dominant OHC Atp2b2-mut1 sgRNA complex to rescue OHC function, we injected lipid-mediated delivery of Cas9:Atp2b2-mut1 sgRNA complexes into neonatal (P0-P2) Atp2b2Obl/+ mice and analyzed the DPOAEs from 4 weeks onwards. Wild type 1-month-old C3H mice, with the same genetic background as the Atp2b2Obl/+, were included for the measurement as additional controls. Four weeks after injection, Atp2b2Obl/+ untreated ears showed elevated DPOAEs thresholds ranging from 75–80 dB SPL across all frequencies, demonstrating a virtually complete lack of the OHC function , which assays the sound evoked neural output of the cochlea. Uninjected Atp2b2Obl/+ inner ears showed elevations in cochlear neural responses at 4 weeks of age, with ABR thresholds ranging from 70–90 dB, compared with the 30–40 dB for wild-type C3H mice from 5.66 to 45.24 frequencies  startle responses following stimulus at 110 and 120 dB SPL compared to the uninjected mice, with the startle response amplitudes that were similar or equal to those observed in the wild-type C3H mice  improvement in DPOAE thresholds was still detected at 11.32 and 16 kHz 16 weeks after injection  in Atp2b2Obl/+ mice and observed no significant improvements in ABR or DPOAE thresholds , in which the Obl mutation primarily affects the OHC whereas the Bth affects the IHC. We performed liposome-mediated delivery of Cas9 complexed with individual Atp2b2-mut1 sgRNA, Tmc1-mut3 sgRNA or the mixture of both into the scala media of neonatal (P1) double mutant mice and tested the hearing 1 month after the injection. We have previously demonstrated that Cas9:Tmc1-mut3 sgRNA:Lipo2000 injection recovered hearing in the Tmc1Bth/+ model14. Uninjected double mutant ears exhibited severely damaged auditory function, shown by large shifts in ABR and DPOAE thresholds across all frequencies   was also detected in the injected inner ears  Fig. . In the 11) Fig. . Lower Dars Fig. . Injectiing Fig. . Taking 21. Outer hair cells are known to be the most vulnerable components of the auditory system and their malfunction is implicated in sensory hearing loss pathologies. Without proper functional OHC, hearing sensitivity and frequency selectivity are impaired34. Mutations in the Atp2b2 have been associated with dominant progressive hearing loss in humans18. An Atp2b2 variant has also been shown to be a modifier that contributes to the severity of hearing loss20. In mice, Atp2b2 encodes the resident Ca+ pump of hair cell stereocilia. The Obl mutation causes dysfunction in the transmembrane domain 6, which significantly reduces the nonactivated Ca2+ exporting ability. The dysfunction of Ca2+ export by the mutated PMCA2 pump causes progressive hearing loss21. Given the importance of PMCA2 in the OHC function and hearing loss, and the dominant transmission pattern, the Obl mouse model is a good model to study using editing to disrupt the mutation and to recover hearing.PMCA2 is essential to OHC function and is required for hearing14. In that model, however, the mutation Beethoven in the Tmc1 gene primarily affects inner hair cells. Furthermore, DPOAE thresholds in the treated inner ears were elevated compared to uninjected control ears. This raised a question if our approach is suitable to target OHC to edit mutations for hearing recovery. In addition to PMCA2, OHC mutations have been associated with the most common forms of dominant genetic hearing loss, including KCNQ435.We have previously demonstrated the feasibility of liposomal-mediated RNP delivery of editing agents in hearing recovery in a mouse model of human genetic hearing loss DFNA36Obl mutation in the Atp2b2 gene, restoring the functions of OHCs with improved cell survival and recovery of hearing in dominant genetic mouse model Oblivion with OHC deficit. The recovery of auditory function quantified here using physiological and behavioral assays suggests that transient lipid-mediated delivery of RNP complexes may be a good therapeutic strategy to target OHC for editing, and recover hearing of dominant genetic OHC dysfunction. Combined with our previous work on editing for hearing recovery in the inner hair cells14, it supports that liposomal-mediated RNP delivery can be applied to recover hearing in dominant hearing loss models of inner and/or outer hair cell origin.The current work shows that liposome-mediated delivery of Cas9-sgRNA RNP complexes in the inner ear leads to editing that abolishes the Atp2b2Obl/+ inner ear, with reductions of 9–32 dB SPL depending on the frequency. Based on our results for 8 and 16 weeks after injection, the treatment effect starts to diminish at 16 weeks post injection. While the underlying mechanism of the diminished hearing recovery is unknown, one hypothesis is that the duration of hearing recovery is related to the equilibrium between edited and unedited hair cells. In our study, we detected editing in ~20% of hair cells, leaving more hair cells unedited. If unedited hair cells start to degenerate, the edited hair cells may not be able to sustain the cochlear function due to the death of unedited HC which may impact both the cochlear structure and function. In addition, the diminished hearing recovery could be gene specific in that the production of 50% of normal PMCA2 protein after editing of the mutant allele may not be sufficient to maintain the health and function of the cochlea long-term. To sustain hearing recovery, improved editing efficiency in more OHC will likely be critical. Another possibility is that the mutant hair cells may have started to degenerate at the time of injection (p0-p2) whereas editing delays the degeneration process. Indeed, significant elevation of DPOAE and ABR thresholds was seen at 1 month of age in the Atp2b2obl/+ ear, suggesting hair cells may have undergone some degeneration at an early stage. In this case, intervention at an even earlier age  may improve the recovery effect. The significant difference between early and later inventions has been illustrated in most hearing recovery studies in which the treatment was only effective when the injection was done in the neonatal stage whereas it would fail if the intervention were done even a few days later 36.Our study shows recovery in DPOAE and ABR thresholds after injection of editing agents in the Obl allele. By NGS and indel analysis from the Atp2b2Obl/+ adult primary fibroblasts treated by nucleofection of the RNP complex in vitro or from the Atp2b2Obl/+ inner ears injected with the complex in vivo, the overall indel rate is relatively low (~1%), which suggested that the analysis underestimated the editing efficiency. A few factors may have contributed to the low indel rate detected. First, editing in the Atp2b2 locus in adult fibroblasts may be intrinsically less efficient than in other rapidly dividing cell type. After we used the transposon to introduce the DNA fragment harboring the Obl mutation into an organ of Corti cell line that is rapidly dividing, we detected an indel rate of 40% after nucleofection of the same RNP complex, suggesting cell type influences editing efficiency. Second, in the cochlear samples collected from in vivo study for indel analysis, the hair cells only consist of a small portion of all the cochlear cells. Third, we identified large deletions from the samples of in vivo studies, which a standard indel analysis failed to detect. As we did not exhaustively characterize large deletions, our selective PCR-based assay is likely to miss other deletions or DNA organizational changes.The hearing recovery is likely due to efficient editing at the 15. By single-cell whole-genome amplification of purified hair cells for NGS, we detected a reduction in the allele frequency ratio from Obl to the WT after RNP delivery. While it is expected that in uninjected Atp2b2Obl/+ inner ear, the allele frequency ratio of Obl:WT should be 50%:50%. The detected frequency ratio of Obl:WT, however, was 53%:47%, which was likely the result of the bias introduced during genome amplification that favored the Obl allele. After the injection, the Obl allele decreased by over 6.4% compared to untreated hair cells, which supports editing in 12.8% of the Obl allele. The lower Obl allele frequency after editing is consistent with the data that only the Obl allele was edited by RNP delivery of Cas9-Atp2b2-mut1 in vitro and in vivo  with profound hearing loss at 1 month. It is significant that injection of Cas9 complexed with the double gRNAs of Atp2b2-mut1 and Tmc1-mut3 recovered hearing shown by ABR thresholds significantly, whereas no hearing was recovered by a single gRNA delivery. The results suggest that in some hair cells both mutant Obl and Bth alleles have been edited that led to hearing recovery. This result is interesting given the fact that the delivery of each gRNA alone resulted in the editing efficiency of ~10% for Bth14 and for Obl, thus a double editing of two mutations in the same hair cell is calculated around 2%. One possible explanation for hearing recovery in the double mutant mice is that the editing by the two gRNAs is not a random event, i.e., a hair cell may be preferentially edited by both gRNAs that gives rise to a skewed outcome. It is also possible that editing efficiency by each gRNA is higher than our data that was estimated to be the baseline for editing efficiency, which may contribute to higher number of hair cells being edited at both alleles. Another explanation is that editing in one hair cell may restore some function whereas collectively, there is an additive effect when both gRNAs were delivered. Despite the hearing recovery in the two-gRNA injected digenic mutant inner ears is much smaller compared to each gRNA injected to Atp2b2Obl/+ or Tmc1Bth/+ individually, the partial hearing improvement observed after RNP delivery supports further development to improve the efficacy, which should open the possibility for the approach as a potential treatment of hearing loss of multigenic origin. Our work provides evidence on the recovery of hearing based on the auditory function tests of ABR and DPOAE. We did not address how hearing recovery may affect mouse behavior. For the digenic mouse model, it is likely that more efficacious hearing recovery  will be needed to benefit the mice in behaviorally significant ways.We further provide evidence that the liposome RNP delivery system has the potential to be applied to partially improve hearing in mice carrying separate mutations in two genes. Genetic hearing loss due to digenic mutations is well documentedAtp2b2Obl/+ mice with the effect that is reduced long term. We have shown that RNP delivery is suitable for the intervention of dominant mutations in the auditory hair cells. Combined with the evidence of the partial recovery effect in the digenic mouse mutants with hearing loss, we have further widened the path for future development of editing base therapy for genetic hearing loss.Our work demonstrates the feasibility of RNP delivery of editing agents into the inner ear to target outer hair cell mutations and recover hearing in the Linear DNA fragments containing the T7 promoter binding site followed by the 20-bp sgRNA target sequence were obtained by PCR using Q5 High-Fidelity DNA Polymerase (NEB) with the primers listed in the supplementary information and 40 ng of pFYF1320 (EGFP sgRNA expression plasmid) as a template according to the manufacturer’s instructions. PCR products were purified using QUIAquick PCR purification kit (Qiagen) and transcribed in vitro using the T7 High Yield RNA Synthesis Kit (NEB) according to the manufacturer’s instructions. sgRNA products were purified using the MEGAclear Transcription Clean-Up Kit (Ambion), quantified by Nanodrop and stored at −80 °C.Atp2b2+/+ and Atp2b2Obl/Obl genomic DNA was isolated from mouse tail snips using the Agencourt DNAdvance Genomic DNA Isolation Kit (Beckman Coulter) according to the manufacturer’s instructions. 956-bp Atp2b2 DNA fragments were amplified from the genomic DNA and used as substrates for the in vitro cleavage reaction . Typically, the Atp2b2 DNA fragment (150 nM) was incubated for 15 min at 37 °C with 300 nM of purified Cas9 protein and sgRNA in Cas9 cleavage buffer  in a total volume of 20 µL in each reaction. Reactions were quenched by adding 500 µL of PB wash buffer (Qiagen), purified on a QIAprep spin column and eluted in 20 µL 1X TE buffer. 10 µL of each reaction were loaded onto a 2% agarose gel and electrophoresed to separate starting DNA and cleaved products. Cas9-induced cleavage bands and the uncleaved band were visualized on an ChemiDoc MP and quantified using ImageJ software. The peak intensities of the cleaved bands were analyzed as previously described39.Atp2b2 gene fragment (2-kb) harboring the Obl mutation was amplified by PCR from Atp2b2Obl/Obl mouse genomic DNA using primers . The PCR products were ligated into the restriction sites (HindIII and NotI) in the PiggyBac donor backbone (PB-CAG-mNeonGreen-P2A-BSD-polyA)40. The constructed donor plasmid was co-transfected with PiggyBac transposon vector  in HEI-OC1 cells. Cells with inserted Atp2b2 gene fragments carrying the Obl mutation were cultured and selected in the medium containing 10 µg/mL Blasticidin. Successful insertion was confirmed by PCR  and sequencing analysis.An TM X Kit). 400 ng of pmaxGFP Control Vector (LONZA) was added to the nucleofection solution to assess nucleofection efficiencies in HEI-OC1, Obl-OC1 cells and primary fibroblasts. To deliver plasmids, cells were transfected using 1000 ng Cas9 plasmid (pCas9), 500 ng sgRNA plasmid (pAtp2b2-mut1 sgRNA). To deliver protein complex, purified sgRNA was incubated with Cas9 protein for 5 min before transfection. Media was replaced ~16 h after nucleofection and cells were harvested for genomic DNA extraction after ~96 h.Transfection programs were optimized following manufacturer’s instruction  background were obtained from Wellcome Trust Sanger Institute. Atp2b2Obl/+ mice were bred with Tmc1Bth/Bth to obtain Pmca2Obl/+Tmc1Bth/+ mice.All in vivo experiments were carried out in accordance with NIH guidelines for the care and use of laboratory animals and were approved by the Massachusetts Eye & Ear Infirmary IACUC committee. Isogenic heterozygous Atp2b2Obl/+ mice (P0-2) of either sex were used for injections. The mice were randomly assigned to the different experimental groups, and at least three mice were injected in each group. Both surgical procedures and injections were performed as described previously14. Briefly, 25 μM of Cas9 and sgRNA was mixed with Lipofectamine 2000 for 20 min at room temperature, and injected into the scala media of the cochlea at three different sites . The volume for each injection was 0.3 μl with a total volume of 0.9 μl per cochlea. The release rate was 69 nl/min, controlled by a MICRO4 microinjection controller (WPI).A total of 112 14. The acoustic tests were performed in mice up to 4 months old anesthetized intraperitoneally with a mix of xylazine (10 mg/kg) and ketamine (100 mg/kg). Three subcutaneous needle electrodes placed at vertex, the ventral edge of the pinna and the tail (ground reference), were used for the recordings. Stimuli consisted in 5 ms tone pips  presented at frequencies 16, 22.64, 32, and 45.25 kHz and delivered in 10 dB ascending steps from 20 to 90 dB . The response was amplified 10,000-fold, filtered (100 Hz–3 kHz passband), digitized, and averaged (1024 responses) at each SPL. Following visual inspection of stacked waveforms, ABR threshold was defined as the lowest SPL level at which any wave could be detected. Wave 1 amplitude was defined as the difference between the average of the 1-ms pre-stimulus baseline and the Wave 1 peak (P1). The cubic distortion product for DPOAE measurements was quantified in response to primaries f1 and f2. The primary tones were set as previously described14. Threshold was computed by interpolation as the f2 level required to produce a DPOAE at 5 dB SPL.Auditory brainstem responses (ABR) and distortion product otoacoustic emissions (DPOAE) were performed in a soundproof chamber as described previouslyMice were placed into a small, acoustically transparent cage resting atop a piezoelectric force plate in a sound-attenuated booth. Acoustic stimuli and amplified force plate signals were encoded by a digital signal processor  using LabView scripts . Mice were placed in silence for 5 min to acclimate to the test environment before real measurements. A 16-kHz tone was presented at randomized intervals from an overhead speaker . Twelve repetitions were recorded for each of the intensities per test subject. Startle response amplitude was measured as the root mean square (RMS) voltage of the force plate signal shortly after sound presentation.2 inhalation. Temporal bones were fixed in 4% paraformaldehyde at 4 °C overnight, and then decalcified in 120 mM EDTA for at least 1 week. When the bone was decalcified, the organ of Corti was dissected in pieces for whole-mount immunofluorescence. Nonspecific labeling was blocked with blocking solution (PBS with 8% donkey serum and 0.3−1% Triton X-100) for 1 h at room temperature and followed by overnight incubation at room temperature with the primary antibody 1:300 mouse anti-Parvalbumin (Sigma P3088), 1:200 rabbit anti-pmca2 (PA1-915 ThermoFisher scientific). Tissues were incubated with the secondary antibody for 1 h after three rinses with PBS. All Alexafluor secondary antibodies were from Invitrogen: donkey anti-rabbit Alex488 (A21206) and donkey anti-mouse Alex594 (A32744) was used as a 1:500 dilution. All specimens were mounted in ProLong Gold Antifade Mountant medium . Confocal images were taken with a Leica TCS SP8 microscope using a 20X or 63X glycerin-immersion lens, with or without digital zoom. We acquired z-stacks by maximum intensity projections of z-stacks for each segment by imageJ (NIH image), and composite images showing the whole cochlea.Injected and non-injected cochleae were removed after animals were sacrificed by COAtp2b2Olb/+ mice were injected with Cas9-GFP:Pmca2-mut1 sgRNA:Lipofectamine 2000 at P1 and were euthanized at P5. Cochleae were harvested with the sensory epithelia (GFP+) dissociated using needles under the microscope , and immersed in 1 μM FM 1-43FX  dissolved in HBSS (ThermoFisher) for 30 s at room temperature in the dark. The sensory epithelia were then transferred to 100 μl TrypleE Express Enzyme  with the GFP+ cells isolated using a 1 μl pipette, Isolated hair cells were subjected to whole-genome amplification by MALBAC Single Cell WGA Kit  to isolate DNA for NGS analysis.Treated cells or tissues were collected after four days and genomic DNA was isolated using the Agencourt DNAdvance Genomic DNA Isolation Kit (Beckman Coulter) according to the manufacturer’s instructions. On-target and off-target genomic regions of interest were amplified by PCR with flanking primers . PCR amplification was carried out with Q5 High-Fidelity DNA Polymerase (NEB) according to the manufacturer’s instructions using 250 ng genomic DNA as a template. PCR products were purified using QUIAquick PCR purification kit (Qiagen). Samples were sequenced and analyzed to detect CRISPR variants from NGS reads using a custom algorithm developed by the Massachusetts General Hospital Center for Computational and Integrative Biology DNA Core. Individual SEQ files containing the variants were visualized by SnapGene.Obl-OC1 cells. A sample nucleofected with dsODN only served as a negative control. Cells were harvested for genomic DNA extraction after 5 days and ~400 ng of genomic DNA for each sample was sheared acoustically using a Covaris E220 sonicator to an average of 500 bp in 130 µL TE buffer. The GUIDE-seq sequencing libraries were prepared and sequenced on an Illumina Miseq as previously described27, with reads were mapped to the mouse reference genome (GRCm38). Off-target data analysis was initially performed with the standard pipeline, mapping the start position of the amplified sequences using a 10-bp sliding window, then retrieving the reference sequence around the site. Given the size of some of the deletions, the number of base pairs used as the flanking sequence was also increased to up to 10,000 bp. The retrieved sequences were aligned to the Cas9 target sequence using a Smith-Waterman local-alignment algorithm. The negative control sample treated with the dsODN but no Cas9 or sgRNA was used to assess background.To identify off-target sites, the GUIDE-seq method was performed essentially as previously described (GUIDE-seq ref) with the following modifications. Briefly, 100 nmol Cas9 , 800 ng sgRNA , and 1 pmol of the double-stranded oligodeoxynucleotide (dsODN) were nucleofected into 2 for 1.5–2 h at room temperature on a tissue rotator. Samples then were washed three times with distilled water. After that, samples were rinsed three times for 10 min with 0.1 M sodium cacodylate buffer and treated with 1% osmium tetroxide (one) for one hour, then a thorough rinse three times for 10 min each with distilled water. And samples were treated with saturated thiocarbohydrazide (two) in distilled water for 30 min. Repeat treatment was followed by an additional 1% osmium tetroxide step, making a treatment order of one-two-one-two-one. Then, samples were transferred to 20 ml scintillation vials for dehydration (in 2 ml distilled water) with ethanol, adding 50 µL of 100% ethanol to the vial, followed by doubling the added volume every 10 min. When the vial was full, samples were transferred to 100% ethanol, dried from liquid CO2 (Tousimis Autosamdri 815) in terms of critical point. Then, samples were mounted with a carbon tape on aluminum specimen stubs, spatter-coated with 4.5 nm of platinum using Leica EM ACE600 and operated with Hitachi S-4700 SEM.After cochlea dissection, tissues were harvested and immersed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (EMS) supplemented with 2 mM CaClStatistical analyses were performed by two-way ANOVA with Bonferroni corrections for multiple comparisons for ABRs and DPOAEs, and by Student t-test for quantification of hair cell survival using the Prism 6.0 statistical analysis program (GraphPad).Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary