MR ≤ 2 at TMEER completion was the strongest predictor for survival  whereas rMR ≤ 1 at discharge was independently associated with a lower risk of the combined endpoint . MVPG was not associated with adverse events. We conclude that the assessments for rMR grade and MVPG, at the completion of TEEMr and at discharge, should be distinctly reported. Improved outcome is expected with rMR ≤ 2 at TEEMr completion and rMR ≤ 1 at discharge. Higher MVPG is not associated with unfavorable outcomes.The correlation between residual mitral regurgitation (rMR) grade or mitral valve pressure gradient (MVPG), at transcatheter edge-to-edge mitral valve repair (TEEMr) completion and at discharge, is unknown. Furthermore, there is disagreement regarding rMR grade or MVPG from which prognosis diverts. We retrospectively studied 82 patients that underwent TEEMr. We tested the correlation between rMR or MVPG and evaluated their association, with outcomes. Moderate or less rMR (rMR ≤ 2) at TEEMr completion was associated with improved survival, whereas mild or less rMR (rMR ≤ 1) was not. Patients with rMR ≤ 1 at discharge demonstrated a longer time of survival, of first heart failure hospitalization and of both. The correlation for both rMR grade ( Transcatheter edge-to-edge mitral valve repair (TEEMr) is a minimally invasive treatment for patients, suffering from symptomatic moderate-to-severe or higher mitral regurgitation (MR), who are deferred from surgery owing to either high or prohibitive surgical risk. Patients who are treated with TEEMr report improved functional capacity, exhibit reduced heart failure symptoms, and possibly have improved clinical course with less heart failure admissions and reduced mortality ,2,3. EffThe objectives of the present study were: (1) to examine the relationship between post TEEMr TEE and TTE assessments of rMR or MVPG and long-term clinical outcomes; and (2) to evaluate the correlation between rMR and MVPG assessments, following TEEMr, by TEE at the completion of the procedure and by TTE at discharge.A retrospective analysis was conducted among patients who underwent TEEMr at Poriya Medical Center (PMC) between March 2015 and November 2020. The study complied with the ethical guidelines of the Declaration of Helsinki, was approved by the Ethical Review Board at PMC and each patient provided written informed consent before the intervention. Data were recorded prospectively during the index hospitalization and follow-up visits. All patients were diagnosed with either moderate-severe MR (MR-3) or severe MR (MR-4) and remained symptomatic despite optimal medical therapy and cardiac resynchronization therapy, when appropriate. Patients were evaluated by a multidisciplinary heart team and were deferred from surgical intervention due to high or prohibitive surgical risk. For those patients who required a repeated procedure, we included only the first procedure in the analysis and the additional procedure was registered as an adverse event.® device  using the standard technique previously described [TEEMr was performed under general anesthesia, using fluoroscopic and 2-dimensional (2D) and 3-dimensional (3D) TEE guidance. All procedures were performed with the Mitraclipescribed . After tA board-certified non-invasive cardiologist performed and interpreted echocardiography images using commercially available ultrasound systems . The assessments were performed by 2D TTE and TEE at baseline, by 2D and real-time 3D TEE during TEEMr, after the last clip was placed while the patient was still under the influence of general anesthesia, as well as by 2D-TTE again at discharge and at follow-up visits. Left ventricle (LV) volume and LV ejection fraction (LVEF) were assessed using the biplane Simpson’s method. LV end-diastolic diameter (LVEDD), LV end-systolic diameter (LVESD) and left atrial (LA) diameter were measured on M-mode in the parasternal long axis view. Systolic pulmonary pressures (SPAP) were estimated from measuring the pressure gradient between the right ventricle (RV) and right atrium (RA) in systole and estimated RA pressure using inferior vena cava size and collapsibility . MR etioTechnical success was assessed at exit from the catheterization laboratory while device and procedural success were measured after 30 days. All were defined according to the Mitral Valve Academic Consortium (MVARC) criteria . Patientp-value ≤ 0.05. Continuous data with normal distribution were reported as the mean and standard deviation (SD) and were compared using either the dependent or independent samples t-test. Continuous variables with a non-normal distribution were reported as medians and interquartile ranges (IQR) and were compared using the Mann–Whitney U test for independent samples or the Wilcoxon signed-rank test for repeated measures. Categorical variables were reported as absolute numbers and percentages and were compared using the Chi-Square test for independent samples. McNemar’s Chi-square test was used to compare paired proportions. The correlation between TEE and TTE measurements of rMR grade and MVPG was assessed by Spearman’s rank correlation coefficients. Events-free survival was estimated using the Kaplan–Meier method and compared by the log-rank test. Univariable and multivariable Cox proportional hazards regression analyses were performed to evaluate for predictors of mortality and a combined endpoint of time to mortality, or first heart failure hospitalization with reported hazard ratios (HR) and 95% confidence intervals (CI). For multivariable analyses, an optimized model was calculated according to the results of the univariable analyses. Risk variables that were determined to be significant in the univariable analyses were tested subsequently with the multivariable modeling. A 2-sided p-value of ≤0.05 was considered statistically significant.Data were analyzed using SPSS software, Version 18.0 . The Shapiro–Wilk test was used to test the normality of distribution. The hypothesis of normality was rejected when the Eighty-two patients had undergone TEEMr at PMC between the years 2015 and 2020 and were included in the analysis. Baseline clinical characteristics are summarized in Most patients (87.8%) were elderly (age > 65 years), and they suffered from multiple co-morbidities. All patients exhibited poor functional capacity and 43% had multiple heart failure related hospital admissions during the preceding year. Eight patients suffered from myocardial infarction complicated by severe mitral regurgitation unresponsive to PCI and medical therapy and they underwent urgent TEEMr during their index hospitalization.Pre-procedural echocardiography parameters are presented in Fifty one (62.2%) patients had left ventricle (LV) systolic dysfunction and 19 patients (23.2%) had left ventricle ejection fraction (LVEF) of 30% or less. PMR was the underlying pathology in 23% of the patients with MR-4 and 5% of the patients with MR-3.® devices were deployed in 56 (68.3%), 22 (26.8%) and four (4.9%) patients respectively. Technical success was achieved in all cases. Seven patients underwent concomitant tricuspid valve repair. There were no intraprocedural deaths. One patient suffered from a large right to left shunt accompanied by hypotension and hypoxemia upon retraction of the delivery sheath from the left to the right atria. An atrial septal defect (ASD) occluder was immediately deployed with restoration of vital signs to normal. An ASD occluder was also used in another case to secure a large mobile mass that appeared after removal of the delivery system and was attached to the left side of the atrial septum. In another case, a patient suffered from sudden severe hypotension shortly after deployment of the device that necessitated cardio-pulmonary resuscitation with subsequent hemodynamic stabilization. TEE and fluoroscopy demonstrated a stable implanted device, rMR-1, low MVPG and no signs of cardiac tamponade. He was later discharged in a stable state. One patient suffered from a minor stroke that was recognized several minutes after extubation. Computed tomography did not demonstrate acute pathology. The patient was treated conservatively and over the course of several days, had resolution of all neurologic deficits.One, two and three Mitraclipp < 0.001). A quarter of the patients died within one year, mostly from heart failure related causes. Overall, during the follow-up period, 23 patients died and 36 patients had either died or were hospitalized for worsening heart failure related symptoms.Median follow-up was 433 (IQR 133–841) days. At 30 days, there were no cases of device embolization or single leaflet device attachment. Device success and procedural success were achieved in 66 (80.5%) and 63 (76.8%) of patients, respectively. Overall, four patients (4.9%) died within 30 days of TEEMr. One of them died after suffering from acute renal failure followed by dialysis and septic shock. Another one died from complications related to prolonged mechanical ventilation. The remaining two patients were discharged to their residencies and the cause of death was unknown. One patient who was discharged with aspirin and oral anticoagulant therapy, suffered from a hemorrhagic stroke 31 days following TEEMr, and later died 54 days after it. Another patient, that had procedural failure and ongoing heart failure symptoms, underwent surgical mitral valve replacement 67 days after the procedure. Three of the patients underwent repeated TEEMr at 47 days, 220 days and 323 days following their initial procedure. At the 1-year follow-up, 42.6% of surviving patients exhibited NYHA FC III and none of them had NYHA FC IV . While 6MR and rMR grades prior to TEEMr, immediately after the completion of the procedure (by TEE) and the results of TTE assessments prior to discharge and at 1-year follow-up are presented in p < 0.001). At discharge, 65.9% of the patients had rMR ≤ 1 and 86.6% of the patients had rMR ≤ 2. rMR grades at discharge were also significantly lower compared to pre-procedure grades (p < 0.001). EROA and regurgitant volume at discharge were almost 50 percent lower compared to pre-TEEMr values (from 0.42 ± 0.23 cm2 to 0.22 ± 0.11 cm2 (p < 0.001) and from 58 ± 25.4 mL to 25.5 ± 24.6 mL (p < 0.001), respectively). There was no significant difference between rMR grades at completion of TEEMr and those at discharge (p = 0.936). Compared to rMR grade at the completion of TEEMr, rMR grade at discharge did not change in 57.3% patients and distributed almost equally between patients with higher (22%) or lower (20.7%) rMR grades. The correlation between rMR grades at completion of TEEMr and those at discharge was moderate . One year after TEEMr, most surviving patients (90.9%) retained lower levels of rMR grade compared to their pre-procedure grade (p < 0.001) Approximately two thirds of them (63.6%) had rMR ≤ 2 and about half of them had rMR ≤ 1.At TEEMr completion, rMR ≤ 1 and rMR ≤ 2 was achieved in 64.6% and 92.7% of patients, respectively. The vast majority (90.5%) of the patients with baseline MR-4 and all the patients with baseline MR-3 had acute reduction of MR severity to rMR ≤ 2. More than half (55.6%) of the patients with baseline MR-4 and most (89.5%) of the patients with baseline MR-3 had acute reduction of MR severity to rMR ≤ 1. Compared to MR grades at baseline, rMR grades were significantly lower at completion of TEEMr (p = 0.002)  .r = 0.51, p < 0.001),  of patients, was higher in 27.8% patients and was lower in 6.9% patients. The correlation for MVPG assessments was also moderate (Spearman’s coefficient of rank correlation  0.001), .p = 0.008), (p = 0.238), nor in the time to the first heart failure hospitalization  or in the time to the combined endpoint of all cause death, or first heart failure hospitalization . Patients with rMR ≤ 1 at discharge, compared to patients with rMR-2 at discharge, demonstrated longer time to all-cause death  (p = 0.047) (p = 0.003) , a. There = 0.037) b, time t= 0.047) c and in = 0.003) d.p < 0.001) (p = 0.012)  rMR ≤ 2 at completion of TEEMr and rMR ≤ 1 at discharge were associated with improved mid-term clinical outcomes. (2) MVPG was not associated with an unfavorable outcome. (3) Following TEEMr, the correlation between either rMR grade or MVDG, assessed by TEE immediately at completion of the procedure and by TTE at discharge, is modest.r = 0.51) [r = 0.66) [rMR, following TEEMr, has been strongly associated with poorer prognosis. However, the grade of rMR from which prognosis is diverted is still uncertain with conflicting reported results ,15,16,21 = 0.51) . The ass = 0.66) . Potenti = 0.66) ,30,31,32 = 0.66) ,29,33. T = 0.66) ,29. Like = 0.66) ,35. Decr = 0.66) . Large p = 0.66) . Additio = 0.66) . Both ofSince this is a single-center, retrospective, observational study, it is possible that confounding factors have influenced the results. The study is of limited sample size and may be underpowered to accurately evaluate the clinical endpoints, thus affecting the results. A study with a larger sample size and higher statistical power may yield stronger correlations for either rMR grade or MVDG, assessed by both TEE immediately at completion of the procedure and by TTE at discharge. The clinical effect of elevated MVPG in a range compatible with severe mitral stenosis, following TEEMr, was not evaluated in this study. The regurgitant fraction was assessed only in cases where additional information was needed in order to determine MR level. Divergences in heart rate, blood pressure, hemoglobin level and the presence of post trans-septal atrial septal defect (ASD), with accompanying shunt, that may contribute to the differences between echocardiography assessments, were also not included in the present analysis. A more extensive study with a larger sample is needed in order to validate our results.rMR grade, as well as MVPG, assessed by TEE at the completion of TEEMr and by TTE at discharge, correlate modestly and therefore should be distinctly reported. Future reports of these parameters are encouraged to include information regarding the timing and the modality of assessment. Achieving rMR ≤ 2, immediately at the completion of TEEMr may be a sufficient result, while encountering higher MVPG does not seem to be associated with an unfavorable outcome. These observations may aid with practical decisions taken in real-time during TEEMr. Additional larger studies are needed to test the association between rMR grade, MVPG or their combination, at TEEMr completion, and long-term outcomes. The association of rMR ≤ 1, by TTE, at discharge, with improved clinical course was further validated by this report.


Mycoplasma hyopneumoniae (M. hyopneumoniae) is the etiological agent of enzootic pneumonia, a highly infectious swine respiratory disease that distributed worldwide. The pathogenesis and virulence factors of M. hyopneumoniae are not fully clarified. As an important virulence factor of bacteria, nicotinamide adenine dinucleotide (NADH) oxidase (NOX) participates in host-pathogen interaction, however, the function of NOX involved in the pathogenesis of M. hyopneumoniae is not clear.M. hyopneumoniae differed in virulence were identified, suggesting that NOX may be correlated with M. hyopneumoniae virulence. The nox gene of M. hyopneumoniae was cloned and expressed in Escherichia coli, and polyclonal antibodies against recombinant NOX (rNOX) were prepared. We confirmed the enzymatic activity of rNOX based on its capacity to oxidize NADH to NAD+. Flow cytometry analysis demonstrated the surface localization of NOX, and subcellular localization analysis further demonstrated that NOX exists in both the cytoplasm and cell membrane. rNOX was depicted to mediate adhesion to immortalized porcine bronchial epithelial cells (hTERT-PBECs). Pre-neutralizing M. hyopneumoniae with anti-rNOX antibody resulted in a more than 55% reduction in the adhesion rate of high- and low-virulence M. hyopneumoniae strains to hTERT-PBECs. Moreover, a significant difference appeared in the decline in CCU50 titer between virulent (168) and virulence-attenuated (168L) strains. NOX not only recognized and interacted with host fibronectin but also induced cellular oxidative stress and apoptosis in hTERT-PBECs. The release of lactate dehydrogenase by NOX in hTERT-PBECs was positively correlated with the virulence of M. hyopneumoniae strains.In this study, significant differences in NOX transcription expression levels among different strains of M. hyopneumoniae and may play a significant role in mediating its pathogenesis.NOX is considered to be a potential virulence factor of The online version contains supplementary material available at 10.1186/s12917-022-03230-7. Mycoplasma hyopneumoniae (M. hyopneumoniae) infection, which is characterized by nonproductive, dry coughing, severe respiratory distress, growth retardation, and inefficient feed conversion [M. hyopneumoniae is considered to be one of the primary pathogens involved in porcine respiratory disease complex (PRDC) [Pasteurella multocida [M. hyopneumoniae pathogenesis are not fully understood. Some reports have suggested that the onset of EP depends on virulence factors of M. hyopneumoniae that enable the pathogen to override host defense mechanisms by inducing a biochemical host response that promotes cell adhesion and immunomodulation [M. hyopneumoniae pathogenesis.Porcine respiratory diseases, which are characterized by high morbidity and mortality, medications cost, feed efficiency and growth rate decreasing , and lower quality of pig carcasses, led to a considerable impact on the pig industry . Enzootinversion . M. hyopx (PRDC) , 3. Infeultocida . This codulation . TherefoMycoplasma bovis have shown possible participation in virulence [M. hyopneumoniae strains that differed in degree of virulence [2O2 or H2O by simultaneously oxidizing NADH to NAD+. Various organisms contain these enzymes, which play important roles in regulating cellular redox and osmotic pressure balance to maintain normal cell growth and development. In addition to its effect on metabolic processes, NOX also plays a vital role in bacterial oxidative stress response, bacterial membrane formation, and virulence regulation [M. hyopneumoniae has already been reported to reside on the cell surface and is suspected to bind to fibronectin, heparin, actin, and plasminogen [M. hyopneumoniae may be linked to its virulence.Several studies have also demonstrated that mycoplasma metabolic enzymes play more than a basic role by possibly serving as an important determinant of pathogenicity –9. Amongirulence . Moreoveirulence , 11. NOXgulation . More imsminogen . Proteinsminogen . Severalsminogen . Thus, NM. hyopneumoniae. NOX specifically adhered to immortalized porcine bronchial epithelial cells (hTERT-PBECs) and recognized host fibronectin. NOX not only shows its enzyme characteristics but also induces obvious cytotoxicity, oxidative stress damage, and apoptosis in hTERT-PBECs. Our findings support the notion that NOX may be a potential novel virulence factor of M. hyopneumoniae, and these findings will provide new ideas and theoretical support for studying the pathogenic mechanisms of M. hyopneumoniae and other mycoplasmas.As a novel virulence factor candidate identified in this study, NOX was found in both the cytoplasm and cell membrane of nox gene between M. hyopneumoniae (Mhp) strains of differing virulence, relative quantitative reverse transcription polymerase chain reaction (RT-PCR) was performed. As shown in Fig. nox gene were significantly upregulated in the highly virulent strains Mhp 168, LH and JS compared to the virulence-attenuated strain Mhp 168L (p < 0.001). In addition, the mRNA expression levels of the nox gene in low virulent strain J were higher than that in the virulence-attenuated strain Mhp 168L, but with no significant difference (p > 0.05).To investigate mRNA expression differences of the M. hyopneumoniae were retrieved from the NCBI and UniProt Protein databases (Genbank accession numbers were shown in supplementary file), and the homologies among them were analyzed. All sequences were aligned using the CLUSTAL W program, as shown in supplementary file Fig. Snox gene of M. hyopneumoniae contains an open reading frame of 1383 base pairs (bp) encoding 460 amino acids. The results of comparative sequence analysis indicated that the amino acid sequences of NOX among all M. hyopneumoniae strains shared more than 99.34% homology with each other. It also showed that NOX is a highly conserved protein, and there were few differences among various M. hyopneumoniae strains. Based on codon optimization, the prokaryotic expression plasmid pET-21a-nox was constructed and expressed in Escherichia coli BL21 (DE3). Expression of purified recombinant NOX protein (rNOX) with a size of 50.6 KDa was shown in panel “a” of Fig. SEighteen amino acid sequences of NOX from M. hyopneumoniae, flow cytometry analysis was performed. The results showed that the outer membrane-localized NOX was surface-accessible to NOX-specific antibodies in Mhp strains 168 and 168L, initially suggesting that NOX antigen was present on the bacterial cell surface of M. hyopneumoniae strains. There was no significant difference in the mean fluorescence intensity (MFI) between the M. hyopneumoniae strain 168L treated with anti-rNOX serum and strain 168L incubated with pre-immune serum -stained cell nuclei in negative controls  were used to determine whether ols Fig. . MoreoveM. hyopneumoniae adhesion to hTERT-PBECs, an antibody inhibition assay was performed. Compared with the control group treated with negative serum, the anti-rNOX serum could reduce the adherence of M. hyopneumoniae (both strains 168 and 168L) to hTERT-PBECs. The adherence degree was shown as the adhesion rate compared with the adherence of M. hyopneumoniae in the absence of antibody. Incubation with anti-rNOX antibody resulted in 52.73% (strain 168) and 57.47% (strain 168L) (p < 0.001) reductions in the adhesion rate of M. hyopneumoniae to hTERT-PBECs  and 1.25  reduction in the 50% color changing unit (CCU50) of M. hyopneumoniae, and there was a significant difference in CCU50 titer decline between virulent strain 168 and virulence-attenuated strain 168L (p < 0.001)  and the unrelated protein  , 56.45% (J), 69.10% (168) and 69.35% (JS). The cells treated with lipopolysaccharides (LPS) and dimethyl sulfoxide (DMSO) (positive controls) induced cytotoxicity at 156.43% and 183.14%, respectively.As shown in Fig. M. hyopneumoniae strain was 27045 RLU (168L), 271068 RLU (J), 313941 RLU (168), and 535685 RLU (JS), indicating that virulence-attenuated M. hyopneumoniae strain 168L-induced ROS release against cells was significantly lower than the three virulent strains .rNOX could induce oxidative stress damage in hTERT-PBECs, as measured by the average luminescence expressed in relative luminescence units (RLUs). As shown in Fig. M. hyopneumoniae strains 168 or 168L in hTERT-PTECs was significantly decreased when rNOX was blocked with an anti-rNOX antibody. The late-stage apoptotic cell percentage (upper right quadrant) decreased by 58.7% and 46.2%, respectively . More importantly, rNOX alone produced significantly lower levels of oxidative stress than the attenuated 168L strain. This result may indicate that M. hyopneumoniae contains other factors that lead to higher oxidative stress in infected host cells. Moreover, flow cytometry results indicated that rNOX could induce apoptosis in hTERT-PBECs as well, and the late-stage apoptosis rate was upregulated by 87.4% compared with the negative control group (4.9%) after 12 h. Late-stage apoptosis induced by high virulence M. hyopneumoniae strain 168 or low virulence strain 168L in porcine bronchial epithelial cells was significantly decreased when rNOX was blocked with anti-rNOX antibody, with the late-stage apoptotic cell percentage decreasing by 58.7% and 46.2%, respectively. In conclusion, the above results further indicate that NOX, like the positive control (various M. hyopneumoniae strains), may function as a mediator of virulence in M. hyopneumoniae strains.Lactate dehydrogenase (LDH) is a stable cytoplasmic enzyme that converts lactate to pyruvate. Measurement of LDH release by damaged cells offers a method for detecting cell viability from the perspective of cell membrane integrity . We fount of ROS , 29. In t of ROS . Researct of ROS . When int of ROS . We founnox genes between high and low virulence strains, suggesting that NADH oxidase may be related with M. hyopneumoniae virulence. In addition, NADH oxidase was found in both the cytoplasm and cell membrane of M. hyopneumoniae and it has strong binding affinity with fibronectin. Besides, NADH oxidase had the ability to adhere to immortalized porcine bronchial epithelial cells and it could also induce host cell cellular oxidative stress, cytotoxicity, and apoptosis. In conclusion, NADH oxidase is considered to be a potential virulence factor candidate of M. hyopneumoniae, which may play a pivotal role in mediating its pathogenesis.The study showed that significant differences exist in the transcription levels of M. hyopneumoniae strains in this study were thawed from frozen stocks and subcultured three times before use. M. hyopneumoniae strain J (ATCC25934), which was subcultured once to establish frozen stocks from ATCC. Strain 168 was isolated from a pig exhibiting typical characteristics of EP in Gansu Province, China [th passage through KM2 cell-free liquid medium (modified Friis medium) containing 20% (v/v) swine serum at 37 °C, to yield strain 168L [M. hyopneumoniae strains were cultured in modified Friis’ medium designated KM2 cell-free medium containing 20% (v/v) swine serum  piglet after irradiation sterilization) at 37 °C in a humidified incubator. Titers of M. hyopneumoniae strains were quantified using a CCU50 assay [All e, China . This fiJ ATCC259, which wain 168L . Strain ain 168L . These f50 assay , which wM. hyopneumoniae infection in vitro by our laboratory [Immortalized porcine bronchial epithelial cells (hTERT-PBECs) were established for boratory . The celM. hyopneumoniae strains  were cultured in KM2 cell-free liquid medium containing 20% (v/v) swine serum at 37 °C for 48 h, and the titer of these five M. hyopneumoniae strains were adjusted to 1 × 108 CCU/mL before the next experiment. And then total RNA was extracted using a Total RNA Extraction Kit (Omega). No less than 1 μg of total RNA was reverse transcribed in a 10 µL reaction volume using HiScript® III RT SuperMix for qPCR (+gDNA wiper)  before running on an ABI 7500 Real Time PCR System using the ChamQ Universal SYBR qPCR Master Mix . The reverse transcription reaction was followed the manufacturer’s protocol, and the qRT-PCR was performed using cDNA under specific conditions for the nox gene with the following procedure: samples were heated at 95 °C for 30 s, and a two-step cycle  was repeated for 40 cycles. The MHP7448_0333 gene of M. hyopneumoniae was used as the internal control [nox gene between different M. hyopneumoniae strains was determined using the 2-ΔΔCT method [Five  control , 39. TheM. hyopneumoniae were retrieved from the National Center for Biotechnology Information (NCBI) and UniProt protein databases, and the homologies among them were analyzed. All sequences were aligned with CLUSTAL W program (open source). Molecular Evolutionary Genetics Analysis version 10 (MAGE10) was used for assessing phylogenetic inference according to the neighbor-joining criterion. The robustness of the hypothesis was evaluated with 1000 nonparametric bootstrap analyses.Eighteen amino acid sequences of NOX from M. hyopneumoniae nox gene (MHP168_RS00400) was synthesized by GenScript Biotech Corp.  and then expressed using the pET-21a vector in the BL21(DE3) E. coli strain. The rNOX protein was purified using high affinity Ni-Charged resin and identified by Western blot analysis. Detailed protocols are provided in supplementary file The Two 1-month-old New Zealand white rabbits were immunized with rNOX protein. After three immunizations at 2-week intervals, sera were collected and purified using a HiTrap Protein G HP antibody purification column. Detailed protocols are provided in supplementary file + at a temperature of 25 °C as previously described with modifications [340) was measured. The specific activity of rNOX was calculated as follows.The enzymatic activity of purified rNOX was determined by measuring the oxidation of NADH to NADications , 41. TheM. hyopneumoniae strains and to probe NOX surface distribution differences between highly virulent strain 168 and virulence-attenuated strain 168L, fluorescence intensity was measured using a flow cytometer as previously described with modifications [M. hyopneumoniae strains 168 and 168L (each 1 × 108 CCU/mL) were incubated with anti-rNOX serum at a 1:100 dilution (1:100 diluted pre-immune serum was used as a negative control). M. hyopneumoniae strains were then stained with fluorescein isothiocyanate (FITC)-conjugated anti-IgG , and the fluorescence intensity was measured using a BD Accuri C6 flow cytometer.To investigate whether NOX is present on the surface of ications . BrieflyM. hyopneumoniae were obtained using a membrane protein and cytoplasmic protein extraction kit  according to the manufacturer’s instructions. At the same time, the washed bacterial precipitate was resuspended in PBS and sonicated to prepare whole bacterial protein. Protein concentration was determined by the BCA Protein Assay Kit . Subcellular localization of NADH oxidase in various M. hyopneumoniae strains that differed in virulence was assessed by Western blot. Detailed protocols are provided in supplementary file Membrane proteins and cytoplasmic proteins of Aeromonas hydrophila) [hTERT-PBECs were grown to confluence in 24-well plates with DMEM/F12 medium supplemented with 2% (v/v) fetal bovine serum plus the abovementioned growth factors. Cultured hTERT-PBECs were washed with PBS three times and fixed with 4% paraformaldehyde for 10 min at room temperature. Subsequently, the cells were treated with 0.1% Triton X-100 and then blocked for 2 h in 3% (w/v) BSA in PBS. After incubation with different concentrations of purified rNOX (from 5 to 500 μg), the cells were washed three times with PBS and incubated with a 1:250 dilution anti-rNOX antibody for 2 h at 37 °C. Following three PBS washes, cells were incubated with tetraethyl rhodamine isothiocyanate (TRITC)-tagged anti-IgG  at a 1:100 dilution for 1 h at 37 °C. Finally, nuclei were stained with DAPI  before cells were observed using a fluorescence microscope (Zeiss). BSA and rGroEL (the cytoplasmic chaperone protein of rophila)  were useM. hyopneumoniae bacteria  were collected and washed three times with PBS before resuspension in 500 µL PBS. The samples were preincubated with polyclonal antibody raised against rNOX or pre-immune sera (1:20 dilution) at 37 °C for 30 min. hTERT-PBECs with a confluence of 85% in 24-well cell plates were washed with PBS three times before the experiment. Bacteria suspended in DMEM/F12 medium were added to cells seeded in 24-well cell plates, and the cell plates were centrifuged at 800 × g for 10 min and then incubated for 2 h at 4 °C. After washing with PBS three times, the cells in each group were treated with lysis buffer containing 0.1% trypsin. Bacterial counting, including bacterial genome extraction and real-time PCR, was performed according to a previous method [M. hyopneumoniae were quantified using a CCU50 assay as mentioned above. These tests were performed in triplicate, and data were analyzed using GraphPad Prism 6 and SPSS 20.0.s method . The rea2SO4). OD readings were obtained at a wavelength of 450 nm with an ELISA microplate reader .As previously described with some modifications , the recEach 20 μg sample of rNOX was resolved by 10% SDS-PAGE and then transferred to a PVDF membrane . The membranes were washed three times with PBS and then blocked with 5% skimmed milk in TBST (TBS containing 0.5% Tween 20) at 37 °C for 2 h. Subsequently, the membranes were incubated with 15 μg/mL fibronectin  for 2 h at 37 °C. Following three washes using TBST, the membranes were incubated with anti-fibronectin antibody  in blocking solution at 37 °C for 2 h, after which they were washed three times with TBST followed by incubation with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG  at 37 °C for 2 h. Finally, the membranes were developed with Electro-Chemi-Luminescence  substrate using a ChemiDoc XRS+ system (Bio–Rad). BSA was used as a negative control rather than rNOX.M. hyopneumoniae strains  were used as positive controls. All groups were performed with three independent replicates. After 6 h, culture supernatants were collected, and LDH activity was measured using a CytoTox 96® Non-Radioactive Cytotoxicity Assay  according to the manufacturer’s instructions.hTERT-PBECs were incubated for 6 h and grown to confluence in 24-well plates. After three washes with PBS, the cells were then incubated with purified rNOX protein at different concentrations . PBS was used as a negative control rather than rNOX, and LPS (5 μg/mL), DMSO (1% diluted in DMEM/F12 medium), and four M. hyopneumoniae strains  were used as positive controls. All tests were performed with five independent replicates. Reactive oxygen species (ROS) were measured using a ROS-Glo™ H2O2 Assay  according to the manufacturer’s instructions, and RLUs were recorded using a plate reader.hTERT-PBECs were seeded and grown to confluence in 24-well plates. The cells were washed with PBS followed by incubation with purified rNOX protein of different concentrations (from 5 to 20 μg) at 37 °C for 6 h. At the same time, PBS was used as a negative control, and cells treated with LPS (5 μg/mL), DMSO (1%), and four 5 cells/well in 500 µL of culture medium. Then, hTERT-PTECs were incubated with 20 μg purified rNOX at 37 °C for 12 h. Cells cultured in DMEM/F12 plus 2% FBS culture medium supplemented with epithelial growth factors F12 without the reagents mentioned above were used as a negative control, and cells incubated with M. hyopneumoniae strains  were used as positive controls. At the same time, to explore whether antiserum to rNOX, which could block the apoptosis induced by M. hyopneumoniae in hTERT-PBECs, we pre-incubated M. hyopneumoniae strains (168 and 168L) with anti-rNOX antibody (1:20 dilution) at 37 °C for 30 min before inoculation with hTERT-PBEC seeds in 24-well plates at 37 °C for 12 h. All groups were performed with three independent replicates. Apoptosis rates were detected with the dual staining Annexin V- fluorescein isothiocyanate / propidium iodide (Annexin V- FITC/PI) method using an Annexin V-FITC/PI Apoptosis Detection Kit .hTERT-PBECs were seeded in 24-well plates at 2×10nox gene between the virulence-attenuated strain 168L and the virulent Mhp strains 168, JS, LH and J were assessed via one-way analysis of variance (ANOVA) using SPSS Statics v20.0 software and GraphPad Prism 6 software. The adhesion rates between anti-NOX serum or negative serum and groups of the high-virulence strain 168 as well as the adhesion rates between the low-virulence strain 168L and anti-NOX serum or negative serum were compared by Student’s t test. P < 0.05 was considered statistically significant, P < 0.01 was considered highly significant, and P < 0.001 was considered extremely significant.All experiments were repeated in triplicate, except for the detection of ROS, which was conducted in five independent replicates. Data represent the mean ± standard deviation (SD) of three or five individual experiments. Relative mRNA expression differences of the Additional file 1: S1.Table S1. Primers used in this study. Fig. S1. (A) Multiple sequence alignment of proteins NOX from different M. hyopneumoniae strains. (B) Phylogenetic analysis based on the NOX proteins of different M. hyopneumoniae strains by the neighbor-joining (NJ) method with the sequences available in GenBank and Uniprot Database. The numbers at the phylogenetic branches indicated the bootstrap values (1000 replicates) in percentage supporting each group. The bar represents the genetic distance. Fig. S2. Prokaryotic expression and purification of recombinant protein, WB verification of prepared polyclonal antibody. “a”, Lane M is prestained protein mass markers, with recombinant protein rNOX size of 50.6 KDa. The prepared polyclonal antibody effect of the recombinant protein rNOX was tested by Western Blot. “b”, a clear band appeared at position of 50.6 KDa. Lane 1, purified recombinant protein rNOX. Lane 2, unpurified E. coli whole bacteria protein before purification. Lane M, prestained protein mass marker. Fig. S3. SDS–PAGE analysis of purified recombinant protein of rNOX, other proteins of M. hyopneumoniae (Mhp) and BSA. Lane M, prestained protein mass marker. Lane 1, purified recombinant protein rNOX. Lane 2, bovine serum albumin (BSA). Lane 3, purified recombinant nicotinamide adenine dinucleotide-dependent flavin oxidoreductase (NFOR) protein of Mhp. Lane 4 and 6, blank. Lane 5 and 7, purified recombinant leucine aminopeptidase (LAP) protein of Mhp. Fig. S4.M. hyopneumoniae proteins with fibronectin interaction analysis by far Western blot. Lane M, prestained protein mass marker. Lane 1, purified recombinant protein rNFOR. PVDF membrane with transferred Mhp NFOR protein incubated with fibronectin and the anti-fibronectin antibody. Lane 2, 4 and 8, BSA, PVDF membrane with transferred BSA (negative control) incubated with fibronectin and the anti-fibronectin antibody. Lane 3, purified recombinant protein rNOX. PVDF membrane with transferred Mhp NOX protein incubated with fibronectin and the anti-fibronectin antibody. Lane 5, 6, 7 and 9, purified recombinant protein rLAP. PVDF membrane with transferred Mhp LAP protein incubated with fibronectin and the anti-fibronectin antibody.


Hearing loss can have a major impact on children’s language development, academic success and hearing comprehension. The aim of the present study was to determinate risk factors for severe and profound hearing loss in child candidates for cochlear implantation in southeast of Iran during 2014–2020.This case-control study consisted of 400 children referring to a cochlear implant center (in southeastern Iran) from Bandar Abbas, Zahedan and Kerman during the years 2014–2020 as cases. The subjects were selected using the random sampling method; 200 children hospitalized in Shafa and Afzalipour hospitals were selected as controls.p < 0.05), hospitalization in NICU (OR = 7.21: p < 0.05), family history of hearing loss (OR = 11.47: p < 0.05), Gestational age over 35 (OR = 9.63: p < 0.05), intracranial hemorrhage (OR = 5.18: p < 0.05), consanguineous marriage (OR = 12.48: p < 0.05) and high fever and seizures (OR = 3.02: p < 0.05) were recognized as risk factors for sensorineural deafness in children.Based on the results of the multivariate logistic regression, weight less than 1500 g (OR = 4.40: Most of the risk factors for deafness are preventable, and hereditary factors play an important role in congenital deafness in children. Therefore, genetic counseling before consanguineous marriage, early diagnosis, timely intervention can prevent many cases of hearing loss in children. Hearing is known to be the main sensory access of human at the beginning of life which enables him to identify, pay attention, trace sounds and integrate essential hearing experiences to develop speech and language . The intThis case-control study aimed to elucidate the role of risk factors related to hearing loss and deafness in children. The case sample consisted of 400 children referring to a cochlear implant center (in southeastern Iran) from Bandar Abbas, Zahedan and Kerman during the years 2014–2020. The subjects were selected using the random sampling method; 200 children hospitalized in Shafa and Afzalipour hospitals were selected as hospital controls.Inclusion criteria for the case group were diagnosis of severe (not hearing sounds between 71 and 90 dB) and profound hearing loss (not hearing sounds more than 91 dB) by the physician or undergoing cochlear implantation.P-value of less than 0.05 was considered significant.The controls of this study consisted of children hospitalized in other wards or outpatient clinics of Shafa and Afzalipour hospitals who had no hearing loss or deafness at the time of the study and had no history of this disease in their past. Data was collected through face-to-face, telephone interviews and medical records using a questionnaire. This questionnaire contained information about age, gender, consanguineous marriage and family history that collected from interviews. The other information about medical care gathered from medical records. In order to comply with the principles of medical ethics, oral consent was obtained from patients that interviewed by telephone and written consent was obtained from patients interviewed through face-to-face. Univariate and multivariate logistic regression models were used to analyze the data, and the risk of hearing loss or deafness was expressed as odds ratio calculations and estimates (95% confidence). First, univariate regression was used; the significant variables were then entered into multivariate regression. Data was analyzed using SPSS software (version 24). In this study, 600 children were examined . Out of 400 children, 216 were boys (54.5%) and 184 were girls (46.0%), with the average age of 8.91 ± 4.39. In the control group, 93 were boys (46.5%) and 107 were girls (53.5%), with the average age of 6.73 ± 3.26.p > 0.05)) were significant factors affecting sensory-neurological deafness in children (Table p < 0.05), hospitalization in NICU (OR = 7.21: p < 0.05), family history of hearing loss (OR = 11.47: p < 0.05), Gestational age over 35 (OR = 9.63: p < 0.05), intracranial hemorrhage (OR = 5.18: p < 0.05), consanguineous marriage (OR = 12.48: p < 0.05) and high fever and seizures (OR = 3.02: p < 0.05) were recognized as risk factors for sensorineural deafness in children  . A systeThe Joint Committee on Infant Hearing has noticed family history of hearing loss as risk factor for permanent congenital, delayed onset, or progressive hearing loss since 1973 . In our Consanguinity is common in some regions of Asia and Africa because of socioeconomic, cultural, and religious factors. Historical studies report a strong preference consanguineous marriages in Iranians. Consanguineous marriage is an important risk factor of genetic disorders . Our stuOur study revealed that pregnancy at the age of 35 and higher is associated with increased risk of hearing loss in child. Leslie et al. also reported maternal age as a risk factor for hearing loss . HoweverA pontine intracranial hemorrhage (ICH) evokes several neurological symptoms, due to the various nuclei and nerve fibers; however, hearing loss from a pontine ICH is rare , howeverOur study also revealed that fever and seizures is associated with increased risk of hearing loss in children. This finding was similar to the results of studies by Karanja et al. . Murray Hereditary factors play an important role in congenital deafness in children especially in families with a family history of hearing loss and genetic counseling prior to consanguineous marriage, which is common in Asian countries, are crucial factors to be considered. In addition, early diagnosis, timely intervention, vaccination to prevent meningitis, increased birth care and preventing the use of ototoxic medications can prevent many cases of hearing loss in children and may dramatically reduce the cost of education related to hearing loss.The present study had some limitations. First, Incomplete medical records of some patients. Second Lack of parental responsibility and due to the data were collected retrospectively, which might cause to recall bias. Third, Change patients’ phone number so we couldn’t contact them.


Early speech perception abilities may impact CHH outcomes and guide future intervention. The objective of this study was to examine the use of a conditioned head turn (CHT) task as a measure of speech discrimination in CHH using a clinically feasible protocol. (2) Methods: Speech perception was assessed for a consonant and vowel contrast among 57 CHH and 70 children with normal hearing (CNH) aged 5–17 months using a CHT paradigm. (3) Results: Regardless of hearing status, 74% of CHH and 77% of CNH could discriminate /a-i/, and 55% of CHH and 56% of CNH could discriminate /ba-da/. Regression models revealed that both CHH and CNH performed better on /ba-da/ at 70 dBA compared to 50 dBA. Performance by hearing age showed no speech perception differences for CNH and children with mild hearing loss for either contrast. However, children with hearing losses ≥ 41 dB HL performed significantly poorer than CNH for /a-i/. (4) Conclusions: This study demonstrates the clinical feasibility of assessing early speech perception in infants with hearing loss and replicates previous findings of speech perception abilities among CHH and CNH. This is a study about behavioral speech perception among hard-of-hearing infants whose hearing losses were identified through universal newborn hearing screenings. Infants in this study were fit with hearing aids by 3 months of age and participated in early intervention through a system of care called Early Hearing Detection and Intervention (EHDI) in the United States. Universal newborn hearing screenings and EHDI systems have radically improved age at identification, diagnosis, and early intervention for children who are hard-of-hearing (CHH), allowing speech perception testing at very young ages. While EHDI programs have led to significant improvements in language outcomes, much remains unknown about early speech perception abilities in young infants with hearing loss or how such abilities contribute to language outcomes compared to their normal hearing peers . CHH whoClinical assessment of speech perception is recommended as part of the Pediatric Minimum Speech Test Battery during the first year of life ; howeverNumerous studies among CHH and CNH have examined speech perception abilities longitudinally ,24,25,26Among CNH, speech perception has been assessed in research settings for over 40 years and can be assessed behaviorally around 6 months of age using a conditioned head turn (CHT) paradigm. CHT is similar to a commonly used audiologic assessment tool, visual reinforcement audiometry (VRA). VRA is used to assess an infant’s ability to detect the presence/absence of sound and is clinically useful for establishing an infant’s audiometric thresholds . WhereasAssessment of speech perception among CNH has demonstrated that infants transition from being universal language learners and become better at discriminating their native languages between 6–12 months of age ,44,45,46p = 0.004), but the same was not true for the CNH (p = 0.45). While there is still more to learn about the development of speech perception among infants with normal hearing, even less is known about how development is impacted by the presence of hearing loss. Much of the work on speech perception development has been done in infants who use cochlear implants. Little is known about speech perception among infants with mild to severe permanent hearing loss between 6 to 17 months of age who benefited from early identification and hearing aid use. One finding is that CHH in the mild to severe hearing loss range discriminate vowel sounds more accurately than consonant sounds ,48,50,51The majority of speech perception studies for CHH have involved young deaf children who receive cochlear implants between 6–24 months of age and assessed speech perception between 9–30 months of age using CHT measures ,52,53 viWhat proportion of CHH and CNH can demonstrate discrimination of each a vowel and consonant contrast as measured by CHT, at 50, 60, and/or 70 dBA SPL in a single test session while accounting for hearing age?Is there a difference in the ability of CHH (who met 1–3–6 benchmarks) and CNH in their demonstration of vowel and consonant discrimination assessed through a CHT task?Is there a relationship between aided SII measured at 50 dB SPL and performance on a CHT speech perception task for each contrast?In this study, we extend our work towards a tool that could be clinically useful to assess speech discrimination in infancy ,39. A toData were collected for 129 children (58 CHH and 71 CNH) aged 5–17 months  = 2.34 months. Two children (one CNH and one CHH) were lost to follow up prior to collecting speech perception data resulting in 127 children contributing to speech perception data . 110 children completed speech perception testing for both consonant and vowel contrasts (/a/ vs. /i/ and /ba/ vs. /da/) the remaining 17 children completed either /a-i/ or /ba-da/. Specifically, nine CNH and two CHH only completed /a-i/ and three CNH and three CHH only completed the /ba-da/ contrast. The reasons for data loss were as follows: failure to condition to the task for the second contrast , loss to follow-up after completing one contrast , and modification in the presentation of the stimuli level for /ba-da/ 50 versus 65 dBA SPL for the first level assessed . The data for 43 children with CNH  and 11 cInclusion criteria were the same as in Uhler  (a) no eAll participants used their own hearing aids during the CHT procedure. Following diagnosis, children received individualized care from their managing audiologist following best practices for amplification fitting, verification, and validation . ChildreTo verify amplification, simulated real-ear response measurements were compared to age-specific DSL targets using real-ear to coupler differences (RECDs). When RECDs were unable to be recorded from at least one ear from the child, average age specific RECD values were substituted. Before laboratory testing, all devices were evaluated to assure proper function. An electroacoustic test box measure was completed to assess hearing aid function. To assess hearing aid output either, measured or simulated real ear coupler differences. The aided SII was automatically calculated at 50, 60, and 70 dBA SPL using the Audioscan Verifit.Hearing aid use was determined by reading the average daily hearing aid use from the child’s hearing aid. The mean hearing aid use was 6.62 h (SD = 4.08), see Parents were paid $15 per hour for their child’s participation for each study visit.For this experiment, two speech sound contrasts were used, /a-i/ and /ba-da/. Contrasts were selected based on difficulty levels, with vowels being the easiest and consonant contrasts being more difficult for both CHH and CNH ,50,51,57All testing was conducted in an acoustically treated sound booth over two sessions. Session one included the case history , tympanometry, hearing screening, and, if time allowed, a threshold search for /a/ using CHT. Session two included a threshold search for /a/ (if not completed at the first visit) and the CHT assessment protocol. During CHT testing, one of the speech sounds for each stimulus pair, /a/ or /i/ and /ba/ or /da/, served as the background stimulus, repeated with 1200 ms interstimulus interval throughout the session. The other speech sound served as the target. The member of the pair serving as the target stimulus was counter-balanced across participants. The infant learned to respond by turning their head when the target stimulus was presented. The infant was accompanied by their caretaker into the sound booth for the CHT assessment. The infant was seated either on the caretaker’s lap or in a highchair in the center of the room to minimize distractions or task fatigue. Care was taken to ensure that the infant was comfortable and the location and distance to the speaker remained constant regardless of how the infant was seated. The background stimulus was on when the infant and caretaker entered the room. The speaker and visual reinforcement video screen were 90º to the right of the infant’s midline. An assistant who centered the infant’s gaze was positioned in front of the infant, slightly to the left. The caretaker and the assistant listened to music through supra-aural headphones to prevent them from hearing the sounds presented to the infant and inadvertently reinforcing the child or alerting the child to a contrast stimulus. The evaluator was seated outside the sound booth in a test room and observed the infant through a window. The evaluator in this study was one of five audiologists. The perception task consisted of two phases: conditioning and testing. During the conditioning phase, only change trials were presented so infants could learn to associate a change in the sound and the reinforcer. To facilitate learning during conditioning, the target sound was presented at 6 dBA louder than the background sound to draw the infant’s attention to the sound change. Initially, in conditioning trials, the reinforcer turned on following two target sounds to “teach” the task to the infant. The infant learned to pair a change in the sound with the reinforcer. After the infant made two consecutive head turns that occurred before the end of the first two presentations of the target sounds the intensity cue was removed. Once testing began, the evaluator could not hear the stimuli and was blinded to stimulus type—trials were initiated by the evaluator by pressing a button when the child’s attention was directed toward the midline.Each of the 15 trials had an equal probability of being a change or no change trial; the trial type was randomly selected by the computer program. If the trial was a no-change trial, the background sound was presented three times. When a change trial was presented, the target sound was presented three times. Once the trial ended, the background sound continued. When head turns were observed by the evaluator, a button was pressed indicating the response. Correct responses were determined by the CHT software, and were rewarded by automatic presentation of an animated video. If the child’s head turn was incorrect, it was considered a false positive because no change had occurred. z-score:Fifteen trials were administered during each contrast assessment. Performance on speech perception was calculated using d-prime (d’) . The advIf the child achieved a d’ value of at least 1.21 at 50 dBA, then testing was complete ,36,38. WHearing age was calculated by subtracting age at hearing aid fitting (average age at hearing aid fitting was 3 months) from chronological age at test time. CNH hearing age was the same as chronological test age. Age was stratified as younger or older than nine months at time of testing, chosen as it matches the hearing age of CHH with the earliest age category of CNH that completed CHT testing (6–7 months) and better reflects an equal amount of auditory access between groups. Hearing status was categorized as normal hearing, mild hearing loss, and moderate or greater (moderate+) hearing loss. Hearing status and hearing age were analyzed as categorical variables instead of continuous variables given our intent to replicate previous literature with interpretable findings ,60. t-tests for continuous measures and chi-squared or Fisher’s exact tests for categorical measures, as appropriate. Descriptive summaries are provided as mean  for continuous measures and as frequency (%) for categorical measures. Normality of the outcomes was evaluated with the Shapiro-Wilk test and graphically, with results suggesting normality. Comparisons of between group demographics used Linear regression models for the outcome of speech perception performance reported as d’ scores  were evaScatterplots with speech perception scores across presentation level and hearing status were created with regression fits based on models including PTA category  and presentation level, with points jittered within a given intensity level  to better visualize the data. All analyses and figures were conducted using R Foundation for Statistical Computing v3.6.3 .t-test p > 0.05). For analysis, we compared the average speech perception performance for both /a-i/ and /ba-da/ contrasts stratified by hearing age group (<9 months vs. >9 months), presentation level, and hearing status. There were no differences between CHH and CNH across conditions (p = 0.03) such that children with moderate+ hearing losses had d’ scores that were 0.45 lower ) than CNH. There were no group differences for /ba-da/ . Next, we categorized speech perception performance as a function of hearing status  while considering hearing age. Performance on /a-i/ as a function of presentation level is shown in p = 0.002). Among CHH cohort, To examine the role of presentation level for vowel and consonant speech perception abilities, we fit linear regression models for the overall sample of CHH and CNH for the outcome of speech perception performance across all presentation levels . For the overall sample in p > 0.05 for all).To examine the potential association of aided SII on speech perception abilities, the regression models were refit and restricted to only the CHH group. Aided SII at 50 dB input ranged from 0.21–0.98. Aided SII is not significantly associated with speech perception for /ba-da/ or /a-i/ when adjusting for age, mild versus moderate+ hearing loss, and presentation level , presentation level, hearing age, and hearing status. Both CHH and CNH were able to discriminate each contrast, however more children, regardless of hearing status, were able to reach criterion on the vowel contrast /a-i/ compared to the consonant contrast /ba-da/. CHH and CNH had higher scores on the /ba-da/ contrast at 70 dBA compared to 50 dBA; the same benefit was observed for the CHH when analyzed as a separate cohort. We also found that children with moderate+ hearing losses performed significantly poorer than CNH across multiple intensity levels for /a-i/. However, children with mild hearing loss did not differ in their performance from CNH for the contrasts tested in this study. Finally, aided SII was not a significant predictor of speech perception performance.Children with moderate+ hearing losses performed significantly poorer than CNH across multiple intensity levels for /a-i/ when considering hearing age. We were surprised that no differences in speech perception abilities were observed between CHH and CNH for /ba-da/ based on our previous research findings. Of note, all CHH benefited from meeting the EHDI benchmarks. Both groups, when considering chronological age, performed similarly, as 74% of CHH and 77% of CNH were able to successfully discriminate /a-i/ and 56% of CHH and 55% of CNH were able to discriminate /ba-da/. Forty-four percent of CHH and 45% CHH did not reach criterion for /ba-da/ at any level. Among CNH this is poorer than previously reported where 29% Uhler  and 28% When looking at our data for /ba-da/ discrimination, 56% of CHH and 55% of CNH reached a criterion, which is poorer than performance on /a-i/ discrimination. The finding that vowel discrimination is easier for infants, regardless of hearing status, is also similar to previously reported speech perception abilities found in the ,48,49,61We speculate that better performance on /a-i/ than place of articulation contrasts such as /ba-da/ ,49,61,62CHH and CNH performed better on the /ba-da/ contrast at 70 compared to 50 dBA. The same benefit was observed for the CHH when analyzed as a separate cohort, while adjusting for age and severity. This difference in overall performance between 50 and 70 dBA agrees with the previous findings from Nozza  where inAs in our previous work, we examined the relationship between speech perception abilities and aided SII, because of the role it plays in hearing aid fittings ,66. AmonThere is a great deal of variability in performance of infant speech perception. We sought to test a large number of CHH and CNH, which we achieved. However, even when tripling our sample size of CHH, there is an uneven distribution of children with each degree of hearing loss ranging from mild to severe. The high variability can be seen from Furthermore, while we do not anticipate that CNH are unable to discriminate /ba-da/, our study design did not allow us to explore what it means that 44% of CNH were unable to reach criterion. However, this is the first work of its kind to report conditioning in a single test session and to report data for all infants who demonstrated successful conditioning to the task regardless of test performance. Future work should include some measure of test-retest reliability across multiple visits. Of note, a subset of our CNH was previously reported in Uhler et al. , for whiThe majority of CHH (74%) and CNH (77%) were able to discriminate /a-i/ at intensity levels between 50–70 dBA in a single testing session. Reported data included all children who demonstrated successful conditioning regardless of their speech perception scores. However, there was a slight decline for CHH who were able to discriminate the /a-i/ contrast compared to our previous studies in which 95% of CHH could achieve criterion. When comparing discrimination performance of the consonant contrast, these findings are again similar among CHH such that 56% of CHH were able to discriminate /ba-da/ compared to our work published in 2017. The same pattern of performance did not hold true for CNH on the /ba-da/ contrast. Similar findings with a larger sample size contribute to the generalizability of our results among a population of CHH who met EHDI benchmarks. CHH can perform similarly to CNH in a quiet environment. This work contributes to the speech perception literature among infants with mild to severe permanent hearing loss and demonstrates the feasibility of using this clinically viable protocol.


Neurofilament light chain (NfL) levels have been suggested as reflecting axonal damage in various inflammatory and neurodegenerative disorders, including acquired peripheral neuropathies. We aimed to investigate if serum NfL (sNfL) levels can be a biomarker of disease activity and treatment response in patients with chronic inflammatory demyelinating polyneuropathy (CIDP).The sNfL levels of eleven newly diagnosed patients with CIDP were retrospectively assayed and compared with seven healthy volunteers. The levels were assayed before and after intravenous immunoglobulin treatment in patients with CIDP and were also assayed in the remission period.Baseline sNfL levels in patients with CIDP before treatment were significantly higher than those in healthy controls. The levels significantly decreased overtime after one month of treatment and in remission period. There were significant negative correlations between the sNfL levels and the disease duration , and weak correlations between the sNfL levels and overall neuropathy limitations scale.sNfL may be a potential biomarker reflecting the disease activity in patients with CIDP. Serum neurofilament light chain (sNfL) levels were assayed in patients with newly diagnosed chronic inflammatory demyelinating polyneuropathy (CIDP). We found that the levels in CIDP patients were significantly higher than those in healthy controls, and that the levels significantly decreased over time after treatment and in remission period. sNfL may be a potential biomarker reflecting the disease activity in patients with CIDP. CIDP tyNeurofilaments are the primary cytoskeletal proteins of neurons in both the central nervous system (CNS) and the peripheral nervous system (PNS), and form a lattice comprising neurofilament light (NfL), medium, and heavy chains . 2. Post‐treatment group: patients with CIDP one month after the first treatment with IVIg (n = 7). 3. Remission group: patients in long‐term remission who had been off treatment or oral prednisolone or immunosuppressant for longer than six months (n = 9). This study was performed with the approval of the Ethics Committee of the University of Toyama . Written informed consent was obtained from all patients.Eleven patients who have been diagnosed with definite CIDP for the first time according to the EFNS/PNS 2010 criteria were studied retrospectively . Treatment was considered effective when the symptoms improved by one or more on ONLS within four weeks after IVIg treatment.2.3All patients underwent nerve conduction studies to confirm the diagnosis of CIDP. As an electrophysiological proxy marker of axonal damage, we used the lowest (left or right) summated distal compound muscle action potential (CMAP) negative peak area of the median, ulnar, and tibial nerves , which runs ultra‐sensitive paramagnetic bead‐based enzyme‐linked immunosorbent assays.2.5t test were used for the patients with CIDP and controls, correlations were determined using nonparametric Spearman's correlation coefficient, and the corresponding plots were drawn on logarithmic scale. Longitudinal time‐effects were analyzed by trend test based on linear mixed regression model. Statistically significant differences were determined at a 5% level of probability. The statistical analysis was performed using JMP®14 (SAS Institute Inc.), or R version 4.0.2 .A rank‐based nonparametric test (Mann–Whitney's U test) and Welch's 3n = 7), MADSAM (n = 3), and pure sensory (n = 1). The median disease duration before blood sampling was 16.0 weeks (interquartile range [IQR]:8.0–28.0). No patients received prior therapy for CIDP, though one patient had been received 2.5 mg of prednisolone for the treatment of idiopathic thrombocytopenic purpura at pre–IVIg sampling. The median ONLS of the pretreatment, post‐IVIg, and remission groups were 5.0 (IQR: 3.0–6.0), 3.0 (IQR: 1.5–5.5), and 2.0 (IQR: 1.0–3.0), respectively   pg/mL than in the healthy controls (mean 12.2 ± 6.3 pg/ml)  )     Figure . There w4In the present study of patients with CIDP in three different phases, significant increase in the sNfL levels was observed in patients with treatment‐naïve CIDP who were supposed to have high disease activity. This result is in line with a previous report  are different from those in conventional CIDP patients with macrophage‐induced demyelination (Koike et al., This study had several limitations in that it was a hospital‐based cross‐sectional study, and the sample size was small.In summary, our results suggest that sNfL may be a potential biomarker for assisting the diagnosis and evaluating the activity of CIDP. Further studies are needed to clarify the significance of sNfL as a biomarker for CIDP.The authors declare no conflicts of interest associated with this manuscript.T.H, T.N., J‐L.P, and Y.N. took part in the study design. R.A., N.M., M.Y., H.K., and N.D. performed the clinical evaluation. T.H. and T.N. analyzed the data. T.S. performed the statistical evaluation. All authors contributed to the interpretation of data. T.H. wrote the manuscript draft, which was critically revised and finally approved by all authors. Y.N. supervised the study.https://publons.com/publon/10.1002/brb3.2084.The peer review history for this article is available at


Is the increase of hearing loss among shipyard workers more rapid at 12.5 kHz than at lower frequencies, and do any auditory processing deficits occur before there is significant hearing loss?In this cross-sectional study of 7890 shipyard workers, hearing loss at 12.5 kHz was more rapid than at lower frequencies. Greater cumulative noise exposure was associated with auditory processing deficits among those with near-normal hearing.These findings suggest that a 12.5-kHz threshold and auditory processing disorders could serve as early indicators of noise-induced hearing impairment. This cross-sectional study examines whether hearing loss among shipyard workers increases more rapidly at extended higher frequencies and whether subtle auditory processing deficits are present in those with extensive noise exposure but little or no hearing loss. It is important to determine what frequencies and auditory perceptual measures are the most sensitive early indicators of noise-induced hearing impairment.To examine whether hearing loss among shipyard workers increases more rapidly at extended high frequencies than at clinical frequencies and whether subtle auditory processing deficits are present in those with extensive noise exposure but little or no hearing loss.This cross-sectional study collected audiometric data (0.25-16 kHz), survey questionnaires, and noise exposure levels from 7890 shipyard workers in a Shanghai shipyard from 2015 to 2019. Worsening hearing loss was evaluated in the group with hearing loss. Speech processing and temporal processing were evaluated in 610 participants with noise exposure and clinically normal hearing to identify early biomarkers of noise-induced hearing impairment. Data analysis was conducted from November to December 2020.Linear regression was performed to model the increase in hearing loss as function of cumulative noise exposure and compared with a group who were monitored longitudinally for 4 years. Auditory processing tests included speech-in-noise tests, competing sentence tests, dichotic listening tests, and gap detection threshold tests and were compared with a control group without history of noise exposure.P < .001).Of the 5539 participants (median [interquartile range (IQR)] age, 41.0 [34.0-47.0] years; 3861 [86.6%] men) included in the cross-sectional analysis, 4459 (80.5%) were hearing loss positive and 1080 (19.5%) were hearing loss negative. In younger participants , the maximum rate of increase in hearing loss was 0.40  dB per A-weighted dB–year (dB/dBA-year) at 12.5 kHz, higher than the growth rates of 0.36  dB/dBA-year at 4 kHz, 0.32  dB/dBA-year at 10 kHz, 0.31  dB/dBA-year at 6 kHz, 0.27  dB/dBA-year at 3 kHz, and 0.27  dB/dBA-year at 8 kHz. In the 4-year longitudinal analysis of hearing loss among 403 participants, the mean (SD) annual deterioration in hearing was 2.70 (2.98) dB/y at 12.5 kHz, almost twice as that observed at lower frequencies . The auditory processing scores of participants with clinically normal hearing and a history of noise exposure were significantly lower than those of control participants (eg, median [IQR] score on speech-in-noise test, noise-exposed group 1 vs control group: 0.63 [0.55-0.66] vs 0.78 [0.76-0.80]; These findings suggest that the increase in hearing loss among shipyard workers was more rapid at 12.5 kHz than at other frequencies; workers with clinically normal hearing but high cumulative noise exposure are likely to exhibit deficits in speech and temporal processing. Advocates point out the advantage of EHF in early identification of hearing loss due to ototoxic drugs and noise exposure.16 However, a systematic review failed to arrive at a robust conclusion regarding the advantage of EHF vs conventional audiometry.15 Most of the conclusions have been derived from a pool of participants with unclear durations and/or doses of noise exposure.17 In addition, many studies lacked longitudinal measurement of hearing. Therefore, the utility of EHF audiometry to test for early onset NIHL remains an open question.The most common biomarker used to identify NIHL is the shape of the audiogram.19 This subclinical hearing damage, called hidden hearing loss, is not easily detected by routine hearing tests focused on thresholds, but it could contribute to temporal processing deficits or difficulty detecting or interpreting sounds in background noise.22 Some of these functional deficits are believed to be due to noise-induced synaptic damage.23 Several studies have tried to verify hidden hearing loss in human participants with mixed results.27 One problem with many studies is the lack of documented history of noise exposure, heterogeneity of participants, and a limited sample size from which to draw firm conclusions.32 With these limitations in mind, this study aimed to assess the growth of hearing loss in the clinical and EHF ranges and to identify auditory processing disorders (APDs) among workers with noise exposure to identify early biomarkers of noise-induced hearing impairment.Traditionally, NIHL was defined by the permanent threshold shifts (PTS), while noise exposures that do not cause PTS were considered relatively safe. However, this idea has been challenged by data from animals in which significant noise-induced damage to the synapses between inner hair cells and spiral ganglion neurons occurred without PTS.ChiCTR-RPC-17012580). Potential consequences and benefits were explained, and written informed consent was obtained from each participant before this study. This study adheres to the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline.Cross-sectional physical examination data were obtained from 7890 sanding, welding, metal, and cutting workers between June 2015 and June 2019. A questionnaire was filled out by each participant with demographic features; noise exposure history; type of work; smoking and alcohol drinking habits; history of major diseases, including genetic and drug-related hearing loss; and the use of hearing protection devices. This study was approved by the institutional ethics review board of the Shanghai Sixth People’s Hospital, affiliated with Shanghai Jiao Tong University, and was registered in the Chinese Clinical Trial Registry (33 Sound levels were measured using A-weighted dB (dBA). The long-term equivalent (leq) noise level was adopted as the primary exposure metric and measured 3 times at each spot. The mean value of the leq in each spot was transformed into an 8-hour continuous equivalent sound pressure level (leq-8h). Cumulative noise exposure  was calculated using the leq-8h during the years of on-duty time, as follows: CNE = leq-8h + 10 × log(T), where the T is the career length in years.Industrial noise was measured with an ASV5910-R digital recorder (Aihua Instruments) across the work areas of different jobs according to the national standard of China.Audiologic evaluations were performed on 7890 workers on site by qualified medical assistants in soundproof chambers. The tests were performed at least 12 hours after the participant’s last shift in the noise-exposed job. Otoscopic inspection and tympanometry were performed on each participant to establish the normal function of their external and middle ears. Tympanograms were measured using a TympStar tympanometer (Grason-Stadler). The passing criterion was a type A tympanogram (peak between −100 and 100 daPa). PTA was measured separately for each ear using a Type 1066 manual audiometer  with Sennheiser HDA-300 headphones for the clinical and EHF audiometric ranges. All thresholds were calculated in dB hearing level, and audiometers were calibrated annually according to the ISO 389-5-2006 standard. If a participant did not respond to at the maximum output of the audiometer at the EHFs , the data were removed to eliminate saturation effects.34 The dichotic listening test (DLT) was obtained from both the left ear and right ear. The gap detection threshold (GDT) was measured using a 3-interval forced-choice procedure designed with MATLAB software. The procedures used for these measurements have been described in previous studies.36 Speech recognition and DLT were scored as the percentage of correct responses. The GDT threshold (in milliseconds) was calculated from the mean value of the last 8 reversals.A battery of 5 auditory processing tests was performed in 2 soundproof rooms using an audiometer equipped with TDH-39 headphones; stimuli were presented at 40 dB sensation level. The speech-in-quiet test (SIQT), speech-in-noise test (SINT), and competing sentences test (CST) were assessed using the Mandarin version of the hearing-in-noise test that was developed with MATLAB version R2012a software.2 test. A linear regression line with a forced intercept of 0 was fit to the data to determine the rate of hearing loss increase (slope) from 3 to 12.5 kHz, which was compared using the Z test in a younger group  and older group . Repeated measures analysis of variance with 2 factors (year and frequency) were used to compare the difference between hearing thresholds from 2015 to 2019. A 2-tailed P < .05 was considered statistically significant. Data analyses were performed using R version 4.02  and Prism version 8.4 (GraphPad Software).The baseline characteristics of participants were summarized as number and percentage for categorical variables and mean and SD for most continuous variables. Median and interquartile range (IQR) were used for skewed continuous variables. Differences in demographic characteristics and results of auditory processing measurements were analyzed by the nonparametric Kruskal-Wallis test and post hoc pairwise comparison with Bonferroni correction if the data were not normally distributed. Categorical data were compared using Pearson χP = .02; NG 2 vs control group: H = −4.675; P < .001), while there was no statistical difference between NG 1 and NG 2. The median (IQR) PTAs at 3 to 6 kHz and 10 to 16 kHz did not differ between the NG 1 group  and the control group CG , while there was a significant difference between the NG 2 group  and the control group . The mean (SD) age in the 2015 of the 403 workers in the longitudinal study was 31.7 (3.2) years, and the mean (SD) career length was 5.7 (1.7) years.The z = 33.78; P < .001; 12.5 kHz vs 4 kHz: z = 20.39; P < .001; 12.5 kHz vs 6 kHz: z = 30.87; P < .001; 12.5 kHz vs 8 kHz: z = 32.96; P < .001; 12.5 kHz vs 10 kHz: z = 20.90; P < .001). The same frequency-dependent trend was present in the older group (>40 years) , the largest slopes were 0.40  dB/dBA-year at 12.5 kHz, 0.36  dB/dBA-year at 4 kHz, 0.32  dB/dBA-year at 10 kHz, and 0.31  dB/dBA-year at 6 kHz . The sloLongitudinal progression of hearing loss was plotted as a function of frequency . During P < .001; NG 2 vs control: H = 14.29; P < .001), but there was no significant difference between the NG 1 and NG 2 groups. The median (IQR) CST score . There was no significant difference between the NG 1 and NG 2 groups. The median (IQR) score on the DL left ear was 0.75 (0.65-0.89) in the control group vs 0.60 (0.50-0.75) for the NG 1 group and 0.60 (0.50-0.80) for the NG 2 group , but there was no significant difference between the NG 1 and NG 2 groups. In the right ear . There was no significant difference between the NG 1 and NG 2 groups. The median (IQR) GDT values in the control group were 2.75 (2.25-3.25) ms compared with 3.08 (2.67-3.67) ms in the NG 1 group and 3.17 (2.75-3.68) ms in the NG 2 group , but there was no significant difference between the NG 1 and NG 2 groups.The NG 1, NG 2, and control groups had similar scores on the SIQT, with no overall or between-group differences A. HoweveST score C in the  2 group D. The DLight ear E, the me 2 group F. The GDAmong shipyard workers, we found that hearing thresholds were greater and increased more rapidly at EHFs compared with conventional audiometric frequencies. These trends were observed in both the cross-sectional and longitudinal analyses. Importantly, we found that workers with noise exposure in the NG 1 group  as well as workers in NG 2  performed worse than the control group on the SINT, DLT, CST and GDT.38 suggest that EHF audiometry (from 9-20 kHz) could identify the first signs of NIHL, especially at 12.5 to 18 kHz. We conducted a large-scale study on shipyard workers in China aged 20 to 60 years, on the assumption that people in this age range would be old enough for noise exposure effects to be measurable but young enough that aging effects would be minimized. The univariate linear regression showed that the maximum increase of hearing loss as a function of career length, type of work setting, and CNE occurred at 12.5 kHz, not only in young workers but even in those aged of 40 years and older in the group with hearing loss.Hearing loss has long relied on hearing tests that evaluate sensitivity in speech frequencies. While the equipment for conducting clinical audiometric tests is widely available, that for assessing hearing at the EHFs is more expensive and not widely available. The use of EHF audiometry as an early indicator of NIHL is still controversial, but our results, as well as previous studies,In the longitudinal analysis , workers41 many others have reported little or no correlation between the amount of noise exposure and APD.47 In our study, the suprathreshold deterioration in hearing function was seen in challenging speech processing tasks such as the SINT, CST, and DLT as well as the GDT. This result was observed even in the NG 1 group, in which the PTAs in both the conventional frequency range and EHFs showed no difference from participants in the control group. One possible source of these APDs is subclinical damage that disrupts the transmission of information from the inner hair cells to the afferent auditory nerve fibers or damage to the spiral ganglion neurons, as suggested by previous human and animal studies.50 However, in the absence of histopathological data, this interpretation remains speculative.Considerable effort has been made to identify APDs in humans with a history of noise exposure. While some studies have identified APD  in individuals with a history of noise exposure,51 Further work is needed to better control for the effects of aging, but finding participants with minimal exposure to noise is becoming increasingly difficult in a world filled with numerous sound-generating devices. The definition of normal hearing in most studies is a threshold of 25 dB hearing level or less. While this may be a practical definition in a clinical setting, only those with thresholds of 0 dB (±10 dB) presumably have normal hearing. Also, there may be some bias in using self-reported data to calculate important variables.This study has limitations. Some of the changes observed could be the result of aging, noise alone, or the interaction between early NIHL and aging.Our findings suggest that growth of hearing loss among shipyard workers was more rapid at 12.5 kHz than at 4 kHz, suggesting that 12.5 kHz could be a sensitive biomarker of early NIHL. Workers with high CNE values, even those with normal hearing at both clinical frequencies and EHFs, were more likely to exhibit APD on SINT, DLT, CST and GDT, especially when CNE values exceeded 80 dBA-year. This was observed even among workers with normal hearing in both the clinical and EHF ranges.


P = 0.118). According to age, in the ≥ 70-year-old group, those with hearing impairment had 11% lower rates of previous statin use than those in the control group . According to sex, in the male group, 12% lower rates of previous statin use were observed among those with hearing impairment than among those in the control group . Previous statin use might have an effect on reducing the prevalence of hearing impairment in elderly individuals and men.This study aimed to investigate the association of previous stain use with hearing impairment in an adult population. Data from the ≥ 40-year-old population in the Korean National Health Insurance Service Health Screening Cohort were used. The hearing impairment group was classified based on the national registry of hearing-impaired persons. Control participants were randomly selected and matched for age, sex, income, and region of residence. The number of days of statin prescription during the 2 years before the diagnosis of hearing impairment was compared between the hearing impairment group and the control group using conditional logistic regression analysis. Additional analyses were conducted according to age and sex. The number of days of previous statin use was not different between the hearing impairment group and the control group  = 0.86–1.02,  By competitively inhibiting HMG-CoA reductase, statins suppress a critical step of cholesterol synthesis1. The reduction in the activity of cholesterol synthesis pathways has therapeutic and protective effects on dyslipidemia and other cardiovascular diseases2. Beyond the lipid-lowering properties via HMG-CoA inhibition, statins have ancillary activities that make them beneficial to cardiovascular systems3. Statins directly activate the expression of vasodilators and reduce the expression of vasoconstrictors, thereby improving endothelial function3. In addition, inflammatory responses could be reduced by reducing the synthesis of proinflammatory molecules3. These pleiotropic effects of statins have broadened the possible clinical indications of statins from cardiovascular disorders to neurologic disorders such as dementia4 and Parkinson’s disease5.Statins, 2-hydroy-3-methyl-glutaryl-coenzyme A reductase (HMG-CoA) inhibitors, are commonly prescribed lipid-lowering agents worldwide6. However, no effective therapies are available in the clinic for sensorineural hearing impairment. Both cardiovascular compromise and neural degeneration are risk factors for hearing impairment9. Because statins are suggested to be effective for cardiovascular and neural disorders, hearing impairment could be another ancillary target disorder for statins. A recent clinical study demonstrated the otoprotective effects of atorvastatin in patients treated with cisplatin for head and neck cancer10. A few previous studies investigated the association of statin use with sudden sensorineural hearing loss (SSNHL) and yielded controversial results12. A case–control study proposed an association of increased SSNHL risk with statin use11. On the other hand, our previous study demonstrated no significant association between SSNHL and statin use by attenuating possible confounding effects using a matched control group and adjusting for many cardiovascular comorbidities12. Moreover, there has been little clinical research on the effects of statins on hearing impairments in the general population.Hearing impairment is a prevalent disorder whose incidence tends to increase with age and is the disease responsible for the third largest health burden worldwide  = 25.3–50.9] years lived with disability)15. To encompass all these types of hearing impairment, this study collected data from a nationally registered hearing-impaired population and compared the previous statin use in this population to that in the control population. Few previous studies have evaluated the impact of statin use on hearing impairment in such a large population.We postulated that previous statin use may have a protective effect against hearing impairment. Hearing impairment in the adult population could have multiple etiologies, such as age-related degenerative processes, noise exposure, ototoxic drug exposure, smoking, adiposity, and chronic diseasesThe ethics committee of Hallym University (2019–10-023) approved this study. The requirement for written informed consent was waived by the Institutional Review Board. All analyses adhered to the guidelines and regulations of the ethics committee of Hallym University.16. Participants with hearing impairment were selected from 514,866 participants with 615,488,428 medical claim codes from 2002 through 2015 . Individuals were included in the control group if they were not defined as having hearing impairment from 2002 through 2015 . Participants who were diagnosed with other disabilities were excluded . To assess the history of only the previous 2 years, we excluded participants with hearing impairment who were diagnosed with hearing impairment before 2003 . Hearing impairment participants were 1:4 matched with control participants for age, sex, income, and region of residence. To minimize selection bias, the control participants were selected with a random number order. The index date of each hearing impairment participant was set as the time of diagnosis of hearing impairment. The index date of control participants was set as the index date of their matched hearing impairment participant. Therefore, each matched hearing impairment participant and control participant had the same index date. During the matching procedure, 447,019 control participants were excluded. Ultimately, 4,387 hearing impairment participants were 1:4 matched with 17,548 control participants  before the index dates in both hearing impairment and control participants. The statins investigated in this study included atorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin. Pravastatin and rosuvastatin were categorized as hydrophilic statins, and atorvastatin, fluvastatin, lovastatin, pitavastatin, and simvastatin were categorized as lipophilic statins.17. In Korea, to be registered as a hearing-impaired person, individuals must be checked 3 times using a pure tone audiometry test (PTA) and 1 time using an auditory brainstem response17. The average hearing threshold of PTA was calculated as follows: (500 Hz + 2 * 1000 Hz + 2 * 2000 Hz + 4000 Hz)/617.Hearing impairment participants were defined as those who were registered as hearing impairment persons in the Ministry of Health and Welfare. Among them, we excluded participants with other disabilities. These individuals were divided into 2 groups based on the degree of impairment: severe hearing impairment  and profound hearing impairment (hearing threshold ≥ 90 dB in both ears)18. Tobacco smoking, alcohol consumption, and obesity according to body mass index  were categorized in the same way as in our previous study19. The records of total cholesterol (mg/dL), systolic blood pressure , diastolic blood pressure , fasting blood glucose (mg/dL), and hemoglobin (g/dl) were used. Missing values for fasting blood glucose (n = 2 [0.009%]), total cholesterol (n = 3 [0.013%]), and hemoglobin (n = 3 [0.013%]) were replaced by the mean values of each variable from the final group of selected participants.Age was divided into 5-year intervals: 40–44, 45–49, 50–54…, and 85 + years old . Income was classified into 5 classes (class 1 [lowest income]-5 [highest income]). The region of residence was grouped into urban and rural areas following the method used in our previous study20. In our study, we excluded dementia from the CCI score.The Charlson Comorbidity Index (CCI) has been used widely to measure disease burden using 17 comorbidities as a continuous variable (0 [no comorbidities] through 29 [multiple comorbidities])Regarding statin medication, a dyslipidemia diagnosis (ICD-10 codes: E78) was additionally assigned if participants were treated ≥ 2 times.t test for continuous variables.The general characteristics of the hearing impairment and control groups were compared using the chi-square test for categorical variables and the independent To analyze the odds ratios (ORs) with 95% confidence intervals (CIs) of statin prescription during a period of 1 year for hearing impairment, conditional logistic regression was used in the groups matched for age, sex, income, and region of residence. In these analyses, a crude model (simple), model 1  and model 2  were used. The analysis was stratified by age, sex, income, and region of residence.For the subgroup analyses, we divided the participants by age and sex  to confirm these associations according to age and sex. The division of the age groups was determined by the median value of the total number of participants. Additionally, we performed analyses according to the type of statin (hydrophilic or lipophilic). We further analyzed the ORs of statin prescription over a period of 1 year for hearing impairment by severity of hearing impairment.Two-tailed analyses were performed, and significance was defined as P values less than 0.05. SAS version 9.4  was used for statistical analyses.P = 0.525, Table P = 0.031 and P = 0.010, respectively). The distributions of obesity, smoking status, SBP, DBP, fasting blood glucose, hemoglobin, CCI score, and dyslipidemia were not different between the hearing impairment and control groups.The durations of statin use were not significantly different between the hearing impairment and control groups . According to sex, men showed a lower rate of hearing impairment related to long durations of statin use .According to age, the older subgroup (≥ 70 years old) demonstrated a lower rate of hearing impairment associated with long durations of statin use . Lipophilic statins were not related to the rate of hearing impairment in the total population or in any age or sex subgroups (S2 Table). According to the degree of hearing impairment, neither hydrophilic nor lipophilic statins were associated with severe or profound hearing impairment (S3 Table and S4 Table).Statins were divided according to hydrophilic and lipophilic types. Hydrophilic statin use was not associated with the rate of hearing impairment in the total population (S1 Table). However, in the ≥ 70-year-old group, a lower rate of hearing impairment was associated with a long duration of hydrophilic statin use 21. Another prospective study reported a decrease in hearing thresholds at 6 kHz and tinnitus after 6 months of statin use  in 84 adults with hyperlipidemia22. The Blue Mountains Hearing Study including individuals aged ≥ 50 years old described that participants with self-reported statin use showed 48% decreased odds of hearing impairment compared with the odds in statin non-users (OR = 0.52 [95% CI = 0.29–0.93])23. On the other hand, a case report suggested hazardous effects of statin use on hearing impairment, although causality has not been established24. Bilateral, progressive, middle-frequency hearing impairment (“cookie bite”) was induced following 18 months of atorvastatin use in a 32-year-old man and three additional anonymous cases24. The authors of the study suggested potential side effects of coadministered drugs without sufficient supportive evidence24. The potential drug-drug interactions of statins with other medications and related adverse drug reactions could be linked with hearing impairment associated with atorvastatin use25.Several prior studies have suggested protective effects of statin use on hearing impairment28. The lipid-lowering effects and reduction in atherosclerosis could mediate the otoprotective effects of statins28. Together with the reduction in atherosclerotic lesions, hearing impairment was prevented in apolipoprotein E gene knockout mice when they were treated with simvastatin28. The anti-inflammatory and antioxidative responses associated with statin use could be linked with the otoprotective effects of statins27. Cisplatin-induced hearing loss, which is accompanied by inflammation and oxidative stress, was reduced by lovastatin administration in adult rats27. The investigators demonstrated that lovastatin administration induced heat shock protein expression in the cochlea, protecting hair cells against cisplatin-induced injuries27. Noise-induced hearing loss, which is also related to inner ear inflammation and oxidative injuries, was attenuated by a low dose of atorvastatin (5 mg/kg) in mice26. However, the otoprotective effects of statins could be variable according to the dose of statins. A higher dose of atorvastatin  did not show otoprotective effects in noise-induced hearing loss26. These variations might explain the lack of a significant association of previous statin use with hearing impairment in this study.A number of experimental studies have suggested otoprotective effects of statins30. For instance, statins reduced the risk of cardiovascular events and mortality in elderly individuals with type 2 diabetes 29. The subgroup analyses also described the protective effects of previous statin use in the male group in the present study. This negative association between previous statin use and hearing impairment in men could be attributed to the higher rate of cardiovascular risk factors, including cardiovascular diseases, obesity, and smoking, in men than in women. In addition, sex differences in adherence to statin medication might affect the association of previous statin use with hearing impairments31. It was reported that women showed lower compliance with statin medication than men31.Only the old-age group and male group showed protective effects of previous statin use in this study. The high proportions of cardiovascular comorbidities in the elderly population could mediate the risk of hearing impairments. The lipid-lowering and anti-inflammatory effects of statins might be more effective in older patients with more cardiovascular comorbidities than younger patients32. In addition, a previous study reported superior anti-inflammatory effects of hydrophilic statins compared with lipophilic statins, with higher levels of adiponectin and lower levels of C-reactive protein in atherosclerosis patients33. The lower rate of side effects and greater anti-inflammatory effects of hydrophilic statins could explain the lower rate of hearing impairment in elderly individuals in the present study.According to the types of statins, only hydrophilic statins showed an association with a lower rate of hearing impairment in the ≥ 70-year-old population. Hydrophilic statins have lower tissue absorption and lower dependency on cytochrome P450 metabolism than lipophilic statins, so hydrophilic statins might result in fewer adverse reactions than lipophilic statins1. Last, although we considered numerous possible confounders, there might be confounding variables, such as noise exposure and stress levels, that were not assessed.This study included a large number of participants. The initial study population was 514,844 which represented the Korean population. To minimize the potential confounder effects, the participants with missing data were excluded and matched hearing impairment and control groups were selected. Finally, the 5,487 of hearing impairment group and 17,548 of control group were analyzed. The degree of hearing impairment was based on PTA and auditory brainstem response threshold tests. Because the hearing-impaired person registry is managed by the Korean government, which provides medical cost assistance for hearing aids, the likelihood of possible misclassification was not high in the present study. In addition, the potential confounding effects were attenuated by adjusting for past medical history, smoking, alcohol consumption, obesity, blood pressure, total cholesterol, and hemoglobin. However, a few limitations should be considered when interpreting the present results. The types of hearing impairment could not be differentiated in the present study. The pathophysiology of hearing impairment could be different according to the type of hearing impairment, such as noise-induced impairment, ototoxicity, and presbycusis. Previous statin use was assessed based on prescription data in this study. Thus, compliance with the statin prescription could influence the associations in the present study. Moreover, the dose of statins could not be determined in this study. More than seven types of statins are available in the clinic and have some variations in pharmacodynamics in terms of bioavailability, cytochrome P-450-linked metabolism, and cellular transportationIn conclusion, previous statin use was not related to hearing impairment in the adult Korean population. However, the old-age and male groups showed a relationship between previous statin use and a low rate of hearing impairment.Supplementary Information.