Hearing loss is a chronic condition affecting 12 million individuals in the UK. People with hearing loss regularly experience difficulties interacting in everyday conversations. These difficulties in communication can result in a person with hearing loss withdrawing from social situations and becoming isolated. While hearing loss research has largely deployed quantitative methods to investigate various aspects of the condition, qualitative research is becoming more widespread. Grounded theory is a specific qualitative methodology that has been used to establish novel theories on the experiences of living with hearing loss.The aim of this systematic review is to establish how grounded theory has been applied to investigate the psychosocial aspects of hearing loss. Methods are reported according to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis Protocols 2015 checklist. Studies included in this review will have applied grounded theory as an overarching methodology or have grounded theory embedded among other methodologies. Studies included will have adult participants (≥18 years) who are either people with an acquired hearing loss, their family and friends (communication partners), or healthcare practitioners including audiologists, general practitioners, ear, nose and throat specialists and hearing therapists. The quality of application of grounded theory in each study will be assessed using the Guideline for Reporting and Evaluating Grounded Theory Research Studies.http://www.crd.york.ac.uk/PROSPERO). Findings will be disseminated via peer-reviewed publications and at relevant academic conferences. Findings may also be published in relevant professional and third sector newsletters and magazines as appropriate. Data will inform future research and guideline development.As only secondary data will be used in this systematic review, ethical approval is not required. No other ethical issues are foreseen. This review is registered with the International Prospective Register of Systematic Reviews (CRD42019134197. This systematic review is the first to provide a comprehensive critique of the use of grounded theory to investigate hearing loss.Methodological quality of studies is assessed using the Guideline for Reporting and Evaluating Grounded Theory Research Studies.Because experiences and articulations of hearing loss are influenced by age, only adult (≥18 years) participants  will be considered.The search will not include grey literature.Hearing loss is a chronic condition that involves a decrease in an individual’s ability to hear sounds. It can occur at mild, moderate, severe or profound levels, and typically worsens over time.2 3The annual global societal cost of unaddressed hearing loss is $750 billion4PHL experience functional limitations such as communication difficulties in day-to-day conversations, where speech is often misheard and becomes challenging to follow.14–17The impact of hearing loss is not only psychological, as the interpersonal relationships of PHL are also affected. Not being able to fully communicate and establish successful interactions can also lead to feelings of frustration.22’ in 2012. Qualitative methods allow researchers to understand the experiences, opinions and perspectives associated with hearing loss, which experimental measures are not designed to uncover.23–25Investigations into the experience of hearing loss are crucial for understanding the implications it has for the person, and their care. To date, most studies in hearing loss research have used quantitative methods. Due to the evident psychosocial impacts of hearing loss, there has been a significant increase in the adoption of qualitative methodologies in hearing loss research,There are five main qualitative methodologies; phenomenology, ethnography and narratives which mainly focus on investigating descriptive characteristics of a phenomenon, and case studies and grounded theory which facilitate both descriptive and explanatory understandings of a phenomenon.26 27Grounded theory was first established by Glaser and Strauss in 1967.et al initially formed the methodology to investigate fatal chronic illnesses,Assessing the methodological applications of grounded theory is essential for determining the trustworthiness and credibility of the theories developed, as suggested by researchers in various fields such as psychology,There has been an increase in the application of grounded theory methodology in the field of hearing loss.No methodological systematic review has yet been conducted to assess the quality of studies that use grounded theory to investigate hearing loss. This is essential to inform future applications of grounded theory for improved and trustworthy hearing loss research. Therefore, this review primarily aims to (1) critically appraise the execution of grounded theory methodology in hearing loss research in terms of methodological quality and (2) produce recommendations for future grounded theory methodological applications pertaining to hearing loss research. A secondary aim is to (3) describe how grounded theory methodology has been applied within the field of hearing loss.The methods and analysis of this systematic review will be reported in accordance to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) 2015 checklist.56Initial searches were conducted in June 2019 but will be updated 1 month prior to submission of the manuscript for review.et alStudies included in this review will have used grounded theory methodology with an appropriate peer-reviewed reference for that adopted approach. For a study to be included, it can apply grounded theory as an overarching methodology, or be embedded into it among other qualitative methodologies such as case studies, ethnography, narratives and phenomenology. During full-text screening, if a study explicitly states that grounded theory was used during data collection and/or analysis, then they will be eligible for inclusion. Studies that do not explicitly state use of grounded theory methodology will be excluded. Purely quantitative studies, studies that have not applied grounded theory methodology, articles reporting expert opinions, case reports, practice guidelines, case series, conference abstracts and book chapters will be excluded. Only studies published in English will be included. Any studies published prior to 1967  guideline for assessing the application of grounded theory methodology in research.Any research setting will be included.Participants will include people with mild to profound, acquired or congenital, hearing loss. Studies involving individuals with no residual hearing will be excluded. This is because people with no residual hearing often identify differently (and have very different experiences) to people with some residual hearing.Only studies including adults (≥18 years) will be included. The levels of hearing loss for eligibility are mild, moderate, severe or profound, according to the British Society of Audiology.A systematic search strategy will be employed to identify peer-reviewed journal articles that meet the eligibility criteria. The following databases will be used: PsycINFO (1800s–current), Applied Social Sciences Index and Abstracts (1987–current), Global Health . Web of Science (1899–current), PubMed (1996–current), British Nursing index (1994–current), Cumulative Index to Nursing and Allied Health Literature (1961–current), MEDLINE , Scopus (1983–current) and EBSCO (1944–current). Google Scholar will be used for forward citation tracking.Additionally, snowballing of the reference lists of articles shortlisted for inclusion will be undertaken, and related articles from shortlisted authors will be screened in an attempt to identify any relevant articles which may not have been returned by the database searches. The electronic database searches will be updated just before the final analyses and any eligible studies retrieved will be included. At the end of the study selection process, the search strategy produced for each database will be reported, in addition to a PRISMA flow diagram.To check the feasibility of this systematic review, a scoping search was conducted across multiple databases including Medline, PubMed and PsycINFO. This indicated that there were sufficient relevant papers, approximately 30, to perform a useful synthesis. The searches also informed the key terms for use in the formal search strategy.The search strategy was formed from free text and controlled vocabularies , consultations with an information specialist at the University of Nottingham, and the testing of search results.Hearing Loss, Noise-Induced/ or Hearing Loss/ or Hearing Loss, Sensorineural/ or Hearing Loss, Unilateral/ or Hearing Disorders/ or Hearing Loss, Sudden/ or Hearing Loss, Bilateral/ or Hearing Tests/ or Hearing Loss, Conductive/hearing loss*.mp. or exp Hearing Loss/1 or 2Amplifiers, Electronic/ or amplifier*.mp.Hearing Aids/ or listening device*.mp.Cochlear Implants/ or cochlear implant*.mp. or Cochlear Implantation/exp Hearing Aids/ or hearing aid*.mp.4 or 5 or 6 or 7hard of hearing*.mp.communication partner*.mp.audiologist*.mp. or Audiologists/ or Audiology/exp Persons With Hearing Impairments/9 or 10 or 11 or 12grounded theory*.mp.exp Grounded Theor*/14 or 153 or 8 or 1316 and 17YHKA will be responsible for management of the data. A digital record will be kept for all identified articles, and the process of data screening and extraction will be tracked in Covidence. A unique ID code will be assigned to the included articles for access to the full text and data collection sheet.This systematic review will follow the Centre for Reviews and Dissemination guidance for undertaking reviews in healthcareAfter piloting the data extraction form by an independent author, YHKA will extract data for all studies and 100% of the studies will be extracted by a second researcher(s) in line with published guidance.Extracted data will include:The study’s title, authors, date of publication, number of citations and country.Date(s)/time period of data collection.Aims, objectives and/or research questions.Source(s) of funding.Statement of conflict(s) of interest.Participant characteristics: for example, age, sample size, gender, country and occupation status.Key participant characteristics: type and severity of hearing loss, hearing aid usage and whether they have a cochlear implantation.Type of participants included .Methods of recruitment of participants.Data collection methods.Particular type of experience of hearing loss being investigated.School of grounded theory followed if mentioned.Ethical standards/approval.Attempts to establish qualitative rigour or trustworthiness.Study/methodology limitations.Advantages and disadvantages of using grounded theory if explicated by authors.Study design: methodology and methods.Key findings.Conclusions.Recommendations.Quality appraisal of studies will be conducted using the GUREGT,A thematic synthesis approach as established by Thomas and Harden66This process will consist of coding the data extracted from the included articles and applying initial codes which are close to the analysed data. This will involve establishing which aspects of hearing loss were investigated, how they were investigated and how grounded theory was used as a methodology in its applications of central principles such as theoretical sampling, constant comparative analysis and theoretical saturation. for investigating the specific hearing loss area. The results of the critical appraisal will then illustrate where grounded theory has been used rigorously and less so, to investigate hearing loss. From descriptive themes, analytical themes will follow, and these will inform a set of guiding statements for researchers wishing to use grounded theory to investigate hearing loss in future studies. Overall, thematic synthesis was chosen as it enables the greater generalisability of results.The data synthesis will be 100% completed by YHKA and 100% by a second author, ensuring inter-rater reliability as required for qualitative rigour.Within the research team, two are the qualitative experts (NW and DC), with the remaining four members having experience of conducting qualitative research investigating aspects of hearing loss . DC and YHKA have experience in the application of grounded theory methodologies. To avoid bias during the conduction of this systematic review, each author will reflect on their theoretical predispositions, biases, decisions made during the research process and any problems faced. The risk of bias during analysis will be avoided by assessing studies using the GUREGT guidelines, the GRADE-CERQual tool to assess confidence in the data synthesis, and by considering the funding and conflict(s) of interest statements outlined by each study during data extraction to evaluate whether selective reporting of outcomes was present.http://www.crd.york.ac.uk/PROSPERO) holds the registration record of this systematic review.As only secondary data will be used in this systematic review, no ethical approval is required. No other ethical issues are foreseen. The International Prospective Register of Systematic Reviews (The results from the systematic review will be disseminated via peer-reviewed publications and relevant academic conferences. Findings may also be published in relevant professional and third sector newsletters and magazines as appropriate. Data will be used to inform future research and guideline development.The primary outcome of this review will be a comprehensive understanding of how grounded theory methodology has been used to investigate hearing loss. This will be presented in a detailed table displaying the main themes and subthemes reached after the critical appraisal process using GUREGT and the qualitative thematic synthesis of data. Another primary outcome will be a set of recommendations highlighting the main downfalls of studies that have investigated hearing loss using grounded theory, and guidance for future researchers on how to apply the methodology most effectively. These will be presented in a finalised list based on the main themes and subthemes established through critical appraisal and thematic synthesis. A secondary outcome will be a table summarising the topics of hearing loss that were investigated using grounded theory, as established from data extraction.This systematic review will be the first to establish how grounded theory has been used to investigate hearing loss. A critical appraisal of all hearing loss studies that have investigated hearing loss within adult populations using grounded theory will be performed. This novel systematic review will aid in implementing more precise applications of grounded theory in future research. This will also enable more refined understandings of the experiences of hearing loss to be established and facilitate better care for those who have hearing loss.


This study described the decrement in accelerations, decelerations and dynamic stress load (DSL) between quarters in elite hurling. GPS (10-Hz) were used to record data from 42 players over 22 games (2018–2020 season). The number of accelerations and decelerations and DSL between quarters were assessed. Accelerations and decelerations were greater in Q1 than Q2 , and Q4 , and in Q3 compared to Q4 . The DSL was 56 ± 21 AU in Q1, 56 ± 20 AU in Q2, 52 ± 20 AU in Q3 and 56 ± 24 AU in Q4. There was a decrease in DSL in Q3 compared to Q1 (ES = −0.20) and Q2 (ES = −0.20). Each position experienced a temporal decrease in at least one quarter (ES = 0.43–1.46) in all metrics except full-backs’, half-backs’ and full forwards’ accelerations, midfielders’ decelerations and midfielders’ and half forwards’ DSL. Current data show temporal decrements in running performance in Q2 and Q4 and DSL in Q3. Players should be conditioned to minimize the drop-off in running performances following the third quarter. All players in the current study were members of the county’s squad that season (2018–2020). Players were chosen from the elite squad as this is the highest possible level that players can compete at. All participants had completed at least a 6-week preseason conditioning program (gym and pitch sessions). Those players on the squad who were injured were not included in the study. Data were only included if a full match  was completed. The elite hurlers were divided into five playing positions: full backs, half backs, midfielders, half forwards and full forwards. All games (n = 22) took place between 14.00 and 21.00 hours. The specific running performance variables were collected with GPS. After ethical approval, the purpose, procedures and potential risks involved in the study were explained. The subjects were informed that they could withdraw from the study at any time. A medical declaration and written informed consent were completed in line with the procedures set by the Limerick Institute of Technology Research Ethics Committee. All procedures were approved by the local Ethics Committee, and the study was conducted according to the Declaration of Helsinki (1975) for studies involving human subjects.Forty-two  and Seca Weighing Scales , respectively. A 10 Hz GPS unit and 100 Hz triaxial accelerometer  was used to collect the running performances ,16. The −2) and decelerations (>3 m·s−2) [GPS units were used to collect the number of accelerations (>3 m·s3 m·s−2)  and the 3 m·s−2) . The dyn3 m·s−2) . Once thStatistical analysis was performed using a statistical software package . Descriptive analysis and assumptions of normality were verified prior to parametric statistical analysis. The analysis was performed using a two-way (quarter × position) mixed-model design. A Bonferroni post-hoc correction was used to detect differences between positions  and match quarters when an interaction occurred . The depDuring the full game, players performed 49 ± 11 accelerations and 58 ± 14 decelerations, with a dynamic stress load of 220 ± 78 (AU). The number of accelerations and decelerations and dynamic stress load per quarter are presented in The number of accelerations was 13 ± 4 in Q1, 12 ± 4 in Q2, 13 ± 4 in Q3 and 11 ± 4 in Q4. A higher number of accelerations were performed in Q1 compared to Q2 (ES = 0.28), and Q4 (ES = 0.57) and in Q3 compared to Q4 (ES = 0.50). The number of decelerations was 16 ± 5 in Q1, 14 ± 4 in Q2, 15 ± 4 in Q3 and 13 ± 5 in Q4. A higher number of decelerations were performed in Q1 compared to Q2 (ES = 0.44), and Q4 (ES = 0.60) and in Q3 compared to Q4 (ES = 0.44). The dynamic stress load was 56 ± 21 AU in Q1, 56 ± 20 AU in Q2, 52 ± 20 AU in Q3 and 56 ± 24 AU in Q4. There was a decrease in dynamic stress load in Q3 compared to Q1 (ES = −0.20) and Q2 (ES = −0.20).p = 0.001). A main effect was found for quarter (p < 0.001), and position (p = 0.005). Midfielders  and half forwards  performed a greater number of accelerations in Q1 compared to Q2 and Q4. There were no other differences (p > 0.05) between quarters in the number of accelerations. Full backs (ES = 0.95), and half backs (ES = 0.85) performed a greater number of decelerations in Q1 compared to Q2.The differences between quarters for each playing position are presented in p < 0.001). A main effect was found for quarter (p < 0.001), and position (p < 0.001). Full backs (ES = 0.57), and full forwards (ES = 0.57) performed a greater number of decelerations in Q1 compared to Q3. Full backs (ES = 0.66), half backs (ES = 0.78), half forwards (ES = 0.88) and full forwards (ES = 0.85) performed a lower number of decelerations in Q4 compared to Q1. Half backs and half forwards performed a greater number of decelerations in Q3 compared to Q2  and Q4 . There were no other differences (p > 0.05) in the number of decelerations between quarters in any position.A quarter × position interaction was found for the number of decelerations (p = 0.004). A main effect was found for quarter (p = 0.001), and position (p = 0.003). The dynamic stress load was lower in Q3 compared to Q1 (ES = −0.42) and Q2 (ES = −0.33) full backs and Q1 (ES = −0.26) and Q4 (ES = 0.37) in half backs. Full forward’s dynamic stress load was higher in Q2 compare to Q1 (ES = 0.59).A quarter × position interaction was found for dynamic stress load  . The halIt has been suggested that dynamic stress load, a measure of the mechanical stress on the body recorded from the number of impacts and forces applied through ground contacts, can be used to measure external load in team sport athletes . For the−2) and decelerations (>3 m·s−2) and dynamic stress load between positions across quarters in hurling, making direct comparisions difficult. However, previous research has found similar trends between quarters in hurling, where the high-speed running- and high metabolic load-distance were greater in Q1 compared to Q2, Q3 and Q4 across most positions [There were between-quarter differences observed for the number of accelerations and decelerations and dynamic stress load within positions. Midfielders and half forwards performed more accelerations in Q1 compared to Q2 and Q4, whereas full backs and half backs performed more decelerations in Q1 compared to Q2, Q3 and Q4. In addition, the number of deceleratons in Q1 within full forwards was also more demanding compared to Q3 and Q4. Less differences between quarters was observed for dynamic stress load within positions, yet Q1 was the highest in full backs, half backs and full forwards. No other research is available which described the number of accelerations (>3 m·sositions . HurlersThe present study comes with some acknowledged limitations. Firstly, the duration of when the ball was in-play was not recorded in this study. When the ball is out-of-play, this limits the opportunities that players have to accelerate and decelerate. Differences in the ball-in-play time between quarters may have affected the results. Future studies should quantify the number of accelerations and decelerations per min of ball-in-play and ball-out-of-play between quarters. Secondly, no attempt was made to account for the technical and tactical differences between quarters. Knowledge of when and where the accelerations and decelerations occur in relations to the ball would be interesting so that hurling-specific activities could be developed. Finally, the dynamic stress load metric measures the force and impact when the feet contact the ground. As players tire, their foot contacts may be longer on the ground, causing an increase in dynamic stress load. Therefore, although this metric is valuable in providing an overview of the stress placed on the player during a game, it can vary between players depending on their fitness level. It must be noted that comparing the dynamic stress load between positions can be challenging. Due to the potential variability between players’ fitness levels, the between-position analysis of the dynamic stress load metric was not included in the present study.Some important practical applications appear considering the current findings. The results show that players are required to accelerate and decelerate throughout all four quarters of the game. Therefore, the conditioning of hurlers needs to include activities that challenge the players to increase and decrease their speed frequently. Coaches should consider the exercise selection that take place towards the third and fourth quarters of training. Players may not be required to perform maximal accelerations and decelerations during certain activities especially towards the end of training. This may leave them underprepared for the maximal changes in speed that occur during a game. Including activities where the players have to compete against each other may help to motivate them to change speed quickly. Conditioning for midfielders and half forwards should ensure that they can repeat the number of accelerations in each quarter. Coaches should consider the distances of activities that they set-up so that the players can perform maximum accelerations especially in hurling drills. The distance of the activity may be too short, or the ball might be approaching the player to quickly, this may limit the opportunity for him to maximally accelerate and therefore decelerate.The current study was the first to provide an insight into the temporal decrements in the number of accelerations and decelerations and dynamic stress load between quarters in elite hurling. The findings showed that the number of accelerations and decelerations decreased in Q2 and Q4 compared to Q1 and Q3. The dynamic stress load experienced a decrement in Q3. Full backs, half backs and full forwards performed a similar number of accelerations in each quarter, whereas only midfielders performed a similar number of decelerations in each quarter. The results of this study provide coaches with new information about the fluctuations in running intensity that occurs during the game. This knowledge may be used to help in the design of specific conditioning programs for elite hurlers.


The aim of the present study is to report the outcomes of round window reinforcement surgery performed with the application of a Vibrant Soundbridge middle ear implant  in a patient with superior semicircular canal dehiscence (SSCD) who presented with recurrent vertigo, Tullio phenomenon, Hennebert's sign, bone conduction hypersensitivity, and bilateral moderate to severe mixed hearing loss. Vestibular evoked myogenic potentials (VEMPs) and high-resolution computed tomography (HRCT) confirmed bilateral superior semicircular canal dehiscence while this was not seen in magnetic resonance imaging. The surgical procedure was performed in the right ear as it had worse vestibular and auditory symptoms, a poorer hearing threshold, and greatly altered HRCT and VEMPs findings. With local-assisted anesthesia, round window reinforcement surgery (plugging) with perichondrium was performed with simultaneous positioning of a VSB on the round window niche. At the one and 3 months follow-up after surgery, VSB-aided hearing threshold in the right ear improved to mild, and loud sounds did not elicit either dizziness or pain in the patient. Superior semicircular canal dehiscence (SSCD) was first described by Minor et al. , and is Both high resolution computed tomography (HRCT) with reconstruction on the plane of the superior canal and vestibular evoked myogenic potentials (VEMPs) are mandatory to confirm the diagnosis of SSCD.Surgical approaches are not mandatory in SSCD. The decision to treat the pathological canal side is based on various assessments including severity of symptoms, surgical candidacy of patients, and their desire for an improvement in quality of life. Conventional surgery provides direct treatment of the canal fistula through either a transmastoid or middle cranial fossa approach: the most common and well-described surgical treatments include capping, resurfacing, and plugging of the superior semicircular canal , 4.Despite the demonstrated efficacy, these interventions are invasive and imply a potential risk of persistent hearing deterioration and vestibular loss. Both the middle fossa and transmastoid approaches require general anesthesia which might increase the overall surgical and anesthesiologic risk. Recently, it has been proposed that a two-window inner ear system can be restored by directly plugging the round window (RW). This procedure has been demonstrated to be safe, fast, and effective compared to classical SSCD surgical treatments , 6.The Vibrant Soundbridge middle ear implant  is an implantable hearing aid that transduces sounds into electromechanical vibrations to the ossicular chain or directly to the RW. It is indicated for the treatment of conductive or mixed moderate to severe hearing loss .We describe the first case in literature of RW plugging and VSB positioning performed simultaneously under local anesthesia in a patient with bilateral SSCD and severe mixed hearing loss.Figures 2a–c).A 78-year-old woman was referred to our clinic with bilateral hearing loss with sound distortion, tinnitus and auditory hypersensitivity, recurrent vertigo/dizziness induced by loud noises (Tullio phenomenon), and a diagnosis of bilateral SSCD at the temporal bone HRCT performed in the emergency room  were discussed with the patient. Comorbid cardiopulmonary conditions represented major contraindications for general anesthesia, so the latter procedure was the only possible option to pursue.Since the patient did not show bilaterally any benefit from an air conduction hearing aid but rather had deteriorating auditory and vestibular symptoms on the right, we decided to perform the surgical procedure in the right ear as it had worse vestibular and auditory symptoms, a poorer hearing threshold, and greatly altered HRCT and VEMPs findings.With local-assisted anesthesia, we performed a transcanal approach with elevation of the tympanomeatal flap and preservation of the chorda tympani nerve with a minimally invasive retroauricular incision. Ossicular mobility and continuity were assessed, we excluded the stapedial fixation, and no cerebrospinal fluid (CSF) leak was observed during the surgical procedure. After identification and reshaping of the RW niche, a vibroplasty was performed paying particular attention to correctly plugging the round window and coupling it with the floating mass transducer (FMT) of the VSB . We opteThe plugging of the round window was achieved using cartilage and perichondrium (tragus). This autologous tissue also helped to seal off the FMT in the round window niche. Furthermore, VSB hearing outcomes were monitored with electrocochleography using a cotton-wick recording electrode placed on the hypotympanum  Figure .The wire of the VSB was housed in a canal tunnel drilled up to the tympanic attic. Minimal drilling of the cortical temporal bone posterosuperior to the external auditory meatus was necessary to house the implant receiver and extra wire . The earThis study received an exemption from the ethics committee of the University Hospital of Siena  on 10/21/2019 for publication.Surgery was uncomplicated, the patient did not complain of any post-operative vestibular symptoms. Sutures and external meatus packing were removed on the 10th postoperative day. At the 1-month follow-up, the patient underwent VSB activation and hearing and vestibular examination. She reported a significant improvement in auditory hypersensitivity and reduced sound distortion although tinnitus remained unchanged. No disabling vestibular symptoms were reported. Neither dizziness nor nystagmus could be observed in response to loud sounds or increased external ear pressure on the right side. The postoperative pure tone audiogram revealed a mild increase at 500, 1,000, and 2,000 Hz and a mild decrease at 250 and 4,000 Hz for bone conduction thresholds . An imprTo the best of our knowledge, this is the first report in the literature of local-assisted anesthesia with simultaneous RW reinforcement surgery and VSB positioning in a patient suffering from SSCD. The outcome of this new procedure confirmed the results in terms of safety and improvement in auditory and vestibular symptoms related to SSCD using the minimally invasive procedure for RW plugging reported by Silverstein et al.  and SuccThe major improvements in terms of vestibular symptoms over auditory symptoms have been described in the literature for RW plugging vs. superior canal plugging. Several studies , 6, 9–11The main limitations of the present study are that it is a report on a single case, and there was only a short follow-up period.In conclusion, simultaneous RW plugging and VSB positioning may be an effective, safe, and rapid surgical approach for SSCD associated with severe mixed hearing loss.Immediately after the surgical procedure the patient reported an improvement of vestibular symptoms due to loud sounds. She also reported a significant reduction in auditory hypersensitivity and sound distortion. Although tinnitus remained unchanged, the significant improvement in hearing threshold (VSB-aided) led to a higher quality of life.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Written, informed consent, including all data and images, was obtained from the patient for the publication of this case report. Surgical procedure was performed in accordance with the Code of Ethics of the World Medical Association (Helsinki Declaration). This study received approval from the ethics committee of the Universitary Hospital of Siena  for the publication on 10/21/2019.MM performed the surgery. All authors contributed to the preparation and revision of manuscript and analyzed the data.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Children suffering from conductive or mixed hearing loss may benefit from a bone-anchored hearing aid system (BAHA Attract implantable prosthesis). After audiological rehabilitation, different aspects of development are improving. The objective of this case report is to propose a comprehensive framework for monitoring cortical auditory function after implantation of a bone-anchored hearing aid system by using electrophysiological and neuropsychological measurements.We present the case of a seven-year-old boy with a congenital hearing loss due to a plurimalformative syndrome, including outer and middle ear malformation. After the diagnosis of hearing loss and the audiological rehabilitation with a BAHA Attract implantable prosthesis, the cortical auditory evoked potentials were recorded. We performed a neuropsychological evaluation using the Wechsler Intelligence Scale for Children – Fourth Edition, which was applied according to a standard procedure.The P1 latency was delayed according to the age . The neuropsychological evaluation revealed that the child's working memory and verbal reasoning abilities were in the borderline range comparing with his nonverbal reasoning abilities and processing abilities, which were in the average and below-average range, respectively.Cortical auditory evoked potentials, along with neuropsychological evaluation, could be an essential tool for monitoring cortical auditory function in children with hearing loss after a bone-anchored hearing aid implantation. Hearing loss, especially in its most severe forms, is associated with a developmental risk in the area of speech and language skills  and alsoWhen cortical regions do not receive the appropriate sensory stimulus , they are more likely to be recruited by other sensory senses , resulting in a cross-modal reorganization . Sharma In children with hearing loss and associated disabilities, an aspect that should be considered is the benefit obtained from the audiological intervention , 11.Patients suffering from conductive or mixed hearing loss may benefit from implantable hearing devices. The bone-anchored hearing aid system (BAHA System) is an implanted device that uses bone conduction in order to stimulate the cochlea, bypassing the outer and middle ear. Sound signals are converted into electrical impulses and transmitted to the brain. In individuals with congenital ear malformations , the BAHA System represents a good rehabilitation alternative , 13.After the audiological intervention, different aspects of development are improving. The neuropsychological evaluation needs to establish a comprehensive framework for monitoring different dynamics of development beyond typical language skills.We report a case in which we assess the cortical auditory function by combining electrophysiological and neuropsychological measurements. The audiological intervention was performed with the BAHA Attract implantable prosthesis.We present the case of a 7-year-old boy who was diagnosed with plurimalformative syndrome at birth, presenting clinical features that could not be included in a specific syndrome. He was born at term after an uneventful pregnancy (mother followed isotretinoin therapy in the first two weeks of pregnancy), spontaneous delivery, birth weight = 3380 grams, APGAR score 10. A craniofacial dysmorphism was discovered: agenesis of the right auricle, severe dysplasia of the left auricle, bilateral external auditory canal atresia, microretrognathia, broad nose implantation – hypertelorism, mongoloid palpebral fissures, severe hypoplasia of the right and moderate hypoplasia of the left upper jaw, palpebral fissures asymmetry (left < right), lagophthalmia , nasolabial asymmetry (more significant on the right), oculomotricity deficiency , clinodactyly of the fifth finger, bilateral flat foot.Temporal bone computed tomography scan revealed dysplastic ossicles, unidentifiable stapes, and oval window, hypopneumatization of mastoid cells, hypoplasia of the tympanic cavity, without changes in the internal ear. The contour of the facial nerve in the mastoid segment was not clearly defined.Following the investigations, the diagnosis of moderate conductive hearing loss in the left ear and severe mixed hearing loss in the right ear was established. On auditory brainstem response, the V wave was present at 70 dB HL in the right ear and 80 dB HL in the left ear. From 1 month and a half, hearing rehabilitation was made with bilateral BAHA Softband. Audiological reevaluation at age 4 revealed the following audiological profile: Auditory brainstem responses (ABR) on bone conduction – V-wave present at 10 and 20 dB HL in the left ear (LE) and at 20 and 30 dB HL in the right ear (RE), auditory steady-state response (ASSR) – between 60 and 70 dB HL in the LE and 80 and 90 dB HL in the RE.SmartEP Intelligent Hearing System (IHS) equipment  was used for ABR and ASSR. Air conduction was tested with headphones, for atretic ears, and with a B-71 transducer for bone conduction, held by one finger on the mastoid.At 6 years and 8 months, a BAHA Attract was implanted on the left ear and three months later on the right ear. At 7 years and one month, we recorded the cortical auditory evoked potentials on the aided condition to quantify the maturation of the central auditory pathways.The P1 CAEP records were obtained after 90 minutes of testing in a soundproofed room. The patient was placed on a chair, and he watched cartoons without sound during the procedure. The patient was using a bilateral BAHA Attract implantable prosthesis. The electrodes were placed according to the norms of the International Electrode System 10-20: the active Cz electrode was connected to the positive input of the amplifier, the reference electrode was positioned on the mastoid of the ear, and the ground electrode was placed at Fpz. To minimize the ocular artifacts, a supraorbital electrode was used, paired with an infraorbital reference electrode placed ipsilaterally. The level of impedance of the electrodes was maintained between 1-3 kOhms. A calibrated loudspeaker placed at 1 m distance in 0° angle emitted a speech stimulus, the “ba” syllable, at 70 dB nHL intensity. The stimulus rate was 1.10/s, duration 114875 µsec, for 512 sweeps, artifact rejection criterion at ± 100 µV. The stimulus CAEPSs recorded in response was analyzed by a SmartEP USB software from the Intelligent Hearing System.The check-up audiogram performed at 7 years and 4 months of age showed hearing thresholds between 20 and 30 dB HL for the RE.Neuropsychological functioning was performed at the age of 7 years and one month, by a clinical neuropsychologist using the Wechsler Intelligence Scale for Children – Fourth Edition (WISC-IV), adapted in Romania in 2012 .The assessment was based on oral/acoustic modality, which refers to the use of spoken language with auditory amplification (Baha Attract System), without visual instructions; the administration was possible without modifications, and all the tests were applied according to a standard procedure. The patient was administered ten subtests of the Wechsler Intelligence Scale for Children – Fourth Edition (WISC IV). WISC-IV measures general intelligence and specific indexes, including verbal comprehension, perceptual reasoning, working memory, and processing speed.Verbal Comprehension Index (VCI) measures verbal concept formation. The subtests included are Similarities, Vocabulary and Comprehension.Perceptual Reasoning Index (PRI) measures non-verbal and fluid reasoning. The subtests included are Block Design, Picture Concepts, and Matrix Reasoning.Working Memory Index (WMI) measures working memory. The subtests included are Digit Span and Letter-Number Sequencing.Processing Speed Index (PSI) measures the speed of information processing. The subtests included are Coding and Symbol Search.The P1 wave was observed with normal morphology and a delayed latency (175 ms) for this age . No artiThe objective of this study was to assess the cortical auditory function in a child with a BAHA implantation using electrophysiological and neuropsychological measurements.The P1 biomarker represents an objective method of assessing the development of the central auditory pathway in children with hearing loss . We obseCortical auditory evoked potentials originate from the cortical-thalamic projections, the latter being involved in working memory .Delayed latency of the P1 component can be correlated with working memory. The cortex development and cognitive abilities are interdependent from one another .In our patient's case, there were no CAEPs measurements before the BAHA implantation; therefore, we could not compare the latencies before and after the results.The patient's general cognitive ability is below the average range of intellectual functioning, as measured by the Full-Scale Intelligence Quotient (FSIQ). His overall thinking and reasoning abilities exceed only approximately 12% of children having his age  .His ability to think with words is lower than his ability to reason without the use of words. His verbal reasoning abilities, as measured by the Verbal Comprehension Index (VCI), are within the borderline range and below those of approximately 93% of his peers  . The VerThe patient's nonverbal reasoning abilities as measured by the Perceptual Reasoning Index (PRI) are below the average range and below those of approximately 67% of his peers  . The PRIThe subject's working memory abilities, as measured by the Working Memory Index (WMI), are within the borderline range, below those of 93% of his peers  . The patth percentile, 95% CI = 94-106) (The patient's speed of processing abilities as measured by the Processing Speed Index (PSI) is within the average range and above those of approximately 50% of his peers  . ProcessWolff and Thatcher et al. have shown using electrophysiological studies that in deaf children, there is a delay in maturation of the left frontotemporal and bilateral frontal regions . Lack ofDaza et al. have shown that vocabulary knowledge and non-verbal cognitive processes such as selective attention, visual-spatial memory, abstract reasoning, and sequential processing are predictive factors for reading comprehension in deaf children .The purpose of this case presentation is to come in support of the need and importance of a comprehensive assessment of developmental evolution in children with hearing loss after a BAHA implantation. The neuropsychological evaluation needs to establish a comprehensive framework for monitoring the child's performances in each developmental area. With this standardized and normed tests for the Romanian population, researchers will be able to investigate outcomes and provide accurate information to offer guidance towards the best intervention in clinical practice. However, more extensive studies are necessary to confirm the findings presented here.Cortical evoked potentials, along with neuropsychological evaluation, could be an essential tool for cortical auditory functionality and a useful clinical instrument for quantifying the outcomes of auditory rehabilitation in children with hearing loss after a BAHA implantation.The authors confirm that there are no conflicts of interest.


Tolypocladium inflatum in Switzerland  and therefore inhibits the activation of genes required for proliferation such as IL-2, IL-4, as well as CD40L , 6. InhiCyclosporine also has an effect on the innate immune cells including dendritic cells, macrophages and neutrophils. Dendritic cells are essential in linking the innate and adaptive immune response, and it is believed that cyclosporine can modulate cytokine production in these cells and lead to altered induction of T cell responses .Cyclosporine is an inhibitor of the P-glycoprotein multidrug efflux pump, also known as Multidrug Resistance Protein 1 (MDR1). It is a competitive inhibitor of MDR1. As cyclosporine is lipophilic, not only is it a substrate for MDR1 , but it While cyclosporine is now a commonly used immunosuppressant, its use is not without side effects. Nephrotoxicity, hypertension, neurotoxicity, hepatotoxicity, hirsutism, gingival hyperplasia, lymphoproliferative neoplasms, opportunistic infections, hyperuricemia, hyperkalemia, and hypomagnesemia are some of the adverse effects described –12.The drug is erratically absorbed from the gastrointestinal tract which leads to variability in blood concentrations and unpredictable pharmacokinetics , 13. CycIt has been suggested that cyclosporine, in combination with methylprednisolone, can cause microangiopathy  and ischCyclosporine is a calcineurin inhibitor, and calcineurin has been detected in the cochlear nerve and inner hair cells of rats . We knowHearing loss can result from ischemic events in the cochlea , and perAnother possible contributing factor is the fact that cyclosporine is a lipophilic molecule , and lipAs a result of these observations, it is therefore reasonable to investigate whether cyclosporine can in fact cause hearing loss or vestibular symptoms. Hence, microangiopathy, neurotoxicity and/or hypomagnesemia may play a role in cyclosporine induced hearing loss or vestibulopathy.n = 19/80) of the patients had hearing loss, however, 47% had sensorineural hearing loss (SNHL) and 53% had conductive hearing loss (CHL). Between the children with and without hearing loss, there was no significant difference in the median cyclosporine treatment duration. Of note, 31% (n = 25/80) of the patients had also received cyclophosphamide, and the authors report that cyclophosphamide intake was significantly greater in patients with CHL vs. SNHL. They suggest this may be due to recurrent ear infections caused by immunosuppression. It is important to remember that the most common cause of pediatric CHL is otitis media with effusion, a highly prevalent condition in children which is generally reversible . They also observed that second-hour serum cyclosporine levels were significantly higher in patients with hearing loss and/or decreased speech understanding when compared to the group without hearing impairment. No vertigo was reported. They suggest that the hearing impairment following transplantation may be a dose-dependent cyclosporine toxicity .Fortes et al. evaluated adult patients undergoing liver transplantation with a hearing test, before and after transplantation. Patients received either cyclosporine or tacrolimus (FK-506) as part of their immunosuppressive regimen. For the cyclosporine group, the time elapsed between transplantation and the post-transplant hearing test was 635.83 days (range 65–939). No clinically significant changes were seen on pure tone audiometry. They do, however, report statistically significant changes with the use of tacrolimus .Rifai et al. sent out a questionnaire to liver transplantation recipients, with a mean follow-up period of 8 ± 5 years since the transplant. Excluding patients with a prior history of hearing loss, they observed that 15% reported hearing loss, and 11%, tinnitus. Of interest, the mean time period from transplantation to onset of hearing impairment was 4 ± 4 years. Self-reported hearing loss was considered severe in 62%, and sudden hearing loss was the cause of hearing impairment in 26% of these patients. Hearing loss was associated with the use of tacrolimus immunosuppression, but not with cyclosporine .Groothoff et al. performed a cross sectional investigation of patients who underwent renal transplantation. While their cohort is quite large, with 249 subjects, the cross-sectional analysis was completed for patients who were alive, with a functioning graft, and for which health and social status could be obtained. Of 106 patients that answered the questionnaire, 8% reported hearing problems, and 69.2% have used cyclosporine as part of their treatment at some point in time. There was no association between hearing problems and the use of cyclosporine .n = 10/356; microemulsion: 5.7%, n = 42/737). Mild and moderate hearing and vestibular disorders were more frequent in the microemulsion group (Finally, Cole et al. compared long-term safety and tolerability of cyclosporine and cyclosporine microemulsion for adult renal transplant patients. As previously mentioned, cyclosporine pharmacokinetics can be tricky, and the microemulsion formulation was created in order to obtain a more consistent drug exposure, showing a significant increase in the area under the concentration-time curve and maximum plasma concentration. By 18 months, they observed that overall, 4.7% had hearing loss. This was more common in the microemulsion group (cyclosporine: 2.8%, on group .To the best of my knowledge, four case reports have been published with regards to hearing impairment due to cyclosporine treatment. Arinsoy et al. reported the case of a 22-year-old male patient that underwent a renal transplant. Cyclosporine treatment began before the surgery and was maintained, he also received methylprednisolone and azathioprine. On post-op day seven, the patient presented with a unilateral SSNHL, with a flat curve on the audiogram, and 12% speech discrimination. No dizziness was described. It was believed to be a vascular event, and was treated with vasodilator and antiaggregant therapy. Seven days later, he had regained his hearing. Initially, his blood levels of cyclosporine were at 200 ng/ml, the dosage was decreased, and by the time he had regained his hearing, blood levels were at 50.8 ng/ml . Porges The literature regarding the ototoxic potential of cyclosporine is lacking. The published studies include patients with nephrotic syndrome , 27, renThe opposite is true as well, cyclosporine can alter tissue distribution of P-glycoprotein substrates. As an example, Saito et al. showed that mice receiving doxorubicin did not develop hearing loss as it is extruded by the P-glycoprotein multidrug efflux pump from the inner ear. Yet, when given in combination with cyclosporine, cochlear damage and dysfunction of the auditory pathway was seen, resulting from a significant accumulation of doxorubicin in the inner ear . On the Cyclosporine presents intra and inter-individual variability in blood concentrations and unpredictable pharmacokinetics , 13. It Literature is currently lacking with regard to risk factors for developing hearing loss when receiving a treatment with cyclosporine. For instance, the synergistic effect of impaired renal function. Most of the patients included in the reviewed studies had some degree of renal insufficiency, or even underwent renal transplantation. Other known ototoxic drugs are known to affect the kidney as well, causing nephrotoxicity, such as cisplatin and aminoglycosides , 45. We Because of the lack of evidence, I cannot elaborate further on a potential ototoxicity monitoring program, however, clinicians dealing with patients receiving cyclosporine as part of their treatment regimen should be alert to the possibility of ototoxicity and screen their patients for hearing loss accordingly. And also, try to avoid the use of a combination of ototoxic medications in these patients.While cyclosporine is not a commonly used drug in all medical specialties, it is an important drug in transplantation, and in 2019, close to 40,000 patients underwent organ transplantation in the US , and mosThe author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Here, we aimed to explore the genome-wide effects of PPARγ on EVCTs and VCTs via treatment with the PPARγ-agonist rosiglitazone. EVCTs and VCTs were purified from human chorionic villi, cultured in vitro, and treated with rosiglitazone. The transcriptomes of both types of cells were then quantified using microarray profiling. Differentially expressed genes (DEGs) were filtered and submitted for gene ontology (GO) annotation and pathway analysis with ClueGO. The online tool STRING was used to predict PPARγ and DEG protein interactions, while iRegulon was used to predict the binding sites for PPARγ and DEG promoters. GO and pathway terms were compared between EVCTs and VCTs with clusterProfiler. Visualizations were prepared in Cytoscape. From our microarray data, 139 DEGs were detected in rosiglitazone-treated EVCTs (RT-EVCTs) and 197 DEGs in rosiglitazone-treated VCTs (RT-VCTs). Downstream annotation analysis revealed the similarities and differences between RT-EVCTs and RT-VCTs with respect to the biological processes, molecular functions, cellular components, and KEGG pathways affected by the treatment, as well as predicted binding sites for both protein-protein interactions and transcription factor-target gene interactions. These results provide a broad perspective of PPARγ-activated processes in trophoblasts; further analysis of the transcriptomic signatures of RT-EVCTs and RT-VCTs should open new avenues for future research and contribute to the discovery of possible drug-targeted genes or pathways in the human placenta.Trophoblasts, as the cells that make up the main part of the placenta, undergo cell differentiation processes such as invasion, migration, and fusion. Abnormalities in these processes can lead to a series of gestational diseases whose underlying mechanisms are still unclear. One protein that has proven to be essential in placentation is the peroxisome proliferator-activated receptor  The human placenta serves as a critical bridge between mother and fetus and thus plays a crucial role in maternal and fetal physiology. The placenta is composed mainly of trophoblast cells, which derive from the outer layer of the blastocyst. Certain trophoblasts can be further distinguished as villous cytotrophoblasts (VCTs), whose development progresses along with that of the placenta. In the process of embryo implantation and placenta formation, VCTs that invade the maternal uterus are known as extravillous cytotrophoblasts (EVCTs); these anchor the chorionic villi. Other VCTs differentiate and fuse to form the syncytiotrophoblast layer, which has critical functions in gas and nutrient exchange between the fetus and the mother. Defects in EVCT invasion and VCT differentiation and fusion contribute to a series of gestational diseases, such as fetus-related miscarriage , pretermγ regulates many downstream target genes involved in lipid metabolism, cell differentiation, and tumorigenesis. PPARγ functions by forming a heterodimer with the nuclear receptor retinoid X receptor α (RXRα) and then binding to the PPAR response element (PPRE) of target genes [γ leads to defects in trophoblast differentiation and abnormal vasculogenesis in mice [γ−/− embryonic lethality can be rescued via PPARγ transfection in the trophoblast [γ inhibited the invasion of first-trimester EVCTs, which implicates PPARγ in the regulation of invasion of the decidua [γ can also induce the differentiation of VCTs isolated from term placenta [γ on the regulation of EVCTs and VCTs suggests that it plays a critical role in trophoblast invasion and differentiation but also that these effects differ dramatically among different subtypes of trophoblast. PPARγ thus appears to play a crucial but poorly understood role in placental development.As a member of the ligand-dependent nuclear receptor superfamily, PPARet genes . It has  in mice , 6, and phoblast . To be m decidua , 9. Furtplacenta . Taken tγ in biological processes, the PPARγ-agonist rosiglitazone has been widely applied to various tissues. In human placenta, rosiglitazone has been used for the study of placental metabolism [In vitro treatment with rosiglitazone has been shown to reverse inflammation of the placenta that is mediated by the PPARγ-NF-κB pathway [γ pathway [γ [γ in both first-trimester EVCTs and term VCTs, as described in the research cited above on the influence of PPARγ in trophoblast invasion and differentiation, was also accomplished by this PPARγ-agonist. Taken together, these studies show the enormous potential and benefit of rosiglitazone use in studies of the placenta.To explore the role of PPARtabolism , 12, inftabolism , 14, anttabolism , 16, andtabolism . In vitr pathway . Similar pathway . Other ithway [γ , 18. Furγ is exclusively located in the nuclei of EVCTs during the first trimester and of VCTs throughout pregnancy [γ in these tissues and during these developmental periods. Therefore, our purpose here was to investigate the performance of PPARγ-activated trophoblasts by analyzing the transcriptomic signatures of rosiglitazone-treated EVCTs (RT-EVCTs) and VCTs (RT-VCTs). In this study, we isolated EVCTs and VCTs from first trimester and term human chorionic villi, respectively; cultured these cells with rosiglitazone; and quantified the transcriptome of each type of cell using microarray analysis, as shown in γ, as well as on the specific genes and pathways targeted, and constitute an invaluable knowledge base for future research.In the human placenta, PPARregnancy –21. To dth gestational weeks and at term . Our ethics committee (CCPRB Paris Cochin no. 18-05) approved the collection of placentas from legal and voluntary terminations of pregnancy in the first trimester as well as of the normal term placentas.Placenta samples in this study were collected with patients' written informed consent, in compliance with the Declaration of Helsinki. Placenta tissues were collected from women with normal pregnancies during the 8-92+-, Mg2+-free Hanks' balanced salt solution for membrane removal. Mononucleated VCTs were isolated using digestion with trypsin-DNase and fractionation on a discontinuous Percoll gradient according to the protocol of Kliman et al. [4, 0.25% (wt/vol) trypsin powder (Difco), 100 IU/mL penicillin, 25 mM HEPES, and 100 μg/mL streptomycin , and monitored under invert microscopy. The initial digested solution (consisting mostly of red blood cells) was discarded while the subsequent digested solution (clearly consisting of EVCTs) was retained. A discontinuous Percoll gradient (5–70% in 5% steps) was used to stratify the digested solutions; the middle layer (which included EVCTs) was retained for further analysis. The purified EVCTs were diluted with Dulbecco's modified Eagle's medium (DMEM), with 2 mM glutamine, 100 IU/mL penicillin, 100 mg/mL streptomycin, and 10% decomplemented fetal calf serum (FCS), to a final density of 0.9 × 106 cells/mL in 60 mm diameter plastic tissue culture dishes . In preparation for culturing, culture plates  were coated with Matrigel™ , then seeded with EVCTs at a density of 5 × 104 cells/cm2. To maintain continuous culture conditions, DMEM-F12 medium was used that contained 10% heat-inactivated fetal calf serum (FCS), Glutamax, 100 μg/mL streptomycin, and 100 IU/mL penicillin (Invitrogen). Plates were incubated for 2 h at 37°C and 5% CO2; then, nonadherent EVCTs were rinsed off. At this point, fresh medium with or without 1 μM rosiglitazone (Cayman) dissolved at 1 mM in ethanol (treatment) or 0.1% ethanol (vehicle) was added for another 24 h of incubation.As previously described , five efn et al.  and Alsan et al. . In brie6 cells/mL were placed in a humidified incubator at 37°C under 5% CO2 for 3 h. Nonadherent VCTs were rinsed off, fresh medium with or without 1 μM rosiglitazone (Cayman) dissolved at 1 mM in ethanol (treatment) or 0.1% ethanol (vehicle) was added, and dishes were incubated for another 24 h.VCTs were isolated from five term placentas using the following procedures. Placentas were oriented with the maternal side facing upwards, and tissues were sampled at a depth of 1.5 cm, half the distance from the edge to the centre. Villous tissues were rinsed, minced, digested, and purified using the steps described above. Culture dishes containing 0.9 × 10After 24 h of incubation, RT-EVCTs and control EVCTs were harvested for microarray experiments. Cell RNA was extracted using TRIzol® reagents (Invitrogen) and purified using RNeasy® Mini Kits (Qiagen). RNA integrity and purity were examined with a 2100 Bioanalyzer with the RNA 6000 LabChip kit (Agilent Technologies). The U133A 2.0 GeneChip  was used for gene expression detection according to the manufacturer's manual. From the 22,000 probe sets on the gene chip, 14,500 genes were detected.μg total RNA, for RNA amplification, and 3 μg amplified RNA were then labeled with Cy-dye using the 26 CyScribe first-strand cDNA labeling kit (Amersham Biosciences). Amplified RNA from rosiglitazone-treated VCTs was labeled with Cy5, and amplified RNA from control VCTs was labeled with Cy3. A Microcon YM 30 column (Millipore) was used to purify and concentrate the labeled mixture (Cy5 and Cy3) after additional modifications with human cot-1, yeast tRNA, and poly A. The probes were denatured and the mixture was hybridized at 65°C overnight in a sealed humidified hybridization chamber, then rinsed with 1XSSC, 2XSSC, 0.03% SDS, and 0.2% SDS solutions for 2 min each. Arrays were scanned with a GenePrix 4000A microarray scanner (Axon Instruments).RT-VCTs and control VCTs were likewise harvested after 24 h of incubation for microarray experiments; RNA extraction, purification, and quality control were performed as described. The SHDZ gene chip (Stanford University) was used for gene detection as described in : for eacSince gene expression in EVCTs and VCTs was detected using different microarray platforms, different procedures were followed for data processing. For EVCT gene expression, which was quantified using the GeneChip  application, data processing used the following filter thresholds: (i) percentage of missing data was no more than 50%, (ii) threshold to identify up- and downregulated genes for statistical comparison was set to a fold change of 1.5, (iii) maximum false discovery rate (FDR) was set to 5%, and (iv) fold change of one gene was equal to mean of treated groups minus mean of control groups and then divided by the minimum value from all (fold change = (mean (treated)–mean (control))/minimum ) . For VCTP values lower than 0.05 identified significant enrichment.ClueGO is a Cytoscape plug-in application for the functional classification of genes . Our anahttps://string-db.org) was used to analyze the interactions of DEG-encoded proteins and construct a PPI network. For this, the significant confidence score was set to greater than 0.4. Cytoscape was used to visualize and organize the PPI network. Proteins interacting with PPARγ or RXRα were indicated by different colors, and shapes were used to represent different groups. Binding site interactions between transcription factors and target genes were predicted by the Cytoscape plug-in iRegulon . Putative regulatory regions were defined as 10 kb around transcription starting sites. The FDR was set to 0.1% to verify the interaction. The resulting chart was modified in Cytoscape using red to indicate upregulated genes and blue to indicate downregulated genes.The STRING database . Gene expression profiles of the rosiglitazone-treated (TRT) samples of EVCTs and VCTs were normalized (P < 0.05), of which 114 genes were upregulated (red) and 25 genes were downregulated (blue). In RT-VCTs, a total of 197 genes were identified as DEGs (P < 0.05), of which 181 genes were upregulated (red) and 16 genes were downregulated (blue)  .The entire set of DEGs from RT-EVCTs and RT-VCTs was separated by cell type of origin and submitted independently to ClueGO with the default parameters. GO and pathway enrichment were set up for analysis. DEGs were classified by three ways: by GO biological process, GO molecular function, and GO cellular component. Enriched pathways were identified through a search of the KEGG and Reactome databases. The results are visualized in Among the DEGs identified in RT-EVCTs, the main GO biological processes represented were “negative regulation of epithelial cell apoptotic process,” “long-chain fatty-acyl-CoA biosynthetic process,” and “phosphatidylcholine biosynthetic process.” For the same group of DEGs, the GO molecular functions were mainly classified as “alpha-tubulin binding,” “wide pore channel activity,” “positive regulation of cold-induced thermogenesis,” “glutathione transferase activity,” “long-chain fatty acid binding,” “regulation of cell adhesion mediated by integrin,” and “positive regulation of non-motile cilium assembly.” Finally, the GO cellular component that was most associated with these DEGs was “desmosome.” In the pathway enrichment analysis of RT-EVCTs, DEGs were mainly associated with the terms “HIF-1 signaling pathway,” “p53 signaling pathway,” “glutathione metabolism,” “NRAGE signals death through JNK,” “PPAR signaling pathway,” “plasma lipoprotein assembly,” and “remodeling and clearance”.In the analysis of GO terms associated with the RT-VCT dataset, DEGs were mainly involved in the following biological processes: “regulation of receptor biosynthetic process,” “negative regulation of nucleotide metabolic process,” “cyclic nucleotide biosynthetic process,” and “negative regulation of B cell apoptotic process.” The molecular functions of this same group of DEGs were mainly linked to “negative regulation of DNA replication,” “regulation of protein deacetylation,” “ubiquitin-like protein conjugating enzyme activity,” “Hsp90 protein binding,” “negative regulation of intracellular protein transport,” and “positive regulation of phosphoprotein.” With respect to GO cellular components, DEGs were mainly associated with the terms “NuRD complex,” “cellular metabolic compound salvage,” and “immunological synapse.” Finally, the pathway enrichment analysis of RT-VCTs revealed that DEGs were mainly involved in “tight junction,” “regulation of HIF by oxygen,” “unfolded protein response,” “HIF-1 signaling pathway,” “nuclear receptor transcription pathway,” and “plasma lipoprotein remodeling”.DEGs that were upregulated in RT-EVCTs and RT-VCTs were submitted separately to ClueGO following the same procedure as described above. The results are visualized in Instead, from the DEGs that were upregulated in RT-VCTs, no significant GO biological process was identified. In the classification of GO molecular functions, these DEGs were mainly linked with “positive regulation of cell cycle” and “mitotic DNA damage checkpoint,” and the most significant GO cellular component was “transcription factor complex.” In the pathway enrichment analysis, upregulated DEGs in RT-VCTs were mainly associated with the terms “mTOR signaling pathway,” “cell cycle checkpoints,” “DNA repair,” “developmental biology,” “metabolism,” and “vesicle-mediated transport”.DEGs that were downregulated in RT-EVCTs and RT-VCTs with respect to controls were submitted to ClueGO using the same procedure as described above. Results are visualized in Instead, DEGs that were downregulated in RT-VCTs were mainly involved in the GO biological processes “cyclic nucleotide biosynthetic process,” “negative regulation of nucleotide metabolic process,” “ncRNA 3'-end processing,” and “O-glycan processing;” the GO molecular functions “nuclear receptor activity,” “histone deacetylation,” “regulation of TOR signaling,” “Hsp90 protein binding,” “ion channel regulator activity,” “nuclear envelope organization,” and “peptidyl-threonine modification;” and the GO cellular component “organellar ribosome.” In the pathway enrichment analysis of RT-VCTs, downregulated DEGs were mainly associated with the “HIF-1 signaling pathway,” “transfer of ubiquitin from E1 to E3,” “cell-cell communication,” “transcription regulation of RUNX3,” and “formation of NR-MED1 coactivator complex”.P values less than 0.05.Next, we wanted to determine the extent to which the cellular processes affected by rosiglitazone treatment were specific to either EVCTs or VCTs, and which instead were present in both tissue types. To do this, we characterized the up- and downregulated DEGs of RT-EVCTs and RT-VCTs separately using clusterProfiler, using information from the GO and KEGG databases, as well as the Disease Ontology (DO) and Disease Gene Network (DisGeNET) databases. Terms appearing in at least three columns were thought important in both, while terms appearing only in the RT-EVCT or RT-VCT dataset were labelled tissue-specific; significance was determined by In both RT-EVCTs and RT-VCTs, the GO biological processes “regulation of endothelial cell migration,” “non-canonical Wnt signaling pathway,” “receptor metabolic process,” “negative regulation of protein phosphorylation,” and “metabolism process” appeared to play important roles. Instead, processes specific to RT-EVCTs included “glycogen catabolic process,” “cellular carbohydrate catabolic process,” “embryo implantation,” “fatty acid derivative biosynthetic process,” and “long-chain fatty-acyl-CoA biosynthetic process,” while those specific to RT-VCTs were “cytoplasmic mRNA processing body assembly,” “ribonucleoprotein complex biogenesis,” “positive regulation of phosphoprotein phosphatase activity,” and “negative regulation of nucleotide metabolic process” .The GO molecular functions “nuclear hormone receptor binding,” “long-chain fatty acid binding,” “fatty acid binding,” “nuclear activity,” and “transcription factor activity” seemed to be important in both RT-EVCTs and RT-VCTs. Functions specific to RT-EVCTs included “steroid hormone receptor binding,” “eicosanoid receptor activity,” “phosphatidylinositol phosphate kinase activity,” and “fatty acid ligase activity,” while those specific to RT-VCTs were “Wnt-activated receptor activity,” “cyclin-dependent protein kinase activity,” “transferase activity,” and “ubiquitin-specific protease activity” .Both tissue types shared the significant GO cellular components “smooth endoplasmic reticulum,” “ruffle,” “transcription factor complex,” “apical plasma membrane,” “lumen,” and “cell-cell junction.” Instead, the component terms “beta-catenin destruction complex,” “M band,” “integral component of lumenal side of endoplasmic reticulum membrane,” and “A band” were found only in RT-EVCTs, while “spliceosomal complex,” “Wnt signalosome,” “pronucleus,” “microtubule end,” and “autophagosome membrane” appeared to be specific to RT-VCTs .Through a search of the KEGG database, the following pathways appeared to be important in both tissue types: “protein processing in endoplasmic reticulum,” “glucagon signaling pathway,” “Epstein-Barr virus infection,” “PPAR signaling pathway,” “HIF-1 signaling pathway,” “progesterone-mediated oocyte maturation,” and “mTOR signaling pathway.” Pathway terms specific to RT-EVCTs included “primary immunodeficiency” and “fatty acid metabolism,” while those specific to RT-VCTs were linked with “bacterial invasion of epithelial cells” and “parathyroid hormone synthesis, secretion, and action” .From the Disease Ontology database, the terms “preeclampsia,” “HELLP syndrome,” “spinocerebellar ataxia,” “familial hyperlipidemia,” “lipid metabolism disorder,” and “musculoskeletal system cancer” were important in both EVCTs and VCTs. Terms specific to RT-EVCTs included “breast benign neoplasm,” “thoracic benign neoplasm,” “lipomatous cancer,” “amyloidosis,” and “vein disease,” while those specific to RT-VCTs were “alveolar rhabdomyosarcoma,” “osteopetrosis,” “giant cell tumor,” and “germ cell and embryonal cancer” .From a search of the DisGeNET database, the terms “preeclampsia,” “hypertrophic cardiomyopathy,” “immunologic deficiency syndromes,” “diabetes mellitus,” “vascular inflammations,” “hematopoietic neoplasms,” “non-alcoholic fatty liver disease,” “vascular disease,” “ischemic cardiomyopathy,” and “triploidy syndrome” were significant for both tissue types. Instead, “chronic neutrophilic leukemia,” “glycogen storage disease,” and “myeloid, chronic, atypical, and BCR-ABL negative leukemia” were specific to RT-EVCTs, and “alport syndrome” and “aggressive non-Hodgkin lymphoma” were specific to RT-VCTs .γ by rosiglitazone, we next attempted to predict (i) the protein-protein interactions (PPI) of PPARγ with DEG-encoded proteins and (ii) the transcription factor-target gene (TF-TG) interactions of PPARγ with DEG promoters. In RT-EVCTs ; concentrations of up to 100 μM of this compound have been reported to activate only PPARγ . Moreovinvasion . In thisγ in trophoblasts, in this study, we also analyzed gene expression changes in VCTs using microarray profiling, and, through various approaches, identified the enriched processes that were linked with these DEGs in RT-EVCTs and RT-VCTs. We were thus able to compare the similarities and differences between EVCTs and VCTs affected by activated PPARγ. In total, there were 139 DEGs in RT-EVCTs and 197 DEGs in RT-VCTs, and these were associated with enrichment in more than 200 GO and pathway terms  and NCoR (nuclear receptor corepressor), which block transactivation or the transcriptional coactivators CREB-binding protein (CBP), histone acetyltransferase p300 (p300), and PPAR-binding protein (PBP), which have the opposite effect [γ might affect the transcription complex formed by PPARγ and RXRα, their cofactors or transcription partners, which could then lead to alterations in the regulation of different transcription circuits. Our results provided evidence for direct protein-protein and protein-promoter interaction with the PPARγ complex. Among the proteins that appear to interact directly with PPARγ, several have been experimentally verified, including ANGPTL4 [γ-MAPK-MMP signaling pathway. Commonly, phosphorylated PPARγ stimulates the MAPK-activated pathway, leading to the activation of extracellular signal-regulated kinases (ERKs) that then induce the upregulation of matrix metalloproteinase (MMP) [γ activation. In addition to the direct target genes predicted here, the genes in secondary relationships should be paid equal attention in terms of potential regulation by other target genes. For example, our previous research has shown the key role of LOX1, through secondary interactions, in cytotrophoblast invasion [Finally, in order to facilitate study of the mechanisms behind the molecular interactions, we attempted to predict the protein-protein interactions between the DEGs recovered here and PPARe effect . We prop ANGPTL4 –61, ABCG ANGPTL4 , APOB [6 ANGPTL4 , CCNE1 [ ANGPTL4 , CPT1B [ ANGPTL4 , FABP4 [ ANGPTL4 –69, HMOX ANGPTL4 , and SER ANGPTL4 , 72. Manse (MMP) –75. Herese (MMP) , which iinvasion .γ activation in EVCTs and VCTs, highlighting extensive changes in gene expression and the biological processes and pathways affected. This study provides a broad perspective of PPARγ-influenced biological processes in trophoblasts and facilitates further study, particularly into potential drug-targeted genes or pathways in the human placenta.To our knowledge, our results reveal for the first time the widespread effects of PPAR


GJB2 was sequenced and out of 150 patients with normal results at MLPA, 29.33% had abnormal results: variants in heterozygous state: c.71G>A (28%), c.457G>A (20%), c.269T>C (12%), c.109G>A (12%), c.100A>T (12%), c.551G>C (8%). Out of 26 patients with c.35delG in heterozygous state, 38.46% were in fact compound heterozygous. Conclusions: We identified two variants: c.109G>A and c.100A>T that have not been reported in any study from Romania. MLPA is an inexpensive, rapid and reliable technique that could be a cost-effective diagnosis method, useful for patients with hearing impairment. It can be adaptable for the mutation spectrum in every population and followed by Sanger sequencing can provide a genetic diagnosis for patients with different degrees of hearing impairment.Background: We have investigated the main genetic causes for non-syndromic hearing impairment (NSHI) in the hearing impairment individuals from the North-Eastern Romania and proposed a cost-effective diagnosis protocol. Methods: MLPA followed by Sanger Sequencing were used for all 291 patients included in this study. Results: MLPA revealed abnormal results in 141 cases (48.45%): 57 (40.5%) were c.35delG homozygous, 26 (18.44%) were c.35delG heterozygous, 14 (9.93%) were compound heterozygous and 16 (11.35%) had other types of variants. The entire coding region of  Hearing impairment (HI) is the most common and heterogeneous sensory deficiency. It is defined by a unilateral or bilateral decrease in hearing acuity, more precisely a decrease in the hearing threshold in decibels (dB), at different frequencies. World Health Organization  estimates that HI affects 466 million people around the world (6.1% of the world’s population), of which 34 million children. It is considered that 1/1000 newborns have a form of congenital hearing impairment More than 50% of cases of deafness are due to genetic causes  out of wIn the last 5 years, progress has been made in identifying new hearing impairment genetic causes, due to research and new technology. Approximately 121 loci for NSHI have been currently mapped: 49 autosomal dominant, 76 autosomal recessive and 5 X-linked [GJB2 (NM_004004.5) or Gap Junction Protein β 2, situated on chromosome 13q12 (DFNB1 locus), is the most common cause of congenital hearing loss in many populations [GJB2 have been reported. The most frequent variant in the Caucasian populations is c.35delG, representing about 60% of all cases of NSHI [ulations  includinulations ,8,9. Mor of NSHI ,10,11,12GJB3 (Gap Junction Protein β 3) and GJB6 (Gap Junction Protein β 6) are the next frequent genes that can cause hearing impairment but they are less common, with less than 10 mutations cited [ns cited ,14,15.GJB2 being accountable for a large proportion of NSHI.The aim of this study was to identify and investigate the main genetic causes for NSHI in the hearing impairment subjects from the North-Eastern Romania and to convince other specialties to advice for genetic testing and counseling. Subsequently we verified a possibility to use Multiplex Ligation-dependent Probe Amplification (MLPA) as a cost-effective diagnosis protocol for developing countries and as a first intention genetic method. Genetic screening is feasible, MLPA is a technique that can analyze in a single reaction up to 50 DNA sequences and detect copy number variations of several human genes, including small intragenic rearrangements but also single-nucleotide polymorphisms or aberrant DNA methylation. To our knowledge, the studies of hearing loss in Romania are based on RFLP (restriction fragment length polymorphism), ARMS-PCR (amplification refractory mutation system-polymerase chain reaction) analysis and Sanger Sequencing. Our Genetic Centre has experience in MLPA for other pathologies since 2012, so we tried to implement it as a screening method for hearing impairment individuals.In patients with hearing impairment, the diagnostic approach starts with personal medical history, physical examination and family history for at least three generations and should continue with genetic tests and appropriate management.The patients included in the study were registered under a numerical code in order to maintain anonymity. The use of the results was done according to a protocol approved by the Ethics Commission of “Grigore T. Popa” University of Medicine and Pharmacy Iasi  and the Ethics Commission of “Saint Mary” Emergency Children’s Hospital Iasi . Informed consent was signed by from patients, parents or legal guardians before beginning the research. All subjects included in this study were offered voluntary entrance.In the study (2015–2019), were enrolled 395 subjects with mild to profound and bilateral hearing impairment from the Iasi Regional Center for Medical Genetics and Audiology Department of Iasi Rehabilitation Clinical Hospital. All the subjects were clinically characterized by physical and auditory examinations. A number of 104 individuals were excluded from this study based on: syndromic or environmental/infectious etiology for hearing loss. The limitation of this study is that it investigates only the hearing loss subjects in order to assess the prevalence of certain type of mutations, so it does not include a control group.Auditory functional assessment was performed only in the absence of the pathology of the middle ear, confirmed by otomicroscopy and wideband tympanometry. In cases identified with otitis media, the appropriate treatment was recommended and the child was rescheduled for repeated controls until the condition of the middle ear allowed audiological testing .The audiological evaluation was adapted to the age and to the psycho-intellectual development of children. Thus, in children over 6 years of age, the auditory thresholds were measured by standard liminal tonal audiometry (air and bone conduction for 250 to 8000 Hz). In those under 6 years of age, as well as in some children over 6 years of age but who could not collaborate in subjective audiometric testing, the identification of hearing thresholds was done by objective audiological assessment of cross check type. The auditory steady state response and brainstem evoked response audiometry using insert headphones and distortion product otoacoustic emissions were measured in natural sleep. We performed visual reinforced audiometry or/and free field audiometric examination for subjective threshold confirmation in children who collaborated. In these cases, the conduction hearing loss was excluded based on normal otomicroscopy accompanied by type A wideband tympanometry. The audiologic evaluations were performed in soundproof rooms, using Interacoustics equipment (Equinox audiometer and Eclipse EP25). The audiologic follow-up was made periodically with the same methods adapted to each child’s particularity , mainly at 4-, 6- or 12-months intervals, in order to identify the dynamic evolution of hearing impairment and for the fitting of the conventional hearing aids. Children with progressive hearing loss received the indication for cochlear implantation when they were included in the category of severe or profound hearing loss.All subjects had cranial computed tomography (CT) scan and none showed ear malformation.a. DNA genomic extractionDNA was extracted from 3 mL of peripheral blood samples stored with EDTA agent, using Wizard Genomic DNA Purification Kit .b. MLPAGJB-WFS1-POU3F4 was used for the detection of deletions or duplications in the GJB2, GJB3, GJB6, POU3F4 genes, genomic microdeletions upstream of POU3F4 and the presence of six specific variants in the GJB2 gene: c.313_326del14, c.235delC, c.167delT, c.101T>C and c.35delG.The probe mix P163 The MLPA analysis was performed according to the manufacturer’s protocol. Briefly, 100 nanograms of genomic DNA was denatured and hybridized with SALSA probes at 60 °C for approximately 17 h. PCR was performed after 15 min ligation at 54 °C, using Cy5 labeled primers. Fluorescent amplification products were separated based on their length by capillary electrophoresis in a CEQ 8000 GeXP Genetic Analysis System  and the results were analyzed using Coffalyser.NET V9 program .The probe ratio of deletion and duplication were fixed at 0.7 and 1.3 respectively.GJB2 gene were sequenced bidirectionally in heterozygous or normal individuals.Genomic regions of the c. Sanger SequencingAll samples were analyzed at the University of Medicine and Pharmacy “Grigore T. Popa” Iasi. The amplification using 125ng genomic DNA (25 µL reaction volume) was performed in a Sensoquest Thermocycler , using Herculase II Fusion DNA Polymerase . PCR conditions included: initial denaturation (10 min at 95 °C), followed by 35 cycles of denaturation (30 s at 94 °C), annealing (30 s at 57 °C) and elongation (60 s at 72 °C), with a final elongation at 72 °C for 5 min, as described by M. RamShankar .® system, Brea, CA USA). The final products were subsequently separated and detected on a CEQ 8000 GeXP Genetic Analysis System (Beckman-Coulter). Sequences were analyzed in both directions (forward and reverse) and compared with the NCBI reference sequence NM_004004, using Mega6 software. The variants were verified for pathogenicity in Mutation taster, ClinVar and PolyPhen for the evaluation of disease-causing potential of sequence alterations [The sequencing was performed using primers previously described  and Genoerations ,19,20.d. Statistical AnalysisExperiment results were analyzed in Excel and presented in descriptive statistics.A total sample of 291 patients from North-Eastern Romania were collected between 2015–2019. HI was reported to be congenital and without other accompanying clinical features. All patients included in this study showed different pathologic levels of auditory thresholds, from mild to profound bilateral hearing impairment. The patients’ age ranged between 1 month to 52 years (median age 12.31).Among the 291 probands, 74.6% (217/291) were sporadic cases of HI (simplex probands)  and 25.4% (74/291) had at least one first degree affected relative with bilateral HI (multiplex probands), of which 4 with parental consanguinity.GJB2, GJB3, GJB6, POU3F4 and WFS1 genes were analyzed by MLPA that revealed abnormal results in 141 cases (48.45%). Out of the total of 141 abnormal cases, 4 (2.84%) had variants in WFS1 gene and 137 (97.16%) in GJB2 gene: 57 (40.43%) were c.35delG homozygous, 26 (18.44%) were c.35delG heterozygous, 30 (21.28%) were compound heterozygous and 28 (19.86%) had other types of variants. No mutations were identified by MLPA in GJB3, GJB6 and POU3F4 genes.Mutations in WFS1 gene, all of the 4 patients with variants in this gene had exon 8 deletion . Of these, 57 patients (58.76%) had the c.35delG variant in homozygous state, 26 (26.84%) in heterozygous state and 14 (14.4%) were compound heterozygous for 3 different 35delG/non-35delG variants. Regarding the GJB2 was sequenced in all individuals included in this study. Out of 26 patients with c.35delG variant in heterozygous state, 10 patients (38.46%) were in fact compound heterozygous. Among 150 patients with normal results at MLPA, 44 patients (29.33%) had abnormal results: 25 patients with variants in heterozygous state: 7 with c.71G>A (28%), 5 with c.457G>A (20%), 3 with c.269T>C (12%), 3 with c.109G>A (12%), 3 with c.100A>T (12%), 2 with c.551G>C (8%). All the patients with c.35delG in homozygous state were confirmed with Sanger Sequencing.The entire coding region of GJB2 variant spectrum found in this study is listed in The GJB2. Out of 18 patients with mild hearing loss, only 2 of them had c.35delG in homozygous state (diagnosed before age 4) and 4 patients had c.35delG in heterozygous state (diagnosed after the age 4). 81 patients had GJB2 biallelic mutations and severe or profound hearing impairment: 20 (24.6%) of them had severe HI and c.35delG in homozygous state, 28 (34.5%) had profound HI and c.35delG in homozygous state, while 8 patients (9.87%) with severe HI had c.35delG in compound heterozygous state and 14 patients with profound HI had c.35delG in compound heterozygous state.Genotype-phenotype correlation was performed based on the distribution of the severity of HI in c.35delG and non-35delG genotype categories as shown in Hearing impairment is one of the most heterogeneous conditions of considerable concern in medicine nowadays. Each population has a different etiologic profile based on ethnic, geographic, social and medical background. It is diagnosed in 1–2 of 1000 newborns , geneticGJB2 gene mutations can vary according to ethnicity: more than 50% in the European population [GJB2 gene [The prevalence of pulation , 16% in pulation  and Iranpulation , 9.6% inpulation  and 6.1%JB2 gene ,28. In oJB2 gene ,30, c.16JB2 gene , c.71G>AJB2 gene  are prevRomania is a Latin country from Central-Eastern Europe and it is heterogenous from an ethnic point of view. The variant frequency and spectrum is different compared to other countries. The most important minorities in Romania are the Hungarian minority in North-West region, followed by Roma and other minorities.GJB2 gene (responsible for the major etiologies of hereditary hearing impairment among Romanians), GJB3, GJB6, POU3F4 and WFS1 genes. Genetic diagnosis was confirmed in 174 (59.7%) of the 291 patients with different degrees of hearing impairment, most of them being accounted for GJB2 gene. This data is in accordance with the literature, GJB2 mutations are frequent in all studied populations [GJB2 mutations are prevalent due to consanguineous marriages. In Turkey autosomal recessive inheritance is responsible for 76.9 % of the studied cases [We performed a genetic screening of ulations ,36,37,38ed cases .In this study we did not found any significant difference in the severity and evolution of hearing impairment when comparing the 74 multiplex probands with 217 simplex probands.GJB2 gene sequence variants [The c.35delG variant (rs80338939) is responsible for approximately 70% of autosomal recessive NSHI and is the most common cause of hearing loss in Caucasian populations . The carvariants .GJB2 gene, it has become the first intention genetic investigation for patients with non-syndromic hearing loss.Because c.35delG is the most frequent variant in the coding region of the GJB2 gene.In our study, the 35delG variant was present in 97 out of 291 (33.3%) patients with different degrees of hearing impairment. These results are in accordance with the findings previously reported in other Romanian and Central European studies ,46. All The study revealed that subjects with 35delG in homozygous state present more severe hearing impairment, compared with the 35delG/non-35delG compound heterozygotes. The subjects with two non-35delG variants have an even less degree of hearing impairment. This observation is in accordance with other studies which conclude that c.35delG in homozygous state is associated with a higher risk for severe hearing impairment ,47,48,49GJB2 have determined that the c.101T>C variant is more frequent in individuals with mid-west American, UK [The next frequent variant was c.101T>C (rs35887622), accounting for 19 out of 291 (6.5%) patients. In the Caucasian population the frequency of the c.101T>C variant was determined to be up to 6.5 %  and was ican, UK  and Germican, UK  origins,More than 50% of our patients with c.101T>C variant had moderate to profound hearing impairment. Also, at the time of the diagnosis, the age of the patients with c.101T>C was greater than the age of the patients with c.35delG. The results of a large study on the UK population affirmed that this variant is associated with mild/moderate HI . The lowThe incomplete penetrance of c.101T>C variant was not confirmed because the study included only subjects with HI. The phenotype of the patients with hearing impairment was variable: the individuals with biallelic c.101T>C and c.35delG had moderate to profound hearing loss and the heterozygous c.101T>C had mild to moderate forms of hearing loss. No individuals with c.101T>C in homozygous state were found. GJB2 gene and disrupts the integrity of connexons. In many European populations, this variant has been identified previously with a frequency of pathogenic alleles from 0.47% to 28.3% [The c.313_326del14 variant (rs111033253), called in the past c.310del14, c.312del14 or c.314del14, truncates the The frequency of the c.313_326del14, variant in our group of participants was another finding in our study. The genotype was found in 8 of 291 patients . A number of 6 patients were compound heterozygous with moderate to severe hearing impairment and the age of diagnosis being under 18 years and 2 patients had c.313-326del14 in heterozygous state with mild to moderate hearing impairment.The c.71G>A variant is the fourth most common in our study and it had over five times lower frequency than the c.35delG variant. This mutation, previously called W24X, was first described in a Pakistani family  and lateThe c.71G>A frequency in different Roma subgroups is variable: it ranges from 0.0% to 26.1% in Slovak subgroups  and up tOur data is not concordant with other Central European series or the study from North-Western Romania, where 35delG and c.71G>A were the most common mutations , one expThe findings in Roma population confirm the ethnic origin of this mutation. Due to the fact that the sample of Roma patients is small, we cannot compare with other studies. The degree of addressability to medical care of the Roma-population from North-Eastern Romania is even lower than in North-Western Romania.GJB2 gene in patients with hearing impairment. The mutation  was dete et al. ,64. In oe et al.  and profe et al. . To dateIn 6 patients of 291 included in the study were identified two variants: c.109G>A and c.100A>T that have not been reported in any study from Romania. Out of these, 3 had c.109G>A variant and presented the same pattern of HI  while the other 3 had c.100A>T .GJB2 mutations did not explain their hearing impairment and the patients with variants in WFS1 gene.Our results contribute to define the mutation spectrum in the Romanian individuals with hearing impairment. Despite the genetic heterogeneity of NSHI, 217 patients were diagnosed out of a cohort of 291 patients. MLPA confirmed the genetic diagnosis in 141 cases (48.45%). We selected for further study the patients to which the Regarding our second aim of the study, we concluded that MLPA can be used as first intention genetic test for patients with HI due to some advantages over the Sanger Sequencing method: it is time saving, has a low price for consumables, the initial investment is lower for the platform than for Sanger Sequencing, the interpretation is much faster and it could easily detect the number copies variation and most frequent pathogenic variants. A description of the advantages and disadvantages is presented in This is the first report of the utility of MLPA and Sanger sequencing of HI in Romania; the results show notable findings in comparison to other European populations. However, some limitations should be noted: the samples included in this study are not truly representative for the entire Romania as all samples were collected from individuals with different degrees of hearing impairment, born in North-Eastern Romania, we did not have access to all of the parental samples to confirm compound heterozygosity. Our results need further studies on larger patient groups, especially Roma-population, in order to estimate the real incidence of the disease and to make more accurate predictions about the genotype phenotype correlation in our population.GJB2 gene is feasible because this is a small gene, with only 2 exons and the costs are reasonable and extra equipment are not necessary.We recommend genetic investigations in all subjects with hearing impairment that cannot be explained by other factors. In our knowledge, this is the first report on the utility and cost-effective of genetic testing in a cohort of Romanian patients with congenital NSHI. Sanger sequencing for The genetic diagnosis in hearing impairment is important for many reasons: allows us to determine the etiology of deafness, offers the possibility to provide genetic counseling and prenatal diagnosis and not at least, based on the genotype-phenotype correlation provides prognostic information and facilitates an adequate management.In this study, 217 patients had pathogenic/likely pathogenic variants, 141 being confirmed by MLPA. We identified two variants: c.109G>A and c.100A>T that have not being reported in any study from Romania. The most common variant in our study is c.35delG followed by c.101T>C, c.313_326del14 and c.71G>A.GJB2 gene in cases of normal results.All of the patients had been confirmed with Sanger Sequencing, proving that MLPA can be a cost-effective diagnosis method, useful for every patient with hearing impairment. MLPA is an inexpensive, rapid and reliable technique that could help as first intention genetic test for every individual with NSHI. Moreover, it can be adaptable for the mutation spectrum in every population and can be followed by Sanger sequencing for