Thesis (PhD)--Stellenbosch Uni versity, 2000. / ENGLISH ABSTRACT: Intracytoplasmic sperm injection (ICSI) introduced a revolutionary way of treatment for male factor infertility. With the exception of some cases of non-obstructive azoospermia, all other male factor infertility cases have the potential to be successfully treated with ICS!. The only prerequisite seems to be the presence of a motile or viable immotile sperm cell for each oocyte. In this study we report on our own experience with the development and implementation of the ICSI method in the Reproductive Biology Unit at Tygerberg Hospita!. An analysis of 5 years of ICSI experience showed that semen parameters, sperm morphology, motility and concentration did not influence fertilization and pregnancy rates adversely. In most cases, patients who could not be treated with in vitro fertilization (IVF) and gamete intrafallopian transfer (GIFT), due to poor semen parameters or fertilization failure, were treated successfully with ICS!. Even a case of globozoospermia was treated successfully with ICS!. Testicular spermatozoa, fresh or frozen-thawed, also resulted in excellent fertilization and pregnancy rates. Cryopreservation of testicular samples facilitated the management of the infertile couple, aiding the coordination of the recovery of vital gametes from both partners and also limiting the repetition of testicular biopsies. Incubation (maturation) of testicular spermatozoa also induced an enhancement in pregnancy rates. It can be concluded that ICSI proved to be a treatment method with success similar to that of in vitro fertilization (IVF) and gamete intrafallopian transfer (GIFT), in spite of a severe male factor. The study also indicated transfer route and embryo quality (viability) to be very important factors in the success of ICS!. The tubal transfer route was shown to be a significant contributor to the pregnancy success (compared to uterine transfer) as was the transfer of embryos that showed early division to the 2-cell stage, 26 hours post injection. The transfer of early dividing embryos into the fallopian tube resulted in a pregnancy rate of almost 40%, a result similar to that of GIFT with a mild male factor. The role of the oocyte in fertilization and pregnancy success was also revealed indirectly by the introduction of ICS!. Visual observation of denuded oocytes was possible and many morphological features, normal and abnormal, can be observed. Immature oocytes can also be identified and it was shown that they could be successfully matured in vitro before injection. In this study transmission electron microscopy (TEM) was used to study abnormalities in oocyte morphology. The standard method was adapted and modified for single cell TEM. The abnormalities observed included lysosomal and non-lysosomal degeneration (yellowish or darkly coloured oocytes), degeneration and vacuole formation (vacuolated oocytes), large secondary lysosomes filled with multiple small lipid droplets - lipofuscin body (refractile body) and a fragmented oocyte. It was also possible to study at ultrastructural level, possible reasons for fertilization failure in ICS!. Different stages of oocyte activation failure, cytoplasmic immaturity, sperm cell extrusion, abnormal sperm cell decondensation, female spindle abnormalities and technique related factors were observed. TEM was also successfully implemented to elucidate the reason for infertility in a patient with a longstanding, unexplained history of infertility. TEM evaluation of two of the patient's unfertilized oocytes revealed a spindle abnormality with contributing cytoskeletal anomalies at ultrastructural level. The modified TEM technique offers a valuable tool to study this small, but important group of patients with unexplained infertility. ThisTEM study opened up a new, valuable and interesting avenue of research with both diagnostic and prognostic value for patients with unexplained infertility. ICSI is therefore a valuable method in the treatment of especially male factor infertility. It is the most advanced fertilization technique developed in the last decade in this field. Not only can almost all male factor patients be treated, but unexplained female infertility can also be exposed, studied and hopefully in future also be treated with micromanipulation methods. / AFRIKAANSE OPSOMMING: Die ontwikkeling van die mikromanipulasie tegniek "Intracytoplasmic sperm injection" (ICSI) het die behandeling van die manlike faktor in infertiliteit, revolusionêr verander. Met die uitsondering van sommige gevalle van nie-obstruktiewe asoospermia, kan potensieel alle ander manlike infertiliteits faktore suksesvol met ICSI behandel word. Die enigste voorvereiste blyk "n bewegende of "n nie-bewegende, maar bewese lewende spermsel te wees. In hierdie studie word verslag gedoen oor die ontwikkeling en toepassing van die ICSI metode in die Eenhed vir Reproduktiewe Biologie by Tygerberg Hospitaal. 'n Analise van 5 jaar se resultate na die implementering van die ICSI metode het gewys dat die semen parameters, sperm morfologie, motiliteit en konsentrasie, nie "n effek op bevrugting- en swangerskapsyfers gehad het nie. Pasiënte wat, as gevolg van ontoereikende semen parameters, nie met die klassieke metodes, in vitro bevrugting (IVB) of gameet intrafallopiusbuis terugplasing (GIFT) behandel kon word nie, kon suksesvol met ICSI behandel word. Daar was selfs "n geval van manlike infertiliteit as gevolg van globosoospermie, wat suksesvol met ICS behandel is. Die ICSI metode het dit ook moontlik gemaak om uitstekende bevrugting- en swangerskap resultate met testikulêre spermatosoa .(vars en gevries) te bereik. Die bevriesing van testisweefsel het ook bygedra tot beter hantering van sulke pasiënte. Herhaalde testisbiopsies word uitgeskakel en die koórdinasie van die verkryging van die manlike en vroulike gamete, word ook vergemaklik wanneer testisweefsel in gevriesde vorm beskikbaar is. Die studie het verder getoon dat wanneer testikulêre weefsel geïnkubeer word (om spermatosoa te laat matureer), die swangerskapsyfers verhoog was. Dit is dus duidelik dat die ICSI metode net so suksesvol soos die IVB en GIFT metodes toegepas kan word, selfs en veral in gevalle van erge manlike faktor infertiliteit. Die studie het ook verder getoon dat die plek waar embrios teruggeplaas word, asook die embriokwalitiet van teruggeplaasde embrios, belangrike bydraende faktore in die ICSI swangerskapsukses was. Embrioterugplasing in die buis van fallopius en terugplasing van embrios wat vroeë 2-sel deling, 26 uur na ICSI getoon het, is uitgewys as faktore wat ICSI swangerskap betekenisvol verbeter het. Dit was moontlik om "n swangerskapsyfer van ongeveer 40%, sootgelyk aan die van GIFT sonder "n erge manlike faktor, te bereik met die terugplasing van ten minste een vroeë deler embrio in die fallopiese buis. Die ICSI tegniek het ook indirek bygedra tot nuwe insigte met betrekking tot die rol wat die vroulike eiersel (oësief in ICSI bevrugting speel. Oósiete word gestroop van hulomringende selle vir die ICSI proses en kan dan maklik vir hul normale en abnormale morfologiese eienskappe evalueer word. Oësiete wat immatuur is kan ook so geïdentifiseer word en dit is moontlik om hulle suksesvol te matureer voor mikro-inspuiting. Transmissie-elektronmikroskopie (TEM) is in die studie gebruik om die ultrastruktuur van onbevrugde en abnormale oësiete te bestudeer. Hiervoor is "n bestaande tegniek gemodifiseer vir die hantering van "n enkele sel, in hierdie geval die oosiet. Lisasomale en nie-lisosomale degenerasie (oósiete wat geelof donker van kleur voorkom), degeneratiewe tekens en vakuole (oësiete met vakuole), groot sekondêre lisosome gevul met klein lipieddruppels ('refractile body') en 'n gefragmenteerde oosiet was van die morfologies abnormale eienskappe wat ultrastruktureel geïdentifiseer is. Moontlike faktore wat 'n rol kan speel in nie-bevrugting na ICSI kon ook op ultrastrukturele vlak met die tegniek geïdentifiseer word. Hierdie faktore het die volgende ingesluit: die onvermoë van verskillende stadiums van oosiet aktivering, sitoplasmatiese immaturiteit, uitwerping van die spermsel na die periviteliene spasie, abnormale spermsel dekondensasie, vroulike spoelvormings abnormaliteite en tegniekgekoppelde faktore. Die TEM tegniek is ook suksesvol aangewend om die infertiliteitsprobleem van 'n pasiënt wat vir etlike jare aan onverklaarbare infertiliteit gely het, te identifiseer. TEM het op die ultrastrukturele vlak gewys dat daar 'n spoel abnormaliteit in twee van haar onbevrugde oëslete was. TEM kan dus baie vrugbaar gebruik word in hierdie groep pasiënte om onverklaarbare infertiliteit, wat andersins ongeïdentifiseerd sou bly, te verklaar. Die ICSI metode is die mees revolusionêre tegniek wat die afgelope dekade vir die behandeling van veral manlike infertiliteit ontwikkel en baie suksesvol toegepas is. Die metode ook kan 'n bydraende rol speel in die hantering van onverklaarbare infertiliteit veral ten opsigte van die vroulike gameet. In die toekoms is dit moontlik dat selfs hierdie probleem met nuwe mikromanipulasietegnieke opgelos sal kan word.