Evaluation of spermatozoa DNA tests for an assisted reproductive techniques (ART) program : correlation with semen parameters and ART outcome

Burger, Riana (2013-03)

Thesis (MScMedSc)--Stellenbosch University, 2013.

Thesis

ENGLISH ABSTRACT: CHAPTER 1 A review of the application of traditional semen parameters for the investigation and diagnosis of male infertility and the role of predictive values in assisted reproductive techniques (ART) is presented. The importance of sperm morphology, with special emphasis on sperm morphology evaluation, is discussed. Also presented is an overview of the physiology of sperm DNA, the process of spermatogenesis, as well as the contribution of the spermatozoon to the embryo. The different causes of sperm DNA damage and techniques to determine DNA damage in spermatozoa are described. A survey is presented of the correlation of sperm DNA with sperm morphology. CHAPTER 2 All the materials and methods applicable to this study are described. Sperm morphology assessment and two different sperm DNA tests, the chromomycin A3 (CMA3) staining test and the terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end labelling (TUNEL) assay, are discussed in detail. CHAPTER 3 Results obtained in this study are presented. Results include the prevalence of abnormal sperm DNA and association with sperm morphology, specifically in the p-pattern and g-pattern morphology groups. Further results include the correlation of sperm morphology and sperm DNA with fertilization in vitro, embryo quality and pregnancy outcome. The percentage CMA3 positive spermatozoa (abnormal DNA) and percentage TUNEL positive spermatozoa (abnormal DNA) had a significant negative association with normal sperm morphology. P-pattern and g-pattern morphology groups differed significantly from each other for both CMA3 and TUNEL. A significant positive association between CMA3 and TUNEL was observed. No association between the percentage normal sperm morphology, percentage CMA3 positive spermatozoa and percentage TUNEL positive spermatozoa and IUI pregnancy outcome was observed. A significant negative association between the percentage TUNEL positive spermatozoa and IVF/ICSI pregnancy outcome was established. The percentage CMA3 positive spermatozoa had a significant positive (unexpected) association with IVF/ICSI pregnancy outcome. There was no association between the three variables and IVF/ICSI fertilization rates. A significant positive association between the percentage normal sperm morphology and IVF/ICSI embryo quality was found. There was a significant positive association between the percentage CMA3 positive spermatozoa and IVF/ICSI embryo quality (unexpected). The percentage TUNEL positive spermatozoa and IVF/ICSI embryo quality was negatively associated. CHAPTER 4 Interpretation of the results and future perspectives are discussed. The CMA3 staining test and TUNEL assay has a limited ability to distinguish between the p-pattern and g-pattern morphology groups. P-pattern spermatozoa are more likely to possess poor chromatin packaging and show increased levels of DNA fragmentation, but some p-pattern patients also may have normal DNA and g-pattern patients abnormal DNA. It is recommended that a sperm DNA test should be implemented routinely in andrology laboratories for the clinical diagnosis of sperm DNA damage in patients.

AFRIKAANSE OPSOMMING: HOOFSTUK 1 'n Samevatting wat handel oor die toepassing van tradisionele semen parameters vir die evaluasie en diagnose van manlike infertiliteit, asook die rol van voorspellingswaardes in kunsmatige voortplantingstegnieke word voorgelê. Die belangrikheid van sperm morfologie, met die klem op sperm morfologie evaluering, word ook bespreek. 'n Oorsig van sperm DNS fisiologie, die proses van spermatogenese, sowel as die sperm se bydrae tot die embrio word hier aangebied. Die verskillende oorsake van sperm DNS skade en die tegnieke om sperm DNS skade vas te stel, asook die die korrelasie tussen sperm DNS en sperm morfologie word ook bespreek. HOOFSTUK 2 Alle materiale en metodes wat van toepassing is op hierdie studie word beskryf. Sperm morfologie evaluering en twee verskillende sperm DNS toetse, die chromomycin A3 (CMA3) kleuringstoets en die “terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end labelling (TUNEL)" toets, word ook in meer besonderhede aangebied. HOOFSTUK 3 Resultate wat verkry is tydens hierdie studie word hier uiteengesit. Resultate behels die voorkomsyfer van abnormale DNS en die assosiasie met sperm morfologie, spesifiek in die p-patroon en g-patroon. Verdere resultate sluit die korrelasie van sperm morfologie en sperm DNS met bevrugting in vitro, embriokwaliteit en swangerskap uitkomste in. Die persentasie CMA3 positiewe sperme (abnormale DNS) en persentasie TUNEL positiewe sperme (abnormale DNS) het 'n betekenisvolle negatiewe assosiasie met normale sperm morfologie getoon. P-patroon en g-patroon morfologie groepe het betekenisvol van mekaar verskil vir beide CMA3 en TUNEL. 'n Betekenisvolle positiewe assosiasie is tussen CMA3 en TUNEL waargeneem. Geen assosiasie is tussen die persentasie normale sperm morfologie, persentasie CMA3 positiewe sperme en persentasie TUNEL positiewe sperme en IUI swangerskap uitkomste waargeneem nie. 'n Betekenisvolle negatiewe assosiasie is tussen die persentasie TUNEL positiewe sperme en IVB/ICSI swangerskap uitkomste vasgestel. Die persentasie CMA3 positiewe sperme het 'n betekenisvolle positiewe (onverwags) assosiasie met IVB/ICSI swangeskap uitkomste opgewys. Daar was geen assosiasie tussen die drie veranderlikes en IVB/ICSI bevrugting nie. 'n Betekenisvolle positiewe assosiasie is tussen die persentasie normale sperm morfologie en IVB/ICSI embryo kwaliteit waargeneem. Daar was 'n betekenisvolle positiewe assosiasie tussen die persentasie CMA3 positiewe sperme en IVB/ICSI embrio kwaliteit (onverwags). Die persentasie TUNEL positiewe sperme het 'n negatiewe assosiasie met IVB/ICSI embrio kwaliteit getoon. HOOFSTUK 4 Interpretasie van die resultate en toekomstige vooruitsigte is bespreek. Die CMA3 kleuringstoets en TUNEL toets het 'n beperkte vermoë om tussen die p-patroon en g-patroon morfologie groepe te onderskei. P-patroon spermatozoa sal heel waarskynlik oor swakker chromatien verpakking en meer DNS fragmentasie beskik. Sommige p-patroon pasiënte mag egter normale DNS toon, terwyl g-patroon pasiënte abnormale DNS het. Die implementering van 'n sperm DNS toets in andrologie laboratoriums, vir die kliniese diagnose van sperm DNS skade in pasiënte, word aanbeveel.

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