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\begin{document}

\title{Evidence of Immune disorder served as the main pathogenesis in SSNHL:
elevated serum concentration of MMP-9 following treatment was associated
with favorable outcomes in patients}



\author[1]{Tao Lu}%
\author[1]{Hong Chen}%
\author[1]{Jiaqiu Dai}%
\author[1]{Siji Wang}%
\author[1]{Ziqi Chen}%
\author[1]{Houyong Kang}%
\affil[1]{The first affiliated hospital of Chongqing Medical University}%


\vspace{-1em}



  
  \date{June 11, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Objectives: Sudden sensorineural hearing loss (SSNHL) is an emergency
disease with undefined pathogenesis in the otolaryngology department. In
our previous study, we found patients with SSNHL had lower serum
concentration of Matrix metalloprotease 9 (MMP-9) than healthy controls,
and the result was accordant with auto-immune diseases. This study aimed
to reveal the correlation between changes in serum MMP-9 concentration
following treatment with the outcomes of patients and to provide further
evidence that immune disorder was the main pathogenesis of SSNHL.
Design, setting, and participants: Fifty-two patients with SSNHL,
hospitalized in The First Affiliated Hospital of Chongqing Medical
University from March 2019 to August 2019, were enrolled. The serum
concentration of MMP-9 was detected by enzyme-linked immunosorbent assay
(ELISA). Main outcome measure: The mean concentration of MMP-9 before
treatment was compared with the post-treatment concentration by the
Mann-Whitney U test. The correlations between favorable outcomes of
patients and clinical characteristics were measured with the Chi-squared
test and binary multiple logistic regression analysis. Results: In
treatment responders, mean serum concentration was elevated from
106.85\selectlanguage{ngerman}±41.40ng/ml to 144.03±37.65 ng/ml following treatment (P\selectlanguage{english}<0.001),
while in non-responders it decreased from 132.09+-59.21 ng/ml to
106.82+-49.93 ng/ml (P=0.142). Changes in MMP-9 concentration was the
only factor associated with favorable outcomes (P=0.008, OR=5.13, 95\%
CI: 1.53-17.28). Conclusions: Elevated MMP-9 concentration is a
potential prognosis biomarker in patients with SSNHL. These findings are
in line with auto-immune diseases and indicate immune disorder is mainly
pathogenesis in SSNHL. Keywords Sudden sensorineural hearing loss;
Matrix metalloprotease 9; Immune disorder; prognosis; Enzyme-linked
immunosorbent assay%
\end{abstract}\selectlanguage{ngerman}%



\sloppy


\textbf{1.~~} {}\textbf{Introduction}

Sudden sensorineural hearing loss (SSNHL) is defined as an acute
sensorineural hearing loss occurring within 72 hours and characterized
by a hearing loss of 30 dB or greater over in at least three contiguous
audiometric frequencies {[}1{]}. The incidence of SSNHL varies from 5 to
30 per 100000 people {[}2,3{]}, and the pathogenesis of SSNHL remains
unclear. Autoimmune disorders, viral infection, and microvascular injury
have been described as one of the etiologies of SSNHL, but none of them
are defined definitely {[}4,5{]}.

~ MMP-9 is one of the Matrix metalloproteinases (MMPs), which is a large
family of zinc-containing calcium-dependent endopeptidases. MMP-9 is
mainly distributed in the extracellular matrix and has a function to
degrade multiple extracellular components, such as collagen and elastin
{[}6{]}. Several studies have shown MMP-9 is involved in the
pathogenesis of diseases including tumor, cardiovascular, and autoimmune
disease. Intriguingly, the results in different diseases were in
contrast. The mean serum concentration of MMP-9 in patients with tumors
or cardiovascular diseases was higher than in healthy controls, whereas
MMP-9 level in patients with autoimmune diseases such as Systemic Lupus
Erythematosus (SLE) was lower than in healthy controls {[}7-11{]}. In
our previous study, we delineated that the serum level of MMP-9 in
patients with SSNHL was lower than in healthy controls, and the result
was in line with autoimmune diseases rather than tumors or
cardiovascular diseases {[}12{]}. Therefore, to further provide evidence
that immune disorder was the main pathogenesis of SSNHL, we collected
serum from 52 patients with SSNHL prior to and following treatment with
detecting the serum concentration of MMP-9 by ELISA. We hypothesized the
mean serum concentration of MMP-9 following treatment elevated in
treatment responders and the elevation was associated with favorable
outcomes of patients with SSNHL.

\textbf{2.~~} {}\textbf{Materials and methods}

\textbf{2.1 Audiometric Evaluation}

All the patients received pure-tone audiometry (PTA) before
treatment\begin{CJK}{UTF8}{gbsn}、\end{CJK}\selectlanguage{english}following a one-week and two-week treatment. Mean pure-tone
audiograms was defined as the average of air-conduction threshold
measured at 500,1000,2000,4000HZ and was employed to describe outcomes
of patients.

Four types of audiograms were identified according to the initial
pattern of hearing loss with PTA: ascending (the average threshold of
0.25 to 0.5 kHz was 20 dB higher than the average threshold of 4 to 8
kHz.), descending (the average threshold of 4 to 8 kHz was 20 dB higher
than the average threshold of 0.25 to 0.5 kHz), horizontal (Less than
20dB threshold difference observed across all frequency and the mean
hearing threshold of 0.5,1,2 and 4KHZ was no more than 80dB), profound
(similar audiogram shapes to the horizontal group, whereas the average
threshold of 0.5,1,2 and 4KHZ was no less than 81dB).

As indicated in Chinese clinical practice guidelines of SSNHL {[}13{]},
patients were stratified into four groups based on their responses to
treatment (criteria were shown in Table. I): Nonrecovery (NR), partial
improvement (PI), marked improvement (MI) and complete recovery (CR).
Patients in the NR group were defined as the treatment non-responders,
while patients in the other three groups were defined as the treatment
responders.

\textbf{2.2 Patients and serum samples}

Fifty-two patients with SSNHL, admitted to {[}removed for blind peer
review{]} from March 2019 to August 2019, were enrolled in this study.
Eligible patients for this study met the following criteria: 1) had
sensorineural hearing loss of 30 dB or greater over in at least three
contiguous audiometric frequencies within 72 hours; 2) had no history of
retrocochlear disease; 3) received treatment in 3 days after onset and
had no treatment in other hospitals. Patients were excluded if they had
the following diseases: 1) hypertension; 2) diabetes mellitus; 3)
autoimmune disease; 4) cancer; 5) any other contraindications for
glucocorticoid. Patients were discharged if they met the criteria of CR
or had a two-week time of hospitalization. Blood samples were collected
before and following treatment and serums were separated after
centrifugation and stored at -80 immediately.

\textbf{2.3 Treatment Protocol}

All the patients received following therapy: 1) oral prednisolone at a
dose of 1mg/kg per day in the first week of therapy; 2) extract of
ginkgo biloba leaves intravenous injection (i.v) at a dose of 20 ml per
day; 3) oral mecobalamin tablets at a dose of 0.5mg three times a day;
4) batroxobin i.v at a dose of 10BU for the first time and 5BU every
other day; 5) intratympanic corticosteroids at a dose of 40mg every
other day in the second week if patients did not meet the criteria of CR
following treatment with oral prednisolone.

\textbf{2.4 ELISA}

Elisa kits for MMP-9 were purchased from R&D System Inc(Catalog No:
DM-900). Serum samples were thawed under room temperature for 10
minutes, diluted at 1:100, and then transferred to polypropylene tubes.
The wells added with 100ul of Assay Diluent were incubated for 2 hours
at room temperature on a horizontal orbital microplate shaker with 100ul
of standard or samples. After washing with Wash Buffer for 4 times,
200ul of Human MMP-9 Conjugate was added per well. Following one-hour
incubation, the wells were washed and added with 200ul of Substrate
Solution to react for 30 minutes. Ultimately, each well was added with
50ul of stop solution, and the optical density was determined with a
microplate reader set to 450nm.

\textbf{2.5 Statistical analyses}

After examining variance equivalence and normal distribution, a
nonparametric Mann-Whitney U test was applied to assess the difference
between pre-treatment and post-treatment serum concentration of MMP-9,
while Chi-squared test was employed to evaluate the correlation between
outcomes of patients and clinical characters. Parameters that were
statistically significant in the Chi-squared test were further carried
out in the binary multiple logistic regression analysis. All analysis
was performed with SPSS version 22.0 and the figures were completed with
GraphPad version 7.04. A P value \textless{}0.05 was considered to be
significant.

\textbf{2.6 Informed Consent}

The protocol was approved by {[}removed for blind peer review{]} and in
concordance with the Helsinki Declaration. All patients provided written
informed consent.

\textbf{Results}

\textbf{3.1 ClinicalCharacteristics of Patients with SSNHL}

A total of 52 patients were enrolled in our study, including 29
(55.77\%) male and 23 (44.23\%) female, with an average age of 47.12
years (SD:16.93, range 15-76). All patients suffered from unilateral
hearing loss, while 84.6\% and 17.3\% of patients accompanied with
tinnitus and vertigo, respectively. The left to right ratio was 25:27.
The PTA average in patients prior to treatment was 65.07dB (SD 27.61,
range 10.00-121.50), while the average following treatment turned to
43.22dB (SD 29.89, range 2.50-116.25). Based on the initial PTA, 9, 8,
19, and 16 patients were divided into the ascending, descending,
horizontal and profound group, respectively. And refer to their final
PTA, 20 patients got NR, whereas 7, 14, 11 patients had CR, MI, PI,
respectively. (data shown in Table. II)

\textbf{3.2 Changes of serum MMP-9 concentration prior to and following
treatment in different types of SSNHL.}

Patients with SSNHL had a higher concentration of MMP-9 following
treatment as compared with prior-treatment concentration, but it was not
statistically significant (116.56\selectlanguage{ngerman}±50.01ng/ml vs 131.45±45.33 ng/ml,
p=0.054). Next, we compared the mean prior-treatment serum concentration
of MMP-9 with post-treatment concentration in ascending, descending,
horizontal, and profound group. (prior to treatment vs post-treatment,
99.76±23.44ng/ml vs 125.82±35.50ng/ml, 129.20±58.33ng/ml vs
148.91±66.43ng/ml, 112.42±66.59ng/ml vs 128.65±45.13ng/ml and
124.69±30.95ng/ml vs 129.20±43.01ng/ml, respectively) .An increase was
found in all four groups, however, none of them had statistical
significance (Fig.\selectlanguage{english}I, P=0.094, P=0.574, P=0.191, P=0.867, respectively).

\textbf{3.3 Changes of serum MMP-9 concentration prior to and following
treatment in different outcomes of patients with SSNHL.}

Given an increase of MMP-9 concentration in patients following treatment
was identified, we further detected whether changes in MMP-9
concentration had a significant difference in the outcomes of patients.
In treatment responders, mean MMP-9 concentration was elevated from
106.85+-41.40ng/ml to 144.03+-37.65ng/ml following treatment, while it was
decreased from 132.09+-59.21ng/ml to 106.82+-49.93 ng/ml in
non-responders. Anecdotally, a difference was found in responders
(P\selectlanguage{english}<0.001), however, the decrease of MMP-9 concentration in
non-responders did not have statistically difference (P=0.142). Next, we
examined if differences existed in each subgroup of treatment
responders. Patients in PI, MI and CR subgroup all gained an increase of
MMP-9 concentration following treatment (pre-treatment vs
post-treatment, 115.62+-40.52ng/ml vs 161.23+-44.95 ng/ml,
98.45+-49.84ng/ml vs 138.46+-32.34ng/ml, 109.87+-20.47ng/ml vs
140.99+-25.72ng/ml, respectively). And with the nonparametric
Mann-Whitney U test, we found significant differences in each group
(Fig. II, P=0.047, P=0.005, and P=0.038, respectively).

\textbf{3.4 Correlation between outcomes of patients with SSNHL and
Clinical characteristics}

Based on the final PTA, patients were dichotomized into treatment
responders and treatment non-responders. A chi-squared test was
performed to disclose the potential correlation between clinical
characteristics and outcomes of patients. As indicated in Table. III,
patients with increased concentration of MMP-9 following treatment were
more frequently accompanied by favorable outcomes (P=0.007, \selectlanguage{greek}χ\selectlanguage{english}2=7.436).
However, the incidence of tinnitus or vertigo had no significant
difference between the two groups (tinnitus vs vertigo, P=0.332 vs
P=0.239, \selectlanguage{greek}χ\selectlanguage{english}2=0.724 vs \selectlanguage{greek}χ\selectlanguage{english}2=1.213). Moreover, treatment responders had
similar trends in gender or side of hearing loss as compared with
treatment non-responders (gender vs side, P=0.353 vs P=0.474, \selectlanguage{greek}χ\selectlanguage{english}2=0.439
vs \selectlanguage{greek}χ\selectlanguage{english}2=0.123). Whereas, patients with favorable outcome was younger than
those suffered from treatment resistance (P=0.03, \selectlanguage{greek}χ\selectlanguage{english}2=4.623).

Though the Chi-squared test did not reveal the incidence of tinnitus or
vertigo was related with the outcomes, we still put them into binary
multiple logistic regression analysis, given several pieces of
literature reported the opposite results. Interestingly, the result
disclosed changes in MMP-9 concentration was the only factor closely
associated with favorable outcomes (P=0.008, OR=5.13, 95\% CI:
1.53-17.28, Table. III).

\textbf{Discussion}

To the best of our knowledge, this is the first study about the
correlation between changes of MMP-9 concentration and outcomes of
patients with SSNHL. In our previous study, we found the concentration
of MMP-9 was decreased in patients with SSNHL, as compared with in
healthy controls, and the difference had no correlation with the types
of SSNHL {[}12{]}. The same conclusion is formulated in this further
study. Though in these 4 types of SSNHL, the mean concentration of MMP-9
seemed to be elevated following treatment, the increment did not have a
significant difference. Besides, this increment seemed more due to the
variational therapy responses in different types of SSNHL. Patients with
a profound type of SSNHL are known to accompany with the worse outcomes,
while patients with ascending type, the prognoses are in contrast.
Hence, the post-treatment concentrations of MMP-9 in patients with
profound type were almost the same as the pre-treatment concentrations,
whereas patients with an ascending type of SSNHL gained significant
increment in MMP-9 concentrations following treatment.

The correlations between age, vertigo, or tinnitus with the favorable
outcomes of patients with SSNHL are in debate {[}14-16{]}. In our study,
we displayed that age and the increment of MMP-9 concentration following
treatment were correlated to the hearing gain by the Chi-squared test.
However, age failed to show the association with outcome in the binary
multiple logistic regression test and the increment of MMP-9 was the
only factor closely related to favorable outcomes. MMP-9 is recognized
as a metalloproteinase to degrade the extracellular matrix and to induce
the infiltration of inflammatory cells {[}17{]}. Recently, several
studies have shown that hyperhomocysteinemia was associated with SSNHL
and can induce an increase in the cochlear expression of MMP-2 and MMP-9
in mice models {[}18-19{]}. In consideration of these studies, we
speculated that MMP-9 might deposit in different sites of the inner ear
and induce inflammation by degrading the extracellular matrix. When the
damage occurred at the top inner hair cells of the cochlea, patients
suffered from ascending type of SSNHL, whereas the damage occurred at
the bottom inner hair cells of the cochlea, patients developed
descending type of SSNHL. Following treatment, especially with the
glucocorticoid, the deposition of MMP-9 in the inner ear decreased and
then induced the relief of the inflammatory reaction in the inner ear.
Due to this nonspecific deposit, the outcome of patients had no
significant difference with the types of SSNHL but with the changes in
the concentration of MMP-9 following therapy.

The etiology of SSNHL is still unknown. The immune disorder is
considered as one of the major causes of SSNHL, but there is no direct
evidence to prove it. Several studies have revealed the correlations
between MMP-9 and other diseases. Robaka, et al found serum MMP-9 level
was decreased in patients with active SLE, and the level was elevated
when SLE turned to an inactive state {[}20{]}. Vira H, et al obtained
the same conclusion with detecting the polymorphisms of MMP-9 in
patients with SLE {[}21{]}. In contrast, studies in cardiovascular
diseases or tumors indicated that serum conc-entration of MMP-9 was
positively associated with the activity of the diseases {[}22,23{]}. In
our study, the altering level of MMP-9 was accordant with the changes in
patients with autoimmune disease {[}24{]}, thus provided further
evidence that autoimmune disorder was one of the major causes of SSNHL.

Altogether, we identified that elevated serum concentrations of MMP-9
was not associated with different types of SSNHL, stratified by the
different shapes of the audiogram, but closely correlated to favorable
outcomes in patients with SSNHL, therefore it might serve as a prognosis
prediction factors of patients with SSNHL. Given the increment of MMP-9
concentration following treatment was accordant with patients with the
auto-immune diseases rather than patients with cardiovascular diseases
or tumors, it proved another indirect evidence that immune disorder was
one of the major pathogenesis in SSNHL.

Limitations in our study are mainly the small case number of patients
with SSNHL. We ascribed this limitation to several patients admitted to
our hospital with treatment in outpatient department or other hospitals.
Whereas, there were still several patients refused to take audiometry
test following treatment, which leaves the clinical characteristics
uncompleted. Furthermore, we only performed an ELISA test to provide the
indirectly evidence, the mechanism of the altering MMP-9 concentration
in patients with SSNHL needed to be further proved.

\textbf{Conclusions}

Our study revealed that elevated serum concentration of MMP-9 following
treatment in patients with SSNHL was closely correlated with favorable
outcomes. The result is accordant with patients with auto-immune
diseases, therefore to provide an indirectly evidence that immune
disorder was one of the main pathogenesis in SSNHL. Further study was in
need to investigate the underlying mechanism in the increment of MMP-9
concentration following treatment with patients benefited from hearing
gained.

\textbf{Conflicts of interest}

The authors declared there are no conflicts of interest in this work.

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