Investigation of The Effectiveness of Antioxidant Therapy in Oligoasthenoteratozoospermic Infertile Men

Primary outcomes

1. Total Antioxidant Capacity

   Time frame: 3 months

   Total Antioxidant Capacity (TAC) was measured by the colorimetric assay using the antioxidant Assay Kit (Cayman Chemical, Michigan, USA). All seminal plasma samples were diluted prior to analysis. Standards were diluted sequentially. All samples and standards were placed in duplicate. Chromogen and metmyoglobin were added to both samples and standards. Hydrogen peroxide was added and then the plate was incubated. 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

2. Sperm DNA Fragmentation

   Time frame: 3 months

   Sperm DNA Fragmentation (SDF) was analysed with TUNEL using the commercial In situ Cell Death Detection Kit. All samples were fixed with 4% paraformaldehyde (PFA). Fixed sperm samples were added onto polylysine-coated slides. Slides were kept in freshly prepared permeabilization solution on ice. For TUNEL reaction, label solution was mixed with enzyme solution, and 50 µl of the mix was dropped. Slides were incubated, afterward, they were washed three times and a mounting medium with DAPI was added. Samples were immediately examined and photographed using a fluorescent microscope. Photographs were analysed with the Image J program and at least 500 cells were evaluated from each sample. 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.

3. % of histone-rich spermatozoa

   Time frame: 3 months

   Sperm pellets were washed then spread on clean slides and the smears were air dried. Dried smears were fixed with 3% glutaraldehyde and they were immersed in a 5% aniline blue solution in 4% glacial acetic acid. 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.