The effects of chronic exposure to high sound pressure levels (SPLs) are widely studied in the industry environment. However, the way that SPLs affect music students has not been thoroughly examined. In this paper, we study the SPL exposure of batucada students and we propose an assisted protection headphone as a part of e-health system. We measured the SPL reached during a regular percussion class. Pure-tone audiometries were performed to a set of percussion students. The gathered data were statistically analyzed. The assisted protection headphones and their operation are detailed and tested during a regular class. Our results show that 35% of the musicians present with a noise-induced hearing loss, considered as two frequencies with hearing loss of 25 dB or more or one frequency with a hearing loss of 30 dB or more. Our data also shows that those students that do not use any protection have a greater hearing loss. However, the students that use protection headphones are also suffering hearing loss. There might be a problem in the way that musicians are using the protection headphones. Our assisted protection headphones as a part of e-health measures can diminish the negative effects of percussion instruments for musicians. The effects of chronic exposure to high sound pressure levels (SPLs) are widely studied. It is well known that the occupational exposure to high SPL produces noise-induced hearing loss (NIHL) . NIHL isThe exposure to high SPLs has been widely studied in the work environment. Nevertheless, in the leisure environments, it has been less studied. Recent studies have focused on the effect of personal hearing devices or headphones , 3. HoweThe e-health systems are becoming more and more useful, and many authors are developing applications for monitoring different issues . Some exThe aim of this paper is to (i) study the effects of SPL reached while playing batucada music in the hearing capability of the musicians, (ii) design an assisted protection headphone, and (iii) evaluate its function. To evaluate the effects of SPL, first, we perform some records during a regular batucada class in “Borumbaia drum school” in Valencia. For the measurements, sound pressure meters of Brüel & Kjær were used. Specifically, the models 2250 and 2270 were employed. The advanced FFT analysis and the frequency analysis modules were employed. Then, pure-tone audiometries are done to the students and teachers in the ear, nose, and throat department of the University General Hospital of Valencia. We will analyze the response of people to frequencies from 100 Hz to 8000 Hz. The use of protections, the period of exposition, and the played musical instrument are other studied variables. Moreover, we will try to relate the frequencies where there are NIHL in the students with the frequencies where the sound level meters registered the maximum SPL. In addition, the design of an assisted protection headphone is shown. This protection headphone will be a part of e-health system for the prevention of NIHL for musicians. It will gather the SPL received by the musicians and send this information to the medical center after each class. The motivation to design these headphones is that many musicians that are using the headphones are reporting hearing loss. For this reason, we suspect that some musicians may not be using them properly. The assisted protection headphone is composed by on-sound sensor in the internal part on each side. The headphone has a vibrating element that alerts to the user that it is not well adjusted. Moreover, the designed protocol for generating individual or collective alarms when the SPL detected is above the established threshold is presented. Finally, the assisted protection headphones are tested during a regular class to evaluate its performance.The rest of this paper is structured as follows, In this section, we will review the current knowledge about sound exposure in professional and nonprofessional musicians. We will look for differences and similarities with our present study and break down the auditory aspects studied both objective and subjective (hearing symptoms).Starting with nonprofessional musicians, Schmuziger et al.  comparedn = 11). They gathered information about SPL during individual and group playing. The information was obtained with personal sound exposure meters (4436 and 4443 of Brüel & Kjær). The difference of SPL between instruments was statistically significant, with the percussionists taking the worst part. Furthermore, the hearing threshold at 4 kHz to 8 kHz was not statistically significant between the students and a 67-people control group. They compared their data with the International Standard Organization standard ISO 1999 : 2013 finding poorer hearing results in their sample at 6 kHz and better results at 3 kHz and 4 kHz. Symptoms as tinnitus and hyperacusis were also found in greater proportions in the musical student group compared with the group that was not exposed to loud sounds (11.3% versus 4.5% in tinnitus and 36.3% versus 11.9% in hyperacusis).Pawlaczyk-Łuszczyńska et al.  studied Størmer et al.  studied Rock professional musicians, along with pop and jazz musicians, were studied by Halevi-Katz et al. . They stPatil et al.  did not Schink et al.  studied The presented papers show the current knowledge about the NIHL in the musicians. Some authors point that musicians that play different instruments present different NIHL. In some studies , 7, 19, In this section, the material and methods used are detailed. Firstly, the equipment used to gather data about the sound levels in the batucada class is shown. Secondly, the medical equipment employed in the audiometries is presented. The data related to the studied population and the characteristics of the class is detailed. Finally, the musical instruments and the protection measures that the school offers are shown.In this subsection, the employed equipment to perform the records is shown. Two different sound level meters were used. Both of them were manufactured by Brüel & Kjær. The used models are the 2250 and the 2270. The windscreen was used in all the measures. The reason why we use the windscreen is that in future work, we want to record in the streets during a live performance and not only in the classrooms. In the streets, we will use the windscreen. As we want to maintain the same equipment in order compare the records in both cases, we will use the windscreen in the rooms even that it is not necessary. Both devices and the windscreen can be seen in One of the sound level meters is gathering data related to the advanced FFT analysis. The analysis is performed from 50 Hz to 20,000 Hz with increments of 50 Hz. Three different records were done during different moments of the class. The records have an average time of 35 s. For each record, it is possible to obtain the mean data and the maximum data for each studied frequency. Data were obtained using A-weighting.The other sound level meter is gathering data related to the frequency analysis. It provides real-time analysis of the 1/1- and 1/3-octave filter band. The scanned frequency goes from 12.5 Hz to 20,000 Hz, with 33 analyzed frequencies. The A-weighting was used for data.In this case, two records were gathered about three minutes each, during different moments of the class. For both measures, a tripod was used. The height of the sound level meter was 1.50 cm from the floor. The sound level meters were placed together in the middle of the class.In this subsection, we detail the studied population and the characteristics of the place where they do the classes.We took our sample from the members of “Borumbaia drum school” in Valencia (teachers and students). Inclusion in the sample was based on voluntary participation, excluding people with a history of chronic ear diseases or surgeries (with the exception of transtympanic tubes during childhood). All patients were invited to participate in this study via email and group request in their weekly rehearsal. The main reason for not taking part was conflicting time schedules. We grouped a sample of 56 people. From 56 people, 43 of them completed the audiometries and questionnaires. From 43 people that form the studied population, 60.4% (26) are women and 39.6% (17) are males.2. It is rectangular shaped with dimensions of 9.075 m per 5.71 m and a height of 2.97 m. The room had been soundproofed to avoid the acoustic pollution to the surrounding buildings.The room where the classes are performed and where the records of sound pressure levels were done is 14.785mIn this subsection, the information of the medical instruments used in the test is shown. In addition, some information of the used questionnaire is detailed.The subjects were studied using clinical examination, pure-tone and high-frequency audiometry, and a questionnaire. We asked about otological and head trauma background, number of years playing, weekly hours playing, and type of drum. Moreover, we ask them if they use or not protection measures during classes. Also, we recollected information about the presence of tinnitus, hyperacusis, or diplacusis. We tried to relate subjective hearing loss with the hearing thresholds found. We had questions about all their other noise exposure through their daily work, concerts, and other noisy hobbies.Both tests and questionnaires were completed at the ear, nose, and throat department of the University General Hospital of Valencia. Clinical examination including otomicroscopy and cerumen was removed when needed to.We used a MADSEN Adstera2 audiometer (Otometrics) in a sound-attenuating room. Audiometry was bilaterally performed using the ascending method  with a random first ear in the frequencies of 125, 250, 512, 1000, 2000, 4000, 6000, and 8000 Hz. The audiometer used can be seen in In this subsection, we present the different musical instruments and protections used by the students.First, we show the different instruments. The instruments can be seen in Now, we detail the protection measures that the school offers to the teachers and students. They can use ear plugs and protection headphones. The earplugs are used by 19 students; the protection headphones by 16 and three of the students use both protections. Finally, there are five students that do not use any protection.In this subsection, we present the hypotheses that motivate our work. Our first hypothesis is that the SPL reached during a batucada class is higher than in other music classes. Because of this, the musicians and students can suffer from NIHL. This possible NIHL must be monitored in order to include this information in an e-health program to prevent the NIHL.Our second hypothesis is that many musicians that are using the protection measures, especially the ones that are using the headphones may not use them properly. There are several reasons that can cause the protection headphones to not correctly protect the users. The main issue is the characteristics of the protection headphones; different headphones offer different acoustic isolation depending on their quality. If the headphones are not well adjusted to the skin of the users, its acoustic isolation decreases.In this section, we show the developed assisted protection headphones. First, we describe the protection headphones. Then, its operation is described and its algorithm is shown.We suspect that some musicians could be using the headphones incorrectly. For this reason, it is necessary, for musicians and other applications, to have protection headphones that help people to use them in a proper manner. We propose a new headphone that can inform the users if they are using it correctly. The assisted protection headphone can be seen in This assisted protection headphone will help to prevent NIHL by the use of sound sensors. They use an Arduino module to gather the data from the sensor and activate the vibrating element. The threshold value of the sound sensor will be established in 80 dB. The protection headphones must be activated with a switch button, and a green LED will indicate that it is operating. This system can assist different musicians, and other professionals, to use properly the protection headphones to avoid NIHL as a part of an e-health program for musicians.The operation of the assisted headphones is described below. The proposed assisted protection headphones will be a part of a collaborative system. The proposed protection headphones will communicate with the headphones of the rest of the musicians. The objective is to differentiate two situations that can produce an increase in the SPL inside the headphones. Those situations are the inappropriate use of the protection headphones or an exceptional increase in the SPL outside of the protection headphones. The protection headphones attenuate part of the noise, in this case, 35%. If the external noise is too high, the protection headphones can be not enough. Those high noise levels can be caused by peaks in the music during the classes.To be able to differentiate between both situations, the following algorithm will be used, see If the data is higher than the threshold, it is necessary to identify if this high value is caused by the incorrect use of the protection headphones or because there is a peak of noise. Then, the Arduino using its Wifi connection requests the data of its neighbors. If the data of the neighbors are below the threshold, then an individual alarm is generated. This individual alarm will produce vibrating elements that will turn on in order to advise the musician to properly adjust the protection headphones. Moreover, the alarm will trigger the node to send a message to the coordinator node (CN). If the data of the neighbors are also above the threshold, then a collective alarm is generated. This alarm will trigger the node to generate a list of the neighbors affected, and this information is sent to the CN. In this case, the vibrating element will not be triggered. The objective of generating a list of the affected musicians is to bring information in order to assess whether new protective measures are necessary for a specific group, by either their location or instrument. All this information is shown in real time in the smartphone of the teacher, which owns the CN. The teacher can assess their students and show them how to adjust the headphones according to the information of the generated individual alarms. If many collective alarms are generated they must consider using protection headphones with higher sound isolation properties.Moreover, the proposed system stores all the data gathered during the class. This information and the generated alarms are sent after the classes to the medical center where the students and professors are attending to monitor its NIHL see . This inIn this section, we present, on the one hand, the gathered data with the sound level meters during a regular session of batucada class. On the other hand, we present the data of the audiometries performed to the musicians. The statistical analysis shows the relationship between the obtained results in the audiometries and the protection measures employed or the musical instrument played. Moreover, the results of the test performed with the assisted protection headphones are presented.In this subsection, the obtained data of the records with the sound level meter presented in the previous section is detailed. First, the results of the advanced FFT analysis are presented. The mean values for each frequency and the maximum value for each frequency in A-weighting is presented in Figures While in the maximum values, the profiles were similar in the three records, the profiles of the mean values are different in each record see . The maxNow, the results of the frequency analysis with A-weighting as the sound level in decibels equivalent to the total A-weighted sound energy measured over a stated period of time, LAeq, are shown in Figures In this subsection, we show the obtained results of the audiometries performed to the musicians. First, we will show the NIHL at different frequencies. It can be seen in The hearing loss is defined when the hearing threshold showed in the audiometry is greater than 25 dB in two or more frequencies in the same ear or with one result greater than 30 dB. Furthermore, we have analyzed the NIHL of different subgroups. The relationship between NIHL and the different instruments played is shown in Now, we are going to analyze the hearing loss at each frequency statistically and its relationship to the exposure period, instrument, and protection employed. For this analysis, we have used the statistical software Statgraphics Centurion . The anaAt 125 Hz, the observed differences are related only to the played instrument. The people that play caixa, surdo, and cutter surdo present greater hearing loss than the others. At 250 Hz, the differences are explained because of the instrument and the protection. People that play caixa and surdo have a greater hearing loss at 250 Hz than the others. Moreover, the use of protections produces differences on hearing loss at 500 Hz. Moreover, the protection measures explain part of the difference. The people that do not use any protection present the worse hearing loss, followed by the ones that use the protection headphones. Again, the use of earplugs of both measures seems to be the best option to avoid the hearing loss. These differences at 2000 Hz, which can be seen in In this subsection, we are going to analyze the data gathered by two prototypes of assisted protection headphones and an external sensor during one batucada class.Two musicians use the prototypes during one regular batucada class. While one of the musicians (musician 1) does not take care of adjusting the protection headphones, the other one takes care and adjusts the headphones properly (musician 2). Moreover, an external sensor is placed between the musicians in order to have a reference. The system was used during the first 60 s of the class. In The generated alarms during this class can be seen in In this subsection, we will analyze the observed differences in the audiometries with the obtained data of the sound meters.From the obtained data, we can see that SPL higher than 120 is reached during the batucada class and these levels are considered as a dangerous SPL and can cause NHIL. This confirms our first hypothesis that the SPL reached in the batucadas is dangerous and some protection must be used to prevent NIHL. The SPL gathered during a batucada class are similar to the ones detected in rock bands , 5.According to the measures taken in the classroom, the peak frequencies are at 500 Hz. The frequencies that present sound pressure level considered as dangerous are between 500 and 6300 Hz. We detected that during different moments of the class, the results were different. For this reason, it is possible to affirm that different instruments can reach different peak frequencies and produce different alterations.The statistical analyses of the medical test show that there is a hearing loss in many members of the studied musicians. From the 43 studied individuals, 15 present NIHL. The NIHL is different in people with different exposure period, people that play different instruments, and people that use different protection measures. This data is confirmed by the performed ANOVAs per frequencies. At 250 Hz, the hearing loss is related to the protection measures and the played instrument. Similar results are found at 512, 1000, and 2000 Hz. No data show that the hearing loss is related to the exposure period. This data confirms our second hypothesis; the musicians are suffering from NIHL even when they are using protection headphones.We can relate the frequencies where we detected SPL (greater than 100 dB) with the frequencies where musician groups have hearing loss. The frequency analysis has shown greater SPL between 125 Hz and 2000 Hz. Statistical analysis in the audiometry has shown a significant difference between different groups between 250 Hz and 2000 Hz.The developed assisted headphones show how the internal SPL when the headphones are not well adjusted is higher. In the test, the musicians that do not adjust properly the headphones reach peak internal SPL higher than 100 dB.The NIHL is a common problem in different sectors of the human population. It is well studied in different professional sectors. However, it is less studied in the leisure environment. In this paper, we developed different tests with musicians of the “Borumbaia drum school” in Valencia. They generally play batucada. The results show that, during a regular class, the peak values are 122.60 dB at 300 Hz and 121.75 at 500 Hz. From the 400 analyzed frequencies, 88 of them have SPL higher than 100 dB. The maximum SPL is found from 125 Hz to 2250 Hz. The audiometries show that 35% of the studied musicians present NIHL. Some of the musicians that use protection headphones as protection measure present NIHL. We suspect that some musicians do not use the protections properly. For this reason, an assisted protection headphone and its operation protocols are designed to ensure the correct adjustment of protection headphones as a part of the e-health program to diminish the effects of high PSL on HINL. A simple test developed during a regular class shows how when a musician does not adjust properly the headphones the peaks of SPL reached in the internal part of the headphones are higher than 100 dB, while if the headphones are properly adjusted they are reduced to 83 dB.Our future work will be focused on studying the sound pressure levels reached by each instrument individually. The data will be gathered by the same methodology employed in this paper but using the A-weighting and C-weighting. Moreover, we will include a higher population with and without high levels of noise in their daily lives. This is aimed to define the observed differences in NIHL between musicians that play different instruments. Moreover, we pretend to evaluate the use of assisted protection headphones in other cases. We will also include indoor self-location systems in order to detect the placement of each headphone , 23.


Different types of bone conduction transducers with different physical and electro-acoustic properties are available for audiometric hearing threshold measurements. The reference equivalent threshold vibratory force levels (RETVFL) specified in ISO 389–3 are based on measurements conducted with the B71 and KH70 transducers but apply to all types of transducers available for bone conduction audiometry. The objective of this study was to compare bone conduction hearing thresholds measured by different transducers.In a prospective study the hearing thresholds were measured psychometrically between 125 Hz and 8000 Hz using the Radioear B71, B81 and Präcitronic KH70 transducers.Twenty-one normal hearing participants and fifteen hearing impaired participants.In both groups significant differences were found between the thresholds measured with the different transducers at the low frequencies 125 Hz and 250 Hz and the high frequencies 3000 Hz, 4000 Hz, 6000 Hz and 8000 Hz. In the normal hearing group, deviations from the reference threshold 0 dB HL towards lower thresholds were observed for the B71 and B81 at 125 Hz and at the high frequencies 3000 Hz, 4000 Hz, 6000 Hz and 8000 Hz.RETVFL-values should be reassessed and provided specifically for the different transducers. In bone conduction (BC) audiometry different types of BC transducers, which have different physical and electro-acoustic properties, can be used for hearing threshold measurements. The recently introduced BC transducer type B81  was found to generate higher output levels and less distortion than the widespread type B71 (Radioear) for frequencies up to 1500 Hz . PhysicaTherefore, reference zeros for the calibration of audiological equipment, such as the reference equivalent threshold vibratory force levels (RETVFL) for BC transducers, are specified in standards and International Electrotechnical Commission (IEC) specifications , 4. The How BC hearing thresholds are influenced by the type of transducer has been investigated on normal hearing participants in previous studies. Brinkmann and Richter  measuredThese results from the literature show that the reliability of BC hearing thresholds obtained with different transducers is a contentious issue. The purpose of this study was to systematically measure hearing thresholds with the conventional B71, the new B81, and the KH70 types of BC transducers and compare the results. The measurements were performed on a group of normal hearing (NH) and a group of hearing impaired (HI) participants.The participants in this study signed an informed consent form before the onset of the experimental procedures. The study protocol, laboratory protocols, informed consent form, and participant information sheets were submitted to the Ethical Review Board of the Martin Luther University Halle-Wittenberg and written approval was given for the study .In the NH group, 21 adults  participated aged between 20 and 32 years (mean 25.5 years). The inclusion criteria were for air conduction (AC) hearing thresholds to not exceed 10 dB HL at the frequencies 125 Hz, 250 Hz, 500 Hz, 750 Hz, 1000 Hz, 1500 Hz, 2000 Hz, 3000 Hz, 4000 Hz and 15 dB HL at the higher frequencies 6000 Hz and 8000 Hz.The HI group consisted of 15 participants  aged between 47 and 81 years (mean 66.6 years). All participants were patients of the University Hospital Halle , so that their pure tone audiograms already existed. The inclusion criteria were for BC thresholds to be 15 dB HL or worse in all audiometric BC test frequencies in the existing clinical audiogram 250 Hz, 500 Hz, 750 Hz, 1000 Hz, 1500 Hz, 2000 Hz, 3000 Hz, 4000 Hz, and 6000 Hz.Before commencement of the study, the frequency response and total harmonic distortion of the B71, B81, and KH70 were measured in order to verify the functionality of the transducers. The set-up and procedure used to obtain these data are described by Fredén Jansson et al. [All measurements were conducted in an anechoic chamber, where the ambient sound pressure levels did not exceed the permissible levels specified in ISO 8253–1 . In the The experimental set-up consisted of the transducers connected to the sound field output of an SD-50 audiometer  in order to increase the output levels compared to the conventional BC output. No additional amplifier was used. The BC hearing thresholds were then measured psychometrically between frequencies of 125 Hz and 8000 Hz for mastoid coupling using pulsed warble tones. The transducers were coupled to the mastoid by headbands made of spring steel. The KH70 comes along with a headband which is provided with a setting screw that allows the adjustment to the individual head size, whereas the B71 and B81 were used with the Radioear P333 headband . The headbands are designed to couple the BC transducers to the head with a static force of 5.4 N ± 0.5 N. The presentation level was increased in steps of 5 dB until the participant responded to hear the tone. Afterwards, the level was decreased by 15 dB so that the test tone was inaudible. Then the presentation level was increased again in steps of 1 dB. The procedure was repeated until the participant repeatedly answered to hear the tone at the same level for three times. This level was defined as the BC hearing threshold. The procedure was conducted using the B71, the B81, and the KH70 in a randomized order. The BC transducer left a mark on the participant’s skin so that the following transducer could be placed at the exact same position as the previous one. This ensured an identical stimulation site for each transducer. The better ear was chosen as the test side for the NH participants, so that no masking was applied. For the HI participants, based on the clinical audiograms the worse side was chosen as the test side determined by the BC 4-PTA . The clinical audiograms were obtained with the KH70 transducer as this is the standard used for BC threshold testing at the University Hospital Halle. If there were unmeasurable thresholds due to exceedance of the audiometer limit, the better side was chosen as the test side. The purpose of the procedure was to ensure obtaining BC thresholds at preferably high presentation levels. The purpose of the study was not to measure the participants’ actual hearing threshold but to compare the results among transducers. Therefore, no masking was applied in both experimental groups and the thresholds were measured independently from the actual perception side.t-tests were used to compare the BC thresholds with the AC thresholds. The level of significance was reduced to 0.002 for multiple comparisons using Bonferroni correction . A two-way analysis of variance (ANOVA) for repeated measures analyzed the results with respect to the study objective, i.e., the effect of the within-subject factors transducer  and frequency  on the BC thresholds. For significant effects post hoc t-tests were used to further analyze the results. The level of significance was reduced to 0.002 in the t-tests for multiple comparisons as explained above.The mean thresholds were statistically analyzed by parametric statistics as normal distribution of the data was confirmed by a Kolmogorov-Smirnov test. For the NH group, dependent t-tests due to missing data from some participants at some frequencies  in dB for each test frequency. The voltage level was converted into the corresponding force level in dB relative to 1μN by considering the pad correction function P(jω) = Vout/Fin of the artificial mastoid. The force level was then converted into hearing levels (dB HL) using the RETVFLs from ISO 389–3. Thus, the actual corresponding hearing level for the respective audiometer level was known for each transducer at each test frequency which represents the calibration of the transducers connected to the sound field output. Due to the linearity of the audiometer it was possible to convert any audiometer level into the actual hearing level. This conversion was done after the threshold recordings.Since the experimental set-up consisted of the transducers connected to the sound field output of an SD 50 audiometer, it was necessary to calibrate the threshold levels obtained in the experiments. The transducers were attached to an artificial mastoid B&K 4930  with a static force of 5.4 N. The output voltage level from the artificial mastoid RMS and Panel B shows the total harmonic distortion of the transducers while they were driven by an input voltage of 1VRMS.In All data underlying the findings of this study, i.e., the BC threshold values measured in this study on normal hearing and hearing impaired participants, can be found in t-tests showed that at the frequencies 125 Hz, 3000 Hz, 4000 Hz, 6000 Hz and 8000 Hz, the BC thresholds measured with the B71 and B81 were significantly lower than the AC thresholds. With the KH70 the BC thresholds were significantly lower than the AC thresholds at 125 Hz and 6000 Hz .transducer on the mean hearing thresholds  = 34.3, p<0.05) as well as a significant effect of the frequency  = 29.6, p<0.05). Also the interaction transducer × frequency was significant  = 20.5, p<0.05). Since the assumption of sphericity was violated for the factors transducer (χ2(2) = 9.43, p<0.05) and frequency (χ2(54) = 93.01, p<0.05) as well as the interaction transducer × frequency (χ2(209) = 420.37, p<0.05), the degrees of freedom were corrected using the Greenhouse-Geisser correction . Post hoc t-tests showed significant differences  between the B71 and B81 at 125 Hz, where the threshold obtained with the B71 was significantly lower than with the B81, and at 8000 Hz, where the threshold was significantly higher with the B71. However, comparing the B71 and B81 with the KH70, the hearing thresholds obtained by the KH70 were significantly higher than with the B71 and the B81 at 125 Hz, 250 Hz, and at frequencies above 3000 Hz. However, at 1500 Hz, the threshold measured with the KH70 transducer was significantly lower than with the B81. This is illustrated in Regarding the influence of the BC transducer, the two-way repeated measures ANOVA showed a significant effect of the t-tests showed significant differences between the B71 and B81 only at 125 Hz where the threshold was lower with the B71 and at 8000 Hz where the threshold was lower with the B81. Comparing the B71 to the KH70, significantly lower thresholds were found for the B71 at 125 Hz, 250 Hz, and 4000 Hz but significantly higher thresholds at 1500 Hz. Similarly, for the B81 in comparison to the KH70, significantly lower thresholds were found with the B81 at 250 Hz, 3000 Hz, 4000 Hz and 8000 Hz, whereas at 1500 Hz the thresholds were significantly higher than with the KH70.The results from the measurements on HI participants are similar to those from the NH participants. However, the BC hearing thresholds exceeded the measurement limit of the transducers at some frequencies for some participants. In order to be able to compare the results of the different transducers only those thresholds were considered for a participant at certain frequencies if the thresholds were measurable with all transducers. For instance, if a threshold was measurable at 3000 Hz with the B71 and B81 but unmeasurable with the KH70 due to the lower maximum output levels of the KH70, the data obtained with the B71 and B81 at 3000 Hz was omitted. The number of HI participants who had measurable thresholds with all transducers was 12 at 125 Hz and 250 Hz, 15 between 500 Hz and 2000 Hz, 13 at 3000 Hz, 12 at 4000 Hz, 11 at 6000 Hz, and 10 at 8000 Hz. In 8 of the 15 measurements the better side has to be chosen see  section The results show statistically significant differences ranging from 4 to 15 dB see  between An influence of the coupling force on the results is unlikely. The coupling force was controlled by using standard headbands (P333 and the KH70 headband) which are designed to couple the transducers to the head with a static force of 5.4 N ± 0.5 N. Deviations of the BC hearing thresholds resulting from BC transducer coupling forces outside the range of 5.4 N ± 0.5 N can only occur for very small or large heads. These deviations would be systematic though, as the results from the transducers were compared, i.e., the difference between the BC thresholds obtained with different transducers was the objective of the study. Each of the three transducers was tested in one participant, respectively (repeated measure).Instead, the different electro-acoustic properties of the transducers might be responsible for the deviations between the results obtained with the different transducers and the deviations from the AC hearing thresholds of NH participants towards lower thresholds. At low frequencies, harmonic distortion is a problem, while at high frequencies acoustic radiation could have influenced the results. Eichenauer et al.  measuredAcoustic radiation is dominant for frequencies of 3000 Hz and higher and varies among different transducers. Frank and Crandell  measuredBesides these deviations between the results with different transducers in the NH group, the BC hearing thresholds were found to be significantly lower than the AC hearing thresholds at 125 Hz, 3000 Hz, 4000 Hz, 6000 Hz and 8000 Hz. Generally, normal hearing does not require the AC thresholds to be the same as the BC thresholds. Studebaker  reportedIn this study significantly different BChearing thresholds were observed between the KH70 compared to the B71 and B81 at 125 Hz and higher frequencies such as 3000 Hz, 4000 Hz and 8000 Hz in NH and HI participants showing a poor reliability of thresholds obtained with different transducers. Results of audiometric BC threshold measurements should not be dependent on the type of transducer used in the test procedure. Therefore, the RETVFL-values used for the calibration of clinical audiometers should be reassessed and provided specifically for the different transducers. To obtain reliable new RETVFL values, the sample size has to be extended and the experiments should be conducted by a larger number of independent institutions.The static force by which the transducers are coupled to the mastoid should be measured for each individual in those studies and the electroacoustic properties of each transducer  should be reported.The differences between the results obtained with the different transducers found in this study are assumed to result from the different electro-acoustic properties of the transducers. In particular, the amount of harmonic distortion and the influence on BC hearing thresholds has to be further analyzed for the low frequencies 125 Hz and 250 Hz by measuring the exact output levels of the second harmonic at several fundamental frequency levels for the B71, B81 and KH70. Furthermore, the occurrence and behaviour of acoustic radiation of the different transducers B71, B81 and KH70 has to be further analyzed in future studies. For this, BC hearing thresholds need to be measured while the ear canal is occluded by an earplug to guard against airborne sound and again without occlusion. Normal hearing and hearing impaired participants who suffer from pure sensorineural hearing loss should be included in the study.S1 TableThe table shows the BC hearing thresholds of all normal hearing and hearing impaired participants in this study (raw data). The thresholds were obtained with the B71, B81 and KH70 BC transducers.(XLSX)Click here for additional data file.


Almost one third of the three million people in China suffering severe deafness are children, and 50% of these cases are believed to have genetic components to their etiology. Newborn hearing genetic screening can complement Universal Neonatal Hearing Screening for the diagnosis of congenital hearing loss as well as identifying children at risk for late-onset and progressive hearing impairment. The aim of this joint academic and Ministry of Health project was to prototype a cost effective newborn genetic screen in a community health setting on a city-wide level, and to ascertain the prevalence of variation at loci that have been associated with non-syndromic hearing loss. With the participation of 143 local hospitals in the city of Wuhan, China we screened 142,417 neonates born between May 2014 and Dec. 2015. The variants GJB2 c.235delC, SLC26A4 c.919-2A>G, and mitochondrial variants m.1555A>G and m.1494C>T were assayed using real time PCR. Newborns found to carry a variant were re-assayed by sequencing in duplicate. Within a subset of 707 newborns we assayed using real-time PCR and ARMS-PCR to compare cost, sensitivity and operating procedure. The most frequent hearing loss associated allele detected in this population was the 235delC variant in GJB2 gene. In total, 4289 (3.01%) newborns were found to carry at least one allele of either GJB2 c.235delC, SLC26A4 c.919-2A>G or two assayed MT-RNR1 variants. There was complete accordance between the real-time PCR and the ARMS PCR, though the real-time PCR had a much lower failure rate. Real-time PCR had a lower cost and operating time than ARMS PCR. Ongoing collaboration with the participating hospitals will determine the specificity and sensitivity of the association of the variants with hearing loss at birth and arising in early childhood, allowing an estimation of the benefits of newborn hearing genetic screening in a large-scale community setting. Hearing loss is one of the most common human disorders, and genetic causes contribute to more than half of congenital hearing loss cases, 2. AlthDeafness is characterized by its etiological heterogeneity. It is estimated that about 50% of cases of childhood hearing loss are associated with genetic factors. Loci in more than 70 genes have been found to be associated with NSHL, 2. AddiEarly-detection is of vital importance in addressing the needs of newborns with hearing loss. The costs of identifying newborns with hearing loss via universal screening are relatively low–28, and In the present study we performed one of the largest scale genetic screenings in a joint academic and Ministry of Health project, assaying 142,417 neonates born between May 2014 and December 2015 in Wuhan, China. 98.7% of newborns at the contributing 143 local hospitals participated in this screening during the time of the project. Deafness-related variants in three genes ) were assayed using real time PCR. The aim was to develop a low-cost, fast, accurate and high-throughput platform for large scale genetic screening applicable to a community health care setting and to compare to other methodologies. This also allows a comparison of the frequency of the chosen variants in Wuhan to other regions and to better assess the variants best suited for this assay.The population enrolled in our study consisted of 142,417 newborns in Wuhan, which is located in the central of China. From May 2014 to Dec. 2015, 142,417 newborns were recruited from 143 hospitals, including both maternity and general hospitals. The participation rate was 98.7%. The screening protocol was approved by the Health and Family Planning Commission of Wuhan Municipality and all the participating hospitals. Three to four heel blood spots  were collected within 72 hours after birth according to standard protocol with FTA cards. The protocol was as follows: the heel of the foot was cleaned with an alcohol wipe, punctured at the edge of the plantar surface using an automated lancet, the first drop of blood was discarded, and a single blood spot was collected per card, and the cards were air dried four hours. The sampling card also contained the participants’ information . All newborns’ information were gathered into a newborn deafness genetic database, and unused blood spots were stored as a biobank. Samples with inadequate number or size of blood spots, samples that were stored improperly, or samples with incomplete information were disqualified from analysis. Written informed consent was obtained from all the neonates’ parents or guardians who participated in the project. The study protocol was reviewed and approved by the internal review board of Huazhong University (#[S189]).A review of published meta-analyses was used to nominate candidate variants. Criteria for inclusion were repeated strong associations with risk of NSHL, potential actionability upon diagnosis, and prevalence in East Asian populations. Variants in GJB2, SLC26A4, MTRNR1 were among the most commonly related to non-syndromic hearing loss–38. The ™ 7 system on 384-well plates . The genotypes of each allele were determined via amplification curves and the Ct value. Newborns found to carry a variant were confirmed by sequencing in duplicate. Heteroplasmy and homoplasmy of the mitochondrial variants were identified by the presence or absence of unique amplification curves.Genetics variants with known associations with hearing loss were assessed. The variants GJB2 c.235delC, SLC26A4 c.919-2A>G, and mitochondrial variants in the MT-RNR1 12S gene m.1555A>G and m.1494C>T were assayed using three independent fluorescent PCR kits. All variants were detected using a real time fluorescent PCR method with a commercial kit . The detection kit is comprised of genomic DNA extraction reagents and the fluorescent PCR reagents, including positive and negative control, and amplification reaction mixtures. All reactions for each variant were performed in a single well. Amplification was performed with ViiAThe specific Taqman-MGB probe sets for  with wild and mutant type were designed using Primer 3.0 and validated by Sanger sequencing. Each loci required a pair of primers common to both wild type (P1) and the mutant sequences (P2), as well as two different MGB (Minor Groove Binder) probes for each assay. The probe for the normal sequence was labeled with the FAM fluorophore, and the probe for the mutant sequence was labeled with the VIC fluorophore. A pair of regular PCR primers  for each mutation is also designed at the same time.Each PCR reaction was carried out in a total volume of 5 μl. The appropriate concentration of a mixture of probes and primers  was added at a volume of 0.25μl, along with 2.5 μl 2X PCR Buffer , 1μl genomic DNA template and 1.25μl ddH2O  .PCR amplification was commenced with an initial denaturation step at 94 °C for 10 min, followed of the cycling conditions and annealing temperatures indicated in Additionally, all variants cases detected by these two PCR assays were validated via Sanger sequencing using sequencing primers designed with Primer 3.0 . Three pThe inter-city differences in frequency of overall and single site carriers were compared using the two-tailed Chi-square test. A P-value less than 0.05 was considered statistically significant.A total of 142,417 neonates were enrolled into the newborn deafness genetic screening study, and a high throughput genetic screening standard procedure was needed to assay their genotypes. Via discussion with the Health and Family Planning Commission and a collaborating otolaryngologist, a workflow was proposed and tested on a pilot project of 500 participants. As the initial results were favorable, the project was expanded to the total size reported in this study.The protocol was comprised of four stages, including blood specimen collection, genetic screening, result interpretation, and follow-up intervention . All parGenetic screening data of four deafness-associated loci in three genes is shown in 37 neonates carried multiple screened variants. GJB2 c.235delC was the variant most frequently found in combination with other variants : 33 neonA total of 4289 neonates were found to be carriers of at least one of the four variants via real time PCR. The targeted regions of these variants were amplified using appropriate specific primers and sequenced in duplicate via Sanger sequencing. Approximately 53 samples were found to not carry a mutation when sequenced, giving a concordance of 98.7% (1-53/4289).Studies in more than 20 Chinese cities have assayed common loci associated with NSHL. We compared the results of this study with those of several studies, collected in cities or provinces in China from 2007 to 2015 . The totWe used real-time PCR with Taqman technology and ARMS-PCR to assay the variant sites simultaneously in 707 neonates. The results for GJB2 c.235delC, m.1555A>G, m.1494C>T in these 707 newborns showed complete accordance. With SLC26A4 c.919-2A>G, 378 samples were consistent between methods, the remaining 314 newborns had no results at this site with the ARMS-PCR . Three rThe time required for real time PCR was significantly lower than for ARMS-PCR, primarily due to differences in the requirements for DNA preparation and visualization of the amplification product. Processing each batch of ARMS-PCR entailed in total six hours, vs two hours for real time PCR. In addition, the reagents costs were 25% higher for ARMS-PCR.Hearing loss is a relatively common disorder, featured by high heterogeneity and phenotypic diversity. Early identification of infants with NSHL can avoid social and language difficulties that can occur in infants with undiagnosed hearing loss, 60, 61.More than 70 genes have been identified to harbor variations linked to non-syndromic hearing loss. Previous studies have identified GJB2 as in important contributor to hereditary NSHL, and mutations in GJB2 can be detected in nearly 50% of patients with autosomal recessive hearing loss. The c.2The m.1555A>G and m.1494C>T are the main pathogenic mutations of 12S rRNA gene. These rare variants have been shown to have a direct relationship with aminoglycoside induced hearing loss. There wSeveral methods for the detection of genetic variants related to hearing loss have been employed in the clinic. ARMS-PCR offers consistent and accurate results with inexpensive equipment. However, the running time for every target is about three hours. Additionally, different loci require different reaction conditions with this methodology. MALDI-TOF mass spectrometry high-throughput genotyping  and chipThe next stage of this study is a comparison between the variants identified via PCR and the results of physical hearing screening. The neonates carrying the positive variants will be tested again by otoacoustic emission testing or automated auditory brainstem response to identify associations with late onset hearing loss. In addition, it is hoped that the identification of alleles which are contraindicative for the use of aminoglycoside antibiotics will prevent their use. Further collaboration with the participating hospitals will determine the specificity and sensitivity of the association of the studied variants with hearing loss at birth and arising in early childhood, allowing an estimation of costs and benefits of delivering newborn hearing genetic screening in a large-scale community setting.S1 Table(XLSX)Click here for additional data file.


Jagged1 or Notch2 in neural crest-derived cells (NCCs) of the pharyngeal arches display a malformed stapes. Heterozygous Jagged1 knockout mice, a model for Alagille Syndrome (AGS), also display stapes and incus defects. We find that Jagged1-Notch2 signaling functions early to pattern the stapes cartilage template, with stapes malformations correlating with hearing loss across all frequencies. We observe similar stapes defects and hearing loss in one patient with heterozygous JAGGED1 loss, and a diversity of conductive and sensorineural hearing loss in nearly half of AGS patients, many of which carry JAGGED1 mutations. Our findings reveal deep conservation of Jagged1-Notch2 signaling in patterning the pharyngeal arches from fish to mouse to man, despite the very different functions of their skeletal derivatives in jaw support and sound transduction.Whereas Jagged1-Notch2 signaling is known to pattern the sensorineural components of the inner ear, its role in middle ear development has been less clear. We previously reported a role for Jagged-Notch signaling in shaping skeletal elements derived from the first two pharyngeal arches of zebrafish. Here we show a conserved requirement for Jagged1-Notch2 signaling in patterning the stapes and incus middle ear bones derived from the equivalent pharyngeal arches of mammals. Mice lacking  In a fascinating evolutionary transition, these tiny middle ear bones are thought to have arisen by modification of the ancestral fish jaw-support skeleton, with the malleus, incus, and stapes being homologous to portions of the Meckel’s, palatoquadrate, and hyomandibular elements of fish, respectively2  pharyngeal arch, and the stapes bone is derived from the second (i.e. hyoid) archly2 Fig. . In a geJAGGED1 (JAG1) have been found in 94% of AGS patients7, with a small proportion of AGS patients harboring heterozygous loss-of-function mutations in NOTCH28, which encodes a receptor for JAG1. Clinical diagnosis of AGS is based on reduced numbers of intrahepatic bile ducts in the liver, accompanied by cholestasis, a characteristic facial appearance, and defects in the heart, eyes, and skeleton9. Although not part of the clinical diagnosis for AGS, there are some reports of hearing loss in patients with AGS and/or mutations in JAG1. Work in mouse and chick has uncovered roles for Jagged-Notch signaling in the patterning of the prosensory domain, which gives rise to the hair and support cells of the cochlea12. These findings would appear to suggest that hearing loss in AGS is primarily due to sensorineural defects. However, there are also a few isolated reports of conductive hearing loss in AGS, which indicates potential structural defects of the middle ear. In one large AGS kindred, mild conductive hearing loss was noted13. Another kindred, with missense mutations in JAG1 yet only the cardiac defects typically associated with AGS, displayed mixed hearing loss 14. In a separate post-mortem analysis of the temporal bone, two AGS individuals were described as having a “bulky” stapes, with one also displaying a “bulky” incus, yet the precise morphological changes of these middle ear bones were unclear15. These studies raise the possibility that defects in not only the neural components of the inner ear but also the structural components of the middle ear might contribute to hearing loss in AGS patients.In humans, heterozygous loss-of-function mutations in Jag1 or Notch2 results in early embryonic lethality in mice17. We had previously reported that conditional deletion of Jag1 in NCCs (using Wnt1-Cre) resulted in a persistent foramen in the frontal bone18, and an independent group reported midfacial hypoplasia in these Wnt1-Cre; Jag1f/f conditional knockout (Jag1-CKO) mice, reminiscent of the facial characteristics of AGS20. Micro-computed tomography (uCT) scans confirmed these previously reported phenotypes in Jag1-CKO mice, and revealed a similar persistent foramen and midfacial hypoplasia in Wnt1-Cre; Notch2f/f (Notch2-CKO) mice , Alizarin Red staining of bone in Jag1f/f controls shows that the stapes consists of two cruces with a prominent foramen. In 100% of Jag1-CKO (n = 8/8) and Notch2-CKO (n = 8/8) mice, the stapes was narrower than in controls and had a reduced or absent foramen  of Notch2-CKO mice and no Jag1-CKO mice having an ectopic process extending from the anterior medial edge of the incus body.Homozygous deletion of ice Fig. . We thermen Fig. . This siJAG1 result in AGS, we also examined the effects of removing just one copy of Jag1 throughout the whole mouse , and an ectopic process extending from the posterior medial edge of the incus body in 75% of animals (n = 15/20). As with Jag1-CKO mice, the malleus was unaffected in Jag1 heterozygotes. These findings indicate that development of the stapes and incus is especially sensitive to reduced dosage of Jag1-Notch2 signaling in mice.As heterozygous loss-of-function mutations in human Jag1f/f, and Wnt1-Cre; Jag1f/+ controls, the stapes cartilage had a prominent foramen. In contrast, Jag1+/− and Wnt1-Cre; Jag1f/f mice had a smaller stapes cartilage with a reduced or absent foramen, consistent with the later columellar phenotype of the ossified stapes bone. We next examined the stapedial artery, which normally passes through the stapes and serves as a bridge connecting the external and internal carotid arteries. To visualize this artery in conjunction with the stapes cartilage, we bred the conditional Jag1 mutants onto a Rosa26-Tomato reporter background and injected India ink into the artery after dissecting out the intact middle and inner ear from newborn mice. As in controls, we still observed a prominent stapedial artery in Jag1-CKO mice (n = 3/3), which curved around the misshapen stapes cartilage  (Table Twist1+/− but not Jag1+/− single mutants. The penetrance of this phenotype increased from 11% (n = 2/18) to 68% (n = 13/22) in compound heterozygotes  Table . The retes Table . These f32. To test whether the ossicular defects of Jag1-CKO mice result in hearing loss, we measured auditory brainstem response (ABR) at P18. In a click stimulus test, in which a large range of frequencies is presented simultaneously, we observed an approximately 30 decibel shift in the sound pressure level (dB SPL) in Jag1-CKO mice compared to Wnt1-Cre; Jag1f/+ or Jag1f/+ controls   of JAG1 that alters the splicing consensus sequence and leads to heterozygous JAG1 loss-of-function. He also had normal compliance of the tympanic membrane in both ears. These findings are consistent with partial defects in the ossicular chain resulting in high-frequency hearing loss38. We also observed an ectopic process extending from the left incus towards the posterior wall of the tympanic cavity in a 9-year-old subject with sensorineural hearing loss in the right ear, and irregular orientation of the petrous part of the temporal bone, which was sloping upwards in a lateral to medial manner, in an 8-year-old subject with mixed hearing loss in both ears  scans of the temporal bone to visualize middle ear structures (summarized in Table es) Fig. , nearly rs Table . This leJag1 or Notch2 are consistent with findings in zebrafish that Jag1b functions in NCCs to pattern the homologous hyomandibular cartilage3. Whereas hyomandibular defects in zebrafish lacking jag1b, or notch2 and notch3, correlate with ectopic expression of the cartilage condensation marker barx1 in the hyoid arch4, we observed no differences in Barx1 expression in the hyoid arches of Jag1-CKO mice at E10.5 and E11.5  in zebrafish jag1b mutants is similarly lost, despite this foramen being associated with the facial nerve instead of an artery3. Thus, rerouting of the stapedial artery appears to be a secondary consequence of the reduced stapes anlagen and not vice versa. Nonetheless, additional studies are clearly needed to elucidate the developmental basis of stapes defects in Jag1-deficient mice.Our findings indicate a conserved role for Jag1-Notch2 signaling in NCCs for the patterning of the stapes and incus bones of the mammalian middle ear. The fully penetrant stapes defects seen upon NCC-specific deletion of 1.5 Fig. . HoweverWnt1-Cre; RBP-Jf/f mice with global loss of Notch signaling in NCCs display similar defects in the frontal bone as we observe upon NCC-specific deletion of Jag1 or Notch241. Although middle ear bones were not examined in Wnt1-Cre; RBP-Jf/f mice, the similarity of stapes defects in Jag1-CKO and Notch2-CKO mice suggest that JAG1 and NOTCH2 are the likely major ligand and receptor for both calvarial and middle ear bone development. Of note, global but not NCC-specific heterozygous loss of Jag1 results in partially penetrant stapes defects, as well as incus defects, suggesting additional requirements for Jag1 in non-NCC tissues for ossicle patterning. One candidate tissue is the first endodermal pouch, which displays strong Jag1 expression in both mice42 and zebrafish3 and develops in close association with the stapes and incus anlagen. Jag2 and Notch3 may also partially compensate for Jag1 and Notch2 in other aspects of NCC skeletal differentiation. Jag2 is required for development of the NCC-derived palate in mice43, and notch3 reduction enhances craniofacial defects in notch2 mutant zebrafish4. However, deletion of Notch1 in neural-crest-derived cells does not cause midfacial hypoplasia characteristic of Alagille Syndrome20, and a previous study did not detect expression of either Notch1 ortholog in the developing zebrafish face3. Interestingly, the liver and heart defects of AGS have not been observed in the analogous Jag1 heterozygous mice44, yet we did observe similar stapes defects in both mice and humans lacking one copy of JAG1. These results indicate that organs have independent dosage sensitivities to JAG1 across species.JAG1 mutation, the presence of conductive or mixed hearing loss but apparently normal middle ear bones in other AGS patients suggests that middle ear bone defects alone cannot account for conductive hearing loss in this syndrome. NCC-specific Jag1 mutant mice have midfacial hypoplasia, and the development and function of the Eustachian tube is closely associated with the craniofacial skeleton46. Thus, other factors such as Eustachian tube dysfunction may contribute to hearing loss in subjects with apparently normal ossicles. Similarly in NCC-specific Jag1 mutant mice, we cannot rule out the possibility that defects in other NCC-derived ear structures besides the stapes contribute to hearing loss, especially as the ABR test we performed does not distinguish between conductive and sensorineural components. For example, NCC derivatives contribute to the early cochleovestibular ganglion and utricle, as well as a few rare cells in the semicircular canals and stria vascularis47. While the cochleovestibular ganglion and stria vascularis are involved in auditory functions, the utricle and semicircular canals are responsible for balance. Indeed, studies on three mouse mutants – Slalom33, Headturner48, and Ozzy34 – have described specific point mutations in Jag1 resulting in malformations of the semicircular canals, and we and others observe frequent losses and anomalies of the superior and posterior semicircular canals in AGS patients37. Further, the role of Jagged-Notch signaling in the development of the vestibular system also appears to be conserved in zebrafish, as jag1b mutants were independently isolated based on semicircular canal defects50. However, we found that NCC-specific loss of Jag1 in mice causes hearing loss without affecting canal morphology, consistent with defects in the stapes and/or other middle and inner ear structures being responsible for hearing defects in these mice. Nonetheless, superior semicircular canal dehiscence can lead to autophony and conductive hearing loss, and dehiscence of the canal can cause disruption of the normal endolymph flow, resulting in a lower bone conduction and higher air conduction threshold51. Although there are currently no reports linking Jag1 and AGS to canal dehiscence, we cannot rule out a possible canal dehiscence component in AGS-associated hearing loss. In addition, Jag1 has a well-known requirement in patterning the ectodermal sensory placode from which the hair and support cells of the inner ear derive12, consistent with the finding of sensorineural hearing loss in several AGS patients. These observations indicate a complex etiology of hearing loss in AGS, likely affecting multiple structures in both the middle and inner ear, particularly for those patients presenting with mixed hearing loss. However, for those patients with isolated middle ear bone defects, surgical correction might be an option to improve hearing52.While we found a correlation between stapes defects and hearing loss in one individual heterozygous for a NOGGIN53 and ANK54, have been linked to congenital conductive hearing loss in humans. A contribution of JAG1 mutations to congenital conductive hearing loss thus expands our knowledge of human middle ear development. However, as AGS is relatively rare, we were only able to examine the middle ears of a small number of patients. Thus, the extent to which stapes and/or incus defects occur in this syndrome and contribute to hearing loss remains to be determined. While the similarity in stapes defects between Jag1 mutant mice and an individual with heterozygous loss of JAG1 strongly supports the view that human stapes malformations are due to JAG1 deficiency, we cannot rule out that second site mutations act synergistically with JAG1 mutations to cause middle ear bone defects. For example, conditional deletion studies in mice have revealed requirements for Tbx1 in middle and inner ear structures55, and DiGeorge Syndrome, which is associated with heterozygous deletion of TBX1, encompasses an array of defects that overlap with Alagille Syndrome, including tetralogy of fallot and hearing loss. On the other hand, the patient with stapes defects did not display the multi-organ defects typically associated with AGS, despite having a child with the same JAG1 mutation and typical AGS features. Together with a previous study showing mixed hearing loss in a kindred with a JAG1 missense mutation but only a subset of AGS features14, and the presence of JAG1 mutations presenting with only one or two AGS features56, our results suggest that reduced JAG1 function can cause hearing loss largely independently of other AGS features. It will therefore be informative to examine the extent to which family members of AGS patients who exhibit hearing loss despite the lack of an AGS diagnosis, as well as unrelated patients with defects in the stapes and/or incus bones, carry JAG1 or NOTCH2 mutations.To date, mutations in only a few genes, such as Jag117, Twist157, Jag1-flox58, Notch2-flox59, Wnt1-Cre60, and Rosa26-Tomato61 mouse lines.Animal experiments were approved by the University of Southern California IACUC committee, and all methods were performed in accordance with the relevant guidelines and regulations. Genotyping was performed as previously published for 62.Imaging was performed using a MicroCT 50 , scanning at high resolution  and a field of view of 20.4 mm. Scans were conducted at an energy setting of 70 kVp, current intensity of 200 µA, and an integration time of 500 ms/projection. Two-dimensional slices taken at 10-micron increments were rendered into three-dimensional reconstructions using Exposure Render63 with minor modifications. Washes in tap water pre and post bone staining were omitted; Alizarin Red S at 0.1% was used at 1:50 dilution in potassium hydroxide (KOH); decolorizing treatment after bone staining was performed with 1% KOH overnight; and the dehydration process was completed through sequentially increasing proportions of 100% glycerol:1% KOH . Samples were imaged using a Leica S8 APO stereo microscope.The heads of three-week-old mice were skinned and cleared with 1% KOH for 1 to 2 days, stained with 2% Alizarin Red S in 1% KOH until mineralized bone is red, and stored in 100% glycerol. Middle ear ossicles were then dissected out for analysis. The heads of P0 newborn mice were skinned and double stained with Alcian Blue and Alizarin Red S for cartilage and bone as describedWnt1-Cre; Jag1f/+; Rosa26-Tomatoki/+ and Jag1f/f; Rosa26-Tomatoki/ki mice were bred to generate Wnt1-Cre; Jag1f/f; Rosa26-Tomato mutants. The heads of P0 newborn mice were skinned, fixed overnight in 4% paraformaldehyde, and stored in phosphate buffered saline (PBS). Whole ear structures were carefully dissected out, and the stapedial artery was injected with India ink using glass capillary needles. The malleus, incus, and other tissues were removed after injections and before imaging to better reveal the stapes. Image z-stacks were taken using a Zeiss Axiozoom and processed using extended depth of field by Zeiss LSM software.ABR was performed through inserted earphones, using closed-field acoustics. The sound pressure level (SPL) of the stimuli ranged between 20 and 105 decibels (dB). In determining ABR thresholds, 300 responses with artifacts less than 30 microvolts were averaged. Presentation of stimuli and averaging of responses were both controlled by BioSig software. When ABR threshold was above maximum output range, it was classified as 105 dB. Hearing level thresholds were measured at specific frequencies of 4, 8, 12, 16, 24, and 32 kilohertz (kHz).Studies on human patients were approved by the Institutional Review Board at the University of Southern California Keck School of Medicine, and all methods were performed in accordance with the relevant guidelines and regulations. Written informed consent to obtain samples for genetics research was obtained from each subject and/or subject’s parent or guardian. Hearing tests were performed at the Alagille Alliance meetings of 2011 and 2014. Otoscopy was performed to check for occluding wax, fluid, or infection, as well as wellness of the canal and middle ear system. The testing used was tympanometry and behavioral audiometry. For tympanometry, a small probe tip is inserted into the ear canal to create a seal, and then air pressure is directed into the external auditory ear canal. The mobility of the eardrum is recorded to confirm the health of the middle ear system. For behavioral audiometry, thresholds of hearing are tested from 250 to 8000 Hz. Test procedures are based on the age of the test subject. If a subject is able to test conventionally by raising their hand only when a test frequency is heard, the standard 10 dB up and 5 dB down testing procedure is performed. Frequencies are tested in conventional order − 1000, 2000, 4000, 8000, 250, 500 Hz. If the difference between octaves is greater or equal to 20 dB, then inner octave threshold is measured. In cases where a subject is too young, unreliable or distracted to complete this testing procedure, conditioned play audiometry is completed. In this form of testing, the subject is trained to perform an action in response to sound, such as dropping a block in a bucket. Frequencies are not presented in any standard order to keep the subject interested. If the patient becomes unreliable, then thresholds are not recorded. In standard air and bone conduction tests, a hearing level of -10 to 25 dBHL (decibels hearing level) is classified as normal, 25 to 40 dBHL as mild, 40 to 70 dBHL as moderate, 70 to 90 dBHL as severe, and 90 dBHL and above as profound. Conductive hearing loss is indicated by decreased air but normal bone conduction, sensorineural hearing loss by decreased air and bone conduction thresholds, and mixed hearing loss by decreased air and bone conduction thresholds with the bone conduction threshold being 10 dB higher than for air. Temporal bone CT scans on subjects 1 and 5 were acquired at the University of Southern California Keck School of Medicine using standard clinical procedures. Temporal bone CT scans for subjects 2, 3, and 4 were kindly provided by the patients’ physicians.All data generated or analyzed during this study are either included in this published article or available from the corresponding author on request.Supplementary Information


This study is aimed at finding out the relationship between long working hours, one of major job stress elements, and hearing impairment in unexposed workers to occupational and environmental noise.This study was performed on 1628 regular, full-time wage workers between the age of 25-64 who indicated in the survey of having no experience of exposure to noise, normal otoscopic findings, and not suffering from diabetes based on the data from the fifth Korea National Health and Nutrition Examination Survey (KNHANES 2010–2012). The average working hours per week was categorized into 40 h and lower group, more than 40 to 48 h group, more than 48 to 60 h group, and more than 60 h group. The groups were defined as suffering from low or high frequencies hearing impairment if the average hearing threshold for 0.5, 1, 2 kHz or 3, 4, 6 kHz in both ears exceeds 25 dB based on the pure tone audiometry. The association between average weekly working hours and hearing impairment was analyzed using logistic regression after gender stratification.The prevalences of low and high frequencies hearing impairment in male workers were 4.3 and 28.6 %, respectively, which were much higher than female’s prevalence of 2.7 and 11.1 %. For male workers, no significant association was found between average weekly working hours and low and high frequencies hearing impairment. For female workers, odds ratios (OR) of low and high frequencies hearing impairment were 4.22  1.09–16.27) and 4.49 (95 % CI 1.73–11.67), respectively, after controlling for several related factors, such as, age, Body Mass Index (BMI), socio-economic status, health-related behavioral, and occupational characteristics variables, in the final model in the group working more than 60 h compared to the group working 40 h and lower. In addition, a dose-response relationship was observed that ORs of low and high frequencies hearing impairment were increased according to increasing average weekly working hours.The association between long working hours and hearing impairment in both low and high frequencies was significant in Korean female workers with a dose-response relationship. Therefore, the law to change the culture of long working hours should be enacted in order to protect the workers’ health and improve the quality of life in Korean workers. The International Labor Organization (ILO) announced the first agreement limiting daily working hours to 8 h and weekly working hours to 48 h for the manufacturing sector in 1919. Around 100 years have passed since then, and the statutory working hours have decreased gradually with many countries currently limiting it to 40 h per week . In the The results of previous studies suggested that long working hours have had influence in various fields. In the short term, long working hours increase fatigue through growing job demands, interference with life outside work, and shortening sleep time and cause negative health behaviors, such as, smoking and alcohol abuse . These rMeanwhile, hearing impairment creates problems, such as, difficulty in formation of relationships, social isolation, and limited career choices and it has become an important social problem that causes mental illness like depression . IncreasIn addition, some studies presented the association between stress and hearing problems. In a study conducted on symphony orchestra musicians occupationally exposed to loud noise frequently, it has been observed that there is a significant correlation between stress-related symptoms and hearing problems, with most hearing problems starting off gradually after a stressful life event . MeanwhiHowever, most previous studies did not differentiate hearing impairment and tinnitus or hyperacusis, based on a self-reported survey regarding hearing impairment, and not excluding well-known hearing impairment related factors. Therefore, this study is aimed at revealing the association between one of major stress elements, long working hours, and hearing impairment by using the data from the fifth Korea National Health and Nutrition Examination Survey (KNHANES) and analyzing the working hours which have not been presented as the cause of hearing impairment until now, although being one of the most important stress factors in the occupational health field .The KNHANES is a study conducted on Koreans nationwide by the Korea Centers for Disease Control and Prevention with the first survey conducted in 1998 and the sixth survey currently in progress. The data from the fifth KNHANES carried out from 2010 to 2012 was used in this study. The survey was constructed with independency and homogeneity through the introduction of the Rolling Sampling Survey in each survey year. In the fifth KNHANES, 192 sampling areas were chosen each year representing Koreans and all members of the 3800 households were surveyed, therefore, 11,400 households from a total of 576 areas surveyed in 3 years.This study was performed on regular, full-time wage workers between the age of 25–64 among the total study participants of the fifth KNHANES. Among them, 1815 workers unexposed to noise who answered ‘no’ to ‘have experience using earphones at a noisy place’, ‘have experience being exposed to noise at workplace’, and ‘have experience being exposed to instantaneous noise’, and also have been diagnosed normal for both ears in otoscopic examination conducted by a otolaryngologist using 4 mm degree endoscope were chosen. The final study population was determined to 1628 study participants by excluding workers diagnosed with diabetes to rule out the impact of diabetes on hearing impairment , 20, solThe International Labor Organization (ILO) stated that working more than 48 h a week is considered a major job stress and that the occurrence of cerebro-cardiovascular diseases are highly associated with working hours exceeding 60 h per week . TherefoBased on these standards, this study made evaluations by using the value of the response to the question ‘how many hours do you work per week on average at work place including overtime and night overtime, excluding lunch hours?’ and classifying them into group working 40 h and lower, group working more than 40 to 48 h, group working more than 48 to 60 h, and group working more than 60 h.To evaluate the hearing level of the study participants, a pure tone audiometry at 0.5, 1, 2, 3, 4, 6 kHz was conducted on both ears through the Entomed SA 203 in a double wall audiology booth. World Health Organization (WHO) had defined normal hearing as the average result recording below 25 dB at 0.5, 1, 2, 4 kHz for the good-ear side , and it In this study, some related factors that can affect hearing impairment suggested by previous studies were included. Some studies showed relatively consistent results that cerebro-cardiovascular risk factors, such as obesity and smoking, have a negative effect on hearing , 27. The2 classified as underweight, BMI of 18.5–25 kg/m2 normal weight, and BMI of over 25 kg/m2 overweight. Socio-economic status included level of educational attainment, marital status, and household income. The level of educational attainment was classified into less than high school, graduated from high school, and graduated from junior college or higher educational level. The marital status was classified into single, married and living together, and others . The household income was classified into four classes of low, middle-low, middle-high, and high in accordance with the monthly average equivalent household income (monthly household income divided by the square root of number of household members).The related factors were constructed as follows to consider socio-economic status, health-related behavior characteristics, and occupational characteristics of the study participants. The age was classified into units of 10 years with Body Mass Index (BMI) of less than 18.5 kg/mHealth-related behavior characteristic variables include smoking, alcohol consumption, and exercise status. Smoking was classified into non-smoker for participants who have smoked less than 100 cigarettes in their lifetime, ex-smoker for those who smoked more than 100 cigarettes in their lifetime but currently not smoking, and current smoker for those currently smoking and have smoked more than 100 cigarettes in their lifetime. Alcohol consumption was classified into non-drinker for participants who did not drink at all in the past year, light drinker for participants drinking less than twice a week or drinking less than seven glasses per occasion for male , and high-risk drinker for those drinking more than twice a week or drinking more than seven glasses per occasion for male . The exercise status was classified into exercise group for participants having conducted intense physical activities more than 3 days a week and over 30 min per occasion resulting in experiencing body pain or breathing heavily than usual while those not applicable to such activities were classified as non-exercise group.Occupational characteristic variables include job type and shift work status. The job type was classified mainly into non-manual jobs for ‘manager’, ‘specialist and related business employee’, ‘office worker’, and ‘service industry employee’ and manual jobs for ‘workers skilled in agriculture and fishery’, ‘technician or related skill employee’, ‘machine operating or assembly worker’, and ‘simple labor employee’. The shift work status was classified into day work group and shift work group for participants applicable to work pattern other than day shift .The fifth KNHANES is designed with all Koreans living in Korea as the target population and it is a complex sampling design data extracted after conducting the initial area-stratification and then the secondary stratification of households within the area. In this study, analysis was carried out considering weight, stratified variables, and cluster variables so that the sample represents the population and prevents biased outcomes.p < 0.05.To examine the general characteristics of the study population, the frequency and the average of each independent variable were presented through stratification by gender while chi-square tests were conducted to compare the distribution and t-tests were conducted to figure out significant mean difference. Taking into consideration previous study outcomes showing significant health impacts of long working hours on female workers –35, the p = 0.174). The proportion of educational attainment level of college or higher was significantly higher in male (p = 0.003) with the proportion of manual job in male at 31.0 % and higher than that of female of 16.8 % (p < 0.001). The proportion of shift work in female was slightly higher but not statistically significant (p = 0.239). The average working hours per week in male was 49.2 h, showing significantly longer than female’s 44.5 h (p < 0.001) while the percentage of group working more than 60 h in male was significantly higher than that in female, with male accounting for 12.6 % and female 7.2 % (p < 0.001).The general characteristics of the study population are presented in Table Average working hours per week according to the characteristics of study population are presented in Table The prevalences of low and high frequencies hearing impairment for each independent variable was presented after gender-stratified analysis in Tables p = 0.003, high frequency: p < 0.001) while the prevalence of high frequency hearing impairment was particularly high for low educational attainment (p < 0.001), others  marital status (p < 0.001), manual job (p < 0.001), and shift work (p = 0.012). The prevalences of hearing impairment by average weekly working hours showed no significant difference in both low and high frequencies .The results of the analysis conducted on the prevalences of hearing impairment according to independent variables in male showed that the prevalences of low and high frequencies hearing impairment increased by age , low educational attainment , and manual job . The characteristics showing any association with hearing impairment in only high frequency were non-drinkers (p = 0.044), others  marital status (p < 0.001), and shift work (p = 0.041). The prevalences of hearing impairment by average weekly working hours in both low and high frequencies increased significantly as the average weekly working hours increased .The prevalences of hearing impairment in female in both low and high frequencies increased by age  1.65–19.34), while OR of high frequency hearing impairment for the group working more than 48 to 60 h was 2.62 (95 % CI 1.34–5.15) and that in the group working more than 60 h was 4.91 (95 % CI 2.06–11.68). Also, in the final model after controlling for all related factors, the dose-response relationship was maintained between average weekly working hours and hearing impairment and ORs of low and high frequencies hearing impairment in the group working more than 60 h was 4.22 (95 % CI 1.09–16.27) and 4.49 (95 % CI 1.73–11.67) with statistical significance.This study explored the risk of hearing impairment resulting from long working hours unexposed to occupational and environmental noise in Korean full-time wage workers based on the data from the fifth KNHANES. The risk of hearing impairment in female workers increased significantly in low and high frequencies while there was no significant result in male workers. Besides, a dose-response relationship was observed between average weekly working hours and the risk of low and high frequencies hearing impairment based on the group working 40 h and lower in female workers. The risk of hearing impairment significantly increased especially when the average weekly working hours exceeded 60 h in low frequency and exceeded 48 h in high frequency.In the previous studies, the prevalence of hearing impairment was low in female workers  because Although the prevalence of hearing impairment was low in female workers in this study, the association between long working hours and hearing impairment was significantly high in female workers. These results are consistent with the outcomes of previous studies showing that the negative impact on health of long working hours is greater for female than male workers . The gen2 drops an average of 40 % when the cochlear blood flow is reduced up to 35 % and a significant hearing impairment occurs [The mechanisms of long working hours raising the risk of hearing impairment can be explained by several aspects. First suggesting mechanism is the activation of the sympathetic nervous system caused by long working hours, changing and damaging the function of the cochlear blood flow . The coct occurs . Also, tt occurs . Juhn ett occurs . Also, Ht occurs . Throught occurs .Another mechanism of long working hours causing hearing impairment can be explained through oxidative stress. Oxidative stress is an element which can create pathophysiology of hearing impairment, damage the DNA, and have a negative impact on hearing impairment by damaging the cochlear hair cells through protein and lipid degradation and increased apoptosis . There iOn the other hand, some animal studies present the outcome that short-term stress can show a hearing protection effect based on the explanation that glucocorticoid is produced through hypothalamic-pituitary-adrenal axis (HPA axis) and the glucocorticoid receptor (GR) which becomes activated as a result of this will start working by using heat shock proteins and antioxidants as mediators . In a reIn this study, the association between long working hours and hearing impairment was observed significantly in low frequency as well as in high frequency. Considering that hearing impairment by the major causes, such as age increase and noise exposure, was mainly observed in high frequency, it can be suggested that another pathological mechanism of hearing impairment may exist which is different from the mechanism of hearing impairment resulting from age increase or noise exposure. In a previous study, authors classified presbycusis as sensory, neural, strial, and cochlear conductive types according to pathogenesis. They reported that strial type was characterized by flat loss on pure tone audiometry and associated with ischemic changes of stria vascularis supplying the cochlea . EspeciaAlthough this study presents that long working hours may cause hearing impairment, another Korean study analyzing the association between stress and hearing impairment reported that there was no significant difference in the level of stress and hearing . HoweverAs far as we know, this study is the first study to explore the association between long working hours and hearing impairment in noise unexposed workers. This study secures representativeness and credibility using the data from the fifth KNHANES, performing hearing test and interviewing by trained researchers. This study aimed to reveal the association between long working hours and hearing impairment after controlling several related factors, such as, age, BMI, socio-economic status, health-related behavioral, and occupational characteristics, excluding not only noise exposure in the working area but also the possibility of noise exposure in everyday life, such as, the use of earphones at noisy places. Nevertheless, this study had some limitations as follows. Firstly, there was the limitation of not being able to include the past work history of the study population. Secondly, information bias may exist because the study relied on the self-reported survey instead of not measuring the actual level of noise exposure. Furthermore, study participants could not recall the whole exposure history to noise in their lifetime. Therefore, there may be a possibility that the association between long working hours and hearing impairment in this study has been biased. Thirdly, this study did not include all of the factors that could lead to hearing impairment like the histories of congenital diseases, middle ear diseases and exposures to physical trauma, medication, and toxic substances because these information was not surveyed in the original dataset, KNHANES. Fourthly, this study could not confirm the causal relationship that long working hours may cause hearing impairment because this study has a cross-sectional design.In order to overcome the limitations of this study, a cohort study can be performed on selected workers unexposed to occupational and environmental noise through not only thorough history taking of work history, past medical history, exposures to noise, physical trauma, medication, and toxic substances, but also noise measurement of the working places and other additional tests to exclude middle ear diseases.The association between long working hours and hearing impairment in both low and high frequencies was significant in Korean female workers with a dose-response relationship. The findings of this study may add some evidence to the body of knowledge of the risk of long working hours, one of well-known risk factors of health. Therefore, the law to change the culture of long working hours should be enacted in order to protect the workers’ health and improve the quality of life in Korean workers.


Peptide drugs for antineoplastic therapies usually have low oral bioavailability and short in vivo half-lives, requiring less preferred delivery methods. Lanreotide depot is a sustained-release somatostatin analog (SSA) formulation produced via an innovative peptide self-assembly method. Lanreotide is approved in the USA and Europe to improve progression-free survival (PFS) in patients with unresectable gastroenteropancreatic neuroendocrine tumors (GEP-NETs) and also approved in Europe for symptom control in carcinoid syndrome associated with GEP-NETs. This review discusses how the distinct molecule and formulation of lanreotide depot provide advantages to patients and health care providers, as well as the most recent clinical evidence demonstrating the safety and efficacy of lanreotide depot in inhibiting tumor growth and controlling hormonal symptoms in GEP-NETs.The lanreotide depot formulation confers a remarkable pharmacokinetic profile with no excipients, comprised only of lanreotide acetate and water. Of note, lanreotide depot constitutes an example for peptide self-assembly based formulations, providing insights that could help future development of sustained-release formulations of other antineoplastic peptides. Most patients with GEP-NETs will present with inoperable or incurable disease; thus, medical management for symptoms and tumor control plays a crucial role. Recent long-term clinical studies have demonstrated that lanreotide depot is well tolerated, prolongs PFS in GEP-NET patients, and significantly reduces symptoms related to carcinoid syndrome.The unique depot formulation and delivery method of lanreotide confer advantages in the treatment of metastatic GEP-NETs, contributing to improvements in NET-related symptoms and PFS without reducing quality of life in this patient population. Peptides are attractive therapeutic molecules due to their significant activity and low toxicity profile. It is not surprising that the number of peptide-based drugs being evaluated in clinical trials has been steadily increasing over the years. Currently, many peptide drug candidates are in clinical or preclinical development phases, and many are being evaluated in the oncology setting . HoweverFortunately, an intrinsic biophysical property of some peptides may help overcome the problems associated with peptide drug delivery. Under specific conditions of pH, concentration, and salts, many peptides and proteins will self-assemble into filamentous structures such as hollow nanotubes or densely packed fibers that have all the properties of an ideal sustained-release formulation , 4. FirsLanreotide is an analog of human somatostatin, and lanreotide depot  is the first marketed sustained-release formulation produced via peptide self-assembly. Lanreotide depot has been used by patients with acromegaly for more than 13 years  and has NETs are a group of malignancies that arise from the secretory cells of the neuroendocrine system. NETs can develop in virtually any organ but are predominantly found in the gastrointestinal system, particularly the pancreas and gastrointestinal tract . GEP-NETWhile indolent in nature, advanced well-differentiated NETs remain incurable with overall poor prognosis, and only a few treatment options can effectively inhibit their growth –13. GoalIn most patients with advanced or metastatic GEP-NETs, curative surgery may not be an option, and systemic management is an important part of clinical care. Somatostatin analogs (SSAs) play a prominent role in systemic management of GEP-NETs. GEP-NETs frequently overexpress receptors for the inhibitory hormone somatostatin, and SSAs have been shown to be effective in controlling symptoms of hormone hypersecretion in a number of functioning GEP-NETs , 13. FurThis review discusses how the formulation of lanreotide depot provides potential advantages to both patients and health care providers, as well as the most recent clinical evidence demonstrating the safety and efficacy of lanreotide depot in the control of tumor growth and hormonal symptoms in GEP-NETs.The first long-acting formulation of lanreotide  comprised a polymeric microsphere system and was intended for dosing every 7, 10, or 14 days. Polymeric microsphere-based formulation systems have been shown to be effective and have also been used in other long-acting SSAs. However, microsphere-based formulations have inherent disadvantages, including the need for reconstitution before injection, a complex manufacturing process requiring the use of organic solvents that are often toxic, potential burst release of the drug, the generation of acidic metabolites during the polymer degradation process, and possible degradation of the peptide , 16–18. w/w), the semi-solid gel is primarily composed of an organized hexagonal lattice of nanotubes ,bes Fig.  27]. Th. Thw/w),Tmax, 7 h). The subcutaneous depot provided a controlled, sustained release of the drug over extended periods of time with a low peak serum concentration  and long terminal half-life (30.1 days). The mean serum concentration 4 weeks after injection was 1.69 ng/mL   GEP-NETs.The primary end point was PFS, defined as the time to disease progression or death. Secondary end points included overall survival, quality of life, and safety .P < 0.001). The risk of disease progression or death within 96 weeks of first dose was significantly reduced with lanreotide   participated in the open-label extension of CLARINET. A median PFS of 32.8 months was reached for the lanreotide group during the extension. No new safety concerns were identified .A study in 85 patients with low-grade (grade 1), midgut NETs demonstrated that octreotide LAR  prolongs time to tumor progression in the studied patient population (PROMID study) . This stAlthough CLARINET included only patients with nonfunctioning tumors, earlier open-label studies of lanreotide depot have suggested activity in all patients with GEP-NETs regardless of tumor-functioning status , 42. TheIn summary, CLARINET showed that lanreotide depot prolonged PFS in patients with advanced, grade 1 to 2, somatostatin receptor-positive GEP-NETs regardless of hepatic tumor volume.n = 115). Patients were randomized to receive subcutaneous lanreotide depot 120 mg every 4 weeks or placebo. Because assessment of symptom control in carcinoid syndrome has always been a difficult study outcome to evaluate, a novel primary end point was used: mean percentage of days requiring rescue medication (MPDR). Patients were allowed to use a rescue medication  to control breakthrough symptoms throughout the study, and MPDR was significantly lower in lanreotide depot vs placebo arms, at 34 vs 49 % , multinational, cross-sectional, observation study to evaluate patient satisfaction with symptom control during lanreotide depot treatment in patients with GEP-NETs who experienced carcinoid syndrome-related diarrhea [SYMNET was a large (diarrhea . At routInterest in peptide-based antineoplastic drugs has been increasing; however, administration of these drugs has historically posed significant challenges in terms of sustaining a therapeutic dose over a long period of time. Peptide self-assembly-based formulations provide an innovative solution to these challenges.Lanreotide depot is a chemically distinct SSA and the first marketed drug with a self-assembly-based sustained-release activity. Extensive research on the lanreotide peptide self-assembly process provides insights that could help future development of self-assembly-based formulations for therapeutic peptides or the bottom-up design of new peptide families that self-assemble into complex architectures with desired properties.SSAs have been used for the treatment of symptoms associated with carcinoid syndrome, and now, based on evidence from a phase 3 study, lanreotide depot is the first, and currently the only, SSA that is approved in the USA for the treatment of metastatic GEP-NETs to improve PFS. Moreover, lanreotide depot’s formulation and method of delivery provide the potential to improve quality of life and tumor management in patients with GEP-NETs.