Triclosan (TCS) is included in various healthcare products because of its antimicrobial activity; therefore, many humans are exposed to TCS daily. While detrimental effects of TCS exposure have been reported in various species and cell types, the effects of TCS exposure on early embryonic development are largely unknown. The aim of this study was to determine if TCS exerts toxic effects during early embryonic development using porcine parthenogenetic embryos in vitro. Porcine parthenogenetic embryos were cultured in in vitro culture medium with 50 or 100 µM TCS for 6 days. Developmental parameters including cleavage and blastocyst formation rates, developmental kinetics, and the number of blastomeres were assessed. To determine the toxic effects of TCS, apoptosis, oxidative stress, and mitochondrial dysfunction were assessed. TCS exposure resulted in a significant decrease in 2-cell rate and blastocyst formation rate, as well as number of blastomeres, but not in the cleavage rate. TCS also increased the number of apoptotic blastomeres and the production of reactive oxygen species. Finally, TCS treatment resulted in a diffuse distribution of mitochondria and decreased the mitochondrial membrane potential. Our results showed that TCS exposure impaired porcine early embryonic development by inducing DNA damage, oxidative stress, and mitochondrial dysfunction. Triclosan (TCS) is synthetic antimicrobial agent that is included in many healthcare products used on a daily basis, such as soap, hand sanitizer, mouthwash, toothpaste, and children’s toys . DespiteMaintaining appropriate hormone levels in the reproductive system is critical for blastocyst formation and implantation . ExposurEarly embryonic development is crucial for subsequent implantation and pregnancy. In particular, blastocyst quality is important for successful implantation. Various factors have been reported to affect blastocyst quality during the preimplantation period. Reactive oxygen species (ROS) regulate cell growth and death  and are It is well known that TCS exhibits toxicity in a variety of cell types, including human-like JEG-3 choriocarcinoma cells  and gingTo determine the effects of TCS on early embryonic developmental competence, parthenogenetically activated (PA) porcine embryos were cultured in the presence of TCS at concentrations of 0, 50, and 100 µM during IVC. We assessed cleavage and blastocyst formation rates on days 2 and 6, respectively A. AlthouAs shown above, TCS treatment had a negative effect on developmental parameters. Therefore, we compared the rate of apoptosis in blastocysts treated with and without TCS. Terminal deoxynucleotidyl transferase-mediated dUTP-digoxygenin nick end-labeling (TUNEL) staining showed that TCS treatment at 100 µM significantly increased both the number of apoptotic cells and the rate of apoptosis compared to the control group; at 50 µM TCS, only the rate of apoptosis was significantly increased A–C. In a2DCFDA during early embryogenesis, including in the 2-cell, 4-cell, morula, and blastocyst stages -mediated estrogen receptor (ER) pathway [catalase, SOD1, and SOD2 were significantly downregulated following treatment with TCS [2O and O2 to limit oxidation injury [catalase serves as a marker of increased levels of ROS [SOD1 and SOD2 indicate oxidative stress status, and convert superoxide anion free radicals into O2 and H2O2 [ROS are necessary for cell development and act as secondary messengers. ROS can be formed and degraded quickly via enzymatic reactions . Howeveroli K-12 , goldfisoli K-12 , rat PC1oli K-12 , and humoli K-12 . Consist pathway . The che pathway . Thus, T pathway . Althougwith TCS . Catalasn injury . Downregs of ROS . TCS migs of ROS . Similarand H2O2 . TCS migand H2O2 . ConsistMitochondria are best known for their involvement in ATP synthesis . HoweverAll chemicals and reagents were purchased from Sigma Aldrich Korea , unless otherwise specified.2 in air. During the first period of maturation (0–22 h), the IVM I medium consisted of 10% porcine follicular fluid, 0.57 mM cysteine, 25 μM β-mercaptoethanol, 10 ng/mL epidermal growth factor, 10 IU/mL pregnant mare serum gonadotropin , and 10 IU/mL human chorionic gonadotropin (Prospec). After the first maturation period, a second period (from 22 to 44 h) was initiated. The same media was used (without hormone).Porcine ovaries were obtained from a local slaughterhouse and transported to the laboratory in 0.9% saline containing 0.75 µg/mL benzyl-penicillin potassium  and 0.5 µg/mL streptomycin sulfate salt at 37–38 °C. After the ovaries had been washed, cumulus oocyte complexes (COCs) were aspirated from follicles (diameter: 3–8 mm) using a 10 mL syringe with an 18-gauge needle. COCs with three or more layers of cumulus cells and homogeneous cytoplasm were selected and washed three times in 0.9% saline with 1 mg/mL bovine serum albumin (BSA). Washed COCs were incubated in IVM I medium for 22 h at 38.5 °C and 5% COTCS was dissolved in DMSO and diluted with culture medium to a final concentration of 50 or 100 µM. The test concentrations  used to determine the effects of TCS on early porcine embryonic development correspond to the TCS concentration range reported in the serum of pregnant women .4·7H2O, 0.1 mM CaCl2·2H2O, 0.5 mM HEPES, and 0.01% polyvinyl alcohol (PVA). Oocytes were immediately activated with 110 V DC for 50 µs using an electro cell fusion generator . Oocytes activated by electricity were incubated in activation medium  for 4 h at 38.5 °C in 5% CO2 in air. After 4 h, the oocytes were washed with IVC medium and transferred to fresh IVC medium at 38.5 °C in 5% CO2 in air. The medium used for IVC was porcine zygote medium-3 containing 0.4% BSA and 0, 50, or 100 µM of TCS. Cleavage and blastocyst formation were evaluated on days 2 and 6. The developmental rate of each stage  of embryo was evaluated at 30 h, 48 h, and 96 h of IVC after activation, respectively.Matured oocytes were placed in a 1 mm wire chamber  overlaid with 10 µL of 0.28 M mannitol solution containing 0.1 mM MgSOv/v) paraformaldehyde on day 6. Fixed blastocysts were washed three times with Dulbecco’s phosphate-buffered saline  supplemented with 0.1% PBS-PVA, followed by incubation in PBS containing 1% (v/v) Triton X-100 for 1 h at room temperature for membrane permeabilization. Subsequently, the blastocysts were washed three times in PBS-PVA and stained with fluorescein-conjugated dUTP and terminal deoxynucleotidyl transferase for 1 h at 38.5 °C. As a negative control for the TUNEL reaction, a group of blastocysts was incubated in fluorescein dUTP at 38.5 °C without terminal deoxynucleotidyl transferase. As a positive control for TUNEL reaction, blastocysts were incubated with 1000 units/mL DNase I  for 15 min. Subsequently, the blastocysts were washed three times in PBS-PVA and mounted on slide glasses  with mounting solution containing 1.5 g/mL 4,6-diamidino-2-phenylindole . DAPI-labeled or TUNEL-positive nuclei were observed under a fluorescence microscope, and the numbers of apoptotic and total nuclei were counted.To determine the rate of apoptosis in porcine blastocysts, an In Situ Cell Death Detection Kit  was used for a TUNEL assay. Blastocysts were fixed in 4%  was used for detection of ROS as green fluorescence. At least 10 embryos from each group were incubated for 20 min in a solution of PBS/PVA mixed with 1 µM CM-H2DCFDA. As a positive control for the CM-H2DCFDA reaction, embryos at each stage were incubated with 200 μM of H2O2 for 30 min. Then, the embryos were washed with PBS/PVA and transferred into 40 μL droplets of PBS/PVA. Samples were observed under a fluorescence microscope  with ultraviolet filters (460 nm). Fluorescent images were saved as graphic files in tagged image file format (TIFF) and the fluorescence intensities of individual embryos in green were analyzed using ImageJ software . The mean of fluorescence intensity per unit area of the target area was calculated by ImageJ. The mean value of fluorescence intensity in each group was normalized to that of 2-cell-stage embryos in the control group.Measurement of intracellular ROS in embryos was performed as described previously . BrieflyExtraction of poly(A) mRNA and cDNA synthesis was conducted as described previously. Briefly, mRNA samples were extracted from 20 2-cell embryos in each group, using the Dynabeads mRNA Direct Micro Kit  according to the manufacturer’s instructions. Next, 100 μL of lysis/binding buffer was used to lyse the 2-cell embryos, 20 μL of Dynabeads oligo (dT)25 was added to separate the mRNAs, and the beads were hybridized for 5 min and separated from the binding buffer using a Dynal magnetic bar (Invitrogen). The beads with poly(A) mRNAs were washed using buffers A and B. After washing, 8 μL of Tris buffer was added to each tube for separation and the resulting poly(A) mRNAs were reverse-transcribed. Genomic DNA was eliminated by treatment for 5 min at room temperature with gDNA Eraser, which degrades DNA. Then, a reverse transcription reaction reagent including a component that inhibits DNA degradation activity was added. For cDNA synthesis, samples were incubated at 37 °C for 15 min and 85 °C for 5 s, and used as a template for PCR amplification . qRT-PCR was performed using the Mx3000P qPCR system  with SYBR premix Ex Taq (Takara Bio Inc.). The threshold cycle (Ct) was defined as the fractional cycle number at which the fluorescence passed a fixed threshold above baseline. For comparative analyses, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used for normalization; gene expression was expressed in terms of the fold change, and the 2−(SΔCT−CΔCT) method was used to analyze gene expression. The primers used in the present study are listed in ® Deep Red FM (Invitrogen) staining was used. Two-cell-stage embryos were fixed with 4% (v/v) paraformaldehyde for 30 h. Fixed embryos were washed with PBS/PVA three times and incubated with PBS/PVA containing 5 µM MitoTracker for 30 min. After incubation, embryos were washed with PBS/PVA three times and mounted on slides. A confocal laser scanning microscope  was used to detect fluorescence with ultraviolet filters at 644/655 nm. Fluorescent images were saved as graphic files in TIFF. The fluorescence intensities of the embryos were analyzed using ImageJ software and normalized to those of control embryos.To determine the distribution of mitochondria in embryos, MitoTrackerThe distribution of mitochondria in 2-cell embryos was quantified using a modified version of a previously described method . All embv/v) paraformaldehyde for 30 h and incubated with PBS/PVA mixed with 200 nM TMRM for 30 min. After incubation, the embryos were washed with PBS/PVA 3 times, mounted on slides and observed under a fluorescence microscope  with ultraviolet filters (575 nm). Fluorescent images were saved as graphic files in TIFF. The fluorescence intensity of red individuals in 2-cell-stage embryos was analyzed using ImageJ software and normalized to control embryos.MMP was measured with tetramethylrhodamine methyl ester . Briefly, for detection of MMP as red fluorescence, at least 10 embryos in the 2-cell stage from each group were fixed with 4% . P-values less than 0.05 were considered statistically significant.The present study revealed toxic effects of TCS on early embryonic development using parthenogenetically activated porcine embryos. TCS increased oxidative stress via ROS generation and induced mitochondrial dysfunction, which might be related to apoptosis in blastocysts. We also found that TCS had a negative effect on the developmental kinetics of 2-cell-stage embryos and blastocysts, which could explain the lower blastocyst formation rates and total number of cells. Therefore, TCS exposure resulted in a decrease in developmental competence compared to the control group during IVC; the mechanisms underlying TCS-induced developmental retardation require further study.


Acute lung injury is a serious form and major cause of patient death and still needs efficient therapies. The present study evidenced that co‐transplantation of mesenchymal stem cells (MSCs) and telocytes (TCs) improved the severity of experimental lung tissue inflammation, edema, and injury, where TCs increased MSCs migration into the lung and the capacity of MSCs proliferation and movement. Of molecular mechanisms, Osteopontin‐dominant networks were active in MSCs and TCs, and might play supportive and nutrimental roles in the interaction between MSCs and TCs, especially activated TCs by lipopolysaccharide. The interaction between epidermal growth factor and its receptor from MSCs and TCs could play critical roles in communications between MSCs and TCs, responsible for MSCs proliferation and movement, especially after inflammatory activation. Our studies provide the evidence that TCs possess nutrimental and supportive roles in implanted MSCs, and co‐transplantation of MSCs and TCs can be a new alternative in the therapy of acute lung injury. TCs can accelerate the migration of MSCs from the intraperitoneal space into injured lung tissues through intercellular signals and/or cell‐cell interaction.Pre‐activated MSCs and TCs with LPS show more efficient in cell‐cell communication by OPN‐ or EGFR‐dominated signal pathways ARDS/ALI‐associated mortality remains more than 50% due to the lack of effective therapies, despite the improvement of supportive cares.Mesenchymal stem cells (MSCs) were suggested as an alternative of potential therapies for ALI to prevent acute lung inflammation, tissue edema, and injury, although there are still a large number of obstacles to be overcome.+ T cells from lungs, or CD8+ T cells from bronchial lymph nodes.Telocytes (TCs) were coined and suggested as a type of interstitial cells with podoms, podomers, and long telopodes (Tp).The present study first proved the therapeutic effects of intraperitoneal co‐transplantation of MSCs and TCs in ALI induced by an intratracheal instillation of lipopolysaccharide (LPS). We defined morphological features and phenomes of isolated and cultured TCs from lungs and investigated how TCs supported and attracted MSCs migration and distribution into the injured lung in animals with or without ALI. Therapeutic effects of co‐transplantation were furthermore evaluated in ALI after intraperitoneal injection with cells or conditioned medium, for example, MSCs, TCs, or both at high or low doses, or the corresponding medium at high or low doses. According to the principle of “two‐hit” model,22.1The animal experiments of the present study were approved by Ethical Committee of Zhongshan Hospital Fudan University. 6–8 weeks Female C57BL /6J mice with the weight of 20–25 g and 6–8 weeks old, were provided by Animal Facility, Zhongshan Hospital Institute of Clinical Science, Fudan University. LPS (Product Number L9143) was originated and phenol‐extracted from serotype 10 Pseudomonas Aeruginosa .2.25 cells/cm2 and maintained at 37°C in a humidified atmosphere (5% CO2) until becoming 80% confluent. Culture medium was changed every 48 h during TCs were expanded and the passages 6–8 were used for cell transplantation. MSCs were obtained, thawed, and expanded from bone marrow of 6–8 weeks C57BL/6J mice.g for additional 5 min to acquire the cell mass. After washing thrice, the cells were resuspended and cultured with DMEM/F12 medium, containing 10% fetal bovine serum (FBS) and 1% penicillin‐streptomycin in cell incubator. Anti‐Sca‐1 antibodies (E13–161.7), anti‐CD34 (RAM34), anti‐CD31 (MEC13.3), anti‐VCAM‐1 (429 [MVCA‐M.A]), anti‐CD11b (M1/70), and anti‐CD45R (RA3‐6B2) and non‐specific isotype IgG as controls  were used for cellular surface labeling, and analyzed by BD FACS Aria III flow cytometer (BD).Lung TCs were isolated from lung tissues of 6–8 weeks C57BL /6J mice and cultured.2.36 MSCs, TCs, or both at 1:1 were cultured to about 80% confluences for 24 h after stimulation with or without LPS. After media and cells were collected separately, cells were rinsed thrice and cultured in serum‐free DMEM/F12 for 24 h. The above‐conditioned medium was removed and cellular debris was removed using a 50‐μm filter.TCs was confluent at 80‐90% after about 10 days to reach 5 × 102.42O and fixed for histopathology analysis.Mice were anesthetized with Isoflurane in a plastic box with controlled ventilation and put in a supine position of head‐up on the board tilted at 30°. LPS at 1000 μg/mL was intratracheally administered to mice at a volume of 1 mL/kg mice body weight (BW).2.56 MSCs alone (n = 18), TCs alone (n = 18), or MSCs combined with TCs (n = 18). Mice were then terminated at 24, 48, or 72 h after the intraperitoneal administration of cells (n = 6 per time point).To define the localization and number of implanted cells, MSCs and TCs were labeled with a red fluorescent dye  and a green fluorescent dye , respectively. The efficacy and viability of labeling were > 98% and > 96%, respectively, evaluated by trypan blue exclusion. C57BL/6J mice were randomized into six groups with the intratracheal administration of PBS or LPS at the volume of 1 mL/kg body weight 1 h before the intraperitoneal injection of 10® II enzyme at 3.6 U/mL .6 was recorded. The right lung was fixed using 4% paraformaldehyde, and the left lung was infiltrated with PBS containing 30% sucrose under the pressure of 20 cm H2O and then fresh‐frozen in liquid nitrogen with Tissue‐Tek OCT Compound . Sections of five μm and 50 μm thick were stored at −80°C before the analysis and then fixed in ice‐cold acetone for 10 min for use. Nuclei was stained with 4′, 6′ ‐diamidino‐2‐phenylindole dihydrochloride hydrate (Sigma‐Aldrich). Images were recorded with a TCS SL laser scanning confocal microscope .Cells were isolated from the left lung using dispase2.66 or 5 × 106, TCs at 106 or 5 × 106, or MSCs combined with TCs at 106 or 5 × 106 each, respectively. Potential effects of mediators produced from cells were investigated by an intraperitoneal injection of the conditioned medium. Another 80 animals were used, including native controls, manipulation controls, or positive controls, or groups were intraperitoneally injected with 0.5 mL MSCs‐conditioned medium at 25% or 100% of the origin, TC‐conditioned medium at 25% or 100%, or MSCs/TCs‐conditioned medium at 25% or 100% for 24 h, respectively, followed by the intratracheal administration with LPS.The therapeutic effects of cell co‐transplantation were evaluated by either MSCs or TCs alone or together at low and high concentrations  after a pilot study. Animals without any manipulation were used as negative controls, intraperitoneally injected with vehicle at the volume of 0.5 mL per animal for 24 h, and intratracheally administered with PBS at 1 mL/kg BW as the manipulation controls, or with LPS at 1 mg/mL/kg BW as positive controls (ALI). Animals were intratracheally challenged with LPS 24 h after the intraperitoneal injection with vehicle at the volume of 0.5 ml per animal, with MSCs at 102.72O. The bronchoalveolar lavage fluid (BALF) was collected into tubes on ice and the volume was recorded. The BALF was centrifuged at 1200 rpm for 5 min and the supernatant was stored at ‐80°C for further analyses. Pulmonary endothelial barrier dysfunction was measured by the plasma exudation from the peripheral blood into the alveolar space. Total protein in BALF were measured with an enhanced BCA Protein Assay Kit  and albumin with a murine‐specific albumin ELISA kit  at the detection limit of 1.5 ng/mL, respectively. The cells were harvested from BALF and re‐suspended in 0.25 mL PBS, and the number of neutrophils and white blood cells was measured using Auto Hematology Analyzer . Cytokine levels of interleukin (IL)‐6 and IL‐1β in BALF or plasma were evaluated with cytokine‐specific Quantizing ELISA kits (R&D Systems).The lung was intratracheally washed with sterile PBS under the pressure of 20 cm H2.82O, fixed with 4% paraformaldehyde, embedded in paraffin, and cut into sections of 5 μm thick. After stained with hematoxylin and eosin, photos were obtained with a BX51 microscope DP71 camera . Twenty random high‐power fields (X400) were independently scored. Images were evaluated for the lung injury score evaluation as reported previously.Lung tissue damage and injury induced by LPS were evaluated by pathological scoring. The lungs from additional animals in each group (n = 6) were washed with PBS under the pressure of 20 cm H2.95, as compared with MSCs or TCs alone. We also investigated potential effects of activated TCs or MSCs stimulated with LPS on MSCs or TCs, as compared with MSCs or TCs alone, or co‐culture of TCs and MSCs without activation. Gene expression profiles were measured as previously described.To define alterations of gene expression profiles during MSCs‐TCs interaction, gene expression profiles were evaluated in co‐culture of MSCs and TCs at the concentration of 102.105 cells/well were cultured alone or co‐cultured with or without the challenge of LPS at doses of 10, 100, or 1000 ng/mL for 4, 8, 24, or 48 h, respectively. MSCs or TCs were detached and collected with Trizol  for RNA isolation. Total RNA was isolated with TRIZOL(Invitrogen) and measured with NanoDrop One (Thermo) at 260 nm. RNA was reversed and transcribed to cDNA using the Super‐Script First‐strand Synthesis System (Invitrogen). Quantitative RT‐PCR was carried out with the two‐stage program parameters using an ABI 7000PCR instrument , as follows: at 95°C for 1 min, then at 95°C for 30 s, and at 60°C for 40 cycles of 5 s. The sequences of the primer sets are as follows: mouse OPN: 5′‐GAGGAAACCAGCCAAGGTAA‐3′ (forward [F]) and 5′‐GCAAATCACTGCCAATCTCA ‐3′(reverse [R]); mouse EGF: 5′‐GGCAGACAGAGCCAGTTCA‐3′ (F) and 5′‐ AGCAGTGATTAGCCGTGGAA ‐3′ (R); mouse Connexin 43: 5′‐GAACACGGCAAGGTGAAGAT‐3′(F) and 5′‐GAGCGAGAGACACCAAGGAC‐3′(R); and mouse glyceraldehyde‐3‐phosphate dehydro‐genase (GAPDH): 5′‐GGTGAAGGTCGGTGTGAACG‐3′(F) and 5′‐CTCGCTCCTGGAAGATGGTG‐3′(R). Dissociation reaction plots were examined for the confirmation of specificity of the amplification product. Each sample had six wells and each well was tested in triplicate.Two potential driven factors OPN and EGF were selected for the further investigation to understand potential mechanisms of MSCs‐TCs interaction. MSCs or TCs were plated into 12 well plate 24 hrs and then transfected with RNA interference OPN siRNA , EGF siRNA , and the fluorescein‐conjugated control siRNAs  using lipofectamine 2000 . Knockdown of OPN or EGF was identified by quantitative PCR and western blot 48 h after expression of the siRNAs. β‐actin was used as an internal control. OPN or EGF‐positive or negative MSCs or TCs at the concentration of 102.11Cells were detached in 1 mM EDTA solution and then added lysing buffer (Sigma–Aldrich). Proteins were boiled for 5 min with β‐mercaptoethanol, and separated by electrophoresis in 10% (wt/vol) SDS‐polyacrylamide gel. Proteins were loaded in each lane and transferred onto polyvinylidene difluoride (PVDF) membrane. The membrane with proteins were incubated with TBST buffer  containing 5% (wt/vol) milk powder for 60 min at room temperature to block nonspecific binding. Membranes with protein were incubated with the primary antibodies, including anti‐mouse EGF monoclonal antibody , anti‐mouse Connexin 43 (Cx43) monoclonal antibody  at 4°C overnight, and then mouse or goat anti‐mouse IgG‐HRPs (sc‐2005) as the secondary antibody. Revelation was performed with the ECL plus Western blotting detection kit , and Chemocapt software using Chemi‐Smart 2000  were used for image acquisition.2.124 cells. The low chamber was coated with TCs at 5 × 104 cells in serum‐free DMEM/F12. The complete culture medium was collected 24 h after MSCs, TCs, or both were challenged with vehicle or LPS at 1000 ng/mL. Cells in upper chambers were washed with pre‐cold PBS for twice, and were stained with May‐Grünwald‐Giemsa. The total number of cells migrated from the upper chamber to the low ones were counted under light microscopy (×200). The influence of external OPN in the interaction between MSCs and TCs on cell migration was also evaluated by neutralizing intracellular OPN with anti‐mouse OPN antibody . Concentrations of anti‐OPN antibody used were selected and chosen on basis of our studies.Indirect interactions between MSCs and TCs were measured with the Trans‐Well migration assay. Migration ratios were examined in transwell dishes with the diameter of 6.5 mm and 8‐μm pore filters . The upper chamber was first coated with 0.1% (wt/vol) bovine gelatin (Sigma‐Aldrich) for 1 h at 37°C and then with MSCs at 5 × 104 cells were placed on 24‐well plates and cultured in an incubator for 24 h. The plates were then incubated in Cell‐IQ incubator after the treatment. Analysis was performed using a freely distributed Image software , with the Manual Tracking plugin supported by Fabrice Cordeliéres . The cell bio‐behaviors were examined using a real‐time cell monitoring system equipped in a Cell‐IQ cell‐culturing platform (Chip‐Man Technologies). Images of each visible through a microscope were captured at 60‐minute intervals for 72 h. Total cell number of each image was measured automatically by Cell‐IQ system. Each group contained three wells and four replicate microscopic fields were measured in each. To investigate the connexin 43‐dependent influence of TCs on MSCs proliferation, CFSE‐labeled MSCs at 105 cells per well were directly co‐cultured with PKH‐26 labeled TCs at the ratio of 1:1 or alone in serum‐free DMEM/F12 for 24 h. Cells were harvested and MSCs proliferation was determined by flow cytometry analysis of CFSE fluorescence 24, 48, and 72 h after 1000 ng/mL LPS was added in MSCs or co‐cultured with TCs, with or without the appearance of carbenoxolone disodium , a gap‐junction (connexin 43) inhibitor, at doses of 1, 10, or 100 nM.Direct interactions between MSCs and TCs were assayed by measuring cell bio‐behaviors by Cell‐IQ , for example, cell morphology, proliferation, death, differentiation, and movement. MSCs, TCs, or both at 102.13P<.05 was considered statistically significant.Values were expressed as means ± SEM. Differences between groups were assessed by one‐way ANOVA with post hoc comparisons and Dunnett's test with statistic software (GraphPad Prism version 6.0). All in vitro experiments were performed in triplicate and repeated three to six times. A value of 33.1+, or VCAM+,Of TCs biomarkers,3.2Therapeutic effects and distributions of transplanted cells depend upon delivery locations and processes.The number of labeled MSCs in animals with both cells significantly higher than those in animals with MSCs, with a peak at 48 h, as compared with that at 24 or 72 h, respectively. The number of labeled MSCs in lungs was significantly higher than that of TCs, especially under LPS challenge, mainly located in the interstitial space and inflammatory area in a small form of cell clusters Figure , and a f3.3To investigate if the combination of MSCs and TCs may have therapeutic effects through cells per se or cell‐produced mediators, we pretreated animals with LPS‐induced ALI by MSCs, TCs, or both at low or high doses, or by the corresponding conditioned medium at low or high doses, respectively, as explained in Figure To investigate if nutritional factors produced from MSCs, TCs, or both may play the dominate roles in cell therapies as suggested previously,Therapeutic effects of MSCs‐TCs co‐transplantation were further evidenced by pathological characteristics and scores of compromised lungs induced by LPS, according to the scoring systems in Figure 3.4To investigate whether TCs can increase the migration of MSCs from the peritoneal cavity to the lung, we observed the indirect interaction between MSCs and TCs by measuring migration of MSCs pre‐activated with LPS or vehicle from the upper chamber to the low one where TCs pre‐activated with LPS or vehicle. The better efficacy of MSCs and TCs may depend upon cell migration and specific homing towards the site where the cells are needed.Effects of LPS‐activated TCs or MSCs on dynamic proliferation and movement of MSCs were furthermore evaluated during 48 hrs using the dynamic monitoring system of cell behaviors. To assess direct effects of TCs on MSCs, we mixed and cultured MSCs alone or with TCs with or without pre‐activation in the system Figure . The dir3.5TCs could benefit MSCs function both directly and indirectly in either pre‐activated or activated condition, although the exact mechanism remains unclear. To investigate the interaction between MSCs and TCs, we measured gene expression profiles of MSCs or TCs, respectively, after culture of either or both challenged with LPS Figure . The int3.6OPN– or TCs OPN–, siRNA against OPN gene inhibited OPN mRNA and protein expression in MSCs . MSCs‐origin OPN is more dependent on the regulation of MSCs movement .OPN was found to regulate expansion, migration, and subsequent transformation of MSCs in the tissue.s Figure  or TCs  in TCs are higher than in MSCs or fibroblasts,4Stem cell transplantation was suggested as an efficient alternative of clinical therapies for ALI /ARDS in translational medicine, although there are still some challenges to be solved.TCs could influence MSCs movement to the lung tissue and increase the appearance of MSCs in local tissues through direct interactions with MSCs or mediators released from activated TCs. Previous studies have postulated that TCs can support tissue stem cells through their extensive telopodes and 3D network.TCs communicate with multi‐cells and support other cell functions through the connections of telopodes.that the EGF‐EGFR axis signal pathway was one of mechanisms by which EGF regulated the interaction between MSCs and TCs in the therapeutic and repairing processes, although there are other signal pathways involved.It is still a challenge to define how TCs interact with and benefit MSCs directly or indirectly. Previous studies demonstrated that TCs might interact with tissue stem cells through paracrine effects of extracellular vesicles released from TCs.There are limitations of using adult bone marrow‐origined MSCs for therapy, e.g. variations of MSCs quality, stem cell senescence, and low proliferative potency. The parental pluripotent stem cells (PSC)‐derived MSCs can possess better cell quality, batch to‐batch consistency, and higher proliferative potential.In conclusion, therapeutic effects of MSCs‐TCs co‐transplantation on the severity of acute lung tissue inflammation, edema and injury critically depended upon MSCs proliferation and migration, and upon supportive roles of implanted TCs. Activated TCs promoted the proliferative and movement capacity of MSCs, and support MSCs function through the regulation of multiple factors. OPN‐dominant networks were active in both MSCs and TCs and could play supportive and nutrimental roles in cell‐cell interaction, especially from activated TCs. EGF‐EGFR axis in MSCs and TCs might play important roles in MSCs proliferation and movement during interaction, especially from activated MSCs. Our studies provide the evidence that TCs possess nutrimental and supportive roles in implanted MSCs and MSCs‐TCs co‐transplantation can be a new alternative in treatment of acute lung injury.DZH, DLS, LS, and XRS designed the study and completed the experimental process, literature search, and generation of figures. DZH, DLS, and XDW wrote and edited the manuscript. DLS, LS, XRS, YHZ, and XDW completed generation of figures. LS, XRS, and YMZ performed bioinformatics and statistical analysis. All authors reviewed the manuscript. All authors read and approved the final manuscript.The authors declare that they have no conflict of interest.Supporting Figure S1Click here for additional data file.Supporting Figure S2Click here for additional data file.Supporting Table S1Click here for additional data file.Supporting Table S2Click here for additional data file.Supporting InformationClick here for additional data file.


A definitive study on the prevalence of adult unilateral hearing loss and hearing aid rehabilitation is lacking in Korea. The purpose of our study was to investigate the prevalence of adult unilateral hearing loss and the factors associated with hearing aid use in patients with unilateral hearing loss in South Korea. We obtained data from 2009 to 2012 from the Korea National Health and Nutrition Examination Surveys (KNHANES), a cross-sectional, nationwide and population-based survey in the Republic of Korea. We analyzed the prevalence and associated factors of unilateral hearing loss and hearing aid adoption by univariable and multivariable analysis. Unilateral hearing loss was defined as pure tone average ≥ 41 dB in the worse hearing ear, and < 41 dB in the other ear assessed at 0.5, 1.0, 2.0, and 3.0 kHz. From 2009 to 2012, 33,252 individuals participated in the KNHANES. Among them, the number of patients with unilateral hearing loss was 1632 (5.55%) and the prevalence of hearing aid adoption in unilateral hearing loss was 1.56%. We also compared the factors between hearing aid users and non-users. Occupational status , the hearing threshold in the better ear , and hearing threshold in the worse ear  were found to affect the adoption of hearing aids. The prevalence of noise exposure at work in hearing aid users was significantly lower than the prevalence of noise exposure at work in those with no hearing aid. The prevalence of hearing aid use in patients with unilateral hearing loss in Korea is very low compared to other countries. Public health education is needed to increase public awareness of unilateral hearing loss, hearing aid adoption and its continued usage. Auditory rehabilitation should be actively recommended to patients with unilateral hearing loss. Sensorineural hearing loss (SNHL) affects approximately 1 in 500 newborns . The truBilateral hearing loss that occurs with aging is known to be associated with dementia and falls , 6. LittAuditory deprivation is one reason why patients with UHL use hearing aids. Auditory deprivation can progress systematically over time and is associated with the reduced availability of acoustic information . Many stData were obtained from the 2009–2012 KNHANES and thus the study involved the secondary analysis of a large data-set. The KNHANES is a cross-sectional, nationwide, population-based survey designed to assess national health and nutrition levels accurately that has been conducted by the Division of Chronic Disease Surveillance under the Korea Centers for Disease Control and Prevention since 1998. It is composed of three different components; a health interview, a physical examination, and a nutrition survey. Every year 10,000–12,000 individuals in approximately 4000 households are selected from a panel to represent the population using a multistage clustered and stratified random sampling method that is based on Korean national census data. Since the Korean Society of Otolaryngology-Head and Neck Surgery began to participate in the survey in 2008, it has included otolaryngologic interviews and examinations performed by well-trained otolaryngologists in a mobile unit containing an endoscopic system and audio booth. Survey sample weights are used in all analyses to generate estimates that are representative of the non-institutionalized civilian Korean population. A detailed description of the KNHANES data collection methods has been published previously , 15. AllPure tone audiograms were conducted in participants aged ≥ 12 years who were eligible for the survey. Only air conduction thresholds were measured. Data from the otolaryngologic survey and examinations were evaluated, including tympanic membrane perforation, hearing loss, dizziness, and vestibular dysfunction. Potential associations between factors derived from the basic health examination and interview and UHL and hearing aid use were assessed. To determine the prevalence of tympanic membrane perforation and cholesteatoma, as well as the retraction pocket and otitis media with effusion, an ear examination was conducted with a 4-mm 0-degree angled rigid endoscope attached to a charge-coupled device camera in all participants. Pure tone audiograms were measured by a trained otolaryngologist using an automatic audiometer  in a soundproof booth. Pure tone thresholds were obtained independently at 6 frequencies in each ear; 0.5, 1.0, 2.0, 3.0, 4.0, and 6.0 kHz. Pure-tone average (PTA) was calculated as the average threshold at 0.5, 1.0, 2.0, and 3.0 kHz in accordance with the recommendations of the American Academy of Otolaryngology-Head and Neck Surgery . UHL wasp values were two-sided, and p < 0.05 was deemed to indicate statistical significance.The prevalence of UHL was calculated and reported as a weighted percentage with standard error. In the univariable analysis, the Rao-Scott Chi-square test and logistic regression analysis were used to test associations between UHL and potentially related factors in complex sampling design. In multivariable analysis, adjusted odds ratios (ORs) with 95% confidence intervals (CIs) were calculated via logistic regression analysis. To reflect the national population estimates, sample weights were applied in all analyses. Statistical analysis was performed using SAS version 9.4 . All p < 0.0001).A total of 33,252 individuals participated in the KNHANES from 2009–2012. Of these, the number of participants with UHL was 1,632 . PTA ≥ 41 dB and < 55 dB, PTA ≥ 55 dB and < 70 dB, and PTA ≥ 70dB accounted for 56.86%, 21.89%, and 21.25% respectively. The prevalence of UHL in the eight different age groups are shown in In participants with UHL, the prevalence of hearing aid adoption was 1.56% (standard error 0.37). Bilateral PTAs in hearing aid adopters with UHL are shown in Univariable and multivariable logistic regression analyses were performed to identify factors associated with hearing aid adoption. In univariable analysis occupational status , the hearing threshold in the better ear , and hearing threshold in the worse ear  were significantly associated with hearing aid adoption. In multivariable logistic regression analyses occupational status , hearing threshold in the better ear , and hearing threshold in the worse ear  were significantly associated with hearing aid adoption . There wt-tests and Chi-square tests were performed to compare hearing aid users and non-users. In that analysis noise exposure at work was associated with hearing aid use (p = 0.0240). None of the other factors analyzed were statistically significant.The prevalence of hearing aid use was 0.86% (standard error 0.28) in participants with UHL, and it was 55.31% (standard error 11.97) in participants who reported initial hearing aid adoption. To identify factors associated with hearing aid use,  aid use . The preIn the present study conducted in South Korea, the prevalence of UHL determined via KNHANES data was 4.91%. This is lower than the prevalence of 7.9%–13.3% in the general population , 18 and In the present study, in participants with UHL, the prevalence of hearing aid adoption was 0.86% and the prevalence of hearing aid use was 1.56%. Hearing aid use prevalence of 14.2% in the USA  and 21.5Occupational status, the hearing threshold in the better ear, and hearing threshold in the worse ear were associated with hearing aid adoption in the present study. Patients with UHL may not feel discomfort in their daily life. If they attend meetings or are required to spend time in a noisy environment however, problems can arise from UHL. Patients with UHL experience difficulty with sound localization and speech recognition in noisy environments . AdditioPeople who adopt hearing aids tend to have more severe hearing loss in both the better ear and the worse ear than non-adopters. In a previous investigation, the severity of hearing loss was a major determinant of hearing aid adoption . In anotNoise exposure during work was the only factor that was significantly associated with hearing aid use in the present study. Hearing aid users were exposed to less noise at work than non-users. In noisy environments patients with hearing impairment can be free from noise without hearing aids. Unlike bilateral SNHL, it is thought that patients with unilateral SNHL have a lower need for hearing aids and that it is not necessary to wear hearing aids even in noisy situations. There have been recent advances in hearing aid technology. Specifically, noise reduction systems have been incorporated into hearing aids to improve the signal-to-noise ratio and listening comfort. These advances may have resulted in improved noise tolerance in hearing aid users.The current study had some limitations. The KNHANES data it utilized were acquired from 2009–2012 and thus were ≥ 7 years old, and hearing aid technology has advanced rapidly in the last 5 years. With regard to financial considerations, in November 2015 the hearing aid subsidy provided by government health insurance in Korea was raised to KRW 1,310,000. As a result, hearing aid adoption and use may have increased since the 2009–2012 KNHANES was conducted. Second, the KNHANES defined UHL as a PTA hearing level ≥ 41 dB in one ear and < 41 dB in the other, whereas other studies defined UHL as a PTA hearing level > 25 dB. Another study limitation is that the KNHANES questionnaire components pertaining to hearing aids are not detailed, thus the scope for analyzing factors associated with the adoption and use of hearing aids was restricted. Notably, however, the present study was based on a very large national cross-sectional survey of a representative sample based on Korean national census data, and by design, the study had the capacity to reliably determine the prevalence of UHL and overall hearing aid use.In conclusion, the prevalence of hearing aid use in patients with UHL is very low in South Korea compared to other countries. Public health education is needed in an effort to increase public awareness of UHL and hearing aid adoption and continued use, and auditory rehabilitation should be actively recommended to patients with UHL. 2 Jan 2020PONE-D-19-32331Big data analysis of unilateral hearing loss and hearing aid use in South KoreaPLOS ONEDear Kim,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.We would appreciate receiving your revised manuscript by Feb 16 2020 11:59PM. 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Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: PartlyReviewer #2: Partly**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: I Don't KnowReviewer #2: No**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: 1. REVIEWER SUMMARY: This is a report of unilateral hearing loss prevalence and also hearing aid use in South Korea. It uses the KNHANES, which should be representative of the actual population through sample weighting. Reporting of accurate, unbiased prevalence statistics is very important. While this has been done, for example, in the US, it is important to replicate in other countries because prevalence may differ. This study contains these important data, but there are a number of limitations detailed below. For example, multivariable regression was used but there was no obvious mention of what the variables were that were adjusted for in the model, which is critical information. In addition, the definition of unilateral hearing used a 40 dB cutoff, which is different from other papers that were mentioned in the discussion. While the authors are free to chose whatever defintion they wish, they should (a) justify the definition and (b) not make comparisons to studies that use a different definition unless they explicitly discuss this.https://www.ncbi.nlm.nih.gov/pubmed/25216153 and https://www.ncbi.nlm.nih.gov/pubmed/28196098 . Both, in fact, reported unilateral hearing loss, although different definitions may have been used. This should be mentioned.2. UNIQUE ASPECTS: There have been a few studies on hearing loss prevalence using the KNHANES that were not mentioned. For example: 3. STUDY DESIGN: Cross-sectional4. WRITING STYLE: Mostly understandable English. Abstract needs proofreading by a native English speaker. Many wording issues, mostly in the abstract:Line 37-38. What does "occupation presence" mean. Does that mean having an occupation ?Line 34. Do not begin a sentence with And.Lines 40-42 "In comparison of hearing aid users and non-users, noise exposure during work in hearing aid users was significantly lower than hearing aid non-users." Needs rewording.Lines 42-43. "The prevalence of hearing aid use in patients with unilateral hearing loss in South Korea is very low compared as other countries." Do you mean that the low use is similar to other countries? In that case, you want to write "comparable to". Otherwise it should be "compared with" or "compared to".Line 43-45. "Public health education is needed for increased insight and auditory rehabilitation for unilateral hearing loss should be recommended actively." Needs rewording.Lines 104-106. "To determine the prevalence of tympanic membrane perforation and cholesteatoma, including retraction pocket and otitis media with effusion" Needs rewording. Retraction pockets and otitis media with effusion are not types of tympanic membrane perforations or cholesteatoma.5. SPECIFIC COMMENTS:Big data is sort of a buzz word. This is a relatively big dataset, but there are many studies that use KNHANES or the US NHANES and they normally don't use the term big data, I would at least remove from the title. You could maybe replace with something like "Nationally representative" to indicate it wasn't just based on a single hospital's audiology clinic.Line 26-27. Do you mean lacking in South Korea? There are other studies using population-level data, e.g., reference #13.Line 48-51. You state the incidence of unilateral hearing loss and then in the next sentence state that the true incidence is not known. In between, you should explain the limitations of the existing methods, i.e., why they are not accurate.Line 57. It is Purcell, not Percell.Line 113-115. Your definition of PTA >= 41 dB for hearing loss is different from many of the recent American epidemiology papers which use >=26 dB. As I am familiar, 25-40 dB is often considered mild hearing loss, which is still hearing loss. How you define hearing loss is a matter of debate and certainly varies. I would place your definition of unilateral hearing loss in the abstract to make this clear . I would also place this definition as a footnote in all of your tables.I would recommend not double-spacing the tables so they are not spread across four pages .Table 1. You present categories, but the total data. It would be helpful to have an overall column that is not divided into a severity category. i.e, >=41 dB. It would also be helpful to have an age >= 12 row to represent "any age."Line 121. It seems you presented the prevalence of UHL with standard eror, not 95% CI Line 170. "In multivariable logistic regression..." You have to state what the other variables that you adjusted for were! This should also be clear in Table 3, e.g., in a footnote. This should also be in the methods.The Table title and results text (and methods) should all match and reflect what was actually done. E.g., Table 3 title states the outcome is hearing aid adoption or cochlear implantation. The results and methods text do not mention cochlear implantation. In fact, nowhere else in the paper is cochlear implant mentioned as far as I see.I am very confused by the different method used to assess factors associated with hearing aid use versus factors associated with hearing aid adoption. Hearing aid use and hearing aid adoption are VERY similar, with the later also including people who have a hearing aid but rarely use them. Why do you use regression to look for variables associated with hearing aid adoption, but then you use T-tests or Chi-squared tests for variables associated with hearing aid use.Lines 193-197. This is very important. If you are comparing your findings of unilateral hearing loss to other papers measuring hearing loss, your definitions must be the same! You cite ref #16 Agrawal but she used >25 dB to define hearing loss, but you used >40 dB. Those will lead to VERY different numbers! Also ref #13 Golub et al used > 25 dB. I think ref #17 Chia et al also used >25 dB.The similarity of the terms hearing aid use and hearing aid adoption is a little confusing. In the results and the discussion, I would clarify that use means something like current active use. And adoption means something like current active or rare use.Methods - you must define how all variables were measured. This includes all variables in the table. For example, how was noise exposure during work measured and what does it even mean? It can mean extremely loud noise such as explosions or factory noise. This could injure your hearing. It can also mean background noise like people talking. This would not injure your hearing but can make hearing with a mild hearing loss more difficult.Line 249 "In conclusion, the prevalence of hearing aid use in patients with UHL is very low in South Korea compared to other countries." This is your key concluding statement, but I am not sure I can trust it because your definition of UHL may be different from the studies you compared to (see above)Reviewer #2: � Line 8; This article did not focus on congenital UHL. So, introduction like this is inappropriate.� Line 63; What does “development” mean? Development of what?� Line 100; Why did you exclude children younger than 12 years-old?� Line 113-4; Was this definition of PTA suitable for UHL or asymmetric HL? General criteria for “hearing loss” is 26dBHL. 40dBHL is used as the criteria of “disabling hearing loss”, NOT “hearing loss” for adults. The criteria of “disabling hearing loss” is 30dBHL for children. Please, provide the reference of authors’ criteria for UHL. I think that authors should have used other term rather than UHL if they insisted on the 40dBHL-criteria.� Line 142-3; Authors told that they analyzed the hearing use in people with UHL, but their data concluded the hearing aid was more used in asymmetric HL. How do you think about my point-out?� To identify the factors associated with hearing aid adoption, logistic regression analyses were performed. But to identify factors associated with hearing aid use, t-tests and Chi-square tests were performed. Why did you use different statistical methods?� Table 1; What was the “weighted percentage”? On what was it weighted?� Table 2; Why was PTA average of better ear classified into {≤ 20dB}, {> 20dB, ≤ 30 dB} and {> 30 dB, ≤ 40 dB}?� Table 2; please, rewrite the data correctly.� Table 3; Cochlear implantation? What is this?**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  9 Feb 2020February 10, 2020Joerg HeberEditorPLOS ONEDear Dr Heber,Please consider our revised manuscript, “National representative analysis of unilateral hearing loss and hearing aid usage in South Korea”, for publication in PLOS OneWe appreciate the interest that the editors and reviewers have taken in our manuscript and the constructive criticism they have given. We have addressed the major concerns of the reviewers. We have also included a point-by-point response to the reviewers in addition to making the changes in the manuscript. Changes to the text in the manuscript are marked in red. Our main findings remain unchanged.This manuscript has not been published or presented elsewhere in part or in entirety and is not under consideration by another journal. The study design was approved by the appropriate ethics review board. We have read and understood your journal’s policies, and we believe that neither the manuscript nor the study violates any of these. There are no conflicts of interest to declare.Bo Gyung Kim, MD, PhDDepartment of Otorhinolaryngology-Head and Neck Surgery, Soonchunhyang University College of MedicineAddress: 170 Jomaru-ro, Wonmi-gu, Bucheon 14584, KoreaTel: +82-32-621-6951Fax: +82-32-621-5018bgkim@schmc.ac.krE-mail: AttachmentResponse_letter_final.docxSubmitted filename: Click here for additional data file. 23 Mar 2020PONE-D-19-32331R1National representative analysis of unilateral hearing loss and hearing aid usage in South KoreaPLOS ONEDear Kim,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.Please carefully review all recommendations for revision in the original submission and re-submission.  While the authors did address the comments in their response, a number of critical clarifications were not incorporated into the revised manuscript.  The reviewer for revised submission has very carefully pointed out some essential changes that clarify how the current manuscript differs in some definitions from others in the literature, and how variables in their analysis were defined in KHANES data. https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.We would appreciate receiving your revised manuscript by May 07 2020 11:59PM. When you are ready to submit your revision, log on to If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocolsTo enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: Please include the following items when submitting your revised manuscript:A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.We look forward to receiving your revised manuscript.Kind regards,Clifford R. Hume, MD PHDAcademic EditorPLOS ONE[Note: HTML markup is below. Please do not edit.]Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.Reviewer #1: (No Response)**********2. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: (No Response)**********3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: (No Response)**********4. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: (No Response)**********5. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: (No Response)**********6. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: Thank you for the revisions and the detailed responses to my comments. Several of my comments from the prior submission remain unaddressed in the manuscript. In general, when a reviewer has a comment that requires a clarification, they would like the clarification to be inside the manuscript, not just in the response to reviewer comments. If the reviewer had a question, then many readers may have the same question.1. NEW COMMENTS. (Line numbers refer to the new submission version with tracked changes)Line 42 "...noise exposure at work in users with no hearing aid" should be "...noise exposure at work in those with no hearing aid"2. PRIOR COMMENTS that were not addressed by modifications to the manuscript. NOTE that line numbers here refer to the **first submission**) Your comments are denoted by >>. My new comments are denoted by >>>Line 26-27. Do you mean lacking in South Korea? There are other studies using population- level data, e.g., reference #13.>>Of course, several studies have been published, such as reference # 13, however, there are very few studies on the use of UHL and hearing aids in Korea as well as in other countries, compared to studies on bilateral SNHL.>>>Thank you, I understand. Please make this more clear in the manuscript by rewording the first sentence of the abstract to add "in Korea" to the end of this current sentence: "A definitive study on the prevalence of adult unilateral hearing loss and hearing aid rehabilitation is lacking."Line 113-115. Your definition of PTA >= 41 dB for hearing loss is different from many of the recent American epidemiology papers which use >=26 dB. As I am familiar, 25-40 dB is often considered mild hearing loss, which is still hearing loss. How you define hearing loss is a matter of debate and certainly varies. I would place your definition of unilateral hearing loss in the abstract to make this clear . I would also place this definition as a footnote in all of your tables.>>Thank you for your valuable comment and suggestion. Many studies define unilateral hearing loss as PTA >= 26 dB. However, the KNHANES defined unilateral hearing loss as PTA >=41 dB. Moreover, the prevalence of unilateral hearing loss in our study is much lower than other studies, which define unilateral hearing loss as PTA >=25 dB. If we change the definition of PTA >=26dB rigidly, there would be no one who used a hearing aid.>>>Thank you for the explanation. You justify why you use >=40 dB. I understand this. However, because this is not the worldwide standard definition of hearing loss, I recommend doing this (repeated from above): Place your definition of unilateral hearing loss in the abstract to make this clear . I would also place this definition as a footnote in all of your tables."Line 170. "In multivariable logistic regression..." You have to state what the other variables that you adjusted for were! This should also be clear in Table 3, e.g., in a footnote. This should also be in the methods.>>The independent variables of the multivariable logistic regression model are the occupational status, the hearing threshold in the better ear, and the hearing threshold in the worse ear. There are no calibration parameters other than those shown in the table. In the univariable analysis results, the significant variables were considered as independent variables of the multivariable analysis. Among the five variables that were significant in univariable analysis, otitis media and dizziness were difficult to estimate due to an insufficient number of patients.>>>I understand. Often in multivariable regression, there are other variables that are non-significant . Since all 3 variables you describe are significant, it is not clear that you did not include any other variables in the model. To make this clear, at the end of the sentence "In multiple logistic regression..." please insert another sentence and explain that there were no other variable present in the multivariable model. Please also, like I said, put this information in the methods and in a Table 3 footnote.Lines 193-197. This is very important. If you are comparing your findings of unilateral hearing loss to other papers measuring hearing loss, your definitions must be the same! You cite ref #16 Agrawal but she used >25 dB to define hearing loss, but you used >40 dB. Those will lead to VERY different numbers! Also ref #13 Golub et al used > 25 dB. I think ref #17 Chia et al also used >25 dB.>>As mentioned earlier, the KNHANES defined unilateral hearing loss as PTA >=41 dB. Moreover, the prevalence of unilateral hearing loss in our study is much lower than other studies, which define unilateral hearing loss as PTA >=25 dB. If we change the definition of PTA >=26dB rigidly, there would be no one who used a hearing aid. The criteria are less strict than other studies, but with lower prevalence, we believe our results are meaningful.>>>Thank you and I understand why you used >40 dB: you had no choice because this is what KHANES used. However, please modify the text here to explain that your definition of hearing loss is different from the papers you are comparing it to. The following statement is completely misleading without an explanation: "In the present study conducted in South Korea, the prevalence of UHL determined via KNHANES data was 4.91%. This is lower than the prevalence of 7.9%–13.3% in the general population  and the prevalence of UHL was 7.2% in the US [13]." You must insert a sentence here to say something like: "However, our definition of hearing loss was >40 dB which is different from the previously cited studies, which used >25 dB to define hearing loss. Thus we can not make direct comparisons."Methods - you must define how all variables were measured. This includes all variables in the table. For example, how was noise exposure during work measured and what does it even mean? It can mean extremely loud noise such as explosions or factory noise. This could injure your hearing. It can also mean background noise like people talking. This would not injure your hearing but can make hearing with a mild hearing loss more difficult.>>We used the data obtained from the 2009-2012 KNHANES. Participants were asked for the presence of noise but there was no query on the degree of noise.>>>Thank you. Please address my comment. Please define how all the variables were measured or defined, briefly according to the KHANES manual, in the methods. If there is no room, place this in a supplement or a reference to the KHANES manual.**********what does this mean?). If published, this will include your full peer review and any attached files.7. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  7 Apr 2020We thank you and the reviewers for your thoughtful suggestions and insights. The manuscript has benefited from these insightful suggestions. I look forward to working with you and the reviewers to move this manuscript closer to publication in Plos One.The manuscript has been rechecked and the necessary changes have been made in accordance with the reviewers’ suggestions. This included editing the entire document for readability and organization. Additions to the manuscript are marked with red text. The responses to all comments have been prepared and attached herewith.Thank you for your consideration. I look forward to hearing from you.Attachment2ndrevision_responseletter.docxSubmitted filename: Click here for additional data file. 8 Apr 2020National representative analysis of unilateral hearing loss and hearing aid usage in South KoreaPONE-D-19-32331R2Dear Dr. Kim,We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact With kind regards,Clifford R. Hume, MD PHDAcademic EditorPLOS ONE 13 Apr 2020PONE-D-19-32331R2 National representative analysis of unilateral hearing loss and hearing aid usage in South Korea Dear Dr. Kim:I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. For any other questions or concerns, please email Thank you for submitting your work to PLOS ONE.With kind regards,PLOS ONE Editorial Office Staffon behalf ofDr. Clifford R. 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Recently, Kato et al. reported recurrent activating mutations in the SET domain of histone methyltransferase G9a, driving an oncogenic cascade in melanoma. The authors also reported correlations between G9a expression and the regulation of the canonical WNT pathway. Although we could not observe such mutations in human colorectal adenocarcinoma, newly reported findings are of high relevance to colorectal cancer, as WNT target gene signatures were closely associated with G9a expression. Here, we put into perspective such new results on G9a expression in colorectal cancers and the potential relationship with tumor heterogeneity and acquisition of neoplastic stemness. Colorectal cancer is a heterogeneous disease associated with accumulation of various genetic and epigenetic aberrations. The epigenetic landscape in colorectal tumors is key to establishing an intratumoral cellular hierarchy that dictates tumorigenesis . Thus, cCancer Discovery, Kato et al. identified recurrent activating mutations and copy gains in EHMT2, which encodes G9a, and such alterations were shown to drive oncogenesis in human melanoma [In a recent issue of melanoma . MoreoveSimilar to the authors, we used the cBioPortal platform to run EHMT2/G9a mutation and copy number analysis on TCGA PanCancer Atlas colorectal adenocarcinoma cohort (524 samples) . We coulWe expanded our analysis to other malignancies using TCGA PanCancer Atlas cohorts and assessed the EHMT2/G9a mutation status in breast 1084 samples), glioblastoma (592 samples), pancreatic (184 samples) and lung tumors (1053 samples). We could not identify recurrent G1069 mutations in the G9a SET domain as reported by Kato et al. Thus, to the best of our knowledge, reported activating mutations in G9a seem to be a distinct characteristic of melanomas. Yet, overexpression of G9a protein and/or associated H3K9 methylation levels is not anecdotic in colorectal tumors and has been reported by several groups [4 samplesDespite clear connections between G9a activating mutations, Kato et al. established a correlation between this histone methyltransferase and repression of DKK1 in colorectal tumors. This complements the body of knowledge on canonical WNT hyperactivity as a hallmark of colorectal cancer linked to tumorigenesis, self-renewal and maintenance of neoplastic stem phenotype. The interplay between G9a and soluble WNT inhibitors was previously documented in neuroendocrine tumors, where DKK1 and DKK3 promoters are silenced by G9a-dependent H3K9 methylation ,12. In aRemarkably, Kato et al. addressed the issue of a significant number of patients experiencing resistance to immunotherapeutic approaches. Indeed, the use of immune checkpoint inhibitors has currently yielded no major clinical benefits for most colorectal cancer patients . The autThe findings reported by Kato et al. are setting the stage for the use of the G9a/GLP inhibitors as an opportunity to evaluate G9a inhibition as a therapeutic avenue to target colorectal cancer-initiating cells in vivo. As aforementioned, the small molecule UNC0642 was used to block G9a histone methyltransferase activity in mouse syngeneic and human-to-mouse xenograft models. The authors highlighted the importance of developing novel and more effective therapeutics targeting epigenetic determinants, emphasizing the particular “druggability” of G9a in cancer. Recently, Casciello et al. also used UNC0642 to show that inhibition of G9a in breast cancer cells decreases tumor growth and lung metastasis in vivo . These a


Cochlear implantation in patients with functional residual low-frequency hearing is performed according to an established hearing-preserving surgical technique in order to cause minimal trauma of inner ear structures. Due to the increasing number of cochlear implants in children, the preservation of residual hearing is becoming increasingly important in this patient collective.Short- and mid-term hearing preservation outcome in pediatric patients is investigated.A group of 9 children (12 ears) between 5 and 12 years of age were examined after hearing-assisted cochlear implantation with respect to the pure tone audiometric thresholds. Retrospectively, short-term hearing preservation (up to 3 months after surgery) was examined. In a subgroup of 5 children, mid-term hearing preservation (7.5 to 16 months after surgery) was also analyzed. The mean values of hearing preserved (HL%) and hearing loss (HL) due to electrode insertion were calculated as measured values.low were 29.8 dB and the short-term postoperative PTAlow 42.6 dB. The mean value of the HL% was 73.6%, corresponding to an HL of 9.4 dB. In the subgroup, the mean PTAlow postoperatively was 46.0 dB in the mid-term and the HL% at 80.7% with a HL of 6.6 dB.In the whole group, the mean values of the preoperative PTAThe results in children are consistent with the results in adults. Electric-acoustic stimulation (EAS) should be used in the treatment of children with existing low-frequency residual hearing, as good residual hearing preservation can also be achieved in children after implantation. If low-frequency residual hearing in subjects with severe high-frequency hearing loss (HL) cannot successfully be treated with hearing aids, electric-acoustic stimulation (EAS) can be used in the same ear, creating a synergistic effect.This has a positive impact on speech understanding –10, 15. Due to the constantly improving clinical diagnosis of hearing disorders in children, frequency-specific hearing thresholds can be assessed with precision in an increasing number of children. Thus, the number of hearing preservation cochlear implantations is steadily increasing in children and residual hearing is gaining in importance in this patient population. Considering the long life expectancy of this patient population and the first experimental achievements in the development of new approaches for the improvement of the electrode/auditory nerve interface  or the nThe international HEARRING Group expert group recommends applying the HPCI standard in all cochlear implantations in children, regardless of the extent of residual hearing .All children with low-frequency residual hearing implanted with electrode arrays specifically designed for hearing preservation at the Medical University Clinic of Mainz between 2012 and 2016 were included in the retrospective data analysis. Pure tone audiometry was performed in a group of 9 children (12 ears) aged between 5 and 12 years after hearing preservation cochlear implantation . The Flex24 electrode array features 12 platinum contacts  over a total length of 20.9 mm with an electrode spacing of 1.9 mm. The insertion depth is 24 mm with an electrode diameter of 0.36–0.48 mm at the apical end and 0.8 mm at the basal end.One ear was implanted with a CI522 implant  with a Slim Straight electrode array. The Slim Straight electrode array features 22 one-sided platinum contacts on a total active spacing of 20 mm with maximum full insertion of 25 mm. The electrode diameter is 0.3 mm at the apical end and 0.6 mm at the basal end.The decision on the type of implant system was made after neutral counselling to the parents. The decision on the type of electrode array was made based on an interdisciplinary agreement within the CI team of the ENT clinic, taking into account the anatomy of the cochlea using computed tomography (CT) and magnetic resonance imaging (MRI) and the audiogram.All implanted electrode arrays are suitable and approved for hearing preservation surgery , 8, 19. low (pure tone average for low frequency) was calculated as a mean value of the audiometric thresholds at 0.125, 0.250, and 0.5 kHz at the different times of observation. Additionally, hearing preservation was calculated in percent (HL%) following Skarzynski et al. [mdh variable describes the maximum output level of the audiometer for the measured audiometric frequencies.First, the short-term hearing preservation (“postoperative short-term”) was analyzed between 2 weeks and 3 months (mean: 6 weeks) postimplantation to detect potential hearing defects caused during implantation. In a subgroup of 5 children, data for the evaluation of the medium-term hearing preservation (“postoperative medium-term”) were available for the period between 7.5 and 16 months (mean: 12.6 months) postimplantation to detect potential progressive hearing loss. For all 12 ears, the preoperative PTAP-values < 0.05 were considered statistically significant.The variables of the total group were tested for normal distribution using the Kolmogorov–Smirnov test for the “preoperative” and the “postoperative short-term” times of observation and analyzed for significant differences using the paired sample t‑test. Due to the small number of cases, the data of the subgroup for the “postoperative medium-term” follow-up were not included in the statistical analysis for significant differences. The statistical analysis was performed using the SPSS statistics 23 software . Audiometric thresholds of 9 children 12 ears) aged between 5 and 12 years were measured before and after hearing preservation cochlear implantation. The averaged audiometric thresholds of the three different times of observation, “preoperative”, “postoperative short-term”, and “postoperative medium-term”, are presented in Fig. 2 ears aglow averaged low-frequency thresholds are presented in Table p = 0.006) deteriorated by 12.8 dB between “preoperative” (PTAlow1 29.8 dB) and “postoperative short-term” (PTAlow2 42.6 dB) due to the surgical insertion of the CI electrode array. The measured values varied between the subjects from a deterioration of 31.6 dB (E07) to an improvement of 8.4 dB (E01). The short-term change in the averaged low-frequency thresholds PTAlow1 measured postoperatively is presented in Fig. The PTA1) was 73.6% (variation: 39.8 to 104%). This corresponds to a mean HL1 of 9.4 dB across all frequencies (range: −2.3 to 22.7 dB).The mean percentage of hearing preservation in the low frequencies based on Eq. Medium-term hearing preservation results were available for a subgroup of 5 children with a mean age of 7.3 years (E01–E05). Analyzing the results of the subgroup over time, the averaged threshold deteriorated in the low frequencies between the preoperative measurement with 39.0 dB and the postoperative short-term follow-up with 45.6 dB and the postoperative medium-term follow-up with 46.0 dB. The most distinct deterioration (6.7 dB) of the auditory threshold was measured at the postoperative short-term follow-up. In the medium term, the auditory threshold only deteriorated by 0.4 dB (Table 1 was 74.7% at the postoperative short-term follow-up (range: 66.7–104.0%) and 80.7% at the postoperative medium-term follow-up (range: 27.5–98.0%). This results in a mean HL1 of 6.4 dB (range: −2.3 to 13.9 dB) across all frequencies at the postoperative short-term follow-up and of 6.6 dB (range: 0.9–22.7 dB) across all frequencies at the postoperative medium-term follow-up.The mean percentage of hearing preservation HL%The individual audiograms of all subjects are presented in Fig. The hearing preservation cochlear implantation has become an established procedure in the treatment of patients with severe to profound hearing loss with usable low-frequency residual hearing. Before implantation, it should be verified if the best possible state-of-the-art hearing aid provision was offered .This study showed that a high percentage of hearing preservation can be achieved in children after cochlear implantation following the HPCI standard in 73.6% of all cases and in up to 80.7% of all cases in the analyzed subgroup.low ≤ 10 dB) in 31.1%, “partial hearing preservation” (definition: deterioration of 10–30 dB) in 47.6%, and “minimum hearing preservation” (definition: deterioration of PTAlow ≥ 30 dB) in 13.6%. Complete HL was detected in 7.8% of all patients. No correlations between the hearing results and etiology, design of the electrode array or surgical approach were identified.Many studies on adult patients have shown that low-frequency hearing preservation can be achieved and that EAS is an option in cochlear implantation in the medium and long term. Helbig et al. , for exaCompared to the data presented in this study, the study of Mertens et al.  showed slow) after 5 years of observation in 32 ears and provision with Cochlear Nucleus Hybrid L24 electrodes [Roland et al. showed no significant deterioration of the postoperative low-frequency thresholds  ). This mlow deteriorated by 11.1 dB from 52.0 to 63.9 dB. Compared to our study, the preoperative baseline HL was clearly more distinct (13 dB higher). Because of the distinct HL, the Flex20/Flex24 electrode arrays specifically developed for EAS and HPCI was only used for implantation in 5 patients of the Bruce et al. study. The other 9 patients were implanted with the considerably longer FlexSoft (31.5 mm) or the Flex28 electrode arrays; therefore, the results of Bruce et al. cannot be directly compared to the data presented in our study.In a retrospective study Bruce et al. showed hearing preservation in 14 adolescents (mean age: 13.5 years), achieved through the soft surgery procedure [Benghalem et al. reported on cochlear implantations in 7 children (mean age: 4.5 years) which were performed using the Mid-Scala Electrode from Advanced Bionics  designed for hearing preservation cochlear implantation according to the HPCI standard . As somelow deteriorated by 25.3 dB from 54.2 dB to 79.5 dB at frequencies of 250 Hz and 500 Hz at the postoperative measurement of auditory thresholds 10.7 months after implantation. The calculated hearing preservation of the low-frequency residual hearing following Skarzynski [low deteriorated by another 9.7 dB on average.Carlson et al. also presented the results after cochlear implantation in 35 children (43 ears) with a mean age of 8.6 years [Further improvements in hearing preservation could probably be achieved by using new procedures to insert the electrode array. Stuermer et al. , for exaIntracochlear pressure waves, caused by the insertion of the CI electrode array into the cochlea, can be reduced by an appropriate design of the electrode array. Pressure measurements by Mittmann et al.  in an arThe main goal of the present study was to analyze the hearing preservation results in children with EAS indication provided with a cochlear implant. Speech understanding outcomes are therefore not part of the retrospective analysis. Due to the retrospective study design, the availability of postoperative audiograms is limited in some cases, and the times of analysis inevitably vary to a certain extent. This limits the conclusions that can be drawn with regard to the actual hearing preservation in the observed cohort.The present study provides evidence that hearing preservation can be achieved after cochlear implantation in children. To achieve this, the implantation should be performed following the HPCI standard. Providing children with low-frequency residual hearing with electric-acoustic stimulation (EAS) should be aimed for as a high level of hearing preservation can also be achieved in children over the medium and long term.Low-frequency hearing preservation can be achieved after cochlear implantation in children.Hearing preservation surgical techniques and hearing preservation electrode array designs must be used.Children should be informed about the expected success of EAS provision with regard to better speech understanding, even though speech audiometry in early childhood is not an option for early performance measurement.


The hearing function at a given age seems to have improved in more recent born cohorts in industrialized countries. But the reasons for the improvement have not yet been explained.We investigated the extent to which better hearing in Norway is attributed to modifiable risk factors by using representative demographic and audiometric data from two cohorts of the Trøndelag Health Study, HUNT2 (1996–1998) and HUNT4 (2017–2019). We estimated natural indirect effects using causal inference methods in order to assess whether cohort improvement in hearing thresholds (HTs) was mediated by occupational noise exposure, recurrent ear infections, smoking and education.The improvement in HTs from HUNT2 to HUNT4 was 2.8 and 3.0 dB at low respectively high frequencies. Together all risk factors mediated this improvement by 0.8 dB (95% CI 0.7–0.9) and 0.8 dB (95% CI 0.7–0.9) respectively, corresponding to mediated proportions of 27 and 28%. Substantial mediation was specifically found for occupational noise in men and recurrent ear infections in women .Increased education, less occupational noise exposure, ear infections and smoking contributed considerably to better hearing in Norway the last two decades.The online version contains supplementary material available at 10.1186/s12889-021-10301-1. Sense organ diseases, among which hearing loss is the most common, was the leading cause of years lived with disability for elderly in 2015 –5, SwedeSeveral environmental and behavioral risk factors for hearing loss may have declined in the twentieth century potentially contributing to this generational improvement. Important risk factors for hearing loss are occupational noise exposure –16, recrThe aim of this paper is to examine to what extent the better hearing in recent birth cohorts is attributed to change in the modifiable risk factors for hearing loss occupational noise, ear infections and smoking. This by estimating natural indirect effects using causal inference methods.HUNT2 Hearing (1996–1998) and HUNT4 Hearing (2017–2019) were part of a large general health-screening study for the entire adult population of Nord-Trøndelag County (HUNT).HUNT2 Hearing included 17 of the 24 municipalities in the county. The participation rate was 63%, and altogether, 51,529 persons attended. HUNT4 Hearing took part in the six larger municipalities, representing about two thirds of the county. The participation rate was 43%, and altogether, 28,388 persons attended. The hearing studies are described in detail elsewhere  and 0.4 dB [95% CI 0.0–0.8] at low respectively high frequencies controlling for age and sex. The influence of list wise deletion on the cohort effect was however less than 1 %.Our study showed that the better hearing in the more recent birth cohort of Norwegian adults to a large extent is attributed to secular trends in education, occupational noise exposure, recurrent ear infections and smoking. While occupational noise was the most important mediator in men, recurrent ear infections was most important in women.The better hearing in the more recent born cohort is in agreement with other studies that suggest cohort improvements in hearing ability among adults –6. HowevSelf-reported occupational noise exposure attenuated the cohort difference among men, especially at high frequencies. This agrees with a suggested reduction of noise-induced hearing loss in the industry in recent decades because of hearing conservation programs . It is oA history of recurrent ear infections has been associated with poorer hearing thresholds . Our stuDespite a large decline in smoking consumption, smoking explained only a minor part of the change in hearing. We believe that this finding is plausible, since smoking has been associated with hearing with modest effect sizes , 39–41.Our study showed that increased educational attainment explained some of the cohort difference, which agrees with the study by Zhan et al. 2011 . SocioecA recent follow-up of the studies in Beaver Dam assessed generational decreases in 5-year hearing loss incidence and 10-year cumulative hearing loss incidence. The study failed to ascribe the decreases to changes in any of the measured known risk factors for hearing loss including work related noise, smoking status and educational level . They thReports from the US National Health and Nutrition Examination showed improvement of hearing between 1959 and 1962 and 1999–2004 and between 1999 and 2004 and 2011–2012 for adults aged 25 to 64 years and 20 to 69 years respectively , 4. As tOur results may be compared with a Swedish study that showed improved hearing in 70-year-old subjects born in 1944 compared with those born in 1922 and 1901–1907 . AlthougOther factors may also have contributed to better hearing in the present study. For example, focus on hearing protection and improved regulation in hunting and sports-shooting may have had an effect. There has also been an improvement in life-style factors other than smoking that we did not measure, such as lack of physical activity/exercise. The improvement may as well be explained by factors related to prenatal and early childhood development such as reduction in infections due to introduction of antibiotics and vaccination, head traumas or use of ototoxic drugs. On the contrary, there are factors that have worsened, such as diabetes, body mass index, and exposure to music through earphones.The major strength of our study is the large population-based design with cohorts separated 20 years apart, and the use of standardized audiometric procedure and contemporary mediation approach.There are also limitations. With measures at only two time points we cannot derive the point at when hearing started to improve, or how it changed. Using the data to forecast future hearing status of the population is therefore limited. Second, we cannot fully reject possible influences of selection bias. As in most observational studies, our recent study wave experienced falling response rates and perhaps a healthier population. The observed birth cohort difference in hearing has previously been shown to be little effected by bias due to nonparticipation differences between the two study waves , nor wasThis study showed that increased education, less occupational noise exposure, ear infections and smoking contribute substantially to improved hearing in Norway the last two decades. Strategies for prevention of these risk factors seem to be successful and might have a strong impact for reducing hearing loss at a population level.Additional file 1.Additional file 2.