Thesis (MMed)--Stellenbosch University, 2105. / ENGLISH ABSTRACT: Intracytoplasmic sperm injection (ICSI), as well as other micromanipulation assisted reproductive technology methods, such as physiologic ICSI (PICSI) and intracytoplasmic morphologically selected sperm injection (IMSI), are routinely used in many fertility laboratories around the world. An integral part of these methods is the manipulation of spermatozoa in preparation of the injection into the oocyte. It is common practice to place prepared spermatozoa in a viscous holding medium to facilitate the handling, manipulation and slowdown of spermatozoon movement during the immobilization and injection processes of ICSI. The possible effect of these holding mediums on basic semen parameters, as well as the sperm deoxyribonucleic acid (DNA) and structural integrity of spermatozoa, is of importance.
Hamilton Thorne IVOS® developed an automated software solution for live sperm morphology evaluation under high magnification, called IMSI StrictTM. It combines Tygerberg Strict Criteria morphological classification of human spermatozoa with motile sperm organelle morphology examination (MSOME) and provides software-based categorization. The IMSI StrictTM software was developed to aid in the IMSI spermatozoon selection process that enables objective classification of spermatozoa to remove inter-technician variation. For good optics and spermatozoon evaluation in IMSI StrictTM, spermatozoa need to be moving very slowly or be immotile, but still viable. This can be achieved by placing spermatozoa in a viscous holding medium, either polyvinylpyrrolidone (PVP) or SpermSlowTM, sometimes for a substantial time period. Before marketing the clinical use of IMSI StrictTM, the possible toxicity or deleterious effect of PVP and SpermSlowTM on spermatozoa needs to be excluded.
The primary objective of this study was to evaluate the effect of PVP and SpermSlowTM on human spermatozoa after different exposure times using a viability stain, CASA motility and kinetic parameters, chromatin packaging analysis (CMA3 staining analysis) and DNA fragmentation analysis (TUNEL analysis). The secondary objective was to evaluate the effect of PVP and SpermSlowTM on human spermatozoa‟s ultrastructure with Transmission Electron Microscopy.
This prospective analytical study was conducted at Drs Aevitas Fertility Clinic (Vincent Pallotti Hospital, Cape Town, South Africa) as well as the Fertility Unit at Tygerberg Hospital (Cape Town, South Africa) between July 2013 and October 2014. A total of 90 separate (no duplication) semen samples were analysed for the quantitative analysis (primary objective) and 1 sample for the descriptive analysis (secondary objective).
Results showed that although PVP and SpermSlowTM treated sperm outcomes often differed significantly after typical statistical analysis, clinically these two mediums were shown to be equivalent (using a specific statistical test for equivalence) for the tested outcomes. PVP and SpermSlowTM had no detrimental effect clinically on sperm viability, motility parameters, chromatin packaging and DNA fragmentation rate. The secondary investigation indicated that SpermSlowTM might exert a disintegrating effect on various sperm membranes, and as a secondary consequence of the eventual necrotic process, alteration of chromatin and cytoskeletal components. PVP medium on the other hand did not show these disintegrating effects. This finding needs to be further investigated since only one semen sample was evaluated.
Based on this study‟s results, either PVP or SpermSlowTM can be used for IMSI StrictTM purposes. However, the study did not include the technical aspects of the usage of PVP and SpermSlowTM. / AFRIKAANSE OPSOMMING: Intrasitoplasmiese sperm inspuiting (ICSI), sowel as ander mikro-manipulasie voortplantings tegnieke, soos fisiologiese ICSI (PICSI) en intrasitoplasmiese morfologies geselekteerde sperm inspuiting (IMSI), word in baie fertiliteitsklinieke regoor die wêreld gebruik. 'n Integrale deel van hierdie metodes is die manipulasie van spermatosoa ter voorbereiding van die inspuitproses. Dit is algemeen om voorbereide spermatosoa in 'n viskose medium te plaas om die hantering, manipulasie en vertraging van spermatosoön beweging tydens die immobilisasie en inspuitproses van ICSI te fasiliteer. Die effek van hierdie mediums op basiese semenparameters, sowel as die sperm deoksiribonukleïensuur (DNS) en strukturele integriteit van spermatosoa, is van belang.
Hamilton Thorne IVOS® het 'n sagteware oplossing, IMSI StrictTM, vir lewende sperm morfologie evaluering onder hoë vergroting ontwikkel. Hierdie sagteware bied sagteware-gebaseerde morfologiese klassifikasie deur die Tygerberg streng kriteria morfologiese klassifikasie met beweeglike spermorganel morfologie ondersoek (MSOME) te kombineer. Die IMSI StrictTM sagteware is ontwikkel om die objektiewe klassifikasie van spermatosoa vir IMSI spermatosoön seleksie moontlik te maak. Spermatosoa moet baie stadig beweeg of immotiel, maar steeds lewensvatbaar wees om goeie optika en spermatosoön evaluering vir IMSI StrictTM te verseker. Dit sal bereik kan word deur spermatosoa in 'n viskose medium, hetsy PVP (“polyvinylpyrrolidone”) of SpermSlowTM, vir 'n aansienlike tydperk te inkubeer. Voordat IMSI StrictTM vir kliniese gebruik bemark kan word moet die moontlike toksisiteit of nadelige effek van PVP en SpermSlowTM op spermatosoa uitgesluit word.
Die primêre doel van hierdie studie was om die effek van PVP en SpermSlowTM op menslike spermatosoa na verskillende inkubasie tye te evalueer deur ʼn lewensvatbaarheid kleuring toets, twee sperm DNS toetse (CMA3 en TUNEL) en rekenaar geëvalueerde sperm beweeglikheid toetse te gebruik. Die sekondêre doel was om die effek van PVP en SpermSlowTM op menslike spermatosoa se ultrastruktuur deur middel van Transmissie Elektronmikroskopie te evalueer. Hierdie studie is by Drs Aevitas Fertiliteitskliniek (Vincent Pallotti Hospitaal, Kaapstad, Suid-Afrika) sowel as die Fertiliteitseenheid by Tygerberg Hospitaal (Kaapstad, Suid-Afrika) tussen Julie 2013 en Oktober 2014 uitgevoer. 'n Totaal van 90 semenmonsters vir die kwantitatiewe analise (primêre doel) en een vir die beskrywende analise (sekondêre doel) is ontleed.
Resultate het getoon dat alhoewel PVP en SpermSlowTM geïnkubeerde spermuitkomste dikwels na ʼn tipiese statistiese analise betekenisvol verskil, hierdie twee mediums vir die geëvalueerde uitkomste klinies ekwivalent (bepaal deur middel van spesifieke statistiese toetse vir ekwivalensie) is. Die mediums het ook nie klinies 'n nadelige effek op sperm lewensvatbaarheid, beweeglikheid parameters, chromatien verpakking en DNS fragmentasie koers getoon nie. Die sekondêre ondersoek het getoon dat SpermSlowTM hoofsaaklik 'n effek van disintegrasie op verskeie spermmembrane getoon het. Hierdie nekrotiese proses kan lei tot verandering van chromatien en sitoskelet komponente. PVP medium het egter nie dieselfde disintegrerende effek getoon nie. Hierdie bevinding moet egter verder ondersoek word, aangesien slegs een semenmonster geëvalueer is.
Alhoewel hierdie studie nie die tegniese aspekte van die gebruik van PVP en SpermSlowTM geëvalueer het nie, kan aanbeveel word dat óf PVP óf SpermSlowTM op grond van geëvalueerde uitkomste tydens die IMSI StrictTM sperm seleksie proses gebruik word.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/97028 |
| Date | 03 1900 |
| Creators | Nel, Marlize |
| Contributors | Windt De Beer, Marie-Lena, Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Obstetrics and Gynaecology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | 145 pages : illustrations |
| Rights | Stellenbosch Universitty |
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