Direct SARS-CoV-2 infection of the human inner ear may underlie COVID-19-associated audiovestibular dysfunction.

BACKGROUND: COVID-19 is a pandemic respiratory and vascular disease caused by SARS-CoV-2 virus. There is a growing number of sensory deficits associated with COVID-19 and molecular mechanisms underlying these deficits are incompletely understood.
METHODS: We report a series of ten COVID-19 patients with audiovestibular symptoms such as hearing loss, vestibular dysfunction and tinnitus. To investigate the causal relationship between SARS-CoV-2 and audiovestibular dysfunction, we examine human inner ear tissue, human inner ear in vitro cellular models, and mouse inner ear tissue.
RESULTS: We demonstrate that adult human inner ear tissue co-expresses the angiotensin-converting enzyme 2 (ACE2) receptor for SARS-CoV-2 virus, and the transmembrane protease serine 2 (TMPRSS2) and FURIN cofactors required for virus entry. Furthermore, hair cells and Schwann cells in explanted human vestibular tissue can be infected by SARS-CoV-2, as demonstrated by confocal microscopy. We establish three human induced pluripotent stem cell (hiPSC)-derived in vitro models of the inner ear for infection: two-dimensional otic prosensory cells (OPCs) and Schwann cell precursors (SCPs), and three-dimensional inner ear organoids. Both OPCs and SCPs express ACE2, TMPRSS2, and FURIN, with lower ACE2 and FURIN expression in SCPs. OPCs are permissive to SARS-CoV-2 infection; lower infection rates exist in isogenic SCPs. The inner ear organoids show that hair cells express ACE2 and are targets for SARS-CoV-2.
CONCLUSIONS: Our results provide mechanistic explanations of audiovestibular dysfunction in COVID-19 patients and introduce hiPSC-derived systems for studying infectious human otologic disease.

Introduction

Viral infections are a common reason for hearing loss and vestibular dysfunction. Viruses known to cause audiovestibular dysfunction include members of the Herpesviridae (cytomegalovirus, herpes simplex virus, varicella zoster virus, Epstein-Barr virus), Paramyxoviridae (parainfluenza viruses, mumps virus, measles virus), polio virus, hepatitis viruses, human immunodeficiency virus, rubella virus, and influenza viruses[1]. Presumed mechanisms of virally induced sensorineural hearing loss (SNHL), tinnitus, vertigo, dizziness, or imbalance include: direct invasion and damage of inner ear structures, including the organ of Corti (OC) and the vestibulocochlear nerve[2]; immune-mediated damage and inflammation, including neuroinflammation[3,4]; and reactivation of latent virus within the inner ear[5]. In addition, viruses can infect the middle ear and typically cause conductive hearing loss due to a middle ear effusion. While coronaviruses are a common cause of middle ear infection[6], their role in inner ear infection has not been systematically studied.

Coronavirus disease 2019 (COVID-19) is a contagious respiratory and vascular disease caused by SARS-CoV-2. The virus is also known to cause anosmia[7] and ageusia[8], highlighting its tropism for sensory systems. Although there are several recent reports of audiovestibular symptoms in COVID-19 patients, these rely on self-reported hearing loss[9], do not comment on hearing outcome after COVID-19 resolution[10-13], lack documentation of objective SARS-CoV-2 testing[10], or only include single patients[10,12-17]. Here we provide the largest series to date of patients with documented SNHL and audiovestibular symptoms during SARS-CoV-2 infection, along with audiograms at 2–4 months after the resolution of COVID-19. To investigate whether these symptoms might be due to direct infection of audiovestibular structures, we examined the expression of SARS-CoV-2 cell entry-related genes and proteins in human and mouse inner ear tissue, and we infected human vestibular tissue to identify target cell types of SARS-CoV-2. Finally, in order to study the pathogenesis of COVID-19 audiovestibular symptoms, we derived otic prosensory cells (OPCs) (the precursors of hair cells and their supporting cells), Schwann cell precursors (SCPs) (the progenitor of Schwann cells), and inner ear organoids (three-dimensional (3D) organized structures mimicking inner ear epithelium) from human induced pluripotent stem cells (hiPSCs) and demonstrated differential expression of host-derived SARS-CoV-2 entry cofactors that might influence tropism of the virus in the inner ear.

Methods

Human subjects

The study of patients with COVID-19-associated sudden audiovestibular symptoms was approved by the Massachusetts General Brigham Institutional Review Board (2020P002900). Verbal informed consent was obtained from all subjects and all procedures were conducted in accordance with the Helsinki Declaration. Verbal informed consent was obtained to publish the detailed case information contained within the Supplementary Information. To evaluate the expression of SARS-CoV-2 receptors in the human inner ear, fresh inner ear tissue was collected during surgical labyrinthectomies and translabyrinthine resections of vestibular schwannomas (N = 6). This research was reviewed by the Massachusetts General Brigham Institutional Review Board (IRB) and determined to be Exempt from IRB approval (2020P003329) and from the need for informed consent.

Generation of hiPSC

For the generation of hiPSC line SK8-A, we recruited healthy subjects and isolated primary dermal fibroblasts from them. The protocols for research involving human subjects and for stem cell research were approved by the Institutional Review Board of Massachusetts Eye and Ear and Partners Human Research Committee. The study participants provided written informed consent.

For establishing fibroblast lines from skin biopsies, tissue was manually dissected into pieces of approximately 1 cm2 in size and digested with Trypsin (Sigma) for 20 min at room temperature. Digested tissue was centrifuged and incubated with 0.04 mg/ml DNase (Sigma) for 10 min at room temperature. Tissue was collected by centrifugation and further disaggregated by incubation with 20 mg/ml collagenase type II (Gibco) for 30 min at room temperature. Cells were plated onto plates in fibroblast medium containing DMEM (Gibco, Cat# 11995073) supplemented with 10% fetal bovine serum (FBS; Gibco, Cat# 26140079) and 2 mM L-glutamine (Gibco, Cat# 25030081).

One day before transduction, 150,000 mycoplasma-free fibroblasts were seeded per well in a 6-well plate previously coated with 0.1% gelatin and cultured in fibroblast medium. On the day of transduction, cells were transduced with the CytoTune™-iPS 2.0 Sendai Reprogramming Kit (Invitrogen, Cat# A16517) in fibroblast medium at a multiplicity of infection (MOI) of 3 for Klf4 and 5 for Klf4–Oct3/4–Sox2 and cMyc. The medium was replaced with fresh fibroblast medium every day. Five days after transduction, 250,000 cells were transferred onto irradiated mouse embryonic fibroblasts (MEFs) in a 10 cm culture dishes. After culturing overnight in fibroblast media, the medium was replaced daily with DMEM/F12 (Gibco, Cat# 11330057) supplemented with 20% KnockOut Serum Replacement (Gibco, Cat# 10828028), 2 mM L-glutamine, 1× MEM Non-Essential Amino Acids Solution (Gibco, Cat# 11140050), 55 μM β-Mercaptoethanol (Gibco, Cat# 21985023), and 10 ng/ml FGF-2 (Gibco, Cat# PHG0360). The hiPSC colonies were picked for expansion and characterization from 18 to 25 days after transduction.

Karyotyping/mycoplasma test

Karyotype analyses were performed at WiCell, according to the International System for Human Cytogenetic Nomenclature. The cell lines were tested at passage 5, with 20 cells in metaphase counted for the analysis. Mycoplasma test was performed using the MycoAlert Mycoplasma Detection Kit (Lonza, Cat# LT07-318) to ensure that all cells are mycoplasma free.

Embryoid body (EB) formation

hiPSC were differentiated as EBs by detaching ~80% confluent hiPSC colonies from MEF feeders using Gentle Cell Dissociation Reagent (StemCell Technologies, Cat# 07174) and a cell scraper. Gently detached cells were transferred to a 15 ml centrifuge tube and the cell clumps were allowed to sink for 10–15 min. EBs were transferred to ultra-low attachment 6-well plates (Corning, Cat# 3471) in DMEM/F12 supplemented with 10% KnockOut Serum Replacement and 10 μM Y-27632 (Calbiochem, Cat# 688001); the medium was replaced every other day. Eight days after plating, EBs were transferred to a gelatin-coated 10 cm cell culture dish in DMEM supplemented with 10% FBS and 2 mM L-glutamine. After 7 days of culturing, EBs were harvested for further analysis.

hiPSCs culture

hiPSC lines SK8-A and UCSD112i-2-11 (WiCell) on Matrigel hESC qualified matrix (Corning, Cat# 354277) were cultured in mTeSR Plus medium (StemCell Technologies, Cat# 100-0276). hiPSC colonies were treated with ReLeSR (StemCell Technologies, Cat# 05872) and detached by tapping the side of the plates. Detached cell clumps were plated on the Matrigel-coated plate. hiPSC were maintained under mTeSR Plus medium and used before passage number 50.

OPC differentiation

We modified the previously published protocol for differentiation of hiPSCs into OPCs using a monolayer culture system[18]. In our study, undifferentiated hiPSC lines UCSD112i-2-11 and SK8-A were dissociated with ReLeSR and seeded at 30,000 cells/cm2 onto laminin-coated plates (R&D Systems, Cat# 3401-010-02) and cultured in DMEM/F12 (Gibco, Cat# 11330032) supplemented with 1× N2 (1% (v/v) final concentration, Gibco, Cat# 17502048), 1× B27 (2% (v/v) final concentration, Gibco, Cat# 17504044), 50 ng/ml FGF-3 (R&D Systems, Cat# 1206-F3), and 50 ng/ml FGF-10 (R&D Systems, Cat# 345-FG). The medium was replaced on day 1 and changed every other day. The concentration of Y-27632 (TOCRIS, Cat# 1254) was maintained at 10 μM throughout days 0–3. On day 14, the cells were transferred onto growth factor reduced (GFR) Matrigel (Corning, Cat# 356230)-coated plates at 80,000 cells/cm2 and cultured in DMEM/F12 supplemented with 1× N2, 1× B27, 5 μM Dibenzazepine (DBZ; TOCRIS, Cat# 4489), and 10 μM Y-27632. The medium without Y-27632 was replaced every other day from day 15 to day 20.

SCP differentiation

For SCP differentiation, we modified a previously published protocol[19]. hiPSC line SK8-A (~10,000 cells/cm2) was replated onto GFR Matrigel-coated culture dishes with 10 μM of Y-27632. The next day, the culture medium was switched from hiPSC culture medium to 10 μM Y-27632 and 10 μM valproic acid (VPA; Sigma, Cat #P4543) supplemented Neuronal Differentiation Medium (NDM). Supplemented NDM contained 1× N2, 1× B27, 0.005% bovine serum albumin (BSA; Sigma, Cat# A8412), 2 mM GlutaMAX (Gibco, Cat# 35050061), 0.11 mM β-mercaptoethanol, 3 μM CHIR99021 (TOCRIS, Cat# 4423), 20 μM SB-431542 (Selleckchem, Cat# S1067) in Advanced DMEM/F12 (Gibco, Cat# 12634028), and Neurobasal medium (Gibco, Cat# 21103049) (1:1 mix). After 2 days of differentiation, the medium was replaced with NDM and freshly changed every other day. After ~2 weeks of differentiation, the cells were dissociated with Accutase (StemCell Technologies, Cat# 07920) and cultured in Neuregulin-1 (NRG-1; R&D Systems, Cat# 5898-NR-050) containing NDM (=SCPM). The cells were split and replated every 3~4 days by using Accutase. The hiPSC-derived SCPs were generated after approximately 20~30 days of differentiation.

Inner ear organoid differentiation

We modified the previously published protocol for the generation of inner ear organoids containing vestibular hair cell-like cells from human pluripotent stem cells[20-22]. In our study, hiPSCs from SK8-A line were dissociated with ReLeSR and distributed 5,000 cells per well onto low-adhesion 96-well U-bottom plates in mTeSR Plus medium containing 20 μM Y-27632 and 100 μg/ml Normocin (Invivogen, Cat# ant-nr-2). The plate was centrifuged at 300 g for 3 min to aggregate the cells. On day 2, the medium was changed into 100 μl of chemically defined medium (CDM) containing 4 ng/ml FGF-2 (R&D Systems, Cat# 233-FB), 10 μM SB-431542 (TOCRIS, Cat# 1614), 2.5 ng/ml BMP4 (R&D Systems, Cat# 314-BP), and 2% GFR Matrigel. CDM contained a 50:50 mixture of F-12 Nutrient Mixture with GlutaMAX (Gibco) and Iscove’s Modified Dulbecco’s Medium with GlutaMAX (IMDM; Gibco, Cat# 31980030) additionally supplemented with 0.5% BSA, 1× Chemically Defined Lipid Concentrate (Invitrogen, Cat# 11905031), 7 μg/ml Insulin (Sigma, Cat# I9278), 15 μg/ml Transferrin (Sigma, Cat# T8158), 450 μM Mono-Thioglycerol (Sigma, Cat# M6145), and Normocin. On day 6, 25 μl of CDM containing a 250 ng/ml FGF-2 (50 ng/ml final concentration) and 1 μM LDN-193189 (200 nM final concentration; Stemgent, Cat# 04-0074-02) was added to the preexisting 100 μl of media in each well. On day 10, 25 μl of CDM containing a 18 μM CHIR99021 (3 μM final concentration; Stemgent, 04-0004-02) was added to the preexisting 125 μl of media. On day 14, the aggregates were pooled together and washed with freshly prepared Organoid Maturation Medium (OMM) containing a 50:50 mixture of Advanced DMEM/F12 and Neurobasal medium supplemented with 0.5× N2 Supplement, 0.5× B27 without Vitamin A (Gibco, Cat# 12587010), 1× GlutaMAX, 0.1 mM β-Mercaptoethanol (Gibco), and Normocin. The aggregates were plated individually into each well of a 24-well low-cell-adhesion plate in OMM containing 3 μM CHIR99021 and 1% GFR Matrigel. On day 17, the medium was changed completely with OMM containing 3 μM CHIR99021. On day 20, the medium was changed with OMM. The 24-well plates were maintained on an in-incubator stir plate at 65 RPM for up to 90 days.

Virus and infections

The SARS-CoV-2 strain, WA1/2020, was obtained from BEI resources (NR-52281) and propagated in Vero E6 (ATCC) cells in DMEM (Corning) with 2% FBS (Gibco) with penicillin and streptomycin (Gibco, Cat# 15140122). Virus-containing cell supernatants were harvested after the appearance of cytopathic effect and clarified by centrifugation. Conditioned media was collected from uninfected Vero E6 cells grown in parallel. Viral titer was determined by plaque-forming assay in Vero E6 cells: cells were infected with dilutions of viral stock and overlaid with 3.2% carboxymethylcellulose solution mixed at 1:1 with DMEM containing 4% FBS. At 4 days post infection, cells were fixed in methanol and stained with 0.5% crystal violet.

Adult human vestibular tissue was maintained in explant media: Neurobasal Medium supplemented with 1× N2, 2× B27, 1 mM GlutaMAX, 0.5 mM dibutyryl‐cyclic AMP (Santa Cruz Biotechnology, Cat# sc-201567B), 10 ng/ml human brain‐derived neurotrophic factor (R&D Systems, Cat# 248-BDB), 10 ng/ml human neurotrophin‐3 (R&D Systems, Cat# 267-N3), 10 ng/ml insulin-like growth factor‐1 (R&D Systems, Cat# 291-G1), and 50 μg/ml Normocin. For infection of explants, at 1 day post tissue harvest, media was exchanged for SARS-CoV-2 inoculum at a viral concentration of 5e+04 pfu/ml (a total of 2.5e+04 pfu per explant). Mock infections were performed with an equivalent dilution of conditioned media. The tissues were incubated for 2 h at 37°C with manual rocking after which viral inoculum was removed and replaced with fresh explant media. At 48 h after media change (48 hours post infection, hpi), the tissues were fixed in 4% paraformaldehyde (PFA) for 4 h. The tissues were washed three times in phosphate-buffered saline (PBS) and then dehydrated overnight in 10% sucrose followed by in 20% sucrose and 30% sucrose. The tissues were embedded in OCT compound (Fisher HealthCare, Cat# 23-730-571) and frozen for sectioning.

For 2D infections, OPCs and SCPs were seeded in black-walled 96-well plates (Corning, Cat# 3603) and infected at day 23 and 21–31 post differentiation, respectively. Virus was added to cells in minimal volume at indicated MOIs diluted in DMEM/F12 (mock controls received equal volume of conditioned media) and incubated with intermittent rocking for 1 h at 37°C. Inoculum was then removed and replaced with fresh OPC/SCP medium. For immunofluorescence studies, at the indicated time post infection, cells were fixed in 3.2% PFA (Electron Microscopy Sciences, Cat# 15714) in PBS for 30 min at room temperature. After fixation, cells were washed three times with PBS. All work with infectious materials was performed in the BSL3 facility at the Ragon Institute in accordance with approved protocols at that facility.

For 3D infections, organoid media was partially exchanged for diluted SARS-CoV-2 at a final viral concentration of 5e+04 pfu/ml (a total of 2.5e+04 pfu per organoid) in fresh organoid media. Mock infections were performed with an equivalent dilution of conditioned media. Organoids were incubated with inoculum overnight at 37°C with orbital shaking (65 rpm), after which the organoids were moved to fresh media. At 48 h after media change (72 hpi), organoids were fixed in 4% PFA for 45 min. Organoids were washed three times in PBS and then dehydrated overnight in 15% sucrose followed by 5 days in 30% sucrose. Organoids were embedded in OCT compound and frozen in 2-methylbutane for sectioning.

Viability assays

The CellTiter-Glo® Luminescent Cell Viability Assay (Promega, Cat# G7570) was used according to the package protocol. Cells were lysed in situ in the 96-well plate in which they were grown, and then the material was transferred to a 96-Well Solid White Polystyrene Microplate (Corning, Cat # 3912) for luminescence reading.

Mouse inner ear collection

Wild-type CBA/CaJ mice were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). Animals of either sex were used for experimentation in an estimated 50:50 ratio. Six weeks old mice were sacrificed through cardiac perfusion, decapitated, and inner ears were removed. Inner ears were then fixed in buffered 4% PFA (ThermoFisher Scientific, Cat# AAJ19943K2) for 2–3 h after piercing both the round and oval windows, decalcified in 0.12 M EDTA at room temperature for 4 days, serially dehydrated in sucrose, embedded in OCT compound, and sectioned into 12 µm cryosections on a Leica CM-1860 cryostat. This research was approved by the Institutional Animal Care and Use Committees at Massachusetts Eye and Ear.

qRT-PCR

To isolate RNA from human vestibular specimens, the tissue was disrupted and homogenized using the TissueRuptor and RNeasy Plus Micro Kit (QIAGEN, Cat# 74034). RNA from the cell cultures in vitro was isolated using the ReliaPrep™ RNA Cell Miniprep System (Promega, Cat# Z6011). Single-stranded complementary DNA was synthesized from 1 μg of the RNA sample, or less than 1 μg of RNA in case of human surgical specimens, using GoScript Reverse Transcription System (Promega, Cat# A5000). The quantitative reverse transcription PCR (qRT-PCR) was performed with the GoTaq qPCR Master Mix (Promega, Cat# A6002) in a QuantStudio 3 real-time PCR system (Applied Biosystems). The reaction parameters were as follows: 95°C for 2 min to denature the cDNA and primers, 40 cycles at 95°C for 15 s, annealing/extension at 60°C for 60 s. A comparative Ct method was used to calculate the levels of relative expression, whereby the Ct was normalized to the endogenous control glyceraldehyde-3-phosphate dehydrogenase (GAPDH). This calculation gave the ΔCt value, which was then normalized to a reference sample (i.e., a negative control), giving the ΔΔCt. The fold change was calculated using the following formula: 2-ΔΔCt. Primers are listed in Supplementary Table 1.

To extract viral RNA, after initial attachment of virus and inoculum removal, a sample of the freshly added media was collected as a 0 hpi supernatant. Following this, media was harvested and changed daily during infection such that each supernatant time point contained viral particles released over the prior 24 h. Viral RNA was extracted from 140 μl of each sample (2 samples per time point per experiment, though 0 hpi samples were only available from one experiment) using the QIAamp Viral RNA Mini Kit (Qiagen #52904) per manufacturer’s protocol, and eluted in 35 μl nuclease-free water passed twice through the column.

qRT-PCR was performed using the universal EXPRESS One-Step SuperScript qPCR kit (ThermoFisher Scientific, Cat# 11781-200) with 2 μl input RNA. The CDC N1 primers and FAM-labeled probe were used for SARS-CoV-2 detection (https://www.cdc.gov/coronavirus/2019-ncov/lab/rt-pcr-panel-primer-probes.html) (IDT #10006713), and eukaryotic 18s rRNA primers and VIC-labeled probe (ThermoFisher Scientific #4319413E) were added to evaluate cellular RNA in the supernatant. For each sample, qRT-PCR was performed in technical triplicate using the ABI StepOnePlus™ Real Time System.

Immunocytochemistry

Cells grown on coverslips were fixed with 4% PFA for 10 min at room temperature. For permeabilization, cells were washed three times with PBS (Gibco, Cat# 14080-055) and incubated in PBST, which is 0.1% Triton X-100 (Sigma, Cat# T8787) 1× PBS solution, for 10 min at room temperature. Unspecific binding was blocked with 5% normal horse serum (Abcam, Cat# ab7484), 5% goat serum (Gibco, Cat# PCN5000), or 3% BSA (Sigma, Cat# A9647) in PBST for 1 h. Samples were then incubated overnight at 4°C with specific primary antibodies (Supplementary Table 2) diluted in 1% BSA in PBST, washed three times with PBS, and incubated with secondary antibodies (Supplementary Table 3) in PBST. Vectashield (Vector Laboratories, Cat# H1000) with DAPI (Cell Signaling, Cat# 4083) was used to mount the samples and visualize cellular nuclei. Negative control experiments without the primary antibodies were processed in parallel. Microscopy was performed using a Leica SP8 confocal microscope (Leica Microsystems) and Olympus FV1200 Laser Scanning Confocal Microscope. Microscopy images were analyzed using Fiji software. Percent of infected OPCs were quantified using 10× magnified images of three independent fields per condition. DAPI-stained nuclei were counted using automated cell counting on Fiji software and infected cells stained for SARS-CoV-2 nucleoprotein (NP) or dsRNA were counted manually.

Immunohistochemistry

Human vestibular tissue was fixed with 4% PFA for 4 h at room temperature. The fixed specimens were cryopreserved with a graded treatment of 10%, 20%, and 30% sucrose and then embedded in tissue freezing medium OCT compound. Frozen tissue blocks were sectioned into 12 μm cryosections on a Leica CM-1860 cryostat. For immunostaining, a 5% goat or horse serum in 0.3% Triton X-100 1× PBS solution was used for blocking, and a 1% BSA and 0.3% Triton X-100 1× PBS solution was used for primary/secondary antibody incubations.

Statistical analysis and reproducibility

All statistics were performed using GraphPad Prism 9.1.0 software and R version 3.5.1. Statistical significance was determined using an unpaired t-test with Welch’s correction for multiple comparisons to a control group. For the analysis of SARS-CoV-2 RNA in the supernatant of OPC, we averaged technical triplicate Ct values for each sample. Normalized and relative expression values were determined for each sample with and without normalization to cell-free 18s rRNA copies using standard relative quantification (RQ) calculations (from ΔΔCt and ΔCt values, respectively) in Microsoft Excel. Sample sizes and number of biological or technical replicates are indicated in each figure legend.

Table 1

Otoacoustic emission test results reflecting cochlear outer hair cell function.

Patient	Hearing loss	Otoacoustic emission test results by frequency
		Ear	2 kHz	2.5 kHz	3 kHz	3.5 kHz	4 kHz	5 kHz	6 kHz	7 kHz	7.5 kHz	8 kHz
5	R profound SNHL	Right	Absent	Absent	Absent	Absent	Absent	Absent	Absent	Absent	Absent	Absent
		Left	Present	Present	Present	Present	Present	Present	Present	Present	Present	Present
6	R profound SNHL	Right	Absent	Absent	Absent	Absent	Absent	Absent	Absent	Absent	Absent	Absent
		Left	Present	Present	Present	Present	Present	Present	Present	Present	Present	Absent
7	L moderate low-frequency SNHL	Right	Present	Present	Present	Present	Present	Present	Present	Present	Present	Present
		Left	Present	Present	Present	Present	Present	Present	Present	Present	Present	Present
8	B severe to profound SNHL	Right	Absent	Absent	Absent	Absent	Absent	Absent	Absent	Absent	Absent	Absent
		Left	Absent	Absent	Absent	Absent	Absent	Absent	Absent	Absent	Absent	Absent
9	L mild high-frequency SNHL	Right	Present	Present	Present	Present	Present	Present	Present	Absent	Absent	Absent
		Left	Present	Present	Absent	Absent	Absent	Absent	Absent	Absent	Absent	Absent
10	B moderate SNHL	Right	Absent	Absent	Absent	Absent	Absent	Absent	Absent	Absent	Absent	Absent
		Left	Absent	Absent	Absent	Absent	Absent	Absent	Absent	Absent	Absent	Absent

L left, R right, B both, SNHL sensorineural hearing loss.