Department of Biomedical Sciences

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Browsing Department of Biomedical Sciences by Subject "Abstinence"

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    Basic semen parameters assisted by Computer-Aided sperm analysis (CASA) and their correlations with advanced semen parameters in normozoospermic men with different abstinence periods
    (Stellenbosch : Stellenbosch University, 2018-03) Ayad, Bashir Mohamed; Du Plessis, Stefan S.; Van der Horst, Gerhard; Stellenbonsch university. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences : Medical Physiology
    Introduction: Affordable basic semen analysis remains a fundamental procedure to be performed routinely during the diagnosis of male infertility. Advanced semen analyses, provide valuable clinical insights in treatment related decision-making, but these are highly expensive and lack universal standardization. The World Health Organization (WHO) guidelines for semen analysis have been adopted by most human andrology and fertility laboratories around the world for more than thirty years. According to the most recent prescribed guidelines of the WHO, subjects must remain abstinent for a minimum period of two days, but not longer than seven days before collecting a sample for a standard semen analysis. Several studies have sought to determine the optimal period for ejaculatory abstinence. However, the results are often found to be contradictory. The aims of this study are two-fold: Aim I: To investigate the effect of short (4 hours) and long (4 days) abstinence periods on sperm quality based on functional and biochemical parameters in a population of normozoospermic men, in addition to the prediction of various basic and advanced semen parameters of the second (4 hours) ejaculate from a set of basic parameters obtained from the first (4 days) ejaculate. Aim II: Establishing a correlation between basic semen parameters assisted by Computer-aided sperm analysis (CASA) and a set of advanced semen analysis tests. To determine cut-off values for advanced semen parameters from various basic parameters based on WHO defined reference values. Methods: Semen samples were collected from one hundred potentially fertile, normozoospermic men (20 to 30 years) who abstained for a period of exactly 4 days and 4 hours prior to collection of the first and second ejaculates respectively. Semen samples were analysed according to the WHO guidelines. Sperm concentration, total sperm count (T.S.C.), total and progressive motility and kinematic/velocity parameters were analysed by CASA. Sperm viability was performed by dye exclusion and morphology via SpermBlueTM staining techniques using Computer Aided Sperm Morphology Analysis (CASMA). Sperm acrosome status was evealuated by fluorescence microscopy. Sperm DNA fragmentation and intracellular superoxide (O2−•) levels were assessed by flow cytometry. Seminal antioxidant status [superoxide dismutase (SOD), catalase (CAT), thiobarbituric acid reactive substances (TBARS)] were measured by means of spectrophotometry. Statistical comparisons between short and long abstinence periods were performed using paired Student’s t-tests on GraphPad Prism™ software, while the prediction of various basic and advanced semen parameters of the second ejaculate from a set of basic semen parameters of the first ejaculate was performed using linear regression models. Correlations were performed using Spearman rank correlation coefficients, while receiver operating characteristic (ROC) curves were used to determine cut-off values. Statistical significance was set at p<0.05. Results I: A significant increase in total and progressive motility as well as in the velocity parameters were observed after short (4 hours) abstinence compared to long (4 days) abstinence periods. DNA fragmentation and intracellular O2−• levels were not significantly different between short and long abstinence periods. Despite the observed decrease in semen volume, sperm concentration and T.S.C. after the short abstinence period, all mean values of the conventional semen parameters still remained above the lower reference limits as recommended by the WHO 5th edition. We were also able to make predictions of various basic (semen volume, sperm concentration, total motility, progressive motility, viability and normal morphology) and advanced (DNA fragmentation, seminal plasma CAT activity and TBARS) parameters of the second ejaculate from a set of basic semen parameters obtained from the first ejaculate with relative certainty. Results II: The proportions of total and progressively motile as well as rapid spermatozoa were positively correlated with CAT activity (p<0.05). A significant negative correlation was observed between VCL, VSL, VAP and both intracellular O2−• and TBARS levels. ALH was significantly and negatively correlated with intracellular O2−• levels and DNA fragmentation, while its correlation with SOD activity was positive (p < 0.05). A negative correlation was also found between the percentage of viable spermatozoa and both O2−• levels and DNA fragmentation, whereas the percentage of normal morphology was negatively correlated with O2−• levels and positively with CAT activity (p < 0.05). The optimal intracellular O2−• cut-off value to differentiate between asthenozoospermic and normozoospermic men was calculated to be 227 median DHE fluorescence intensity [MFI] (p < 0.01). At this cut-off value, the test was 80% sensitive and 86% specific. Sperm viability was associated with a seminal plasma TBARS cut-off value of 9.86 Umol/L (p = 0.02) with sensitivity and specificity of 81% and 80% respectively. Conclusion: Our data challenges the generally accepted guidelines regarding the prescribed prolonged abstinence periods since the results show that 4 hours of sexual abstinence yielded significantly better samples from a sperm functional point of view. The results obtained from this study further support the validity of some CASA parameters as sensitive indicators of changes in sperm oxidative status and DNA integrity. This study also enabled defining the cut-off values and prediction of certain advanced variables from the basic semen analysis.