Intra-Day and Inter-Day Reliability of Measurements of the electromyographic signal on masseter and temporal muscles in patients with Down syndrome

The aim of the present study was to evaluate intra-day (test) and inter-day (re-test) reliability of surface electromyography (sEMG) signals of the masseter and temporal muscles in patients with Down syndrome (DS). We determined the reliability of sEMG variables in 33 patients with DS. EMG signals were recorded at rest as well as during maximum voluntary clenching and maximum habitual intercuspation (MHI). The signals were analyzed considering the amplitude in the root mean square (RMS), mean frequency (MNF), median frequency (MDF) and approximate entropy (ApEn). The intraclass correlation (ICC2,1) for the three trials recorded during MHI in the two sessions (test and retest) revealed excellent intra-session and inter-session reliability (ICC2,1 = 0.76 to 0.97) for all sEMG variables and muscles. In the rest position, excellent reliability was found for RMS and ApEn (ICC2,1 = 0.75 to 1.00) and good to excellent reliability was found for MDF and MNF (ICC2,1 = 0.64 to 0.93). The intra-session (test) and inter-session (re-test) analyses demonstrated the reliability of nonlinear sEMG variables of the masticatory muscles in adults with Down Syndrome.

evaluation of the complexity of sEMG variables of the masticatory muscles and can enable a better understanding of neurophysiological conditions. The root mean square (RMS) is a linear variable used to evaluate the excitability and activation of muscles before and after therapy 11,12 . Median frequency (MDF) is another linear variable widely used to interpret spectral characteristics of sEMG signals, reflecting muscle electrophysiology in different clinical conditions such as temporomandibular disordes 13 , cerebral palsy 14 and low back pain 15 .
Nonlinear time-series analyses indicate features of motor control that are important for clinicians to measure 16 . Information entropy has been proposed as a measure of irregularity (nonlinear behavior) in biological signals [16][17][18][19] . The measurement of entropy is reported to be a reliable method for characterizing neuromuscular changes 20 and approximate entropy (ApEn) enables a general understanding of the complexity of sEMG data 21,22 . In statistics, ApEn is used to establish the uncertainty or variability in a system. ApEn calculated from sEMG signals is dependent on the variability in both amplitude and frequency and better represents this aspect than amplitude or frequency alone 21,22 . As studies have demonstrated that nonlinear time-series analysis is more sensitive to changes in myoelectrical signals in comparison to linear methods 23,24 , the use of ApEn to quantify the irregularity or complexity of sEMG signals of the masseter and anterior temporal muscles in patients with DS may be important to the quantification and understanding of the neurophysiological conditions of these muscles.
However, the usefulness of sEMG data is dependent on the reproducibility of the signal detection method both within and between recording sessions 25 . This is a key factor to evaluating sEMG data in rehabilitation programs and clinical trials. Factors such as the preparation of the skin and positioning of the electrodes can exert an influence on the results when sEMG data are collected on different days 26,27 , as occurs when signals are collected before and after a clinical intervention.
Although a number of studies have demonstrated the reliability and reproducibility of sEMG signals for the evaluation of the masticatory muscles in both healthy and disabled patients 17,21,28 , to the best of our knowledge, no studies have assessed the reliability of sEMG signals of the masticatory muscles or the reproducibility of sEMG variables (linear and nonlinear) of the masseter and anterior temporal muscles in adults with DS. It is important to determine the reproducibility of measuring sEMG signals (intra-session and inter-session reliability) prior to suggesting the use of this technique as a tool for evaluating the efficacy of therapy designed to improve masticatory muscle function in this population.
Therefore, the aim of the present study was to determine the intra-session (test) and inter-session (re-test) reliability of sEMG signals of the masseter and anterior temporal muscles in adults with DS.

Results
The sample was composed of 23 adults with DS (15 men and eight women) with a mean age of 22.7 ± 6.5 years, mean body mass index (BMI) of 28.5 ± 6.8 kg/m 2 and mean neck circumference of 40.3 ± 4.2 cm ( Table 1).
The reliability of the sEMG variables was determined in all participants. Tables 2 and 3 displays the mean (SD), ICC and SEM values for each sEMG measurement during MIH and in the rest position for each muscle. The ICCs for all sEMG parameters and muscles during MHI in the two sessions (test and retest) revealed excellent intra-session and inter-session reliability (range: 0.76 to 0.97) ( Table 2). In the rest position, excellent reliability was found for RMS and ApEn (range: 0.75 to 1.00), whereas good to excellent reliability was found for MDF and MFN (range: 0.64 to 0.93).

Discussion
The present study confirmed the reliability (calculated using the ICC and SEM) of different sEMG variables of the masseter and anterior temporal muscles recorded during MHI and in the rest position in adults with DS. The results of the linear (amplitude and frequency) and non-linear (ApEn) analyses revealed excellent intra-session and inter-session reliability of sEMG signal readings during MIH (Table 2) as well as good to excellent reliability in the rest position (Table 3).
These results are similar to those reported in a previous study involving patients with cerebral palsy, i.e., high reproducibility for the data recorded during MIH and good to excellent reproducibility for data recorded in the rest position 17 . A high degree of reliability during MHI has also been found for the anterior temporal and masseter muscles in healthy patients 29,30 . These results demonstrate that sEMG activity recorded during MIH and at rest can be used to quantify the effects of clinical interventions on the masseter and anterior temporal muscles.
The sEMG evaluation is diagnostically useful in identifying patients with pain-related temporomandibular disorders 31,32 or for the observation of muscle activity in the clinical setting 30 . Multiparameter sEMG analysis has been used as a strategy to investigate the performance of the masticatory muscles following a stroke 33,34 , assess pain patterns in the masticatory muscles 35 and investigate the reproducibility of the evaluation of the masticatory muscles in patients with cerebral palsy 17 .

Variables
Baseline mean/SD www.nature.com/scientificreports www.nature.com/scientificreports/ Electromyographic components calculated in the time and frequency domains are the most widely used variables for the study of masseter and anterior temporal muscle activity, whereas nonlinear components, such as ApEn, remain under-investigated in clinical studies. This is an issue that needs to be better explored, as a change in the entropy of the signal also reflects a qualitative change in the physiological system 22 . Specifically for ApEn, the result is given between 0 and 2, with higher values reflecting greater irregularity within the time-series [36][37][38] . Thus, values near 2 indicate healthy muscle function. The excellent reliability found for ApEn under both test conditions (MIH and rest) could be an important stimulus for future studies to use this sEMG parameter in the evaluation of the effects of therapeutic interventions performed on the masseter and anterior temporal muscles.

conclusion
The present findings demonstrate the excellent reliability of surface electromyography variables (root mean squared, mean frequency, median frequency and approximate entropy) of the masseter and anterior temporal muscles in adults with Down Syndrome during maximum clenching effort as well as good to excellent reliability of the median and mean frequency recorded in the rest position.

Materials and Methods
Subjects. A convenience sample of adult patients with Down syndrome were evaluated at the Center of Bioscience Applied to Persons with Special Care Needs (CEBAPE), Institute of Science and Technology -UNESP/SJC, Brazil. The inclusion criteria were adult patients aged between 18-35 years-old, presenting snoring and/or mild to moderate apnea/hypopnea index, preserved cognitive function (ability to understand and respond to verbal commands necessary to perform this study, such as "open the mouth", "close the mouth", " bite", "relax"), agreed to participate by free will and, the Awareness Term along with signed by patient or patient's responsible. The exclusion criteria were body mass index (BMI) greater than 30, unsatisfactory dental health, not able to reach the University when is needed, present psychiatric disease and have been submitted to physiotherapy, speech therapy and/or orthodontic treatment at least 6 months before the beginning of this study.
This study received approval from the Human Research Ethics Committees of the Institute of Science and Technology-UNESP/SJC, Brazil, CEPh 2.127.141 (process number CAAE 64173616.4.0000.0077).
Instrumentation. The device used for the EMG acquisition was an eight-channel module (EMG System do Brasil Ltda ® ) consisting of a conditioner with a band pass filter with cut-off frequencies at 20 to 500 Hz, an amplifier gain of 1000 times and a common mode rejection ratio >120 dB. All data were acquired and processed using a 16-bit analog to digital converter (EMG System do Brasil Ltda ® ) with a sampling frequency 2 kHz. Active bipolar electrodes with a pre-amplification gain of 20x are included. interval between visits. Sessions were held in a silent, well-lit recording room. The participant was instructed to remain seated comfortably erect on a chair with eyes open, feet apart, shoulders relaxed and hands resting on thighs, with the head on the Frankfort plane parallel to the ground. During the procedures, the participants did not receive any visual feedback of the signals displayed on the computer. A short training period was conducted prior to the tests to familiarize the participant with the tasks. Explanations concerning the procedures and electrode placement were given and the participant was trained to bite as hard as possible with maximum voluntary clenching (MVC) effort with a cotton role between the arches and maximum habitual intercuspation (MHI) effort with no material interposed between the teeth.
The sites for the placement of the electrodes were shaved and cleaned with a cotton ball soaked in 70% alcohol to diminish impedance between the skin and electrode. Pre-gelled, self-adhesive, bipolar, disposable, Ag/AgCl surface electrodes (MediTrace ® ) measuring 10 mm in diameter were positioned over the right masseter (RM), left masseter (LM), right temporal (RT) and left temporal (LT) muscles with an inter-electrode distance of 20 mm in the region of greatest tonus after the participant performed moderate intercuspation 13 . Bandage tape was used to secure the electrodes further, with care taken to avoid micro movements. A rectangular metallic electrode measuring 3 ×2 cm coated with Lectron II conductive gel (Pharmaceutical Innovations) to increase the conduction capacity and impede interference from external noise was attached to the left wrist as a ground reference.
Readings were performed under three conditions: at rest, during MVC and during MHI. In session 1 (test), three readings (10 seconds each) were performed in the rest position, followed by three readings (five seconds each) during MVC with a two-minute interval between readings. After three minutes, three readings (five seconds each) were performed during MIH with a two-minute interval between readings. The same procedures were repeated after one week in session 2 (retest). Data processing. The sEMG signals obtained for the RT, RM, LT and LM muscles were processed using traditional linear analysis in the time (amplitude) and frequency domains as well as nonlinear analysis through the calculation of the complexity of the sEMG signal 17 . For the analysis of MVC and MIH, the first and last seconds of the five-second signals were discarded, resulting in three-second readings considered for each test. In contrast, all 10 seconds of the signals captured in the rest position were considered (Fig. 1).
The amplitude of sEMG signal was determined considering root mean square (RMS) using a 200-ms moving window. The mean RMS values of the three readings at rest and during MIH were normalized by the highest RMS values obtained during the three MVC readings (µV/µV × 100: % MVC).
The power spectral density of the sEMG signals was calculated using Welch's averaged periodogram with a Hamming window length zero-padded to the length of 2048 points. Overlap was 50% of the window length. The mean frequency (MNF) and median frequency (MDF) of the power spectrum were calculated for each