The impact of oocyte meiotic spindle and automated zona pellucida score and sperm chromatin packaging on fertilization and pregnancy for assisted reproduction techniques

Raubenheimer, Kara (2016-03)

Thesis (MSc)--Stellenbosch University, 2016.

Thesis

ENGLISH ABSTRACT: Non-invasive selection of developmentally competent human oocytes may provide information on the true fertilization potential of the spermatozoon in the absence of oocyte limitations. The objective was to measure 856 oocyte’s competence by assessing oocyte metaphase 2 (M2) maturity, zona pellucida score (ZS) and presence of the meiotic spindle (SPp) using birefringent imaging software. ICSI was performed and fertilization (n=90 patients) and pregnancy rates (n=89) were measured and compared to the oocyte competence and sperm DNA chromatin via Chromomycin A3 (CMA3) (n=89). Fisher’s exact and odds ratio’s (OR) were used to determine effect. In total, 856 oocytes were harvested of which 568 (66%) were M2 stage of development. SPp oocytes were (384/730) 52.60%, OR was performed on the SP to determine its relevance to fertilization, its presence in the oocyte prior to ICSI, resulted in fertilization 1.5:1 times more than when it was absent, p=0.01. SPp embryos selected for embryo transfer resulted in a 65% expanded blastocyst rate, full blastocyst rate of 58% and early blastocyst rate of 54%. A negative development competence on day 5 was also correlated to absence of meiotic spindle (SPa) prior to ICSI with 56% of day 5 embryos transferred reaching only the compacted morula stage; while 50% of SPa embryos reached the morula stage at time of embryo transfer on day 5. Although there were no statistical differences between the pregnancy rates of SPp and SPa embryos, there were slight tendencies for better embryo quality. The SPp had a pregnancy rate (PR) of 40.91% (36/88). Random effects logistic regression OR performed on 768 oocytes from 90 patients indicated pregnancy to succeed 1.4:1 when SPp (p=0.89). The mean average automated ZS was 18.96 μm (95% CI: 15.75; 22.16; n=625 positive ZS from 90 patients). The ZS revealed a GLS linear regression with p=0.04 to fertilize when the ZS was 19.20 μm (16.60; 21.79). There was no statistical difference between ZS of the pregnant and non-pregnant groups. The main objective was to prove that when oocyte quality is optimized, that fertilization rates and by implication, pregnancy rates would be improved. If not, failure to fertilize or implant would most probably be due to decreased spermatozoa capacity to fertilize possibly due to damaged chromatin packaging. The chromatin packaging (CMA3) of the study population was 74% semen samples with >40% immature DNA. The OR for CMA3 underlines the hypothesis, that when oocyte competence for fertilization is controlled to a degree, the success or failure of treatment can be indicated by the CMA3 value. In this instance, the OR is highly predictive for success with pregnancy when ICSI is performed on CMA3 values that are immature (≥41%). Logistic regression calculated the OR for immature DNA (CMA3≥41%) to predict pregnancy to be likely with odds of 1.6:1, p=0.39. Hypothesis OR: If ICSI is performed on an oocyte with: M2 and SPp, and this embryo develops to day 5 for embryo transfer, the odds of pregnancy, if working with a semen sample with >40% immature DNA, would be 1.9: 1.

AFRIKAANSE OPSOMMING: Nie indringende seleksie van oösiete se potensiële ontwikkelingsbevoegtheid mag inligting voorsien met betrekking tot sperm DNA chromatien. Die doelwit was om 856 oösiete se doeltreffendheid te meet deur oösiet metafase 2 (M2) volwassenheid te asseseer, zona pellucida telling (ZS) en die teenwoordigheid van die meiotiese spool (SPp) deur die gebruik van lig deurdringbare sagteware. Deur middel van intrasitoplasmiese sperm inspuiting (ISSI) is bevrugting (n=90 pasiënte) en swangerskap syfers (n=89) gemeet en vergelyk met die oösiet doeltreffendheid en sperm DNA chromatien via Chromomycin A₃ (CMA₃) (n=89). Fisher se ‘exact and odds ratio’s’ (OR) is gebruik om die effek te bepaal. In totaal was 856 ge-oes waarvan 568 (66%) op M2 vlak van ontwikkeling was. SPp oösiete was (384/730) 52.60%. OR was toegepas op die SP om die relevansie hiervan te bepaal met bevrugting. SPp teenwoordigheid in die oösiete voor ISSI het, 1.5:1 kere meer bevrugting tot gevolg gehad, as wanneer dit nie teenwoordig was nie, p=0.01. SPp geselekteerde embrios vir embrio oorplasing het 65% uitgesette blastosiste tot gevolg gehad, volledig ontwikkelde blastosiste van 58% en vroeë blastosiste van 54%. ‘n Negatiewe ontwikkelings doeltreffendheid op dag 5 is ook gekorreleer met die gebrek aan meiotiese spoel (SPa) voor ISSI met 65% van dag 5 embrios oorgeplaas wat slegs op die kompakte morula fase was terwyl 50% van SPa embrios die morula fase bereik het teen embrio oorplasing op dag 5. Hoewel daar geen statistiese verskille tussen die SPp en Spa gevind was nie, was daar klein tendense vir beter embrio kwaliteit in die SPp met ‘n swangerskap syfer (PR) van 40.91% (36/88). Steekproewe van logistiese regressie van OR uitgevoer op 768 oösiete van 90 pasiënte het aangedui dat swangerskap sal slaag 1.4:1 wanneer SPp(p=0.89). Die gemene gemiddelde automatiese ZS was 18.96 μm (95% CI:15.75;22.16;n=625 positief ZS van 90 pasiënte). Uit die ZS blyk dit dat GLS linieêre regressie om te bevrug ZS 19.20μm (16.60;21.79) was, p=0.04. Daar was geen statistiese verskil tussen die ZS van die swanger en die nie swanger groepe. Die hoofdoel was om te bewys dat wanneer oösiet kwaliteit optimaal is dat bevrugting syfers en by implikasie swangerskap syfers verbeter kan word. Indien nie, sal onvermoë om te bevrug of te implanteer heel waarskynlik veroorsaak word deur die verminderde spermatozoa kapasiteit of vermoë om te bevrug as gevolg van beskadigde chromatien integriteit. Die chromatien integriteit (CMA₃) van die studie populasie was 74% semen monsters met >40% onvolwasse DNA. Die OR vir CMA₃ bevestig die hipotese dat wanneer oösiet doeltreffendheid vir bevrugting beheer word tot ‘n mate, die sukses of mislukking van behandeling aangewys kan word deur die CMA₃ waarde. In hierdie geval, is die OR hoogs voorspelbaar vir sukses met swangerskap wanneer ISSI uitgevoer word op CMA₃ waardes wat onvolwasse is (≥41%). Logistieke regressie bereken dat die OR vir immature DNA (CMA₃ ≥41%) om swangerskap te voorspel met die waarskynlikheid van 1.6:1,p=0.39. Hipotese OR: wanneer ISSI uitgevoer word op ‘n oösiet met: M2 en SPp, en die embrio ontwikkel tot dag 5 vir embrio terugplasing, sal die waarskynlikheid van swangerskap, wanneer die semen monster ‘n onvolwasse DNA CMA3 waarde van >40% het, 1.9:1 wees.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/98420
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