Methods and Materials

Methods and Materials

Patients consented to their participation in the study particularly the use of their semen. Samples of male ejaculate were obtained from 200 males aged between 20 and 45. The participants had abnormal to normal sperm counts. The samples were tested for CT using a urethral smear test -u A commercial kit that uses direct immunofluorescence manufactured by Trinity Biotech was used. 64 samples returned positive whereas 136 returned negative. A random sampling method was used to select 50 positive samples and 25 negative samples that would be the control group for the study. Some of the participants who tested positive volunteered information on how long they had been infected with the disease and this was noted down. The participants were required to abstain from sexual activity 48 hours before the commencement of the study. On the day of semen collection, the men masturbated into vials that were appropriately numbered numerically (black ink for positive cases and blue ink for negative cases). Semen analysis was carried out using the standard procedure prescribed by the World Health Organization (WHO). Equal parts from each vial were analyzed for DNA fragmentation and the result was recorded appropriately on the sticker. Seminal plasma was also examined for antibodies using an enzyme-linked immunosorbent assay (ELISA) test.

The first step in the SCSA test was diluting an aliquot of semen from each sample to about 10 million. The diluting agent used was a pH 1.2 buffer of phosphate and salt (PBS). This treatment was allowed to hold for about 35 seconds (It is expected that fragments of DNA with 2 strands break (denature) while those with whole double strands remain unchanged.) The sperm cells were then stained with an acridine orange dye which produces contrasting stains in fragmented and intact DNA (red and green respectively). Flow cytometry analysis was then carried out for the stained samples in which blue light excitation of each sample was carried out and the sample observed for color changes (green to red). The flow cytometry allowed the measurement of the extent of sperm chromatin damage by quantifying the extent of metachromatic shifts from green to red in the cytogram intensity patterns. Denatured DNA was observed as the DNA fluorescent Intensity (DFI) or the ratio of denatured DNA to the total DNA present in a sample. The SCSA Diagnostics software was used to analyze the flow cytometry data and a DFI histogram modeled to present the percentage of denatured DNA strands per sample. The differences between samples from participants who tested positive for CT and those who tested negative for CT were averaged and statistically tested for reliability and validity. Also, the extent of DNA damage as revealed by the SCSA was compared across positive samples to observe whether long-term sufferers had a higher prevalence of fragmented DNA. SCSA has been used successfully to identify sperm with fragmented DNA from those sperm with whole DNA. The test reveals fragmented DNA as a plot with dots. The more the dots the higher the prevalence of SDF.