# METHODS

The patient (NC) was a 27-year-old Caucasian male working as a general medical practitioner at the time of the acoustic shock. Written informed consent was obtained from the participant for the publication of this case report. On November 10th 2013, in a leisure shooting stand, without wearing any hearing protection, he was exposed to a unique and unexpected gunfire at an approximate 7 m distance from his right side. As bothersome symptoms progressively emerged after this acoustic incident, he attended several ENT clinics. Audiograms at week 3 and 7 after ASI (thresholds <15 dBHL at all tested frequencies from 0.125 to 8 kHz) and all other exams (tympanometry, blood check, and cerebral MRI) were normal. Later, he was asked to assess the severity of his symptoms, using a 0–10 visual analog scale, for each ear several times a day (from May 1st to May 9th 2015). The symptoms were tingling, otalgia, ear tension, tinnitus loudness, neck pain/tension, temporal pain/tension, and pain/blocked nose. He also managed to photograph his eardrums using a video-otoscope (Firefly DE500 v1.1, 4 mm speculum). For analysis purpose, the images were transformed into numeric values estimating the "redness" of the eardrum. The number of red pixels was normalized according to the eardrum surface captured by the video-otoscope. In March 2017, NC visited our laboratory in Marseille to assess the psychoacoustic properties of his tinnitus and explored the middle ear function (Multifrequency Tympanometer Zodiac, Otometrics). Static admittance was obtained at four frequencies (226, 678, 800, and 1,000 Hz). Admittance variation while the patient voluntarily (but not forcefully) eye blinked was investigated over time both at the pressure at which the admittance is maximal and at ±50 daPa from this value. Symptom severity from each ear was analyzed using principal component analysis (PCA). The PCA was performed on all symptoms, excluding neck and temporal pain as these symptoms did not affect the left side.

### RESULTS

In regard to the clinical course, immediately after ASI, the patient felt a clicking in the right ear and thereafter reported a subjective perception of ear tension and fullness. At week 2 emerged a bilateral high-pitched fluctuating tinnitus. These auditory symptoms were associated with an erratic acute pain (sting or electrical shock) located deep in either ear. More than 3 weeks after the acoustic shock, the patient reported additional painful sensations starting around the concha and irradiating to the midface (constriction, blocked nose, and clear nasal discharge) and to the temporal region or the neck. Interestingly, the laterality and severity of the pain was correlated with the amount of tension perceived in the ipsilateral (right) ear. The psychoacoustic properties of the tinnitus, its amplitude modulation (tremolo), also varied according to the ear tension level. From low to mid tension, the tremolo varied from low frequency ("morse code") to high frequency ("cricket-like sound"). For high tension, tinnitus was described as a high-pitched whistling. The pitch of this tinnitus was measured at 12 kHz and the tremolo, estimated from an amplitude modulated stimulus at the tinnitus frequency, was found at 32 Hz (the severity of ear tension and tinnitus loudness were 4 and 3, respectively). The loudness of this high-pitched tinnitus is reported in **Figure 1**. The patient also reported a low-pitched tinnitus associated with a sensation of fluttering in the ear. The low-pitched tinnitus was enhanced when something (earplug, stethoscope) was inserted in the ear canal. The high- and low-pitched tinnitus could be absent when the ear was completely relaxed. In contrast the tinnitus was not modulated by forceful head and neck contractions (3).

The temporal evolution of symptoms severity (**Figure 1**) was investigated using PCA. The first principal component (48% of the total variance) represents the severity of the symptoms and does not discriminate between them. On the other hand, the second principal component (18% of the total variance) separates two symptom clusters, namely "tension," "blocked nose," and "tympanum redness" for symptom cluster 1 and "tingling," "tinnitus," and "otalgia" for symptom cluster 2 (**Figure 2**, upper panels). The feeling of pain in the temporal region and the neck (plotted on the first two PCA axes) represent a third group of symptoms (symptom cluster 3), different from cluster 1 and 2. The symptom cluster 3 is reported in the right side only, is moderate in severity, and is present only occasionally compared to the other symptoms. The PCA indicates that the correlation between symptom cluster 3 and both symptom cluster 1 and 2 is low. **Figure 2** (middle panels) shows the multi-dimensional data (symptom severity) plotted on the new coordinate system defined by the first two principal components at each time point. The symptoms are usually more severe in the right ear than in the left ear, and the maximum severity is also greater in the right ear. Interestingly, the two symptom clusters are reported in the right ear, while only symptom cluster 1 is reported in the left ear. The temporal dynamics of symptom severity (first principal component) in the left and right ear (**Figure 2**, bottom panel) is also clearly anti-correlated across the two ears.

The sensation of tension in the ear was associated with tympanum hyperemia (**Figure 1**, also see Video S1 in Supplementary Material). This hyperemia was also present during jaw muscle contraction (hypothetically involving the mylohyoid muscle) and was associated with an increase of the high-pitch tinnitus (see Video S2 in Supplementary Material). This phenomenon was reported to be only present when the sensation of tension in the ear was high and coincidentally, the tympanum was always showing dilated vessels.

The tympanometry measurements demonstrated different middle ear function for the two ears (**Figure 3**). Indeed, for the right ear, the static admittance was larger (**Figure 3**, upper panel) and the resonant frequency (estimated from the frequency at which the susceptance is null) was abnormally low (<678 Hz) (**Figure 3**, middle panel). Moreover, the admittance was modulated by eyelid closure, especially in the right ear (**Figure 3**, bottom panel). Finally, the stapedial reflexes were present and clinically normal in both ears at 500 Hz and 4 kHz at 95–100 dB HL.

#### DISCUSSION

This report completes and adds to previous studies on acoustic shock (1). First, the symptoms reported after the acoustic shock, including their temporal dynamics and characteristics, have been described both qualitatively and quantitatively. This case reports all the symptoms that are commonly described after ASI, except vertigo and/or dizziness (1), and additional symptoms (nasal congestion, rhinorrhea, and tympanum hyperemia). Second, the functional integrity of the middle ears was also investigated

were taken).

using multifrequency tympanometry and direct examination of the eardrums. Our findings provide further insights into the mechanisms involved in ASI, and in particular they question and extend the framework previously developed by others (1, 2).

This study is the first to provide experimental support suggesting that middle ear muscles (MEM) can behave abnormally after ASI. Indeed, middle ear admittance is changed by simple eyelid closure, eardrum movements in the right ear can be large enough to be visible by the naked eye and the fluttering sensation (likely accompanying MEM contraction) is enhanced by cutaneous stimulation of the ear canal (4–7). This abnormal behavior of MEM may result from a dysfunction and/or injury of MEM caused by an exaggerated contraction in response to the acoustic incident. While stapedius muscle contraction is activated by loud

sounds, TTM contraction can be triggered by unexpected sounds through the general startle reflex (8). The low resonant frequency and large admittance in the most symptomatic (right) ear may reflect an abnormal ossicle arrangement, possibly caused by the exaggerated contraction of the MEM. The sensation of tension in the ear reported immediately after the acoustic shock may result from an abnormal contraction of the MEM signaled by the muscle spindles (9) and tympanum mechanoreceptors due to eardrum deformation (10). The traumatic acoustic episode may lead to anxiety and sensory hypersensitivity, which may in turn reduce the startle reflex threshold and/or potentiate the startle reflex (11), possibly through a descending serotoninergic innervation (12). This central feedback may facilitate tonic and phasic TTM contraction.

The putative exaggerated contraction of the MEM, eventually followed by tonic contraction, may be associated with MEM injury and inflammatory processes. In particular, the TTM is innervated by fibers containing substance P and calcitonin gene-related peptide, which may play a key role in the noiseinduced inflammation of the middle ear (13–15). Inflammation can then diffuse from the MEM to the tympanum and middle ear mucosa and can even become neurogenic as these tissues are rich in mastocytes (16–18). The diverse pain feelings in the ear (otalgia and tingling) likely result from middle ear inflammation and activation of the trigeminal nerve (TGN), which innervates the middle ear mucosa, the eardrum, and the TTM (15, 19). Intriguingly, tinnitus loudness is correlated to the severity of otalgia and tingling in the ear, suggesting that middle ear inflammation plays a role in tinnitus generation and/or severity. One can propose that antidromic activation of the TGN may change the vascular permeability of the blood vessels in the stria vascularis (20), which may eventually increase the endocochlear potential and produce tinnitus.

The MEM dysfunction/injury and the chronic inflammatory processes following ASI may be associated with neural hyperactivity in the trigeminal pathways. This neural hyperactivity may

in turn lead to plastic changes (sensitization) at several levels of the trigeminal pathways (21). Pain in the neck and the temporal region may result from referred pain as a consequence of central sensitization, possibly at the trigemino-cervical complex (TCC) level where multimodal neurons are present (22). Sensitization may also contribute to shift the pathophysiology from acute to chronic (21).

Finally, the PCA suggests that the symptoms from cluster 1 originate from tightly coupled mechanisms. The trigeminal sensory inputs from regions of the head, including middle ear mucosa, eardrums, and the TTM (14, 15, 19), are collected by the TCC that has a reflex connection with the superior salivatory nucleus (SSN). Interestingly, the SSN provides a parasympathetic innervation to the head region *via* the sphenopalatine ganglion. This reflex loop between the TGN and the parasympathetic pathway of the head has been suggested to account for the autonomic symptoms associated with trigeminal-autonomic cephalalgia (TAC) (23). The activation of the trigeminal-autonomic reflex in this case may account for the nasal congestion, rhinorrhea, and the tympanic hyperemia. The autonomic nervous system, which innervates the middle ear (18, 24–26), may also contribute to enhance the MEM tonus and the feeling of tension and aural fullness (27).

In summary, this case suggests that a complex pathophysiology can be involved in ASI. Initially, the acoustic shock may trigger an exaggerated MEM response causing muscle dysfunction/injury and inflammatory processes, which may later diffuse to the tympanum and middle ear mucosa. The activation of the trigeminal pathways due to inflammation may account for the diverse pain feelings in the ear and tinnitus (symptom cluster 2) and also for referred pain in the neck and the temporal region (symptom cluster 3) after induction of central sensitization. Finally, nasal congestion and tympanum hyperemia, which are tightly coupled to the feeling of ear tension (symptom cluster 1), may result from the activation of a trigeminal-autonomic reflex. Interestingly, the pathophysiology of ASI is strongly reminiscent of that of post-traumatic TAC (28). In this context, the case reported here may define a new clinical entity that we suggest calling post-traumatic trigeminal-autonomic otalgia. The clinical picture presented here, i.e., with numerous, diverse, and severe symptoms, could be an extreme form of this clinical entity and may be relatively rare. However, the prevalence of this clinical entity may be more prevalent than expected at first sight, as less symptomatic forms may also exist. Many patients with tinnitus and/or hyperacusis report one or more additional symptoms such as ear fullness, otalgia, tympanic flutter, and/or pain in the neck (2). This framework opens new and promising perspectives on the understanding and medical management of ASI and beyond, i.e., tinnitus and hyperacusis (23).

### ETHICS STATEMENT

The case reported in the study gave written informed consent in accordance with the Declaration of Helsinki.

#### AUTHOR CONTRIBUTIONS

AL, NC, DP, PF, LP, and AN collected data. LP and AN analyzed data and made figures. AN wrote a first version of the manuscript. All authors contributed to the final version of the manuscript.

#### ACKNOWLEDGMENTS

The authors would like to thank Dirk De Ridder, Anne Donnet, and Lénaic Monconduit for their useful comments on earlier versions of the manuscript. We also wish to thank Otometrics for their help and support. This work has been conducted with the

#### REFERENCES

financial assistance of CNRS, Aix-Marseille Université, B2V, and the supplementary pension institution of Klesia.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at http://journal.frontiersin.org/article/10.3389/fneur.2017.00420/ full#supplementary-material.

Video S1 | The video shows the right eardrum when the feeling of tension in the ear is high and when it is low.

Video S2 | The video shows the right eardrum, when the tension in the ear is high, before, during, and after the mylohyoid muscle contraction.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2017 Londero, Charpentier, Ponsot, Fournier, Pezard and Noreña. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Tinnitus in Normal-Hearing Participants after Exposure to Intense Low-Frequency Sound and in Ménière's Disease Patients

*Margarete Anna Ueberfuhr1,2, Lutz Wiegrebe2,3, Eike Krause1,4, Robert Gürkov1,4 and Markus Drexl1 \**

*1German Center for Vertigo and Balance Disorders, University Hospital Munich, Ludwig-Maximilians Universität München, Munich, Germany, 2Graduate School of Systemic Neurosciences, Ludwig-Maximilians-Universität München, Martinsried, Germany, 3Division of Neurobiology, Department Biology II, Ludwig-Maximilians-Universität München, Martinsried, Germany, 4Department of Otorhinolaryngology, Head and Neck Surgery, Grosshadern Medical Centre, Ludwig-Maximilians Universität München, Munich, Germany*

Tinnitus is one of the three classical symptoms of Ménière's disease (MD), an inner ear disease that is often accompanied by endolymphatic hydrops. Previous studies indicate that tinnitus in MD patients is dominated by low frequencies, whereas tinnitus in non-hydropic pathologies is typically higher in frequency. Tinnitus of rather low-frequency (LF) quality was also reported to occur for about 90 s in normal-hearing participants after presentation of intense, LF sound (120 dB SPL, 30 Hz, 90 s). LF sound has been demonstrated to also cause temporary endolymphatic hydrops in animal models. Here, we quantify tinnitus in two study groups with chronic (MD patients) and presumably transient endolymphatic hydrops (normal-hearing participants after LF exposure) with a psychophysical procedure. Participants matched their tinnitus either with a pure tone of adjustable frequency and level or with a noise of adjustable spectral shape and level. Sensation levels of matching stimuli were lower for MD patients (mean: 8 dB SL) than for normal-hearing participants (mean: 15 dB SL). Transient tinnitus after LF-exposure occurred in all normal-hearing participants (*N* = 28). About half of the normal-hearing participants matched noise to their tinnitus, the other half chose a pure tone with frequencies below 2 kHz. MD patients matched their tinnitus with either high-frequency pure tones, mainly above 3 kHz, or with a noise. Despite a significant proportion of MD patients matching low-pass (roaring) noises to their tinnitus, the range of matched stimuli was more heterogeneous than previous data suggested. We propose that in those participants with noise-like tinnitus, the percept is probably generated by increased spontaneous activity of auditory nerve fibers with a broad range of characteristic frequencies, due to an impaired ion balance in the cochlea. For tonal tinnitus, additional mechanisms are conceivable: focal hair cell loss can result in decreased auditory nerve firing and a central auditory overcompensation. Also, normal-hearing participants after LF-exposure experience alterations in spontaneous otoacoustic emissions, which may contribute to a transient tonal tinnitus.

#### Keywords: tinnitus, low-frequency, Ménière's disease, bounce phenomenon, endolymphatic hydrops

#### *Edited by:*

*Pim Van Dijk, University Medical Center Groningen, Netherlands*

#### *Reviewed by:*

*Abhineet Lall, Holy Spirit Hospital, India Paul Avan, University of Auvergne, France*

*\*Correspondence:*

*Markus Drexl<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Neuro-otology, a section of the journal Frontiers in Neurology*

*Received: 14 July 2016 Accepted: 12 December 2016 Published: 05 January 2017*

#### *Citation:*

*Ueberfuhr MA, Wiegrebe L, Krause E, Gürkov R and Drexl M (2017) Tinnitus in Normal-Hearing Participants after Exposure to Intense Low-Frequency Sound and in Ménière's Disease Patients. Front. Neurol. 7:239. doi: 10.3389/fneur.2016.00239*

**Abbreviations:** BP, bounce phenomenon; DPOAE, distortion product otoacoustic emission; ICC, intra-class correlation coefficients; IHCs, inner hair cells; LF, low-frequency; MD, Ménière's disease; NH, normal-hearing; OAE, otoacoustic emission; OHCs, outer hair cells; SOAE, spontaneous otoacoustic emission.

# INTRODUCTION

Tinnitus is defined as the perception of sound in the absence of external acoustic stimulation and can take various forms from pure tones to more atonal percepts (1, 2).

Tinnitus can be distinguished into two main classes: objective tinnitus and subjective tinnitus. Objective tinnitus is caused by sounds originating from internal sources, e.g., from the patient's inner ear such as prominent spontaneous otoacoustic emissions (SOAEs) (3, 4). Subjective tinnitus is characterized by an abnormal spontaneous activity within the auditory periphery or the central auditory pathway in the absence of any acoustic stimulation, which is interpreted by the brain as sound (5, 6). A psychophysical characterization of tinnitus serves to quantify loudness and pitch/timbre, which can, in the second step, contribute to the understanding of the underlying pathology (7). For the sake of simplicity, in the following, the term pitch will be applied to pure tones and noises, although the term pitch refers to pure tones only and the equivalent for noises would be timbre. Pitch and loudness of a tinnitus sensation are typically characterized by matching a synthesized sound to the tinnitus percept [see Ref. (8, 9) for an overview]. Pitch matches of tinnitus patients are usually in the rather high-frequency region above 3 kHz and only rarely below 1 kHz (10).

However, patients with Ménière's disease (MD) seem to be an exception regarding tinnitus pitch. MD is classically characterized by a triad of symptoms: fluctuating hearing loss, episodes of vertigo, and tinnitus (11). In early stages of the disease, tinnitus seems to be related to episodes of vertigo, being more intense before and during attacks. With progression of the disease, which is typically associated with increasing hearing loss and ceasing of vertigo, tinnitus stays and its intensity increases (12–14). Descriptions of tinnitus in MD patients range from roaring over buzzing to ringing (13, 15); except for few MD patients presumably in later stages of the disease, who describe their tinnitus as tonal and containing high-pitched components (12, 13). Overall, studies concluded that tinnitus in the majority of MD patients was confined to lower-frequency ranges below 1 kHz and in particular between 125 and 250 Hz (16–21).

An excess of endolymph volume, termed endolymphatic hydrops, is often suggested to cause MD and specifically symptoms such as tinnitus. A transient endolymphatic hydrops was experimentally induced in rodents by an intense low-frequency (LF) sound (22).

In human participants, transient endolymphatic hydrops induced by an intense LF sound has not been shown directly. However, a post-LF-exposure phenomenon called "Bounce Phenomenon" (BP), lasting only a few minutes, was found in humans. The BP includes a transient tinnitus percept, fluctuating hearing thresholds with transient improvement and subsequent worsening (23–26), and biphasic amplitude level changes in otoacoustic emissions (OAEs) (27–31). As OAEs are sounds emitted by the cochlea due to active amplification processes by outer hair cells (OHCs) (32), the origin of the BP is thought to be cochlear (33). Increased endocochlear potentials in the cochlea have been recorded after presentation of LF sound (22) and were suggested to lead to an increase in the spontaneous firing rate of auditory nerve fibers, resulting in a "rate tinnitus" (34), presumably in the absence of any structural cochlear impairment.

In BP studies, human participants reported a transient "roaring" tinnitus immediately after LF sound exposure (23, 26, 27). The time course and relative loudness of the transient tinnitus percept were characterized with psychophysical studies (26, 30), but tinnitus pitch has not been systematically described with psychophysical measures yet. This study employed a tinnitusmatching procedure to quantify and compare tinnitus percepts in MD patients and normal-hearing (NH) participants after exposure to intense LF sound. Both study groups presumably presented with impaired cochlear ion balance that might manifest in endolymphatic hydrops in MD patients and shows indirectly as BP after LF sound in NH participants.

# MATERIALS AND METHODS

#### Subjects

The study population consisted of two groups: a group of NH participants and a group of patients with MD. In the following, members of both groups will be referred to as participants. NH participants comprised 18 females and 10 males (age range 20–29, mean age 23.7) with no reported hearing problems, no ear surgery, no recent ear infections, and no tinnitus. All NH participants had hearing thresholds of less than 25 dB HL between 0.25 and 8 kHz, tested with a Matlab-based automated procedure [Automatic Pure Tone Audiometry APTA-HF 2012 V2.28 (HörTech, Oldenburg, Germany)]. One ear was pseudorandomly chosen (14 left ears, 14 right ears) and exposed to a LF sound. The transient tinnitus was lateralized and perceived on the exposed side only.

The group of MD patients consisted of nine females and nine males (age range 26–74, mean age 53.1) diagnosed with definite MD according to the criteria recently formulated by the Bárány Society joint with several national and international organizations (14). Additionally, endolymphatic hydrops had been detected with magnetic resonance imaging after intra-tympanic gadolinium injection (35) before study participation in 15 of 18 patients.

Ménière's disease patients were included when they were unilaterally affected only (10 left ears, 8 right ears) and when they reported tinnitus localized to the affected ear.

Patients with middle ear disorders, pathologies of the auditory nerve or recent ear infections were excluded from the study. Patients with obvious noise-related hearing damage (i.e., notched audiograms) as well as patients with a history of noise exposure were not eligible either. All MD patients were required to be in a stage of the disease with fluctuating symptoms, with at least one vertigo attack during the 6 months preceding the experiment. In the contralateral, non-affected ear patients were required to have hearing thresholds better than 40 dB HL below 2 kHz, and better than 70 dB HL at higher frequencies.

This study was approved by the ethics committee of the University Hospital of the Ludwig-Maximilians-Universität Munich, Germany, in agreement with the Code of Ethics of the World Medical Association for experiments involving humans (Declaration of Helsinki) and all participants (MD and NH) gave their written informed consent.

Experiments with NH participants were conducted in a double-walled, sound-attenuated booth at the Department of Biology, Ludwig-Maximilians-Universität Munich, Martinsried. Experiments with MD patients were carried out in a sound-attenuated booth at the ENT Department at the University Hospital of the Ludwig-Maximilians-Universität Munich, Germany.

#### Signal Generation and Data Acquisition

Signal generation and data acquisition was implemented with scripts written in MATLAB 7.5 (MathWorks, Natick, MA, USA). Sound generation and acquisition was done with an RME Fireface UC 24-bit external sound card (RME, Audio AG, Haimhausen, Germany). The sampling rate was 44.1 kHz. For sound stimulation, SoundMexPro (HörTech GmbH, Oldenburg, Germany) was employed, which enables low-latency multi-channel Audio Stream Input/Output and interactive changes of stimuli properties within the MATLAB environment.

#### SOAEs Recording

In NH participants, SOAEs were recorded with the ER-10C distortion product otoacoustic emission (DPOAE) probe microphone (Etymotic Research Inc., Elk Grove Village, IL, USA). The recorded signal was amplified 30 dB by the preamplifier of the external sound card. Level and frequency of SOAEs were recorded in the control trial for 120 s (measured in 21 of 28 NH participants) and after LF stimulation for 240 s (measured in 15 of 28 NH participants). In an artificial ear (B&K 4157, Brüel & Kjaer Sound and Vibration Measurement A/S, Denmark), no artifacts exceeding the noise floor of the system could be detected during recording. A probe-fit-check procedure preceded and concluded each measurement by presenting a band-stop noise consisting of a low- and a high-frequency band and analyzing the ear response using a Fourier transform analysis. If the probe-fit-check procedure at the end of a trial indicated that the probe position had changed, the trial was rejected and repeated.

### Tinnitus-Matching Procedure

In experiments with MD patients, the output of the sound card was sent to HDA 200 headphones (Sennheiser, Wedemark-Wennebostel, Germany). In NH participants, two different sound systems were used for the two ears: an ER4 insert ear phone (Etymotic Research Inc., Elk Grove Village, IL, USA) was used to present the matching stimuli to one ear. An ER-10C DPOAE probe system with an additional tube coupled to an external transducer was used to present LF sounds (30 Hz sine wave, 120 dB SPL, 90 s, including 0.1 s raised-cosine ramps) to the other ear in order to elicit a tinnitus sensation.

The external transducer was a small broadband unit (NSW1- 205-8A, Aura Sound Inc., Santa Fe Springs, CA, USA) driven by a RB-960BX power amplifier (Rotel, Worthing, UK). This transducer was connected to a 50-cm long polyethylene tube (inner diameter 1 mm), the tip of which was fed through the foam ear tip of the ER-10C DPOAE probe. The ER-10C includes a microphone for recording sound pressure in the ear canal enabling the examiners to calibrate the LF sound. The amplitude response of the DPOAE probe microphone was compared with the amplitude response in an artificial ear (B&K 4157, Brüel & Kjaer Sound and Vibration Measurement A/S, Denmark) and corrected for deviations. The level of the first harmonic of the LF sound was at least 50 dB lower than the level of the desired LF frequency.

Participants (NH and MD) were given standardized, written and illustrated instructions for the tinnitus-matching procedure. In NH participants, each trial was started with LF-sound exposure to one ear, thereby inducing a transient tinnitus percept in the exposed ear (see **Figure 1A**). Since the transient tinnitus lasted only for about 90 s after LF exposure (30), NH participants were allowed to restart the LF stimulus playback if necessary. As tinnitus was a unilateral symptom in both groups, matching stimuli were presented to the contralateral, unaffected ear (see **Figures 1A,B**). Participants were able to interactively adjust matching stimuli regarding loudness and pitch with a gamepad (Bigben Interactive GmbH, Bergheim, Germany). Matching stimuli were continuous and generated in real time by a SoundMexPro plug-in.

The matching procedure was split into two stages. The first stage served to determine whether the participants' tinnitus was matched best either with a pure tone or with a noise. Therefore, 10 different synthesized stimuli were presented. The stimuli comprised pure tones of five different frequencies, equally spaced on a logarithmic frequency axis between 0.25 and 8 kHz (0.25, 0.595, 1.414, 3.364, and 8.0 kHz) and five noises derived from Gaussian noise with different filter slopes (see **Figure 1C**). In the following, those slopes are referred to as spectral tilts with units of decibel per octave. They ranged from negative values with dominant LF components (−12 and −6 dB/octave) over white Gaussian noise (0 dB/octave) to positive values with dominant high-frequency components (+6 and +12 dB/octave). Stimuli were filtered such that linear phase and frequency distortions of the transducers (NH participants: ER4, Etymotic Research; MD patients: HDA 200, Sennheiser) were exactly compensated for. Participants started the playback of the individual stimuli successively by choosing from a graphical representation of the stimuli on a user interface. No information regarding the physical properties of the stimuli was available from the graphical representation. The 10 stimuli were identical but reordered on the user interface for each trial.

After having listened to all stimuli, participants were asked to select the stimulus best matching their tinnitus. After stimulus selection, participants had to adjust the stimulus level, such that the selected tone matched their tinnitus as well as possible. Selecting 1 out of the 10 stimuli and adjusting its loudness was counted as one trial in the first stage of the matching procedure. In NH participants, trials were repeated until the same stimulus class was selected in two successive trials. In MD patients, due to time constraints, only two trials were carried out regardless of whether participants selected the same stimulus or different stimuli in those two trials.

In the second stage, participants carried out fine adjustments of level and pitch of either a pure tone or a noise. Matching stimuli were presented with a starting frequency or starting spectral tilt corresponding to the selected stimulus of the first stage, respectively. In MD patients selecting two different stimuli in the first stage, the matching stimulus was chosen based on the patient's statement of which of the two selected stimuli was the better fit.

The starting sound level was the mean sound level derived from the last two trials of the first stage.

During presentation of the matching stimulus, participants could continuously adjust loudness and frequency or spectral tilt (see **Figure 1D**). When participants found an adjustment for the matching stimulus that resembled their tinnitus, they stopped the adjustment procedure. Subsequently, the adjusted matching stimulus was again presented continuously and participants were required to adjust its sound level such that it was just audible. Similar to a Békésy tracking procedure, participants decreased the level of their adjusted matching stimulus until they did not perceive it anymore and then increased its intensity until they heard the stimulus again. This was done to estimate the sensation level of the matching stimulus. Adjusting the matching stimulus pitch and level and tracking the corresponding hearing threshold was considered as one trial in the second stage of the matching procedure.

In this second matching stage, NH participants always ran five trials and MD patients two to five trials depending on how many they were capable of doing due to time constraints and cognitive load.

After each trial, NH participants and MD patients were asked to describe their transient tinnitus in their own words. In NH participants, measurements were taken on two to four different days and lasted between 25 and 60 min each. In MD patients, testing was embedded in their clinical routine and carried out on 1 or 2 days. Measurements lasted 40–60 min.

All data analysis and statistics were carried out with scripts written in MATLAB 7.5 (MathWorks, Natick, MA, USA). Visualizations were done either with MATLAB 7.5 or Inkscape 0.91 (The Inkscape Team, http://www.inkscape.org).

#### RESULTS

# Estimates of Level and Pitch of Tinnitus in NH Participants after LF Sound Exposure

All 28 NH participants exposed to the LF sound experienced a transient tinnitus percept and were able to match its pitch and level with a matching stimulus presented to the contralateral ear. Matching stimuli were adjusted after offset of the LF exposure, on average within 68.7 ± 32.5 s (mean ± SD, *N* = 28). This suggests that most NH participants concluded the matching while still hearing the transient tinnitus, which lasts about 90 s on average (30).

Conclusions drawn from questioning the NH participants after the software-based adjustment procedure are summarized as follows: 7 of the 28 NH participants described their tinnitus as tonal, 8 NH participants as a noise. The remaining 13 NH participants reported a hybrid percept consisting of noise and one or more tones. During the matching procedure, 8 out of those 13 NH participants experiencing a hybrid percept selected a matching tone and only 5 selected a matching noise. Altogether, 15 NH participants selected and adjusted a tone and 13 NH participants selected a noise (**Figure 2A**). Subjective descriptions of tinnitus perceived by NH participants after LF exposure are summarized in **Table 1**.

The loudness of the transient tinnitus percept was reported to be faint up to clearly audible with the tinnitus starting to fade out after a minute. Some participants also described qualitative changes in their tinnitus percept over time. Most of those participants reported a hybrid percept of pure tone(s) and noise. In those cases, the relative contribution of noise and tones to the tinnitus percept shifted over time. The tonal components seemed to fade out over time, while the noise serving as a background noise at the beginning of the BP got more prominent at later time points. Participants did not complain about the percept and its loudness. At most participants stated the tinnitus to be irritating, especially in combination with a feeling of aural fullness.

# Tonal Tinnitus Percepts in NH Participants after LF Exposure

Fifteen NH participants selected pure tones to characterize their tonal tinnitus. The pure tone frequency was determined by averaging all adjustments per subject. The selected matching tone frequencies (see **Figures 3A,B**) show an accumulation between 0.1 and 2 kHz, where 14 out of 15 participants selected matching tones below 2 kHz.

The mean frequency of the 15 averaged pure tones chosen was 0.96 ± 0.89 kHz (mean ± SD, *N* = 15). As the SD across adjustments in hertz is only of limited value, frequency deviations were also expressed logarithmically as fraction of an octave in cent,

TABLE 1 | Qualitative description of transient tinnitus after intense lowfrequency sound exposure (30 Hz, 120 dB SPL, 90 s) by normal-hearing participants (*N* **=** 28).

where 100 cents equal a semitone, so that 1,200 cents correspond to an octave.

In 13 of 15 NH participants, SDs for single participants comprised less than 1,200 cents, and for some participants, the SD was as low as 30 cents. The inter-subject mean of the sound pressure level of the adjusted matching tone was around 49.5 ± 15.9 dB SPL (mean ± SD, *N* = 15).

Sound levels for matching tones decreased with increasing frequency from 90 dB SPL at 70 Hz to 30–35 dB SPL at 2–4 kHz. Matched pure tone levels followed equal-loudness contours for human hearing within a loudness level range of 25–60 phons (see **Figure 3A**). Thus, for NH participants, matching tones at lower frequencies were not generally perceived louder than at higher frequencies, despite the difference in sound pressure levels. For an interpretation of how loud participants (NH and MD) perceived their tinnitus, sensation levels were calculated (see **Figure 4A**). The sensation level is the difference (in decibel) between the presented sound level and the absolute hearing threshold for the same sound. The mean sensation level for tonal tinnitus matches of NH participants was 15.2 ± 6.7 dB SL (mean ± SD, *N* = 15).

# Noise-Like Tinnitus Percepts in NH Participants after LF Exposure

Thirteen NH participants chose noises to match their LF-induced transient tinnitus. Adjusted spectral tilts showed a bimodal distribution slightly shifted toward negative tilts with an average adjusted spectral tilt of −1.8 ± 7.9 dB/octave (mean ± SD, *N* = 13) (see **Figures 5A,B**). Except for one participant choosing white noise (mean tilt: ~0 dB/octave) to match the tinnitus percept, all NH participants selected either noises with a clearly positive tilt (mean ± SD = 6.7 ± 3.3 dB/octave, *n* = 5) or with a clearly negative tilt (mean ± SD = −8.1 ± 3.1 dB/octave, *n* = 7).

FIGURE 3 | Adjustments of level and frequency of externally presented pure tones to match a tonal tinnitus percept. (A,B) Normal-hearing participants (*N* = 15). (C,D) Ménière's disease patients (*N* = 7). (A,C) Plots show adjusted pure tones of individual participants. Data from individual participants are represented by different colors. Square symbols represent the mean of the two to five individual adjustments of one participant (indicated by cross symbols). Error bars represent the SD regarding sound pressure level (dB SPL) (vertically) and frequency (Hz) (horizontally). Lines in (A) represent equal-loudness contours for loudness levels at 25 and 60 phons. (B,D) Distribution of the selected matching tone frequencies of data shown in (A,C), respectively (bin width = 1,249 cents).

Within-participant variability between adjustments was quite small (SD between 0.02 and 4.4 dB/octave).

Sound levels of the matching noises were within the range of 36–70 dB SPL with a mean of around 49.5 ± 12.9 dB SPL (mean ± SD, *N* = 13). Matching noises with highest sound pressure levels were noises with the steepest spectral tilts, either negative or positive. The mean sensation levels of matching noises were 15.3 ± 5.6 dB SL (mean ± SD, *N* = 13).

Sensation levels of both selected matching tones and matching noises in NH participants were summarized (see **Figure 4A**), and the mean was calculated as 15.2 ± 6.1 dB SL (mean ± SD, *N* = 28).

### Estimates of Level and Pitch of Tinnitus in MD Patients

In all 18 MD patients, the tinnitus was a permanent sensation, sometimes varying in loudness over time. In eight patients, the loudness of the tinnitus was apparently correlated to the vertigo attacks increasing right before and/or during an attack. Three patients reported not only a loudness change correlated to the attacks but also an increase of tinnitus pitch. In this case, patients were asked to match their current tinnitus percept. Seven patients reported their tinnitus to be a noise, and five patients reported it to be a pure tone. The other six patients reported to hear a hybrid percept of pure tone(s) and noise. In the tinnitus-matching procedure, two of those patients perceiving a hybrid tinnitus matched their tinnitus with a pure tone and four of them with a noise (see **Figure 2B**). Qualitative tinnitus descriptions by the patients are summarized in **Table 2**. Descriptions included low-pitched noise, modulated noise, and high-frequency whistling.

#### Tonal Tinnitus in MD Patients

Seven MD patients selected pure tones in the matching procedure. Thereby, data did not show the previously suggested correlation that older patients or patients in later stages of the disease were more likely to experience tonal tinnitus of high-frequency quality instead of noise-like tinnitus (linear correlation coefficients, age: *r*=−0.12, *p*-value = 0.64, stage of disease *r*= 0.08, *p*-value = 0.76).

FIGURE 5 | Adjustments of level and frequency content of externally presented noises to match a noise-like tinnitus percept. (A,B) Normal-hearing participants (*N* = 13). (C,D) Ménière's disease patients (*N* = 11). (A,C) Plots show adjusted noises of individual participants. Error bars represent the SD regarding sound pressure level (dB SPL) (vertically) and frequency content (dB/octave) (horizontally). (B,D) Distribution of the matching noise tilts of the data shown in (A,C), respectively, within the range of −17.5 to +17.5 dB/octave (bin width = 5 dB/octave). Color coding and symbols as in Figure 3.

TABLE 2 | Qualitative description of tinnitus by Ménière's disease patients (*N* **=** 18) with unilateral disease and one-sided, permanent tinnitus.

Selected matching tone frequencies showed a bimodal distribution with peaks around 500 Hz and around 5 kHz (see **Figures 3C,D**). The mean frequency was 4.6 ± 3.3 kHz (mean ± SD, *N* = 13).

Matched sound levels increased with increasing frequency from 20 dB SPL at 0.6 kHz to 80–100 dB SPL at 7–8 kHz. Sensation levels of matching tones, however, were small (mean ± SD = 7.0 ± 7.8 dB SL, *N* = 7).

# Noise-Like Tinnitus Percepts in MD Patients

Eleven of 18 MD patients selected noises to describe their tinnitus percept. The matching noise properties were averaged across the two to three trials for each patient.

Across patients, the average spectral tilt of matched noises was −2.9 ± 6.3 dB/octave (mean ± SD, *N* = 11). Matching noises showed adjusted tilts between −13 and +4 dB/octave and resulted in an almost normal distribution with a shift toward negative tilts (see **Figures 5C,D**).

On average, the noise level adjusted by MD patients was 50.4 ± 17.3 dB SPL (mean ± SD, *N* = 11), which was comparable to noise levels of noises matched by NH participants with transient tinnitus. Due to hearing loss in the majority of MD patients, the sensation levels of the matching tones were with 8.5 ± 4.9 dB SL (mean ± SD, *N* = 11) significantly lower than in NH participants (Mann–Whitney test, *p*-value = 0.0002).

Sensation levels of both matched tones and matched noises in MD patients were summarized (see **Figure 4B**), and the mean was 7.9 ± 6.0 dB SL (mean ± SD, *N* = 18).

#### Comparison of Tinnitus Percepts in MD Patients and in NH Participants after LF-Sound Exposure Tonal Tinnitus Percepts

#### Pure tones matched by MD patients and matched by NH participants differed in their distribution (see **Figure 3**) (twosample Kolmogorov–Smirnov test, *p*-value = 0.019). While the majority of MD patients chose pure tones above 3 kHz, which is comparable to most tinnitus percepts of tinnitus patients (10, 16), the majority of NH participants selected pure tones below 2 kHz.

#### Noise-Like Tinnitus Percepts

Comparing the two subgroups of MD patients selecting noises and NH participants with BP-induced tinnitus selecting noises did not show pronounced differences. Although the distribution appeared bimodal for NH participants and rather normally distributed in MD patients, the null hypothesis stating that the two subgroups were from equal distributions could not be rejected (two-sample Kolmogorov–Smirnov test; *p*-value = 0.86). Both subgroups had a tendency to select noises with a negative spectral tilt, which corresponds to previous reports from the literature (16–18, 23, 30).

#### Test–Retest Reliability

In general, the pitch and loudness matches obtained with the current two-stage procedure showed good test–retest reliability. Intra-class correlation coefficients (ICC) were calculated for the five trials of fine matching run in one to three different sessions in NH participants (pitch matching: ICC = 0.92; loudness matching: ICC = 0.85). In MD patients, data were usually collected in one session only, but for the two to three different adjustments ICC indicated very strong test–retest reliability, too (pitch matching: ICC = 0.93; loudness matching: ICC = 0.92).

#### SOAE Measurements in NH Participants

In 15 of the 28 NH participants, SOAEs were measured in the same ear that was exposed to the LF sound. The first measurement was run before LF exposure to find SOAEs. The second SOAE measurement was carried out immediately after LF exposure during the BP, while NH participants perceived the corresponding transient tinnitus. During the BP, "new" SOAEs could develop and existing SOAEs increased in level. Occasionally, those SOAEs exceeded the hearing threshold determined for the same ear by audiometric testing at the beginning of the experiments. In 10 NH participants, SOAEs could be found. In all of those participants, SOAEs were slightly altered when recorded during the BP compared to baseline before LF exposure. In eight NH participants, SOAE levels exceeded the individual hearing threshold at the SOAE characteristic frequency during the BP. From these eight participants, five matched their tinnitus with a pure tone, three with a noise. **Figure 6** shows SOAEs, hearing threshold and selected tinnitus-matching tones from three participants with "audible" SOAEs. These data suggest that some NH participants might possibly have matched their tinnitus to a transiently audible SOAE.

#### DISCUSSION

The current study, by applying a comprehensive tinnitusmatching procedure, compared tinnitus in two groups with

chronic (MD patients) and transiently (NH participants after LF exposure) challenged cochlear homeostasis presumably resulting in hydropic conditions.

Our results show that sensation levels of tinnitus percepts were on average between 8 dB SL (MD patients) and 15 dB SL (NH participants after LF sound). Tinnitus pitch varied substantially both within and across MD patients and NH participants. While MD patients matched their tinnitus mainly with either high-pitched pure tones or noises with a tendency to low-pitched (roaring) noises, NH participants perceiving a transient tinnitus after LF-exposure matched that tinnitus with high- or low-pitched noises or low-pitched pure tones. In the few studies available in the literature, MD patients were found to perceive mainly LF tinnitus (16–21), albeit reports on MD patients with high-frequency tinnitus exist (12, 13, 36). Tinnitus percepts of MD patients in the current study were more diverse, and this is probably due to the following reasons: first, the matching procedures employed in the available literature differed from our advanced matching paradigm, in that participants could adjust matching stimuli on their own without any interference or bias caused by the examiner. Furthermore, participants could choose between two matching stimulus classes (pure tones and noises). Second, participating MD patients were heterogeneous regarding age (26–74 years). As the onset of MD symptoms generally occurs around 30–50 years (37, 38), younger MD patients without age-related hearing loss are underrepresented in our patient population. Nevertheless, contralateral, unaffected ears of all participating patients were within the normal hearing range when considering the age-corrected pure-tone average at 2, 3, and 4 kHz. This is important as the matching stimulus was delivered to the unaffected ear. All patients, despite their age, were in a stage of the disease with fluctuating symptoms.

In the following, we will dissect tinnitus percepts into tonal and noise-like tinnitus and discuss tinnitus loudness for both kinds of percepts in the end. Underlying mechanisms possibly generating the tinnitus percepts in both MD patients and NH participants after LF exposure will be proposed for tonal and noise-like tinnitus separately.

#### Tonal Tinnitus Generation

MD patients mainly chose high-frequency matching tones when their tinnitus was predominantly tonal. This is comparable to the tinnitus quality typically related to inner hair cell (IHC) and OHC damage (39). Here, it was suggested that the tinnitus frequency roughly corresponds to characteristic frequency of the impaired hair cells or to the frequency at the boundary between intact and impaired hair cells (40, 41). This tinnitus mechanism might generate the tonal tinnitus percepts MD patients perceive. Audiograms of MD patients often show LF hearing loss but high-frequency hearing loss also occurs, mostly in later stages of the disease (12, 13). The majority of MD patients in this study showed greatly reduced DPOAE levels as judged from measurements acquired during the clinical routine. This indicates some degree of OHC function impairment, but it is unclear if this is age-related or due to the presence of endolymphatic hydrops. OHC loss might not suffice to induce tinnitus. Therefore, damage to IHCs or the auditory nerve, which cannot be detected with DPOAE measurements, might also be required. Given the presence of both IHC and OHC dysfunction, tonal tinnitus could also be the result of endolymphatic hydrops (or its underlying pathology) combined with the pre-existing hair cell dysfunction.

Tonal tinnitus in NH participants after LF exposure showing frequencies below 2 kHz is presumably triggered by transiently challenged cochlear homeostasis. Intense, LF sound causes broad excitation patterns peaking at the apical part of the cochlea. Thus, effects on cochlear homeostasis are not restricted to the characteristic frequency region of LF sound and higher-frequency regions can be affected as well.

Temporary tinnitus in humans is generally thought to be caused by temporary decrease of OHC amplification, which results in increased firing in the central auditory system and tinnitus generation (42). Although this generation mechanism cannot be ruled out, especially for participants showing temporary worsening of hearing threshold after LF exposure, it seems to be unlikely because of concurrent, increasing OAE levels typically following LF exposure due to enhanced OHC amplification (30). Temporary enhanced amplification and increased OAE levels might contribute to a different generation mechanism.

In three participants, it could be shown that frequencies of the selected matching tones roughly corresponded to the recorded SOAE frequencies. Besides the fact that permanent tinnitus percepts originating from an SOAE are rare [between 2 and 4.5% (4, 43)], SOAEs seem to be a plausible explanation in the case of transient tonal tinnitus after LF sound exposure for the following reasons:

Tinnitus frequencies selected by NH participants in the present study correspond to typical SOAE frequencies. SOAEs in human adults are usually in the range between 0.9 and 4 kHz with distribution maxima near 1.5 and 3 kHz (44, 45).

Under normal conditions, SOAEs are relatively stable in both frequency and level (46). After intense LF exposure, however, frequency and level of SOAEs slowly cycle for a time period of about 2 min in a stereotypic manner with a level maximum typically occurring about 50 s after LF sound offset (27, 31). SOAEs presumably buried in the noise floor become detectable during that period. Typically, SOAEs are not perceived by their owner (47). It has been shown, however, that induced frequency or level changes of SOAEs can result in SOAEs becoming transiently audible (47–49), as the inhibition of SOAE perception at higher stages of auditory processing (45) might not be active anymore. Not all NH participants perceiving tonal tinnitus had SOAEs with frequencies similar to the chosen matching tone frequency. NH participants with recordable SOAEs often showed more than one SOAE, which would have resulted in a percept difficult to match with a single pure tone.

In NH participants without recordable SOAEs, SOAE levels might be significantly underestimated by external recordings after OAE back-propagation, compared to sound pressure level of OAEs within the cochlea (47). This is strongly supported by the comparison of DPOAE sound levels measured in the gerbil meatus and the electrophysiological response to the same DPOAE in the gerbil cochlear nucleus (50). Thus, for SOAEs to be perceived by their owner, it might not be necessary that SOAE levels, as measured in the meatus, exceed the individual hearing thresholds at the SOAE characteristic frequency.

#### Noise-Like Tinnitus Generation

In both subject groups, a major proportion of tinnitus matches indicated a noise-like tinnitus. In experimental animals, it has been shown that the endocochlear potential increases temporarily with intense LF exposure (22, 51), resulting in a depolarization of IHCs and consequently in an increased spontaneous activity of the auditory nerve (34).

While direct recordings of the endolymphatic potential in MD patients are not feasible, a similar mechanism is nonetheless conceivable. Increased spontaneous activity of the auditory nerve could lead to the perception of a tinnitus, for which Patuzzi (34) coined the term "rate tinnitus." If a large expanse of the cochlea was affected by the above mechanism, a noise-like percept would result.

MD patients perceiving noise-like tinnitus percepts chose mainly noises with negative tilts (low pitches). This is in line with reports that MD patients typically show LF hearing loss at frequencies below 3 kHz (14), suggesting that, for hitherto unknown reasons, the apical part of the cochlea is most affected, resulting in a LF rate tinnitus. But, presumably depending on the duration of the disease, audiograms of MD patients reveal a broad range of affected frequencies (15), which is also reflected in our study population. This might explain why MD patients selected matching noises not necessarily limited to the LF range.

During intense LF stimulation, cochlear excitation is not restricted to the characteristic frequency, but the whole cochlea is excited and individual differences in cochlear excitation patterns might exist. LF stimulation affects the apical end of the cochlea strongest and LF components should be more prominent in noise-like tinnitus after intense LF exposure in NH participants (corresponding to selected noises with negative tilt). Those NH participants that chose high-pitched noises to match their LF-induced tinnitus may have done so as a compromise to represent hybrid tinnitus percepts consisting of both noise and tonal components or cochlear locations other than the apex dominate the tinnitus percept.

#### Tinnitus Sensation Levels

Sensation levels of tinnitus percepts were estimated with previously selected matching stimuli. In MD patients, hearing thresholds even on the contralateral, unaffected ear were slightly elevated at higher frequencies due to age-related hearing loss. Therefore, for MD patients choosing a high-pitched pure tone to match their tinnitus, loudness matches might have resulted in lower values than loudness matches using frequencies at which hearing was normal (8, 52). Loudness recruitment could also contribute: for participants suffering from cochlear hearing loss, low to moderate sensation levels may be much louder than for NH participants because the lack of cochlear compression decreases the overall dynamic range of loudness perception dramatically (52, 53). Furthermore, low sensation levels of matching tones in MD patients can be due to tinnitus loudness fluctuations. Patients were measured between vertigo attacks when tinnitus loudness was typically lower than immediately before or during attacks.

On the other hand, high loudness values in NH participants could be explained with a shift of attention toward the suddenly occurring tinnitus percept. Tinnitus sensation levels after LF exposure were on average higher than tinnitus sensation levels in MD patients and higher than tinnitus sensation levels in most patients with pathologies other than MD. Studies showed that tinnitus loudness was generally matched with stimuli below 10 dB SL, even in participants referring to their tinnitus as loud (8, 10, 54).

#### Procedural Limitations

Participants were presented with a limited range of sounds from which they had to select. Although this limited range of sounds was chosen to facilitate the matching procedure, participants complained about not being able to adjust amplitude modulations or about being restricted to one stimulus class when hearing hybrid percepts with both pure tones and noises. Furthermore, for both MD patients and NH participants with an LF-induced transient tinnitus, tinnitus loudness, and sometimes even tinnitus pitch were varying over time. In case of MD patients, these variations can take place over days or weeks depending on vertigo attacks or the current stage of the disease (12, 13). In NH participants, fluctuations appear within the 1–2 min of tinnitus duration. This is inherent to the transient percept after LF exposure. To guarantee consistency, NH participants were able to retrigger the tinnitus percept by turning on the LF stimulation again.

In either case, tinnitus-matching results can only represent approximations of actual tinnitus percepts, constrained by both choice of offered stimuli and experimental procedures.

# CONCLUSION

Estimates of tinnitus pitch reported in the literature are heterogeneous and depend heavily on the methods used. Here, we implemented a tinnitus-matching procedure with fewer constraints than in previous, comparable studies, including noises with adjustable spectral shapes. As a consequence, our results revealed a relatively large proportion of participants with noiselike tinnitus, which might have not been detected in previous studies mostly employing sinusoidal matching tones. Contrary to reports in the literature, tinnitus pitch in NH participants after LF sound exposure and in MD patients is not exclusively LF, and hybrid percepts with noise-like and tonal components can occur.

Noise-like tinnitus cannot easily be explained with localized damage to cochlear regions. Rather, a mechanism affecting a broad frequency range is needed. An increase of spontaneous activity of the auditory nerve was suggested to cause the noise-like tinnitus observed here. However, further experiments are now required to identify unusual patterns in auditory nerve spontaneous activity as a potential tinnitus generator in chronic and induced hydropic conditions.

### AUTHOR CONTRIBUTIONS

MU contributed to study design, performed data acquisition, statistical analysis and interpretation of results, drafting of the manuscript, revised the manuscript, and approved the final manuscript. LW contributed to study design and data acquisition, critically reviewed and approved the final manuscript. EK contributed to data acquisition, revised and approved the final manuscript. RG contributed to data acquisition, revised and approved the final manuscript. MD conceptualized and designed the study, contributed to data acquisition, interpretation of results, drafting of the manuscript, critically reviewed and approved the final manuscript. All authors are agreeable to be accountable for the content of the work, integrity, and accuracy of the data.

#### REFERENCES

#### ACKNOWLEDGMENTS

The authors wish to thank Benedikt Grothe for his valuable support of the study and Kathrin Kugler for critically reading earlier versions of the manuscript. Parts of this work have been presented at the 39th MidWinter Meeting of the Association for Research in Otolaryngology in San Diego, CA, USA.

#### FUNDING

This work was funded by a grant (01EO1401) from the German Ministry of Education and Research (BMBF) to the German Center for Vertigo and Balance Disorders (DSGZ) (project TRFII-6).

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2017 Ueberfuhr, Wiegrebe, Krause, Gürkov and Drexl. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Analysis of Audiometric Differences of Patients with and without Tinnitus in a Large Clinical Database

*Dominik Gollnast1†, Konstantin Tziridis1 \*† , Patrick Krauss1,2, Achim Schilling1,2, Ulrich Hoppe3 and Holger Schulze1*

*1Experimental Otolaryngology, Department of Otorhinolaryngology, Head and Neck Surgery, Friedrich Alexander University Erlangen-Nürnberg (FAU), Erlangen, Germany, 2Department of Physics, Center for Medical Physics and Technology, Biophysics Group, Friedrich-Alexander University Erlangen-Nürnberg (FAU), Erlangen, Germany, 3Audiology, Department of Otorhinolaryngology, Head and Neck Surgery, Friedrich Alexander University Erlangen-Nürnberg (FAU), Erlangen, Germany*

Human hearing loss (HL) and comorbidities like tinnitus pose serious problems for

#### *Edited by:*

*Winfried Schlee, University of Regensburg, Germany*

#### *Reviewed by:*

*Tobias Kleinjung, University of Zurich, Switzerland Roland Schaette, University College London, UK*

*\*Correspondence:*

*Konstantin Tziridis<EMAIL_ADDRESS>
*† These authors have contributed equally to this work.*

#### *Specialty section:*

*This article was submitted to Neuro-otology, a section of the journal Frontiers in Neurology*

*Received: 29 July 2016 Accepted: 24 January 2017 Published: 09 February 2017*

#### *Citation:*

*Gollnast D, Tziridis K, Krauss P, Schilling A, Hoppe U and Schulze H (2017) Analysis of Audiometric Differences of Patients with and without Tinnitus in a Large Clinical Database. Front. Neurol. 8:31. doi: 10.3389/fneur.2017.00031*

people's daily life, which in most severe cases may lead to social isolation, depression, and suicide. Here, we investigate the relationship between hearing deficits and tinnitus. To this end, we conducted a retrospective study on anonymized pure tone and speech audiometric data from patients of the ENT hospital Erlangen in which we compare audiometric data between patients with and without tinnitus. Overall data from 37,661 patients with sensorineural (SHL) or conductive HL (CHL) with (T, 9.5%) or without (NT, 90.5%) a tinnitus percept in different age groups and with different tinnitus pitches were included in this study. The results of the pure tone audiometry comparisons showed significant differences in T patients compared to NT patients. In young patients, we generally found lower hearing thresholds in T compared to NT patients. In adult patients, differences were more heterogeneous: hearing thresholds in T patients were lower in low frequency ranges, while they were higher at high frequencies. Furthermore, lower thresholds were more often found in CHL patients and could rarely be detected in SHL patients. In speech audiometry, only CHL patients with high-pitched tinnitus showed lower thresholds compared to NT patients' thresholds. The results of this study may point to a biologically plausible functional benefit on hearing thresholds in HL tinnitus patients. We hypothesize that the physiological mechanism of stochastic resonance counteracts HL by adding neuronal noise to the system. This neuronal noise may induce changes in the auditory pathway and finally—as a side effect of threshold improvement—lead to the development of a tinnitus percept. We propose a general model of changed hearing thresholds in T patients, being either decreased or increased compared to NT patients.

Keywords: human, pure tone audiometry, speech audiometry, sensorineural hearing loss, conductive hearing loss, tinnitus model

# INTRODUCTION

Hearing is crucial for audio-verbal communication in humans and by that essential for social interactions. Consequently, hearing impairment pose serious problems for the people's daily life, which in most severe cases may lead to social isolation, depression, and suicide (1, 2). The fact that our

western societies face increasing noise exposure in daily routine, work, and spare time adds to the problem and results in continuously increasing numbers of people suffering from hearing impairments. Studies in the United States revealed about 9% of the general population being affected by hearing deficits in 2000 (3)—with an overrepresentation of 15% in children (4)—and an increase of cases over 10 years when assessed with more advanced testing procedures (5).

In general, factors leading to this high prevalence of hearing impairments are diverse. Consequently, the cause for hearing impairment may be located within different anatomical structures. Hearing disorders may have their origin in outer or middle ear, causing conductive hearing loss (CHL), or sensorineural structures (starting in the cochlea and including all structures along the auditory pathway), causing sensorineural hearing impairments. Of course patients can also suffer from a combination of conductive and sensorineural hearing loss (HL), either with unilateral or bilateral impairments. Probably, the most common cause for sensorineural hearing impairments is noiseinduced HL (NIHL), which can lead to a number of secondary symptoms like tinnitus [for review, see Ref. (6)], depression (7), or hyperacusis (8). With increasing HL over time, the capability of speech discrimination is affected, generating difficulties in everyday life communication (9). As Cruickshanks and colleagues (10) demonstrated that the risk of HL is increased by almost 90% every 5 years, the demographic changes in most industrialized western countries will make age-dependent HL even more relevant in the future.

Tinnitus is a widespread, but poorly understood symptom often seen in HL patients. The prevalence in the general population is assumed to be at around 10–15% (11). Men seem to be more often affected then women up to the age of 75, when prevalence is about equal for both genders (12). About 1–2% of the tinnitus patients state their quality of life being significantly decreased by their phantom percept (13). Although HL and the prevalence of tinnitus are increasing with age (14), it is still under debate if age-related HL or age-related changes in physiological processes in the central and peripheral auditory system are the source of the increasing prevalence of tinnitus (15).

Population studies revealed that individuals with tinnitus on average suffer from stronger HL in high frequencies; patients with low-pitched tinnitus (below 1,500 Hz) show stronger low frequency HL than patients with middle- or high-pitched tinnitus [e.g., Ref. (16–19)]. In any case, the question remains why in some patients with HL subjective tinnitus is developing at all. In a recent study (20), we put forward a model for the physiological improvement of hearing thresholds, which as side effect also explains the development of tinnitus. The model is based on the idea that the auditory system tries to compensate for a HL by means of stochastic resonance (SR) at the receptor level. SR refers to the phenomenon that weak signals that are subthreshold for a given sensor still can be detected and transmitted by that sensor if noise (internal or external) is added to the sensor input, both in technical and physiological systems (21–24). We further assume that HL leads to an unequal distribution of spectral input into the auditory system, with a reduced input from the affected spectral ranges. Our model proposes that the impaired hearing thresholds within those frequency channels may be improved again (at least to a certain degree) by means of SR. Obviously, for SR to work, internal noise has to be generated within the auditory system and fed back to the receptor level (20). We propose that this internal noise is reflected in neuronal hyperactivity. If the HL is permanent, the neuronal hyperactivity that enables SR to compensate for increased thresholds may subsequently cause neuronal plasticity along the auditory pathway and finally may lead to the development of a phantom percept, i.e., subjective tinnitus. In that sense, the model views tinnitus as a side effect of a mechanism within the auditory system that seeks to optimize signal transmission at the receptor level. If this model would be true, we would expect that, in tinnitus patients, initial HL should be compensated to a certain degree, resulting in overall better hearing thresholds in tinnitus patients compared to non-tinnitus patients with comparable damage in the auditory system.

In this retrospective study, we search for data in support of this hypothesis by performing a fine-grained analysis of the audiometric data of over 37,000 patients with different forms of HL, and with or without a tinnitus percept.

# MATERIALS AND METHODS

We performed a retrospective study on anonymized audiometric data from patients with HL who came to the ENT hospital in Erlangen for medical examination. Therefore, no declaration of consent was required by German law. All data were collected between the years 2000 and 2015, HL patients who complained about experiencing pure-tone tinnitus percepts were classified as tinnitus patients (group T) and patients without complains about any form of tinnitus were classified as non-tinnitus patients (group NT). Patients with other forms of tinnitus (e.g., noise-like tinnitus) were not included in this study. Applied audiometric methods were pure-tone and speech audiometry for multisyllabic numbers. Data from 37,661 patients (74,976 ears) including all groups of age [median (25, 75% quantil): 42 (21, 58)] were investigated. Patients were not characterized by their gender or by former or current pathologies not affecting hearing.

#### Testing Procedures

Standardized audiometric testing instruments of an audiological clinic were used for this study. All devices fulfilled the necessary requirements according to ISO 8253-1 and 8253-3. The following audiometric methods were performed: pure-tone audiometry: air conduction hearing level thresholds were measured for both ears separately for every patient. Analyzed frequencies were 250; 500; 750; 1,000; 1,500; 2,000; 3,000; 4,000; 6,000; and 8,000 Hz and HL was calculated (range: −10–130 dB). Speech Audiometry: using the Freiburger test, multisyllabic numbers were presented to each ear separately. For these data, acoustic levels at 50% understanding were calculated and used for further analysis (range: 0–120 dB). Audibility was directly linked to the acoustic level (range: 0–100%). The Freiburg multisyllabic numbers were used since it belongs to our standard procedure to determine the level of 50% understanding. This value usually correlates with the hearing threshold in the low frequency region and is less affected by high-frequency HL. We did not use the monosyllabic words since monosyllabic perception is usually measured at higher levels.

Tinnitus characteristics were determined in terms of signal type and signal level measured in decibel HL by comparison of the internal tinnitus with external sound from the audiometer. Three types of signals were possible: broadband noise, narrow band noise (1/3 octave bandwidth), and pure tones between 0.25 and 8 kHz. Tinnitus loudness was determined by increasing the signal level above hearing threshold slowly in steps of 1 dB and asked the subjects for a comparison with their tinnitus percept.

## Data Preprocessing and Statistical Analysis

Data preprocessing was performed with a custom-made Matlab 2008 program (MathWorks, MA, USA). For statistical analysis Statistica 2007 (StatSoft. Inc., OK, USA) was used. Patients were classified within the preprocessing into groups based on their audiometric data: mild to medium symmetric sensorineural HL in adults [SHL, mean air-bone–gap ≤5 dB across all frequencies, HL difference between both ears <20 dB, maximal HL ≤40 dB; number of ears: *n*(T) = 4,390, *n*(NT) = 26,142], symmetric CHL in adults [CHL, mean air-bone gap >5 dB across all frequencies, HL difference between both ears <20 dB, maximal HL ≤40 dB; *n*(T) = 2,538, *n*(NT) = 24,726], and a group of children and adolescents under 18 years with symmetric HL [HL difference between both ears <20 dB, maximal HL ≤40 dB; *n*(T) = 234, *n*(NT) = 16,946] without further subdivision in SHL or CHL patients.

Furthermore, adult subjects were grouped by age: young adults 18–39 years [SHL: *n*(T) = 1,418, *n*(NT) = 8,540; CHL: *n*(T) = 630, *n*(NT) = 7,642], elder adults 40–60 years [SHL: *n*(T) = 2,164; *n*(NT) = 11,236; CHL: *n*(T) = 1,182; *n*(NT) = 9,254], and seniors >60 years [SHL: *n*(T) = 808; *n*(NT) = 6,366; CHL: *n*(T) = 726; *n*(NT) = 7,830]. The subjective pure-tone tinnitus frequencies were grouped by pitch: low-pitched [<1,000 Hz; *n*(SHL) = 507; *n*(CHL) = 387], medium-pitched [1,000–4,000 Hz; *n*(SHL) = 875; *n*(CHL) = 662], and high-pitched [>4,000 Hz; *n*(SHL) = 3,008; *n*(CHL) = 1,489].

In pure tone audiometry, we aimed to quantify the potential threshold decrease or increase tinnitus may have on hearing thresholds in each single tested hearing frequency. To this end, the difference of mean HL in each frequency was calculated for each age and tinnitus pitch group and its age matched NT patients (mean HL difference, where positive values indicate a threshold decrease, negative values indicate an increase of hearing thresholds by tinnitus in decibel) and compared by multifactorial ANOVAs. Generally, for statistical population analysis, parametrical tests like Students *t*-test and multifactorial ANOVAs were used. Tukey *post hoc* tests enabled detailed analysis within the ANOVAs. Additionally, we compared the paired pure tone and speech audiometry data of 2,548 patients in which both audiometric methods were allied by multiple linear regressions and 2-factorial ANOVA (Figure S1 in Supplementary Material).

### RESULTS

### Pure-Tone Audiometry in Patients with and without Tinnitus Percepts

#### Hearing Thresholds of Children and Adolescents

The HL of young patients without (NT) and with reports of tinnitus (T) was compared. To rule out any age bias due to audiometric limitations in infants, we tested first if any difference in hearing thresholds of very young children (aged 1–9 years) and adolescents (aged 10–17 years) could be found. Neither in NT (*t*-test, mean ± SD: children 20.5 ± 17.3 dB, adolescents 19.2 ± 21.6 dB, *p* = 0.15) nor in T patients group (*t*-test: children 16.0 ± 12.6 dB, adolescents 14.3 ± 19.8 dB, *p* = 0.17) any significant differences were found. Therefore, all subjects were pooled and HL was analyzed by a 2-factorial ANOVA with the factors frequency and group.

**Figure 1A** depicts this variance analysis. The upper panel shows an increase of mean HL toward higher frequencies in all patients (**Figure 1A**, upper panel). Interestingly, young people with tinnitus generally suffered less from HL than non-tinnitus patients (**Figure 1A**, inset), overall showing about 5 dB lower mean hearing thresholds. They were especially less affected at frequencies below 4,000 Hz and above 6,000 Hz (**Figure 1A**, lower panel, Tukey *post hoc* tests, always *p* < 0.05). In **Figure 1B**, the mean HL difference of NT and T patients is given as a function of frequency. Each green bar indicates the significant difference (tested by single sample *t*-tests) of the HL at a given stimulation frequency. Again, all frequencies except 4 and 6 kHz show significantly positive values indicating lower hearing thresholds in T compared to NT patients.

Note that this young patients group is not representative for patients affected by tinnitus in the "general population" where usually only adults are considered (1, 25). For that reason, further analyses were focused on adult groups with SHL and CHL. In these patients, we were able to analyze a significantly larger number of patients' ears.

#### Hearing Thresholds in Adults with and without Tinnitus

In a first overview, NT and T patients' audiograms separated for their cause of HL (sensorineural or conductive) were analyzed by 2-factorial ANOVAs with the factors frequency and group as depicted in **Figures 2A,C** and further illustrated by the HL difference in **Figures 2B,D**, respectively.

In adult patients, we generally found that T patients' thresholds in lower frequency ranges were lower compared to NT patients, while in other frequency ranges there was no difference or, especially above 2 kHz, those patients showed higher thresholds compared to NT patients: a detailed analysis revealed that for both groups, SHL (**Figure 2A**, upper panel) and CHL (**Figure 2C**, upper panel), HL significantly increased from low to high frequencies. HL in general was higher for CHL patients (30.9 ± 20.0 dB) than for subjects suffering from SHL (18.3 ± 16.7 dB; *t*-test, *p* < 0.001).

In patients with SHL, the analysis revealed the opposite result seen in young patients, namely mean thresholds across all

Figure 1 | Hearing loss (HL in decibel) in young patients (aged 1–17 years) with and without tinnitus. (A) Results of a 2-factorial ANOVA on HL with factors frequency (upper panel) and patient group (inset), interaction plot at the bottom. Symbols depict mean values; whiskers give 95% confidence intervals. Asterisks depict significance value of the 1-factorial ANOVA part: \*\*\**p* < 0.001. Gray-shaded area indicates two frequencies not significantly different between groups (Tukey *post hoc* tests). (B) Mean differences between the HL of NT [blue in (A)] and T patients [red in (A)] quantifying the threshold difference referred to as mean HL difference, where positive values indicate lower, negative values higher hearing thresholds in decibel. Values significantly different from 0 are colored green (single sided *t*-tests).

frequencies being higher in patients with tinnitus compared to patients without such a mispercept by roughly 1 dB (**Figure 2A**, inset). In the interaction of both factors, it became clear (Tukey *post hoc* tests, *p* < 0.001) that T patients only showed significantly higher thresholds compared to NT patients at frequencies above 2 kHz (**Figure 2A**, lower panel). This result is further illustrated in **Figure 2B** showing the mean HL difference, which also revealed a significant threshold increase at frequencies above 2 kHz for SHL patients with tinnitus.

In patients with CHL, the analysis of the factor group showed no significant difference between T and NT patients when thresholds were averaged across all frequencies (**Figure 2C**, inset). Nevertheless, the interaction analysis (**Figure 2C**, lower panel) revealed significantly lower thresholds in T patients for frequencies below 750 Hz but higher thresholds compared to NT patients in the range of 3–4 kHz (Tukey *post hoc* tests, *p* < 0.05), which was further supported by the HL difference analysis (**Figure 2D**).

Next, we analyzed different aspects of our patient cohort to outline some additional characteristics of the threshold differences between T and NT patients. First, we focused on the age dependency of thresholds as a function of the existence of a tinnitus percept: SHL patients of all ages showed higher hearing thresholds when affected by tinnitus, while CHL patients with tinnitus only showed such higher thresholds in the age group of 40–60 years. This was assessed by 3-factorial ANOVAs with the factors frequency, age and group as depicted in **Figures 3A,C**. Lower hearing thresholds in T patients only reached significance when analyzed across frequency ranges (low =< 1 kHz, mid =< 4 kHz, high = ≥ 4 kHz; green shaded areas in **Figures 3A,C**) but could be further identified when analyzed by HL differences for specific age groups (**Figures 3B,D**).

Patients with SHL (**Figure 3A**) replicated the results shown in **Figure 2** averaged over all three age groups when factors frequency, group, or the corresponding interaction were analyzed. For the factor age [(F2, 291,140) = 11,515.0, *p* < 0.001], we found lower HL in 18- to 39-year-old adults (12.0 ± 0.2 dB) compared to 40- to 60-year-old adults (19.6 ± 0.15 dB) and adults above 60 years (30.4 ± 0.2 dB). The interaction of age and frequency is given in the upper left panel of **Figure 3A** and the interaction of age and group in the right upper panel. There, T patients showed generally higher thresholds than NT patients (Tukey *post hoc* tests), which is carved out in the three-way interaction in the lower part of **Figure 3A**: in SHL, Tinnitus patients' higher frequencies showed significantly larger thresholds than those of non-tinnitus patients (cf. red shaded areas; statistical tests were performed separately for every stimulation frequency). When 2-factorial ANOVAs [factors frequency range (low, mid, high) and group] were performed, significantly lower hearing thresholds in T patients could be detected for low frequencies in the above 60 years group only (green shaded area in **Figure 3A**). The HL difference (**Figure 3B**) for the three different age groups showed corresponding frequency ranges affected, with higher values at high frequency ranges of the tinnitus patients and lower values at 750 Hz in the above 60 years tinnitus patients.

In CHL patients (**Figure 3C**), we found a similar agedependent HL as above [F(2, 262,589) = 5,794.5, *p* < 0.001; 18- to 39-year-old adults: 18.7 ± 0.15 dB, 40- to 60-year-old adults: 31.7 ± 0.3 dB, adults above 60 years: 46.5 ± 0.4 dB]. Again, a significant interaction of age and frequency as well as age and group was found (**Figure 3C**, upper panels). Across all frequencies, CHL T patients' hearing thresholds differed by age, as we found higher threshold in 40- to 60-year-old adults but lower threshold in 18- to 39-year-old adults. No overall significant differences were seen in adults above 60 years (Tukey *post hoc*

tests; **Figure 3C**, upper right panel). In the three-way interaction (**Figure 3C**, lower panel) higher thresholds in T patients were found at frequencies above 1 kHz in 40- to 60-year-old adults (cf. red shaded area, statistical tests were performed separately for every stimulation frequency), while significantly lower hearing thresholds in T patients could be detected at low frequencies in 18- to 39-year-old adults and the adults above 60 years of age only [2-factorial ANOVAs for frequency ranges (low, mid, and high) and group, green shaded area in **Figure 3C**]. Correspondingly, in the analyses of HL differences (**Figure 3D**) of CHL patients, we found significantly lower values for low frequency thresholds in 18- to 39-year-old adults and adults above 60 years of age with

tinnitus while 40- to 60-year-old adults with tinnitus showed significantly higher values in the mid- to high-frequency range.

From **Figures 2** and **3**, it became clear that potential effects of the perception of tinnitus on hearing thresholds follow a frequency-specific pattern: in T compared to NT patients, lower thresholds were only observed in the low-frequency range while higher thresholds were exclusively seen in the high-frequency range. As pitches of tonal tinnitus percepts most frequently are located in the high-frequency range, we hypothesized that the higher hearing thresholds in T patients may be due to masking of the perception of tones in that frequency range. To test this hypothesis, we analyzed if there is a relation between the perceived

thresholds than NT patients (Tukey *post hoc* tests). (B,D) Red bars indicate significantly higher thresholds, green bars significantly lower thresholds.

Figure 3 | Age dependency of hearing loss (HL in decibel) in sensorineural (A,B) and conductive HL (C,D) patients. (A,C) show the interaction plots of the 3-factorial ANOVAs of HL with factors age, patient group, and frequency. Asterisks depict significance levels of the Tukey *post hoc* tests in the interaction plot of age X patient group: ns, not significant, \**p* < 0.05, \*\*\**p* < 0.001. Red shaded areas indicate frequencies with T patients exhibiting higher hearing thresholds compared to NT patients (Tukey *post hoc* tests separately for each frequency). Green shaded areas indicate frequency ranges (low: <1 kHz, mid: <4 kHz, high: ≥4 kHz) with T patients exhibiting lower hearing thresholds compared to NT patients (2-factorial ANOVAs for frequency ranges and patient groups). (B,D) give the mean HL difference of NT and T patients with red bars indicating significantly higher thresholds, green bars significantly lower thresholds in T patients compared to NT patients.

tinnitus pitch and the hearing thresholds of patients by 2-factorial ANOVAs with the factors frequency and perceived tinnitus pitch (NT, low, medium, and high tinnitus pitch). The results of these analyses are depicted in **Figure 4**. We found higher thresholds in SHL T patients rather being independent of tinnitus pitch. Lower threshold in CHL T patients on the other hand were most prominent in patients with high-pitched tinnitus percepts.

In detail, in SHL patients' data (**Figure 4A**, center panel), we found higher thresholds in T compared to the NT patients being most prominent but frequency unspecific in low-pitch tinnitus patients (Tukey *post hoc* tests), but also significant in the medium and high-pitch tinnitus patients, especially for frequencies above 2 kHz (**Figure 4B**). In CHL patients, a somewhat different picture emerged (**Figure 4C**). While low- and medium-pitch tinnitus patients showed generally higher thresholds than NT patients when averaged across all stimulation frequencies, the high-pitch tinnitus patients did show lower thresholds compared to the NT patients' thresholds (**Figure 4C**, center panel). This difference (**Figure 4D**) was most prominent for frequencies below 1,500 Hz in high-pitched T groups, while the higher values in tinnitus patients with medium-pitched tinnitus frequencies started at 1,500 Hz and reached up to the highest tested frequency of 8 kHz (Tukey *post hoc* tests). For low-pitched T patients, thresholds were again generally higher compared to NT patients.

In summary, the results of the pure tone audiometry comparisons showed significant differences in hearing thresholds of T compared to NT patients in both directions. In young patients with tinnitus, we generally found significantly lower thresholds almost independent of stimulation frequency (**Figure 1**) or perceived tinnitus pitch (not shown). In adult patients (**Figures 2**–**4**), data were more heterogeneous: in T compared to NT patients, lower hearing thresholds were found at low frequencies only, while higher thresholds were generally found at high frequencies. Furthermore, lower thresholds in T patients were more often found in CHL patients and could rarely be detected in SHL patients.

## Differences in Speech Audiometry between Patients with and without Tinnitus

In order to examine the potential differences in speech comprehension between both patient groups, we analyzed the results of the Freiburger test, which examines the intelligibility of multisyllabic numbers. **Figure 5** shows the 2-factorial ANOVA of the speech reception thresholds with factors age group and tinnitus pitch group, tested for the two major patients groups separately.

Variance analysis of speech audiometry in SHL patients (**Figure 5A**) showed the expected dependency of speech reception thresholds on age (left panel), but in addition, a stronger impairment of speech intelligibility in patients with low tinnitus pitch in comparison to all other groups (Tukey *post hoc* tests), the medium and high tinnitus frequency perceivers were not significantly different in their understanding of numbers compared to each other and to the NT patients. We did find a significant interaction of both factors (age group and tinnitus pitch), with Tukey *post hoc* tests revealing stronger impairments in adults above 39 years of age and with tinnitus pitches below 1 kHz (**Figure 6**, right panel).

In CHL patients (**Figure 5B**), we found a comparable age dependency of the speech reception thresholds (**Figure 5B**, left panel) as in SHL patients as well as a decrease of speech intelligibility in low-pitch tinnitus patients. In contrast to SHL patients, CHL high-pitched tinnitus patients showed an increase of speech intelligibility thresholds of 7 dB compared to NT patients' thresholds (**Figure 5B**, middle panel). No interaction of both factors was found, indicating a threshold decrease in high-pitched tinnitus patients at all ages (**Figure 5B**, right panel).

To control for any direct effect of the individual hearing threshold on speech reception threshold, we analyzed the relation of both thresholds by multiple linear regressions, center of gravity comparisons, and a 2-factorial ANOVA of the individual threshold differences of the two, pooled across all patients. The results of these analyses are depicted in Figure S1 in Supplementary Material and show no qualitative differences between the age or tinnitus groups in the first two analyses. Nevertheless, the differences between low and medium-pitched tinnitus patients and high-pitched tinnitus and NT patients emerge in the 2-factorial ANOVA and further support the finding, that low/medium pitch tinnitus patients show a different pattern of threshold loss compared to high-pitched tinnitus and NT patients, namely a lower distance between speech reception and pure tone thresholds.

#### Analysis of Tinnitus Loudness

The subjective tinnitus loudness perceived by the patients of the two groups relative to their hearing thresholds turned out to be not a good predictor of speech intelligibility. An analysis of this relative tinnitus loudness by a 3-factorial ANOVA with the factors age group, HL group, and tinnitus pitch is given in **Figure 6**. The tinnitus perception was loudest in 40- to 60-yearold adults (**Figure 6**, upper left panel, Tukey *post hoc* tests, always *p* < 0.001) and in the SHL patients (**Figure 6**, upper center panel), particularly in the high-frequency pitched pure tone tinnitus percepts (**Figure 6**, upper right panel, Tukey *post hoc* tests, always *p* < 0.001). No interaction of age and HL group indicated a parallel shift of tinnitus loudness (**Figure 6**, center left panel). Furthermore, we found significant interactions of age and pitch (**Figure 6**, center panel) with a decrease of tinnitus loudness with age for low frequencies and increase for high frequencies. We also found a significant interaction of pitch and HL group (**Figure 6**, center right panel) with louder low-pitched tinnitus percepts in SHL patients (tukey *post hoc* test, *p* < 0.001) and louder highpitched tinnitus in CHL patients (tukey *post hoc* test, *p* < 0.01). Most interestingly, the lack of a significant three-way interaction (**Figure 6**, bottom panel) indicated that the identified differences of tinnitus loudness dependency on age and pitch seemed to be similar in both HL groups.

### DISCUSSION

# Hearing Thresholds in Patients with and without Tinnitus

In this retrospective study, we compared audiometric data of a total of more than 37,000 patients (74,976 ears) with sensorineural

or CHL with or without tinnitus. We hypothesize that tinnitus is an epiphenomenon of a neuronal process normalizing impaired hearing thresholds. The audiometric results and tinnitus frequency distributions of tinnitus patients of different age groups are in line with the literature [e.g., Ref. (17, 26, 27)] even though the sample groups investigated there were much smaller than in our patient cohort. Also, the hearing thresholds of non-tinnitus patients are comparable to other cohorts (28, 29). Only few studies compared both patient groups directly (30, 31) and found either no change or a worsening of speech perception in tinnitus patients. To our knowledge, no peer reviewed study compared the pure tone hearing thresholds of both patient groups the way we did.

Here, we report specific differences in hearing thresholds between T and NT patients that may be interpreted as gains or losses in patients with tinnitus compared to non-tinnitus patients. These differences emerged on the population level of over 37,000 patients and in the paired analysis of a smaller subpopulation of roughly 2,500 patients in both pure tone and speech audiometry that seemed to follow a certain systematic (**Figure 7**, center panel): in T compared to NT adults, lower thresholds were seen in the low frequency range only, while higher thresholds were observed the high frequency range. In addition, lower thresholds were more often seen in CHL patients, while in SHL patients, higher thresholds predominated. Finally, the individually perceived tinnitus pitch seemed to be correlated with systematic alterations to the differences in hearing thresholds (**Figure 4**), which obviously are more complex than the simple masking we had first hypothesized to explain threshold increasing effects (see Hearing Thresholds in Adults with and without Tinnitus). In the case of paired data (Figure S1 in Supplementary Material), it becomes clear additionally that the most frequent (high) tinnitus pitch leads to the strongest improvement in speech intelligibility. Children and adolescents did not seem to follow this systematic, as only lower thresholds were seen there (**Figure 1**).

Based on our observations, we here put forward a model of tinnitus impact on hearing thresholds as shown in **Figure 7**: the basic feature of the model (**Figure 7**, center panel) is that thresholds in the low frequency range are lowered in tinnitus patients, i.e., in the range where HL is typically mild (**Figure 7**, center panel, green shaded area). Here, high frequency thresholds are higher in tinnitus patients, i.e., in the range where HL is typically more severe (**Figure 7**, center panel, red shaded area). This general scheme of hearing threshold change in tinnitus patients may be shifted by two main factors: sensorineural HL can shift the threshold function down (**Figure 7**, upper row)—CHL (**Figure 7**, lower row) has no effect on the function, while tinnitus pitch may shift the function along the abscissa (**Figure 7**, columns), resulting in a larger frequency range of lower thresholds (green shaded areas) for high-pitched tinnitus and a larger frequency range of higher thresholds (red shaded areas) for low-pitched tinnitus.

This model qualitatively reproduces the results of our retrospective data analysis (see **Figures 2B,D** and **4B,D**). Interestingly,

when analyzing the relative frequency of the different tinnitus– threshold-interaction types postulated here (percent values in panels of **Figure 7**), it became obvious that interaction types with predominantly higher hearing thresholds are less frequent (adding up to 25.5%) than those types with lower thresholds (adding up to 74.4%). As these lower thresholds were exclusively observed for pure tone measurements in the low frequency range, we here put forward the hypothesis that *the biological function of the neuronal mechanism that finally also leads to tinnitus is to improve speech perception in the case of hearing loss*. This seems to work particularly well for high-pitched tinnitus which was encountered in about 2/3 of the patients (64.9%). In other words, patients suffering from tinnitus might be affected by this phantom percept as a side effect of their auditory system compensating for HL (cf. below).

# Models of Tinnitus Development

Recent tinnitus models (32–37) all postulate damage to the peripheral receptor epithelium to be etiologic for the development of tinnitus. In response to the decreased input into the auditory system caused by such damage, one class of models further suggest an increased neuronal gain to provide homeostatic plasticity, while other suggest misbalanced lateral inhibition or a failure of a compensatory mechanism as the source of the phantom percept. Both types of models lack explanatory power since the potential biological function of homeostatic plasticity (in terms of information processing) or misbalanced lateral inhibition remains unclear.

To overcome these conceptual problems, we have recently put forward a model for tinnitus-related development of neuronal hyperactivity that is based on SR to restore hearing thresholds after HL (20). SR refers to the phenomenon that weak signals that are sub-threshold for a given sensor still can be detected and transmitted by that sensor if noise is added to the sensor input (21–24). In that way, SR serves to lift signals above a given hearing threshold and most probably is a mechanism that already works in the healthy system (38–40). One could interpret earlier results in a way that SR (while not mentioned explicitly) may also counteract increased hearing thresholds (17, 41). In this interpretation, tinnitus would be a (condoned) side effect of threshold restoration (42). In this context, recent studies have shown that patients with tinnitus show poorer listening performance in noise than patients without tinnitus (30). This is in line with our results, as the researchers focused mainly on sensorineural HL patients with and without tinnitus where we find comparable effects in our large patient cohort.

The analysis of audiometric patient data presented here are perfectly in line with the view of our model: first, the decreasing effects of SR on hearing threshold predicted by our model were observed in all adolescents and child HL patients with tinnitus as well as 65% of the adult HL patients with tinnitus. Second, threshold decreasing effects in tinnitus patients were observed in the low frequency range where HL is mild, that is, in a range where SR should be more effective as the amount of internal noise to be added to the signal to lift it above threshold must not be too high. Third, the threshold decreasing effects were most frequently observed in CHL tinnitus patients and were rather uncommon in SHL patients, which is in line with the idea that an intact neuronal system (as in CHL) more likely is able to compensate for HL by means of a neuronal SR mechanism than a damaged system (as in SHL). Note that still not all CHL patients may show lower thresholds, as patients suffering from CHL and SHL would be classified as CHL based on the diagnostic criteria (mean air-bone gap >5 dB; see Materials and Methods) and, therefore, may reduce the effect in the group. Finally, our model seems biologically plausible in that tinnitus development is condoned as a side effect of threshold restoration, as it particularly improves the speech intelligibility (improved thresholds in the speech relevant frequency range, most frequent (high) tinnitus pitch leads to strongest improvement of speech intelligibility), and communication by speech is crucial for social interaction and communication between

humans (2, 25, 43). This view is supported by the fact that high pitch tinnitus patients show the largest distance of speech and corresponding pure tone thresholds, which are even higher than those of NT patients. An alternative interpretation of the data presented here is that steeper audiogram slopes due to better hearing thresholds at low frequencies and worse hearing thresholds at high frequencies are more likely to lead to the development of tinnitus (17).

One drawback of this retrospective study is the lack of information on etiology, comorbidities or medication of the 37,000 patients pooled together here. As tinnitus is only a symptom resulting from different diseases, its specific characteristic may differ from case to case. The fact that our analysis still yielded highly systematic results further strengthens our interpretation of the functional benefit of tinnitus on hearing thresholds.

In conclusion, we think that within an evolutionary frame of reference it makes perfectly sense to optimize hearing and information extraction ability on the cost of generating a phantom percept off the frequencies of interest, i.e., high-pitched tinnitus. Why some patients suffer from low or medium-pitched tinnitus remains unclear and may have different reasons than the threshold decreasing high pitch tinnitus. Another open question why only a minority of CHL patients is developing a tinnitus percept has to be addressed in a more detailed, possibly prospective, follow-up study.

### AUTHOR CONTRIBUTIONS

DG analyzed the data and wrote a part of the manuscript. KT wrote a part of the manuscript, interpreted the data, and performed the statistical analysis. PK and AS provided assistance in the statistical analysis and interpretation of the data. UH provided the data. HS wrote a part of the manuscript.

# FUNDING

The present work was performed in fulfillment of the requirements for obtaining the degree "Dr. med. dent." at the Friedrich-Alexander University Erlangen-Nürnberg (FAU). PK was supported by the Dr. Willmar Schwabe GmbH & Co., KG, Karlsruhe, Germany. AS was supported by the Interdisciplinary Center for Clinical Research (IZKF, Project E15). We acknowledge support by Deutsche Forschungsgemeinschaft and Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU) within the funding program Open Access Publishing.

#### REFERENCES

# SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at http://journal.frontiersin.org/article/10.3389/fneur. 2017.00031/full#supplementary-material.

putative cause of tinnitus-related neuronal hyperactivity. *Front Neurosci* (2016) 10:597. doi:10.3389/fnins.2016.00597

43. Langguth B, Landgrebe M, Kleinjung T, Sand GP, Hajak G. Tinnitus and depression. *World J Biol Psychiatry* (2011) 12:489–500. doi:10.3109/1562297 5.2011.575178

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2017 Gollnast, Tziridis, Krauss, Schilling, Hoppe and Schulze. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Different Patterns of hearing loss among Tinnitus Patients: a latent class analysis of a large sample

*Berthold Langguth1,2\*, Michael Landgrebe1,3, Winfried Schlee1,2, Martin Schecklmann1,2, Veronika Vielsmeier2,4, Thomas Steffens2,4, Susanne Staudinger2 , Hannah Frick5 and Ulrich Frick1,6*

*1Department of Psychiatry and Psychotherapy, University of Regensburg, Regensburg, Germany, 2 Interdisciplinary Tinnitus Center of the University of Regensburg, Regensburg, Germany, 3 kbo Lech-Mangfall-Klinik Agatharied, Hausham, Germany, 4Department of Otorhinolaryngology, University of Regensburg, Regensburg, Germany, 5Department of Statistical Science, University College London, London, UK, 6HSD University of Applied Sciences, Cologne, Germany*

Background: The heterogeneity of tinnitus is a major challenge for tinnitus research. Even if a complex interaction of many factors is involved in the etiology of tinnitus, hearing loss (HL) has been identified as the most relevant etiologic factor. Here, we used a data-driven approach to identify patterns of hearing function in a large sample of tinnitus patients presenting in a tinnitus clinic.

#### *Edited by:*

*Sergio Carmona, Instituto de Neurociencias de Buenos Aires (INEBA), Argentina*

#### *Reviewed by:*

*Nicolas Perez, Universidad de Navarra, Spain Hideo Shojaku, University of Toyama, Japan*

*\*Correspondence:*

*Berthold Langguth<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Neuro-otology, a section of the journal Frontiers in Neurology*

*Received: 12 December 2016 Accepted: 31 January 2017 Published: 20 February 2017*

#### *Citation:*

*Langguth B, Landgrebe M, Schlee W, Schecklmann M, Vielsmeier V, Steffens T, Staudinger S, Frick H and Frick U (2017) Different Patterns of Hearing Loss among Tinnitus Patients: A Latent Class Analysis of a Large Sample. Front. Neurol. 8:46. doi: 10.3389/fneur.2017.00046*

Methods: Data from 2,838 patients presenting at the Tinnitus Center of the University Regensburg between 2007 and 2014 have been analyzed. Standard audiometric data were frequency-wise categorized in four categories [a: normal hearing (0–20 dB HL); b: moderate HL (25–50 dB HL; representing outer hair cell loss); c: severe HL (>50 dB HL; representing outer and inner hair cell loss); d: no data available] and entered in a latent class analysis, a statistical method to find subtypes of cases in multivariate categorical data. To validate the clinical relevance of the identified latent classes, they were compared with respect to clinical and demographic characteristics of their members.

results: The classification algorithm identified eight distinct latent classes with an excellent separation. Patient classes differed with respect to demographic (e.g., age, gender) and clinical characteristics (e.g., tinnitus location, tinnitus severity, gradual, or abrupt onset, etc.).

Discussion: Our results demonstrate that data-driven categorization of hearing function seems to be a promising approach for profiling tinnitus patients, as it revealed distinct subtypes that reflect prototypic forms of HL and that differ in several relevant clinical characteristics.

Keywords: chronic tinnitus, hearing loss, cluster analysis, latent classes, audiometry

# INTRODUCTION

Tinnitus, the perception of sound in the absence of a corresponding auditory stimulus, is a frequent disorder (1). Clinically, tinnitus can be very heterogeneous with respect to the perceived sound characteristics (e.g., tonal vs. broadband noise), its localization (in one or both ears, in the head, etc.), its time course (continuous, intermittent, fluctuating), its modifying factors (e.g., reduction by masking), and its comorbidities (hyperacusis, depression, insomnia). This clinical heterogeneity is paralleled by heterogeneity in tinnitus pathophysiology. Recent pathophysiological models assume that tinnitus emerges as a clinical symptom as result of abnormal activation of different overlapping and interacting brain networks (2). Abnormally activated networks in tinnitus patients include the auditory, attention, salience, and distress networks. The activation pattern varies from patient to patient and reflects the individuals' symptoms (3, 4). As an example, distressed and not distressed tinnitus patients differ in their activation of the cortical stress-related network (5).

Among other factors, the large heterogeneity of the tinnitus patient population represents a major barrier for the development of effective tinnitus treatments [see, e.g., Ref. (4) for a review]. The heterogeneity of tinnitus can be described on various dimensions such as its etiology, perceptual characteristics of the sound (i.e., pitch and loudness), time since onset, continuous or intermittent, levels of conscious awareness and perceived distress, and comorbidities (6). One approach to address this challenge is the establishment of large databases for enabling data-driven identification of subtypes (7–9).

Even if current etiologic models assume a complex interplay of various factors, several lines of evidence indicate that hearing loss (HL) is the most relevant etiologic factor for tinnitus development (10, 11). First, epidemiological studies have identified HL as a major risk factor for tinnitus (12). Second, induction of HL in animals induces reliably increased neuronal activity and synchronicity (11, 13) as well as behavioral evidence of tinnitus (14). Third, the tinnitus spectrum of most tinnitus patients is clearly related to their pattern of HL (15, 16). If, for example, somebody experiences tinnitus at 4 kHz at the left ear, typically a HL at 4 kHz on the left ear can be detected in the audiogram.

Because of the high etiological relevance of HL for tinnitus, hearing function is presumably one of the relevant criteria for classifying tinnitus patients. Currently, this factor is not receiving major attention in the classification of tinnitus patients. In the description of study samples in tinnitus research rarely details are given about the hearing function of participants. If anything, the mean audiogram (averaged over all participants) is displayed. Whenever statistical analyses are performed relating hearing function to other aspects of tinnitus, typically either HL averaged over both ears and all measured frequencies or the maximum HL is used to characterize patients' incapacities.

Both indicators are of only limited value, as there exist different patterns of the quantity of HL, e.g., related to outer or inner hair cell damage, which might be highly relevant for a comprehensive characterization of a tinnitus patient. However, the information about specific patterns of HL quantity is getting lost, if the audiograms of participants are averaged.

Here, we used a data-driven approach to identify patterns of hearing function in a large sample of tinnitus patients presenting in a specialized Tinnitus Center. The goal of the study was to clarify, whether specific patterns of HL quantity can be established from patients' audiograms and how stable different tinnitus patients can be classified using these patterns of HL. In case, this data-driven approach for pattern identification reveals a statistically sound and medically well interpretable solution, one could assume that this new categorization of hearing function would be a reasonable alternative to current approaches to deal with audiogram data in the characterization of tinnitus patients.

#### MATERIALS AND METHODS

#### Patient Assessment

The analysis has been based on data from all patients who presented between 2007 and 2014 at the Interdisciplinary Tinnitus Center at the University of Regensburg, Regensburg, Germany and who gave informed consent for data collection in the Tinnitus Research Initiative database (TRI Database). The TRI Database is an international patient database that has been established for facilitating research efforts toward the identification of tinnitus subtypes and outcome predictors (8). For these purposes, patients presenting in tinnitus clinics and undergoing specific, well-defined treatment interventions, both in clinical trials or in clinical routine, are systematically assessed and their data are preprocessed for plausibility in standardized protocols (8). In addition to audiometric data, the database includes demographic and clinical data, data about tinnitus handicap, tinnitus severity, and quality of life [for a detailed description of the datasets, see Ref. (8)]. Collection of data for the TRI Database has been approved by the local ethics committee of the University of Regensburg, Germany. All patients completed various tinnitus questionnaires, underwent a microscopy of the ear and received an audiological examination including pure-tone audiometry (125–8,000 Hz). Sample size for this classificatory part of our analysis thus reached *n* = 2,838.

Due to item-wise missing values of the 17 variables used to describe demographic or clinical characteristics of patients beyond the results of the audiometry, effective sample sizes in comparisons of emerging latent classes (see below) vary. Most variables reach effective sample sizes clearly above 2,000 patients. Only Beck Depression Inventory (BDI) (*n*= 1,544) and TBF-total score (*n* = 1,464), which both were added later to the assessment program, and patients' CGI-ratings (*n* = 807), which were assessed only in patients entering a treatment program at their first day of treatment, reached smaller sample sizes.

### Classification of Hearing Function

As HL presenting at different threshold levels can be linked to different possible pathological mechanisms (17), patients' audiogram data were not treated as a "naive" continuous metric of intensity of HL, but classified into four different states. At each of seven pre-specified frequencies (125 Hz, 250 Hz, 500 Hz, 1 kHz, 2 kHz, 4 kHz, and 8 kHz) the measured grade of HL was classified into one of the following four categories: (1) normal hearing (0–20 dB HL); (2) mild/moderate HL (25–50 dB HL), representing mostly outer hair cell loss; (3) severe/profound HL (>50 dB HL), representing outer and inner hair cell damage; and (4) no data available (either due to technical restrictions or because of physician based abbreviations of the audiogram assessment). This categorization tries to better reflect the physiological condition of patients' hearing and to avoid a potentially misleading interpretation of HL as homogenously quantifiable risk factor (18). Additionally, by introducing a fourth category of "not measured" into the analysis, a potential selection bias due to technical equipment or due to physicians' practices in assessing HL can be avoided.

#### Statistical Methods

Latent class analysis (LCA) is a statistical approach for identifying groups of cases in multivariate categorical data. These groups are called latent classes. Our assumption is that HL of the left ear does not predetermine HL for the right side, and *vice versa*. Therefore, a vector of 14 variables (each frequency with 4 HL categories) per patient defines the starting point of statistical analysis. Theoretically, over 250 million combinations (exactly 4\*\*14 = 268,435,456) are possible. Observed were 590 answering vectors in 2,838 patients. LCA tries to reproduce the empirical frequencies of these answering vectors by estimating two different kinds of model parameters: π*g*, i.e., the relative sizes of *G* latent classes (*G* has to be determined *a priori*), and the probability π*isg* for an answer *s* (*s* = 1, …, 4) on each item *i* (*i* = 1, …, 14), given the membership in a certain latent class *g*. The class-specific answering probabilities π*ix*|*<sup>g</sup>* thus indicate the nearness between this specific answer and membership in the respective latent class *g*. LCA therefore results in *G* membership probabilities per person to each of the latent classes (see Supplementary Material for further details). Strong solutions with little overlap between different latent profiles provide for each person one unequivocal high membership probability and *m* − 1 very low membership probabilities. Classification then is based on the modal value of these probabilities.

Visualization of membership probabilities is an intuitively appealing method of model evaluation. Alternatively, so-called fit indices can be calculated for each number of latent classes chosen. Clearly, a perfect model fit must be reached, if (in our case) 590 classes are introduced to the model. By introducing a penalty term for adding new latent classes, a decision for the optimal number of classes can be drawn choosing the model with the best fit. We used the BIC index as criteria to decide on the number of latent classes. Calculations were performed using WinMIRA by von Davier (19).

Differences between latent classes on continuous variables (like age) were assessed using SAS PROC GLM to perform analysis of variance for unequal cell sizes. Differences on qualitative variables (like sex) were assessed using chi-square test (SAS PROC FREQ). Due to the exploratory character of this study, no adjustment for type-I error inflation was performed.

#### RESULTS

The sample comprised 2,838 patients (mean age 51.7 ± 12.9 years, 67.6% male). In 1,925 of them, audiometric data were available.

In order to avoid local maxima of the estimation function, 50 starting values for parameter estimation were randomly chosen for each model covering 2 up to 12 latent classes. According to the BIC fit index, eight latent classes represent an optimal solution for the given data set. Posterior probabilities of class membership display excellent separation of groups of HL as indicated by a mean membership probability above 0.9 for all latent classes (**Table 1**) (see Supplementary Material for details about the calculation of latent classes). Detailed clinical and demographic data of the sample are given in **Table 2**.

The largest class (LC1; **Figure 1** upper left chart) comprises nearly one-third (32.2%) of the sample and represents patients with lacking audiometry. By holding these untested patients in a separate group it is possible to scrutinize potential selection biases between clinical characteristics and audiometry. Therefore, it is meaningful to analyze these patients as a specific "pattern of hearing loss."

The 21.6% of the sample suffers from mild to moderate HL probably due to primarily outer hair cell damage especially for frequencies above 4 kHz (LC2; **Figure 1**, upper right chart). This group was entitled "bilateral high frequency (HF) hearing loss." Tinnitus patients with nearly normal audiogram (LC3; **Figure 1**, lower left chart) comprise about 20.6% of the total sample. Here, in rare cases (about 10% of this group), only frequencies above 4 kHz are involved with mild/moderate HL for both ears. A large proportion of patients with at least moderate HL in higher frequencies (2 kHz and above) for both ears can be observed in LC4. Twenty to thirty percent of this latent class were measured with thresholds over 50 dB above 4 kHz. Lower frequencies (below 500 Hz) are mostly not affected by HL. The proportion of this group is 13% of the total sample. The group was entitled "bilateral medium-high frequency HL."

**Figure 2** displays patterns of HL with much smaller proportion among tinnitus patients (all <5%). LC5 (upper left chart in **Figure 2**) was called "severe pantonal HL" and is characterized by high proportions of at least moderate HL at all measured frequencies. Almost half of the patients of this group have thresholds over 50 dB above 4 kHz. Both ears are concerned quite similarly.

By contrast, latent classes 6, 7, and 8 all display asymmetric patterns of HL. In LC6 (3.8% of total sample; upper right chart in **Figure 2**), most patients have normal hearing at the right ear below 4 kHz but severely impaired hearing at their left ear at all frequencies. Damage on the left ear is mostly a HL between 25 and 50 dB ("mild to moderate"). We therefore named this group "left-sided pantonal medium HL."

It is noteworthy, that LC7 (2.9%, lower left chart of **Figure 2**) is not a symmetrical counterpart to LC6, though in this group mostly the right ear is affected by HL. But whereas LC6 members displayed mild to moderate HL at their affected left ear, members of LC7 have a broadband severe HL at their right ears. Already at 125 Hz, more than 20% of this group suffer from threshold elevations above 50 dB. This proportion is continuously increasing with increasing frequencies up to nearly 60% at 8 kHz. We therefore called this group of patients "right-sided pantonal severe HL."

The smallest group of patients isolated by LCA can be depicted from the lower right chart of **Figure 2** (LC8). In 1.2% of all patients, a pattern was observed with considerable proportion of normal hearing at the right ear in lower frequencies (e.g., 66% at 125 kHz), continuously decreasing to 20% at 8 kHz. But at patients' left ears, nearly nobody was measured with normal hearing at any frequency: mostly, a severe HL (thresholds >50 dB) could be observed across all frequencies, or the left ear was not assessed (especially at higher frequencies). As practically, all patients had a full examination of the right ear, it can be assumed that missing values for left ears in this group do not mean "unknown" degree of HL, but represent a physician's or audiologist's decision to stop the assessment, because complete deafness of the left ear impede

#### TABLE 1 | Mean membership probabilities for latent classes.

a full measurement of all frequencies at the left side. This group therefore was named "left-sided pantonal severe HL."

Latent class 1 (lacking audiometry) during all statistical comparisons of clinical and demographic parameters yielded results very similar to the total average or proportions of the total sample (see **Table 2**). Therefore, no hints are present that the lack of an audiometric test was related to a certain subgroup of tinnitus patients. The relative size of the remaining seven HL-patterns thus could be adjusted to the proportions given in **Table 2**. These reflect the expected latent class sizes in our patient sample, if all patients had received threshold measurements.

Mean age of patients (see **Table 2**) was 51.7 years (SD 12.9) with the youngest patients presenting in LC3 (normal hearing) and the oldest patients in LC5 (severe pantonal HL). Female patients (total: 32.4%, see **Table 2**) were under-represented in LC2 (bilateral HF HL) and LC4 (bilateral medium-HF HL), and at largest over-represented in LC5 (severe pantonal HL) and LC6 (left-sided pantonal medium HL).

Age at onset of tinnitus was on average 43.2 years (SD 13.9; **Table 2**). Thus, there was a mean delay of nearly 9 years until presenting at the Regensburg Tinnitus Center. Patients with more or less normal hearing (LC3) had virtually no delay in help seeking. The small difference between age at onset as compared to biographical mean age (41.7 vs. 41.9 years) is probably confounded by missing values about the onset. Patients with bilateral medium-to-severe HF HL (LC4) and with severe pantonal HL (LC5) reported the latest onset of tinnitus.

Symptom load as measured by the Tinnitus Questionnaire (TQ, 20) was highest in LC5 (severe pantonal HL), and lowest in LC3 (normal hearing). Signs of depression as measured by BDI were also most prominent in LC5, and quite lenient in all other patient groups. For the TBF Questionnaire, a short form of the Tinnitus Handicap Inventory, mean scores 2 points above the total average were found for LC5 and LC8 (left-sided pantonal severe HL). Whereas patients' self-ratings of tinnitus severity on the clinical global impression scale of tinnitus (CGI self-rating) revealed no significant differences between the different latent classes, the answers to the question "How much of a problem is your tinnitus?" differed significantly. But this effect was very small with membership in latent classes of HL explaining only less than 3% of the variance of patients' ratings.

Characteristics of tinnitus manifestation over groups of HL are also given in **Table 2**. The different latent classes differed in most but not all characteristics (see **Table 2**). Whether or not tinnitus started abruptly, differed most between LC4 (43% abrupt start) and LC5 (40%) on the one side, and over 60% for latent classes 6 (left-sided pantonal medium HL) and 7 (right-sided pantonal severe HL). A non-pulsatile tinnitus was in total reported by 79.6% of all patients. Only in LC7 (right-sided pantonal severe HL) this proportion was considerably smaller (66%).

Handedness of patients could not be shown to covary with patterns of HL (and therefore was also not connected to side of HL). On which side patients experienced their tinnitus (exclusively on one side or with dominance of one side vs. bilateral symptoms) was clearly connected to the asymetrical patterns of HL. Members of LC6 (left-sided pantonal HL) as well as members of LC8 (left-sided pantonal severe HL) reported tinnitus more often for their left side. Accordingly, patients in LC7 (right-sided pantonal severe HL) clearly reported more often to experience their tinnitus on the right side (48% as compared to 12.3% in the total sample).

For LC8 (left-sided pantonal severe HL), their tinnitus was more frequently experienced as constant over time. If patients suffered from HF HL (LC2) or had quite normal audiometric results (LC3), they tended more often to describe their tinnitus as a tonal event. Patients with severe pantonal HL (LC5) by contrast had much higher proportions of tinnitus experienced as a kind of noise. The tinnitus pitch did not differ too much across the LCs except a smaller proportion of very high pitch (19% as compared to 28% for the whole sample) in LC7 (right-sided pantonal severe HL), and more prominence of low pitch tinnitus in LC3 (normal hearing) and LC6 (left-sided pantonal medium HL) with 4.4 and 6.3% (total sample: 2.5%).

#### DISCUSSION

In the present study, we performed a data-driven analysis of a large sample to identify clinically meaningful subtypes of HL patterns among tinnitus patients. Information about hearing function was derived from standard pure-tone audiograms, which were all performed in the context of clinical routine assessment according to standard procedures in one clinical center.

A potential limitation of our study is the limited sensitivity of the standard audiogram. The database only contained audiogram data from air conduction measurements, which made it impossible to differentiate cochlear from conductive HL. Moreover measurement of otoacoustic emissions might provide a more exact measurement of outer hair cell function than audiometric

#### TABLE 2 | Patterns of HL and related demographic and clinical data.

#### Description of patterns of HL

(*Continued*)

thresholds and it also remains to be determined whether the cut-offs for categorization of HL used in this study (<20 dB HL, 25–50 dB HL, >50 dB HL) are most appropriate. Finally, cochlear damage in the HF range above 8 kHz (21), dead regions between tested frequencies (22) and synaptopathy of high-threshold fibers (23) can all be relevant for tinnitus development but are not detected by the standard audiogram.