(2024) 24:521
Liu et al. BMC Pediatrics
https://doi.org/10.1186/s12887-024-04999-2

BMC Pediatrics

RESEARCH

Open Access

The therapeutic use of exosomes in children
with adenoid hypertrophy accompanied
by otitis media with effusion (AHOME):
a protocol study
Yixuan Liu1†, Xiaoling Lu1†, Shan Sun1*, Huiqian Yu1* and Huawei Li1,2,3*

Abstract
Background The adenoids act as a reservoir of bacterial pathogens and immune molecules, and they are significantly involved in children with otitis media with effusion (OME). As an essential carrier of intercellular substancetransfer and signal transduction, exosomes with different biological functions can be secreted by various typesof cells. There remains significant uncertainty regarding the clinical relevance of exosomes to OME, especially in its
pathophysiologic development. In this study, we will seek to determine the biological functions of exosomes in children with adenoid hypertrophy accompanied by OME (AHOME).
Methods The diagnostic criteria for OME in children aged 4–10 years include a disease duration of at least 3 months,
type B or C acoustic immittance, and varying degrees of conductive hearing loss. Adenoidal hypertrophy is diagnosed
when nasal endoscopy shows at least 60% adenoidal occlusion in the nostrils or when nasopharyngeal lateral X-ray
shows A/N > 0.6. Children who meet the indications for adenoidectomy surgery undergo adenoidectomy. Peripheral
blood, nasopharyngeal swab, and adenoid tissue will be collected from patients, and the exosomes will be isolated from the samples. Following the initial collection, patients will undergo adenoidectomy and peripheral bloodand nasopharyngeal swabs will be collected again after 3 months.
Expected results This study aims to identify differences in exosomes from preoperative adenoid tissue and peripheral blood samples between children with AHOME and those with adenoid hypertrophy alone. Additionally, it seeks
to determine changes in microbial diversity in adenoid tissue between these groups.
Conclusions The findings are expected to provide new insights into the diagnosis and treatment of OME, to identify novel biomarkers, and to enhance our understanding of the pathophysiology of OME, potentially leading
to the development of innovative diagnostic and therapeutic approaches.
Keywords Exosome, Otitis media with effusion, Adenoid hypertrophy†

Yixuan Liu and Xiaoling Lu contributed equally to this work.

*Correspondence:
Shan Sun
Shansun@fudan.edu.cn
Huiqian Yu
yhq925@163.com
Huawei Li
huaweili2000@163.com
Full list of author information is available at the end of the article© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0
International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as longas you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if
you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article orparts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicatedotherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To
view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

Liu et al. BMC Pediatrics(2024) 24:521

BackgroundOtitis media, also known as middle ear inflammation,
is a spectrum of diseases, including acute otitis media,
chronic suppurative otitis media, and otitis media with
effusion (OME). OME was first described by Politzerzai in 1867 as a non-suppurative inflammatory diseasein the middle ear characterized by hearing loss caused
by loss of transduction of sound waves in the middle ear
due to the presence of middle ear effusion [1]. OME isvery common and is one of the main causes of hearing
loss in children, and about 80% of children suffer one ormore episodes of OME by 10 years of age [2]. However,
because of its inconspicuous symptoms, the incidence
and prevalence are difficult to establish accurately, which
is a significant obstacle to the prevention and treatment
of OME. Treatment for OME includes enhancement of
middle ear ventilation and drainage, removal of middle
ear fluid, infection management, and treatment of related
conditions. For children with OME without high-risk factors, there should be a 3-month watchful waiting period
after diagnosis [3]. Routine systemic use of antibiotics
and hormones is not recommended, and there is a lack oflong-term evidence for the efficacy of nasal corticosteroids and antihistamines. Current therapies, whether surgical or non-surgical, can temporarily alleviate ear cavity
aeration, but the disease’s natural course, long-term
prognosis, and relapse rate remain unclear. For tympanostomy tubes [4], most of the important considerations
are the impact of OME on children’s social interactions
and learning ability. There are no uniform recommendations for the indication of adenoidectomy in nationalguidelines, and the age of the child and whether there is arecurrence are important factors to consider.
OME has a complicated etiology that can be causedby several factors [5, 6]. Dysfunction of the Eustachian
tube, which balances the air pressure within and outside
the middle ear and provides a cleansing function to avoid
retrograde infection, is typically thought to play a majorrole in the etiology of OME [7], and such dysfunction canbe caused by adenoid hypertrophy, chronic sinusitis, or adeviated nasal septum. OME can also be caused by bacterial infection [8, 9], and contemporary detection techniques, such as polymerase chain reaction (PCR), havebeen used to demonstrate the existence of bacterial DNA
in patients’ middle ear secretions.
The microbiome plays a crucial role in airway inflammation and homeostasis. The different microenvironments in the upper airway – including the anterior
nostrils, the nasal cavities, the sinuses, the nasopharynx,
the Eustachian tubes, the middle ear cavities, the oralcavity, the oropharynx, and the larynx – host specificmicrobial communities [10]. Bacterial biofilms have beenfound in the middle ear secretions using fluorescence

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confocal microscopy [11], suggesting that the formation of bacterial biofilms may also be associated with thepathogenesis of OME [12]. Inflammatory reactions also
play an indispensable role in the pathogenesis of OME[13], and it has been suggested that OME is related to
the nasopharyngeal inflammatory and immunologicalresponse. Exudates rich in proteins and inflammatory
mediators are secreted by the inflammatory response,
which generates cytokines. The dilation of blood vessels
in response to cytokines improves gaseous exchange inthe middle ear and lowers endotympanic pressure [14].
The middle ear cavity remains relatively stable, except
for the tympanic membrane, making the pars tensa themost vulnerable area due to its lack of a fibrous layer, andthe pars tensa is frequently the primary site of retraction. If the drop in endotympanic pressure persists, thetympanic atelectasis will lead to injury of the pars tensa,
eventually leading to complete atelectasis of the tympanic
membrane. Long-term middle ear inflammation causes
cellular differentiation and an increase in the numberof mucous cells, which leads to a gradual increase in
exudate that fills the middle ear cavity. The decrease in
upstream pressure in the middle ear due to mucus adhering to the Eustachian tube makes it more difficult to drain
the mucus, which aggravates OME even further. In addition, biofilm production on mucosal membranes has
been observed in OME [15], in which biofilms are created by bacteria creating an "anchor-like" microcolonythat colonizes an adherent matrix containing polysaccharides, nucleic acids, and proteins [16]. Therefore, biofilms
on the surface of adenoid tissue are an excellent candidate for exploring the pathogenesis of OME [11, 12].
The detection of exosomes in these tissues to explore
the pathophysiology of OME has already been performed, with peripheral blood primarily used in the studyof OME and adenoid tissue primarily used in the study of
adenoid hypertrophy. Exosomes were first described in
the late 1960s by Bonucci and Anderson [17], and exosome-related research accelerated greatly in the 1980s[17]. Exosomes are the smallest extracellular vesicles(EVs), with a diameter of 30~200 nm, and they containmultiple cargo types, including protein, DNA, mRNA,and miRNA, and they have an endosomal origin [17, 18]
and can be found in different bodily fluids [19]. Receptor cells take up exosomes through endocytosis to transfer their cargo. The critical functions of exosomes arecell-cell communication, immune modulation, extracellular matrix turnover, stem cell differentiation, neovascularization, and cellular waste removal [20]. Proteins andmiRNA contained in exosomes can regulate cell physiology and can modify the microenvironment around target
cells, indicating that exosomes have potential as diagnostic and therapeutic tools [21].

Liu et al. BMC Pediatrics(2024) 24:521

Exosomes have been found to have pathological roles
in a variety of diseases, including heart disease [22], neurodegenerative diseases [23, 24], cancer [25, 26], andinflammation [27–29], and many studies have shown that
exosomes may be crucial in regulating inflammationbased cells. For example, repeated injection of exosomes
isolated from the peripheral blood of mice fed a high-fat
diet into mice on a regular diet causes the accumulation of activated immature ­CD11b+Ly6ChiLy6G– bonemarrow cells in the liver, thus promoting obesity-relateddiseases such as fatty liver on top of causing chronic
inflammation [27]. Exosome numbers are substantiallygreater in lipopolysaccharide-treated cells compared
to control cells, as seen in electron microscope images[30]. Exosomes can also be used as inflammatory bowel
disease (IBD) biomarkers, and their source can be hosting cells, food, or microorganisms [31]. It has beenfound that samples from cases with malignant common
bile duct stenosis contain significantly higher levels of
EVs than bile samples from healthy controls [29]. BothANXA1 and PSMA7, which are present in exosomes
released by intestinal epithelial cells and oral mucosal
cells, are expressed at significantly different levels in IBD
patients and healthy controls [32, 33], suggesting thatthey might be promising biomarker candidates for early
detection of IBD. In the field of otolaryngology, EVs play
an important role, for example, in head and neck cancer
metastasis [34], lymphocyte regulation [35], angiogenesis [36], microenvironment remodeling [37, 38], anddrug resistance [39, 40]. Related studies have shown thatdisease-associated bacterial products drive the differential expression of miRNAs in human middle ear epithelial
cells in the OME model [41]. Elevated levels of cysteine
protease inhibitors-1 and -2 in EVs [42] in patients withchronic rhinosinusitis (CRS) may serve as markers of
CRS and predict disease phenotype, while EVs may also
lead to polyp formation by upregulating pappalysin andserpins [43]. A previous study found that keratinocytederived exosomal miR-17 led to the upregulation offibroblast protein expression, promoted osteoclast differentiation, and led to bone destruction, which are hallmarks of acquired cholesteatoma [44]. Inner ear cells
release Hsp70-carrying exosomes in response to heatstress, and these interact with TLR4 on hair cells to provide a protective effect [45]. Although limited researchhas been performed to date, studies have shown animportant role for EVs in various otolaryngological conditions [46]. Continued research into the characterizationof EVs in ENT diseases will help develop new diagnostic
biomarkers and therapeutic targets and will provide clinicians in the field of ENT with a new set of tools.
In this study, we are collecting exosomes from peripheral blood and adenoid tissue following adenoidectomy

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and have been evaluating the microbial modifications in
the biofilm on the adenoid surface to better understandthe pathophysiology of OME. Thus, this study will highlight topics for future research and give scientific insight
into the development of drugs for treating OME.

Methods
Study design

The study design for the current prospective trial isdescribed in Fig. 1.
Patient and public involvement
Group

The children will be divided into two groups: the adenoid
hypertrophy accompanied by OME (adenoid hypertrophy accompanied by OME, AHOME) group and the
adenoid hypertrophy alone (adenoid hypertrophy, AH)
group.
The biofilm-positive rate in patients with adenoid
hypertrophy was shown to be 35.5% in the study by
Szalmás et al. [47]. The odds ratio of the AHOME groupcompared to the AH group is projected to be 1.25, and achi-square test was used to estimate the positive rate of
biofilm in the AH group with a statistical power of 80%
and an alpha level of 5%, resulting in sample sizes of 122
for the AHOME group and the AH group.
A total of 244 subjects (n = 122 in the AHOME group
and n = 122 in the AH group) will be recruited from the
Eye & ENT Hospital of Fudan University and from thelocal community. After obtaining informed consent,
nasopharyngeal swabs, peripheral blood, and adenoid tissue samples will be collected.
Inclusion and exclusion criteria

Patients who meet the following inclusion criteria will beconsidered eligible for the AH group:(1) Aged between 4 and 10 years old.
(2) Nasal endoscopy showing adenoidal occlusion in
60% of the nostrils or nasopharyngeal lateral X-ray
showing A/N > 0.6.
Patients diagnosed with AH will also meet at least oneof the following inclusion criteria to be considered eligible for the AHOME group:(1) Diagnosis of OME with a disease course of at least 3
months.
(2) An acoustic immittance of type B or C.
(3) The presence of conductive hearing loss with PTA(the mean hearing threshold at 500 Hz, 1000 Hz,
and 2000 Hz) ≥ 30 dB.

Liu et al. BMC Pediatrics(2024) 24:521

Page 4 of 8

Fig. 1 Technology Road map. A The grouping and sample collection procedure. B The evaluation of exosomes and the examination of microbial
alterations in the samples. Abbreviation: AHOME group, adenoid hypertrophy accompanied by OME; AH group, adenoid hypertrophy alone

Patients who meet any of the following exclusion criteria will be considered ineligible:(1) One or more episodes of otitis media in the previous one year.
(2) Rhinitis (acute or chronic rhinosinusitis or allergic
rhinitis).
(3) Any immunologic diseases, any intrinsic diseasesof the hearing system, or any anatomical or physiological defects of the ear.
Surgery and specimen collection

All of the enrolled children will be eligible for adenoidectomy. Peripheral blood and nasopharyngeal
swabs will be collected before surgery, and adenoid
tissue samples will be collected during surgery. Blood
samples and nasopharyngeal swabs will be collectedagain 3 months after surgery to monitor the changes
in exosome profiles over time and to assess the longterm effects of the surgery on the exosome-mediatedimmune response and microbial alterations.

Isolation and detection of exosomes

Exosomes in the different samples will be extracted byultracentrifugation. The sample will be collected in a
15 mL tube and then centrifuged at 500 × g for 10 min,and the supernatant will be collected in a new tube. The
supernatant will then be centrifuged at 2,000 × g for 15
min at 4 °C, and the resulting supernatant will be transferred to a new centrifuge tube. This supernatant will becentrifuged at 10,000 × g for 30 min at 4 °C. The supernatant will be collected and filtered through a sterile 0.22
μm filter (Thermo Fisher, Invitrogen). Exosome isolation reagent (Thermo Fisher, Invitrogen) will be mixed
with the supernatant at a ratio of 1:2 and incubatedovernight at 4 °C. The next day, the supernatant will becentrifuged at 10,000 × g for 60 min at 4 °C to pellet theexosomes. The supernatant will be removed completely,
and the pellet will be resuspended in PBS and stored at–80 °C. Transmission electron microscopy will be used
to observe the morphology and size of the exosomes, andNanoSight particle tracking will be used to accurately andquickly detect the distribution of vesicle sizes as a whole.
Western blot and flow cytometry will be used to verify

Liu et al. BMC Pediatrics(2024) 24:521

the purity and presence of exosomes with the commonlyused exosome markers CD63, CD81, and CD9.
High‑throughput sequencing and pathway analysis

The molecular mechanisms behind the pathogenesis of
OME can be revealed through high-throughput sequencing, which can suggest new avenues for the developmentof clinical diagnosis and therapeutic methods. Gene
ontology (GO) (http://​www.​geneo​ntolo​gy.​org) and Kyoto
Encyclopedia of Genes and Genomes (KEGG) (http://​www.​genome.​jp/​kegg) analyses will be performed on
the differentially expressed genes. GO is used to analyze the biological processes, molecular functions, andcellular components of differentially expressed genes,
while KEGG is used to identify pathway enrichment
analysis of the differentially expressed genes. Differentially expressed genes will be annotated using the Cluster Profile package in R using the criteria of |Log2 Fold
Change|≥ 1 and adjusted p < 0.05.
Histopathologic examination

According to reports, the combination of hematoxylin
and eosin (H&E) staining and Gram staining protocols
is a viable method to detect the presence of biofilms
and corresponding histopathological changes because
H&E and Gram staining can show microstructures andvarious bacterial elements, respectively [48]. Adenoid
specimens acquired by surgical excision will be preserved in 10% (w/v) formaldehyde and then cut into tissue sections. Conventional H&E and Gram staining will
be used to evaluate two successive 5 μm frozen sections.
A surrounding polysaccharide layer and the presence ofa distinctive shape and the presence of a gram-positive/
negative micro-community of bacteria following lightmicroscopy examination will be the criteria for the histopathological confirmation of microbial biofilms [48]. Two
independent pathologists who are blinded to the clinicaldiagnosis will evaluate both histologic staining protocols
for evidence of bacteria and biofilms.
Detection of microbial alterations

The 16S ribosomal RNA (16S rRNA) is a component of
the 30S subunit in the prokaryotic ribosome. Prokaryotic
16S rRNA sequences feature nine highly variable regions,of which regions V4-V5 offer the highest specificity andprovide database information for bacterial diversity analysis and annotation. To acquire information on the abundance of each species in the colonies, we will amplify
the genome of the V4-V5 region, collect sequence
information using a MiSeq sequencer, and compare thesequences with a known bacterial genome database.

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Statistical methods

R software version 4.0.5 will be used for all statisticalanalyses, and a p-value < 0.05 will be considered statistically significant.
Expected results(1) Exosomal differences in adenoid tissue: We expect
to identify differences in exosomes derived frompreoperative adenoid tissue samples between the
AHOME group and the AH group.
(2) Exosomal differences in peripheral blood: We
expect to identify differences in exosomes derived
from preoperative peripheral blood samples
between the two groups.
(3) Alterations in microbial diversity: We expect to seechanges in microbial diversity in the adenoid tissues
between the two groups.
(4) Diagnostic and therapeutic insights: We expect togain new insights into the diagnosis and treatment
of OME, thus contributing to the development ofnovel diagnostic and therapeutic approaches.
(5) Identification of novel biomarkers: We expect toidentify novel biomarkers for OME, thus laying
an important scientific foundation for the development of preventive measures, diagnostic techniques, and treatment options.
(6) Comprehensive understanding of OME pathophysiology: We expect to gain a more comprehensive understanding of the pathophysiology of OME,
revealing downstream pathways of initiation factorsthat may contribute to OME development.

Discussion
The patients in this study with adenoid hypertrophy arebeing divided into two distinct groups, namely those withisolated adenoid hypertrophy and those with coexisting
OME and adenoid hypertrophy. Peripheral blood samples have been obtained from the participants in bothgroups, and exosomes will subsequently be isolated forfurther analysis. Nasopharyngeal swabs were used todetect microbial alterations. Adenoidectomies are being
performed, and adenoid tissue samples are being collected during the procedures. Three months after thesurgery, another round of blood and nasopharyngeal
swab collection will take place. Exosomes will be isolated
again for subsequent studies, mirroring the pre-surgery
protocol. Our investigation will also encompass proteome research, exosome biomarker exploration, and theapplication of high-throughput sequencing techniques.
These methodologies will allow us to delve into theLiu et al. BMC Pediatrics(2024) 24:521

pathophysiology of OME, explicitly focusing on the etiology related to inflammation. Ultimately, we aim to offer
innovative insights into OME diagnosis and treatment.
OME differs from acute otitis media in that it lacks
the evident signs and symptoms of an acute infection[3], but once symptoms appear conductive hearing loss
occurs due to middle ear effusion. The impact on an individual’s quality of life and the disease burden on children
cannot be exaggerated due to its tenacity and frequent
occurrence [49]. However, estimating the frequency andprevalence of the condition and providing early diagnosis are challenging due to the lack of evident symptoms[1]. Pneumatic otoscopy is the major diagnostic method
currently suggested, and this has high diagnostic accuracy but a poor coverage rate due to significant technical difficulties and equipment issues, making this method
undesirable for mass population screening for OME [3,50–52].
This research project aims to collect adenoid tissue andperipheral blood samples from two groups of patients.
The isolated exosomes obtained from these samples willserve as a valuable material for investigating the underlying etiological mechanisms of OME. The ultimate
objective is to generate novel insights that can contribute to developing innovative screening and treatment
approaches for OME.
Considering the potential interference of rhinitis on
the research results of adenoid hypertrophy and otitis
media [53–55], it is necessary to exclude the presenceof rhinitis when collecting patient samples. The following measures are being implemented to achieve this: (1)We conduct a detailed clinical assessment of the patients,
including nasal and sinus examinations, before collecting the samples. A preliminary judgment can be madeon the presence of rhinitis by observing whether there
are symptoms of nasal inflammation such as nasal congestion, runny nose, itching, etc. (2) We perform nasal
endoscopy [56] using nasal endoscopes or fiber-optic
nasopharyngoscopes to observe the whole nasal cavity. This provides a clearer view to detect abnormalities
in the nasal mucosa such as congestion, secretions, etc.
(3) If necessary, nasal X-rays, CT scans, or MRI examinations can be performed to further evaluate the conditionof the nasal cavity and sinuses [57]. These examinations
can reveal nasal inflammation, obstructions, or otherabnormalities thereby helping to exclude the presence ofrhinitis. By applying these methods together, it is possible
to minimize the interference of rhinitis on sample collection and to ensure the collection of valid samples directly
related to AHOME.
Exosomes have emerged as promising targets forvarious research studies due to their potential roles

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in various diseases, and they can be used in multiple
ways to investigate pathophysiological pathways and toelicit inflammatory responses. Additionally, exosomes’
morphological and quantitative properties can provide insights into their regulatory functions in the cell[27]. Furthermore, the presence of specific components
within exosomes, such as miRNAs, NADPH oxidase,
nitric oxide synthases, and protein disulfide isomerase,
have been found to influence the progression and prognosis of cardiac diseases [58, 59]. These components are
significant in cardiac health and can serve as potential
molecular targets for therapeutic interventions [30].
Additionally, oncogenic proteins, mRNAs, and miRNAs are associated with cancer progression, indicating
the involvement of exosomes in cancer biology [60].
Moreover, specific proteins like ANXA1 and PSMA7
have been identified as biomarkers for IBD, and theirpresence in exosomes can provide valuable information
for diagnosing and monitoring the progression of thisdisease [32, 33]. These examples highlight the diverseroles of exosomes in different disease contexts, emphasizing their potential as molecular targets and diagnostic biomarkers in various research fields.
Exosomes indeed contain a significant amount of biological information that warrants further investigation.
Previous studies have demonstrated the presence ofmiRNA-carrying exosomes in middle ear effusion, andthe abundant miRNAs and proteins in these exosomes
appear to be key mediators of innate immunity andneutrophilia [41]. In this project, high-throughput
sequencing will be employed to delve deeper into the
biological information carried by exosomes. The aim isto uncover the downstream pathways of initiation factors that may play a role in the development of OME.
By identifying these pathways, this research will contribute to a more comprehensive understanding of thepathophysiology of OME. Moreover, this project seeks
to identify and establish a novel biomarker for OME.
Potential biomarkers specific to OME can be identified
by analyzing the biological information obtained fromexosomes using high-throughput sequencing. Thesebiomarkers have promise as essential scientific foundations for the development of preventive measures,
diagnostic techniques, and treatment options for OME.
The use of high-throughput sequencing technology toinvestigate the biological information in exosomes represents an innovative approach that can provide valuable insights into the underlying mechanisms of OME.
This study is a protocol study, and the data will beobtained in the future implementation of the trial. The
purpose of this paper is to present the study design
and methodology and to lay the foundation for future
research. It is anticipated that the outcomes of thisLiu et al. BMC Pediatrics(2024) 24:521

project will contribute significantly to advancements inthe understanding and management of OME.

Conclusions
This study seeks to understand the role of exosomes inchildren with AHOME. The adenoids, which harbor bacterial pathogens and immune system molecules, play asignificant role in OME. This research aims to elucidate
the biological functions of exosomes and their involvement in the pathophysiological development of OME.
We will collect patients’ peripheral blood and adenoid
tissue samples in order to isolate and analyze exosomes.
The results will shed light on differences in exosomes
between the two groups before surgery as well as changes
in microbial diversity in the adenoid tissue. The findings of this study might contribute to the developmentof innovative diagnostic and therapeutic approaches for
OME.
Abbreviations
OM	Otitis media
OME	Otitis media with effusion
AH	Adenoid hypertrophy
AHOME	Adenoid hypertrophy accompanied by otitis media with effusion
PCR	Polymerase chain reactionEVs	Extracellular vehicles
CRS	Chronic rhinosinusitis
Acknowledgements
The authors would like to thank the participants of this study and the patientadvisers for their support.
Authors’ contributions
LYX and LXL were acknowledgement of drafting the article, SS, YHQ and LHW
were final approval of the version to be published. All authors reviewed themanuscript.
Funding
This study is supported by the Natural Science Foundation of China (Nos.
82371146, 82192862), the Science and Technology Innovation Action of
Shanghai (21Y31900500, 20MC1920200), the Excellent Doctors-Excellent
Clinical Researchers Program (SYB202008, SZA202002), the National Natural
Science Foundation of Shanghai (21ZR1411800), and the Research Projects of
the Shanghai Municipal Health Committee (2020YJZX0110 and 2022XD059).
Availability of data and materials
The datasets used and/or analyzed during the current study are available fromthe corresponding author on reasonable request.

Declarations
Ethics approval and consent to participate
This study complies with the Declaration of Helsinki and is approved by the
ethics committee of the Eye and ENT Hospital of Fudan University (No.
2021022–1). For every child participating in the study an informed consent
form was obtained from the parents or legal guardians. The results of thisprotocol will be published in peer-reviewed journals and presented at international conferences.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.

Page 7 of 8

Author details
Department of ENT Institute and Otorhinolaryngology, NHC Key Laboratoryof Hearing Medicine Research, Demin Han’s Academician Workstation, Eye &ENT Hospital, Fudan University, 83 Fenyang Road, Shanghai 200032, People’s
Republic of China. 2 Institutes of Biomedical Sciences, Fudan University,
Shanghai 200032, People’s Republic of China. 3 The Institutes of Brain Scienceand the Collaborative Innovation Center for Brain Science, Fudan University,
Shanghai 200032, China.

1

Received: 28 July 2023 Accepted: 8 August 2024

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