Study population
A randomized controlled trial was conducted for 3 months at the Sendai Medical Center in Sendai City, Miyagi Prefecture, located in northeastern Japan. The study was conducted in compliance with the Declaration of Helsinki and approved by the Institutional Review Board of the Medical Corporation of Shinkokai. All participants provided written informed consent for participating in the study. This study was registered with the University Hospital Medical Information Network (UMIN) Clinical Trial Registry (trial registration number: UMIN000030975).
The study implementation period in the protocol was during 2017; however, due to recruitment issues, it was delayed until the beginning of 2018. The recruitment process started on September 25, 2017, using pamphlets, targeting postmenopausal women who had planned to undergo medical check-ups in January 2018. Until the scheduled date of closure on November 30, 2017, eighty-five women voluntarily participated in the study. Among them, 63 were selected based on the inclusion and exclusion criteria as follows.
Inclusion criteria: (1) Postmenopausal women (natural absence of menstruation over at least 12 months since the last menstrual period or a bilateral oophorectomy procedure performed in a woman (surgical menopause); (2) Those who could visit the clinic every month for interviews or investigations during the study period; (3) Those who could remain compliant with the daily supplementation regimen and record daily notes throughout the study period comprising 12 weeks.
Exclusion criteria: (1) Those with history of allergy to soy foods, dairy products, or Brewer's yeast; (2) Those who reported intake of medications or functional foods that could affect our study results; (3) Those administered hormone therapy or medications that could affect blood hormone levels; (4) Those considered ineligible by the investigators.
The details of the study were explained to these women, and the first 60 women to give consent to participate in the study were selected at the end of December 2017. They were listed in chronological order and assigned into two groups by simple randomization. Odd number group A received a 10 mg equol supplement and lactobionic acid daily, while even number group B received no supplement. The participants received preintervention investigations from January 16 to 18, 2018. The equol supplementation group started taking the supplement on the day after completion of these investigations. All the participants had a regular follow-up every month for 3 months until April 20, 2018. In the equol supplementation group, one woman dropped out, and two women reported that they had occasionally used the equol supplement before the intervention. Consequently, 57 women (48 to 69 years of age) were eventually included for analysis in the study (Figure 1). The nurses at the Sendai Medical Center generated the random allocation sequence, enrolled participants, and assigned participants to interventions. The registered dietitian was responsible for explanation, distribution and monitoring supplements to the intervention group.
Study treatments
Twenty-seven women belonging to the equol supplementation group received 10 mg of oral equol-containing supplement per day, composed of 98% S-equol, 2% daidzein, 0.2% glycitein, and 0.1% genistein extracted from fermented soybeans (product name: FlavoCel EQ-5, Daicel Corporation, Tokyo, Japan).
Determination of equol-producer status
Prior to the intervention, early morning urine samples were collected from all participants. Urinary equol was measured using an immunochromatographic strip (Soy Check, Healthcare Systems Co., Ltd), as described in a previous study [15]. Individuals were considered equol producers if their urinary equol level was higher than 1.0 μM, as described in previous studies [3, 4].
Primary outcome measures
Body height and weight were measured using a height weight scale (A & D Company Limited, Tokyo, Japan). Overnight fasting blood samples were obtained to determine the levels of triglycerides (TG), total cholesterol (TC), low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, hemoglobin A1c (HbA1c), uric acid (UA), intact parathyroid hormone (PTH), and 25-hydroxy vitamin D. To assess the degree of arterial stiffness (arteriosclerosis), brachial-ankle pulse wave velocity (baPWV) was measured using vascular ultrasound (Fukuda Denshi, Tokyo, Japan). Visceral fat area was measured using computed tomography (CT). The level of AGEs was determined by measuring the skin autofluorescence on the volar side of the forearm using an AGE reader (DiagnOptics, Groningen, Netherlands) as described previously [26]. Autofluorescence was defined as the average fluorescence per nm over the entire emission spectrum (420-600 nm) as the ratio of the average fluorescence per nm over the 300-420-nm range [26]. Age-adjusted SAF levels (z scores) were calculated for each woman based on the total population. The above measurement of parameters was performed at baseline and 3 months postintervention.
Secondary outcome measures
Self-administered questionnaires were used for the assessment of climacteric symptoms using the Climacteric Scale developed by the Japan Society of Obstetrics and Gynecology (table 1). The questionnaire contained 21 items scored on a 4-point scale (Never=0, mild=1, moderate=2, severe=3). The total score represented the overall severity of the symptoms. These questionnaires were administered during the monthly follow-ups for 3 months. Starting from the first month of the intervention, the overall improvement in symptoms was also assessed using the following four responses: ‘a lot’, ‘somewhat’, ‘no change’, and ‘worse’. This technique was validated and widely used in the Japanese population.
Table 1. Climacteric Symptom Rating Scale by the Japan Society of Obstetrics and Gynecology
|
Item
|
Score
|
Q1
|
1.Facial skin blushing and upper body (hot flashes)
|
Never=0, mild=1, moderate=2, severe=3
|
Q2
|
2.Easy to sweat (sweating)
|
Never=0, mild=1, moderate=2, severe=3
|
Q3
|
3.Difficulty getting to sleep (insomnia)
|
Never=0, mild=1, moderate=2, severe=3
|
Q4
|
4.Difficulty staying asleep (light sleep)
|
Never=0, mild=1, moderate=2, severe=3
|
Q5
|
5.Irritability
|
Never=0, mild=1, moderate=2, severe=3
|
Q6
|
6.Anxiety
|
Never=0, mild=1, moderate=2, severe=3
|
Q7
|
7.Often irritated by trifles (anxious trifles)
|
Never=0, mild=1, moderate=2, severe=3
|
Q8
|
8.Feeling unhappy or depressed (depressive mood),
|
Never=0, mild=1, moderate=2, severe=3
|
Q9
|
9.Fatigue
|
Never=0, mild=1, moderate=2, severe=3
|
Q10
|
10. Eye strain
|
Never=0, mild=1, moderate=2, severe=3
|
Q11
|
11.Memory problems (forgetfulness)
|
Never=0, mild=1, moderate=2, severe=3
|
Q12
|
12.Dizziness
|
Never=0, mild=1, moderate=2, severe=3
|
Q13
|
13.Palpitations
|
Never=0, mild=1, moderate=2, severe=3
|
Q14
|
14.Chest tightness
|
Never=0, mild=1, moderate=2, severe=3
|
Q15
|
15.Headache
|
Never=0, mild=1, moderate=2, severe=3
|
Q16
|
16.Neck stiffness
|
Never=0, mild=1, moderate=2, severe=3
|
Q17
|
17.Backache and low back pain
|
Never=0, mild=1, moderate=2, severe=3
|
Q18
|
18.Joint pain
|
Never=0, mild=1, moderate=2, severe=3
|
Q19
|
19.Cold hands and feet
|
Never=0, mild=1, moderate=2, severe=3
|
Q20
|
20.Numbness in the legs or arms
|
Never=0, mild=1, moderate=2, severe=3
|
Q21
|
21.Sensitive to sounds
|
Never=0, mild=1, moderate=2, severe=3
|
Q22
|
Overall improvement in symptoms (not included in the baseline questionnaire)
|
Worse=-1, No change=0, Somewhat=1, A lot=2
|
Treatment adherence and monitoring of adverse effects
During the monthly follow-ups, the staff interviewed adherence to equol supplements, such as frequency and dose, as well as clinical signs and symptoms of adverse effects, to ensure compliance and to make timely decisions on cessation of the supplement. No participant reported any adverse effects during the course of the study.
Statistical analyses
All statistical analyses were performed using IBM SPSS 19 statistical software (IBM Japan, Minato-ku, Tokyo, Japan). The Mann-Whitney test was used to compare the differences in continuous data, and the chi-square test was used to compare categorical data between the equol supplementation group and the control group. For the assessment of quantitative changes in the metabolic and aging biomarkers before and after intervention, we used the Wilcoxon sign rank test. To compare the proportions of people with improved metabolic and aging biomarkers between the control and equol supplementation groups, we used the chi-square test and Fisher’s exact test. Additionally, we assessed the change in results with respect to equol exposure by categorizing the groups into four categories: 1) equol producers consuming equol supplements, 2) equol nonproducers consuming equol supplements, 3) quol producers without equol supplements, and 4) equol nonproducers without supplements. The extended Mantel-Haenszel chi square for linear trend was used to examine the association between postintervention improvement (dependent variable) and equol exposure (independent variable).
Changes in the severity (total score) of climacteric symptoms over time, i.e., at baseline and 1 month, 2 months and 3 months post equol intervention, were analyzed using a two-way repeated measure analysis of variance (ANOVA). Analysis of the studentized residuals showed that there was normality, as assessed by the Shapiro-Wilk test of normality and no outliers, as assessed by no studentized residuals greater than ± 3 standard deviations. There was sphericity for the interaction term, as assessed by Mauchly’s test of sphericity (p > .05). Additionally, post hoc pairwise comparisons between equol intervention and control groups as well as between equol producers and nonproducers were conducted using the Bonferroni correction. Improvements in climacteric symptoms in the control and equol supplementation groups were compared using the chi-square test and Fisher’s exact test for proportions. All tests were two-sided, and the statistical significance was set to p<0.05.