Evaluation of gamete dysfunction as a cause of failed human in vitro fertilization

Esterhuizen, Aletta Dorothea (2000-12)

Thesis (D.Phil.)--Stellenbosch University, 2000.

Thesis

ENGLISH ABSTRACT: Chapter 1 provides literature based background information on the clinical importance of sperm morphology as recorded by strict criteria during the diagnostic approach of the infertile couple. Furthermore, the use of a sequential diagnostic schedule for couples in an assisted reproductive programme is emphasized. The author revisited the literature on chromatin packaging of spermatozoa and addresses this issue as an additional semen parameter providing information relating to DNA damaged spermatozoa. The chapter also includes evidence underlining the growing need for the implementation of the acrosome reaction as an important contribution to the assisted reproductive programme. Chapter 2 provides detailed descriptions of the material and methods used during the study. Chapter 3 is sub-divided into 5 sections, each of which represents a separate study that was prepared as a scientific paper. The study included 338 couples consulting for infertility treatment at various gynaecologists in Pretoria and Johannesburg. The diagnostic assisted reproductive laboratory support was provided by the Andrology laboratory of Drs du Buisson and partners from Pretoria. In the first study the role of chromatin packaging as an indicator of in vitro fertilization rates, the semen samples from 72 men were used to record their chromatin packaging quality as well as their sperm morphology classification. Significant different percentages CMA3staining (mean±SE) were recorded among the 2 morphology groups, namely 65.9%±3.5 and 44.5%±1.7 (p=0.001). Using cut off values for chromatin packaging established during the first study, the second study utilized semen from 140 men in the in vitro fertilization (IVF) and intracytoplasmic sperm injection programme (ICSI) to analyze for sperm concentration, motility, morphology and chromatin packaging (CMA3).IVF and ICSI data were stratified using 3 basic cut off values for CMA3staining, namely <44%, >44-60% and >60%. The study concluded that results on the chromatin packaging quality of spermatozoa could be used as an additional parameter of sperm quality since it could provide valuable information on decondensation status of a given sperm population. The third study aimed to establish zona pellucida induced acrosome reaction response (ZIAR) among 35 couples with normal and G-pattern sperm morphology and repeated poor fertilization results during assisted reproduction treatment. Interactive dot diagrams, divided patients into 2 groups i.e. ZIAR<15% and ZIAR>15% with mean fertilization rates of 49% and 79%, respectively. The study concluded that the ZIAR test has diagnostic potential, since it can assist the clinician to identify couples that will benefit from ICSI therapy. The forth study revisited the importance of micro-assay for acrosome reaction determinations in a diagnostic andrology laboratory. The micro-assay not only allows the use of a single zona pellucida, but also facilitates the future possibility of using recombinant zona pellucida proteins in a diagnostic test system. The final study in Chapter 3 includes results obtained from 49 couples (172 oocytes) and aimed to evaluate the role of chromatin packaging and sperm morphology during sperm-zona binding, sperm decondensation and the presence of polar bodies among 170 oocytes that failed in vitro fertilization (IVF). Odds ratio analyses indicated that being in the a group with elevated CMA3 staining i.e. >60%, the risk of decondensation failure increases 15.6 fold relative to normal CMA3 staining <44%. Chapter 4 underlines the validity of the sequential diagnostic approach and summarizes the results and value of a multistep diagnostic scheme. The chapter concludes with the recommendation that both chromatin packaging quality and zona pellucida mediation of the acrosome reaction should be part of the diagnostic tools in the assisted reproductive programme.

AFRIKAANSE OPSOMMING: Die literatuuroorsig in Hoofstuk 1 konsentreer in hoofsaak op die kliniese belang van sperm morfologie en die uitbreiding van die diagnostiese toetse en hantering van die egpaar in die reproduktiewe ondersteuningsprogram. Die kromatien pakkingskwaliteit van die spermsel word onderskryf as In belangrike toevoeging tot die diganostiese program, aangesien ONS skade dikwels saam met kromatiendefekte aangetref word. Die rol van die akrosoomreaksie word ook in detail literatuuroorsigtelik beklemtoon. Hoofstuk 2 bevat volledige inligting omtrent materiaal en metodes wat in die studie gebruik is. Hoofstuk 3 bevat die eksperimentele gegewens wat in 5 afsonderlike sub-afdelings as wetenskaplike publikasies aangebied word. Die studies bestaan uit data van 338 pasiënte, wat deur verskillende ginekoloë van Pretoria en Johannesburg gekonsulteer is waartydens drs. du Boisson en vennote van Pretoria die diagnostiese reproduktiewe laboratoriumdienste verskaf het. Die eerste studie stel dit ten doel om die belang en korrelasie van die spermsel kromatienpakkingskwaliteit van 72 mans te vergelyk met die morfologiese bou van sie sel. Aangesien morfologie reeds gevertig is as 'n kliniese voorspeller van bevrugting was dit nodig om hierdie parameter te vergelyk met die kromatienpakking van die sel. Twee afsnypunte word vir die normo-en teratozoospermiese mans identifiseer naamlik, 44.5%±1.7 en 65.9%±3.5 (p=O.001),respektiewelik. Die tweede studie gebruik die afsnypunte 44% en 66% om die in vitro bevrugting en intrasellulêre sperm inspuiting (ICSI) data te ontleed. Die resultate dui aan dat kromatienpakking In waardevolle bydrae tot die diagnostiek van die pasiënte lewer. Die derde studie stel dit ten doelom die waarde van die zona pellucida geinduseerde akrosoomreaksie (ZIAR) te bepaal. Die studie sluit die data van 35 egpare in wat almal normale of G-patroon morfologie het en verder onverklaarde swak bevrugtings resultate tydens in vitro bevrugtingsterapie. Interaktiewe punt diagram (interactive dot diagrams) verdeel die data in twee groepe naamlik, ZIAR<15% en ZIAR>15% met gemiddelde bevrugtingssyfers van 49% en 79%, respektiewelik. Die studie sluit af met die gedagte dat die ZIAR toets 'n groep pasiënte identifiseer met 'n besondere fisiologiese afwyking d.i. subnormale akrosoom respons op zona pellucida blootstelling. Die vierde afdeling van die hoofstuk onderstreep die belang van die mikro-tegniek vir die bepaling van die akrosoom reaksie, wat tydens die projek gebruik is Die vyfde afdeling van Hoofstuk 3 stel dit ten doelom 170 onbevrugde eierselle van 49 pasiënte te ontleed vir moontlike oorsake vir die mislukte bevrugting. Ondersoeke sluit in die kromatienpakking, sperm-zona binding, sperm dekondensasie en die teenwoordigheid van polêre liggaampies. Statisties blyk dit dat indien 'n kromtienpakking nie normaal is nie (>66%) het die spermsel 'n 15 keer groter kans om nie te dekondenseer nie. Hoofstuk 4 bespreek die noodsaaklikheid van die diagnostiese skedule by die hantering van die onvrugbare egpaar in.

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