Dissertation (PhD)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: Male fertility has for many years been defined in vitro as the ability of sperm to fertilize
oocytes and to obtain early cleavage-stage embryos. Spermatozoa comprise of an
extraordinary high percentage of polyunsaturated fatty acids in their plasma membrane.
Due to an extremely low content of cytoplasm, sperm cells have a particularly low
potential to scavenge reactive oxygen species (ROS), and are therefore highly sensitive
to oxidative processes, which lead to sperm nucleus DNA damage/fragmentation.
Normally, DNA fragmentation occurs in every ejaculate and can be induced by an
excessive ROS production of active leukocytes or the spermatozoa themselves. Under
distressed conditions, DNA fragmentation may also occur in the testis as a result of
oxidative processes in the apoptotic cascade. These DNA fragmentations can be
regarded as late signs of programmed cell death (apoptosis).
Clinically, DNA fragmentation in spermatozoa results in significantly decreased
implantation and pregnancy rates especially in patients with oligo- and/or
teratozoospermia. The p-pattern normal sperm morphology has been shown to give
poorer fertilization rates in vitro than the g- and n-patterns. In this study there is
reported on the significant correlation found between the p-pattern normal sperm
morphology and sperm DNA fragmentation as measured with the terminal
deoxynucleotidyl transferase-mediated dUDP-biotin end labeling (TUNEL) assay. This
finding further explains the lower fertility potential of patients presenting with p-pattern
normal sperm morphology.
In addition, this study explores the intricate relations between ROS in the semen, DNA
fragmentation of the spermatozoa, as measured with the TUNEL assay and the sperm
chromatin structure assay (SCSA ), spermatozoa apoptotic status and sperm
parameters as measured with a standard semen analysis. Positive correlations were
found between ROS and the apoptotic status of the sperm, as well as between sperm
with non-fragmented DNA and sperm concentration and percentage motility. The
results emphasize the importance of sperm selection especially when the treatment of
choice is intracytoplasmic sperm injection (ICSI). An early sign of programmed cell death, also known as apoptosis, is the externalization
of phosphatidylserine (PS) from the inner membrane leaflet to the outer leaflet. PS
shows a high affinity to Annexin V. Apoptotic spermatozoa are able to fertilize oocytes,
but embryo senescence may occur at the time when the paternal genes are activated.
In this study there is reported on a novel method whereby spermatozoa can be
separated on the basis of their apoptotic status through flow cytometry. Results showed
that the normal sperm morphology, according to strict criteria, of the resultant nonapoptotic
sperm fraction is significantly higher than that of the apoptotic counterpart.
With refinement of this technique, it will be possible in future to use these separated
non-apoptotic sperm cells during ICSI for fertilization.
From the above it is apparent that the spermatozoon has to play a vital role in the
development of the embryo from fertilization to implantation and pregnancy. It is,
however, important to note that besides the gametes, there are other critical factors
which contribute to a successful in vitro fertilization (IVF) cycle, among these are the in
vitro culture conditions. In this regard, this study compared two sequential embryo
culture systems. It was found that the more complex medium resulted in better day
three embryo quality and a better blastocyst formation rate and pregnancy rate.
These findings highlight the importance of a holistic perspective towards the complexity
of the factors involved in affecting embryo quality and pregnancy outcome. / AFRIKAANSE OPSOMMING: Manlike fertiliteit is vir baie jare gedefinieer as die in vitro vermoë van ‘n spermsel om ‘n
eiersel te bevrug om sodoende embrios te verkry. Die spermsel se plasmamembraan
bestaan uit ‘n hoë persentasie poli-onversadigde vetsure. As gevolg van die klein
hoeveelhede sitoplasma van die spermsel het dit ‘n beperkte weerstand teen reaktiewe
suurstof spesies (ROS) en is gevolglik baie sensitief vir oksidasie. Oksidasie lei tot
DNS skade/fragmentasie. DNS fragmentasie kom in spermselle van alle ejakulate voor
en is gewoonlik die gevolg van ROS produksie deur die leukosiete in die semen of
vanaf die spermselle self. Onder sekere omstandighede kan DNS fragmentasie ook
voorkom in die testis waar dit deel vorm van apoptose. Hierdie tipe DNS skade word
gesien as laat tekens van geprogrammeerde seldood (apoptose).
In oligo- en/of teratozoospermiese mans lei DNS fragmentasie tot verlaagde
implantasie- en swangerskapssyfers. Die p-patroon normale sperm morfologie groep
gee laer in vitro bevrugting en swangerskapsyfers as die g- en n-patrone. In hierdie
studie doen ons verslag oor die statisties betekenisvolle korrelasie wat gevind is tussen
die p-patroon normale sperm morfologie en DNS fragmentasie soos gemeet met die
‘terminal deoxynucleotidyl transferase-mediated dUDP-biotin end labeling’ of te wel
TUNEL toets. Hierdie bevinding is ‘n verdere verklaring vir die laer fertiliteits potensiaal
van pasiënte wat voordoen met p-patroon sperm morfologie.
‘n Verdere doel van die studie was om die moontlike verband tussen ROS in die semen,
spermatozoa DNS fragmentasie, apoptotiese status van die sperms en die motiliteits
parameters van die spermatozoa te bepaal. ‘n Positiewe korrelasie is gevind tussen
ROS en sperm apoptotiese status. Sperms met ongeframenteerde DNS is ook positief
gekorreleer met sperm konsentrasie en motiliteit. Die resultate beklemtoon die
belangrikheid van spermseleksie veral in pasiënte waar die keuse van behandeling
intrasitoplasmiese sperm inspuiting (ICSI) is.
‘n Vroeë teken van apoptose is die eksternalisering van ‘phosphatidylserine’ (PS) vanaf
die interne oppervlakte van die plasmamembraan na die eksterne oppervlak. PS het ‘n
hoë affiniteit vir Annexin V. Apoptotiese sperms het die vermoë om ‘n oösiet te bevrug, maar kan lei tot die staking van embrio deling wanneer die vaderlike gene ‘n rol begin
speel in embrio ontwikkeling. In hierdie studie het ons ‘n nuwe metode ontwikkel
waarvolgens die spermatozoa in die ejakulaat op grond van hul apoptotiese status
geskei kan word in apoptotiese en nie-apoptotiese fraksies. Die normale sperm
morfologie van die nie-apoptotiese fraksie is betekenisvol beter as dié van die
apoptotiese fraksie. Verdere verfyning van die tegniek kan daartoe lei dat dit in die
toekoms toegepas kan word om vir nie-apoptotiese sperms te selekteer veral voor die
uitvoering van ICSI.
Uit die bogenoemde is dit duidelik dat die spermsel ‘n baie belangrike rol in die
ontwikkeling van ‘n embrio, vanaf bevrugting tot implantasie en swangerskap, speel. Dit
is egter ook belangrik om in gedagte te hou dat daar ander bydraende faktore tot ‘n
suksesvolle in vitro swangerskap is, soos laboratorium toestande en embrio
kultuursisteem. Om hierdie rede is daar ook twee kultuurmedia in hierdie studie
vergelyk. Daar is bevind dat die meer komplekse medium beter kwaliteit embrios op
dag drie lewer, asook meer blastosiste en ‘n hoër swangerskapsyfer.
Dit is dus duidelik dat dit uiters belangrik is om ‘n holistiese perspektief te hê op die
komplekse faktore wat ‘n invloed mag hê op bevrugting, embrio kwaliteit asook die
swangerskapsyfer.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/21626 |
| Date | 12 1900 |
| Creators | Hoogendijk, Christiaan F. (Christiaan Frederik) |
| Contributors | Kruger, T.F., 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 | xxiv, 160 leaves : ill. |
| Rights | Stellenbosch University |
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