The Impact of Antioxidant Food Supplementation on Seminal Antioxidant Capacity, Sperm DNA Fragmentation and Sperm Chromatin Quality in Subfertile Men With Oligoastenoteratozoospermia Randomized Clinical Trial
Phase: NA
Conditions: Male Infertility
Sponsor: Ondokuz Mayıs University

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The Impact of Antioxidant Food Supplementation on Seminal Antioxidant Capacity, 
Sperm DNA Fragmentation and Sperm Chromatin Quality in Subfertile Men with 
Oligoastenoteratozoospermia Randomized Clinical Trial 
 
DATE OF ETHICS COMMITTEE’S APPROVAL: 16 March 2021 
MATERIALS AND METHODS (STUDY PROTOCOL) 
1. Patient Selection 
 
Forty-eight subfertile men diagnosed with idiopathic OAT in the Urology Clinics of 
Ondokuz Mayis Univerity (OMU) Medical Faculty Hospital between March and November 
2021 was included in this study. Ethics committee approval (2021/108) was obtained from 
Clinical Research Ethics Committee at OMU. All participants could not achieve pregnancy 
despite their regular sexual intercourse for 12 months or longer, and their female partners had 
normal fertility examinations. All patients were evaluated with OAT after two semen analyses 
in the last six months. Physical examination data, hormonal, and genetic evaluations of all 
patients were normal and were accepted as idiopathic infertile. Exclusion criteria of the patients 
were undergoing to sterilization procedures such as vasectomy, medical treatment or drug 
affecting the reproductive function or metabolism, taking multivitamins or herbal products, use 
of anticoagulants, sperm concentration <5 million/mL, untreated hypothyroidism or 
uncontrolled diabetes mellitus and medical conditions such as cancer, heart disease or cirrhosis. 
2. Antioxidant Food Supplement  
 
By computer-assisted simple randomization (www.randomizer.org) patients who 
agreed to participate in the study were divided into Group 1 (n=24) and Group 2 (n=24). A food 
supplement containing 2000 mg carnitine, 2000 mg fructose, 932 mg acetyl L-carnitine, 225 
mg vitamin C, 115 mg citric acid, 50 mg coenzyme Q10, 14 mg zinc, 115 µg selenium, 3750 

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µg vitamin B12, 500 µg folic acid (Alfasigma Health Science, Trento, Italy) was given daily as 
an antioxidant to the patients in Group 1. We did not suggest the use of any supplements to 
Group 2. All subjects were recommended to do moderate physical activity for at least 45 
minutes (at least 150 minutes-600 METs per week) 3-4 days a week for three months. 
3. Hormone Analyses 
 
Hormone analyses (FSH, LH, prolactin, estradiol, and total testosterone) were 
performed using the ELISA method by drawing peripheral venous blood samples from the 
patients between 7 am – 11 am after at least 12 hours of fasting. 
4. Body Mass Index (BMI) and Physical Activity Assessment 
 
BMI of the participants was calculated using the formula kg/m2. All patients 
participating in the study were recommended to get moderate physical activity at least 45 
minutes 3-4 times a week for three months. Physical activity assessments were made using the 
International Physical Activity Questionnaire (IPAQ) Short form, in which total scores included 
the sum of walking, moderate-intensity activity, and duration (minutes) and frequency (days) 
of vigorous activity (Meh et al. 2021).  
5. Semen Collection and Preparation 
 
Semen samples were collected from all patients at the 0th and 3rd month of 
examinations after 2-7 days of sexual abstinence. After incubation for 20-30 minutes at 37ºC, 
liquefied samples were analyzed according to the World Health Organization (WHO) 2010 
guidelines (World Health 2010). Semen samples were then treated to evaluate the total seminal 
antioxidant capacity (TAC), sperm DNA fragmentation (SDF) and sperm DNA chromatin 
condensation. For the analysis of SDF, sperm concentrations of samples were adjusted to 
2.5×106 /mL (Sharma et al. 2021). Semen samples were centrifuged at 2000 rpm for 8 minutes 
to separate seminal plasma from the sperm pellet. Then, seminal plasma was centrifuged at 

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8000 rpm for 10 minutes at 4°C and was stored at -70ºC until TAC measurements. A portion 
of the semen pellet was fixed for SDF analysis. The remaining pallet was used to determine 
sperm chromatin condensation with aniline blue staining. 
6. Measurement of Seminal Total Antioxidant Capacity 
 
TAC was measured by the colorimetric assay using the antioxidant Assay Kit (Cayman 
Chemical, Michigan, USA) according to the manufacturer's recommendations (Mahfouz et al. 
2009). All seminal plasma samples were diluted from 1:10 to 1:20 prior to analysis. Briefly, 
Trolox standards were diluted sequentially with assay buffer, and then 10 µl of each was added 
to the 96-well plate. Afterward, 10 µl of each sample was added to the sample wells. All samples 
and standards were placed in duplicate. Chromogen 10 µl metmyoglobin and 150 µl ABTS 
(2,2'-Azino-di-[3-ethylbenzthiazoline sulphonate]) were added to both samples and standards. 
To all wells 40 µl of 441 µM hydrogen peroxide was added and then the plate was incubated 
for 5 minutes on a shaker at room temperature. The absorbances of the standards and samples 
were measured at 750 nm using a microplate spectrophotometer (Multiscan GO, Thermo 
Scientific, Finland) after incubation. 
 
Calculations of each standard and sample were made to evaluate the assay. A standard 
Trolox curve was plotted with the mean absorbance of the standards. The TACs of the samples 
were calculated according to the formula using the linear regression of that standard curve and 
the average of the absorbance of samples: Antioxidant (mM) = [(Sample average absorbance) 
- (y-intercept)/ Slope] x Dilution 
7. Assessment of Sperm Chromatin Condensation by Aniline Blue Staining 
 
After the semen sample of each patient was immediately separated from the seminal 
plasma, some of the sperm pellet was washed twice with 1x phosphate-buffered saline (PBS) 
(Gibco, NY, USA). The spermatozoa were then spread on clean slides and the smears were air 

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dried for approximately 15 minutes. Dried smears were fixed with 3% glutaraldehyde at room 
temperature for 30 minutes. Afterward, all slides were immersed in a 5% aniline blue solution 
(Sigma, Steinheim, Germany) in 4% glacial acetic acid (Sigma, Steinheim, Germany) for 15 
minutes (Hekim et al. 2021). After staining, 200 sperm were counted at least on each slide at 
1000x magnification at a light microscope. Pale blue spermatozoa that received less or no 
staining were considered protamine-rich, and spermatozoa partially or completely stained dark 
blue were evaluated as histone-rich. 
8. Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) 
for Direct Detection of SDF  
 
SDF was analysed with TUNEL using the commercial In situ Cell Death Detection Kit 
(Roche, Mannheim, Germany) according to instructions of the manufacturer (Hekim et al. 
2021). After removing the seminal plasma, the pellet was washed with 1xPBS. For the positive 
control of the analysis, 30% hydrogen peroxide solution was placed on the fresh semen pellet 
of one of the samples and incubated for 15 minutes at 37°C. Afterward, all samples were fixed 
with 4% paraformaldehyde (PFA) (Merck KGaA, Darmstadt, Germany) for 20 minutes at room 
temperature. Then, PFA was removed, and the samples were washed twice with 1xPBS by 
centrifugation at 2000 rpm for 8 minutes. Sperm samples were added onto phosphate-buffered 
(PB) sucrose on polylysine-coated slides. They were kept in a humid and dark environment at 
4°C overnight. The next day, slides were washed twice with 1xPBS for 5 minutes and kept in 
freshly prepared 0.1% sodium citrate and 0.1% Triton X-100 permeabilization solution on ice. 
For TUNEL reaction, label solution was mixed with enzyme solution, and 50 µl of the mix was 
dropped onto the washed slides at the end of permeabilization. Negative controls were obtained 
by adding only label solution. Slides were incubated for 60 minutes at 37°C in a humid and 
dark environment. Afterward, the slides were washed three times with 1xPBS and a mounting 
medium with DAPI (Fluoroshield, Sigma, Missouri, USA) was added and covered with a 

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coverslip. Samples were immediately examined and photographed using an excitation 
wavelength of 461 nm and 519 nm (green) detection under a fluorescent microscope (BX51, 
Olympus Life and Material Sciences). Photographs were analysed with the Image J program 
(LOCI, University of Wisconsin) and at least 500 cells were evaluated from each sample. While 
blue fluorescence stained all nuclei, sperm nuclei showing green fluorescence were considered 
to have fragmented DNA. SDF was calculated as the number of sperm nuclei stained green as 
a percentage of the total sperm nuclei identified as blue in the same area. 
9. Statistical Analysis 
 
The conformity of the dependent and independent continuous variables used in the study 
to the normal distribution was analysed with the Kolmogorov Smirnov test. The comparison of 
the arithmetic mean of independent groups (with and without antioxidants), Student's t-test was 
used for variables with normal distribution, and Mann Whitney U test was performed for 
variables that did not fit a normal distribution. Comparisons of arithmetic means in dependent 
groups (pre-treatment and post-treatment) were analysed with Paired t-test for normally 
distributed variables and Wilcoxon test for variables found to be non-normally distributed. 
Statistical data analysis was performed using IBM SPSS Statistics Version 22.0 (Armonk, NY, 
USA). P<0.05 was considered statistically significant

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