Trial design and participants
The current study was a randomized double-blind placebo-controlled clinical trial, registered in the Iranian registry of clinical trials (http://www.irct.ir: IRCT20171105037262N5), performed at Shahid-Beheshti hospital affiliated to Kashan University of Medical Sciences (KAUMS), Kashan, Iran, among 40 subjects with breast cancer at any stage undergoing neoadjuvant chemotherapy aged >25 years old between January 2020 and June 2021. Exclusion criteria were as follows: having renal and liver failure abnormal blood test.
Firstly, all subjects randomly divided into two groups to receive either melatonin supplements (n=20) or placebo (n=20) for 24 weeks. Every night one hour prior to sleep, patients will orally receive two 5 mg capsules of melatonin for 24 weeks. The appearance of the placebo was indistinguishable in color, shape, size, and packaging, smell and taste from the melatonin capsule. All capsules were produced by Zahravi Pharmaceutical Company (Tabriz, Iran). Subjects were requested not to change their ordinary physical activity and not to take any nutritional supplements during the 24-week trial.
To evaluate the compliance, the remaining supplements were counted and subtracted from the amount of supplements provided to the subjects. To increase compliance, all subjects received short messages on their cell phones every day to remind them about taking the capsules.
Ten milliliters fasting blood samples were collected at baseline and after the 24-week intervention at Kashan reference laboratory in a fasting status. Then, the samples were stored at -80°C before analysis. Serum C-reactive protein (CRP) values were assessed by commercial ELISA kit (LDN, Nordhorn, Germany) with inter- and intra-assay CVs of 4.5 to 6.4%, respectively. The plasma NO using Griess method (14), TAC by the use of ferric reducing antioxidant power developed by Benzie and Strain (15), GSH using the method of Beutler et al. (16) and MDA concentrations by the thiobarbituric acid reactive substances spectrophotometric test (17) were determined. All inter- and intra-assay CVs for NO, TAC, GSH and MDA values were less than 4%.
Using a formula suggested for clinical trials, having 25 subjects in each group were adequate while considering a type one error (α) of 0.05 and type two error (β) of 0.20 (power=80%), 283.7 ng/mL as SD and 290.0 ng/mL as the mean distinction (d) of hs-CRP as the key variable (18). Assuming 5 dropouts in each group, the final sample size was determined to be 20 subjects in each group.
Randomization and allocation were conducted as blindness using computer-generated random numbers.
To evaluate whether the study variables were normally distributed or not, we used the Kolmogrov-Smirnov test. To assess the effects of melatonin supplementation on biomarkers of inflammation and oxidative stress, we used one-way repeated measures analysis of variance. The P-value of <0.05 were considered statistically significant. All statistical analyses used the Statistical Package for Social Science version 18 (SPSS Inc., Chicago, Illinois, USA).