Participants
PCOS outpatients were recruited from the Department of Endocrinology, Shengjing Hospital of China Medical University in 2020 and data collection was completed in January 2021. The inclusion criteria were: age 18-40 years; BMI≥24kg/m2; anovulation; and a diagnosis of PCOS based on the Rotterdam diagnostic criteria. The exclusion criteria were: use of medication therapy that impacting on carbohydrate or lipid metabolism (oral contraceptive pills, insulin-sensitizers, anti-epileptics, anti-psychotics, statins, and fish oil) in the recent 6 months; body weight fluctuations of more than 5% in the past 3 months; in preparation for pregnancy, pregnant or lactating; perimenopausal; night-shift workers; fasting for more than 16 hours a day; hypotension; patients with other diseases (such as congenital adrenal hyperplasia, Cushing syndrome, androgen-secreting tumors, hyperprolactinemia, diabetes, thyroid diseases, severe serious cardiovascular, gastrointestinal, and kidney or liver diseases); alcohol intake of more than 100g per week; smoking within the past 3 months and engaging in high-intensity exercise.
Study Design
The protocol for this study was approved by the Medical Research and New Technology Ethics Committee of Shengjing Hospital of China Medical University (reference: 2020PS682K). The study followed a pre-post non-randomized design and it was pre-registered on ClinicalTrials.gov (NCT04580433). The trial consisted of a 1-week baseline weight stabilization period followed by a 5-week TRF intervention period. After signing the informed consent, the following data were obtained to determine eligibility: (1) Height, weight, and age, (2) Blood pressure, (3) Menstrual cycle, and (4) Medical history. The eligible participants were then invited to attend a baseline assessment visit where they completed the following assessments: (1) Body composition analysis, and (2) Three Factor Eating Questionnaire Revised 21 Item (TFEQ-R21) questionnaire [23]. They also underwent a standard 75 g oral glucose tolerance test and blood samples for measurement of plasma glucose and insulin were drawn prior to and at 60 and 120 min after glucose ingestion. During the baseline (week 0-1), participants continued with their usual diets, exercise, and sleep habits to keep their weight stable. During the TRF period (week 2-6), they were asked to not change the composition of their usual diets but were instructed to eat freely from 8 a.m. to 4 p.m. daily and fast from 4 p.m. to 8 a.m. the next day. Participants were provided with a food diary and were instructed to record their daily food intake from start to finish and use the Boohee software, a diet and fitness app in China to calculate the corresponding calories. Daily dietary calorie intake was required to be approximately consistent with the baseline for as much as possible (fluctuations of no more than 10%). During the 16-hour fasting, only water or calorie-free beverages were allowed, and participants were encouraged to drink enough water throughout the intervention period. We contacted participants via phone at the end of each week to review the protocol, monitor any adverse events, and provide support and guidance to promote compliance with interventions. All the baseline assessments were repeated at the follow-up visit on the 6th week when the diaries and data of time to return to normal menstrual cycle were collected. All the examinations were conducted in the morning while the participants were fasting.
Anthropometric Measurements
For each participant, body weight and height were measured to calculate the BMI [weight (kg) divided by height squared (m2), kg/m2]. Height was measured to the nearest 1 cm using a wall-mounted stadiometer (Seca 711; Seca, Hamburg, Germany). Body weight was determined to the nearest 0.1 kg using a multi-frequency bioelectrical impedance analyzer InBody 770 scanner (In-body Bldg, Seoul, Korea), with high resolution touch screen, frequency 1,5, 50, 260, 500, 1000kHz and measurement time 60 seconds, with the subjects in a standing position after shoes, coats and sweaters were removed, according to the manufacturer’s instructions. Waist-to-Hip Ratio (WHR) was measured by InBody 770 scanner, waist circumstance and hip circumstance were measured by the same nurse. The WHR was calculated using these measurements.
Body Composition
Body composition such as skeletal muscle mass (SMM) (kg), body fat mass (BFM) (kg), body fat percentage (BF%) and visceral fat area (VFA) (cm2) were evaluated by InBody 770 scanner.
Blood Sampling and Analysis
The participant’s fasted blood samples were collected before and after the intervention. Blood samples were taken from antecubital vein and collected into BD Vacutainers Tubes (SSTTM II Advance, REF 367953). Then, samples were centrifuged for 10 min at 3600 rpm at 4 °C. The obtained serum and plasma were aliquoted and stored at –80 °C until analysis. Insulin 0min, 60min, 120min (μU/mL) were measured by radioimmunological assay, luteinizing hormone (LH) (mIU/mL) and follicle-stimulating hormone (FSH) (mIU/mL) were measured by chemiluminescent immunoassay, total testosterone (TT) (ng/mL) was measured by electrochemiluminescent immunoassay, insulin-like growth factor 1 (IGF-1) (ng/mL) and sex hormone-binding globulin (SHBG) (nmol/L) was measured by immunochemiluminescent on Beckman Coulter Unicel Dxl 800. Total cholesterol (TC) (mmol/L) was measured by cholesterol oxidase method, triglycerides (TG) (mmol/L) were measured by deionization & enzyme method, and low density lipoprotein-cholesterol (LDL-C) (mmol/L) was measured by selective solubilization method, alanine aminotransferase (ALT) (U/L) and aspartate aminotransferase (AST) (U/L) were measured by NADH method, uric acid (UA) (umol/L) (umol/L), Glucose 0min, 60min, 120min (mmol/L) and high-sensitivity C-reactive protein (hs-CRP) (mg/L) were measured separately by uricase-PAP method, hexokinase method and rate nephelometry method on ci 16200 Abbott Architect analyzer. Homeostasis model assessment-insulin resistance (HOMA-IR) was calculated according to the formula “FINS (μU/mL) × FG (mmol/L)/22.5”. Free androgen index (FAI) (%) was calculated according to the formula “TT (ng/mL) × 100/SHBG (nmol/L)”.
Questionnaires
The TFEQ-R21 were completed during pre- and post- visits. The TFEQ-R21 covers 3 eating behavior domains: the cognitive restraint scale (6 items) assesses control over food intake to influence body weight and body shape; the emotional eating scale (6 items) measures the propensity to overeat in relation to negative mood states; the uncontrolled eating scale (9 items) assesses the tendency to lose control overeating when feeling hungry or when exposed to external stimuli. It contains 20 questions based on a 4-point Likert-Scale (1 = definitely false, 2 = mostly false, 3 = mostly true, 4 = definitely true) and one question scored between 1–8 on eating restraint (1 = no restraint when eating, 8 = extreme restraint when eating). Higher scores indicate greater cognitive restraint, uncontrolled eating, or emotional eating.
Statistical Analysis
The distribution of continuous data was tested with the Anderson-Darling test. Continuous variables were presented as mean ± standard deviation (mean ± SD) (normally distributed) or median (25th–75th percentile) (non-normally distributed) and analyzed using Student’s test for matched pairs (normally distributed) or Wilcoxon matched pairs signed-rank test (non-normally distributed) to compare parameters before and after 5 weeks of the TRF period. All data analyses were performed in GraphPad Prism (version 8.0.1; GraphPad Software). Significance was considered at a value of P<0.05. For responses with missing values, the values were not included in the analyses.
Compliance with ethical standards
Medical Research and New Technology Ethics Committee of Shengjing Hospital affiliated to China Medical University (reference: 2020PS682K).