Protocol for CHIEF (cochlear implants and inner ear inflammation) study; an observational, cross-sectional study of children and young people undergoing cochlear implantation.

Inflammation, Cochlear implants, Hearing, Hearing outcomes, Macrophages, Fibrosis, Transcriptome ALL Metrics - Views Downloads How to cite this article Hough K, Nichani J, Findlay C et al. Protocol for CHIEF (cochlear implants and inner ear inflammation) study; an observational, cross-sectional study of children and young people undergoing cochlear implantation. [version 1; peer review: 1 approved, 1 approved with reservations]. NIHR Open Res 2025, 5:18 (https://doi.org/10.3310/nihropenres.13879.1) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. Export Citation Sciwheel EndNote Ref. Manager Bibtex ProCite Sente Select a format first ▬ ✚ Study Protocol [version 1; peer review: 1 approved, 1 approved with reservations] Kate Hough https://orcid.org/0000-0002-5160-2517 1, Jaya Nichani2, Callum Findlay1,3, Iain A Bruce2,4, Tracey A Newman1Kate Hough https://orcid.org/0000-0002-5160-2517 1, Jaya Nichani2, [...] Callum Findlay1,3, Iain A Bruce2,4, Tracey A Newman1 PUBLISHED 04 Mar 2025 Author details Author details 1 Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, Southampton, England, SO17 1BJ, UK 2 Royal Manchester Children's Hospital, Manchester University Hospitals NHS Foundation Trust,, Manchester Academic Health Science Centre, Manchester, M13 9WL, UK 3 Department of Otolaryngology, University Hospital Southampton NHS Foundation Trust, Southampton, SO16 6YD, UK 4 Division of Infection, Immunity and Respiratory Medicine, School of Biological Sciences, Faculty of Biology, Medicine and Health, The University of Manchester, Manchester, England, M13 9PL, UK 2 Royal Manchester Children's Hospital, Manchester University Hospitals NHS Foundation Trust,, Manchester Academic Health Science Centre, Manchester, M13 9WL, UK 3 Department of Otolaryngology, University Hospital Southampton NHS Foundation Trust, Southampton, SO16 6YD, UK 4 Division of Infection, Immunity and Respiratory Medicine, School of Biological Sciences, Faculty of Biology, Medicine and Health, The University of Manchester, Manchester, England, M13 9PL, UK Kate Hough Roles: Conceptualization, Methodology, Resources, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing Roles: Conceptualization, Methodology, Resources, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing Jaya Nichani Roles: Conceptualization, Methodology, Project Administration, Writing – Review & Editing Roles: Conceptualization, Methodology, Project Administration, Writing – Review & Editing Callum Findlay Roles: Methodology, Writing – Review & Editing Roles: Methodology, Writing – Review & Editing Iain A Bruce Roles: Conceptualization, Funding Acquisition, Methodology, Project Administration, Writing – Review & Editing Roles: Conceptualization, Funding Acquisition, Methodology, Project Administration, Writing – Review & Editing Tracey A Newman Roles: Conceptualization, Funding Acquisition, Investigation, Methodology, Project Administration, Resources, Supervision, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing Roles: Conceptualization, Funding Acquisition, Investigation, Methodology, Project Administration, Resources, Supervision, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing OPEN PEER REVIEW REVIEWER STATUS Cochlear implantation is a surgical intervention for people with severe-to-profound hearing loss. Electrodes in the cochlea generate electrical currents that stimulate the auditory nerve to elicit hearing. Despite the success of cochlear implants, some people do not receive the expected hearing benefits. One reason for this is that tissues in the cochlea vary in their response to implantation of the electrode array. Many people have a healthy wound-healing response that results in mature scar tissue (fibrosis). However, some individuals have a heightened inflammatory response associated with excessive fibrosis. This leads to greater electrical resistance to the current flow (impedance) and reduces the quality of electrical stimulation, both of which can lead to poorer hearing outcomes with the implant. Excessive inflammation can damage cochlear structures and result in loss of residual hearing. This study will increase our understanding of why some people have a heightened inflammatory response that leads to poorer hearing. We propose that there are detectable individual inflammatory differences between people when they are implanted, which may result in variable hearing outcomes following implantation. If we could understand and identify these differences, we could detect people who may be at risk of less favorable outcomes and use therapies to modulate inflammation and improve outcomes. A cross-sectional study of children and young people undergoing cochlear implantation. On the day of surgery, a middle ear mucosa sample, swabs of the nasopharynx and middle ear, cochlear fluid, and blood samples will be collected. Samples will be analyzed using molecular techniques to determine the inflammatory status of the person at the time of implantation. Clinical hearing data will be collected for up to five years after implantation to explore the relationship between inflammation at the time of implantation and long-term hearing outcomes. Despite the success of cochlear implants, some patients do not achieve the expected hearing benefits. This is detrimental to a person’s well-being and makes it challenging for them to interact daily in the ‘hearing’ world. Poor outcomes with implants are costly to the individual, society, and health system. The factors that contribute to underperformance are not well understood. One factor could be how the body responds to the insertion of the implant into the cochlea. Most people have a healthy tissue response, which results in scar tissue (fibrosis) around the implant. In some individuals, the response is more inflammatory, leading to increased fibrosis in the cochlea. This causes damage to the surrounding tissue and can lead to further hearing loss and poor hearing with the implant. A better understanding of the factors that cause poorer hearing may enable the use of anti-inflammatory drugs before and after surgery to give people better hearing with their implants. CHIEF (cochlear implants and inner ear inflammation) is a cross-sectional study of children and young people undergoing cochlear implantation. On the day of surgery, tissue and fluid samples will be collected, including the middle ear mucosa, middle ear, nasal swab, cochlear fluid, and blood sample. In cases where an implant is removed because of implant failure, the implant and tissue attached will be collected. Following implantation, we will collect clinical outcome measures for up to five years. We will test our hypothesis that the insertion of an electrode array into the cochlea causes an inflammatory tissue response that varies due to individual inflammatory differences at the time of implantation. We aim to characterize the inflammatory state of the ear. We will combine biological data with health and hearing data to investigate the relationship between the inflammatory state and long-term hearing outcomes. Inflammation, Cochlear implants, Hearing, Hearing outcomes, Macrophages, Fibrosis, Transcriptome Corresponding Author(s) Tracey A Newman ([email protected]) Grant information: This project is funded by the National Institute for Health and Care Research (NIHR) under its [‘Research for Patient Benefit (RfPB) Programme’ (Grant Reference Number NIHR203308)]. The views expressed are those of the author(s) and not necessarily those of the NIHR or the Department of Health and Social Care. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Copyright: © 2025 Hough K et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. How to cite: Hough K, Nichani J, Findlay C et al. Protocol for CHIEF (cochlear implants and inner ear inflammation) study; an observational, cross-sectional study of children and young people undergoing cochlear implantation. [version 1; peer review: 1 approved, 1 approved with reservations]. NIHR Open Res 2025, 5:18 (https://doi.org/10.3310/nihropenres.13879.1) First published: 04 Mar 2025, 5:18 (https://doi.org/10.3310/nihropenres.13879.1) Latest published: 15 May 2025, 5:18 (https://doi.org/10.3310/nihropenres.13879.2) The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. There is a newer version of this article available. of this article available. Cochlear implantation is a treatment developed for people with profound deafness. Cochlear implants are effective in helping young children to learn to talk and listen, older children to achieve at school, and children and young people of all ages to socialize. Children who hear well with implants, are likely to meet key developmental milestones in line with their unaided hearing peers1. This contributes to the transition to adults who are better able to succeed in society. Cochlear implantation involves the surgical implantation of a wire (electrode array) into the hearing organ (cochlea) in the deep part of the ear (inner ear). The electrode array carries a signal from an electronics package implanted under the skin (receiver-stimulator package), which itself receives a coded signal from a bespoke hearing aid worn on the ear (audio processor). Electrical signals stimulate hearing pathways in the brain, resulting in sound perception. Some people do not do as well as expected following surgery or the hearing benefit from the implant tail-off over time2–4. This can be detrimental to the well-being of the person, resulting in non-use of the cochlear implant or even the need to undergo another operation to insert a new cochlear implant5. This can mean the potential benefits of restored hearing are lost. How the cochlear implant electrode array interacts with the delicate tissues in the inner ear is crucial to the effectiveness of the electrical stimulation of the hearing pathway to the brain and the preservation of any remaining natural hearing. The immune system and inflammatory response within the inner ear are likely key factors in the interaction between the electrode array and fine structures of the ear6–9. We know that implantation causes an inflammatory response, but most of our understanding of this comes from studies of implantation in animals10,11 and cadaveric temporal bone studies12,13. Importantly, these studies have not enabled us to understand how the inflammatory response might vary between individuals, each of whom has their own immune history14. We need to understand and ideally be able to predict or anticipate the individual inflammatory response, as this could lead to improved clinical management and better hearing outcomes for more people who use cochlear implants. Our hypothesis is that the insertion of an electrode array causes an inflammatory response that varies due to individual differences between people at the time of implantation. There is an urgent need to investigate the effect of the immune state of the inner ear on outcomes following cochlear implantation. To achieve this, we need to study the inflammatory state of the ear in children and young people undergoing implantation. Importantly, we need to see how this varies between children and young people, and how it is associated with hearing outcomes in people with a history of middle ear inflammation (acute otitis media and otitis media with effusion)15,16. The desired outcome from this study would be to understand when targeted treatment (e.g., steroids) before, during, or after surgery is needed to ensure the best outcome for the person with their implant. ALL_EARS@UoS is a patient and public involvement and engagement (PPIE) group that was first established at the University of Southampton in March 2022. The group is committed to improving the understanding of the mechanisms, lived experience, and management of hearing loss by contributing to and influencing hearing loss research17. The chief investigator for this project (TN) and the postdoctoral fellow (KH), who will work on the project, have been central to ALL_EARS@UoS. The CHIEF study was presented and discussed with the group for feedback at our regular PPIE meetings. All documents for the study were shared with members of the group for detailed feedback. One group member attended an ethical approval panel meeting with the chief investigator of the project. A group of young people is beginning to be co-developed to ensure that they are engaged with the project and our wider research. The study will be conducted at the Manchester University Hospital NHS Foundation Trust (MFT) and the University of Southampton. The Manchester pediatric cochlear implant program was established in 1991, and it serves a diverse population and implants in over 60 children per year. It has a sustained record in undertaking research to improve outcomes for children and young people with implants. The University of Southampton has significant expertise in inflammatory biology, microbiology, and proteomics. It is a core partner of the National Biofilm Innovation Center and home to one of the 19 auditory implant services in the UK (University of Southampton Auditory Implant Service). Participant recruitment, sample collection, and clinical management of the participants will be carried out in the MFT. Sample analysis and initial data interpretation will be carried out in Southampton with data sharing between centers. Only fully anonymized patient and sample data will be shared with Southampton, and all identifiable information will be limited to the clinical care team in the MFT. Observational, cross-sectional study of children and young people undergoing cochlear implantation. Children and young people who meet the inclusion criteria and who have consented and/or give assent will be recruited to the study. The surgical protocol and the participants’ routine clinical care will not be altered by being study participants. Figure 1 outlines the participant pathway from determining eligibility and recruitment into the study through sample collection on the day of surgery, followed by the collection of clinical and health data post-implantation. This protocol was written according to the “Strengthening the Reporting of Observational Studies in Epidemiology” (STROBE) guidelines. The STROBE checklist was filled out where applicable to this protocol (https://doi.org/10.5258/SOTON/D3345). Eligibility criteria Children and young people undergoing cochlear implantation under the care of the Manchester University NHS Foundation Trust (MFT) will be screened to determine if they are eligible to participate in the study. See Table 1 for inclusion and exclusion criteria. If the patient meets the eligibility criteria mentioned above, the clinical care team will provide the necessary information about the study to the children and young people and/or their parents or guardians. Recruitment We aim to recruit from children and young people who are eligible within the 24-month period of sample collection. We estimate 75 participants over two years of recruitment to the study. This is based on the recent rates of pediatric cochlear implantation at the MFT. The results of this observational study will provide the sample size and recruitment criteria for future interventional studies. Recruitment will be done by advertising the study to young people, parents, and guardians of children who meet the eligibility criteria. The clinical care team responsible for the care of patients will determine eligibility. The study details will be shared during the clinic visit with children and young people who have been identified by the clinic coordinator as meeting the criteria. All eligible patients and/or their parents or guardians will receive information regarding the study, including age-appropriate participant information sheets, consent, and assent forms. Information about the study will be publicized on posters in the clinic, with links through quick response [QR] codes to more information about the study. The information will be hosted on our publicly available PPIE group, ALL_EARS@UoS, website [https://generic.wordpress.soton.ac.uk/all-ears/2024/11/12/chief-study-cochlear-implants-and-inner-ear-inflammation/]. The study documents were developed with the input and scrutiny of members of ALL_EARS@UoS. Sample collection A sample of the middle ear mucous membranes, swabs of the nasopharynx and middle ear canal, cochlear fluid, and a blood sample will be collected at the time of surgery for cochlear implantation. Samples from the ear will only be collected from the ear or ears being implanted. In cases where an implant is removed because of implant failure, along with the samples listed above, we will collect any tissue attached to the implant being removed. Sample management and storage Samples will be collected and either transferred for routine analysis (blood sample-hematology) or post-collection processing (tissue samples and any explanted arrays) and then stored using the biobanking facility (4°C or -80°C) at MFT. The nasal and middle ear swab, middle ear mucosa, and tissue from the explanted array will be stored before being transferred to the University of Southampton for analysis. The cochlear fluid (CF) will be stored for up to one year at MFT and transferred to Southampton at the end of year one and year two. Sample processing The middle ear mucous membrane and tissue associated with the implant in cases of re-implantation will be collected into pre-prepared collection tubes of 10% neutral buffered formalin18 during surgery for tissue fixation (overnight (12–16hrs at 20–25°C), before being transferred to 70% ethanol for storage (4°C) until processing. The samples will be processed into paraffin wax tissue and prepared as microarrays for histology and spatial transcriptomics. Samples collected through swabs of the middle ear and nasopharynx will be stored before bacterial gene and viral analyses. The samples will be processed to isolate DNA using commercial kits for low (low blood contamination) and high biomass (fluid samples and where blood is present), and the 16S DNA quantity will be determined. Cochlear fluid collected immediately prior to insertion of the cochlear implant array will be analyzed for the presence of proteins. The samples will be processed using an existing technique19 and the data will be analyzed using bioinformatics. Immune cell identification and characterisation from tissue analysis Histological analysis - Using our published method20, antibodies for macrophages, activated macrophages, T cells, fibroblasts, endothelial cells, and a marker of cellular proliferation and appropriate counterstain will be used to identify the gross tissue morphology and distribution of cells within <4μm tissue sections. Appropriate controls will be used, and the samples will be processed in batches to reduce inter-sample variation. Cell counts will be performed on each sample, with the final data expressed as cells/unit area of tissue after image capture and analysis of the tissue sections using quantitative image analysis microscopy with a custom ImageJ plugin. Spatial transcriptomic analysis – Gene expression and spatial transcriptome profiles at single-cell resolution of immune cells (macrophages) from middle ear mucosa tissue samples will be generated in a subset of samples21–23. We will analyze the expression profiles to provide unbiased characterization of the activation state and ‘memory’ of macrophages between, and within, samples using the high-plex, spatial molecular imaging platform, CosMx (manufactured by Bruker Spatial Biology)24. Initial analysis and data visualization will be performed using AtoMx, a cloud-based, fully integrated spatial informatics platform. We will work through a data analysis pipeline, using TileDB as the primary data structure. The data will go through the quality control module to flag any unreliable negative probes, cells, fields of view, and target genes. The data will be normalized before Principal Component Analysis (PCA). This will be used as an input to uniform manifold approximation and projection (UMAP) to enable the visualization of clusters of related cells within the tissue. The InSituType Cell Typing module will be used for cell typing. Differential gene expression testing will be used to determine the genes that were expressed at different levels between and within the samples. The primary aim of this study is to understand whether inflammation is a factor in inter-individual variability in response to cochlear implantation. Macrophages are long-lived cells and have ‘a memory’ of exposure to injury or infection. This ‘memory,’ known as activated or primed6,25,26, can cause the macrophage to generate a larger inflammatory response if it is stimulated by a second injury or infection. The primary inflammatory response in macrophages is essential to drive repair, remodelling, and recovery, as might occur when macrophages respond to clear infections or pathogens in the inner ear. However, an excessive inflammatory response in primed macrophages causes bystander damage to the delicate tissues of the cochlea27,28 and may contribute to the development of scar tissue or fibrosis around the implant. Fibrosis around the array insulates the electrodes and alters the release of electrical current from the electrodes to their intended target for stimulation, the spiral ganglion cells of the auditory nerve. This change in electrical behavior can be measured as an increase in impedance29–31. Evidence suggests that fibrosis is associated with electrode migration32–34. Altered current spread and movement in the electrode array are likely to be perceived and measured as a change or poorer hearing outcome with the implant. The gene expression patterns, determined through bioinformatic analyses, of the macrophages will enable us to achieve our primary outcome for this part of the study, which is to characterize macrophage ‘memory’, and how this differs within and between cases. This phase of the work is a pilot study; if successful, we aim to secure additional funding to characterize the response in all the study cases. A secondary outcome of this phase of the study will be a dataset that captures the gene expression patterns of other cell types in the tissue. This dataset will be used in the development of follow-up studies from this study. The data will be made accessible to other researchers with appropriate ethical approval on request via data curation through the University of Southampton library. We will monitor and consider the most appropriate data-hosting site as the project evolves. If a bespoke discipline-specific externally hosted data repository becomes available, we can store fuller datasets externally. This will be incorporated in future funding applications. Identification of bacterial species Nasal and middle ear swabs will be analyzed using culture-independent 16S ribosomal RNA (rRNA) gene amplicon sequencing to identify the bacterial species within these sites. This method enables the identification of different strains of bacteria on the mucosal surface (swabs) or within middle ear fluid35. The samples will be processed to isolate DNA using commercial kits for low (low blood contamination) and high biomass (fluid samples and where blood is present), and the 16S DNA quantity will be determined. Quantitative PCR will be carried out to determine the bacterial populations and relative proportions of the populations. A known issue with using a non-culture method for the identification of bacterial strains and species is an increased likelihood of false-positive results36. Anonymized positive control samples and sample spiking will be included within our analysis protocols to mitigate the risk of false positives. Identification of viruses Samples collected from swabs of the middle ear and nasopharynx will be stored before viral analysis. A key aim is to understand how bacterial or viral populations relate to the state of inflammation of the tissue in the middle ear in children and young people at the time of cochlear implantation. Bacteria in the middle ear have been studied in children37 and there is some published work on bacterial analysis in adults undergoing implantation35. However, very few children and young people undergoing implantation have been included in this work and the relationship between inflammation and hearing outcomes is not known and has not been systematically studied. This study provides the first data of this type. The secondary aim is to investigate the relationship between the microbiota of the middle ear and nasopharynx. A secondary outcome of this study is to identify whether there is a distinct middle ear microbiota or biomarker associated with a poorer hearing outcome with a cochlear implant. This would be a significant new finding that may influence the clinical management of the middle ear prior to surgery. If the middle ear microbiota are identified as a biomarker for hearing outcomes with a cochlear implant, there would be further limitations and challenges to address, as sampling the middle ear is an invasive procedure. Therefore, if during this study, we find that the nasopharynx microbiota mirrors that of the middle ear it may be that routine nasal swabs could be collected at home from children with a history of middle ear infection and sent to a laboratory for analysis. This could provide a more tolerable, easier to complete pre-surgical test, and one that can be readily repeated to monitor improvements after, for example, treatment with antibiotics. Identification of inflammatory markers in the blood Blood samples will be collected on the day of surgery and analyzed in the hospital hematology department to provide a full blood count on the day of surgery. The counts will be analyzed, along with the differential white blood cell count and neutrophil-to-leukocyte ratio, to determine the presence of systemic inflammation at the time of surgery. Previous studies, including cross-sectional analyses, have investigated the relationship between inflammation and hearing loss38–41. The mechanism linking inflammation to changes in the cochlea, which results in poorer sensorineural hearing loss later in life, is not understood. This study will add new information about these changes in early life. A key aim of this study is to determine whether there is an association between the levels of systemic inflammation at the time of implantation and hearing outcome following implantation. Does increased levels of systemic inflammation correlate with an increased immune/inflammatory state in the middle and inner ear and does this influence the tissue response following cochlear implantation? Identification of inflammatory markers in the cochlea Proteomics will be used to characterize the levels of inflammatory markers in the cochlea19,42–44. Proteomics is an unbiased technique that enables the detection, identification, and quantification of proteins within a sample. The high sensitivity of this technique enables the detection and quantification of very low levels of protein. This unbiased approach to protein detection means that all proteins in the sample above the detection limits can be identified, and their relative expression determined. A limitation to this can be introduced by samples that require large proteins, such as those found in blood, to be stripped prior to analysis. Low expression of proteins, or proteins with a high affinity for blood proteins, may result in the loss or reduction of some proteins. We will use existing techniques to collect and analyze samples19 with specialist technical support from our proteomics and bioinformatics research units. These results will enable us to determine whether the cochlea shows evidence of inflammation, as determined by the proteins present in the cochlear fluid45 prior to the surgical insertion of the implant. A key aim of this study is to understand whether some of the differences in response to cochlear implantation are due to individual differences in inflammation in the cochlea at the time of implantation. There are no published or established techniques to measure or detect these differences in the intact cochlea prior to implantation. However, there is evidence that there may be differences between people, as in some cases, people who have had meningitis, labyrinthitis, and other auto-immune-induced SNHL have fibrosis, or the growth of tissue within the cochlea46 at the time of cochlear implant insertion. Fibrosis occurs in many organs, such as the liver and lungs47 and inflammation is a consistent feature irrespective of which organ is affected. On this basis, we predict that some people will be more inflamed, and that there is a need for a method to anticipate this. We will integrate the information from the cochlea at the time of implantation with the data collected from tissue and swab samples. Together, this may enable us to develop a method of predicting children and young people who have a greater likelihood of inflammation in their cochlea prior to implantation or a stronger or more prolonged inflammatory response after implantation, both of which may mean they hear less well with their implants and/or initially preserved natural low-frequency hearing is not maintained48. In the longer term, it may be possible to identify these children and young people for more aggressive anti-inflammatory management when they have implants. A secondary outcome of this phase of the study will be a dataset that captures the protein expression of the fluid in the cochlea in a cohort of children and young people. This dataset will be developed and used in the development of follow-up studies from this work, and the data will be made accessible to other researchers on request via data curation through the University of Southampton library. Clinical data collection To explore the relationship between the immune state of the middle ear tissue and long-term hearing outcomes, we will request access to anonymized, routine clinical outcome measures for up to five years post-implantation, including post-implantation complications, electrode impedance, deactivated electrodes, hearing, and device measurements over time with the device and hearing measures. We will access data from medical records including date of birth, ethnicity, history and cause of deafness, history of ear disease (infections, surgery), and history of other infections (meningitis, cytomegalovirus, measles). Descriptive statistics will be used to analyze hearing performance and device measurements. Research questions: 1. What is the immune state of the middle ear in children and young people undergoing cochlear implantation? 2. How distinct or similar are the microbiota (bacteria and viruses) of the nasopharynx and middle ear within individuals? 3. What is the relationship between microbiota and the immune status of the middle ear tissue? 4. What is the inflammatory state of the cochlear fluid at time of surgery? 5. What is the inflammatory state of the tissue associated with the cochlear implant electrode array? How does this relate to the other sites tested in this study? 6. What is the relationship between the inflammatory state of the middle ear and the outcome of cochlear implantation?

Use molecular techniques to; 1. identify the immune cells that are present. 2. characterize the inflammatory state of these cells in the samples of the middle ear mucous membrane (MM) or tissue attached to an implant (FT). 3. identify bacteria that are present and the size/distribution of the populations in the middle ear (ME) and nasal swab (NS). 4. identify viruses that are present and the size/distribution of the populations in the middle ear (ME) and nasal swab (NS). 5. detect the presence and levels of inflammatory markers in cochlear fluid (CF) and blood samples (S). Analyse clinical outcomes routinely measured across five years for the study participants. Primary outcome This study aims to identify differences in ear inflammation between children and young people at the time of cochlear implantation. Secondary outcome A secondary outcome of this study is to identify the relationship between the inflammatory status and microbiota of the ear. The secondary outcome of this study is to identify the relationship between the immune state of the ear in children and young people at the time of implantation and hearing outcomes following implantation.

of the study design This study is an observational study. The results of this study will inform the sample size and recruitment criteria of future interventional studies. In addition, the complete analysis and interpretation of the samples in this study will require several highly specialized and expensive techniques. This will require follow-on funding bids informed by data from this study. Improve patient outcomes This study will allow us to combine spatial gene and protein expression data with clinical and hearing data to address the long-term aim of improving patient outcomes by identifying if there is a predictable relationship between inflammatory status and hearing outcomes following implantation. This is the first study that aims to characterize the immune state of the ear at the time of implantation and to correlate it with hearing outcomes with a cochlear implant. Initially, we will determine whether the inflammatory state of the middle ear at the time of implantation is different between children and young people using histology and spatial transcriptomics. We will combine molecular data with clinical and hearing data to determine whether there is an association between hearing and surgical outcomes following implantation. This provides essential pilot data to support further funding applications. We hypothesize that some children and young people who have a heightened inflammatory tissue environment in the ear due to previous inflammatory insults such as recurrent infections may elicit an increased inflammatory tissue response to cochlear implantation6, resulting in increased fibrosis. This could result in poorer hearing outcomes with the implant, both in terms of initial preservation and subsequent maintenance of residual natural hearing after implantation48,49 and worsening of the quality of electrical stimulation over time50,51. If we can identify patients who are at greater risk of an increased inflammatory tissue response to implantation and therefore poor hearing outcomes, we could intervene prior to, or at, surgery with existing and novel anti-inflammatory and/or antimicrobial therapies and ensure closer post-implantation monitoring to improve hearing outcomes following implantation. Build a rich database containing clinical and medical records of children and young people who have undergone cochlear implantation, alongside building a rich tissue bank This longitudinal, observational study design will allow us to collect and bank multiple tissue and fluid samples alongside detailed participant clinical, hearing, and medical data for five years. Using this data, we will produce a rich database that will allow us to ask hypothesis-driven research questions. We will use pilot data obtained from initial histological and transcriptomic tissue analyses to apply for larger funding bids. Contribute novel data sets Our study design, alongside the use of spatial transcriptomics, will contribute novel data sets, including the first spatial transcriptome profile of macrophages in the middle ear tissue of deaf children undergoing cochlear implantation, as well as the transcriptome profile of other cell types, including fibroblasts, which are relevant for cancer and respiratory biology. Full ethical approval was obtained from the Integrated Research Application System (IRAS) [330110] and local ethical approval was obtained from University of Southampton via Ethics and Research Governance Online (ERGO) [89599]. Approval was obtained on 20.09.2024. Written consent will be obtained from the parents or guardians of the children and young people (aged 1–15 years old) that will be recruited into this study. In addition, written assent will be obtained from children aged 5–15 years old. Written consent will be obtained from children aged 16 years old. A preprint of this article is available on MedXRiv52. The results will be submitted to international peer-reviewed journals and presented at conferences. The results will be presented to a lay audience via the patient and public involvement and engagement group (ALL_EARS@UoS) website (https://generic.wordpress.soton.ac.uk/all-ears/). No data are associated with this article. Future datasets from the CHIEF study will be published in the University of Southampton PURE repository. University of Southampton Institutional Repository: STROBE checklist for ‘Protocol for CHIEF (cochlear implants and inner ear inflammation) study;an observational, cross-sectional study of children and young people undergoing cochlear implantation. https://doi.org/10.5258/SOTON/D3345 This project contains the following underlying data: CHIEF_Protocol_-_STROBE-checklist.docx - Other STROBE_Checklist_Readme.txt – Dataset Data are available under the terms of the Creative Commons Zero "No rights reserved data waiver (CC0 1.0 Public domain dedication). TAN, IB, KH and JN conceived the study. TAN and IB previously carried out a small pilot study that confirmed the feasibility of cochlear fluid sampling during routine surgery for cochlear implantation. IB and JN considered the timeline, feasibility, and method of sample collection during the surgical procedure. TAN and KH demonstrated the feasibility of histological analysis and identified inflammation associated with the tissue on an explanted device. CF contributed to the experimental design, including the capture and management of the data. TAN generated the first draft of the protocol and all authors contributed to this draft. The authors are grateful to members of the ALL_EARS@UoS PPIE group who shared their experiences of hearing loss and cochlear implantation, contributed to the discussion, and provided feedback about the CHIEF study. The authors are grateful to the group member SW, who attended the ethical approval panel meeting. The authors are grateful to the Pediatric and Adolescent Cochlear Implant Team of Manchester for supporting this study. We thank the cochlear implant patients who will take part in the study. Faculty Opinions recommendedReferences - 1. 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Publisher Full Text Author details Author details 1 Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, Southampton, England, SO17 1BJ, UK 2 Royal Manchester Children's Hospital, Manchester University Hospitals NHS Foundation Trust,, Manchester Academic Health Science Centre, Manchester, M13 9WL, UK 3 Department of Otolaryngology, University Hospital Southampton NHS Foundation Trust, Southampton, SO16 6YD, UK 4 Division of Infection, Immunity and Respiratory Medicine, School of Biological Sciences, Faculty of Biology, Medicine and Health, The University of Manchester, Manchester, England, M13 9PL, UK 2 Royal Manchester Children's Hospital, Manchester University Hospitals NHS Foundation Trust,, Manchester Academic Health Science Centre, Manchester, M13 9WL, UK 3 Department of Otolaryngology, University Hospital Southampton NHS Foundation Trust, Southampton, SO16 6YD, UK 4 Division of Infection, Immunity and Respiratory Medicine, School of Biological Sciences, Faculty of Biology, Medicine and Health, The University of Manchester, Manchester, England, M13 9PL, UK Kate Hough Roles: Conceptualization, Methodology, Resources, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing Roles: Conceptualization, Methodology, Resources, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing Jaya Nichani Roles: Conceptualization, Methodology, Project Administration, Writing – Review & Editing Roles: Conceptualization, Methodology, Project Administration, Writing – Review & Editing Callum Findlay Roles: Methodology, Writing – Review & Editing Roles: Methodology, Writing – Review & Editing Iain A Bruce Roles: Conceptualization, Funding Acquisition, Methodology, Project Administration, Writing – Review & Editing Roles: Conceptualization, Funding Acquisition, Methodology, Project Administration, Writing – Review & Editing Tracey A Newman Roles: Conceptualization, Funding Acquisition, Investigation, Methodology, Project Administration, Resources, Supervision, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing Roles: Conceptualization, Funding Acquisition, Investigation, Methodology, Project Administration, Resources, Supervision, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing Competing interests No competing interests were disclosed. Grant information This project is funded by the National Institute for Health and Care Research (NIHR) under its [‘Research for Patient Benefit (RfPB) Programme’ (Grant Reference Number NIHR203308)]. The views expressed are those of the author(s) and not necessarily those of the NIHR or the Department of Health and Social Care. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Article Versions (2) Copyright © 2025 Hough K et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. metrics VIEWS $counts.viewCount downloads Citations CITE how to cite this article Hough K, Nichani J, Findlay C et al. Protocol for CHIEF (cochlear implants and inner ear inflammation) study; an observational, cross-sectional study of children and young people undergoing cochlear implantation. [version 1; peer review: 1 approved, 1 approved with reservations]. NIHR Open Res 2025, 5:18 (https://doi.org/10.3310/nihropenres.13879.1) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. track receive updates on this article Track an article to receive email alerts on any updates to this article. Current Reviewer Status: ? Key to Reviewer Statuses VIEW HIDE ApprovedThe paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approvedFundamental flaws in the paper seriously undermine the findings and conclusions Version 1 VERSION 1 PUBLISHED 04 Mar 2025 Views 0 How to cite this report: Tang J and Pan J. Reviewer Report For: Protocol for CHIEF (cochlear implants and inner ear inflammation) study; an observational, cross-sectional study of children and young people undergoing cochlear implantation. [version 1; peer review: 1 approved, 1 approved with reservations]. NIHR Open Res 2025, 5:18 (https://doi.org/10.3310/nihropenres.15081.r35055) The direct URL for this report is: https://openresearch.nihr.ac.uk/articles/5-18/v1#referee-response-35055 https://openresearch.nihr.ac.uk/articles/5-18/v1#referee-response-35055 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Reviewer Report 08 Apr 2025 Jing Pan, Department of Otolaryngology Head and Neck Surgery, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong, China Approved VIEWS 0 This study protocol addresses a critical research gap by systematically investigating the inflammatory response and its association with auditory outcomes in pediatric and young adult cochlear implant recipients. The manuscript is well-organized, supported by a thorough literature review, and adheres ... Continue reading 2. Luo R, Dai J, Zhang J, Li Z: Accelerated Skin Wound Healing by Electrical Stimulation.Adv Healthc Mater. 2021; 10 (16): e2100557 PubMed Abstract | Publisher Full Text 3. Pinheiro-Dardis CM, Russo TL: Electrical Stimulation Based on Chronaxie Increases Fibrosis and Modulates TWEAK/Fn14, TGF-β/Myostatin, and MMP Pathways in Denervated Muscles.Am J Phys Med Rehabil. 2017; 96 (4): 260-267 PubMed Abstract | Publisher Full Text We confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Close This study protocol addresses a critical research gap by systematically investigating the inflammatory response and its association with auditory outcomes in pediatric and young adult cochlear implant recipients. The manuscript is well-organized, supported by a thorough literature review, and adheres to the STROBE guidelines for observational studies. It presents a rigorous methodological framework for specimen collection, laboratory analyses, and longitudinal follow-up, ensuring both reproducibility and scientific validity. The incorporation of patient and public involvement and engagement (PPIE) enhances the clinical relevance and translational potential of the research for the target population. While the study design is methodologically robust, certain aspects could benefit from further elaboration to maximize the study's impact and clarity. This study protocol collects biological samples on the day of cochlear implantation surgery, with the aim of investigating the relationship between the inflammatory state of the inner ear and postoperative auditory outcomes. For these analyses to yield meaningful conclusions, one critical prerequisite is that all patients receive standardized postoperative care, particularly regarding infection prevention and anti-inflammatory management. I recommend explicitly stating these standardized postoperative care protocols in the Methods section, with special emphasis on infection control and anti-inflammatory measures. Additionally, I would like to inquire whether there was consistency in the time interval between device implantation and initial activation across study participants. Emerging evidence suggests that macrophage activity and development of fibrosis may be influenced by electrical stimulation from the implant (Zhang et al., 2023 [Ref 1]; Luo et al, 2021 [Ref 2]; Pinheiro-Dardis CM, et al., 2017 [Ref 3]). If activation timing varied among participants, was this parameter incorporated into the analysis of the relationship between inflammatory status and implantation outcomes? This temporal factor could potentially serve as an important covariate in the investigation. While the study design is methodologically robust, certain aspects could benefit from further elaboration to maximize the study's impact and clarity. This study protocol collects biological samples on the day of cochlear implantation surgery, with the aim of investigating the relationship between the inflammatory state of the inner ear and postoperative auditory outcomes. For these analyses to yield meaningful conclusions, one critical prerequisite is that all patients receive standardized postoperative care, particularly regarding infection prevention and anti-inflammatory management. I recommend explicitly stating these standardized postoperative care protocols in the Methods section, with special emphasis on infection control and anti-inflammatory measures. Additionally, I would like to inquire whether there was consistency in the time interval between device implantation and initial activation across study participants. Emerging evidence suggests that macrophage activity and development of fibrosis may be influenced by electrical stimulation from the implant (Zhang et al., 2023 [Ref 1]; Luo et al, 2021 [Ref 2]; Pinheiro-Dardis CM, et al., 2017 [Ref 3]). If activation timing varied among participants, was this parameter incorporated into the analysis of the relationship between inflammatory status and implantation outcomes? This temporal factor could potentially serve as an important covariate in the investigation. - Is the rationale for, and objectives of, the study clearly described? Yes - Is the study design appropriate for the research question? Yes - Are sufficient details of the methods provided to allow replication by others? Yes - Are the datasets clearly presented in a useable and accessible format? Yes

1. Zhang D, Chen D, Wang K, Pan J, et al.: Electrical stimulation of cochlear implant promotes activation of macrophages and fibroblasts under inflammation.Laryngoscope Investig Otolaryngol. 2023; 8 (5): 1390-1400 PubMed Abstract | Publisher Full Text2. Luo R, Dai J, Zhang J, Li Z: Accelerated Skin Wound Healing by Electrical Stimulation.Adv Healthc Mater. 2021; 10 (16): e2100557 PubMed Abstract | Publisher Full Text 3. Pinheiro-Dardis CM, Russo TL: Electrical Stimulation Based on Chronaxie Increases Fibrosis and Modulates TWEAK/Fn14, TGF-β/Myostatin, and MMP Pathways in Denervated Muscles.Am J Phys Med Rehabil. 2017; 96 (4): 260-267 PubMed Abstract | Publisher Full Text Competing Interests: No competing interests were disclosed. Reviewer Expertise: hearing disorders CITE HOW TO CITE THIS REPORT Tang J and Pan J. Reviewer Report For: Protocol for CHIEF (cochlear implants and inner ear inflammation) study; an observational, cross-sectional study of children and young people undergoing cochlear implantation. [version 1; peer review: 1 approved, 1 approved with reservations]. NIHR Open Res 2025, 5:18 (https://doi.org/10.3310/nihropenres.15081.r35055) The direct URL for this report is: https://openresearch.nihr.ac.uk/articles/5-18/v1#referee-response-35055 https://openresearch.nihr.ac.uk/articles/5-18/v1#referee-response-35055 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. Views 0 How to cite this report: Ching T. Reviewer Report For: Protocol for CHIEF (cochlear implants and inner ear inflammation) study; an observational, cross-sectional study of children and young people undergoing cochlear implantation. [version 1; peer review: 1 approved, 1 approved with reservations]. NIHR Open Res 2025, 5:18 (https://doi.org/10.3310/nihropenres.15081.r34871) The direct URL for this report is: https://openresearch.nihr.ac.uk/articles/5-18/v1#referee-response-34871 https://openresearch.nihr.ac.uk/articles/5-18/v1#referee-response-34871 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Reviewer Report 01 Apr 2025 Teresa Ching, Macquarie University, Sydney, New South Wales, Australia Approved with Reservations VIEWS 0 Individual variability in performance of cochlear implant recipients is a key question in aural (re)habilitation. This study posits that variability in performance may be related to individual variations in inflammatory response to cochlear implantation. Accordingly, the study aims to 1) ... Continue reading I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Close Individual variability in performance of cochlear implant recipients is a key question in aural (re)habilitation. This study posits that variability in performance may be related to individual variations in inflammatory response to cochlear implantation. Accordingly, the study aims to 1) identify differences in ear inflammation between individual pediatric recipients of cochlear implants, and 2) explore the relationship between the immune state of the ear at the time of implantation and hearing outcomes up to 5 years post-implantation. The background section does not include current knowledge about effects of middle ear inflammation on performance with cochlear implants in adult and pediatric recipients. How this may differ between adult and pediatric patients have not been described. The protocol provides a detailed description of the collection and analysis of biologic samples to be collected at the time of (re)implantation to address Aim 1. Research questions and objectives for this part are listed, and methods to address the questions have been clearly spelt out. However, there is very little information on analytical methods to achieve Aim 2. Indeed, the definition of ‘hearing outcomes’ in the protocol appears elusive. The protocol indicated that descriptive statistics will be used to analyze hearing performance and device measurements (pg.8). Descriptive statistics serve to describe, but not to explore relationships. It is not clear what specific ‘device measurements’ will be collected, at what time-points, and how the data will be analyzed in relation to the other biologic-sample-based data to address Aim 2 regarding the relationship between immune status and outcomes. Furthermore, current evidence suggests that a range of biographic, etiologic, device-related and surgical factors influences individual performance with cochlear implants. The way in which this study will examine the extent to which these factors interact with the immune status of the ear at implantation to influence hearing outcomes is not clear. The protocol does not contain information on how the range of known factors would be considered or controlled for when examining the role of immune status at cochlear implantation in influencing hearing outcomes. The specific variables depicted by the term ‘hearing outcomes’ are not explicit, and the method to address the question on relationship is not clear (Aim 2). It appears that there are no patient and public involvement and engagement group that includes parents/ guardians and young people with lived experience at the locality where recruitment and clinical management will occur. Especially when the research study involves children and young people, engagement with parents would be crucial. The current protocol does not contain a plan to disseminate findings to stakeholders. An important part of engagement is to disseminate findings. Maybe this aspect has already been covered in other related documents. The protocol suggests that a potential benefit of the study is that identification of patients at risk of increased inflammatory response to cochlear implantation (if a relation were found between immune status at implantation and ‘hearing outcomes’) could pave the way for intervention prior to or at surgery with anti-inflammatory /antimicrobial therapies and ensure closer monitoring. There is some evidence that use of drugs/steroids contribute to preservation of residual hearing after implantation, and soft surgery techniques have been widely implemented to achieve the goal of preservation of residual hearing. Are these not in use in standard protocols for cochlear implantation at the test site? Overall, the study will make a significant contribution to knowledge about inflammatory responses in pediatric recipients of cochlear implants and create a novel data set. However, the protocol will benefit from a clarification of how other factors affecting hearing outcomes in pediatric recipients will be considered or controlled for when examining the role of immune status in influencing outcomes; and an explanation of how the relationship between immune status and hearing outcomes will be explored. The end. The background section does not include current knowledge about effects of middle ear inflammation on performance with cochlear implants in adult and pediatric recipients. How this may differ between adult and pediatric patients have not been described. The protocol provides a detailed description of the collection and analysis of biologic samples to be collected at the time of (re)implantation to address Aim 1. Research questions and objectives for this part are listed, and methods to address the questions have been clearly spelt out. However, there is very little information on analytical methods to achieve Aim 2. Indeed, the definition of ‘hearing outcomes’ in the protocol appears elusive. The protocol indicated that descriptive statistics will be used to analyze hearing performance and device measurements (pg.8). Descriptive statistics serve to describe, but not to explore relationships. It is not clear what specific ‘device measurements’ will be collected, at what time-points, and how the data will be analyzed in relation to the other biologic-sample-based data to address Aim 2 regarding the relationship between immune status and outcomes. Furthermore, current evidence suggests that a range of biographic, etiologic, device-related and surgical factors influences individual performance with cochlear implants. The way in which this study will examine the extent to which these factors interact with the immune status of the ear at implantation to influence hearing outcomes is not clear. The protocol does not contain information on how the range of known factors would be considered or controlled for when examining the role of immune status at cochlear implantation in influencing hearing outcomes. The specific variables depicted by the term ‘hearing outcomes’ are not explicit, and the method to address the question on relationship is not clear (Aim 2). It appears that there are no patient and public involvement and engagement group that includes parents/ guardians and young people with lived experience at the locality where recruitment and clinical management will occur. Especially when the research study involves children and young people, engagement with parents would be crucial. The current protocol does not contain a plan to disseminate findings to stakeholders. An important part of engagement is to disseminate findings. Maybe this aspect has already been covered in other related documents. The protocol suggests that a potential benefit of the study is that identification of patients at risk of increased inflammatory response to cochlear implantation (if a relation were found between immune status at implantation and ‘hearing outcomes’) could pave the way for intervention prior to or at surgery with anti-inflammatory /antimicrobial therapies and ensure closer monitoring. There is some evidence that use of drugs/steroids contribute to preservation of residual hearing after implantation, and soft surgery techniques have been widely implemented to achieve the goal of preservation of residual hearing. Are these not in use in standard protocols for cochlear implantation at the test site? Overall, the study will make a significant contribution to knowledge about inflammatory responses in pediatric recipients of cochlear implants and create a novel data set. However, the protocol will benefit from a clarification of how other factors affecting hearing outcomes in pediatric recipients will be considered or controlled for when examining the role of immune status in influencing outcomes; and an explanation of how the relationship between immune status and hearing outcomes will be explored. The end. - Is the rationale for, and objectives of, the study clearly described? Partly - Is the study design appropriate for the research question? Partly - Are sufficient details of the methods provided to allow replication by others? Partly - Are the datasets clearly presented in a useable and accessible format? Not applicable Competing Interests: No competing interests were disclosed. Reviewer Expertise: Cochlear implants, pediatric outcomes, adult outcomes, aural rehabilitation for children who are deaf or hard of hearing CITE HOW TO CITE THIS REPORT Ching T. Reviewer Report For: Protocol for CHIEF (cochlear implants and inner ear inflammation) study; an observational, cross-sectional study of children and young people undergoing cochlear implantation. [version 1; peer review: 1 approved, 1 approved with reservations]. NIHR Open Res 2025, 5:18 (https://doi.org/10.3310/nihropenres.15081.r34871) The direct URL for this report is: https://openresearch.nihr.ac.uk/articles/5-18/v1#referee-response-34871 https://openresearch.nihr.ac.uk/articles/5-18/v1#referee-response-34871 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. - Author Response 28 May 2025Kate Hough, Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, Southampton, SO17 1BJ, UK28 May 2025Author ResponseThank you for taking the time to review the study protocol and providing your comments. I have included your comments in bold and my responses below each point. Individual ... Continue reading Thank you for taking the time to review the study protocol and providing your comments. I have included your comments in bold and my responses below each point. Individual variability in performance of cochlear implant recipients is a key question in aural (re)habilitation. This study posits that variability in performance may be related to individual variations in inflammatory response to cochlear implantation. Accordingly, the study aims to 1) identify differences in ear inflammation between individual pediatric recipients of cochlear implants, and 2) explore the relationship between the immune state of the ear at the time of implantation and hearing outcomes up to 5 years post-implantation. The background section does not include current knowledge about effects of middle ear inflammation on performance with cochlear implants in adult and pediatric recipients. How this may differ between adult and pediatric patients have not been described. There is limited current knowledge about the effects of middle ear inflammation on performance with cochlear implants in both adult and paediatric recipients. Hence why this is an important area to investigate. Literature exploring the effect of middle ear inflammation on surgical outcomes (Rak et al., 2018; Alzoubi et al., 2015) has shown a history of otitis media (OM) or active OM can increase the risk of complications and increase the time needed for surgery. However, in most cases – surgical outcomes are fine. Studies in adult cochlear implant patients with a history of chronic otitis media, which have investigated surgical outcomes and rate of complications, have included investigation of performance such as long-term speech comprehension (Rak et al., 2018) and auditory performance (Goh et al., 2020). Improvements in performance were measured compared to pre-CI levels, but no comparisons were made between adults with and without a history of chronic otitis media. We have no found studies that have investigated hearing outcomes/performance over time in a cohort of paediatric cochlear implant patients with and without a history of middle ear inflammation. We are in the process of applying for ethical approval to carry out a retrospective study of this in patients at a single implant centre in the UK. In addition, the data collected through the CHIEF study will enable us to explore this with the study cohort. The text below has been added the introduction: 'There is an urgent need to investigate the effect of the immune state of the inner ear on hearing outcomes following cochlear implantation. To achieve this, we need to study the inflammatory state of the ear in children and young people undergoing implantation. Importantly, we need to see how this varies between children and young people, and how it is associated with hearing outcomes in people with a history of middle ear inflammation (acute otitis media and otitis media with effusion)(15,16). Hearing outcomes is the combination of audiological tests, speech perception tests, language development tests, datalogging and parental assessments that allow cochlear implant professionals to measure and determine how much benefit someone is getting from their cochlear implant. Currently, there are no studies that have investigated hearing outcomes over time in a cohort of pediatric cochlear implant patients with and without a history of middle ear inflammation. The CHIEF study will allow us to investigate this'. The protocol provides a detailed description of the collection and analysis of biologic samples to be collected at the time of (re)implantation to address Aim 1. Research questions and objectives for this part are listed, and methods to address the questions have been clearly spelt out. However, there is very little information on analytical methods to achieve Aim 2. The following text has been added to the section with the sub-heading ‘Secondary outcome’ to clarify this point. 'The characterisation of the immune state of each study participant will inform the analytical plan to achieve this secondary outcome. Due to anticipated variability in immune state it may be that this is the pilot study to informs a larger powered study. This step is necessary as there is no prior equivalent data. Likewise the analysis of the hearing outcome will depend on the age of participants and the battery of hearing, speech and language assessments over time'. Indeed, the definition of ‘hearing outcomes’ in the protocol appears elusive. To clarify what we mean by hearing outcomes, this text has been added to the second to last paragraph of the introduction: 'Hearing outcomes is the combination of audiological tests, speech perception tests, language development tests, datalogging and parental assessments that allow cochlear implant professionals to measure and determine how much benefit someone is getting from their cochlear implant'. To further clarify what we mean by hearing outcomes, the following text has been added to the ‘clinical data collection’ section: 'To measure hearing outcomes, a battery of age-appropriate audiological, speech perception and language development tests are used, in addition to datalogging measurements. To explore the relationship between the immune state of the middle ear tissue and long-term hearing outcomes, we will request access to anonymized, routine clinical outcome measures for up to five years post-implantation of study participants. This will include audiological and device data; aided levels from speech and language tests (ASSE and AB words); datalogging (device usage e.g. average daily use) and electrode impedances and deactivations (number of electrodes and position). Together with a log of any recorded post-implantation complications. All data collected post-implantation will be from routinely collected clinical data. The device measurements will be collected when a patient comes in for an appointment or via remote appointments. The time points of these measurements from the day of implantation may vary but will loosely follow the appointment structure which is: switch on 7 – 14 days post-implantation, 1 month, 3 months, 6 months, 2 years, 4 years, 6 years (new processor). We will access data from medical records including date of birth, ethnicity, history and cause/etiology of deafness, history of ear disease (infections, surgery), and history of other infections (meningitis, cytomegalovirus, measles). Descriptive statistics will be used to describe hearing performance and device measurements'. The protocol indicated that descriptive statistics will be used to analyze hearing performance and device measurements (pg.8). Descriptive statistics serve to describe, but not to explore relationships. It is not clear what specific ‘device measurements’ will be collected, at what time-points, The following device measurements will be collected: Electrode impedance, position of electrode deactivation and number of electrode deactivation. The following text has been added to the manuscript in the Clinical data collection section to clarify the specific device measurement that we will be collecting and at what time-points: '…device measurements including electrode impedances and deactivations (number of electrodes and position). All data collected post-implantation will be from routinely collected clinical data. The device measurements will be collected when a patient comes in for an appointment or via remote appointments. The time points of these measurements from the day of implantation may vary but will loosely follow the appointment structure which is: switch on 7 – 14 days post-implantation, 1 month, 3 months, 6 months, 2 years, 4 years, 6 years (new processor)'. and how the data will be analyzed in relation to the other biologic-sample-based data to address Aim 2 regarding the relationship between immune status and outcomes. As well as using the spatial transcriptomic data to get a detailed understanding of what genes the cells in the middle ear mucosal tissue are expressing and how they are signalling to each other, the biologic sample-based data will be used to split the participants into two groups: inflamed and non-inflamed. The individual hearing outcome measures and device measurements will be analysed to compare the inflamed and non-inflamed group. For example – what are the differences in Aided levels at each frequency over time post-implantation between inflamed and non-inflamed group? What is the impedance over time for the inflamed and non-inflamed group? Furthermore, current evidence suggests that a range of biographic, etiologic, device-related and surgical factors influences individual performance with cochlear implants. The way in which this study will examine the extent to which these factors interact with the immune status of the ear at implantation to influence hearing outcomes is not clear. This is an observational, pilot study where we will be collecting a bank of biological samples as well as building a clinical database of up to 75 paediatric cochlear implant recipients. The study may not be sufficiently powered to carry out regression analysis to examine the relationship between hearing outcomes and the multiple factors that are likely to influence this. However, this study will allow us to combine the detailed understanding of the immune biology in the ear at the time of implantation (for the first time) with a rich clinical dataset containing information about the various known factors that can influence performance following implantation. This will enable us to better understand which factors are interacting with immune status to help us design clinical studies. The protocol does not contain information on how the range of known factors would be considered or controlled for when examining the role of immune status at cochlear implantation in influencing hearing outcomes. Biographic factors – we will have access to medical history so these factors will be considered when examining role of immune status influencing hearing outcomes. These factors will not be controlled for as the inclusion criteria is wide. [This is an observational study not an intervention study]. Etiologic factors – we will assess medical history and consider these factors. We will not know how well reported these factors will be until we have recruited participants and looked at these data. This is important and relevant for otitis media/middle ear inflammation and other immune related etiologies. Device related factors – this is a single site study so all participants will have the same pre-operative protocol. Surgical factors – this is a single site study so all participants will have the same surgical approach, and the surgeries will be done by one of three surgeons. Hearing preservation soft surgery approach is utilised at this site. The specific variables depicted by the term ‘hearing outcomes’ are not explicit, and the method to address the question on relationship is not clear (Aim 2). This is a data collecting study. The research questions and the type of analysis that will be possible will depend on the cohort and the data collected. This is because the inclusion criteria are wide, we are recruiting participants across a wide age range, meaning different audiological, speech and language tests will be used depending on the age of the participants. It appears that there are no patient and public involvement and engagement group that includes parents/ guardians and young people with lived experience at the locality where recruitment and clinical management will occur. Especially when the research study involves children and young people, engagement with parents would be crucial. The current protocol does not contain a plan to disseminate findings to stakeholders. An important part of engagement is to disseminate findings. Maybe this aspect has already been covered in other related documents. We have a thriving PPIE group that includes parents of children who are deaf or hard of hearing. We have discussed the study throughout the design stages and had feedback throughout and we will continue to keep an open dialogue with our PPIE group members. We also have links with key PPIE professionals in Manchester, where we are recruiting participants from. This will enable us to link our PPIE activities with those happening in Manchester and disseminate information about the study in Manchester as well as Hampshire. We were aware that we were missing the voice of young people in our group so in March 2025 we launched ALL_EARS@UoS Young People’s Advisory Group (YPAG) for young people aged 14 – 16 years ago (https://generic.wordpress.soton.ac.uk/all-ears/2025/03/07/join-our-new-young-peoples-advisory-group-ypag/). Young people are being recruited nationally to join this group. This group will support the CHIEF project throughout. The following text has been added to the PPIE section in the manuscript: 'In March 2025, we launched ALL_EARS@UoS Young People’s Advisory Group (YGAG) which is PPIE group for young people aged between 14 – 16 years old (https://generic.wordpress.soton.ac.uk/all-ears/2025/03/07/join-our-new-young-peoples-advisory-group-ypag/). Members of ALL_EARS@UoS YPAG will support this study alongside our wider research'. In the Ethics and dissemination section, we have outlined that we plan to disseminate findings through international peer-reviewed journals and conferences. In addition, we will present findings to a lay audience through our website. The following text has been added in to this section: 'In addition to this, we will share findings and progress of the study through the various public engagement activities that we take part in each year alongside ALL_EARS@UoS members'. The protocol suggests that a potential benefit of the study is that identification of patients at risk of increased inflammatory response to cochlear implantation (if a relation were found between immune status at implantation and ‘hearing outcomes’) could pave the way for intervention prior to or at surgery with anti-inflammatory /antimicrobial therapies and ensure closer monitoring. There is some evidence that use of drugs/steroids contribute to preservation of residual hearing after implantation, and soft surgery techniques have been widely implemented to achieve the goal of preservation of residual hearing. Are these not in use in standard protocols for cochlear implantation at the test site? The following text has been added to a new sub-section in the Methods section titled ‘Standard protocol for cochlear implantation at Manchester University Hospital’ 'Surgeons routinely use soft surgery technique for all patients, even for those who do not have residual hearing. This is so that they preserve neural tissue in all patients. All children get 1 dose of peroperative co-amoxiclav 30mg/kg up to a maximum of 1.2g or an alternative if penicillin allergic. In addition, the patients will also get a peroperative dose of steroids – 0.15mg/kg, up to a maximum of 6.6mg. Post-operatively, all children are discharged on 3 doses of oral dexamethasone, 0.15mg.kg and 3 doses of antibiotic. If there are signs of acute inflammation during surgery in the form of inflamed mucosa and sero-mucinous otitis media, the surgeons will give 1 week’s treatment with oral antibiotics post-operatively'. Overall, the study will make a significant contribution to knowledge about inflammatory responses in pediatric recipients of cochlear implants and create a novel data set. However, the protocol will benefit from a clarification of how other factors affecting hearing outcomes in pediatric recipients will be considered or controlled for when examining the role of immune status in influencing outcomes; and an explanation of how the relationship between immune status and hearing outcomes will be explored. The end.Thank you for taking the time to review the study protocol and providing your comments. I have included your comments in bold and my responses below each point.Competing Interests: No competing interests were disclosed. Close Individual variability in performance of cochlear implant recipients is a key question in aural (re)habilitation. This study posits that variability in performance may be related to individual variations in inflammatory response to cochlear implantation. Accordingly, the study aims to 1) identify differences in ear inflammation between individual pediatric recipients of cochlear implants, and 2) explore the relationship between the immune state of the ear at the time of implantation and hearing outcomes up to 5 years post-implantation. The background section does not include current knowledge about effects of middle ear inflammation on performance with cochlear implants in adult and pediatric recipients. How this may differ between adult and pediatric patients have not been described. There is limited current knowledge about the effects of middle ear inflammation on performance with cochlear implants in both adult and paediatric recipients. Hence why this is an important area to investigate. Literature exploring the effect of middle ear inflammation on surgical outcomes (Rak et al., 2018; Alzoubi et al., 2015) has shown a history of otitis media (OM) or active OM can increase the risk of complications and increase the time needed for surgery. However, in most cases – surgical outcomes are fine. Studies in adult cochlear implant patients with a history of chronic otitis media, which have investigated surgical outcomes and rate of complications, have included investigation of performance such as long-term speech comprehension (Rak et al., 2018) and auditory performance (Goh et al., 2020). Improvements in performance were measured compared to pre-CI levels, but no comparisons were made between adults with and without a history of chronic otitis media. We have no found studies that have investigated hearing outcomes/performance over time in a cohort of paediatric cochlear implant patients with and without a history of middle ear inflammation. We are in the process of applying for ethical approval to carry out a retrospective study of this in patients at a single implant centre in the UK. In addition, the data collected through the CHIEF study will enable us to explore this with the study cohort. The text below has been added the introduction: 'There is an urgent need to investigate the effect of the immune state of the inner ear on hearing outcomes following cochlear implantation. To achieve this, we need to study the inflammatory state of the ear in children and young people undergoing implantation. Importantly, we need to see how this varies between children and young people, and how it is associated with hearing outcomes in people with a history of middle ear inflammation (acute otitis media and otitis media with effusion)(15,16). Hearing outcomes is the combination of audiological tests, speech perception tests, language development tests, datalogging and parental assessments that allow cochlear implant professionals to measure and determine how much benefit someone is getting from their cochlear implant. Currently, there are no studies that have investigated hearing outcomes over time in a cohort of pediatric cochlear implant patients with and without a history of middle ear inflammation. The CHIEF study will allow us to investigate this'. The protocol provides a detailed description of the collection and analysis of biologic samples to be collected at the time of (re)implantation to address Aim 1. Research questions and objectives for this part are listed, and methods to address the questions have been clearly spelt out. However, there is very little information on analytical methods to achieve Aim 2. The following text has been added to the section with the sub-heading ‘Secondary outcome’ to clarify this point. 'The characterisation of the immune state of each study participant will inform the analytical plan to achieve this secondary outcome. Due to anticipated variability in immune state it may be that this is the pilot study to informs a larger powered study. This step is necessary as there is no prior equivalent data. Likewise the analysis of the hearing outcome will depend on the age of participants and the battery of hearing, speech and language assessments over time'. Indeed, the definition of ‘hearing outcomes’ in the protocol appears elusive. To clarify what we mean by hearing outcomes, this text has been added to the second to last paragraph of the introduction: 'Hearing outcomes is the combination of audiological tests, speech perception tests, language development tests, datalogging and parental assessments that allow cochlear implant professionals to measure and determine how much benefit someone is getting from their cochlear implant'. To further clarify what we mean by hearing outcomes, the following text has been added to the ‘clinical data collection’ section: 'To measure hearing outcomes, a battery of age-appropriate audiological, speech perception and language development tests are used, in addition to datalogging measurements. To explore the relationship between the immune state of the middle ear tissue and long-term hearing outcomes, we will request access to anonymized, routine clinical outcome measures for up to five years post-implantation of study participants. This will include audiological and device data; aided levels from speech and language tests (ASSE and AB words); datalogging (device usage e.g. average daily use) and electrode impedances and deactivations (number of electrodes and position). Together with a log of any recorded post-implantation complications. All data collected post-implantation will be from routinely collected clinical data. The device measurements will be collected when a patient comes in for an appointment or via remote appointments. The time points of these measurements from the day of implantation may vary but will loosely follow the appointment structure which is: switch on 7 – 14 days post-implantation, 1 month, 3 months, 6 months, 2 years, 4 years, 6 years (new processor). We will access data from medical records including date of birth, ethnicity, history and cause/etiology of deafness, history of ear disease (infections, surgery), and history of other infections (meningitis, cytomegalovirus, measles). Descriptive statistics will be used to describe hearing performance and device measurements'. The protocol indicated that descriptive statistics will be used to analyze hearing performance and device measurements (pg.8). Descriptive statistics serve to describe, but not to explore relationships. It is not clear what specific ‘device measurements’ will be collected, at what time-points, The following device measurements will be collected: Electrode impedance, position of electrode deactivation and number of electrode deactivation. The following text has been added to the manuscript in the Clinical data collection section to clarify the specific device measurement that we will be collecting and at what time-points: '…device measurements including electrode impedances and deactivations (number of electrodes and position). All data collected post-implantation will be from routinely collected clinical data. The device measurements will be collected when a patient comes in for an appointment or via remote appointments. The time points of these measurements from the day of implantation may vary but will loosely follow the appointment structure which is: switch on 7 – 14 days post-implantation, 1 month, 3 months, 6 months, 2 years, 4 years, 6 years (new processor)'. and how the data will be analyzed in relation to the other biologic-sample-based data to address Aim 2 regarding the relationship between immune status and outcomes. As well as using the spatial transcriptomic data to get a detailed understanding of what genes the cells in the middle ear mucosal tissue are expressing and how they are signalling to each other, the biologic sample-based data will be used to split the participants into two groups: inflamed and non-inflamed. The individual hearing outcome measures and device measurements will be analysed to compare the inflamed and non-inflamed group. For example – what are the differences in Aided levels at each frequency over time post-implantation between inflamed and non-inflamed group? What is the impedance over time for the inflamed and non-inflamed group? Furthermore, current evidence suggests that a range of biographic, etiologic, device-related and surgical factors influences individual performance with cochlear implants. The way in which this study will examine the extent to which these factors interact with the immune status of the ear at implantation to influence hearing outcomes is not clear. This is an observational, pilot study where we will be collecting a bank of biological samples as well as building a clinical database of up to 75 paediatric cochlear implant recipients. The study may not be sufficiently powered to carry out regression analysis to examine the relationship between hearing outcomes and the multiple factors that are likely to influence this. However, this study will allow us to combine the detailed understanding of the immune biology in the ear at the time of implantation (for the first time) with a rich clinical dataset containing information about the various known factors that can influence performance following implantation. This will enable us to better understand which factors are interacting with immune status to help us design clinical studies. The protocol does not contain information on how the range of known factors would be considered or controlled for when examining the role of immune status at cochlear implantation in influencing hearing outcomes. Biographic factors – we will have access to medical history so these factors will be considered when examining role of immune status influencing hearing outcomes. These factors will not be controlled for as the inclusion criteria is wide. [This is an observational study not an intervention study]. Etiologic factors – we will assess medical history and consider these factors. We will not know how well reported these factors will be until we have recruited participants and looked at these data. This is important and relevant for otitis media/middle ear inflammation and other immune related etiologies. Device related factors – this is a single site study so all participants will have the same pre-operative protocol. Surgical factors – this is a single site study so all participants will have the same surgical approach, and the surgeries will be done by one of three surgeons. Hearing preservation soft surgery approach is utilised at this site. The specific variables depicted by the term ‘hearing outcomes’ are not explicit, and the method to address the question on relationship is not clear (Aim 2). This is a data collecting study. The research questions and the type of analysis that will be possible will depend on the cohort and the data collected. This is because the inclusion criteria are wide, we are recruiting participants across a wide age range, meaning different audiological, speech and language tests will be used depending on the age of the participants. It appears that there are no patient and public involvement and engagement group that includes parents/ guardians and young people with lived experience at the locality where recruitment and clinical management will occur. Especially when the research study involves children and young people, engagement with parents would be crucial. The current protocol does not contain a plan to disseminate findings to stakeholders. An important part of engagement is to disseminate findings. Maybe this aspect has already been covered in other related documents. We have a thriving PPIE group that includes parents of children who are deaf or hard of hearing. We have discussed the study throughout the design stages and had feedback throughout and we will continue to keep an open dialogue with our PPIE group members. We also have links with key PPIE professionals in Manchester, where we are recruiting participants from. This will enable us to link our PPIE activities with those happening in Manchester and disseminate information about the study in Manchester as well as Hampshire. We were aware that we were missing the voice of young people in our group so in March 2025 we launched ALL_EARS@UoS Young People’s Advisory Group (YPAG) for young people aged 14 – 16 years ago (https://generic.wordpress.soton.ac.uk/all-ears/2025/03/07/join-our-new-young-peoples-advisory-group-ypag/). Young people are being recruited nationally to join this group. This group will support the CHIEF project throughout. The following text has been added to the PPIE section in the manuscript: 'In March 2025, we launched ALL_EARS@UoS Young People’s Advisory Group (YGAG) which is PPIE group for young people aged between 14 – 16 years old (https://generic.wordpress.soton.ac.uk/all-ears/2025/03/07/join-our-new-young-peoples-advisory-group-ypag/). Members of ALL_EARS@UoS YPAG will support this study alongside our wider research'. In the Ethics and dissemination section, we have outlined that we plan to disseminate findings through international peer-reviewed journals and conferences. In addition, we will present findings to a lay audience through our website. The following text has been added in to this section: 'In addition to this, we will share findings and progress of the study through the various public engagement activities that we take part in each year alongside ALL_EARS@UoS members'. The protocol suggests that a potential benefit of the study is that identification of patients at risk of increased inflammatory response to cochlear implantation (if a relation were found between immune status at implantation and ‘hearing outcomes’) could pave the way for intervention prior to or at surgery with anti-inflammatory /antimicrobial therapies and ensure closer monitoring. There is some evidence that use of drugs/steroids contribute to preservation of residual hearing after implantation, and soft surgery techniques have been widely implemented to achieve the goal of preservation of residual hearing. Are these not in use in standard protocols for cochlear implantation at the test site? The following text has been added to a new sub-section in the Methods section titled ‘Standard protocol for cochlear implantation at Manchester University Hospital’ 'Surgeons routinely use soft surgery technique for all patients, even for those who do not have residual hearing. This is so that they preserve neural tissue in all patients. All children get 1 dose of peroperative co-amoxiclav 30mg/kg up to a maximum of 1.2g or an alternative if penicillin allergic. In addition, the patients will also get a peroperative dose of steroids – 0.15mg/kg, up to a maximum of 6.6mg. Post-operatively, all children are discharged on 3 doses of oral dexamethasone, 0.15mg.kg and 3 doses of antibiotic. If there are signs of acute inflammation during surgery in the form of inflamed mucosa and sero-mucinous otitis media, the surgeons will give 1 week’s treatment with oral antibiotics post-operatively'. Overall, the study will make a significant contribution to knowledge about inflammatory responses in pediatric recipients of cochlear implants and create a novel data set. However, the protocol will benefit from a clarification of how other factors affecting hearing outcomes in pediatric recipients will be considered or controlled for when examining the role of immune status in influencing outcomes; and an explanation of how the relationship between immune status and hearing outcomes will be explored. The end. COMMENTS ON THIS REPORT - Author Response 28 May 2025Kate Hough, Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, Southampton, SO17 1BJ, UK28 May 2025Author ResponseThank you for taking the time to review the study protocol and providing your comments. I have included your comments in bold and my responses below each point. Individual ... Continue reading Thank you for taking the time to review the study protocol and providing your comments. I have included your comments in bold and my responses below each point. Individual variability in performance of cochlear implant recipients is a key question in aural (re)habilitation. This study posits that variability in performance may be related to individual variations in inflammatory response to cochlear implantation. Accordingly, the study aims to 1) identify differences in ear inflammation between individual pediatric recipients of cochlear implants, and 2) explore the relationship between the immune state of the ear at the time of implantation and hearing outcomes up to 5 years post-implantation. The background section does not include current knowledge about effects of middle ear inflammation on performance with cochlear implants in adult and pediatric recipients. How this may differ between adult and pediatric patients have not been described. There is limited current knowledge about the effects of middle ear inflammation on performance with cochlear implants in both adult and paediatric recipients. Hence why this is an important area to investigate. Literature exploring the effect of middle ear inflammation on surgical outcomes (Rak et al., 2018; Alzoubi et al., 2015) has shown a history of otitis media (OM) or active OM can increase the risk of complications and increase the time needed for surgery. However, in most cases – surgical outcomes are fine. Studies in adult cochlear implant patients with a history of chronic otitis media, which have investigated surgical outcomes and rate of complications, have included investigation of performance such as long-term speech comprehension (Rak et al., 2018) and auditory performance (Goh et al., 2020). Improvements in performance were measured compared to pre-CI levels, but no comparisons were made between adults with and without a history of chronic otitis media. We have no found studies that have investigated hearing outcomes/performance over time in a cohort of paediatric cochlear implant patients with and without a history of middle ear inflammation. We are in the process of applying for ethical approval to carry out a retrospective study of this in patients at a single implant centre in the UK. In addition, the data collected through the CHIEF study will enable us to explore this with the study cohort. The text below has been added the introduction: 'There is an urgent need to investigate the effect of the immune state of the inner ear on hearing outcomes following cochlear implantation. To achieve this, we need to study the inflammatory state of the ear in children and young people undergoing implantation. Importantly, we need to see how this varies between children and young people, and how it is associated with hearing outcomes in people with a history of middle ear inflammation (acute otitis media and otitis media with effusion)(15,16). Hearing outcomes is the combination of audiological tests, speech perception tests, language development tests, datalogging and parental assessments that allow cochlear implant professionals to measure and determine how much benefit someone is getting from their cochlear implant. Currently, there are no studies that have investigated hearing outcomes over time in a cohort of pediatric cochlear implant patients with and without a history of middle ear inflammation. The CHIEF study will allow us to investigate this'. The protocol provides a detailed description of the collection and analysis of biologic samples to be collected at the time of (re)implantation to address Aim 1. Research questions and objectives for this part are listed, and methods to address the questions have been clearly spelt out. However, there is very little information on analytical methods to achieve Aim 2. The following text has been added to the section with the sub-heading ‘Secondary outcome’ to clarify this point. 'The characterisation of the immune state of each study participant will inform the analytical plan to achieve this secondary outcome. Due to anticipated variability in immune state it may be that this is the pilot study to informs a larger powered study. This step is necessary as there is no prior equivalent data. Likewise the analysis of the hearing outcome will depend on the age of participants and the battery of hearing, speech and language assessments over time'. Indeed, the definition of ‘hearing outcomes’ in the protocol appears elusive. To clarify what we mean by hearing outcomes, this text has been added to the second to last paragraph of the introduction: 'Hearing outcomes is the combination of audiological tests, speech perception tests, language development tests, datalogging and parental assessments that allow cochlear implant professionals to measure and determine how much benefit someone is getting from their cochlear implant'. To further clarify what we mean by hearing outcomes, the following text has been added to the ‘clinical data collection’ section: 'To measure hearing outcomes, a battery of age-appropriate audiological, speech perception and language development tests are used, in addition to datalogging measurements. To explore the relationship between the immune state of the middle ear tissue and long-term hearing outcomes, we will request access to anonymized, routine clinical outcome measures for up to five years post-implantation of study participants. This will include audiological and device data; aided levels from speech and language tests (ASSE and AB words); datalogging (device usage e.g. average daily use) and electrode impedances and deactivations (number of electrodes and position). Together with a log of any recorded post-implantation complications. All data collected post-implantation will be from routinely collected clinical data. The device measurements will be collected when a patient comes in for an appointment or via remote appointments. The time points of these measurements from the day of implantation may vary but will loosely follow the appointment structure which is: switch on 7 – 14 days post-implantation, 1 month, 3 months, 6 months, 2 years, 4 years, 6 years (new processor). We will access data from medical records including date of birth, ethnicity, history and cause/etiology of deafness, history of ear disease (infections, surgery), and history of other infections (meningitis, cytomegalovirus, measles). Descriptive statistics will be used to describe hearing performance and device measurements'. The protocol indicated that descriptive statistics will be used to analyze hearing performance and device measurements (pg.8). Descriptive statistics serve to describe, but not to explore relationships. It is not clear what specific ‘device measurements’ will be collected, at what time-points, The following device measurements will be collected: Electrode impedance, position of electrode deactivation and number of electrode deactivation. The following text has been added to the manuscript in the Clinical data collection section to clarify the specific device measurement that we will be collecting and at what time-points: '…device measurements including electrode impedances and deactivations (number of electrodes and position). All data collected post-implantation will be from routinely collected clinical data. The device measurements will be collected when a patient comes in for an appointment or via remote appointments. The time points of these measurements from the day of implantation may vary but will loosely follow the appointment structure which is: switch on 7 – 14 days post-implantation, 1 month, 3 months, 6 months, 2 years, 4 years, 6 years (new processor)'. and how the data will be analyzed in relation to the other biologic-sample-based data to address Aim 2 regarding the relationship between immune status and outcomes. As well as using the spatial transcriptomic data to get a detailed understanding of what genes the cells in the middle ear mucosal tissue are expressing and how they are signalling to each other, the biologic sample-based data will be used to split the participants into two groups: inflamed and non-inflamed. The individual hearing outcome measures and device measurements will be analysed to compare the inflamed and non-inflamed group. For example – what are the differences in Aided levels at each frequency over time post-implantation between inflamed and non-inflamed group? What is the impedance over time for the inflamed and non-inflamed group? Furthermore, current evidence suggests that a range of biographic, etiologic, device-related and surgical factors influences individual performance with cochlear implants. The way in which this study will examine the extent to which these factors interact with the immune status of the ear at implantation to influence hearing outcomes is not clear. This is an observational, pilot study where we will be collecting a bank of biological samples as well as building a clinical database of up to 75 paediatric cochlear implant recipients. The study may not be sufficiently powered to carry out regression analysis to examine the relationship between hearing outcomes and the multiple factors that are likely to influence this. However, this study will allow us to combine the detailed understanding of the immune biology in the ear at the time of implantation (for the first time) with a rich clinical dataset containing information about the various known factors that can influence performance following implantation. This will enable us to better understand which factors are interacting with immune status to help us design clinical studies. The protocol does not contain information on how the range of known factors would be considered or controlled for when examining the role of immune status at cochlear implantation in influencing hearing outcomes. Biographic factors – we will have access to medical history so these factors will be considered when examining role of immune status influencing hearing outcomes. These factors will not be controlled for as the inclusion criteria is wide. [This is an observational study not an intervention study]. Etiologic factors – we will assess medical history and consider these factors. We will not know how well reported these factors will be until we have recruited participants and looked at these data. This is important and relevant for otitis media/middle ear inflammation and other immune related etiologies. Device related factors – this is a single site study so all participants will have the same pre-operative protocol. Surgical factors – this is a single site study so all participants will have the same surgical approach, and the surgeries will be done by one of three surgeons. Hearing preservation soft surgery approach is utilised at this site. The specific variables depicted by the term ‘hearing outcomes’ are not explicit, and the method to address the question on relationship is not clear (Aim 2). This is a data collecting study. The research questions and the type of analysis that will be possible will depend on the cohort and the data collected. This is because the inclusion criteria are wide, we are recruiting participants across a wide age range, meaning different audiological, speech and language tests will be used depending on the age of the participants. It appears that there are no patient and public involvement and engagement group that includes parents/ guardians and young people with lived experience at the locality where recruitment and clinical management will occur. Especially when the research study involves children and young people, engagement with parents would be crucial. The current protocol does not contain a plan to disseminate findings to stakeholders. An important part of engagement is to disseminate findings. Maybe this aspect has already been covered in other related documents. We have a thriving PPIE group that includes parents of children who are deaf or hard of hearing. We have discussed the study throughout the design stages and had feedback throughout and we will continue to keep an open dialogue with our PPIE group members. We also have links with key PPIE professionals in Manchester, where we are recruiting participants from. This will enable us to link our PPIE activities with those happening in Manchester and disseminate information about the study in Manchester as well as Hampshire. We were aware that we were missing the voice of young people in our group so in March 2025 we launched ALL_EARS@UoS Young People’s Advisory Group (YPAG) for young people aged 14 – 16 years ago (https://generic.wordpress.soton.ac.uk/all-ears/2025/03/07/join-our-new-young-peoples-advisory-group-ypag/). Young people are being recruited nationally to join this group. This group will support the CHIEF project throughout. The following text has been added to the PPIE section in the manuscript: 'In March 2025, we launched ALL_EARS@UoS Young People’s Advisory Group (YGAG) which is PPIE group for young people aged between 14 – 16 years old (https://generic.wordpress.soton.ac.uk/all-ears/2025/03/07/join-our-new-young-peoples-advisory-group-ypag/). Members of ALL_EARS@UoS YPAG will support this study alongside our wider research'. In the Ethics and dissemination section, we have outlined that we plan to disseminate findings through international peer-reviewed journals and conferences. In addition, we will present findings to a lay audience through our website. The following text has been added in to this section: 'In addition to this, we will share findings and progress of the study through the various public engagement activities that we take part in each year alongside ALL_EARS@UoS members'. The protocol suggests that a potential benefit of the study is that identification of patients at risk of increased inflammatory response to cochlear implantation (if a relation were found between immune status at implantation and ‘hearing outcomes’) could pave the way for intervention prior to or at surgery with anti-inflammatory /antimicrobial therapies and ensure closer monitoring. There is some evidence that use of drugs/steroids contribute to preservation of residual hearing after implantation, and soft surgery techniques have been widely implemented to achieve the goal of preservation of residual hearing. Are these not in use in standard protocols for cochlear implantation at the test site? The following text has been added to a new sub-section in the Methods section titled ‘Standard protocol for cochlear implantation at Manchester University Hospital’ 'Surgeons routinely use soft surgery technique for all patients, even for those who do not have residual hearing. This is so that they preserve neural tissue in all patients. All children get 1 dose of peroperative co-amoxiclav 30mg/kg up to a maximum of 1.2g or an alternative if penicillin allergic. In addition, the patients will also get a peroperative dose of steroids – 0.15mg/kg, up to a maximum of 6.6mg. Post-operatively, all children are discharged on 3 doses of oral dexamethasone, 0.15mg.kg and 3 doses of antibiotic. If there are signs of acute inflammation during surgery in the form of inflamed mucosa and sero-mucinous otitis media, the surgeons will give 1 week’s treatment with oral antibiotics post-operatively'. Overall, the study will make a significant contribution to knowledge about inflammatory responses in pediatric recipients of cochlear implants and create a novel data set. However, the protocol will benefit from a clarification of how other factors affecting hearing outcomes in pediatric recipients will be considered or controlled for when examining the role of immune status in influencing outcomes; and an explanation of how the relationship between immune status and hearing outcomes will be explored. The end.Thank you for taking the time to review the study protocol and providing your comments. I have included your comments in bold and my responses below each point.Competing Interests: No competing interests were disclosed. Close Individual variability in performance of cochlear implant recipients is a key question in aural (re)habilitation. This study posits that variability in performance may be related to individual variations in inflammatory response to cochlear implantation. Accordingly, the study aims to 1) identify differences in ear inflammation between individual pediatric recipients of cochlear implants, and 2) explore the relationship between the immune state of the ear at the time of implantation and hearing outcomes up to 5 years post-implantation. The background section does not include current knowledge about effects of middle ear inflammation on performance with cochlear implants in adult and pediatric recipients. How this may differ between adult and pediatric patients have not been described. There is limited current knowledge about the effects of middle ear inflammation on performance with cochlear implants in both adult and paediatric recipients. Hence why this is an important area to investigate. Literature exploring the effect of middle ear inflammation on surgical outcomes (Rak et al., 2018; Alzoubi et al., 2015) has shown a history of otitis media (OM) or active OM can increase the risk of complications and increase the time needed for surgery. However, in most cases – surgical outcomes are fine. Studies in adult cochlear implant patients with a history of chronic otitis media, which have investigated surgical outcomes and rate of complications, have included investigation of performance such as long-term speech comprehension (Rak et al., 2018) and auditory performance (Goh et al., 2020). Improvements in performance were measured compared to pre-CI levels, but no comparisons were made between adults with and without a history of chronic otitis media. We have no found studies that have investigated hearing outcomes/performance over time in a cohort of paediatric cochlear implant patients with and without a history of middle ear inflammation. We are in the process of applying for ethical approval to carry out a retrospective study of this in patients at a single implant centre in the UK. In addition, the data collected through the CHIEF study will enable us to explore this with the study cohort. The text below has been added the introduction: 'There is an urgent need to investigate the effect of the immune state of the inner ear on hearing outcomes following cochlear implantation. To achieve this, we need to study the inflammatory state of the ear in children and young people undergoing implantation. Importantly, we need to see how this varies between children and young people, and how it is associated with hearing outcomes in people with a history of middle ear inflammation (acute otitis media and otitis media with effusion)(15,16). Hearing outcomes is the combination of audiological tests, speech perception tests, language development tests, datalogging and parental assessments that allow cochlear implant professionals to measure and determine how much benefit someone is getting from their cochlear implant. Currently, there are no studies that have investigated hearing outcomes over time in a cohort of pediatric cochlear implant patients with and without a history of middle ear inflammation. The CHIEF study will allow us to investigate this'. The protocol provides a detailed description of the collection and analysis of biologic samples to be collected at the time of (re)implantation to address Aim 1. Research questions and objectives for this part are listed, and methods to address the questions have been clearly spelt out. However, there is very little information on analytical methods to achieve Aim 2. The following text has been added to the section with the sub-heading ‘Secondary outcome’ to clarify this point. 'The characterisation of the immune state of each study participant will inform the analytical plan to achieve this secondary outcome. Due to anticipated variability in immune state it may be that this is the pilot study to informs a larger powered study. This step is necessary as there is no prior equivalent data. Likewise the analysis of the hearing outcome will depend on the age of participants and the battery of hearing, speech and language assessments over time'. Indeed, the definition of ‘hearing outcomes’ in the protocol appears elusive. To clarify what we mean by hearing outcomes, this text has been added to the second to last paragraph of the introduction: 'Hearing outcomes is the combination of audiological tests, speech perception tests, language development tests, datalogging and parental assessments that allow cochlear implant professionals to measure and determine how much benefit someone is getting from their cochlear implant'. To further clarify what we mean by hearing outcomes, the following text has been added to the ‘clinical data collection’ section: 'To measure hearing outcomes, a battery of age-appropriate audiological, speech perception and language development tests are used, in addition to datalogging measurements. To explore the relationship between the immune state of the middle ear tissue and long-term hearing outcomes, we will request access to anonymized, routine clinical outcome measures for up to five years post-implantation of study participants. This will include audiological and device data; aided levels from speech and language tests (ASSE and AB words); datalogging (device usage e.g. average daily use) and electrode impedances and deactivations (number of electrodes and position). Together with a log of any recorded post-implantation complications. All data collected post-implantation will be from routinely collected clinical data. The device measurements will be collected when a patient comes in for an appointment or via remote appointments. The time points of these measurements from the day of implantation may vary but will loosely follow the appointment structure which is: switch on 7 – 14 days post-implantation, 1 month, 3 months, 6 months, 2 years, 4 years, 6 years (new processor). We will access data from medical records including date of birth, ethnicity, history and cause/etiology of deafness, history of ear disease (infections, surgery), and history of other infections (meningitis, cytomegalovirus, measles). Descriptive statistics will be used to describe hearing performance and device measurements'. The protocol indicated that descriptive statistics will be used to analyze hearing performance and device measurements (pg.8). Descriptive statistics serve to describe, but not to explore relationships. It is not clear what specific ‘device measurements’ will be collected, at what time-points, The following device measurements will be collected: Electrode impedance, position of electrode deactivation and number of electrode deactivation. The following text has been added to the manuscript in the Clinical data collection section to clarify the specific device measurement that we will be collecting and at what time-points: '…device measurements including electrode impedances and deactivations (number of electrodes and position). All data collected post-implantation will be from routinely collected clinical data. The device measurements will be collected when a patient comes in for an appointment or via remote appointments. The time points of these measurements from the day of implantation may vary but will loosely follow the appointment structure which is: switch on 7 – 14 days post-implantation, 1 month, 3 months, 6 months, 2 years, 4 years, 6 years (new processor)'. and how the data will be analyzed in relation to the other biologic-sample-based data to address Aim 2 regarding the relationship between immune status and outcomes. As well as using the spatial transcriptomic data to get a detailed understanding of what genes the cells in the middle ear mucosal tissue are expressing and how they are signalling to each other, the biologic sample-based data will be used to split the participants into two groups: inflamed and non-inflamed. The individual hearing outcome measures and device measurements will be analysed to compare the inflamed and non-inflamed group. For example – what are the differences in Aided levels at each frequency over time post-implantation between inflamed and non-inflamed group? What is the impedance over time for the inflamed and non-inflamed group? Furthermore, current evidence suggests that a range of biographic, etiologic, device-related and surgical factors influences individual performance with cochlear implants. The way in which this study will examine the extent to which these factors interact with the immune status of the ear at implantation to influence hearing outcomes is not clear. This is an observational, pilot study where we will be collecting a bank of biological samples as well as building a clinical database of up to 75 paediatric cochlear implant recipients. The study may not be sufficiently powered to carry out regression analysis to examine the relationship between hearing outcomes and the multiple factors that are likely to influence this. However, this study will allow us to combine the detailed understanding of the immune biology in the ear at the time of implantation (for the first time) with a rich clinical dataset containing information about the various known factors that can influence performance following implantation. This will enable us to better understand which factors are interacting with immune status to help us design clinical studies. The protocol does not contain information on how the range of known factors would be considered or controlled for when examining the role of immune status at cochlear implantation in influencing hearing outcomes. Biographic factors – we will have access to medical history so these factors will be considered when examining role of immune status influencing hearing outcomes. These factors will not be controlled for as the inclusion criteria is wide. [This is an observational study not an intervention study]. Etiologic factors – we will assess medical history and consider these factors. We will not know how well reported these factors will be until we have recruited participants and looked at these data. This is important and relevant for otitis media/middle ear inflammation and other immune related etiologies. Device related factors – this is a single site study so all participants will have the same pre-operative protocol. Surgical factors – this is a single site study so all participants will have the same surgical approach, and the surgeries will be done by one of three surgeons. Hearing preservation soft surgery approach is utilised at this site. The specific variables depicted by the term ‘hearing outcomes’ are not explicit, and the method to address the question on relationship is not clear (Aim 2). This is a data collecting study. The research questions and the type of analysis that will be possible will depend on the cohort and the data collected. This is because the inclusion criteria are wide, we are recruiting participants across a wide age range, meaning different audiological, speech and language tests will be used depending on the age of the participants. It appears that there are no patient and public involvement and engagement group that includes parents/ guardians and young people with lived experience at the locality where recruitment and clinical management will occur. Especially when the research study involves children and young people, engagement with parents would be crucial. The current protocol does not contain a plan to disseminate findings to stakeholders. An important part of engagement is to disseminate findings. Maybe this aspect has already been covered in other related documents. We have a thriving PPIE group that includes parents of children who are deaf or hard of hearing. We have discussed the study throughout the design stages and had feedback throughout and we will continue to keep an open dialogue with our PPIE group members. We also have links with key PPIE professionals in Manchester, where we are recruiting participants from. This will enable us to link our PPIE activities with those happening in Manchester and disseminate information about the study in Manchester as well as Hampshire. We were aware that we were missing the voice of young people in our group so in March 2025 we launched ALL_EARS@UoS Young People’s Advisory Group (YPAG) for young people aged 14 – 16 years ago (https://generic.wordpress.soton.ac.uk/all-ears/2025/03/07/join-our-new-young-peoples-advisory-group-ypag/). Young people are being recruited nationally to join this group. This group will support the CHIEF project throughout. The following text has been added to the PPIE section in the manuscript: 'In March 2025, we launched ALL_EARS@UoS Young People’s Advisory Group (YGAG) which is PPIE group for young people aged between 14 – 16 years old (https://generic.wordpress.soton.ac.uk/all-ears/2025/03/07/join-our-new-young-peoples-advisory-group-ypag/). Members of ALL_EARS@UoS YPAG will support this study alongside our wider research'. In the Ethics and dissemination section, we have outlined that we plan to disseminate findings through international peer-reviewed journals and conferences. In addition, we will present findings to a lay audience through our website. The following text has been added in to this section: 'In addition to this, we will share findings and progress of the study through the various public engagement activities that we take part in each year alongside ALL_EARS@UoS members'. The protocol suggests that a potential benefit of the study is that identification of patients at risk of increased inflammatory response to cochlear implantation (if a relation were found between immune status at implantation and ‘hearing outcomes’) could pave the way for intervention prior to or at surgery with anti-inflammatory /antimicrobial therapies and ensure closer monitoring. There is some evidence that use of drugs/steroids contribute to preservation of residual hearing after implantation, and soft surgery techniques have been widely implemented to achieve the goal of preservation of residual hearing. Are these not in use in standard protocols for cochlear implantation at the test site? The following text has been added to a new sub-section in the Methods section titled ‘Standard protocol for cochlear implantation at Manchester University Hospital’ 'Surgeons routinely use soft surgery technique for all patients, even for those who do not have residual hearing. This is so that they preserve neural tissue in all patients. All children get 1 dose of peroperative co-amoxiclav 30mg/kg up to a maximum of 1.2g or an alternative if penicillin allergic. In addition, the patients will also get a peroperative dose of steroids – 0.15mg/kg, up to a maximum of 6.6mg. Post-operatively, all children are discharged on 3 doses of oral dexamethasone, 0.15mg.kg and 3 doses of antibiotic. If there are signs of acute inflammation during surgery in the form of inflamed mucosa and sero-mucinous otitis media, the surgeons will give 1 week’s treatment with oral antibiotics post-operatively'. Overall, the study will make a significant contribution to knowledge about inflammatory responses in pediatric recipients of cochlear implants and create a novel data set. However, the protocol will benefit from a clarification of how other factors affecting hearing outcomes in pediatric recipients will be considered or controlled for when examining the role of immune status in influencing outcomes; and an explanation of how the relationship between immune status and hearing outcomes will be explored. The end. Alongside their report, reviewers assign a status to the article: - Approved - Approved with reservations - Not approved | Invited Reviewers | || |---|---|---| | 1 | 2 | | | Version 2 (revision) 15 May 25 | || | Version 1 04 Mar 25 | read | read | Sign up for content alerts You are now signed up to receive this alert Alongside their report, reviewers assign a status to the article: Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions Provide sufficient details of any financial or non-financial competing interests to enable users to assess whether your comments might lead a reasonable person to question your impartiality. 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