Thesis (MScMedSc (Biomedical Sciences. Medical Physiology))--University of Stellenbosch, 2010. / Bibliography / ENGLISH ABSTRACT: Sperm morphology remains an important parameter in the prediction of fertility, both
in vivo and in vitro. However, there remains a considerable level of concern
surrounding the true potential of this parameter due to the lack of standardization of
differential staining techniques used for the evaluation of sperm morphology. This
study aimed at investigating two commonly used staining techniques, Rapidiff® (RD)
and Papanicolaou (PAP), along with a new commercially available stain, SpermBlue®
(SB), in the evaluation of sperm morphometry and morphology. Results indicated that
significant differences in sperm morphometry exist due to the use of the staining
techniques. Findings further indicated that RD causes sperm head swelling while PAP
causes sperm head shrinkage. Results obtained using the SB staining technique have
indicated measurements closest to that which would be obtained through the
evaluation of fresh, unstained sperm. The lack of standardization and the different
effects various stains have on sperm structure and overall sperm morphology
evaluation should raise a level of concern, particularly when evaluating patients with
borderline morphology. Based on this, the use of the SB staining technique is
recommended over RD and PAP for effective and accurate morphology evaluation. In
further support of this technique, SB was shown to be quick and simple in method,
and allowed for the easy detection of sperm by computer aided sperm analysis
(CASA) systems such as the Sperm Class Analyzer (SCA®).
The second aim of this study was to examine the concentration, morphology and
motility of the resultant sperm populations following semen preparation using the
PureSperm® density gradient and swim-up techniques. Semen preparation is an
essential step in any fertility treatment protocol, and it is important that the sperm
obtained following semen preparation has sperm morphology and motility
characteristics capable of improving assisted fertility success rates. Currently, the
PureSperm® density gradient and sperm swim-up are the most widely employed
techniques in fertility clinics. Although there is sufficient evidence to suggest they are
each effective at extracting sperm with improved quality from neat semen, there
remains insufficient evidence to suggest which of these two techniques is superior.
The present investigation revealed that both sperm preparation methods were effective at improving sperm morphology and motility, however to varying degrees. The swimup
method yielded a population of sperm with superior motility and morphology
when assessed according to World Health Organisation (WHO) criteria, while the
PureSperm® density gradient technique isolated a higher percentage of normal sperm,
according to both WHO and Tygerberg strict criteria, with motility better than that of
neat semen. Although results obtained via the swim-up method suggest it would be
best for use in in vitro fertilization (IVF), the very low concentration of sperm isolated
via this method remains a significant draw-back. The PureSperm® density gradient
separation technique on the other hand is capable of isolating larger quantities of
sperm, which is likely to be of more benefit with fertility treatments requiring larger
quantities of sperm. Based on these findings, the use of PureSperm® density gradient
technique is recommended, due to its ability to isolate large quantities of good quality
sperm. However, a swim-up may still be of use when performing fertility treatment
using a sperm sample which possesses a high concentration and motility. / AFRIKAANSE OPSOMMING: Sperm morfologie bly ‘n belangrike parameter in die voorspelling van vrugbaarheid,
beide in vivo en in vitro. Tog is daar nogsteeds ‘n aansienklike vlak van kommer
rondom die ware potensiaal van hierdie parameter weens die gebrek aan
standardisering van verskillende kleuringstegnieke wat gebruik word vir die
evaluering van spermmorfologie. Hierdie studie is daarop gemik om ondersoek in te
stel na twee algemeen gebruikte kleurings tegnieke naamlik, Rapidiff® (RD) en
Papanicolaou (PAP), asook ‘n nuwe kommersiëel beskikbare kleurstof, SpermBlue®
(SB), vir die evaluering van spermmorfometrie en morfologie. Resultate dui aan dat
beduidende verskille in sperm morfometriese afmetings ontstaan as gevolg van die
gebruik van die verskillende kleurstowwe. Bevindinge dui verder daarop dat RD
swelling van die sperm se kop versoorsaak, terwyl PAP die spermkop laat krimp.
Resultate wat verkry is met behulp van die SB kleuringstegniek dui daarop dat hierdie
kleurstof aanleiding gegee het tot afmetings naaste aan die verkry tydens die
beoordeling van vars, ongekleurde sperme. Die gebrek aan standardisasie en die
uiteenlopende effekte wat verskillende kleurstowwe het op die spermstruktuur en die
evaluering van sperm morfologie ingeheel is kommerwekkend, veral tydens die
evaluering van pasiënte met grensgeval morfologie. Op grond van hierdie resultate,
word die gebruik van die SB kleuringstegniek, bo die gebruik van RD en PAP, vir
effektiewe en akkurate morfologie evaluering aanbeveel. Verdere ondersteuning vir
die gebruik van die SB kleuringstegniek is die feit dat daar bevind is dat SB ‘n
vinnige en eenvoudige metode is, wat toelaat vir maklike visualisering van sperme
deur rekenaargesteunde sperm analise sisteme soos die Sperm Class Analyzer
(SCA®).
Die tweede doel van hierdie studie was om die konsentrasie, morfologie en die
motiliteit van spermpopulasies te ondersoek, soos verkry tydens die voorbereiding van
semen deur gebruik te maak van die PureSperm® digtheidsgradiënt en op-swem
tegnieke. Die voorbereiding van semen is ‘n noodsaaklike stap in enige
vrugbaarheidsbehandeling protokol, aangesien dit belangrik is dat die sperme wat
deur hierdie prosesse verkry word oor die nodige morfologiese en motiliteit
eienskappe beskik wat in staat is om die sukses van vrugbaarheidsbehandelings te
verbeter. Huidiglik is die PureSperm® digtheidsgradiënt en op-swem tegnieke die mees algemeen gebruikte tegnieke in vrugbaarheidsklinieke. Alhoewel daar
voldoende bewyse is wat voorstel dat elke tegniek effektief is vir die ekstraksie van
sperme met beter kwaliteit vanuit semen, bly daar steeds onvoldoende bewyse wat
daarop dui dat een van hierdie twee tegnieke beter is as die ander een. Huidige
navorsing het getoon dat beide sperm voorbereidings metodes daarin geslaag het om
sperme met normale morfologie en beter motiliteit te selekteer. Die opswem metode
het ‘n spermpopulasie met beter motiliteit en verbeterde morfologie gelewer, soos
getoets volgens die WGO kriteria, terwyl die PureSperm digtheidsgradiënt tegniek
sperme met verbeterde morfologie, volgens beide die WGO en Tygerberg Streng
Kriteria, en ‘n redelike verbetering in sommige motiliteits parameters geselekteer het.
Hoewel die resultate wat verkry word via die op-swem metode voorstel dat dit die
beste metode vir die gebruik tydens in vitro bevrugting sou wees, bly die baie lae
konsentrasie van sperme wat met hierdie metode verkry word ‘n belangrike nadeel.
Die PureSperm® skeidingstegniek laat egter toe vir die isolering van groter
hoeveelhede sperme, wat waarskynlik meer voordelig sal wees vir
bevrugtingsbehandelings wat meer sperme benodig. Gebaseer op hierdie bevindinge,
word die gebruik van die PureSperm® digtheidsgradiënt tegniek aanbeveel, as gevolg
van hierdie tegniek se vermoë om groot hoeveelhede goeie gehalte sperm te isoleer.
Daar kan egter nogsteeds van op-swem metodes gebruik gemaak word tydens
vrugbaarheidsbehandeling indien die semenmonster beskik oor ‘n hoë konsentrasie
sperme met goeie beweeglikheid.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/5137 |
| Date | 12 1900 |
| Creators | McAlister, Debra Ann |
| Contributors | Du Plessis, Stefan, Van der Horst, Gerhard, Maree, Liana, University of Stellenbosch. Faculty of Health Sciences. Dept. of Biomedical Sciences. Medical Physiology. |
| Publisher | Stellenbosch : University of Stellenbosch |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | Unknown |
| Type | Thesis |
| Format | xvii, 105 p. : ill. |
| Rights | University of Stellenbosch |
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