Basic semen parameters assisted by Computer-Aided sperm analysis (CASA) and their correlations with advanced semen parameters in normozoospermic men with different abstinence periods

Ayad, Bashir Mohamed (2018-03)

Thesis (PhD)--Stellenbosch University, 2018.

Thesis

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.

Inleiding: Bekostigbare basiese semenanalise bly ‘n fundamentele prosedure wat roetinegewys uitgevoer word tydens die diagnose van manlike onvrugbaarheid. Gevorderde semenanalises bied waardevolle kliniese insigte in behandelingsverwante besluitneming, maar dit is baie duur en is nie universeel gestandaardiseer nie. Die Wêreldgesondheidsorganisasie (WGO) se riglyne vir die analisering van semen word wêreldwyd al vir meer as dertig jaar deur die meeste menslike andrologie- en vrugbaarheidslaboratoriums aangeneem. Volgens die mees onlangse voorgeskrewe riglyne van die WGO, moet mans hulself onthou van ejakulasie vir ‘n minimum tydperk van twee dae, maar nie meer as sewe dae lank voor die versameling van ‘n monster vir ‘n standaard semenanalise nie. Verskeie studies het probeer om die optimale tydperk vir ejakulatoriese onthouding te bepaal, maar die bevindinge was dikwels teenstrydig. Die doelstellings van hierdie studie is tweevoudig: Doelwit I: Om die effek van kort (4 uur) en lang (4 dae) onthoudingsperiodes op spermkwaliteit te ondersoek, gebaseer op funksionele en biochemiese parameters in ‘n populasie van normozoospermiese mans; en benewens daartoe om verskillende basiese en gevorderde semenparameters van die tweede (4 uur) ejakulaat te voorspel gebaseerop ‘n stel basiese parameters verkry vanaf die eerste (4 dae) ejakulasie. Doelwit II: Die vasstelling van korrelasies tussen basiese semenparameters, bygestaan deur rekenaargesteunde spermanalise (CASA), en ‘n stel gevorderde semenanalise toetse. Om afsnypunte vir gevorderde semenparameters te bepaal uit verskeie basiese parameters gebaseer op WGO gedefinieerde verwysingswaardes. Metodes: Semenmonsters is versamel van een honderd potensieel vrugbare, normozoospermiese mans (20 tot 30 jaar) wat vir ‘n tydperk van presies 4 dae en 4 ure voor die eerste en tweede ejakulasie onderskeidelik onthou het. Semenmonsters is volgens die WGO-riglyne ontleed. Spermkonsentrasie, totale spermtelling (T.S.C.), totale en progressiewe motiliteit en kinematiese / snelheid parameters is deur CASA geanaliseer. Spermlewensvatbaarheid is bepaal deur middel van kleurstofuitsluiting en morfologie met behulp van SpermBlueTM kleuringstegnieke en die gebruikmaking van rekenaargesteunde spermmorfologie-analise (CASMA). Akrosoom status is bepaal deur fluoresensie mikroskopie. DNA-fragmentasie en intrasellulêre superoksied (O2-•) vlakke is gemeet met behulp van vloeisitometrie. Seminale antioksidante status [superoksied dismutase (SOD), katalase (CAT), tiobarbituriensuur reaktiewe substanse (TBARS)] is geassesseer met behulp van spektrofotometrie. Statistiese vergelykings tussen kort en lang onthoudingsperiodes is uitgevoer deur gebruik te maak van gepaarde Student t-toetse op GraphPad PrismTM -programmatuur, terwyl die voorspelling van verskeie basiese en gevorderde semenparameters van die tweede ejakulasie vanaf ‘n stel basiese semenparameters van die eerste ejakulaat uitgevoer is deur gebruik te maak van lineêre regressiemodelle. Korrelasies is uitgevoer met behulp van die Spearman-rangkorrelasie koëffisiënt, terwyl operasionele karakteristieke krommes van ontvanger (ROC) gebruik is om afsnypunte te bepaal. Statistiese betekenisvolheid is vasgestel op p<0.05. Resultate I: ‘n Beduidende toename in totale en progressiewe motiliteit sowel as in die snelheidsparameters is waargeneem na kort (4 uur) onthouding in vergelyking met lang (4 dae) onthoudingsperiodes. DNA fragmentasie en intrasellulêre O2-• vlakke was nie beduidend verskillend tussen kort en lang onthoudingsperiodes nie. Ten spyte van die waargeneemde afname in semen volume, sperm konsentrasie en T.S.C. na die kort onthoudingsperiode, bly alle gemiddelde waardes van die konvensionele semenparameters steeds bo die laer verwysingslimiete soos aanbeveel deur die WGO 5de uitgawe. Ons was ook in staat om voorspellings met relatiewe sekerheid te maak van verskillende basiese (semen volume, spermkonsentrasie, totale motiliteit, progressiewe motiliteit, lewensvatbaarheid en normale morfologie) en gevorderde (DNA-fragmentasie, plasma-CAT-aktiwiteit en TBARS) parameters van die tweede ejakulasie vanaf ‘n stel basiese semen parameters verkry vanaf die eerste ejakulasie. Resultate II: Die verhoudings van totale en progressief motiele sowel as vinnige spermatozoa was positief gekorreleer met CAT aktiwiteit (p <0.05). ‘n Beduidende negatiewe korrelasie is waargeneem tussen VCL, VSL, VAP en beide intrasellulêre O2-• en TBARS-vlakke. ALH was beduidend en negatief gekorreleer met intrasellulêre O2-• vlakke en DNA-fragmentering, terwyl die korrelasie met SOD-aktiwiteit positief was (p <0.05). ‘n Negatiewe korrelasie is ook gevind tussen die persentasie lewensvatbare spermatozoa en beide O2-• en DNA-fragmentering, terwyl die persentasie normale morfologie negatief gekorreleer is met O2-• vlakke en positief met CAT-aktiwiteit (p <0.05). Die optimale intrasellulêre O2-• afsnywaarde om te onderskei tussen astenozoospermiese en normozoospermiese mans is bereken as 227 mediaan DHE fluoresensie-intensiteit [MFI] (p <0.01). By hierdie afsnywaarde was die toets 80% sensitief en 86% spesifiek. Spermlewensvatbaarheid is geassosieer met ‘n seminale plasma TBARS-afsnywaarde van 9.86 Umol / L (p = 0.02) met ‘n sensitiwiteit en spesifisiteit van onderskeidelik 81% en 80%. Gevolgtrekking: Ons data daag die algemeen aanvaarde riglyne uit rakende die voorgeskrewe verlengde onthoudingsperiodes aangesien die uitslae aantoon dat 4 uur seksuele onthouding aansienlik beter semenmonsters oplewer soos beskou uit ‘n spermfunksionele oogpunt. Die resultate soos verkry uit hierdie studie, ondersteun verder die geldigheid van sommige CASA parameters as sensitiewe aanwysers van veranderinge in sperm oksidatiewe status en DNA integriteit. Hierdie studie het ook die definiëring van afsnywaardes en voorspelling van sekere gevorderde veranderlikes vanaf die basiese semenanalise bepaal.

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