This study was a prospective observational study conducted from October 2016 to March 2019. The study was approved by the Institutional Review Board of the Kagawa Prefectural University of Health Sciences, and informed consent was obtained from the participants before the study. Additionally, the study was conducted in accordance with the Declaration of Helsinki. Twenty-nine healthy participants (16 females, 13 males) aged 19–34 years were enrolled. The participants were either students or staff at Kagawa Prefectural University of Health Sciences and Tottori University. Body weight and height were measured, and body surface and body mass index (BMI) were calculated for each participant. Participants with obesity (BMI >25), neuromuscular disease, cardiorespiratory disease, or chronic illness were excluded.
Ultrasound imaging and analysis
Using an ultrasound machine (ARIETTA 60, Hitachi, Chiba, Japan), measurements were taken with the 7.5 MHz linear array transducer placed in the 9th or 10th intercostal space between anterior and midaxillary lines in the zone of apposition. The participants were placed in the supine position for the measurement . Only one focus-level measurement was chosen, and the depth was at diaphragm level. Mean diaphragm thickness (MDT) was measured using the mean intima media thickness (IMT) method both at total lung capacity (TLC) and at functional residual capacity (FRC) . The mean IMT method is often used for measuring plaques automatically in the carotid artery. We calculated a mean of three points using IMT software: the center of the diaphragm thickness, and two surrounding points on each side (1 cm from the center of the diaphragm thickness) (Fig. 1).
Subsequently, we calculated the change ratio of diaphragm thickness (expressed in percentage) using the following formula: MDT at TLC minus MDT at FRC divided MDT at FRC [(MDTtlc - MDTfrc)/ MDTtlc] ×100.
Measurement of pulmonary function test and maximal respiratory pressure
A multifunctional spirometer (Autospiro AS507, Minato Medical Science, Osaka, Japan) was used to evaluate respiratory function. Before recording respiratory pressure data, the spirometric parameters of forced vital capacity (FVC) and forced expiratory volume % in one second (FEV1) were evaluated with participants in the sitting position. Respiratory pressure examinations, as well as the spirometric examination, were also conducted with the participants in the sitting position. First, to measure exhaled air from the participant’s mouth, they had to hold a mouthpiece between their teeth. Second, they held the cylinder between their teeth using light pressure, and a clip was placed on their nose to prevent air leaking out during the respiratory tests. Finally, we recorded maximal respiratory pressure to assess respiratory muscles strength. We measured maximum expiratory pressure (MEP) or maximum inspiratory pressure (MIP) by asking participants to breath in/out as deeply as they could and breath out/in sharply and quickly. The MEP and MIP were recorded three times, and maximal end-expiratory and end-inspiratory potential data were chosen for further analysis.
We compared the age, weight, height, BMI, body surface, mean diaphragm thickness, change ratio of the mean diaphragm thickness, and maximal end-expiratory and end-inspiratory potentials between males and females. We used the Welch t-test for these analyses. Analyses were performed using GraphPad 6 (GraphPad Software, La Jolla, CA, USA). Pearson’s correlation coefficient was used to assess correlation between diaphragm measurements and the maximal respiratory potentials or anthropometric measurements. A p-value <0.05 was considered statistically significant.