Hearing loss is quite prevalent and can be related to people’s quality of life. To our knowledge, there are limited studies assessing the efficacy of hearing interventions on quality of life in adults. Therefore, we aim to conduct a randomized controlled trial (RCT) to determine the impact and cost-effectiveness of community-based hearing rehabilitation on quality of life among Chinese adults with hearing loss.n = 464) will be recruited from Linyi City, Shandong Province. They are randomly assigned to the treatment group or the control group. Those in the treatment group are prescribed with hearing aids, while those in the control group receive no intervention. Reinstruction in use of devices is provided for the treatment group during booster visits held 12 months post-randomization or unscheduled interim visits when necessary. Data are collected at baseline and the follow-up 20 months later. The primary outcome is changes in quality of life over a 20-month study period. Secondary outcomes include sub-dimensions in quality of life, physical functioning, chronic diseases, cognitive function, depression, social support, hospitalizations, falls, and healthcare costs. Finally, we will evaluate whether hearing aids intervention is cost-effective to apply in a large scale.In this two-arm feasibility study, participants aged 16 and above with some degree of hearing loss (The trial is designed to evaluate the impact and cost-effectiveness of a community-based rehabilitation intervention on quality of life among Chinese adults with hearing loss. We hope that it would help improve the well-being for Chinese adults and provide references in policy and practice for China and other countries.ChiCTR1900024739. Registered on 26 July 2019.Chinese Clinical Trial Registry The online version contains supplementary material available at 10.1186/s13063-021-05228-2. Hearing is one of the basic means of perception and communication. With the global aging, hearing loss is the most common sensory dysfunction and is becoming an increasingly serious public health issue . It is sHearing loss can be associated with a series of health problems, such as poor physical and mental health , 8. EmpiAlthough there is a strong correlation between hearing loss and health, it has not received enough attention from the public. First, hearing loss usually has a slow onset and gets worse progressively, which is difficult to detect in time unless by audiometry tests . Our preThe use of hearing aids is a main rehabilitation intervention for people with hearing loss . One stuGiven the high prevalence of hearing loss and low accessibility of hearing aids, it is imperative to conduct high-quality studies to figure out the efficacy of wearing hearing aids. Systematic reviews on hearing and quality of life have found that a majority of studies were cross-sectional and only a few were conducted in developing countries . AlthougIn this protocol, we described the design of a randomized controlled trial to assess the impact and cost-effectiveness of hearing aids intervention on 20-month changes in quality of life. The study is expected to involve 464 Chinese adults aged 16 and older who have some degree of hearing loss and are required to wear a hearing aid by otologists. They are randomly assigned to the treatment group of wearing hearing aids or the control group with no intervention to determine the efficacy of hearing aids in improving the quality of life and to move toward early prevention and treatment of hearing loss.The primary objective is to determine whether hearing aids intervention is effective in improving the quality of life in adults with hearing loss. The secondary objectives are to determine whether hearing aids treatment is cost-effective, including improving the health-related quality of life, decreasing inpatient and outpatient visits, and recovering productivity.Our study is a randomized, controlled trial lasting 20 months with two parallel groups, the treatment group and the control group. According to a list provided by the Hearing Center of Linyi Disabled Persons’ Federation, which records all the hearing-disabled people in Linyi City, 464 people are randomly selected. Participants are prescreened by telephone and complete a hearing screening at baseline (T0). Those in the treatment group are prescribed with hearing aids and receive a post intervention of reinstruction in use of hearing aids 12 months after T0, while the control group receives no interventions. The follow-up surveys are scheduled 20 months after T0 to trace changes in health outcomes , untreatable conductive hearing loss (difference in air audiometry and bone audiometry (“air-bone gap”) > 15 dB in two or more contiguous frequencies in both ears that cannot be medically resolved), or unwillingness to regularly wear hearing aids.Participants in the hearing treatment group are prescribed with hearing aids at baseline, while the control group with no interventions. Reinstruction in use of hearing aids is prescribed during booster visits held 12 months and 20 months post-randomization. Unscheduled interim visits may also be sporadically required . Participants in the control group will not have access to hearing aids. They will be asked to attend twice for this project: baseline and follow-up 20 months later.Study participation and hearing intervention are expected to have a low risk of adverse events. But the age of the participants may naturally lead to certain deleterious health outcomes. In case of any accidental injury, lack of efficacy, or withdrawal of participant consent during the trial, investigators can modify or discontinue the trial with the approval of the principal investigator (PI).Individual assessment and instruction.Participants are asked to bring a communication partner with them to the study visits.Participants who miss a scheduled meeting are contacted by telephone by study staff to encourage continued participation and to evaluate and overcome barriers to participation.Adherence to the study intervention will be assessed at each study visit using questions designed to capture adherence in both the treatment and control groups. Strategies to promote adherence include:Potential intervention benefits are structured given the participant’s level of hearing loss in order to ensure that participants’ expectations are reasonable and realistic.Participants are informed that they are allowed to keep the hearing aids for free if they complete all study visits.For the treatment group, it additionally includes:The primary study outcome is the 20-month changes in quality of life from the 2019 baseline to the 2021 follow-up. Quality of life can be measured by standard tests of Short Form 12 Health Survey (SF-12)  and EuroKey secondary outcomes include changes in sub-scores of SF-12   and EQ-5Participants are required to receive pure tone audiometry at the thresholds of 0.5, 1, 2, and 4 kHz to derive accurate hearing data. Besides pure tone audiometry, standardized questionnaires like Hearing Handicap Inventory for the Elderly—Screening version (HHIE-S) , combineSociodemographic factors such as age, gender, residency, family information, socioeconomic status, and other clinical factors are collected at baseline and the follow-up survey.Power calculations showed that 404 (202 in each group) participants are required in each group to obtain 80% statistical power with a 5% significance level and to detect a 0.28-SD difference in the mean change from baseline in quality of life score to 20-month follow-up . To accoOur recruitment will last 1 month, from July 1 to 31, 2019. Adults with untreated hearing loss will be recruited according to the list of people with hearing loss provided by the hearing center of Linyi Disabled Persons’ Federation. Informed, written consent is obtained from all participants prior to participation.http://www.random.org) to allocate participants in a random sequence to the treatment or control group. In a separate room after completion of the baseline measurements, an independent researcher will tell participants to which group they have been allocated, thereby ensuring concealment of the identity and characteristics of participants.Participants are randomized stratified by severity of hearing loss, so as to avoid uneven distribution of participants. A researcher who is not involved in data collection or analysis will use a random sequence generator  blinding of participants to the study hypothesis, (2) use of standardized protocols for training of data collectors and assessment of study outcomes, and (3) masking of field working staff to block size, to avoid unintentional and possibly unconscious bias by study staff during data collection.Standardized data forms or tablets are used to collect data with paper back up available in case of tablet failure. Participants will fill in all questionnaires in a separate room. To ensure that appropriate help and guidance can be given when needed, one of the researchers will present. Meanwhile, communication partners who communicate with participants on a daily or near-daily basis  are often a key to getting accurate results. Therefore, accompanying adults are also invited to join the study and contribute to the data.To minimize data-entry errors, the questionnaires have inbuilt check-and-skip rules. And the questionnaires were tested on four participants beforehand, who found the questions understandable and possible to complete in 30–45 min. For participants who withdraw from the trial, any data collected up to the withdrawal date will be retained and included in the analyses. Data are collected at the local field prior to randomization (baseline T0), after 12 months (post intervention T1) and 20 months after baseline (follow-up T2). A SPIRIT flow diagram illustrates the data collection in the intervention group and control group  principle is applied with conservative estimates of missing data . ParticiIn addition, some variables such as socioeconomic status and social support may potentially affect the utilization of hearing rehabilitative services, so the interaction between socioeconomic status or social support with the hearing intervention can be analyzed to gain further results. The cost-effectiveness analysis is based on the cost of the intervention, effects in improving health-related quality of life, decreasing inpatient and outpatient visits, and saving healthcare costs and recovering productivity. By these means, we aim to explore the most cost-effective intervention strategy to improve the quality of life for people with hearing loss.The principal investigator (PI) is responsible for the quality and integrity of data collected. During the period of recruitment, interim analyses will be supplied in strict confidence, which may include analyses of data from other comparable trials. In the light of these interim analyses, the PI will advise if the intervention has been proved, or different from expected. Then the PI will decide whether or not to modify the trial. Unless this happens, however, study staff will remain ignorant of the interim results.In our study, adverse events will be collected and recorded after participants have provided consent and enrolled, until the end of the study. If a participant experiences an adverse event after the informed consent is signed (entry) but the participant has not started to receive study intervention, the event will be reported as not related to our intervention. An adverse event that meets the criteria for a serious adverse event (SAE) will be reported to the institutional review board (IRB). And study personnel will document the circumstances.PI will permit study-related monitoring, audits, and inspections by the IRB of all study related documents . PI will ensure the capability for inspections of applicable study-related facilities.This trial is carried out without patient or public involvement. Neither patients nor the public are involved in the development of the objective, design, or implementation of this trial. Patients will not be invited to develop patient-relevant outcomes or interpret the results, or to participate in the writing or editing of the final manuscript for readability or accuracy.The study was approved by Peking University’s Institutional Review Board (IRB00001052-19046). Any modifications to the protocol which may impact on the study, or the potential benefit and safety of the participants, including changes of study objectives, study design, participants, sample sizes, study procedures, or significant administrative aspects, will require a formal amendment to the protocol. Such amendment will be approved by the IRB prior to implementation.Investigators will introduce the trial to participants in light of the information provided in the information sheets. Participants will then be able to have an informed discussion with the investigator. Investigators will obtain written consent from willing participants. For confidentiality, each participant will be given a unique identification number. Other identification information such as names, mobile phone numbers, and addresses will not be recorded in the same form as sensitive data. In case of any accidental injury during the trial, medical treatment and economic compensation will be provided according to the laws and regulations of China.PI will be given access to the cleaned data sets. Results will be disseminated in the clinical and scientific communities and also to the population with hearing loss via peer-reviewed research publications both online and in print, conference and meeting presentations, posters, newsletter articles, website reports, and social media. Results will be reported according to the Consolidated Standards of Reporting Trials (CONSORT) guidelines . ImportaThe quality of life is associated with individuals’ physical health, psychological experience, and social relations . It is rThis will be the first RCT accessing the efficacy of hearing aids intervention on quality of life among Chinese adults. Strengths of this study include the robust randomized controlled trial design, with randomization stratified by hearing loss severity to reduce contamination, the inclusion of follow-up measures, and the rigorous economic evaluation. This study will contribute to building the evidence base around the effectiveness of interventions to improve the well-being for Chinese adults and provide references for other countries.We should note some weaknesses in the design of the trial. First, participants in the control group might also undergo changes in quality of life, which may conceivably be induced by questions about their health behaviors. It has been shown that participants’ behaviors can be increased or changed simply if questions are asked about their behaviors . In our To conclude, whether hearing treatment and rehabilitation can delay quality of life decline in at-risk adults is lacking compelling evidence, but could have substantial clinical, social, and public health impacts for people with hearing loss. When completed in 2021, our study should provide definitive evidence of the cost-effectiveness of hearing aids treatment on quality of life in community-dwelling Chinese adults with hearing loss.The current protocol is version 2, dated July 26, 2019. It took quite a few months to select journals and submit the protocol for review. Recruitment of patients began on July 1 and ended on July 31, 2019. The data collection ran until all participants completed the survey. Follow-up will be conducted after 20 months to test whether the effects of the intervention are still present. The study is expected to run until the end of March 2021. Until then, the intervention effects are unknown.Additional file 1. Trial Registration Data.Additional file 2. Informed Consent Forms.


Paramecium bursaria. Chlorovirus populations rise and fall in indigenous waters through time; however, the factors involved in these virus fluctuations are still under investigation. Chloroviruses attach to the surface of P. bursaria but cannot infect their zoochlorellae hosts because the viruses cannot reach the zoochlorellae as long as they are in the symbiotic phase. Predators of P. bursaria, such as copepods and didinia, can bring chloroviruses into contact with zoochlorellae by disrupting the paramecia, which results in an increase in virus titers in microcosm experiments. Here, we report that another predator of P. bursaria, Bursaria truncatella, can also increase chlorovirus titers. After two days of foraging on P. bursaria, B. truncatella increased infectious chlorovirus abundance about 20 times above the controls. Shorter term foraging (3 h) resulted in a small increase of chlorovirus titers over the controls and more foraging generated more chloroviruses. Considering that B. truncatella does not release viable zoochlorellae either during foraging or through fecal pellets, where zoochlorellae could be infected by chlorovirus, we suggest a third pathway of predator virus catalysis. By engulfing the entire protist and digesting it slowly, virus replication can occur within the predator and some of the virus is passed out through a waste vacuole. These results provide additional support for the hypothesis that predators of P. bursaria are important drivers of chlorovirus population sizes and dynamics.Chloroviruses are large viruses that replicate in chlorella-like green algae and normally exist as mutualistic endosymbionts (referred to as zoochlorellae) in protists such as  Paramecia bursaria. The chloroviruses can attach to paramecia without infecting them and thus the viruses are present if the paramecia are disrupted and release their symbiotic algae. Previous microcosm studies have demonstrated that organisms (predators) such as copepods and didinia feed on paramecia (prey) by different mechanisms that result in the release of the viral host algae; this release leads to an increase in chlorovirus populations. In the current manuscript, we show that another paramecia predator, Bursaria truncatella, with a different feeding behavior, can also cause an increase in chlorovirus titers.Importance: Chloroviruses are found in most freshwater environments around the world and their titers can fluctuate significantly with the seasons. Their algal hosts typically live as symbionts in other protists such as 7 virus particles per mL [31 virus particles in the world’s oceans and this estimate does not include RNA viruses or viruses present in soils and other environments. Most of these viruses infect bacteria, with lesser numbers infecting microalgae and other organisms. Consequently, it is becoming clear that viruses and their hosts play major roles in food webs and in the biogeochemical recycling of materials e.g., [Viruses are the most abundant biological entities on the planet, with ocean water reported to contain ~10s per mL ,3. This ls e.g., ,5.Phycodnaviridae family) are large, plaque-forming dsDNA viruses that infect certain chlorella-like green algae that are endosymbionts (referred to as zoochlorellae) in a variety of single and multicellular aquatic organisms [Chloroviruses  is a ciliate found widely in freshwater environments [ronments  that canronments ,16,17. Ironments ,19. Thusronments ,11, zoocronments .Eucyclops agilis can engulf entire paramecia (referred to as whole-feeding) and release fecal pellets containing viable zoochlorellae that can then be infected by chloroviruses and generate bursts of virus production [Didinium nasutum can disrupt the paramecia by tearing them apart (referred to as messy feeding) and releasing the zoochlorellae into the water where they can be infected [Paramecia are prey for a variety of zooplankton such as crustaceans and other protists ,20. The infected .Bursaria truncatella is another predator that feeds on paramecia [B. truncatella has a different feeding pattern compared to Eucyclops and Didinia in that it consumes whole ciliates such as paramecia by sweeping them inside their buccal cavity and forming a food vacuole. B. truncatella eventually release the waste from a food vacuole through the cell membrane into the surrounding environment [B. truncatella to determine (i) if it also increases chlorovirus concentrations in microcosms by disrupting paramecia and exposing zoochlorellae to chloroviruses during predation, and (ii) the mechanism(s) by which B. truncatella could facilitate virus production.The ciliate aramecia   and cultured in CBS spring water at 23 °C. P. bursaria were collected from a pond at Spring Creek Prairie Audubon Center, near Denton, NE, USA  [2 s−1). B. truncatella was maintained in the laboratory using a mixture of paramecia culture and a standardized hay–wheat medium, prepared by mixing 5 g of dry hay with 5 g of wheat grain in 2 L of autoclaved water collected from Spring Creek. The mixture was boiled for 5 min and filtered through a Whatman filter paper 4 . Sterile pond water from Spring Creek was produced by a series of filtration steps. Pond water was first filtered with Whatman filter paper 4 (pore size 20–25 µm) to remove large debris, followed by subsequent filtration (pore size 0.45 µm). Finally, the pond water was filtered (0.1 µm) and autoclaved at 121 °C for 60 min. Protozoan medium was also prepared by mixing 100 mL of CBS spring water with 900 mL of autoclaved pond water from Spring Creek.B. truncatella and paramecia interactions, and their impact on chlorovirus (strain Osy-NE-ZA1) titers were monitored in two types of experiments. Long (48 h) and short-term (3 h) foraging experiments were conducted in 3 mL of microcosms in Petri dishes (35 mm diameter) with lids. In all of the foraging experiments, the B. truncatella were removed from growing stock cultures, rinsed three times with sterile pond water, and then placed in sterile pond water for one day prior to the start of the experiments to ensure that their buccal cavity and insides were apparently free of nutrients [utrients .B. truncatella for a total of 3 mL. Three treatments were created: (i) five replicates of microcosms with 30 paramecia only (negative control); (ii) five replicates of microcosms where the 30 paramecia were sonicated at the beginning of the experiment using a Tekmar Sonic Disruptor (model number TM100) for ~10 s at an output level of 5 to disrupt the paramecia and release the algae (positive control); and (iii) ten replicates of microcosms with one B. truncatella and 30 paramecia. The B. truncatella were allowed to feed on the paramecia for 48 h. Each day, 0.5 mL of water was removed from the microcosm cultures, ensuring that none of the paramecia or B. truncatella were picked up as well, and replaced with 0.5 mL of autoclaved pond water. Infectious viral titers in all of the experiments were monitored daily from these samples by triplicate plaque assays on lawns of Chlorella variabilis Syngen 2-3 [B. truncatella and paramecia from each microcosm were counted daily at the time of sampling. All trials were performed at 23 °C with constant light.The microcosms for the long-term foraging experiments consisted of 30 paramecia in 1 mL of culture media, 1 mL of protozoan media, and 1 mL of hay–wheat media containing one ngen 2-3 . SamplesB. truncatella: (i) 30 paramecia only ; (ii) 30 sonicated (~10 s) paramecia ; and (iii) 30 paramecia with one B. truncatella (18 replicates). The B. truncatella were allowed to forage on paramecia for ~3 h. During this time, food vacuoles formed inside the B. truncatella. After 3 h, the B. truncatella were removed, rinsed 3 times with sterile pond water, and placed in Petri dishes in 2 mL of sterile pond water. The B. truncatella were transparent and the green paramecia could be seen inside the cell . A possible explanation is that as the predators consume paramecia, there is a feature that the B. truncatella possess which stops them from consuming more paramecia than they can handle until they digest all the contents in their digestive tract. As more paramecia are consumed, a large mass of paramecia accumulates inside the food vacuole, which results in the B. truncatella taking a longer time to digest the contents inside the food vacuole. Presumably, B. truncatella releases digestive enzymes inside the food vacuole, which can inactivate the virus particles and the zoochlorellae. This leads to a negative effect on vital algae and virus particles, resulting in low virus concentrations being released into the microcosm. In short-term foraging, the B. truncatella consume a small number of paramecia, resulting in the faster digestion of the paramecia inside the food vacuole. This results in more algal cells and virus particles surviving and being released into the microcosm.The initial experiments were designed to determine if by day 3 A, while by day 3 B. In theby day 3 A and chlby day 2 B. In theby day 2 A and theby day 2 B. Thus, B. truncatella in the seven microcosms that consumed paramecia showed an increase in chlorovirus titers of about ~300 PFU/culture .In the short-term experiment, seven of the eighteen microcosms showed foraging along with slight increases in chlorovirus populations, while the remaining eleven microcosms had no foraging and no increase in chlorovirus populations. The /culture C. After B. truncatella were simply stripping the infectious chloroviruses attached to the surface of the paramecia. To investigate this possibility, we measured virus titers inside the B. truncatella by disrupting them with Triton at 0, 2, 4, and 6.5 h after the B. truncatella had consumed the paramecia. Virus titers of the Triton-treated B. truncatella remained reasonably constant at 0, 2, and 4 h, but then doubled at 6.5 h, suggesting that the viruses were replicating inside the B. truncatella, as this is about how long the viruses require to begin replicating . An ongoing question has been: how do the chloroviruses replicate in their natural environments? This question is relevant because zoochlorellae are protected from chlorovirus infection while they are in their symbiotic phase due to the physical barrier of the paramecium cell membrane, which prevents chlorovirus–zoochlorellae interactions. There is no evidence for an alternative chlorovirus host and, although zoochlorellae can grow in culture in the laboratory, the zoochlorellae grow poorly, if at all, in native waters free of viruses . It is known, however, that chloroviruses can attach to the external surface of paramecia without infecting them ,18,19. CEucyclops aeglis, can engulf entire paramecia (referred to as ‘whole-feeding’) and release fecal pellets containing viable zoochlorellae, which can then be infected by chloroviruses and generate an ~100-fold increase in chlorovirus titers (20). In a similar manner, the protist Didinium nasutum, a predator of paramecia, can disrupt paramecia by tearing them apart (referred to as ‘messy feeding’) and releasing zoochlorellae into the water where they can be infected, leading to an ~1000-fold increase in virus titers [Didinium can engulf the entire paramecia (whole-feeding) and, when they do so, stimulate less virus production than smaller Didinia.Some understanding of this dilemma has been provided by two recent reports of predator catalysis. Predators of paramecia, such as the copepod s titers . Larger B. truncatella, which has a different feeding pattern than both E. agilis and D. nasutum, also increases chlorovirus titers by about 20-fold  of virus production compared to the ~17% of the theoretical yield for the copepods [B. truncatella, the number of chloroviruses generated was only ~0.3% of the maximum yield (B. truncatella’s feeding process in the microcosms was also slower than the two other predators we examined, as a food vacuole can exist in B. trucatella for up to 7 h, providing enough time for virus replication to occur, as reported in Daphnia, where predators release parasitic fungal spores from infected Daphnia prey into the environment [Three different types of organisms have been described that can feed on paramecia and consequently disrupt the barrier between the chloroviruses and their host zoochlorellae ,21. All copepods ,21. In tum yield . The B. ironment ,30 or whironment ,32.Didinium, and B. truncatella, other aquatic predators, including nematodes, Stentor, and planarians, are reported to feed on paramecia [Didinium, and/or B. truncatella, but the role those larger predators play on chlorovirus titers in natural environments, if any, is unknown.The predator–paramecia–zoochlorellae–chlorovirus relationships are part of larger aquatic food webs. In addition to copepods, aramecia ,34,35,36B. truncatella can disrupt paramecia carrying symbiotic zoochlorellae. There is a period post-ingestion in which chloroviruses replicate inside the food vacuole. We suggest that some of these viruses survive digestion and are then released into the microcosm. This interaction results in slight increases in chlorovirus populations and can help explain the consistent presence of chlorovirus titers in nature.Microcosm experiments establish that


P value for trend shows that urinary caffeine metabolites 137X and AAMU were significantly associated with low-frequency PTA, and that 17X, 137X, AAMU were significantly associated with high-frequency PTA, but when the model was adjusted for sex, age, education level, firearm noise exposure, occupational noise exposure, recreational noise exposure, serum cotinine, body mass index, diabetes, hypertension, these were no longer statistically significant. In conclusion, urinary caffeine metabolites were not associated with the hearing threshold shifts in US adults.Previous studies have reported the relationship between effect of caffeine and many diseases. However, studies to evaluate the association between caffeine and hearing loss are contradictory. To examine the relationship of urinary caffeine metabolites with the hearing threshold in US adults, a total of 849 adults aged 20–69 years who participated in the National Health and Nutrition Examination Survey  were enrolled in this study. Urinary caffeine and its 14 metabolites were applied as biomarkers to assess caffeine exposure. Hearing loss was defined as mean pure tone averages > 25 dB HL at 500, 1000, and 2000 Hz in both ears (low frequency); and 3000, 4000, and 6000 Hz in both ears (high frequency). Univariate and multivariate linear regression analyses were conducted to examine the associations of urinary caffeine metabolites with low- and high-frequency hearing thresholds, respectively. Low-frequency hearing loss were 5.08% and 6.10% in male and female participants, respectively; and high-frequency hearing loss were 31.81% and 15.14% in male and female participants, respectively. In the unadjusted model, the  However, studies investigating the relationship between caffeine and hearing loss are contradictory. Two studies reported lower prevalence of hearing loss in coffee consumers3. Another study reported that chocolate-based diet may protect middle-aged people from hearing loss4. Some studies based on animal models suggested that administration of caffeine can exacerbate cisplatin-induced hearing loss and impair the recovery of hearing after an acoustic over stimulation events (AOSE)6. To date, no large-scale retrospective study has evaluated the relationship between caffeine and hearing threshold shifts among US population.Caffeine is contained widely in dietary consumption of beverages  and foods . Caffeine is also used as a food additive in beverages  and as a drug . As a psychoactive stimulant, its high prevalence in the worldwide diet has developed significant scientific interest in its effects on human health. Studies have reported the relationship between effect of caffeine and many diseases, like diabetes, hyperlipidemia, stroke, heart failure and coronary artery disease7. As the source of most estimates of caffeine intake, food frequency questionnaires or 24-h dietary recalls are usually limited to beverages and do not contain the full caffeine-containing products. Furthermore, the differences in caffeine metabolism among individuals may result in different amounts of circulating caffeine and its metabolites. Testing of urinary caffeine metabolites excretion has been a simple but common way to determine the metabolism and effect of caffeine. In this study, the association of urinary caffeine and 14 caffeine metabolites were determined with hearing threshold shifts in US adults.Assessment of caffeine exposure in epidemiologic studies is conducted mostly by using dietary intake data. However, estimating the caffeine content of dietary sources can be challengingP > 0.01).Table P ≤ 0.01) with low-frequency PTA; and age, sex, education level, firearm noise exposure, occupational noise exposure, recreational noise exposure, BMI, diabetes, hypertension, urinary caffeine metabolites 3-methyluric acid (3U), 7-methyluric acid (7U), 17X, 137X and 5 acetylamino-6-amino-3-methyluracil (AAMU) had a significant univariate correlation (P ≤ 0.01) with high-frequency PTA. Race, PIR and urinary caffeine metabolites 1-methyluric acid (1U), 1,3 dimethyluric acid (13U), 1,7-dimethyluric acid (17U), 3,7-dimethyluric acid (37U), 1-methylxanthine (1X), 3-methylxanthine (3X), 7-methylxanthine (7X), 1,3 dimethylxanthine (theophylline) (13X) and 3,7 dimethylxanthine (theobromine) (37X) had no correlation with either low- or high-frequency PTA hearing threshold shifts , the P value for trend shows there was no significant linear trend among tertiles of urinary 3U, 7U, 137U, 17X, 137X and AAMU and both low-frequency and high-frequency PTA hearing thresholds .Table 10. However, studies to evaluate the effect of caffeine on hearing loss are contradictory. Two studies based on test of guinea pigs showed that a daily dose of caffeine can impair the recovery of hearing after an acoustic overstimulation event11. Another study conducted using a rat model of cisplatin ototoxicity suggested that caffeine consumption may exacerbate cisplatin-induced hearing loss5. Contradictorily, two investigations reported lower prevalence of hearing loss in coffee consumers3. Another study reported the protective effect of chocolate-based diet on hearing loss4. This retrospective study has conducted to evaluate the relationship between caffeine and hearing threshold shifts among US population. To our knowledge, this is the first study examining association between urinary caffeine metabolites and hearing threshold shifts in US adults. Regression models were designed to investigate relationships between 15 caffeine metabolites detected in urine samples and PTA at low and high frequency. The findings of this study differ from those previous studies11. In the present study, there was no effect of caffeine exposure on hearing threshold shifts.Caffeine known to have psychoactive stimulatory effects is widely used in the world. It’s in our beverages  and foods , as well as in certain medications . Its high prevalence of intake in the worldwide diet has arisen significant scientific interest in its effects on human health. Caffeine consumption has been studied as a risk factor for many diseases and conditions, including hypertension, cardiovascular diseases, bone density, various cancers, mental and behavioral disorders, and reproduction and developmental abnormalities12. People have been taking efforts to improve the quality of dietary intake data13. Studies have shown the possibility of using caffeine and caffeine metabolites as biomarkers of caffeine intake17. This study shows that 137U, 17X and 137X had a significant univariate correlation (P ≤ 0.01) with low-frequency PTA; and that 3U, 7U, 17X, 137X and AAMU had a significant univariate correlation (P ≤ 0.01) with high-frequency PTA. These compounds are almost all the ones shown promise as potential biomarkers of caffeine intake in the previous study14. Second, it should be noted that besides the challenge in calculating caffeine intake from food, the common food  for study caffeine contain agents other than caffeine. Coffee has high levels of different polyphenols with antioxidant and anti-inflammatory effects which can protect against oxidative-related disorders, including age-related hearing loss20; polyphenols, phenethyl ester, and other bioactive phytochemicals in coffee have been demonstrated protective effects against ototoxicity in the peripheral auditory system22. However, caffeine was reported to have the opposite effect. Oral administration of caffeine can exacerbate cisplatin-induced hearing loss5. Besides those agents, coffee also contains active compounds like trigonelline, which is determined to be the protective agent in coffee for animals with diabetic neuropathy, but not caffeine23. Furthermore, in the aspect of analysis, the conclusion of “daily coffee consumers had 50–70% less hearing loss than rare coffee consumers” in the previous study was not been calculated using multivariate model. The protective effect of daily coffee consumption is only on a narrow range of people who were aged between 40 and 69 years old with bilateral hearing loss after multivariate analysis8.The results of studies on the effect of caffeine on hearing loss are inconsistent for several reasons. First, caffeine intake from source of food frequency questionnaires or 24-h dietary recalls may be misclassified and underreported, which make assessment of caffeine exposure in epidemiologic studies to be challengingThe data in this study is from the NHANES which contains a large and nationally representative sample of participants in the US. The measurements of demographics data, audiometry data, laboratory examination data, and questionnaire data in the NHANES are standardized and reliable. Despite these strengths, some limitations of this study should be taken into consideration. The temporal relationship between urinary caffeine metabolites levels and hearing loss is not confirmed for the NHANES is a cross-sectional study. Some potential covariates were not calculated in our models. Only 2 years of the NHANES data (2011–2012) were used, thus, the proportion of the adults who met standards in this study is relatively small and larger cohort studies may be needed to confirm the findings. In conclusion, according to the results of NHANES analyses, urinary caffeine metabolites were not associated with hearing threshold shifts in US adults.The NHANES data were approved by the National Center for Health Statistics Institutional Review Board. Informed consents were obtained from all participants before the surveys. All methods were carried out in accordance with the relevant guidelines and regulations of NHANES.24. It is administered by the institutional Review Board of the National Center for Health Statistics of the Centers for Disease Control and Prevention. The survey combines Initial screening questions to determine qualified participants, extensive interviews held at home and physical examination or clinical evaluations performed at mobile examination centers. Detailed questionnaire instruments, brochures, procedure manuals, and consent documents for the 2011–2012 NHANES are publicly accessible from the NHANES website (https://wwwn.cdc.gov/nchs/nhanes/ContinuousNhanes/Default.aspx?BeginYear=2011).The NHANES study is an ongoing cross-sectional survey based on a randomly-selected sample of national residents in the United StatesThe data from the 2011–2012 cycle of NHANES are used for this retrospective study because these were the only years when both urine caffeine samples and audiometry data of participants aged 20–69 years were collected at the time of study. There were 9756 participants in the NHANES dataset from 2011 to 2012. After excluding those with missing data such as audiometry data, urinary caffeine analysis data, and those with abnormal otoscopic results, poor-quality tympanogram results or tympanograms with compliance ≤ 0.3 mL which indicate the likelihood of conductive hearing loss, 849 participants were included in our analysis. Figure 25. Participants who were unable to remove hearing aids for examining and testing and participants with ear pain who could not tolerate headphones were excluded.Audiometry data were collected from participants aged 20–69 years old. The ears of participants were examined by a trained examiner using a Welch Allyn otoscope (model 25020). The Earscan Acoustic Impedance tympanometer (Micro Audiometrics) was used for tympanometry which was performed by measuring the sound pressure level of a 226-Hz probe tone introduced into the ear canal at 85 dB while varying the air pressure in the ear canal from 200 to − 312 daPa. An AD226 audiometer, TDH-39P headphones and EARTone 3A earphones were used for audiometry of both ears. Manual testing was used instead of automated audiometric testing when the subject had difficulties in operating the response switch28.In this study, low-frequency hearing loss was defined as mean PTA at 500, 1000, and 2000 Hz > 25 dB HL in both ears; and high-frequency hearing loss was defined as mean PTA at 3000, 4000 and 6000 Hz > 25 dB HL in both ears29.Urine samples were collected by trained operators at the mobile examination centers and were tested in the National Center for Environmental Health  using ultra-high performance liquid chromatography–electrospray ionization–tandem quadrupole mass spectrometry . Caffeine and 14 of its urinary metabolites, 15 in total, were tested, including 1U, 3U, 7U, 13U, 17U, 37U, 137U, 1X, 3X, 7X, 13X, 17X, 37X, 137X, and AAMU. More detailed procedures are described on the NHANES website30.Potential covariates considered in the analyses included: age as continuous variables; sex, race/ethnicity, education level, PIR, diabetes, hypertension, serum cotinine, BMI, firearm noise exposure, occupational noise exposure and recreational noise exposure as categorical variables. Information on age, sex, race/ ethnicity, education level, PIR, diabetes, hypertension, firearm noise exposure, occupational noise exposure and recreational noise exposure were obtained during in-home interviews. BMI were calculated with data on weight and height measured through physical examination. Serum cotinine level, a marker for both active and passive tobacco exposure, was measured using an isotope dilution-high performance liquid chromatography with an atmospheric pressure chemical ionization tandem mass spectrometry28. Hypertension was defined as “ever been told by a doctor or other health professional had hypertension, also called high blood pressure”28. Firearm noise exposure was defined as “ever used firearms for any reason”, occupational noise exposure was defined as “ever had a job, or combination of jobs exposed to loud sounds or noise for 4 or more hours a day, several days a week”, recreational noise exposure was defined as “ever been exposed to very loud noise or music for 10 or more hours a week”31.Diabetes was defined as “other than during pregnancy, ever been told by a doctor or health professional had diabetes or sugar diabetes”. The answer of “borderline” was also considered as diabetesP value was calculated by weighted linear regression model. The categorical variables were expressed as percentages. P value was calculated by weighted chi-square test. Potential variables were selected and univariate analysis was done (Table P ≤ 0.01 were included in a multivariate linear regression model used to investigate regression coefficients (β) and 95% confidence intervals (CIs) between urinary caffeine metabolites and mean bilateral low-frequency and high-frequency PTA (Table The 2-year C subsample weights (WTSC2YR) of 2011–2012 survey cycle were used in this study to estimate more representative measures for general population in the United States following the analytic guidelines of NHANES. The baseline characteristics of the study participants were divided into two groups by sex and analyzed (Table ne Table . All theTA Table . Three mSupplementary Table 1.


Hearing loss is one of the most common concerns for presentation for a geneticist. Presentation prior to the age of one , profound sensorineural hearing loss (SNHL), and bilateral hearing loss are sensitive and should raise concern for genetic causes of hearing loss and prompt referral for genetic testing. Genetic testing particularly in this instance offers the opportunity for anticipatory guidance including possible course of the hearing loss over time and also connection and evaluation for additional congenital anomalies that may be associated with an underlying syndrome vs. isolated genetic hearing loss. Every 2–3 children out of 1,000 in the United States are born with hearing loss (HL), making it the most common congenital sensory deficit in humans  testing] no later than 3 months of age, and infants that do have confirmed hearing loss should receive appropriate intervention no later than 6 months. Intervention should be from health care and education professionals with expertise in hearing loss and deafness in infants and young children .Infants admitted to the neonatal intensive care unit (NICU) require more in-depth screening based on days of admission. NICU infants admitted to the hospital for more than 5 days should have ABR included as part of their screening . RegardlClinicians should establish potentially reversible causes of hearing loss as soon as possible. Half of all the non-genetic causes of HL are attributed to infectious disease. TORCHES  infections are known risk factors for SNHL. Out of these infectious causes, congenital CMV is the most common cause of non-hereditary SNHL in childhood. Most estimates from studies conducted in Europe, the United States, and Japan show the prevalence of congenital CMV varies from 0.2 to 2.0%, but much higher in developing countries at 6–14% . CMV infOverall in the literature, use of CT in children with hearing loss ranges from 20 to 70% of the time . It is sIf the pediatric HL is considered to be non-genetic and non-infectious in etiology, additional testing should be based on clinical exam findings, imaging, and medical and family history. A “shotgun” approach to obtain an etiologic diagnosis for pediatric HL has been shown to have very low diagnostic yield and to unnecessarily increase cost . HoweverGJB2 and/or GJB6 genes  requires a genetics consultation. Comprehensive genetic testing has the highest diagnostic yield of any single test for bilateral SNHL and up to 60% of cases of congenital HL are due to a genetic etiology , 10. OriB6 genes . IPOG coB6 genes . It is iGenetic etiologies for congenital HL can be divided into syndromic or non-syndromic SNHL (NSSNHL). Approximately 70% of cases of genetic-related hearing loss are NSSNHL, which are often contributed to a single gene mutation. The remaining 30% syndromic SNHL cases are often accompanied by other physical abnormalities or systemic clinical manifestations. https://search.clinicalgenome.org/kb/gene-validity.The Clinical Genome Resource (ClinGen), a National Institutes of Health (NIH)-funded initiative has built a central resource to provide a semiquantitative framework to assign clinical validity to the gene-disease relationships in the hundreds of genes reported in the literature associated with hearing loss. The gene-disease pairs are classified by either being definitive, strong, moderate, limited, disputed, or refuted. For the hearing loss associated genes ClinGen applied the clinical validity framework on 142 genes associated with non-syndromic and syndromic hearing loss that are included on panels from 17 diagnostic testing laboratories from around the world , or point mutations of the gene (In the NSSNHL group, 75–80% are inherited in an autosomal recessive pattern, with 15–24% autosomal dominant, 1–2% X-linked, and <1% mitochondrial , 16. Abnreported , 18. It  descent , 19–21. the gene .The diagnostic yield of genetic testing in children with unilateral SNHL ranges from 1 to 5% in the literature , 24. HowRadiographic imaging can play an important role in diagnosis in genetic or non-genetic etiologies for pediatric HL. See Identifying a genetic etiology has been shown to have many clinical benefits as well as psychosocial and emotional benefits for patient families. Obtaining a genetic diagnosis has been shown to improve parental psychological well-being, mostly by alleviating guilt and accelerating involvement in rehabilitation , 25–27. RB responsible for writing manuscript draft, editing, and figure/table creation. JD, CW, and FV responsible for writing and editing manuscript and creation of figure protocol. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In cochlear hair cells, the activation of mechano-electrical transduction and voltage-gated Ca2+ channels result in a large influx of Ca2+. The intracellular rise in Ca2+ is partly balanced by the mitochondria which rapidly uptakes Ca2+ via a highly selective channel comprised of the main pore-forming subunit, the mitochondrial Ca2+ uniporter (MCU), and associated regulatory proteins. MCU thus contributes to Ca2+ buffering, ensuring cytosolic homeostasis, and is posited to have a critical role in hair cell function and hearing. To test this hypothesis, Ca2+ homeostasis in hair cells and cochlear function were investigated in FVB/NJ mice carrying the knockout allele of Mcu (Mcu/−+ or Mcu−/−). The Mcu knockout allele, which originated in C57BL/6 strain cosegregated along with Cdh23ahl allele to the FVB/NJ strain, due to the close proximity of these genes. Neither Mcu/−+ nor Mcu−/− genotypes affected cochlear development, morphology, or Ca2+ homeostasis of auditory hair cells in the first two postnatal weeks. However, Mcu−/− mice displayed high-frequency hearing impairment as early as 3 weeks postnatal, which then progressed to profound hearing loss at all frequencies in about 6 months. In Mcu/−+ mice, significantly elevated ABR thresholds were observed at 6 months and 9 months of age only at 32 kHz frequency. In three-month-old Mcu−/− mice, up to 18% of the outer hair cells and occasionally some inner hair cells were missing in the mid-cochlear region. In conclusion, mitochondrial Ca2+ uniporter is not required for the development of cochlea in mice, but is essential for hearing and hair cell preservation in congenic FVB/NJ mice.Mitochondrial Ca Predictably, evidence implicates that mitochondrial dysfunction is a lead cause in several nonsyndromic, syndromic, and acquired forms of deafness4. Moreover, mitochondrial DNA mutations (mtDNA) contribute to more than 5% of post-lingual non-syndromic hearing impairment in the Caucasian and Asian populations6. Mutations in the mtDNA or nuclear DNA, which affect mitochondrial function, could result in increased oxidative damage and associated loss of HCs, SGNs, or cells of stria vascularis8. Like in many other cell types in an organism, mitochondria in the cochlear cells are responsible for vital cellular functions, including energy production, apoptosis, cell signaling, and calcium storage. These functions are dependent on the ability of mitochondria to modulate Ca2+ levels15. Particularly, Ca2+ uptake via mitochondrial calcium uniporter (MCU) is critical for the rapid buffering of increases in intracellular Ca2+ loads; an increase in mitochondrial calcium regulates ATP production to fuel Ca2+ extrusion from the cell16. Surprisingly, while slightly smaller in size, mice lacking MCU have a normal life-span and exhibit no adverse phenotype when unchallenged17. These mice were generated on a mixed C57BL/6-CD1 (B6-CD1) background17 and the loss of MCU was not conclusively shown to affect hearing18. Importantly, due to their genetic predisposition, both the C57BL/6 and CD1 strains show progressive hearing loss20, making it impossible to discern the pathogenic contribution of Mcu loss above incumbent hearing impairment. Therefore, a "good hearing" strain, such as the CBA/CaJ or FVB/NJ22 is necessary to transcend the potentially confounding issues associated with the B6-CD1 background. For this purpose, herein, we used marker-assisted breeding, or speed congenics, to transfer the Mcu knockout allele to the FVB/NJ background. Since the Mcu knockout allele is very closely located to the Cdh23ahl allele, which is linked to age-related hearing loss, they cosegregated to the FVB/NJ background. Consequently, the FVB/NJ mice homozygous for the knockout allele of Mcu (Mcu−/−) were also homozygous for the C57BL/6 derived Cdh23ahl allele. We found that the Mcu−/− mice develop substantial hearing loss at one month across all frequencies tested, experience degeneration of cochlear hair cells at 3 months, and are profoundly deaf by 6 months of age. This study demonstrates that Mcu is essential for hearing preservation in congenic FVB/NJ mice.Acquired hearing impairment is mostly associated with deficiency or loss of cochlear cells laden with mitochondria: the inner and outer hair cells (HCs), the spiral ganglion neurons (SGNs), and the cells of the stria vascularisMcu/++), heterozygous (Mcu/−+), and homozygous (Mcu−/−) mice on the mixed B6-CD1 background17 at various ages  and the calcium-binding protein oncomodulin (Ocm) were similar across all three genotypes  in C57BL/6 mice—the Cdh23ahl allelic variant23. Among a panel of 51 mice tested  were also found to be Cdh23ahl/ahl (homozygous for Cdh23c.753A), the Mcu/++ mice (n = 12) were Cdh23/++ (homozygous for Cdh23c.753G), and Mcu/−+ mice (n = 17) were Cdh23/ahl+ (Cdh23c.753G/A). Genomic coordinate analyses of Mcu and Cdh23 in Ensembl genome browser identified the close proximity of these genes in Chr.10qB4, with an intergenic distance of ~ 686 kb  of wild type  frequency compared to the Mcu/−+ or Mcu/++ littermates  the calcium levels are affected in HCs of FVB/NJ Mcu−/− mice at postnatal (P) day P5–P8, after they acquire mechanosensitivity26, and whether (ii) the Ca2+ flow via mechano-electrical transduction (MET) channels will be buffered by HC mitochondria differently in Mcu−/− mice compared to the controls. Initially, the MET function was estimated using FM1-43, a fluorescent molecule that serves as an indicator of mechanotransduction channel function. FM1-43 uptake by HCs of Mcu−/− mice demonstrated no significant difference in MET activity as compared to the Mcu/−+ and Mcu/++ littermates  production28. Within the mitochondria, the primary source of ROS is superoxide31. Therefore, we analyzed the superoxide levels in the mitochondria of HCs in Mcu−/− mice, and found them to be similar to those in Mcu/−+ and Mcu/++ mice . A structural correlate to hearing loss in Mcu−/− mice became apparent in adult cochlea around 3 months of age, when a significant fraction of HCs in the mid-cochlear turn showed signs of degeneration. Considering the Mcu knockout allele originated in the C57BL/6 background, and because of its linkage to the Cdh23ahl loci, the FVB/NJ Mcu−/− mice are also homozygous for the age-related hearing loss linked Cdh23c.753A allele. Thus, MCU deficiency, possibly along with the Cdh23c.753A allele, resulted in hearing loss and degeneration of HCs in the FVB/NJ mice.The uniporter protein MCU traversing the inner mitochondrial membrane is a highly selective CaMcu knockout mice reported originally17. Our observation is concordant with the results reported by a study, where both Mcu−/− and Mcu/++ mice of the same mixed B6-CD1 background had elevated ABR thresholds at high frequency18. However, the authors did not assess ABR thresholds in mice later than 7 weeks of age. In the present study, we recorded the ABR thresholds longitudinally in mice starting as early as 3 weeks to 12 months of age, in B6-CD1 as well as FVB/NJ background. C57BL/6J and CD1 strains are genetically predisposed to progressive hearing loss20, and are known to harbor the Cdh23ahl allele in their genome39. The B6-CD1 background, despite being isogenic for Cdh23ahl, may present with genetic variability and/or heterozygosity at innumerable loci; based on our results, this mixed background turned out to be unsuitable to study the hearing phenotype especially relating to MCU. Another logical assumption is the presence of genomic variants in the mixed background that may mask or compensate for the loss of Mcu. Supporting this notion, Mcu loss was shown to be protective to a certain degree against acoustic stress in the mixed B6-CD1 background, while in the CBA/J mice, siRNA or Ru360 mediated inhibition of MCU was protective against noise-induced OHC loss and permanent hearing impairment18. Our strategy to transfer the Mcu−/− to an inbred strain of mice, like FVB/NJ, minimized genetic variation and aided in resolving the hearing impairment associated with Mcu loss. In contrast to the FVB/NJ background, the Mcu−/− (homozygous) genotype in the C57BL/6J40 or CBA/CaJ resulted in embryonic lethality. Put together, the expression and characteristics of the phenotype associated with the Mcu mutation is strongly influenced by the genetic background. Studying the consequence of Mcu loss in different mouse strains is important to understand the spectrum of genotype–phenotype correlation or variation, and how that might relate to human subjects41.We set out by demonstrating the progressive hearing impairment intrinsic to the mixed B6-CD1 background of Cdh23ahl allele is linked to the Mcu knockout allele may partly explain the accelerated hearing loss observed in the FVB/NJ double homozygous mice. However, we argue that this hearing impairment is a direct result of loss of Mcu based on the following observations: (i) The elevation in ABR threshold in Mcu−/− mice is noticed as early as 3 weeks, a timeline when the effect of Cdh23ahl allele is insignificant on hearing in several inbred mouse strains44. Additionally, the rapid ABR threshold elevation at 8 kHz and 16 kHz in FVB/NJ Mcu−/− mice at 1 month does not correspond to Cdh23ahl—induced phenotype. (ii) Moreover, Cdh23ahl homozygosity is shown to be necessary in the C57BL/6 mice, but not by itself sufficient to account for the hearing loss44; also the C57BL/6J-derived Cdh23ahl allele had little effect on hearing loss in the CBA/CaJ background44. (iii) The threshold elevation noted at 3 months in double heterozygous mice  evidently indicates the importance of Mcu in hearing, given the homozygosity requirement45 of Cdh23ahl. (iv) The postulated synergistic effect of Mcu−/− and Cdh23ahl on hearing loss in the FVB/NJ strain is not apparent in the mixed B6-CD1 background even at high frequency. The above points suggest a greater cumulative influence of the genetic background on the loss of Mcu rather than Cdh23ahl itself. This reiterates that AHL adheres to a quantitative trait model in which a recessive or hypomorphic allele act in concert with a few other recessive/hypomorphic alleles to control the progression and severity of hearing impairment and the age of onset46. For instance, Cdh23ahl has been shown to interact epistatically with Atp2b2dfw/+genotype to accelerate hearing loss, and provides an additive effect by modulating the progression of hearing loss together with Mass1frings/frings or Sod1tm1Leb/tm1Leb mutations48. The effect of Cdh23ahl on Mcu gene mutations has not been studied until now opening up a possibility for further exploration. The dispositional attribution of several genes co-transferred with the congenic Mcu-Cdh23 region  pumps that extrude Ca2+, and organelles including mitochondria and endoplasmic reticulum that serve as Ca2+ reservoirs60. In Mcu−/− cochlea, we analyzed the transcript levels of Pmca1, which is expressed in the basolateral membrane of IHCs, Pmca2 predominantly expressed in the hair bundles of OHCs, and Ocm which codes for oncomodulin, a dominant Ca2+ buffer of the EF-hand superfamily in OHCs63. The transcript levels of these molecules are unchanged despite the significant loss in expression of Mcu. On the other hand, mitochondrial matrix Ca2+ is not completely abolished in the Mcu−/− mice asserting the existence of additional mitochondrial Ca2+ channels as pointed out by several studies64. Debate continues on whether these additional channels allow rapid Ca2+ uptake or a gradual accumulation to sustain mitochondrial Ca2+ homeostasis in the absence of MCU65. The ability of model organisms, specifically selective strains of mice, to survive without any overt phenotype in the absence of MCU supports the idea that MCU is either dispensable or is functionally compensated71. Nonetheless, the crucial role of MCU in several contexts, including acute energy demand, strenuous exercise, pathological conditions, tissue bioenergetics, and longevity cannot be disregarded70. HCs and other cell types in the cochlea likely face acute energy demand after the onset of hearing, due to chronic stimulation from the surroundings. Our results confirm that cochlear development and onset of acoustic function are not significantly affected in the absence of Mcu. The timeline of the hearing loss phenotype observed in Mcu−/− mice in this study recapitulates the crucial role of MCU in acute energy demand. To the best of our knowledge, this is the first report to show the hearing loss phenotype in Mcu−/− mice.The current study indicates that Mcu−/− mice may be the synergistic result of MCU deficiency and Cdh23ahl. The role of MCU in this aspect could be explained partially by the concentration of mitochondria along the lateral wall and in the belt beneath the cuticular plate of HCs73, where MCU potentially contributes to the clearance of Ca2+ entering from the stereocilia during acoustic stimulation49. The integrity of stereocilia in HCs is dependent on chronic Ca2+ influx via MET channels74, and without MCU to handle excess Ca2+, the stereocilia may degenerate. In addition, the presence of Cdh23ahl allele disrupts the normal exon splicing of Cdh23 and causes in-frame exon-skipping46. Because Cadherin 23 is a component of the tip links of hair cell stereocilia along with Protocadherin 1576, the presence of Cdh23ahl variant may affect the preservation of stereocilia function contributing to HC degeneration44. However, the lack of correlation in the timeline corresponding to the onset of hearing loss and the degeneration of HCs in the FVB/NJ Mcu−/− mice indicates that the latter is a secondary manifestation of the hearing deficit. Genetic mouse models carrying mutated Otof, Ocm, or Vglut3 genes79 are good examples to show impairment of hearing function ahead of visible hair cell or stereocilia damage. Alternatively, when compared against ABR, a quantitative technique, SEM imaging may fail to capture small structural changes in hair cells that commensurate with a decline in hearing. Degeneration of stereociliary bundles of OHCs and, less frequently, IHCs in older mice, initiating at the base of the cochlea and gradually progressing to the apex, is a distinctive characteristic of the Cdh23ahl allele80. In contrast, the HC and stereocilia degeneration observed in FVB/NJ Mcu−/− mice at 12 weeks of age, predominantly along the mid-frequency region of the cochlea, might be the outcome of Mcu loss. A burgeoning question is why do only a middle turn fraction of HCs and their stereocilia degenerate in Mcu−/− mice? While further research is warranted, the scientific community is starting to appreciate the tissue-specific or cell-type-specific functions of MCU81. In summary, the present study provides convincing evidence that Mcu is essential for the preservation of hearing and stereocilia maintenance in congenic FVB/NJ mice. Understanding the precise mechanism associated with the loss of Mcu and the primary site of dysfunction that lead to the observed phenotype is important. Since the FVB/NJ Mcu mutant mice used in this study are constitutive knockouts, it is difficult to ascertain the initial site of the genesis of hearing loss in this model. It is also possible that the observed phenotype in Mcu mutant mice is a synergistic effect of mitochondria-rich cell types in the cochlea  and the Cdh23ahl allele. Future studies involving cell-type-specific conditional knockout mice will be necessary to dissect the mechanism of hearing loss induced by the lack of MCU.Although unlikely, the degeneration of HCs and their stereociliary bundles in adult FVB/NJ Mcu/−+ heterozygous mice on a mixed B6-CD1 background were purchased from the Texas A&M Institute of Genomic Medicine17. The FVB/NJ mice and CBA/CaJ mice were purchased from The Jackson Laboratory . After 6 generations of backcrossing through both the female and the male FVB/NJ line, two Mcu/−+ progeny were intercrossed and animals heterozygous for mutant Mcu were selected to generate the experimental mice. All mice had free access to regular feed and water and were maintained under the standard light–dark conditions (12:12 h) at the Animal Resource Centre of Case Western Reserve University (CWRU). An approximately equal proportion of females and males were used in all experiments. All mice experiments and research protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of CWRU. The study was carried out in compliance with the ARRIVE guidelines and all methods were performed in accordance to the IACUC regulations which meets or rather exceeds the requirements of the following agencies: Department of Agriculture Animal Welfare Act, Public Health Service Policy on Humane Care and Use of Laboratory Animals, and Association for Assessment and Accreditation of Laboratory Animal Care International.The Ear punched tissues of weaned mice were collected in sterile Eppendorf tubes and were processed for DNA isolation. Briefly, the tissues were lysed in SNET Lysis buffer, hair and other debris were removed by centrifugation, and the remaining supernatant was mixed with an equal volume of Isopropanol. DNA was pelleted by centrifugation, washed once with 70% Ethanol, and air-dried at RT for 15 min. The DNA was dissolved in 40 µL 0.5× TE buffer at 50 °C for 1 h and then 2 µL of each of the sample was used for PCR genotyping with DreamTaq Green PCR Master Mix (Thermo Fischer Scientific), and the following primers:Mcu WT Fwd: 5′-GGAGTTAAGTCATGAGCTGC-3′.Mcu WT Rev: 5′-CTGGCTTAGTTGGCAGAGTT-3′.Mcu KO Rev: 5′-GACTTGTGGTCTCGCTGTTC-3′.Mcu produced amplicons of 311 bp while the KO allele amplicons were 176 bp in length. The PCR products were resolved in a 1.5% Agarose Gel run in 1X TAE at 100 V, 400 mA for 35 min. Bullseye DNA SafeStain (MidSci) was used to visualize the DNA.The primers were synthesized by Integrated DNA Technologies and were used at a final concentration of 200 nmol/PCR reaction. The total reaction volume of the PCR was 20 µL, and the conditions were initial denaturation at 94 °C for 5 min., followed by 35 cycles of 94 °C for 15 s, 48 °C for 15 s, and 72 °C for 1 min, followed by a final extension at 72 °C for 10 min and 4 °C hold. The wild type allele of Cdh23c.753A/G polymorphism. A segment of exon 7 spanning the single nucleotide variant of interest of Cdh23 was amplified using the following primers:We developed a PCR–RFLP (Restriction Fragment Length Polymorphism) method for genotyping the Cdh23.E7-Fwd 5′-AAGCTGTGTCATTATGTGTGGTAC-3′.Cdh23.E7-Rev 5′-TACTGTAGCACCATCAGGCTC-3′.BsrI restriction enzyme (New England Biolabs) in 1× NEB 3.1 buffer totaling to a final volume of 25 µL. The digestion was performed at 65 °C for 1 h, followed by inactivation at 80 °C for 20 min. and 10 °C hold. Following digestion, the products were resolved in a 1.5% agarose gel with undigested controls and 100 bp DNA ladder. The 534 bp amplicons with Cdh23c.753G allele remain undigested with BsrI, while the Cdh23c.753A allele produces two fragments of 350 bp and 184 bp upon BsrI digestion. Validation was carried out by sequencing the PCR amplicons to verify restriction digestion.PCR was set up to a total volume of 20 µL, with 100 nmol final concentration of each of the primers, 100 ng of genomic DNA and 1× DreamTaq Green PCR Master Mix. The PCR conditions were 95 °C for 2 min, followed by 40 cycles of 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s, a final extension of 72 °C for 2 min, and 10 °C hold. 10 µL of the PCR products were run in 1.5% agarose gel to verify amplification, and the remaining 10 µL was subjected to restriction digestion using 6 units of ® VET Ointment, and the mice were placed in a sound-isolated chamber. The body temperature was maintained at 37 °C through a temperature-controlled heating pad. Subdermal electrodes were inserted at the vertex of the skull (positive), the mastoid region under the left ear (ground), and the mastoid region under the right ear (negative). Pure tone bursts were delivered through earphones using a computer-aided evoked potential system . Thresholds were determined for the 8 kHz, 16 kHz, and 32 kHz frequencies by escalating or deescalating the intensity in 10-dB steps starting at 70 dB, and then in 5-dB steps near-threshold until no organized responses were detected. For each stimulus, 768 sweeps were averaged for a response. Thresholds were defined as the lowest stimulus level where an organized response is observed. The recordings were performed blindly without knowledge of the genotype, and later the recorded values were matched with the genotype and grouped for statistical analyses.The mice were weighed, and anesthetized with an Intra-peritoneal (IP) injection of a cocktail containing ketamine (20 mg/mL) and xylazine (1.75 mg/mL) at a dose of 0.1 mL/20 g. The depth of anaesthesia was tested by the toe-pinch response, eyes lubricated by application of PURALUBElater™ solution (Thermo Fischer Scientific), and stored at 4 °C for a day. The next day, cochleae were transferred to ice-cold PBS to give a quick wash, and TRIzol  was used for total RNA extraction following the manufacturer's recommendation and instructions. The SuperScript IV First-Strand Synthesis System (Invitrogen) was utilized to synthesize cDNA from 2 µg of total RNA and used subsequently at a final concentration of 10 ng/reaction in qPCR. Quantitative PCR was performed using the PowerUp SYBR Green Master Mix  on an ABI StepOnePlus™ Real-Time PCR System (Thermo Fischer Scientific). Details of the primers are provided in Table Hprt and PolR2f and then analyzed using the 2−ΔΔCT method82.The mice cochleae were quickly dissected and isolated in ice-cold PBS, transferred to RNA26. Organs were dissected at postnatal days 3–4 (P3–P4). Briefly, the sensory epithelium was separated first from the lateral wall and then from the modiolus. The most basal “hook” region was cut out and the remaining organ was placed into a glass-bottom Petri dish  and cultured in DMEM medium supplemented with 7% fetal bovine serum  at 37 °C and 10% CO2. The organ of Corti explants were kept in vitro from one to five days before the experiments. The equivalent age (age of dissection plus days in vitro) of the specimens reported in this study was P5–P9. To prevent contamination, 10 µg/mL of ampicillin  was added to the medium. FM1-43 (Invitrogen) was used to evaluate mechanotransduction and to count functional hair cells. FM1-43 was dissolved in DMSO to obtain a stock solution with a concentration of 1 mM. Immediately before the experiment, the stock solution was diluted to 5 μM in Ca2+-free HBSS. Then, all specimens were briefly incubated in FM1-43 for 30 s at room temperature and then carefully rinsed in standard HBSS. MitoSOX Red superoxide indicator (Invitrogen), as well as calcium indicators Fluo-2 LeakRes AM (TEFlabs) and Rhod-2 AM (Biotium), were used according to manufacturer provided protocol. The raw images were gathered using an upright Olympus BX51WI microscope equipped with a 100 × 1 NA objective and a Grasshopper3 CMOS camera (FLIR), using manufacturer provided software. Images were subjected to fluorescence measurements using ImageJ (NIH). A region of interest was used to obtain measurements from the hair cells (Icell) and an area without cells (Ibackground) in the same image. Fluorescence intensity (Iload) for the hair cells were normalized (Iload = Icell − Ibackground).Organ of Corti explants were obtained as described previously25. Temporal bones of mice at the age of 4 weeks and 12 weeks were removed after euthanasia by CO2 inhalation and fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, , supplemented with 2 mM CaCl2. After removing the stapes, and opening the round and oval window, the cochleae were immersed in the fixative for 2 h and then stored at 4 °C in 1/10th fixative diluted with buffer until collected for further preparation. The cochleae were removed, then washed with PBS, and placed in 5% EDTA, pH 7.4 at 4 °C for three days to decalcify. Following decalcification, samples were dissected and organs of Corti were removed and divided into apical, middle, and basal parts. The specimens were then dehydrated in a gradient ethanol series, critical-point dried using CO2, mounted on an SEM stub, and sputter-coated with 10 nm palladium. Cochlear epithelia were viewed and imaged with a high-resolution scanning electron microscope  housed at Swagelok Center for Surface Analysis of Materials (SCSAM), CWRU.Samples were prepared as previously describedt-test. P values < 0.05 were considered significant.All statistical analyses were performed using Microsoft Excel and GraphPad Prism 7. Data are reported as mean ± SEM unless noted otherwise. Two way ANOVA analyses with post hoc pairwise comparisons using Bonferroni adjustments or Kruskal–Wallis test with Dunn’s multiple comparisons were used depending on data distribution. Comparisons between two groups were tested by Student’s


Influenza A viruses (IAV) are widespread viruses affecting avian and mammalian species worldwide. IAVs from avian species can be transmitted to mammals including humans and, thus, they are of inherent pandemic concern. Most of the efforts to understand the pathogenicity and transmission of avian origin IAVs have been focused on H5 and H7 subtypes due to their highly pathogenic phenotype in poultry. However, IAV of the H9 subtype, which circulate endemically in poultry flocks in some regions of the world, have also been associated with cases of zoonotic infections. In this review, we discuss the mammalian transmission of H9N2 and the molecular factors that are thought relevant for this spillover, focusing on the HA segment. Additionally, we discuss factors that have been associated with the ability of these viruses to transmit through the respiratory route in mammalian species. The summarized information shows that minimal amino acid changes in the HA and/or the combination of H9N2 surface genes with internal genes of human influenza viruses are enough for the generation of H9N2 viruses with the ability to transmit via aerosol. Influenza A viruses (IAV) are members of the family Orthomyxoviridae with a segmented RNA genome of negative polarity. IAV are divided into subtypes by the combination of the surface proteins, the hemagglutinin HA, H1–H18) and the neuraminidase NA, N1–N11) –H18 and ,3. IAVs  –H18 andIAVs of the H9N2 subtype are widespread in several species of wild waterfowl, shorebirds, and poultry, such as chickens, turkeys, and quails, among others ,20,21,22Mycoplasma gallisepticum, which can lead to high mortality [H9N2 IAV infections can be mild. However, significant economic losses are associated with H9N2 IAV infections because of delayed growth and lower egg production. Studies have shown that H9N2 virus replication in the oviduct results in poor eggshell quality and deterioration of eggshell ,15,30. Hortality ,35,36,37Phylogenetic analyses of the H9 HA have classified the H9 into two lineages: the American and Eurasian lineages, which are further divided into four sublineages (h9.1 to h9.4) . StrainsOf great significance, H9N2 IAVs have contributed the internal gene segments to more virulent zoonotic strains such as H5N1/N6, H7N9, and H10N8/N3 that have been implicated in human infections and loss of life ,45,46,47Several molecular signatures have been associated with host range restriction and species jump of IAV in mammalian species. Particularly, the segments encoding the HA and the polymerase complex  play a major role in the host range and adaptation of IAVs ,57,58,59In addition, other amino acid signatures can modulate and/or enhance binding of H9 HAs to terminal α2,6SAs, such as the mutation from isoleucine (I) to threonine (T) at position 155  or the pThe viral polymerase subunits PB1, PB2, and PA can also contribute to the adaptation of IAV of avian origin to mammalian hosts. Of interest, the change from E to lysine (K) in position 627 (E627K) in PB2 is a major determinant of host adaptation. This has been the predominant host adaptation marker identified in human cases of H5N1 and H7N9 infections ,82. A hiThe PB2 A588V mutation is also potentially involved in mammalian adaptation. H9N2 IAVs carrying the PB2 V588 signature show enhanced virulence in mice . For PB1According to the World Health Organization (WHO), 58 human cases of H9N2 IAV infection have been reported since December 2015, with 17 of those cases reported during 2021 from the western Pacific region (as of 17 September 2021) . AdditioFor an avian H9N2 virus to successfully transmit in mammals, the virus must evolve to become compatible with the new host environment, allowing effective replication and transmission ,118. ReaMore recent work demonstrated that although most H9N2 IAV isolates possess either Q226 or L226, this position is highly flexible and able to tolerate multiple amino acids (at least 10), some not previously detected in natural H9N2 isolates . In vitrDespite direct-contact transmission in ferrets of the H9N2 IAV field isolates in the study described above, no airborne transmission was observed (in contrast to control ferret studies using human origin H1N1 or H3N2 IAVs) ,126. FurTo confirm the role of reassortment  on the ability of H9 viruses to transmit in mammals, a transfection-based inoculation (TBI) study was performed in ferrets to select airborne transmissible H9 reassortant viruses under host selection pressure. In brief, ferrets were inoculated with cells previously transfected with 15 plasmids: 6 encoding the internal gene segments of the WF10 H9N2 virus, 7 gene segments (excluding the HA) of a prototypic pH1N1 virus and the surface gene segments of the P10 ferret-adapted virus. The resulting virus mixture was then serially passaged in ferrets, allowing for the selection of any possible H9 reassortant that was compatible/fit with respiratory droplet transmission in ferrets. The results show two different H9N1 viruses that were selected and able to transmit by the respiratory route in ferrets . Both H9Experimental infection/transmission with poultry adapted H9N2 IAV have been evaluated also in multiple alternative animal models such as guinea pigs, mice, and others ,113,116.Although H9N2 IAV are LPAIV and cause mostly mild infections, these viruses still result in significant economic losses to the poultry industry. Furthermore, H9N2 IAV have been implicated as the donors of internal genes for prevalent HPAIV outbreaks that have resulted in human infections in some cases. Additionally, cases of H9N2 IAV have been reported in different mammalian species including humans, demonstrating their risk to public health and pandemic potential. Though most infections do not result in mammal-to-mammal transmission, different experiments have demonstrated that transmission of H9N2 IAV in mammals is possible. Nevertheless, adaptation or reassortment with mammalian-adapted internal genes is required for an efficient transmission between mammals, particularly by the respiratory route, although experimental airborne transmission of natural isolates has been observed. Therefore, continued surveillance and research is needed to understand the evolution, pathogenicity, transmission, and antigenicity of H9N2 IAV. Furthermore, understanding the molecular traits that facilitate transmission of H9N2 to and between mammals is crucial to evaluate their pandemic potential and to allow timely identification of viruses with increased potential for interspecies transmission.


Objective: This study was aimed at evaluating improvements in speech-in-noise recognition ability as measured by signal-to-noise ratio (SNR) with the use of wireless remote microphone technology. These microphones transmit digital signals via radio frequency directly to hearing aids and may be a valuable assistive listening device for the hearing-impaired population of Mandarin speakers in China.Methods: Twenty-three adults (aged 19–80 years old) and fourteen children (aged 8–17 years old) with bilateral sensorineural hearing loss were recruited. The Mandarin Hearing in Noise Test was used to test speech recognition ability in adult subjects, and the Mandarin Hearing in Noise Test for Children was used for children. The subjects’ perceived SNR was measured using sentence recognition ability at three different listening distances of 1.5, 3, and 6 m. At each distance, SNR was obtained under three device settings: hearing aid microphone alone, wireless remote microphone alone, and hearing aid microphone and wireless remote microphone simultaneously.Results: At each test distance, for both adult and pediatric groups, speech-in-noise recognition thresholds were significantly lower with the use of the wireless remote microphone in comparison with the hearing aid microphones alone (P < 0.05), indicating better SNR performance with the wireless remote microphone. Moreover, when the wireless remote microphone was used, test distance had no effect on speech-in-noise recognition for either adults or children.Conclusion: Wireless remote microphone technology can significantly improve speech recognition performance in challenging listening environments for Mandarin speaking hearing aid users in China. With the advancement and evolution of current hearing technology, a variety of digital audio electronic devices have become more prevalent in the general population. These accessories which allow wireless transfer of high quality audio, such as smartphone compatible wireless earphones, have enhanced listening experiences for normal-hearing listeners, with the potential of similar accessories improving listening experiences for the hearing-impaired as well .Sensorineural hearing loss not only reduces the sensitivity to sound and dynamic range of auditory perception for hearing-impaired listeners, but also reduces their frequency and temporal resolution that can lead to difficulty in speech comprehension . HearingHearing aids today can provide listeners with a clear, high-quality sound experience in a quiet environment, but they deteriorate in the presence of noise . KochkinWhen hearing aids and their microphones alone do not provide adequate assistance, some of the most beneficial wearable technology comes in the form of assistive listening devices which can also effectively improve the SNR for hearing aid users. For example, remote microphone hearing assistance technology (HAT) is widely used for hearing-impaired children. Amongst various HAT devices, wireless frequency modulation (FM) system is an early development that is still widely used. A typical FM unit consists of a small transmitter and microphone, which picks up the voice of a speaker and sends the clean speech to a radio-frequency (RF) receiver plugged into the hearing aid of a listener. Using a FM system to transmit the teacher’s voice directly to the student is equivalent to reducing the communication distance to within 3–6 inches. Boothroyd showed that using the FM system in a noisy environment resulted in the same speech recognition as in a quiet environment . For FM Mandarin Chinese is a tonal language with four phonologically distinctive tones characterized by syllable-level fundamental frequency (F0) contour patterns. These pitch contours are commonly described as high–level (tone 1), high–rising (tone 2), falling–rising (tone 3), and high–falling (tone 4) . AccordiSD = 18.7) and had relatively symmetric sensorineural hearing loss in both ears. The mean pure-tone hearing thresholds at frequencies of 500, 1,000, 2,000, and 4,000 Hz (PTA0.5 to 4 kHz) across the two ears for the groups of subjects ranged from 40 to 75 dB HL, as shown in Two groups of subjects were recruited in this study. All subjects had a history of digital hearing aids use for more than 1 year but no experience with HAT in combination with their hearing devices. Twenty-three native Mandarin Chinese-speaking adult subjects  participated in the adult group. The subjects were between 19 and 80 years old , and had bilateral sensorineural hearing loss with a PTA0.5 to 4 kHz ranging from approximately 35 to 80 dB HL, as shown in Fourteen native Mandarin Chinese-speaking children  were recruited for the children’s group. These subjects were between 8 and 17 years old  hearing aids, and pediatric participants were fitted with two ReSound UP 988 Behind-the-Ear (BTE) hearing aids. Pediatric participants utilized their own earmolds during the test, which were coupled to hearing aids. Pediatric subjects with good low frequency hearing had earmolds with small vents., which would have negligible effect on the gain of the amplified sound path . A ReSouThe test equipment included five Audioengine2 + active speakers, four of which were used to present noise signals and one was used to present the target speech. The Mandarin Hearing in Noise Test (MHINT) was used to test the speech recognition ability for adults. The HINT is a standardized and recorded test that can be used to estimate the signal-to-noise ratio (SNR) at which the sentences embedded in background noise can be repeated correctly 50% of the time. MHINT is the Mandarin version of HINT. The MHINT materials consist of 12 lists, each containing 20 sentences. Each sentence contains 10 Chinese characters. The presentation level is response dependent; lowered or raised according to a participant’s correct or incorrect response of the test material. Presentation levels were decreased by 2 dB after a correct response and increased by 2 dB after an incorrect response. The reception threshold for sentences (RTSs) was calculated using this adaptive procedure . The ManThe test location was a quiet meeting room measured at 5.5 × 8 × 2.5 m, with ambient background noise levels below 45 dB A. The testing room resembles a typical classroom setting with a reverberation time of 0.46 s. As shown in Adult subjects were fitted with ReSound LiNX2 962 RIE hearing aids on both ears according to their audiograms. The proprietary fitting prescription of Audiogram + in the ReSound Aventa 3 software was used. The hearing aid microphones were set to a fixed directional response, and all other advanced signal processing features  were disabled. The ReSound Mini Microphone was paired with the hearing aids and set at a hearing aid to Mini Microphone ratio of 1:1. . There were nine test conditions consisting of three test distances and three program settings. Each program setting was tested at each distance condition. The program settings included directional microphones active (HA_D), the Mini Microphone active only (MM), and both hearing aid microphones and MM active (HA_D + MM). The nine test conditions were carried out in a randomized order. The remote control was used by the investigator to switch the hearing aid program settings. For each test condition, a list of MHINT materials were presented to obtain the speech recognition threshold in SNR. During the test, the SSN noise level was fixed at 65 dB A.Pediatric subjects were fitted with bilateral Resound UPS988-DLW BTE hearing aids based on their audiograms with a DSL v5 fitting prescription. The programming of the hearing aids were the same as those utilized for the adult subjects, with the addition of an omnidirectional microphone mode (HA_O) program. Hence, for the pediatric subjects, there were twelve test conditions (four test program settings at three distances each) carried out in a randomized order. For each test condition, a list of MHINT-C materials were used to obtain recognition thresholds in SNR.Statistical analysis was carried out using Statistics Package for Social Science (SPSS) 16.0. A repeated-measures analysis of variance (RM-ANOVA) was conducted to determine if there was any overall statistical significance among the outcome SNRs across the three or four device settings at the three test distances for both adult and pediatric groups. The test distances and device settings were considered the independent variables. SNR was considered the dependent variable.F = 267.91, p < 0.05]. There was a significant interaction effect between the distance and device settings . This indicates that SNR is affected by the interaction between the distance and device settings.The SNRs obtained under nine test conditions for adults are detailed in p < 0.05). Performance was significantly better when the MM was active compared to the hearing aid microphone alone (p < 0.05). The performance with the MM alone was significantly better than the performance with HA_D + MM .p < 0.05), with no significant differences in SNR between 3 and 6 m conditions. When the MM was active, the test distance had no effect on SNRs (p > 0.05).Moreover, the results showed that with the hearing aid microphone alone, the SNR for the 1.5 m condition was significantly better than those for the 3–6 m conditions  = 267.91, p < 0.05]. There was a significant interaction effect between test distance and device settings (p < 0.05). This indicates that the SNR is affected by the interaction between the distance and device settings.The SNRs obtained under twelve test conditions for children are detailed in p < 0.05). Performance was significantly better for the two conditions with the MM active, in comparison with the hearing aid microphone alone (p < 0.05) regardless of microphone directionality. The performance for the MM alone was significantly better than the performance with MM + HA . When the test distance was set at 3 and 6 m, there were no differences in performance between HA_O and HA_D.p > 0.05).The results show that using the hearing aid microphone alone, regardless if directional or omnidirectional, the SNR in the 1.5 m condition was significantly better than that for the 6 m condition, with no significant difference in SNR between 1.5 and 3 m, 3 and 6 m. When the MM was active, the test distance had no effect on speech in noise recognition thresholds  has introduced a digital wireless standard for manufacturers of hearing aids and wireless accessories, as well as consumer devices. With the increase of such technology, Bluetooth digital signal coverage in public spaces such as theaters and cinemas may increase, improving accessibility for hearing aid users who may be able to use remote microphones in public spaces to improve communication and listening.In the current study, loudspeakers were used as the source of speech signals. The role of lip-reading and facial expressions in communication was not fully examined. However, in daily communication, lip reading and facial expressions play a vital role in understanding speech, especially for hearing-impaired individuals and children . The resLastly, one of the limitations of the current study is that, only native speakers of Mandarin Chinese were selected. For future studies, bilingual  adults and children could be recruited to evaluate the effect of remote microphone and assistive listening devices in both tonal and non-tonal languages. In addition to the aforementioned wearable assistive listening devices, speech-to-text conversion apps for smart phones have been designed specifically to provide communication redundancy for individuals with hearing loss. These apps have been shown to improve communication for those with hearing loss, especially the profoundly hearing impaired population in certain listening situations .The addition of a wireless remote microphone to bilaterally worn hearing aids compensates for increased distance from the sound source. The use of a wireless remote microphone can significantly improve speech in noise communication performance in Chinese hearing-impaired listeners.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.The studies involving human participants were reviewed and approved by the Institutional Review Board at the Beijing Institute of Otolaryngology and Beijing Tongren Hospital. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.SW, JC, and RD contributed conception and design of the study. JC, RD, ZW, and YW performed the experiments. JC, SW, XF, and RD performed the statistical analyses. JC and SW wrote the first draft of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.