# data[].attributes.body
Axelle Vannieuwenhuyse Aya Bougria

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Neural correlates of efficacy of voice therapy in Parkinson's disease identified by performance-correlation analysis

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https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0184359&type=printable
Helena Massa, Lotte Ost, Ruth Peleman

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Margot De Gand en Sylvie De Rechter

# data[].attributes.body
Jana Steeman, Janne Bulens en Laura Provoost

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Elisabet Cantó,

Emma Roca,

Lidia Perea,

Ana Rodrigo-Troyano,

Guillermo Suarez-Cuartin,

Jordi Giner,

Anna Feliu,

Jose Manuel Soria,

Lexa Nescolarde,

Silvia Vidal

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Citation: Cantó E, Roca E, Perea L, Rodrigo-Troyano A, Suarez-Cuartin G, Giner J, et al. (2018) Salivary immunity and lower respiratory tract infections in non-elite marathon runners. PLoS ONE 13(11): e0206059. https://doi.org/10.1371/journal.pone.0206059

Editor: David M. Ojcius, University of the Pacific, UNITED STATES

Received: April 20, 2018; Accepted: October 6, 2018; Published: November 21, 2018

Copyright: © 2018 Cantó et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and Supporting Information files.

Funding: The authors received no specific funding for this work.

Competing interests: The authors have declared that no competing interests exist.

Introduction

The number of non-elite runners participating in marathons had increased dramatically in the last ten years [ 1]. Several studies have demonstrated that marathons and ultramarathons may alter immune function and increase the risk of respiratory tract infections [ 2- 4]. Within two weeks of completing such strenuous exercise, the risk of infection increased 100% to 500% [ 5] and around 25% of finishers reported respiratory symptoms [ 3].

Salivary Immunoglobulin A (sIgA) is the most common antibody on the mucosal surface. Different studies have analyzed changes in sIgA following strenuous exercises and their potential relationship with respiratory symptoms, with conflicting results [ 6- 8]. Other studies have suggested that immune factors present in mucosal secretion, including antimicrobial peptides (AMPs) and immune mediators such as chemokines, changed after prolonged running. An increase in salivary lactoferrin, one of the most abundant AMPs, has been described in participants in a 50 km ultramarathon [ 9]. Furthermore, chemokines such as Growth-Regulated Oncogene-alpha (Groα or CXCL1), growth-Regulated Oncogene-beta (Groβ) and monocyte chemoattractant protein 1 (MCP-1) have demonstrated an important role in the regulation of local immunity against infections by contributing to the tissue infiltration of leukocytes [ 10]. Experimental studies showed that serum levels of these inflammatory mediators increased markedly in response to exercise in mice [ 11]. However, limited data regarding salivary IgA, AMPs, chemokines and the presence of lower respiratory tract infections (LRTI) in marathon runners are available.

We hypothesized that non-elite marathon runner who experienced changes in their salivary immunity would be more prone to developing a respiratory infection. Therefore, our aim was to determine salivary levels of IgA, AMP and inflammatory markers before and after a marathon and their relationship with the development of LRTI.

Materials and methods

Subjects

Forty seven healthy non-elite marathon runners (28 males and 19 females) completed the 42.195 km of the 2016 Barcelona Marathon. Mean finishing time was 3 hours and 38 min (±41 min). Study participants were recruited using an announcement in the marathon newsletter that all marathon runners received two weeks prior to the race. Participation was voluntary in all cases. The study was approved by the local ethics committee "Comité Étic de Investigació Clínica de la Fundació de Gestió Sanitaria del Hospital de la Santa Creu i Sant Pau de Barcelona", project number IIBSP-SUMMIT-2016-02. All participants gave signed informed consent.

All runners were asked to report any incidence of respiratory symptoms 2 weeks before and after the race. Runners who reported respiratory symptoms 2 weeks before marathon were excluded from the study. LRTI was defined according the clinical definition proposed by the European Respiratory Society and European Society for Clinical Microbiology and Infections disease [ 12]: acute illness (present for 21 days or less) characterized by cough as the main symptom, with at least one other respiratory tract symptom (sputum production, dyspnea, wheeze or chest discomfort/pain) and no alternative explanation. In addition, all runners had to report at least one determination of fever (≥38°C) and duration of symptoms must be higher than 3 days in order to exclude those symptoms induced only by strenuous exercise. All runners with suspected LRTI were follow-up until a breakdown of symptoms was reported.

Saliva analysis

The saliva samples were then thawed and total protein saliva was quantified using the Qubit protein Assay Kit (Thermofisher Scientific). Salivary IgA (Human IgA Platinum ELISA, ebioscience, Affymetrix, Santa Clare, CA), lactoferrin, lysozyme (AssayPro, St. Charles, MO, USA), Groα, Groβ and MCP-1 (Elabscience, Houston, Texas) were measured by ELISA according to the manufacturer's instructions. The limit of detection for IgA was: 1.6 ng/ml; lactoferrin: 0.625 ng/ml; lysozyme: 0.0781 ng/ml; Groα: 15.63 pg/ml, Groβ: 15.63 pg/ml and MCP-1: 15.63 pg/ml. Data were expressed as the concentration of each molecule relative to total saliva protein concentration [ 13]. Those determinations of relative salivary IgA concentration with a ratio higher than 1 were excluded of the study due to possible contaminations [ 14]. All determinations were performed in triplicate.

Results

Study participants

Table 1 shows demographic and laboratory test results prior to the marathon for all participants. Median age was 30 years old, median years of training were around 10 and training hours per week were around 6. All laboratory tests performed were in the normal range.

No differences between males and females were found in baseline characteristics (data not shown), except for Body Mass Index (BMI), which was higher in males than in females (23.6 ±2.1 vs 20.7 ± 1.4, p = 0.01).

Lower respiratory tract infections

Eight participants (17%) reported a LRTI during follow-up. No differences in demographics and laboratory findings between infected and non-infected participants were found prior to the marathon ( Table 1). All runners with LRTI experienced a breakdown of the symptoms before 14 days of their onset and were fully recovered within one month after the race.

Salivary IgA

No differences before and after the marathon were found in total salivary protein concentration (2.34±1.16 mg/ml vs 2.37±0.97 mg/ml, respectively) nor in salivary IgA levels normalized to total salivary protein (0.39±0.25 vs 0.35±0.24) ( Fig 1A). In addition, no differences in IgA levels before and after the race were found between infected and non-infected runners ( Fig 1B), and no differences were observed between male and females before marathon in salivary IgA/protein.

Salivary antimicrobial proteins

No differences before and after the race were observed in salivary lactoferrin (0.013±0.01 vs 0.010±0.007) and salivary lysozyme levels (0.0094±0.007 vs 0.0078±0.007). Furthermore, no differences between the sexes were observed before the marathon.

When runners with and without LRTI were compared, we observed no differences in salivary lactoferrin levels before and after the race. However, those runners who developed a LRTI showed higher levels of salivary lysozyme after the race compared to those who did not develop it (0.012±0.01 vs 0.006±0.004, p = 0.02). In addition, a decrease in lysozyme levels was observed in runners who did not develop LRTI when levels before and after the race were compared (0.010±0.007 vs 0.006±0.004, p = 0.003) ( Fig 2).

Salivary chemokines

No differences before and after the race were observed regarding salivary Groα Groβ and MCP-1. However, runners who did not develop LRTI showed a significant decrease in salivary Groα and Groβ levels after the race (Groα, 0.37±0.15 vs 0.30±0.14, p = 0.02; Groβ 0.47±0.17 vs 0.37±0.18, p = 0.03) ( Fig 3A and 3B). No differences in MCP-1 levels were found.

Systemic correlations

Before the race, salivary lactoferrin levels had a weak but statistical significant negative relationship with blood lymphocyte counts (r = -0.13, p = 0.04) and salivary Groα had a weak positive correlation with the percentage of blood basophils (r = 0.22, p = 0.03) ( Fig 4A and 4B).

After the race, salivary lysozyme correlated positively with the absolute number of blood leukocytes (r = 0.26, p = 0.006) ( Fig 4C).

Discussion

In our study we observed that LRTI is common in non-elite runners following a marathon and we demonstrated that runners who developed a LRTI had different immunological profile in their saliva. Specifically, a decrease in salivary lysozyme, Groα and Groβ levels was observed in those runners who did not develop a LRTI when levels before and after the race were compared. In addition, higher levels of lysozyme were detected after the race in runners with LRTI compared with those without infection. These findings suggest that immunological status may be important for the development of a LRTI after a marathon.

Several data have been reported reflecting the importance of the immune system in the development of LRTI after strenuous exercise. We observed that 17% of marathon runners develop LRTI symptoms within 2 weeks after marathon, which is concordant to other studies [ 7, 15]. Although the effect of close proximity between runners during the race cannot be ruled out, extreme effort has been shown to alter immune defenses by increasing inflammation in the respiratory tract [ 16]. Different studies have postulated that a combination of several immune, biochemical and hematological parameters participate in the severe stress on the body resulting from such exercise and that they may be associated with an increased susceptibility to developing infections [ 17]. Salivary IgA (sIgA) is one of the most widely-studied parameters [ 7]. Some studies have pointed to the usefulness of sIgA as a noninvasive biomarker of mucosal immunity and LRTI risk [ 18, 19]. However, other studies have suggested that reductions in sIgA cannot be solely responsible for the decline in immune function that may lead to LRTI [ 20]. In our cohort of runners, we did not observe differences in sIgA as other authors previously showed [ 21]. We observed that sIgA represented about 40-60% of total salivary protein, which is concordant with previous studies [ 22, 23] but higher than other ones [ 24]. Most of the studies were carried out few hours after exercise [ 25, 26] and we wanted to evaluate two days after finishing the marathon, since few data regarding the recovery period and its influence on the development of LRTI are available [ 7].

Salivary antimicrobial proteins (sAMPs) such as lysozyme and lactoferrin protect the respiratory tract from invading microorganisms and have been linked with an increased infection risk in athletes [ 27]. In our cohort of runners, we observed no differences in the levels of saliva lysozyme before the race. However, runners who did not develop LRTI showed a decrease in lysozyme after the marathon. In contrast, those runners who developed a LRTI showed higher levels of salivary lysozyme after the race compared to those runners who did not develop LRTI. Our findings are in line with other studies showing a decrease in salivary lysozyme immediately after an ultra-marathon, though this decrease was not related to the presence of LRTI [ 5]. However, other studies did not observe differences in salivary lysozyme levels after a 50 km race [ 25]. These discrepancies could be the consequence of different levels of effort during exercise. Interestingly we observed an inverse relation between salivary lactoferrin and blood lymphocyte counts before the race, which may be explained by the mobilization of neutrophils to maintain blood homeostasis. Previously, Inoue H et al. [ 28] described an immediate increase in serum lactoferrin concentrations immediately after running exercise and serum lactoferrin may play an antibacterial role in host defenses before the mobilization of neutrophils into the circulating pool. We also observed a positive correlation between blood leukocyte counts and the content of saliva lysozyme after the marathon. It has been observed that exercise sessions in both humans and animal models resulted in the stimulation of neutrophil degranulation releasing lysozyme from blood neutrophils [ 29]. All of these findings suggested that systemic alterations may affect the content of saliva lactoferrin and lysozyme in runners and may contribute to the development of LRTI after races. Nevertheless, we cannot exclude a process of dehydration during the marathon, since it has been demonstrated that dehydration decreases saliva antimicrobial proteins [ 30, 31]. Dehydration can therefore affect the content of saliva AMPs, and further studies are needed to better elucidate its importance, though we did not observe differences in protein content before and after the race.

Different chemokines have demonstrated a potential role in the regulation of immune response during exercise [ 32]. We also found that Groα and Groβ, which are key components in the attraction of neutrophils to the site of inflammation, decreased after the race in runners who did not develop a LRTI. It has been described that daily moderate exercise suppressed Groα [ 33] and, in experimental models, exercise down regulated multiple inflammatory cytokines and chemokines including Groα and MCP-1 [ 34, 35] and had a systemic anti-inflammatory effect, reducing Groα, Groβ and MCP-1 [ 36, 37]. These findings were observed in runners who did not develop LRTI, suggesting that they had a better inflammatory regulation response that may protect against infection. Therefore, athletic performance is both a stress factor and an adaptive response to exercise that can be modulated by training, reduce inflammation and help to prevent disease. Further studies are needed to better understand the relationship between salivary immunity and systemic inflammation as a key factor in the development of LRTI after exercise.

Interestingly, we observed a positive correlation between blood basophils and salivary Groα level before the race. Sastre B et al, identified basophils as a new player in the status of bronchial inflammation in athletes [ 38]. We observed that non LRTI runners had a higher percentage of systemic basophils, although differences were not statistically significant.

Our study has several limitations. First, due to the small number of runners included, the results should be validated in other studies before generalizing them. Second, since some data reported the effect of feeding in post exercise saliva AMPs proteins [ 39] we cannot exclude feeding related affectations in the determinations of AMPs in our cohort of runners. Third, levels of leukocyte subpopulations after the race were measured 48 hours after the marathon and other determinations at 7 or 14 days would be helpful to better characterize systemic response in marathon runners, and it should be an important point to be taken into account in further studies. And fourth, infectious agents that caused LRTI are not described due to the absence of microbiological studies. Further studies including bacterial and viral determinations would be very helpful to clarify this important issue.

In conclusion, non-elite runners who developed a LRTI after a marathon showed a differential profile of saliva IgA, AMPs and chemokines compared to those runners who did not develop infection. Therefore, exercise training and post-marathon recovery would be important in the immunological profile and the risk of developing a LRTI. Further studies are needed to better understand the underlying mechanisms and the impact of salivary immunity and its regulation in the prevention and development of LRTI in non-elite marathon runners.

References

1. 12. Woodhead M, Blasi F, Ewig S, Garau J, Huchon G, Ieven M, et al. Guidelines for the management of adult lower respiratory tract infections-Full version. Clin Microbiol Infect. 2011;17:E1-59.

2. 32. Terra R, da Silva SAG, Pinto VS, Dutra PML. Effect of exercise on the immune system: response, adaptation and cell signaling. Rev Bras Med do Esporte. 2012;18(3):208-14.

3. 39. Costa R JS, Fortes MB, Richardson K, Bilzon J LJ, Walsh NP. The effects of post-exercise feeding on saliva anti-microbial proteins. Int J Sport Nutr Exerc Metab. 2012

# data[].attributes.attachment_link.title
Salivary immunity and lower respiratory tract infections in non-elite marathon runners

# data[].attributes.attachment_link.description
Rationale Respiratory infections are common after strenuous exercise, when salivary immunity may be altered. We aim to investigate changes in salivary immunity after a marathon and its relationship with lower respiratory tract infections (LRTI) in healthy non-elite marathon runners.

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Amber Hofman, Jilske Maes, Jitske Michiels

# data[].attributes.subject
Amber Hofman, Jilske Maes, Jitske Michiels

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https://dl.acm.org/doi/pdf/10.1145/3132525.3132546

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Lore Verheyden, Eline Vermeulen en Sanne Verfaillie

# data[].attributes.subject
Lore Verheyden, Eline Vermeulen en Sanne Verfaillie

# data[].attributes.body
https://onlinelibrary.wiley.com/doi/full/10.1111/jar.12384

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Hayo Terband , Marjolein C. Coppens-Hofman , Maaike Reffeltrath , Ben A. M. Maassen ,

Centre for Language and Cognition (CLCG), Faculty of Arts, University of Groningen, Groningen, The Netherlands

Research School of Behavioural and Cognitive Neurosciences (BCN), University Medical Center Groningen, Groningen, The Netherlands
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Department of Medical Psychology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands
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Department of Medical Psychology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands

Nijmegen Centre for Evidence-Based Practice (NCEBP), Nijmegen, The Netherlands
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Corresponding Author

Utrecht Institute of Linguistics - OTS, Utrecht University, Utrecht, The Netherlands

Department of Medical Psychology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands

Centre for Language and Cognition (CLCG), Faculty of Arts, University of Groningen, Groningen, The Netherlands
Correspondence
Hayo Terband, Utrecht Institute of Linguistics - OTS, Utrecht University, Utrecht, The Netherlands.
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Abstract

Background

This study investigated the effect of speech therapy in a heterogeneous group of adults with intellectual disability.

Method

Thirty-six adults with mild and moderate intellectual disabilities (IQs 40-70; age 18-40 years) with reported poor speech intelligibility received tailored training in articulation and listening skills delivered in two 3-month periods. Pre- to post-changes in speech intelligibility and receptive vocabulary were assessed using standardized tasks.

Results

The results showed a positive effect of treatment on speech intelligibility and receptive vocabulary, irrespective of severity of intellectual disability, hearing loss and intellectual disability aetiology.

Conclusions

Speech therapy for people with intellectual disability can be effective at adult age and hearing loss should not prevent treatment. Continued attention to speech can help augment verbal communication skills in this population.

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Effectiveness of speech therapy in adults with intellectual disabilities

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Lara Van Den Bergh, Elza Szechtman en Maud Thijbergin

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Objective: The main objective of this study was to assess the associati…

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Paulien Dockx
Liesbeth Destrooper

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Language: English | Slovene

Abstract

Keywords: risk factors, voice disorders, voice quality

IZVLEČEK

Ključne besede: rizični faktorji, glasovne motnje, kakovost glasu

1. Introduction

In modern societies, about one third of the labour force works in professions in which one's voice is the primary tool. Voice problems are common throughout the general population, but they are even more common in professions which require prolonged voice use such as talking in surroundings with background noise, situations where there is a great distance between the speaker and the listener, poor room acoustics, a lack of adequate equipment such as voice amplifiers, etc ( 1).

Professional actors and singers perform a profession that requires a great vocal load and good voice quality at the same time. At work, they expose their vocal tract to various risk factors, which are more likely to cause voice problems. It is to be expected that frequent voice problems can jeopardise their professional careers. Therefore, they are legitimately classified among elite voice users as proposed by Koufman and Isaacson ( 2).

Singers and actors are among the most frequent visitors of voice clinics. In Boston, it was established that subjects working in arts and entertainment have the greatest relative risk for voice disorders requiring special care in a tertiary laryngological centre among all the patients' occupations ( 3).

Only a few surveys on the incidence of voice disorders in singers and actors have been performed. A systematic review and meta-analysis was conducted in order to find the incidence of self-reported voice problems in singers. The overall prevalence of self-reported dysphonia in singers was 46.09% (95% confidence interval: 38.16-54.12), with an incidence of 40.53% in classical singers ( 4).

Goulart and Vilanova conducted a survey in a group of professional actors on the occurrence of voice problems during their everyday vocal load. Thirty-five percent of the included actors reported voice problems, with 16% reporting problems since the beginning of their professional careers ( 5).

In Slovenia, candidates for the study of classical solo singing and (drama) acting must pass a phoniatric ear, nose and throat examination in order to prove that their vocal tracts are healthy and capable of the great vocal load required for their future occupation. The future actors also have a speech therapist's assessment of both speech and articulation before being accepted into the study programme. Therefore, future professional singers and actors must prove that they have healthy vocal tracts before their enrolment in the study programmes for occupations that involve a great vocal load and demand for high voice quality ( 6). There have been no studies on the prevalence of voice problems in elite voice users in Slovenia. The purpose of the study was to determine how frequently professional actors and singers report their voice problems, to compare the two groups in regards to voice problems, and to identify the risk factors for their voice problems.

2. Methods

Only professional actors and singers were included in the study. One hundred and thirty questionnaires were sent to the 5 largest professional theatres in Slovenia and 126 questionnaires to singers in the two largest professional choirs in Ljubljana.

The data on voice problems of professional actors and singers and their possible causes was obtained through this anonymous questionnaire. The questionnaire was similar to the one used for future (dramatic) acting and solo singing students during their phoniatric examinations, which are administered before they can be accepted to their selected study programmes. The questions sought information on age, gender, length of career, daily vocal load, voice rest during voice problems, vocal habits (speaking loudly, shouting frequently and fast speaking rate), factors affecting voice quality (gastroesophageal reflux, frequent throat clearing, allergies and asthma, smoking). All of the questionnaires that were returned were included in the study. In order to establish the prevalence of the voice problems of these elite voice users over different time periods, the professional singers and actors were asked about their voice problems during their studies, over the last year, and throughout their careers. Recurrent voice problems over the entire career with an impact on working ability manifesting more than three times per year were considered "frequent voice problems". They also detailed their opinion of the reasons for their voice problems.

The results of the questionnaires obtained from the professional singers were compared to the results of the professional actors. In order to identify the factors causing voice problems, data on vocal load, vocal habits and certain factors influencing voice quality in a subgroup of singers and actors with frequent voice problems over the entire career was compared to data from those with infrequent or no voice problems.

The data in the completed forms was statistically analysed using the x ² test, the Fisher's exact test, the t-test, and the Mann-Whitney test (in the case of non-normal arrangement of the data) included in the programme package SPSS 20.0 (SPSS Inc., Chicago, USA). All the statistical tests were two-sided and a p-value of 0.05 was considered to be statistically significant.

3. Results

Of the 130 questionnaires distributed to professional theatres in Slovenia, a total of 65 (50%) questionnaires were returned. Out of 126 questionnaires sent to professional choirs, we received 63 (50%). Thus, 128 subjects were included in our study: 65 professional actors (27 females, 38 males; mean age 43.03 years; SD 11.93 years) and 63 professional singers (32 females, 31 males; mean age 45.05 years; SD 11.52 years) There were no statistical differences between the singers and the actors regarding age (t-test, p=0.557) and gender (Fisher exact test, p=0.294).

All included questionnaires were at least 90% completed. Professional actors had worked in their occupation between 1 and 47 years (mean 19.66 years; SD 12.19 years) and professional singers between 1 and 39 years (mean 18.83 years; SD 10.53 years). There were no significant differences between the groups regarding length of career (Mann-Whitney test, p=0.679). Professional actors reported from 2 -16 hours of daily vocal load, while singers' daily vocal load ranged from 1 -7 hours.

The great majority of actors and singers had voice problems following the completion of vocal loading tasks while suffering from an upper respiratory tract infection. Thirteen singers and thirteen actors did not specify the cause of their voice problems. All these elite voice users did not report frequent voice problems or reported no problems. The groups did not differ in regards to the incidence of voice problems in any of the periods of their professional life or in the cause of their voice disorders ().

There were some significant differences between professional actors and professional singers regarding diseases and vocal and other habits influencing voice quality. Professional actors reported speaking loudly more frequently as well as a fast speaking rate, shouting and smoking. At least one inappropriate vocal habit (speaking loudly, fast speaking rate, frequent shouting) was reported in 31 singers and 59 actors (Fisher exact test, p=0.000). On the other hand, professional singers more frequently reported typical symptoms of gastroesophageal reflux (heartburn and/or regurgitation) ().

Comparing professional singers with frequent voice problems during their career to those without frequent voice problems revealed significant differences in two parameters. The professional singers with frequent voice problems reported more often loud speech and allergy problems than the singers without frequent voice problems ().

Comparing the group of professional actors with frequent voice problems (n=8) to the group of professional actors without frequent voice problems (n=56) did not offer up any particular differences (). One of the actors did not provide an answer about frequent voice problems.

4. Discussion

Professional actors and singers depend on their voices to pursue their profession. They need healthy vocal tracts with great endurance to manage the great vocal load at work. In Slovenia, only such candidates are enrolled in study programmes as elite vocal users. In the phoniatric examination performed prior to their acceptance (solo singing or drama acting), they must demonstrate both normal anatomy and function in their vocal tract ( 6). In the event that they report vocal and life style habits that may jeopardize their voice quality over the course of their careers, they receive information on proper voice care. They are also informed of the fact that maintaining harmful habits and an improper life style during their careers can lead to serious voice problems and can even endanger their career in the long run.

Our study did not reveal frequent voice problems in a considerable section of the included professional singers and actors. Only 12.3% of actors and 20.6% of singers admitted having recurrent voice problems that prevent their performance more than three times per year in their career. The data from the literature shows voice problems occurring more often than in our research, although the study design was not exactly the same in other studies. Goulart and Vilanova reported 35% of the actors included in their study with voice problems in the daily voice load. In 16% of actors, the problems started even at the beginning of their careers ( 5). In classical choir singers or professional opera choristers, self-reported voice problems were detected in 38% - 43.59% ( 7, 8). We suppose that one of the important reasons behind the small percentage of professional actors and singers with voice problems in our study is the preventive phoniatric examination and, in the case of future actors, also the speech therapist's assessment before enrolment in the study of drama acting or solo singing in Slovenia. Consequently, only one actor and one singer from our study had frequent voice problems during their study period and also later in his/her career. On the other hand, at the time of their study, the vocal load was not as extensive as later on in their careers, therefore voice problems become more evident after one starts working in an occupation with great vocal load. It is possible that other studies included singers and actors who did not conclude a proper professional study programme, and did not have to demonstrate having a healthy and well-functioning vocal tract. The data for this assumption are lacking in the literature.

Another piece of evidence for the correct selection of candidates as future actors is the fact that only 12.5% of actors had frequent voice problems in their careers in spite of the fact that half of them were smokers, and almost all of them had improper voice habits. There were no significant differences between the singers and the actors regarding frequent voice problems in their educational period, in the last year, and over their entire career. On the contrary, the actors' daily vocal load was significantly greater than the vocal load of the singers.

Another reason for the small percentage of singers with frequent recurrent voice problems in our study could be their style of singing, and proper education. In a group of 100 healthy singers, Koufman et al tried to find out whether factors such as gender, professional or amateur singing, and style of singing influence vocal muscles' tension. The lowest incidence of excessive muscle tension was observed in professional singers, singers with formal singing education, and in singers of classical opera or choir singing ( 9). The singers in our study were members of two professional choirs with a classical singing repertoire. A formal education in singing training is a necessity for employment in a professional choir. Pestana et al. reported in his meta-analysis that the overall prevalence of self-reported dysphonia in singers was 46.09%, with an incidence of 40.53% in classical singers. In their systematic review and meta-analysis, the authors stressed a considerable heterogeneity of the reviewed studies including lacking data on the professional education of the participants of the study ( 4).

The unhealthy vocal and other behaviour noticed among elite voice users in our study is worrying because it can result in benign vocal fold lesions ( 10). The presence of inappropriate speech habits in almost all included actors (90.8%) is understandable, since their work often requires loud speech, fast talking, crying, shouting, singing and dancing at the same time ( 11). The nature of the work of professional choir singers does not demand other harmful speech habits but voice overload is possible. Nevertheless, professional singers expressed inappropriate speaking habits at a rate of almost 50%. Speaking loudly was also one of the risk factors for frequent voice disorders during their career of professional singer. On the other hand, other studies do not report such a high incidence of voice abuse as ours. One of the published voice profile analyses of 16 solo singers revealed voice abuse only in 6% ( 12). It is possible that one of the reasons for our unfavourable results about considering proper voice care is a lack of information on vocal hygiene during the career. Candidates for elite vocal performers receive information on possible improper vocal and lifestyle habits that can influence their voice quality and their careers only at the time of phoniatric examination before being accepted into the academy. Later on, during their studies, they have courses on singing techniques, rhetoric issues and performing, but no lectures on the physiology of phonation, voice disorders, voice care and vocal hygiene. We suppose that supplementing a curriculum for solo singers and actors with such information would be beneficial for the voice health of these elite vocal performers.

The professional singers in our study reported typical symptoms of gastroesophageal reflux more often than the actors. We can find the results of frequent gastroesophageal reflux in singers in the literature ( 13, 14, 15). Even in recent papers, opera singers and solo singers reported heartburn, regurgitation, coughing and hoarseness more often than the controls ( 16). The reason is probably the increased intraabdominal pressure during singing which causes the retrograde flow of gastric content up the oesophagus to the level of the larynx and pharynx ( 17). The occupation of a professional singer or actor combines a lifestyle with working late, inappropriate nutritional habits, late meals in the evening or even at night and a lot of stress. All these are risk factors that influence acid reflux. It is also possible that professional singers report LPR more often because of their need for a high voice quality and are more watchful in terms of voice changes ( 18). In any event, allergy and asthma, but not gastroesophageal reflux, were found to be risk factors for frequent voice problems in singers. Professional singers should be aware of a possible negative influence of these diseases on their voice quality, and should seek early medical help when having such health problems.

In the group of professional actors, we need to highlight the problem of smoking, as almost half of them reported smoking. Other studies also proved smoking to be a problem in the majority of actors ( 19, 20). Another research study showed that the percentage of improper habits (smoking, late meals in the evening, vocal abuse), regardless of voice education and getting information about voice hygiene, remains high ( 21). Smoking is not as frequent among singers as among actors, as was also shown in our study (17% of singers are smokers). Among Broadway musical theatre performers, tobacco use was reported by only 10.4% of them ( 22). The low percentage of smokers among singers was found also in a study of Timmerman ( 12).

Professional singers more often considered voice rest during voice problems (54%) than professional actors (46%). It is possible that they are more sensitive to the quality of their voices because, in singing, even a slight alteration of the voice can influence one's performance capabilities. On the other hand, there are some reports of more abusive voice use during hoarseness in singers as well. A study by Boominathan showed that only 22% of singers considered voice rest during voice problems ( 23).

A person having a voice problem or getting over an acute respiratory infection, should reduce voice load to avoid the development of a functional voice disorder ( 24). Even though professional singers and actors are aware of the necessity for voice rest during voice problems, they cannot always fully consider it due to the nature of their work. The results of our study showed that a great majority of the participants had voice problems following the completion of vocal loading tasks while suffering from a respiratory infection.

In order to further reduce inappropriate lifestyle issues, improper habits and speech abuse, the managers of theatres and professional choirs should organize occasional workshops for their employees, where they will be able to renew their knowledge of the anatomy and functioning of the vocal tract, the causes of voice problems and methods of preventing problems. Such knowledge can help them to avoid the occurrence of voice disorders. In this area of lifelong education for elite voice users, the speech therapist can play a very important role, not only with information on the influence of harmful factors on the voice, but also with practical instructions for the most rational voice use and the correct method of phonation and modulation of phonation when performing and talking.

5. Limitations of the study

One of the limitations of the study is the subjective perception of voice problems reported in the questionnaire. In the case of additional phoniatric and speech pathologist examination of the included professional singers and actors, a better insight would be given into the actual state of the voice apparatus of elite voice users in Slovenia.

On the basis of the questionnaire used we did not obtain information about the exact time of appearance of voice problems. Therefore we cannot exclude the possibility that ageing of the voice or hormonal problems influenced voice quality in the participants.

Another limitation of the study is the small number of participants. In the evaluation of this factor, it should be noted that there are only a few professional choirs and only eleven professional theatres in Slovenia.

6. Conclusion

The present study among elite voice users in Slovenia confirmed the importance of preventive examinations before enrolment in the study of drama acting and solo singing. According to the results, professional actors and singers have insufficient knowledge or disrespect of proper voice care. Worse voice behaviour was demonstrated by actors, whose percentage of inappropriate speech and smoking habits was far too high for a representative of elite voice users. Speaking loudly and having an allergy or asthma proved to be risk factors for frequent voice problems in professional singers. Professional singers and actors should have additional information on the risk factors that impair their voice quality as well as occasional phoniatric or speech and language therapist examinations.

Speech and language therapists can play a crucial role in lifelong learning and monitoring elite voice users, thereby effectively preventing voice problems that might render them unable to pursue their profession.

Footnotes
CONFLICTS OF INTEREST
The authors declare that no conflicts of interest exist.
FUNDING
The study was financed through the tertiary research project of the University Medical Centre Ljubljana No. 20150104 Incidence of voice problems in professional singers and actors.
ETHICAL APPROVAL
This study was approved by the Republic of Slovenia National Medical Ethics Committee (document No. 0120-334/2015-2).

References

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Risk Factors for Voice Problems in Professional Actors and Singers

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The purpose was to determine the incidence of voice disorders in a group of professional actors and singers, to compare the two groups, and to investigate the potential causes of their voice problems.65 actors and 63 singers from professional theatres ...

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Margot Coudron, Manou Knockaert, Chloé Gombert & Paulien Keppens 

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Margot Coudron, Manou Knockaert, Chloé Gombert & Paulien Keppens 

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Mostaert themanummer diagnostiek 2019 (2)

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Femke Van Poucke, Elena Van Muylem & Justine Van Nieuwenhuyse

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Allyssa De Hondt, Lisse Deshommes & Femke De Gols

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Allyssa De Hondt, Lisse Deshommes & Femke De Gols

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Door Noor, Leni en Flore

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Artikel Eline Vandewiele, Elias Abbeel en Lana Lek

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Kyra Platteau, Isabeau Vanden Berghe en Karolien Lambers

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Kyra Platteau, Isabeau Vanden Berghe en Karolien Lambers

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artikel PRA opdracht Anterior open bite and speech disorders in children with Down Syndrome

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Alice Vanhoucke, Marie De Blanger en Linde Van De Velde 

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Alice Vanhoucke, Marie De Blanger en Linde Van De Velde 

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Background

Presbycusis is the most common sensory impairment associated with ageing and it presents with variability of symptoms. Physicians need to recognize early clinical and audiometric signs of presbycusis in order to render adequate and quality care to patients and reduce associated morbidities.

Objective

To characterize the clinical modes of presentation and the typical audiometric tracings among patients with presbycusis.

Methods

This descriptive, prospective hospital-based study was conducted in the Ear, Nose and Throat (ENT) clinic of Olabisi Ona hing Hospital, (OOUTH) Sagamu, Nigeria. Patients with clinical diagnosis of presbycusis confirmed with bilateral sensorineural hearing loss (SNHL) on diagnostic audiometry were administered with questionnaires. Information obtained was analyzed using SPSS statistical package version 17.0 and presented in descriptive forms as percentages, means and graphs.

Results

Sixty-nine patients were diagnosed with presbycusis (M:F =1.6:1). Modal age group was 71-80 years. Hearing loss 88.4%, tinnitus 79.7% and vertigo 33.3% were the major symptoms on presentation. The average duration of symptoms before presentation was 2.6 years. There was positive history of ototoxic drugs usage in 24.6 %, family history in 11.6 %, hypertension in 34.8% and osteoarthritis in 13.0%. The most common type of audiometric pattern was strial. Hearing losses increased with age both at the speech and at the higher frequencies of sounds.

Conclusions

We found hearing impairment affected both speech and higher frequencies and the strial type of audiometric pattern was most common. The need for screening for hearing impairment from early middle age in symptomatic individuals is emphasized.

# data[].attributes.attachment_link.title
Clinical and audiometric features of presbycusis in Nigerians.

# data[].attributes.attachment_link.description
Background Presbycusis is the most common sensory impairment associated with ageing and it presents with variability of symptoms. Physicians need to recognize early clinical and audiometric signs of presbycusis in order to render adequate and quality care to patients and reduce associated morbidities.

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# data[].attributes.headline
Frauke Dedeurwaerder; Ibe Vyncke; Anke Van Lathem

# data[].attributes.subject
Frauke Dedeurwaerder; Ibe Vyncke; Anke Van Lathem

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Your access to the NCBI website at www.ncbi.nlm.nih.gov has been temporarily blocked due to a possible misuse/abuse situation involving your site. This is not an indication of a security issue such as a virus or attack. It could be something as simple as a run away script or learning how to better use E-utilities, http://www.ncbi.nlm.nih.gov/books/NBK25497/, for more efficient work such that your work does not impact the ability of other researchers to also use our site. To restore access and understand how to better interact with our site to avoid this in the future, please have your system administrator contact info@ncbi.nlm.nih.gov.

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Your access to the NCBI website at www.ncbi.nlm.nih.gov has been temporarily blocked due to a possible misuse/abuse situation involving your site. This is not an indication of a security issue such as a virus or attack.

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https://files.eric.ed.gov/fulltext/EJ1098182.pdf

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https://pdfs.semanticscholar.org/9ab1/ce2979172fa5c9f0c48461dba99946a4b5a2.pdf

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https://jnnp.bmj.com/content/jnnp/46/2/140.full.pdf

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Introduction

About one fourth of tinnitus sufferers in Germany considers stress as the main reason for their tinnitus1,2. Similarly, many clinicians relate tinnitus onset and maintenance not only to cochlear mechanisms, but also to stress3. Despite the suggestion of a link between stress and tinnitus, there is little empirical support for this idea to date. Several issues are discussed in the association of tinnitus and stress. First, stress may be a predisposing risk factor of tinnitus. In-patients with tinnitus indicated more stress than a normative healthy control group1 and reported more frequent and more stressful life events than a clinical control group6. Stress also seems to be an important factor in the transition from mild to severe tinnitus7. Second, cognitive maladjustment to stressful situations could favor tinnitus onset and maintenance8,9. In accordance, tinnitus patients used more maladaptive coping strategies than a clinical control group6. Neuroticism, a personality factor associated with increased experienced levels of anxiety, sadness, embarrassment, and guilt, has also been identified as a risk factor for tinnitus severity10. Lastly, tinnitus symptoms themselves may act as a stressor, resulting in higher general physiological arousal and psychological distress11. Consistent with this idea, research showed elevated ratings of subjective strain in response to experimentally induced stress in tinnitus patients compared to controls12.

About 10-15% of the general population is affected by tinnitus and the majority habituates to it13,14. Only in about 2-6% of all tinnitus patients the symptoms cause a considerable amount of psychological distress interfering with their lives14,15. The psychological understanding of why tinnitus becomes problematic in a small proportion of patients suggests that in those cases, it acquires a negative emotional significance through maladaptive cognitive appraisal and dysfunctional processes in the autonomic nervous system (ANS). Thus, the negative, catastrophic interpretations of the tinnitus percept are sustained and habituation to the tinnitus fails. Central to cortical mechanisms associated with tinnitus annoyance is the limbic system. The amygdala, essential for the emotional evaluation of sensory stimuli and in turn also a control structure of the ANS, may be the essential common correlate in the maintenance of tinnitus annoyance and its bodily consequences, e.g. elevated levels of arousal. This idea is supported by the finding that temporarily inactivating the amygdala can transiently reduce tinnitus annoyance20. Additionally, imaging studies demonstrated that limbic structures, including the amygdala, are frequently altered in tinnitus patients21,22,23,24.

Especially the sympathetic branch of the ANS plays an important role in stress and its chronic hyperactivity also seems to be involved in tinnitus25. This idea is corroborated by the old finding that sympathectomy can relieve tinnitus symptoms in patients with Ménière's disease26. Recent studies have observed a positive association between tinnitus-related distress and sympathetic tone27 and a negative association between tinnitus-related distress and parasympathetic tone28. Also, successful suppression of tinnitus has been associated with an increase in parasympathetic tone29. Reports from tinnitus patients indicating higher levels of muscle tension in face, jaw and shoulder in contrast to healthy controls may provide a further hint for hyperarousal in tinnitus, even if this relationship may also be driven by direct somatosensory-auditory interactions. Finally, the frequent co-occurrence of insomnia33,34 and depression35 with tinnitus might be mediated by hyperarousal19.

Taken together, autonomic functioning may be dysregulated in patients with chronic tinnitus, plausibly associated with a state of chronic stress16. In accordance, psychoendocrine research showed attenuated biological stress reactions measured by cortisol levels in tinnitus patients. In contrast, one further study could find no altered psychophysiological stress reactivity in tinnitus as assessed with electromyography and skin conductance level12.

Recent psychophysiological research has advanced the role of heart rate variability (HRV) as another potent indicator of ANS activity and stress in healthy and clinical samples. HRV describes the phenomenon that the duration of intervals between consecutive heartbeats (RR intervals) is subject to spontaneous oscillations42. Alterations in RR intervals may occur before any appreciable change occurs in HR itself43,44. Sympathetic activity induces an increase in HR with a concomitant decrease in HRV, while parasympathetic activity leads to a lower HR and a simultaneous increase in HRV. The two systems are constantly interacting to ensure adaptability41,45. Global HRV is supposed to reflect the interplay of influences from the sympathetic and parasympathetic branch41,46. Generally, high HRV is considered neurologically healthy47. Under acute stress, HR increases, HRV decreases transiently, and high correlations between the reduction in HRV and the perceived stress are observed in healthy samples. Hence, HRV is considered an important marker of psychological stress45.

Few studies have used HR or HRV as markers of basal ANS functioning in chronic tinnitus. Two studies reported decreased HRV and a relative predominance of the sympathetic branch of the ANS in tinnitus patients27,28, whereas one further study did not detect differences in basal ANS activity between controls and elderly tinnitus patients50. We are not aware of any study that has assessed tinnitus patients' ANS reactivity, i.e. stress response, with measures of HR and HRV. However, provocation tests are an essential aspect for stress assessment51. Moreover, increases of subjectively perceived stress are associated with increased tinnitus loudness52. Therefore, in the present study we initially used HR and HRV to investigate the ANS reactivity in patients with chronic tinnitus and healthy participants without tinnitus (controls). We examined ANS activity at rest and the ANS response to stress in two different stress tasks (arithmetic stress and concentration on the own tinnitus). Relaxation/resting phases after each stress task served to investigate the recovery from stress. We included subjective ratings of the perceived stressfulness of each experimental condition to compare physiological data with subjective strain. Additionally, tinnitus patients indicated the extent of the perceived presence of their tinnitus during each condition.

Method and Materials

Participants

Patients with chronic tinnitus out of the database of the Multidisciplinary Tinnitus Center Regensburg were contacted via postal mail and invited to participate in the study. Controls were recruited from the local community via adverts and selected by matching for age, sex, and education. Exclusion criteria were cardiovascular disease or diseases with influence on the autonomic nervous system (e.g. diabetes), intake of psychotropic medication (e.g. antidepressants) and other medication known to alter autonomic functioning up to four weeks before the study and regular usage of illegal drugs. Tinnitus was subjective and chronic in all participants with tinnitus, i.e. had been present for at least six months (mean duration = 139.42 months, SD  = 113.44). Tinnitus was perceived monaurally in 15.8% and binaurally in 84.2% of patients with chronic tinnitus. Tinnitus was described as a tone (84.2%) or noise (15.8%). Tinnitus patients indicated poor hearing (21.1%), noise sensitivity (21.1%), both (31.6%) or no further ear-related problems (26.3%).

Participants gave written informed consent and the study was approved by the Ethics Committee of the University Hospital of Regensburg (reference number: 16-101-0037) and carried out in accordance with the approved guidelines.

Forty-one participants participated in the study. Each participant was tested in an individual session and was compensated with 20€. Two persons were excluded from the final analysis due to difficulties in carrying out the arithmetic stress task. Another participant was excluded because the electrocardiogram (ECG) showed ectopic beats. All three dropouts were patients with chronic tinnitus. The final sample comprised 38 persons, ages 21 to 70 ( M = 49.32, SD = 11.49, 42% female), with 19 persons in each experimental group. Table 1 summarizes sample characteristics. No significant differences in age, sex, education, weight, caffeine consumption, smoking, and alcohol consumption were found. Thus, the comparability of the groups was given. To rule out the possibility that the descriptively higher cigarette consumption in the tinnitus group had influenced our data, we calculated all analyses excluding the smokers in the tinnitus group ( n = 3) and the control group ( n  = 3), which did not change our results.

Table 1 Clinical and Demographic Characteristics.

Procedure

The experiment took place at the Department of Psychiatry and Psychotherapy of the medbo Regensburg from March 2016 till April 2016. Participants were asked not to drink caffeinated beverages or smoke up to one hour before the start of the experiment. Participants underwent a screening interview for personal data, medical history and consumption habits. Tinnitus patients were additionally interviewed about laterality of their tinnitus, type of tinnitus percept, and further problems with their ears. After that, participants were familiarized with the arithmetic stress task. Then, they filled out various questionnaires (Major Depression Inventory (MDI53; German version54), Perceived Stress Questionnaire 20 (PSQ-2055; German version56), NEO-Five-Factor-Inventory (NEO-FFI57; German version:58), Mehrfachwahl-Wortschatz-Intelligenztest for estimation of intelligence (MWT-B59), Tinnitus Questionnaire (TQ8; German version60). Next, participants were seated comfortably in a chair and sensors for HRV assessment were attached. They were instructed not to speak and to restrict all voluntary movements to a minimum during the ECG recordings. The experimental procedure is illustrated in Fig. 1. First, participants underwent a resting measurement with a duration of five minutes (baseline). They were asked to relax as much as possible without closing their eyes to ensure comparability across conditions. Afterwards, the first stress measurement (stress I) of five minutes took place during which participants conducted the arithmetic stress task. Subjects were asked not to communicate their reaction to the stress tasks overtly (e.g. cursing) to prevent interference with ECG data collection. After that, a relaxation phase (relaxation I) identical to the baseline measurement took place. For the second stress measurement (stress II), participants were given earplugs to shield themselves from any noise present in the room. Tinnitus patients were instructed to concentrate on their tinnitus percept, while controls were asked to listen to noises possibly observable in their ears. After five minutes, earplugs were removed and participants passed a final relaxation phase (relaxation II) of five minutes. For each experimental condition, a separate ECG recording was gathered. After each condition, participants had to indicate their subjective level of perceived stress ("How stressed have you been feeling during the last five minutes?") verbally on a 11-point Likert scale (0 = not at all; 10 = very much). Participants with chronic tinnitus also indicated the subjective perception of the tinnitus during each of the five measurements ("How many percent of the time have you been consciously aware of your tinnitus in the last five minutes?"). After the ratings for the second relaxation phase were obtained, sensors for data recording were removed. The entire experiment took about 75-90 minutes.

Arithmetic Stress Task

Mental arithmetic, a mild, but effective environmental stressor previously used in healthy41,61 and clinic samples12,62 was administered to induce stress in participants. To minimize the effects of speaking on HRV61, the arithmetic stress task was carried out silently on a computer screen (HP, Type 1702, screen width 17") that was positioned approximately one meter in front of the participants. The task was written with the stimulus-delivery software Presentation, version 0.71 (Neurobehavioral Systems, Berkeley, CA, USA). Information was presented in white font (font size: 29 pt) on a black screen. Participants were presented with arithmetic problems that required adding or subtracting the number 17 to or from two- or three-digit figures (e.g. 176 + 17; 61-17) which was determined as a potent stressor in preliminary tests in our laboratory. After the arithmetic problem had appeared in the center of the screen for 1000 ms, the screen turned black for 4000 ms and participants had to solve the problem. Using a wireless keyboard on their laps, participants had to select the correct solution from two alternatives that were presented simultaneously for 1000 ms by pressing either the left or right arrow key corresponding to the locations of the solutions on the screen. Immediately after their response or a maximum of 1000 ms, trial-to-trial feedback of 1000 ms was provided as to if the participant had chosen the right solution ("Correct"), the wrong solution ("Incorrect") or took too long (>1000 ms) to answer ("Too slow"). The next trial started automatically after feedback was delivered. The arithmetic stress task with a total of 45 arithmetic problems lasted for 5 min. A training session with five simple items and verbal instructions delivered by the experimenter at the beginning of the experiment (before electrode installation) ensured that participants understood the task and the stimulus response-assignments.

ECG recordings and data analysis

ECG data were recorded using four silver/silver chloride (Ag/AgCl) surface ring electrodes (EASYCAP GmbH, Herrsching, Germany) with an outer diameter of 1.2 cm and an inner diameter of 0.6 cm. The ECG signal was amplified and digitized with a BrainAmp DC amplifier linked to BrainVision Recorder software (both Brain Products GmbH, Gilching, Germany). The sampling rate was set at 1000 Hz. After cleansing the participant's skin with isopropyl alcohol (70%), two active electrodes were attached beneath the left and right clavicle. A reference electrode and a ground electrode were attached in the participant's neck area. The electrodes were covered with electrode cream (Grass, Warwick, RI, USA) to ensure good conductivity.

For preliminary off-line data processing, BrainVision Analyzer (Brain Products GmbH, Gilching, Germany) was used. A high pass filter of 15 Hz and a low pass filter of 45 Hz were implemented, as well as a Notch filter (50 Hz). The recorded data were re-referenced to an averaged ECG signal using linear derivation (left minus right clavicle electrode). For subsequent HRV data analysis according to the guidelines recommended by the Task Force of the Society of Cardiology (1996), we used Kubios HRV63. All recordings were visually examined and manually corrected for artifacts. In two cases, artifacts caused by coughing were located at the beginning or ending of the recording, so only the artifact-free period was analyzed. For one recording with high rates of voluntary movement, different filter settings (high pass: 15 Hz, low pass: 25 Hz) were applied in preliminary data processing to be able to obtain RR intervals series from the data.

Two essential measures in the time domain are mean HR, which is indexed in beats per minute, and the standard deviation of normal to normal RR intervals (SDNN), which is given in units of ms. SDNN estimates global HRV. We calculated mean HR and SDNN for each subject in each of the five experimental conditions. Further analyses using frequency domain measures of HRV (low- and high-frequency power) showed results identical to SDNN and are not reported in this paper.

Design

A two (group factor: tinnitus group vs. control group) by five (condition: baseline, stress I, relaxation I, stress II, and relaxation II) mixed factorial design with repeated measures on the experimental condition variable was administered. Dependent variables were mean HR and global HRV (SDNN) as well as subjective ratings of stress. Additionally, a one-way repeated-measures ANOVA was conducted to explore the effect of condition on the perceived presence of tinnitus during measurements.

Statistical analysis

Statistical analyses were performed with SPSS 22.0 (SPSS/IBM, Chicago, IL, USA). The significance threshold was set at p = 0.05 for the statistical tests. Greenhouse-Geisser-correction was applied if the assumption of sphericity was violated in repeated-measures analysis of variance (ANOVA). Bonferroni correction was used for pairwise comparisons. Effect sizes for the t-tests are reported with Cohen's d calculated with G*Power software, version 3.166. Exploratory correlational analyses for physiological and psychological measures of stress were conducted using Pearson's r. Scatterplots served to detect outliers.

Results

Arithmetic stress task

On average, the tinnitus group solved 25.74 ( SD = 5.01) of the 45 arithmetic problems presented on the arithmetic stress task correctly. The mean number of correct responses in the control group was similar ( M = 27.63, SD = 5.86). The performances did not differ significantly ( t  < 2).

ECG data

Mean HR

Results for mean HR are presented in Fig. 2. Mean HR data was submitted to a mixed-design 2 (group factor) × 5 (condition) ANOVA. Mauchly's test indicated that the assumption of sphericity had been violated, χ ²(9) = 56.08, p < 0.001, therefore degrees of freedom were corrected using Greenhouse-Geisser estimates of sphericity (ε = 0.52). The ANOVA revealed a main effect of group factor, F(1, 36) = 7.07, p = 0.012, η p ² = 0.16, which indicated that, overall, mean HR was lower for patients with chronic tinnitus than for controls. The analysis also yielded a main effect of condition, F(2.07, 74.46) = 39.65, p < 0.001, η p ² = 0.52. The interaction between group factor and condition was also significant, F(2.07, 74.46) = 4.31, p = 0.016, η p ² = 0.11. Bonferroni-corrected post hoc analyses confirmed that controls displayed a significantly higher mean HR during stress I ( p = 0.015, d = 1.06). None of the other comparisons reached significance level (all ps > 0.10.) Thus, the interaction effect can be explained by mean HR values during stress I where tinnitus patients showed specifically lower mean HR than did controls. Overall in both groups, during stress I, i.e. the arithmetic stress task, mean HR was significantly higher than during all other conditions phases (all ps < 0.001). Hence, stress induction using mental arithmetic was successful. Additionally, stress II, i.e. attention allocation to tinnitus/ear noises, differed significantly from baseline ( p = 0.002) and relaxation I ( p = 0.006). None of the other comparisons reached significance level (all p s > 0.114).

SDNN

A 2 (group factor) × 5 (condition) ANOVA was conducted on SDNN data. Neither the group factor nor the experimental condition had a statistically significant impact on SDNN data ( Fs < 2). The interaction of group factor × condition did not reach significance level, either ( F  < 1).

Correlation of mean HR and SDNN

We correlated mean HR with SDNN data for the two groups separately. In the control group, there was a trend towards statistical significance for the association of HR and SDNN during baseline, r(19) = −0.26, p = 0.143, relaxation I, r(19) = −0.21, p = 0.198, stress II, r(19) = −0.23, p = 0.169, and relaxation II, r(19) = −0.30, p = 0.107. In the tinnitus group, the association of HR and SDNN trended towards statistical significance during relaxation I, r(19) = 0.31, p = 0.101. No other trends or statistically significant relationships were found (all p s > 0.389).

Verbal Ratings

Subjective stress ratings

Figure 3 illustrates subjective stress ratings after each experimental condition for tinnitus patients and controls. These results were submitted to a 2 (group factor) × 5 (condition) ANOVA. Mauchly's test indicated that the assumption of sphericity had been violated, χ ²(9) = 30.00, p < 0.001, therefore degrees of freedom were corrected using Greenhouse-Geisser estimates of sphericity (ε = 0.74). The analysis revealed a significant main effect of group, F(1, 36) = 10.37, p = 0.003, η p ² = 0.22, which indicated that, overall, tinnitus patients perceived all five conditions as more stressful than did controls. Additionally, there was a significant main effect of condition, F(2.94, 105.9) = 53.68, p < 0.001, η p ² = 0.60. However, these main effects were qualified by a significant group × condition interaction, F(2.94, 105.9) = 7.22, p < 0.001, η p ²  = 0.17.

On average, a trend towards higher subjective stress was reported by tinnitus patients than by controls during relaxation I, but the comparison did not reach significance, t(36) = 1.83, p = 0.076, d = 0.60. Subjective stress ratings for stress II were significantly higher in the tinnitus group than in the control group, t(18.56) = 4.39, p < 0.001, d = 1.42. Additionally, relaxation II was perceived as significantly more stressful by tinnitus patients than by controls, t(19.81) = 3.47, p = .002, d = 1.13. None of the other comparisons reached significance level (all ts < 1). Bonferroni-corrected pairwise comparisons confirmed that in both groups, stress ratings for stress I were significantly higher than for all other four conditions (all ps < 0.001), providing further evidence that stress induction using mental arithmetic was successful. In addition, ratings after baseline differed significantly from ratings after stress II ( p = 0.011). None of the other comparisons reached significance ( p s > 0.236).

Perceived presence of tinnitus

Results for the ratings of the perceived temporal presence of tinnitus during each condition are shown in Fig. 4. A one-way repeated measures ANOVA was conducted to examine the effect of condition on the perceived presence of tinnitus. The assumption of sphericity was violated as indicated by Mauchly's test, χ ²(9) = 17.82, p = 0.045, thus degrees of freedom were corrected using Greenhouse-Geisser estimates of sphericity (ε = 0.69). The ANOVA showed that ratings of the perceived presence of tinnitus changed within the conditions, F(2.76, 49.68) = 26.63, p < 0.001, η p ²  = 0.59.

Bonferroni-corrected pairwise comparisons indicated that the perceived presence of the tinnitus during stress I was in both groups significantly lower than in relaxation I ( p = 0.001), stress II ( p < 0.001) and relaxation II ( p < 0.001). Additionally, tinnitus was perceived more extendedly in stress II than during baseline ( p < 0.001), relaxation I ( p = 0.002) and relaxation II ( p = 0.006). Moreover, the perceived presence of tinnitus was higher during relaxation II than during baseline ( p = 0.016). None of the other comparisons reached significance level ( p s > 0.060).

Correlation analyses

Stress ratings and HRV data

To explore the association between subjective acute strain and physiological stress reactivity, we correlated self-reported stress ratings with the respective ECG data (mean HR, SDNN) for the two groups separately. There were no significant correlations between stress ratings and mean HR. HRV measured with SDNN was negatively correlated with strain ratings during the mental arithmetic task in the control group, indicating that a reduction in HRV was associated with higher subjective strain, r(19) = −0.47, p  = 0.042. In the tinnitus group, no such association was found neither for the arithmetic stress task nor the stressor "attention allocation to the own tinnitus".

TQ score and HRV data

For the tinnitus group, correlation analyses were used to explore relationships between tinnitus severity as measured with TQ scores and HRV data (mean HR, SDNN). Only non-significant relationships were found.

TQ and subjective stress ratings

Correlations between TQ scores and subjective stress ratings were analyzed. TQ scores were positively correlated with stress ratings for the first relaxation phase, r(19) = 0.62, p  = 0.005. None of the other correlations reached significance level.

Discussion

In this study, we investigated the stress reactivity in patients with chronic tinnitus using HR and HRV. Contrary to previous studies27,28, tinnitus patients and control subjects did not differ significantly in baseline HRV. These results challenge the assumption of a greater basal arousal in patients with chronic tinnitus19. Unexpectedly, baseline levels of mean HR were significantly lower in tinnitus patients than in controls. We expected increased baseline HR levels in tinnitus sufferers as it is known from patients with PTSD67, another disease with hyperarousal as major symptom. It is possible that hyperarousal affects tinnitus patients not as much as PTSD patients. Additionally, as exclusion criteria in the present study were stringent to control for confounders of HRV, the tinnitus sample was rather homogeneous and tinnitus severity was lower than in other investigated clinical tinnitus samples (e.g.68). The rather low tinnitus severity in our sample may have been the reason why we did not detect HRV alterations. Yet, our data are in line with a recent study that reported no group differences in HRV, but significantly lower mean HR in the tinnitus group, using 24h-Holter-recordings to explore ANS functioning in an elderly tinnitus population50. Decreased HR in patients with chronic tinnitus may be interpreted as physiological long-term adaptation of the cardiac system to mild chronic stress, comparable to competitive sports where trained athletes show decreased HR compared to untrained controls69. Psychoendocrine research has suggested tinnitus as a chronic stressor resulting in diminished efficiency of the stress hormone system36,38.

Relative to healthy controls, tinnitus patients displayed a lower mean HR increase during the first stress task. This was confirmed by the significant interaction effect of group factor × condition for mean HR and post hoc analyses that illustrated that group differences were greatest during stress I. Mean HR therefore suggests that tinnitus patients' response to stress was attenuated, indicating maladaptive ANS reactivity. Similarly, previous studies showed blunted cortisol stress responses in patients with chronic tinnitus. Contrary to HR, HRV did not reflect stress exposure - neither in the patient group nor in the control group - even though HRV decreased in both groups on a descriptive, statistically not significant level in response to the arithmetic stress task.

These findings suggest that HRV may not be a valid marker of stress. The reliability of short-term measures of HRV has been questioned70. Moreover, HRV has been shown to be prone to confounding variables such as gender, age, weight, smoking, depression, medication of antidepressants, antihypertensive drugs and antiarrhythmic drugs, and importantly, HR itself. Successful matching and preliminary screening ensured that these confounding variables were controlled for in our study sample, except for HR, which differed significantly between the groups. It is possible that group differences in HR prevented us from detecting subtler differences in HRV. Higher HR has been associated with lower HRV47. We observed this trend in the control group during four phases of measurement. In the tinnitus group, no associations were found, except for relaxation I, where the trend was opposed to common observations, i.e. a higher HR was associated with higher HRV. Speculatively, the reduced HR and the reversal or lack of correlation between HR and HRV reflect a dysfunction of the ANS in tinnitus patients resulting in abnormal regulation of physiological variables observed in healthy controls, i.e. increase in HR with concomitant decrease in HRV in response to stress.

Subjective perceived stress was increased during the relaxation phases and stress II in tinnitus patients compared to controls, whereas the arithmetic stress tasks induced similar levels of subjective stress in both groups. Tinnitus presence may be a factor influencing the subjective stress experience in tinnitus patients. Ratings for the perceived tinnitus presence indicated that during arithmetic stress exposure, patients were distracted from their tinnitus percept and stress levels were similar to the ones of controls. During the following relaxation phase, the tinnitus was subjectively perceived for a longer amount of time due to a discontinuation of distraction and simultaneous re-focusing on the tinnitus percept and stress levels remained high. When tinnitus patients should focus on their tinnitus percept during the second stress phase, no distraction was given and ratings for the perceived tinnitus presence further increased in comparison to arithmetic stress exposure. In the subsequent relaxation phase, tinnitus presence and stress levels again stayed high. The lack of decline in stress ratings during the relaxation phases may signal disturbed recovery from stress in tinnitus patients. Increased tinnitus presence after the stress tasks is in line with anecdotal reports of many tinnitus patients who indicate that tinnitus presence is augmented after phases of intensified stress or physical activity. One could speculate that subjective strain after stress may take longer to abate because physiological adaptation to stress fails during stress exposure. Also, stress ratings for the first relaxation phase and long-term tinnitus severity were positively correlated, indicating that increased levels of life strain related to tinnitus went along with higher levels of experienced stress during relaxation. Negative cognitive appraisal mechanisms associated with chronic tinnitus, e.g. intensified attention allocation to the tinnitus, may impair recovering from acute stress.

Contrary to healthy controls in our and other samples, a lack of correlation between subjective strain ratings and objective physiological data emerged in tinnitus patients, as has been shown previously12. Similarly, there were no significant correlations between chronic tinnitus severity as indexed by TQ scores and physiological data, which also parallels earlier findings12. These findings suggest that physiological stress reactions and the subjective reported short-term and long-term stress perception are de-synchronized, again indicating disturbed physiologic reactivity in chronic tinnitus. Speculatively, some sort of cognitive bias predisposes tinnitus patients to think more negatively about relatively small changes in arousal, enhancing subjective strain, yet the exact mechanisms are still unclear.

Taken together, our results of reduced baseline HR, reduced HR increase during the arithmetic stress task, decoupling of HR and HRV and de-synchronized physiological and subjective perceived stress reactivity confirm a dysfunction of the ANS in chronic tinnitus, as postulated in psychological8,9 and neurophysiological models of chronic tinnitus. Unexpectedly, we were not able to identify hints for ANS hyperarousal in tinnitus patients questioning the validity of HRV as marker for stress reactivity in patients with mild to moderate chronic tinnitus. All our findings suggest a rather blunted ANS activity in the investigated sample. Furthermore, we found subjectively increased stress and tinnitus presence levels after stress exposure, which reflects very well the clinical complaints of many tinnitus patients. Further research is needed to elucidate whether the blunted physiological activity is related to the observed impaired relaxation after stress exposure.

Our findings also demonstrated that physiological findings concerning ANS functioning in chronic tinnitus seem to be more subtle and less uniform than assumed so far, providing a further hint that tinnitus is a pathophysiological heterogeneous condition. More research involving large samples will be needed to better understand the interplay between tinnitus severity, subjective stress and ANS function.

Additional Information

How to cite this article: Betz, L. T. et al. Stress reactivity in chronic tinnitus. Sci. Rep. 7, 41521; doi: 10.1038/srep41521 (2017).

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References

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Affiliations

1. Department of Psychiatry and Psychotherapy, University of Regensburg, Regensburg, Germany

Linda T. Betz, Berthold Langguth & Martin Schecklmann

2. Department of Psychology (Clinical Psychology and Psychotherapy), University of Regensburg, Regensburg, Germany

Andreas Mühlberger

Contributions

"M.S. and L.B. designed the study and LB conducted the experiment. L.B., M.S., B.L. and A.M. wrote the main manuscript text and L.B. prepared all figures. All authors reviewed the manuscript."

Corresponding author

Correspondence to Linda T. Betz.

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The authors declare no competing financial interests.

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Tinnitus is primarily an auditory symptom. Yet not only patients and clinicians, but also current pathophysiological models relate the onset and maintenance of tinnitus to stress. Here physiological and psychological stress reactivity was investigated in 19 patients with subjective chronic tinnitus and 19 comparable healthy controls.

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Introduction

The male-biased prevalence of autism [ 1, 2], together with the finding that autistic girls have a higher mutational load than autistic boys [ 3, 4, 5], suggests that males have a higher likelihood of developing autism. The sex ratio in autism diagnoses persists even after taking into account under- and/or mis-diagnosis, as well as camouflaging in females, with males being three times more likely to have the condition [ 6]. This implicates mechanisms of sexual differentiation in the development of autism. Five recent findings support this inference.

First, autistic women have atypical brain structure in sexually dimorphic regions, when assessed via magnetic resonance imaging and compared to neurotypical controls [ 7]. Second, functional connectivity in the brain of males with autism shows both hypermasculine and hyperfeminine patterns, when assessed in relation to neurotypical sex differences [ 8]. Third, autistic people show a masculinised shift in scores on two key sexually dimorphic psychological traits, empathy and systemising, a finding that has been replicated in a big data study of 36,000 autistic people [ 9, 10]. Fourth, autistic women have elevated androstenedione levels, the precursor to testosterone [ 11]. Finally, fifth, autistic children have hypermasculine facial features, as rated using three-dimensional photogrammetry [ 12].

Although autism is strongly heritable and sex-associated genetic mechanisms could contribute to this implication of sexual differentiation in autism [ 2, 5], prenatal hormone exposure and a brief surge in foetal testosterone are critical for sexual differentiation and masculinisation in humans [ 13, 14]. In line with this, we previously found elevated steroidogenic activity during this prenatal masculinisation window (PMW) in the amniotic fluid of autistic boys [ 15]. Subsequently, three very large epidemiological studies revealed a link between autism and maternal polycystic ovarian syndrome (PCOS), a condition associated with androgenic excess [ 16, 17, 18]. Consistently with this, the 2D:4D digit ratio, a marker of prenatal androgen exposure, is also masculinised in autistic children and their parents [ 19]. Finally, autistic women and their mothers have elevated rates of steroid-related cancers, such as breast cancer and ovarian cancer [ 20].

However, a number of studies that focused on testosterone have not replicated the correlation of hormonal levels with autistic traits. First, umbilical cord testosterone measured soon after birth was not associated with the development of autistic traits [ 21]. Second, salivary testosterone during a brief period of postnatal steroid surge ('mini-puberty') also did not correlate with autistic traits in toddlers [ 22]. In both cases, testosterone was measured postnatally-in the neonatal period-rather than during the PMW, during which foetal testosterone is first produced and masculinisation of the brain and body commences. This would suggest that timing is critical for the effects of testosterone on the brain, with the late first-early second trimester PMW being key, rather than the neonatal period. Finally, univariate assessment of amniotic testosterone in a separate cohort of neurotypical children also failed to reveal an association to autistic traits in childhood [ 23]. This latter finding may reflect that the wider endocrine environment outside testosterone is also significant for autism likelihood.

While prenatal androgens are responsible for masculinisation in humans, prenatal oestrogens also contribute to foetal and neonatal brain development [ 24], and yet these have not been thoroughly investigated for their potential role in autism likelihood. Oestrogens and their receptors are widespread in the developing brain in both males and females and regulate many neurodevelopmental processes, including synaptogenesis, apoptosis and neuronal differentiation [ 25, 26, 27]. Oestradiol in particular supports synapse formation in the cortex by enhancing excitatory GABA activity [ 28]. In autism, synapse formation [ 29], neuronal differentiation [ 30] as well as the GABAergic system [ 31] are all atypical. These provide clues that prenatal oestrogens may be involved in autism. However, we still lack direct evidence of this.

With regard to clinical studies in humans, low oestriol in maternal serum during the second trimester of pregnancy significantly increases the likelihood of autism in the foetus, as demonstrated in a large study of n = 2586 autistic pregnancies [ 32]. This study may have been confounded by a variety of pregnancy complications, such as pre-eclampsia [ 33] and being small for gestational age [ 34], since these are also more frequent in autism [ 35, 36, 37]. Thus, further study of prenatal oestrogenic activity, particularly in foetal circulation, is warranted. In addition, there is a need to compare different prenatal oestrogens to each other, in relation to autism likelihood.

In the present study, we measured prenatal levels of oestriol, oestradiol, oestrone and oestrone sulphate in amniotic fluid of boys with and without autism ( n = 98 and n = 177 respectively) from the Danish Historic Birth Cohort (HBC), in the same samples in which we had found an elevated steroidogenic factor, following principal component analysis of prenatal androgens and other steroid hormones [ 15]. We have expanded on these findings by assaying oestrogens and by assessments of each steroid hormone to autism likelihood via univariate logistic regression. We also investigated potential differences in the aromatising capacity in autism by comparing the ratio between androgens and oestrogens. Finally, we calculated standardised effect sizes for all hormones assayed to date in this cohort, in order to understand which amniotic fluid hormones make the largest contribution to autism likelihood.

Methods

Participants and laboratory methods

The study was approved by the Danish Data Protection Agency and The Danish Ethical Committee of Midtjylland Region. The Danish Historic Birth Cohort was established at Statens Serum Institute, Copenhagen with a grant from The Danish Medical Research Foundation and The Danish Ministry of the Interior and Health (Project no 271-05-0523/09-060179). A full description of the cohort selection procedure is available elsewhere [ 15]. Briefly, cases and controls were drawn from singleton births between the years 1993 and 1999 inclusive, whose amniotic fluid samples were stored in the HBC. These corresponded to amniocentesis procedures performed between 14 and 16 weeks of gestational age. Cases were identified from the Danish Psychiatric Central Register using ICD-10 autism spectrum codes F84.0 (childhood autism), F84.5 (Asperger's syndrome), F84.1 (atypical autism), F84.8 (other pervasive developmental disorder) and F84.9 (unspecified pervasive developmental disorder). Any additional amniotic fluid was assayed for oestradiol, oestriol, oestrone, and oestrone sulphate, using liquid chromatography-tandem mass spectroscopy (Supplementary Table 1). As some individuals did not have sufficient remaining sample for analysis, the sample size was slightly decreased. The same data quality screening criteria that were used in the initial analysis were applied: i.e. removal of outliers >99%, removal of records where duplicate assay values were >3 SD (standard deviation) apart [ 15]. After this step, the final sample with high-quality data for all steroids assayed to date consisted of 98 males with autism and 177 control males. This sample was used for all analyses in this paper, unless otherwise specified (Supplementary Fig. 1).

Statistical analyses

We calculated a correlation matrix for all assayed steroid hormones using Pearson's correlation coefficient. We also examined the univariate distribution of each of the oestrogens. All hormones showed a substantial rightward skew. We transformed the oestrogens using the Box-Cox procedure to reduce their skew, as the distribution of the predictor variable affects the statistical power of logistic regression [ 38]. We used univariate logistic regression models to determine whether each hormone separately increased autism likelihood in this cohort. As the oestrogens considered here vary in their equilibrium association constants to the oestrogen receptors, and therefore their potency [ 39, 40], we hypothesised that if a relationship between amniotic oestrogens and autism was found, oestradiol would have the largest effect size as the most potent oestrogen. Therefore, we also considered each hormone separately, with the goal of establishing accurate effect sizes to enable a comparison between them, and we corrected for multiple comparisons using the Benjamini-Hochberg false discovery rate (FDR). To estimate and compare the aromatising capacity between cases and controls, we calculated the aromatisation ratios, according to previously published recommendations [ 41], by log-transforming the raw concentration values and subsequently subtracting them according to the following formula:

Ratio 1 = log(testosterone concentration in nmol/l)−log(oestradiol concentration in nmol/l)

Ratio 2 = log(androstenedione concentration in nmol/l)−log(oestrone concentration in nmol/l)

We subsequently used nonparametric tests to compare these ratios to each other (Spearman's rank correlation coefficient) and between cases and controls (Wilcoxon rank-sum test).

Results

There were no significant differences between groups in maternal age at birth, paternal age at birth, birth weight, gestational week at amniocentesis or storage time between groups (Table 1). Raw data for each of the oestrogens are presented in Fig. 1. Values for the median and interquartile range as well as raw data categorised by autism diagnosis are available in the Supplemental Information (Supplementary Table 2). Oestradiol levels were the most predictive of an autism diagnosis, as revealed by univariate logistic regression ( β = 0.029, FDR-adjusted q = 0.031). The same was found for the levels of oestriol and oestrone, with both hormones being significantly associated to an autism diagnosis using logistic regression (Oestriol: β = 0.025, q = 0.034; oestrone: β = 0.029, FDR-adjusted q = 0.031) (Table 2). Oestrone sulphate was also a nominally significant predictor of autism likelihood in the logistic regression model but did not maintain statistical significance following false discovery rate correction ( β = 0.033, p = 0.036, q = 0.065) (Table 2).

Table 1 Description of Danish Historic Birth Cohort sample

Beeswarm plots illustrating the distribution of oestriol, oestradiol, oestrone and oestrone sulphate concentrations. Error bars represent the interquartile range, and the black dot represents the median; ( n CTR = 177, n ASC = 98)

Table 2 Results of univariate logistic regression for amniotic steroid hormones

Androgens and other steroids

We revisited the previously assayed concentrations of androgens and cortisol [ 15] in the same subset of samples in which we assayed oestrogens, to understand whether the relationship between oestrogens and autism likelihood was similar to the relationship between androgens and autism likelihood (Table 2, Beeswarm plots of distribution in Supplementary Fig. 2). Of the previously analysed steroid hormones, only progesterone was a significant predictor of autism diagnosis, following univariate logistic regression and correction via FDR in this subset of the cohort ( β = 0.053, FDR-adjusted q = 0.031) (Table 2).

Comparison

Pairwise correlation analysis (Pearson's) revealed varying degrees of similarity between the steroid hormones. The concentrations of oestrogens were significantly correlated with one another at q < 0.05, adjusted for multiple comparisons via FDR (Fig. 2, Supplementary Table 3). Oestrone and oestriol showed higher correlations to the other steroids than did oestrone sulphate and oestradiol. In comparison, the previously analysed steroid hormones formed a distinct group, with weaker correlations to oestrogens and stronger correlations to each other. Androstenedione and progesterone (Pearson's β = 0.59317, FDR-adjusted q < 0.001), oestrone and oestrone sulphate (Pearson's β = 0.589, FDR-adjusted q  < 0.001) were the pairs that were more closely related. Oestradiol did not correlate with either testosterone or oestriol.

Heatmap and dendrogram of the pairwise correlations between the steroid hormones assayed in amniotic fluid. Asterisk denotes statistical significance ( q  < 0.05), following correction via FDR. P progesterone, A androstenedione, T testosterone; P-OH 17-OH-progesterone, C cortisol, E-S oestrone sulphate, E2 oestradiol

We also calculated standardised odds ratios (ORs) for all analysed hormones to determine which hormones had the largest effect on autism likelihood (Fig. 3). Each hormone was standardised by its median and interquartile range, so that a one-unit increase in a hormone corresponded to the movement from the 25th to the 75th percentile of its range. Progesterone and oestradiol had the highest standardised ORs, with a movement from the 25th to 75th percentile of these hormones incurring nearly a 50% increase in autism likelihood. Oestrone and oestrone sulphate also had ORs over 1. In contrast, increases in testosterone or androstenedione levels were not associated with increases in likelihood of diagnosis with statistical confidence.

Standardised odds ratios (ORs) for autism diagnosis for all amniotic steroids assayed to date. Each hormone was standardised by its median and interquartile range, so that a one-unit increase in a hormone corresponded to the movement from the 25th to the 75th percentile of its range

The aromatising of testosterone (Τ) to oestradiol (Ε2) and of androstenedione (Α) to oestrone (Ε1) were indirectly assessed by calculating their ratios, following log-transformation and subtraction of their raw values. The two ratios were highly correlated with each other (Spearman's rho = 0.5855, p < 0.001) but were not significantly different between autism cases and neurotypical controls (T/E2: Wilcoxon rank-sum w = 11186, p = 0.1364) (A/E1: Wilcoxon rank-sum w = 11032, p  = 0.2038).

Discussion

This study reports the first evidence that elevated levels of prenatal amniotic oestradiol, oestriol and oestrone are each associated with autism, with oestradiol levels being the most significant predictor of autism likelihood in univariate logistic regression models. These findings complement earlier observations that elevated steroidogenic activity is associated with autism in the same samples derived from the Danish Historic Birth cohort [ 15]. We also calculated standardised ORs, in order to directly compare the effect sizes of all amniotic steroids measured to date. We found that oestradiol had the strongest positive effect size on autism likelihood, followed by oestrone, oestriol and progesterone (Fig. 3). This finding appears to contradict an earlier report by Windham et al. [ 32] that showed that lower levels of oestriol in second trimester were modestly associated with a later diagnosis of autism in the offspring. However, our samples correspond to a slightly earlier time point in pregnancy compared to Windham et al. (mean gestational week = 14.9 vs. 17.2 respectively) (see Table 1) [ 32], which could potentially better capture the steroid surge during the PMW [ 14]. Furthermore, our samples are of different origin, as Windham et al. assayed maternal serum, rather than foetal amniotic fluid. Steroid hormone levels in maternal serum do not differ relative to the baby's sex and do not correlate to amniotic levels during the PMW [ 42]. Therefore, amniotic oestrogens are arguably more relevant to the current research question than are maternal serum oestrogens.

A discrepancy in oestrogen levels between the mother and child could potentially be attributed to the placenta, which acts as an endocrine regulator of the maternal-foetal interface and the main source of oestrogen production for the foetus via the aromatisation of androgens [ 43]. Several lines of evidence suggest a contributory role for the placenta in the aetiology of autism. First, there is increased placental inflammation in autism [ 44]. Second, there is atypical placental morphology [ 45] and increased placental size [ 46] in cases of autism and at high familial risk respectively. Third, complications related to the placenta (pre-eclampsia [ 47], hypertensive disorders [ 48]) are also more frequent in pregnancies that lead to autism. As with autism, placental dysfunction also disproportionately affects males more than females [ 49].

Given the high pairwise correlations between many of the steroid hormones (Fig. 2, Supplementary Table 3), as well as a lack of difference in aromatisation between cases and controls, our data suggest that an increase in foetal oestrogens is secondary to increased activity along the entirety of the steroidogenic axis in pregnancies that later result in autism [ 15]. Interestingly, oestradiol was not significantly correlated to testosterone (Pearson's β = 0.007, p = 0.9103) despite their proximity in steroidogenesis. This discrepancy may be because oestrogens are also de novo produced by the placenta, in addition to being aromatised from foetal and maternal androgens [ 43, 50]. Thus, a multi-systems approach is needed in order to clarify the causes of elevated foetal oestrogens in autism.

In the brain, oestrogen-mediated signalling on GABAergic neurons in the hypothalamus is required in order to suppress the steroidogenic axis [ 51]. Inefficient suppression of this axis in autism could be due to inefficient aromatisation of androgens in the hypothalamus, resistance to oestrogen signalling and/or dysfunction of the GABAergic system. Prenatally, foetal genetics (e.g. due to mutations in aromatase [ 52] or aromatase activators [ 53]), pregnancy complications (e.g. placental size [ 46]), as well as maternal risk factors (e.g. PCOS [ 18]) could all affect various points in this pathophysiologic pathway. These speculations would require further testing. Specifically for aromatisation, ratios based on circulating hormone levels may not be sufficient to capture tissue-specific activity, since aromatase is differentially regulated by separate promoters in the placenta, the adrenals and the brain [ 54].

High levels of prenatal oestrogens could dysregulate many aspects of prenatal endocrinology and affect prenatal brain development in areas that are not restricted to sexual differentiation. Several lines of evidence support a wider role of oestradiol as a 'neurosteroid' with many regulatory properties [ 55]. For example, disruption of oestrogen signalling in the developing cerebellum of mice reduces Purkinje cell growth in both males and females, but only reduces social behaviour in male mice, suggesting that the cerebellum may react to oestrogenic disruption in a sexually dimorphic way [ 56]. In early development, oestradiol decreases GABAergic signalling [ 57] and mediates its postnatal shift from excitation to inhibition [ 28, 58]. Oestrogens both increase the number of spines on embryonic primary cortical neurons [ 55] and induce the recruitment of proteins necessary for excitatory synapse formation, such as neuroligin-1, NMDA subunit GluN1, and post-synaptic density protein 95 (PSD-95) to the spines [ 59]. Higher levels of prenatal oestrogens might therefore increase the number of excitatory synapses in the cortex, increasing the likelihood for autism, as suggested by the excitatory/inhibitory (E-I) theory of autism [ 60]. The perceptual phenotype in autism is characterised by reduced GABAergic inhibition, as shown using paradigms such as binocular rivalry [ 61] and attention to detail [ 62]. Oestrogen signalling could thus be a significant modulator of neuronal inhibition, particularly during early brain development and the 'critical period' of cortical plasticity, which is heavily reliant on the GABAergic system [ 63].

Although oestradiol (aromatised from testosterone) is the main prenatal masculinising agent in most mammals [ 24], its role in human sexual differentiation remains unclear. Men with aromatase deficiency have typical development of their urogenital tract [ 64], but may have cognitive disabilities, lack a growth spurt, and have atypical secondary sexual characteristics such as feminised body proportions in adulthood [ 65]. Oestrogens may therefore both feminise and masculinise humans, depending on the target tissue and developmental milieu. In autism, cognitive styles and sexually dimorphic neuroanatomy show some masculinised phenotypes [ 7, 9, 10], but functional connectivity and physical growth show a mixed pattern of masculine and feminine shifts [ 8, 66]. Prenatally though, and particularly during the masculinisation window, the process of sexual differentiation is understood to be directionally masculine over an anatomically and physiologically female default. The observed high levels of foetal oestrogens could thus be contributing to developmental cognitive differences [ 10], according to the "extreme male brain" theory of autism.

There was no statistically significant univariate, logistic association between autism and testosterone or androstenedione, which act via the androgen receptor. Mechanisms through which androgenic signalling could increase autism likelihood, which may have been missed in this analysis, include additional androgens or other agonists of the androgen receptor (e.g. neurosteroids like dehydroepiandrosterone [ 67]), interaction effects between androgens and oestrogens (e.g. coactivation of the androgen receptor by oestradiol [ 68]), as well as non-linear associations of androgens to autism likelihood. Consequently, androgenic activity may still be an important feature in the development of autism, as suggested by related clinical comorbidities [ 18, 69] and shown in associations of foetal testosterone to autistic traits in a separate cohort [ 70].

We could not test whether prenatal oestrogens were associated with autism likelihood in females as there were too few diagnosed women in the HBC in this time window. We plan to test this by expanding the time window. Thus, at present, our findings only generalise to males. Furthermore, comparison of the concentrations of androgens and cortisol to oestrogens is potentially confounded by the fact that the latter were analysed at a later time point and underwent an additional freeze-thaw cycle. We have attempted to minimise any potential sources of confounding by using the same assay methodology with the initial analysis (LC-MS/MS), as well as reassessing for any differences in total storage time in this subset of the original cohort (Table 1).

Another limitation of this study is its reliance on clinical diagnoses from the Danish Central Psychiatric Register, which we could not be independently validated. However, a previous validation study of childhood autism diagnoses in the Danish Central Psychiatric Register found that 94% of diagnoses between 1990 and 1999 in the register were valid using a standardised coding scheme [ 71]. Similarly, we cannot be certain about the source of amniotic steroids, as they could be of foetal, maternal or placental origin. Foetal plasma and amniotic fluid are in osmotic equilibrium until the foetal skin is keratinised (typically by 25 weeks of gestation) [ 72]. Therefore, steroid concentrations in amniotic fluid accurately reflect those in foetal circulation.

In conclusion, we have demonstrated that prenatal oestradiol, oestriol and oestrone are elevated in in boys who went on to develop autism. This extends our previous finding of elevated prenatal steroidogenesis in the same cohort and provides further evidence for the prenatal steroid theory of autism [ 15]. High levels of prenatal oestradiol contribute to a greater degree to autism likelihood than other prenatal sex steroids, including testosterone. We conclude that prenatal oestrogenic excess is a characteristic of autism and may interact with genetic predisposition to affect neurodevelopment.

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Greenberg DM, Warrier V, Allison C, Baron-Cohen S. Testing the empathizing-systemizing theory of sex differences and the extreme male brain theory of autism in half a million people. Proc Natl Acad Sci USA. 2018;115:12152-7.

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Kaludjerovic J, Ward WE. The Interplay between Estrogen and Fetal Adrenal Cortex. J Nutr Metab. 2012;2012:837901.

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Acknowledgements

This study was supported by grants to SB-C by the Autism Research Trust, the Medical Research Council and the Wellcome Trust. This study was conducted using the Danish National Biobank resource supported by the Novo Nordisk Foundation. The research was supported by the National Institute for Health Research (NIHR) Collaboration for Leadership in Applied Health Research and Care East of England at Cambridgeshire and Peterborough NHS Foundation Trust. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health. AP was funded by a doctoral scholarship from Trinity College Cambridge. AT was funded by a doctoral scholarship from Peterhouse Cambridge. This study also benefitted from support from the NIHR Cambridge Biomedical Research Centre and the Templeton World Charitable Foundation. We are grateful for discussions with Ken Ong, John Perry, David Dunger, Gordon Smith, Mike Lombardo and Varun Warrier.

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Author notes

These authors contributed equally: Simon Baron-Cohen, Alexandros Tsompanidis

Affiliations

1. Autism Research Centre, Department of Psychiatry, University of Cambridge, Cambridge, UK

Simon Baron-Cohen, Alexandros Tsompanidis, Bonnie Auyeung & Alexa Pohl

2. Department of Psychology, School of Philosophy, Psychology and Language Sciences, University of Edinburgh, Edinburgh, UK

Bonnie Auyeung

3. Department for Congenital Disorders, Danish Center for Neonatal Screening, Statens Serum Institute, Copenhagen, Denmark

Bent Nørgaard-Pedersen, David M. Hougaard & Arieh Cohen

4. Department of Child and Adolescent Mental Health Services, Region of Southern Denmark, Hospital of Southern Jutland, Aabenraa, Denmark

Morsi Abdallah

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Correspondence to Simon Baron-Cohen or Alexandros Tsompanidis.

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# data[].attributes.attachment_link.description
Elevated latent prenatal steroidogenic activity has been found in the amniotic fluid of autistic boys, based on measuring prenatal androgens and other steroid hormones. To date, it is unclear if other prenatal steroids also contribute to autism likelihood. Prenatal oestrogens need to be investigated, as they play a key role in synaptogenesis and corticogenesis during prenatal development, in both males and females.

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https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4228534/

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https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0133758

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Correlation of the Voice Handicap Index and Voice Related Quality of Life

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Charlotte De Smet

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When the voice ages, disorders, risk factors and socio-emotional effects-1

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https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3961131/

# data[].attributes.attachment_link.title
Finetuning of auditory cortex during speech production

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Abstract

Introduction

Cleft lip and/or palate, depending on the form in which they appear, generate many problems, such as difficulties while feeding, nursing and swallowing, impaired hearing, orthodontic anomalies, speech disorder, impaired resonance, aesthetic and psychosocial disorders, social and professional isolation and the like [ 1]. Cleft lip and palate affect all oral functions, and the consequences are especially evident in a speech [ 2].

Children with cleft lip and palate have pathology in verbal communication, and the reason for that is the incomplete closure of the velopharyngeal sphincter, i.e., the soft palate, lateral walls and posterior wall of the pharynx, which means the oral cavity is not separated from the nasal cavity during speech and swallowing [ 3]. In the speech, the closure of the velopharyngeal sphincter separates the oral from the nasal cavity, which prevents nasal air loss in the production of all sounds, except for the nasal consonants /M/, /N/ and /Nj/. The incomplete closure during speech production results with velopharyngeal insufficiency which characterises with hypernasal speech and nasal air emission [ 4]. If there is no closure of the velopharyngeal sphincter, the child will lack the adequate aerodynamic conditions for the adoption of normal articulation during speech development. Thus, the dysfunctional velopharyngeal sphincter, i.e., cleft palate or cleft lip and palate affect the development of speech and compensatory mechanisms greatly during articulation.

Articulation is not a mechanism the child is born with; it is adopted through the process of learning over time when the speech-language system is formed. The conditions for the development of regular speech are anatomical, well-functioning speech and hearing organs and a proper speech model. If these conditions do not exist, the resulting adoption of compensatory mechanisms leads to a pathological and completely incomprehensible articulation. The most noticeable articulatory mistakes in the speech of children with cleft palate occur during the production of consonants that require high pressure in the oral cavity [ 5]. The child's speech can only be understood by his parents, while social contacts are encumbered. The speech disorders impair the individual and disturb their emotions deeply as well. Bearing in mind that speech is an indispensable tool for expressing and conveying thoughts and the most perfect means of communication, it is important that the impairments are resolved from the very beginning, which asks for inclusion of a multidisciplinary specialist team and team approach [ 6].

The basis of impaired speech lies in hypernasality, i.e. nasal air emission, or directing part of the air through the nose during the speech when the intraoral pressure, which is most important for speech, cannot reach its necessary value. Hypernasality is one of the most typical characteristics of children with cleft [ 7], [ 8]. As it is known, nasal air emission often occurs with hypernasality, but it can occur with normal resonance, too [ 9]. There are four types of nasal air emission [ 10]: inaudible nasal air emission, audible nasal air emission, nasal rustle (nasal turbulence) and nasal emission typical for phonemes. Apart from the last one, the three other types of nasal air emission are often and commonly accompanied by severe, moderate or mild hypernasality.

Therefore, establishing a link between hypernasality, i.e., nasal air emission during speech and the type of cleft palate can provide useful information for future diagnostic and therapeutic purposes in the field of speech disorders.

The relationship between the degree of anatomical disruption of the primary and the secondary palates, on which the Veau classification for cleft is based, and nasal air emission in the speech of children with a cleft can be established using perceptual and instrumental (direct and indirect) examinations. Since it is primarily a matter of dealing with a young population from a very early age, it is important to consider non-invasiveness and simplicity during implementation, but fast and precise diagnosis, too. These are just some of the advantages and benefits of perceptual examinations and some other instrumental indirect examinations as well.

Still, the last tool in the chain of procedures for treating speech disorders is not diagnostics. The final correction from an anatomic, morphological and functional aspect is what matters. The success in resolving the existing problem depends on an efficient and appropriate treatment, but palatoplasties as well.

Material and Methods

Results

Discussion

An anatomical and physiologically functional vocal apparatus plays an important role in correct articulation and speech. The relation of cleft palate or cleft lip and palate - nasal air emission - regular speech requires a separate analysis of each of them because, in such a way, unique information will be obtained about their condition and role in the speech of children with cleft palate or cleft lip and palate. Since it is primarily a matter of dealing with a population of the youngest age, it was important to consider the non-invasiveness and simplicity of the examinations, as well as a fast and precise diagnosis during implementation. These advantages and benefits were gained with the application of the perceptual examination method which determined the grade of severity of cleft palate, but also with the application of an instrumental non-invasive indirect method for determining of the nasal air emission in the speech of children with cleft palate or cleft lip and palate.

The results obtained from the examined variables in this research for the respondents with cleft palate or cleft lip and palate and irregular speech (experimental group) differed from those of the respondents with only irregular speech (control group), i.e., the values obtained for nasal air emission during speech are much higher for the experimental group. Specifically, our research results revealed statistically significant more common presence of nasal air emission in the respondents with cleft palate or cleft lip and palate (experimental group) than those with no cleft (control group).

Within the experimental group, our research results revealed that in the speech of respondents with cleft palate or cleft lip and palate the degree of nasal air emission rises with the increase of the severity of cleft and vice versa.

Some authors worldwide have conducted different research in this field, but each of them analyses and presents, with their approach and from their perspective, the importance of the type of cleft and nasal air emission in the speech of children with cleft palate or cleft lip and palate. Taking into consideration all the research that has been done so far, there is no study, or studies, in the international scientific literature identical to this research where the same methods and number of respondents are used so that a complete comparison of the results obtained could be made. Still, there was an opportunity to make limited comparisons and correlations with similar research by other authors, but only about certain defined variables. It has been proven that the results obtained in our research are, to a certain level, compatible with the results obtained in other research, but also different at times.

Kaewkumsan et al., [ 16], who examined the degree of hypernasality and formation of an oronasal fistula in 40 patients, aged between 5 and 6,9 years, with non-syndromic cleft palate with or without cleft lip, established that the patients with cleft Class IV and Class III (according to the Veau classification) had a higher total score for hypernasality than patients with Class I. Garcia-Vaquero et al., [ 17] conducted research about the hearing and speech of 121 patients aged above 6 who had already undergone intervention of the cleft palate. Among other things they also examined the relation between Veau classification and hypernasality. They established a direct association between hypernasality and the Veau grade of cleft palate (p = 0.053).

In our paper, the increasing severity of cleft is related to a higher degree of nasal air emission; while it must also be pointed out that hypernasality was noticed in all respondents where nasal air emission was registered. The See-Scape instrument used in our examination indicates nasal air emission during speech rather than nasality, but in that way, it also indicates towards lack of velopharyngeal competence, which is perceived as hypernasality [ 14]. Also, when it comes to nasal air emission and hypernasality during the speech, Dotevall et al., [ 18] have established a strong link between the results of velopharyngeal function and hypernasality and nasal air emission. They examined the correlation between perceptual assessment of speech variables related to velopharyngeal function and the behaviour of nasal air emission in the phase of velopharyngeal closure in the speech of children with or without cleft palate. According to them, the results for nasal air emission related to velopharyngeal closure during the speech were closely related to the perceptual results for velopharyngeal function and hypernasality.

Marrinan et al., [ 19] had researched 228 patients with cleft palate to determine the relative importance of the surgical technique, the year when it was conducted and the type of cleft on velopharyngeal function. According to them, it was commonly believed that the more severe the cleft was (according to the Veau classification), the weaker the speech results would be. However, according to Marrinan et al., [ 19] the patients with Veau Class II and IV had weaker speech results than those with Class I and III. They suggest that the predicted gradation of the severity of the cleft described with the Veau classification may not be clinically progressive, at least in respect to the speech results. Similar is the attitude of Timmons et al., [ 20], who studied a speech of patients after correction of the isolated cleft palate or cleft lip and palate. They point out to the existence of many studies in which focus is to make a correlation between speech results and the degree of anatomical disruption of the primary and the secondary palates (Veau classification). According to their study, some authors found that there was no correlation between the type of cleft and degree of speech disorders, while others that severe cleft entailed worse speech. Still, the results of Timmons et al. did not reveal a simple correlation between the Veau classification and speech. Lam et al., [ 4] imply that among the studies that examined speech results upon palate correction, certain researchers noticed negative or non-linear association with the severity of the cleft as defined by the Veau classification.

In their experiment, Coston et al., [ 21] assessed the surgical intervention of m.levator veli palatini based on the velopharyngeal competence acquired as a result of the intervention. An important finding which the results of Coston et al., the show is that the more severe the cleft is, the less likely the surgical intervention is to produce favourable results, i.e., have normal nasality and no nasal air emission [ 21].

Our study contains certain limitations that should be pointed out. First, future research should include a greater number of respondents so that the obtained results have greater relevance. The second limitation is that this research only established the existence and classification of cleft (cleft palate or cleft lip and palate) using the Veau classification, but no a measurement of its width. Considering that the types of cleft from the same Veau class can have different width [ 22] which can affect the degree of nasal air emission during the speech, future research should explore this aspect as well.

Given that certain speech aspects are directly related to the condition of cleft palate or cleft lip and palate and nasal air emission establishing unique information about their condition and status is of great importance for correct diagnosis and rehabilitation of speech disorders in children with cleft palate or cleft lip and palate.

Footnotes

References

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The Relationship between the Type of Cleft and Nasal Air Emission in Speech of Children with Cleft Palate or Cleft Lip and Palate.

# data[].attributes.attachment_link.description
Cleft lip and/or palate, depending on the form in which they appear, generate many problems, such as difficulties while feeding, nursing and swallowing, impaired hearing, orthodontic anomalies, speech disorder, impaired resonance, aesthetic and psychosocial disorders, social and professional isolation and the like [ 1].

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https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6691675/

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Is dit een goed artikel? https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0206059

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Elisabet Cantó,

Emma Roca,

Lidia Perea,

Ana Rodrigo-Troyano,

Guillermo Suarez-Cuartin,

Jordi Giner,

Anna Feliu,

Jose Manuel Soria,

Lexa Nescolarde,

Silvia Vidal

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Citation: Cantó E, Roca E, Perea L, Rodrigo-Troyano A, Suarez-Cuartin G, Giner J, et al. (2018) Salivary immunity and lower respiratory tract infections in non-elite marathon runners. PLoS ONE 13(11): e0206059. https://doi.org/10.1371/journal.pone.0206059

Editor: David M. Ojcius, University of the Pacific, UNITED STATES

Received: April 20, 2018; Accepted: October 6, 2018; Published: November 21, 2018

Copyright: © 2018 Cantó et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and Supporting Information files.

Funding: The authors received no specific funding for this work.

Competing interests: The authors have declared that no competing interests exist.

Introduction

The number of non-elite runners participating in marathons had increased dramatically in the last ten years [ 1]. Several studies have demonstrated that marathons and ultramarathons may alter immune function and increase the risk of respiratory tract infections [ 2- 4]. Within two weeks of completing such strenuous exercise, the risk of infection increased 100% to 500% [ 5] and around 25% of finishers reported respiratory symptoms [ 3].

Salivary Immunoglobulin A (sIgA) is the most common antibody on the mucosal surface. Different studies have analyzed changes in sIgA following strenuous exercises and their potential relationship with respiratory symptoms, with conflicting results [ 6- 8]. Other studies have suggested that immune factors present in mucosal secretion, including antimicrobial peptides (AMPs) and immune mediators such as chemokines, changed after prolonged running. An increase in salivary lactoferrin, one of the most abundant AMPs, has been described in participants in a 50 km ultramarathon [ 9]. Furthermore, chemokines such as Growth-Regulated Oncogene-alpha (Groα or CXCL1), growth-Regulated Oncogene-beta (Groβ) and monocyte chemoattractant protein 1 (MCP-1) have demonstrated an important role in the regulation of local immunity against infections by contributing to the tissue infiltration of leukocytes [ 10]. Experimental studies showed that serum levels of these inflammatory mediators increased markedly in response to exercise in mice [ 11]. However, limited data regarding salivary IgA, AMPs, chemokines and the presence of lower respiratory tract infections (LRTI) in marathon runners are available.

We hypothesized that non-elite marathon runner who experienced changes in their salivary immunity would be more prone to developing a respiratory infection. Therefore, our aim was to determine salivary levels of IgA, AMP and inflammatory markers before and after a marathon and their relationship with the development of LRTI.

Materials and methods

Subjects

Forty seven healthy non-elite marathon runners (28 males and 19 females) completed the 42.195 km of the 2016 Barcelona Marathon. Mean finishing time was 3 hours and 38 min (±41 min). Study participants were recruited using an announcement in the marathon newsletter that all marathon runners received two weeks prior to the race. Participation was voluntary in all cases. The study was approved by the local ethics committee "Comité Étic de Investigació Clínica de la Fundació de Gestió Sanitaria del Hospital de la Santa Creu i Sant Pau de Barcelona", project number IIBSP-SUMMIT-2016-02. All participants gave signed informed consent.

All runners were asked to report any incidence of respiratory symptoms 2 weeks before and after the race. Runners who reported respiratory symptoms 2 weeks before marathon were excluded from the study. LRTI was defined according the clinical definition proposed by the European Respiratory Society and European Society for Clinical Microbiology and Infections disease [ 12]: acute illness (present for 21 days or less) characterized by cough as the main symptom, with at least one other respiratory tract symptom (sputum production, dyspnea, wheeze or chest discomfort/pain) and no alternative explanation. In addition, all runners had to report at least one determination of fever (≥38°C) and duration of symptoms must be higher than 3 days in order to exclude those symptoms induced only by strenuous exercise. All runners with suspected LRTI were follow-up until a breakdown of symptoms was reported.

Saliva analysis

The saliva samples were then thawed and total protein saliva was quantified using the Qubit protein Assay Kit (Thermofisher Scientific). Salivary IgA (Human IgA Platinum ELISA, ebioscience, Affymetrix, Santa Clare, CA), lactoferrin, lysozyme (AssayPro, St. Charles, MO, USA), Groα, Groβ and MCP-1 (Elabscience, Houston, Texas) were measured by ELISA according to the manufacturer's instructions. The limit of detection for IgA was: 1.6 ng/ml; lactoferrin: 0.625 ng/ml; lysozyme: 0.0781 ng/ml; Groα: 15.63 pg/ml, Groβ: 15.63 pg/ml and MCP-1: 15.63 pg/ml. Data were expressed as the concentration of each molecule relative to total saliva protein concentration [ 13]. Those determinations of relative salivary IgA concentration with a ratio higher than 1 were excluded of the study due to possible contaminations [ 14]. All determinations were performed in triplicate.

Results

Study participants

Table 1 shows demographic and laboratory test results prior to the marathon for all participants. Median age was 30 years old, median years of training were around 10 and training hours per week were around 6. All laboratory tests performed were in the normal range.

No differences between males and females were found in baseline characteristics (data not shown), except for Body Mass Index (BMI), which was higher in males than in females (23.6 ±2.1 vs 20.7 ± 1.4, p = 0.01).

Lower respiratory tract infections

Eight participants (17%) reported a LRTI during follow-up. No differences in demographics and laboratory findings between infected and non-infected participants were found prior to the marathon ( Table 1). All runners with LRTI experienced a breakdown of the symptoms before 14 days of their onset and were fully recovered within one month after the race.

Salivary IgA

No differences before and after the marathon were found in total salivary protein concentration (2.34±1.16 mg/ml vs 2.37±0.97 mg/ml, respectively) nor in salivary IgA levels normalized to total salivary protein (0.39±0.25 vs 0.35±0.24) ( Fig 1A). In addition, no differences in IgA levels before and after the race were found between infected and non-infected runners ( Fig 1B), and no differences were observed between male and females before marathon in salivary IgA/protein.

Salivary antimicrobial proteins

No differences before and after the race were observed in salivary lactoferrin (0.013±0.01 vs 0.010±0.007) and salivary lysozyme levels (0.0094±0.007 vs 0.0078±0.007). Furthermore, no differences between the sexes were observed before the marathon.

When runners with and without LRTI were compared, we observed no differences in salivary lactoferrin levels before and after the race. However, those runners who developed a LRTI showed higher levels of salivary lysozyme after the race compared to those who did not develop it (0.012±0.01 vs 0.006±0.004, p = 0.02). In addition, a decrease in lysozyme levels was observed in runners who did not develop LRTI when levels before and after the race were compared (0.010±0.007 vs 0.006±0.004, p = 0.003) ( Fig 2).

Salivary chemokines

No differences before and after the race were observed regarding salivary Groα Groβ and MCP-1. However, runners who did not develop LRTI showed a significant decrease in salivary Groα and Groβ levels after the race (Groα, 0.37±0.15 vs 0.30±0.14, p = 0.02; Groβ 0.47±0.17 vs 0.37±0.18, p = 0.03) ( Fig 3A and 3B). No differences in MCP-1 levels were found.

Systemic correlations

Before the race, salivary lactoferrin levels had a weak but statistical significant negative relationship with blood lymphocyte counts (r = -0.13, p = 0.04) and salivary Groα had a weak positive correlation with the percentage of blood basophils (r = 0.22, p = 0.03) ( Fig 4A and 4B).

After the race, salivary lysozyme correlated positively with the absolute number of blood leukocytes (r = 0.26, p = 0.006) ( Fig 4C).

Discussion

In our study we observed that LRTI is common in non-elite runners following a marathon and we demonstrated that runners who developed a LRTI had different immunological profile in their saliva. Specifically, a decrease in salivary lysozyme, Groα and Groβ levels was observed in those runners who did not develop a LRTI when levels before and after the race were compared. In addition, higher levels of lysozyme were detected after the race in runners with LRTI compared with those without infection. These findings suggest that immunological status may be important for the development of a LRTI after a marathon.

Several data have been reported reflecting the importance of the immune system in the development of LRTI after strenuous exercise. We observed that 17% of marathon runners develop LRTI symptoms within 2 weeks after marathon, which is concordant to other studies [ 7, 15]. Although the effect of close proximity between runners during the race cannot be ruled out, extreme effort has been shown to alter immune defenses by increasing inflammation in the respiratory tract [ 16]. Different studies have postulated that a combination of several immune, biochemical and hematological parameters participate in the severe stress on the body resulting from such exercise and that they may be associated with an increased susceptibility to developing infections [ 17]. Salivary IgA (sIgA) is one of the most widely-studied parameters [ 7]. Some studies have pointed to the usefulness of sIgA as a noninvasive biomarker of mucosal immunity and LRTI risk [ 18, 19]. However, other studies have suggested that reductions in sIgA cannot be solely responsible for the decline in immune function that may lead to LRTI [ 20]. In our cohort of runners, we did not observe differences in sIgA as other authors previously showed [ 21]. We observed that sIgA represented about 40-60% of total salivary protein, which is concordant with previous studies [ 22, 23] but higher than other ones [ 24]. Most of the studies were carried out few hours after exercise [ 25, 26] and we wanted to evaluate two days after finishing the marathon, since few data regarding the recovery period and its influence on the development of LRTI are available [ 7].

Salivary antimicrobial proteins (sAMPs) such as lysozyme and lactoferrin protect the respiratory tract from invading microorganisms and have been linked with an increased infection risk in athletes [ 27]. In our cohort of runners, we observed no differences in the levels of saliva lysozyme before the race. However, runners who did not develop LRTI showed a decrease in lysozyme after the marathon. In contrast, those runners who developed a LRTI showed higher levels of salivary lysozyme after the race compared to those runners who did not develop LRTI. Our findings are in line with other studies showing a decrease in salivary lysozyme immediately after an ultra-marathon, though this decrease was not related to the presence of LRTI [ 5]. However, other studies did not observe differences in salivary lysozyme levels after a 50 km race [ 25]. These discrepancies could be the consequence of different levels of effort during exercise. Interestingly we observed an inverse relation between salivary lactoferrin and blood lymphocyte counts before the race, which may be explained by the mobilization of neutrophils to maintain blood homeostasis. Previously, Inoue H et al. [ 28] described an immediate increase in serum lactoferrin concentrations immediately after running exercise and serum lactoferrin may play an antibacterial role in host defenses before the mobilization of neutrophils into the circulating pool. We also observed a positive correlation between blood leukocyte counts and the content of saliva lysozyme after the marathon. It has been observed that exercise sessions in both humans and animal models resulted in the stimulation of neutrophil degranulation releasing lysozyme from blood neutrophils [ 29]. All of these findings suggested that systemic alterations may affect the content of saliva lactoferrin and lysozyme in runners and may contribute to the development of LRTI after races. Nevertheless, we cannot exclude a process of dehydration during the marathon, since it has been demonstrated that dehydration decreases saliva antimicrobial proteins [ 30, 31]. Dehydration can therefore affect the content of saliva AMPs, and further studies are needed to better elucidate its importance, though we did not observe differences in protein content before and after the race.

Different chemokines have demonstrated a potential role in the regulation of immune response during exercise [ 32]. We also found that Groα and Groβ, which are key components in the attraction of neutrophils to the site of inflammation, decreased after the race in runners who did not develop a LRTI. It has been described that daily moderate exercise suppressed Groα [ 33] and, in experimental models, exercise down regulated multiple inflammatory cytokines and chemokines including Groα and MCP-1 [ 34, 35] and had a systemic anti-inflammatory effect, reducing Groα, Groβ and MCP-1 [ 36, 37]. These findings were observed in runners who did not develop LRTI, suggesting that they had a better inflammatory regulation response that may protect against infection. Therefore, athletic performance is both a stress factor and an adaptive response to exercise that can be modulated by training, reduce inflammation and help to prevent disease. Further studies are needed to better understand the relationship between salivary immunity and systemic inflammation as a key factor in the development of LRTI after exercise.

Interestingly, we observed a positive correlation between blood basophils and salivary Groα level before the race. Sastre B et al, identified basophils as a new player in the status of bronchial inflammation in athletes [ 38]. We observed that non LRTI runners had a higher percentage of systemic basophils, although differences were not statistically significant.

Our study has several limitations. First, due to the small number of runners included, the results should be validated in other studies before generalizing them. Second, since some data reported the effect of feeding in post exercise saliva AMPs proteins [ 39] we cannot exclude feeding related affectations in the determinations of AMPs in our cohort of runners. Third, levels of leukocyte subpopulations after the race were measured 48 hours after the marathon and other determinations at 7 or 14 days would be helpful to better characterize systemic response in marathon runners, and it should be an important point to be taken into account in further studies. And fourth, infectious agents that caused LRTI are not described due to the absence of microbiological studies. Further studies including bacterial and viral determinations would be very helpful to clarify this important issue.

In conclusion, non-elite runners who developed a LRTI after a marathon showed a differential profile of saliva IgA, AMPs and chemokines compared to those runners who did not develop infection. Therefore, exercise training and post-marathon recovery would be important in the immunological profile and the risk of developing a LRTI. Further studies are needed to better understand the underlying mechanisms and the impact of salivary immunity and its regulation in the prevention and development of LRTI in non-elite marathon runners.

References

1. 12. Woodhead M, Blasi F, Ewig S, Garau J, Huchon G, Ieven M, et al. Guidelines for the management of adult lower respiratory tract infections-Full version. Clin Microbiol Infect. 2011;17:E1-59.

2. 32. Terra R, da Silva SAG, Pinto VS, Dutra PML. Effect of exercise on the immune system: response, adaptation and cell signaling. Rev Bras Med do Esporte. 2012;18(3):208-14.

3. 39. Costa R JS, Fortes MB, Richardson K, Bilzon J LJ, Walsh NP. The effects of post-exercise feeding on saliva anti-microbial proteins. Int J Sport Nutr Exerc Metab. 2012

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Salivary immunity and lower respiratory tract infections in non-elite marathon runners

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Rationale Respiratory infections are common after strenuous exercise, when salivary immunity may be altered. We aim to investigate changes in salivary immunity after a marathon and its relationship with lower respiratory tract infections (LRTI) in healthy non-elite marathon runners.

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Study of the hearing treshold of dance Teachers

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