Study design and subjects
We performed two studies: a longitudinal study on the changes in the quadriceps femoris muscle thickness in women with normal pregnancy, both during pregnancy and the postpartum period (study 1), and a prospective comparison study on the quadriceps femoris muscle thickness during pregnancy and the postpartum period in women with normal pregnancy and women treated for TPL with bed rest (study 2) between June 2014 and March 2016. The Ethics Committee of Tokushima University Hospital reviewed and approved the study (Approval number 2037-1), and written informed consent was obtained from all participants. All experimental protocols were designed according to the Declaration of Helsinki’s ethical principles and performed in accordance with the Ethical Guidelines for Medical and Health Research Involving Human Subjects.
The study population consisted of 26 pregnant women aged 23–40 years. We recruited the participants from the outpatient clinic of the Department of Obstetrics and Gynecology, Tokushima University Hospital between June 2014 and March 2016 for the longitudinal study. Women with hypertensive disorders of pregnancy, diabetes mellitus, gestational diabetes mellitus, maternal complications requiring medication, prenatally diagnosed severe fetal malformations, and those who were prescribed bed rest for any other reason were excluded. All included women had singleton pregnancies lasting 37 weeks or more. Muscle thickness of the proximal, intermediate, and distal points of the rectus femoris and vastus intermedius muscles were measured using ultrasonography at 11–13, 26, 30, and 35 weeks of pregnancy, and at 3–5 days and 1 month postpartum. We used the food frequency questionnaire software (Excel Eiyoukun FFQg version 3.0; Kenpousha, Tokyo, Japan) for evaluating the caloric and protein intake of the outpatient participants35. Physical activity was assessed using the Japanese version of the international physical activity questionnaire (IPAQ), which was used to evaluate physical activity in different domains36.37. Each domain assesses walking, with moderate and vigorous physical activity performed for at least 10 minutes each day per week. By using the IPAQ, we calculated an average metabolic equivalents (METs) score for total physical activity performed per week in MET min/week38.
Measurement of muscle thickness
The position of the participants during ultrasonographic measurements, and the choice of the site for measurements on limbs were the same as those described in a previous study9. We measured the muscle thicknesses of the quadriceps femoris at the anterior aspect of the right leg using a real-time B mode ultrasound imaging device (Hi Vision Preirus; Hitachi, Tokyo, Japan) with a 7.5 MHz linear array probe (EUP-L74M ). To improve acoustic coupling without compressing the dermal surface, a water-soluble transmission gel was placed over the scan head. The transducer was held perpendicular to the skin surface. Images were captured, stored on the hard disk of the ultrasound machine, and then muscle thickness was measured using on screen calipers (Fig. 3). All measurements were performed with the participants in the supine position, meaning full extension (passively) at the knee and elbow joints. Mean muscle thickness was calculated as the mean of five consecutive measurements of each muscle site, and all measurements were performed by the same experienced investigator.
Sites for ultrasonic measurement
The quadriceps femoris muscle is subdivided into four separate muscles in front of the thigh: vastus lateralis, located in the lateral superficial part; vastus medialis, located in the medial superficial part; rectus femoris, located in the middle superficial part; and vastus intermedius, located between vastus laterals and vastus medialis, in the deep part of the front of the thigh. We measured the muscle thickness of the rectus femoris and vastus intermedius separately at the proximal, intermediate, and distal points. These points were determined as follows: proximal point, on the anterior surface, 25% proximal to the point between the anterior superior iliac spine and upper pole of the patella; intermediate point, on the anterior surface midway between the anterior superior iliac spine and upper pole of the patella; distal point, on the anterior surface, 75% distal to the point between the anterior superior iliac spine and upper pole of the patella (Fig. 4).
We recruited 15 pregnant women who were admitted to our hospital at less than 30 weeks of pregnancy and treated with bed rest for TPL. We compared the muscle thickness in these women with that in the 26 normal pregnant women included in study 1. Cases with regular objective uterine contractions and/or significant cervical changes (dilation and/or effacement and/or short cervical length) were diagnosed as TPL . All 15 women with TPL were treated with bed rest and ambulation restricted to bathroom privileges, and intravenous ritodrine hydrochloride. The bed rest and intravenous ritodrine hydrochloride were continued until 35–36 weeks of pregnancy. Dietary supplement in the hospital included 626 mg of calcium, 1145 mg of phosphorus, 7 μg of vitamin D, and 2000 kcal/day on an average. Women with hypertensive disorders of pregnancy, diabetes mellitus, gestational diabetes mellitus, maternal complications requiring medication, prenatally diagnosed severe fetal malformations and those who had received glucocorticoid treatment or magnesium sulfate treatment were excluded from the study. All women had singleton pregnancies lasting 35 weeks or more. We measured the muscle thickness at the same six sites as in study 1 at 30 and 35 weeks of pregnancy, and then at 3–5 days and 1 month postpartum. We used the same frequency questionnaire software as in study 1 for outpatient participants, and we calculated the intake of protein and calories from the hospital meals given to the inpatients. Physical activity was assessed using the same questionnaire as in study 1.
Test-retest reliability of ultrasound measurements
To assess the reliability of the muscle thickness measurements, the observer made two sets of ultrasonic measurements on 17 pregnant and postpartum women who were not participating in study 1 and study 2. Two sets of measurements were taken 30 min apart. In each set, five measurements were performed at all six muscle sites. The investigator was blinded to the results of the measurements displayed on the ultrasound machine during the tests, and the order of the measurement sites was randomized in order to prevent memory bias. Intraclass correlation coefficients for the proximal point of the rectus femoris, intermediate point of the rectus femoris, distal point of the rectus femoris, proximal point of the vastus intermedius, intermediate point of the vastus intermedius, and distal point of the vastus intermedius were 0.95, 0.99, 0.99, 0.99, 0.99, and 0.99, respectively. Test–retest intra-examiner reliability was considered high for all measurements. Test–retest inter-examiner reliability was also high for all measurements, with the intraclass correlation coefficients for the proximal point of the rectus femoris, intermediate point of the rectus femoris, distal point of the rectus femoris, proximal point of the vastus intermedius, intermediate point of the vastus intermedius, and distal point of the vastus intermedius, being 0.77, 0.7, 0.75, 0.87, 0.99, and 0.84, respectively.
Data were expressed as mean ± standard deviation, medians and IQRs, and proportions (%). Fisher’s exact test, student t-test, and Mann–Whitney U test were used for statistical analysis of the baseline characteristics. Differences in the muscle thickness between the groups were compared by the student’s t-test (in the cross-sectional study) or a repeated measures ANOVA (in the longitudinal study), while multiple comparisons in the longitudinal study were performed using the paired t- test with Bonferroni post-hoc correction. All statistical analyzes were performed with EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan), which is a graphical user interface for R (R Foundation for Statistical Computing, Vienna, Austria). More specifically, it is a modified version of R commander designed to add the statistical functions that are frequently used in biostatistics39. All p-values were two-tailed, and α was set at a significance level of 0.05.