Thesis (MMed)-- Stellenbosch University, 2013. / ENGLISH ABSTRACT: Misoprostol is often used for the purpose of induction of labour. However, its effect on fetal heart rate has not been systematically studied. Objective To assess the effect of misoprostol on fetal heart rate parameters during induction of labour from 38 completed weeks in women with previous intrauterine death or postterm pregnancy. Study design A retrospective descriptive study of 127 women for a period of 18 months. Method Women who underwent induction of labour with misoprostol for either previous intrauterine death or postterm pregnancy at Tygerberg hospital were eligible. The selected process of induction of labour happened according to the departmental protocol. The primary outcomes were changes in fetal heart rate (variability, accelerations and decelerations) pre-and post-administration of misoprostol. Secondary outcomes were neonatal highcare or intensive care unit (ICU) admissions. Results There was no statistical difference in the mean fetal heart rate and baseline variability in relation to time recordings after administration of misoprostol. There were no statistically significant differences in the distribution of accelerations and decelerations in different time intervals before and after administration. There were more reactive patterns at all time intervals after the administration of misoprostol, but these differences did not quite reach statistical significance. In both study groups no neonatal complications or intensive care admissions were reported. Conclusion In the absence of contra indications, 50mcg of oral misoprostol can be given to mothers for induction of labour as no harmful fetal heart tracing abnormalities were found for 45 minutes; however large prospective randomized controlled trials are still needed to confirm effectiveness and evaluate further maternal and neonatal safety issues. Optimal dose and frequency also still need robust interrogation. Based on this thesis it does appear that misoprostol is probably not harmful to the fetus under these circumstances. / AFRIKAANSE OPSOMMING: Misoprostol word dikwels gebruik vir induksie van kraam. Die effek daarvan op fetale hartspoed is egter nie sistematies ondersoek nie. Doel Om die effek van misoprostol op fetale hartspoedparameters gedurende die induksie van kraam van 38 voltooide weke in vroue met vorige intra-uteriene dood or oortyd swangerskap te evalueer. Studei-ontwerp „n Retrospektiewe beskrywende studie van 127 vroue oor „n periode van 18 maande. Metode Vroue wat induksie van kraam met misoprostol ondergaan het vir of vorige intra-uteriene dood of oortyd swangerskap by Tygerberg Hospitaal is ingesluit. Die proses van induksie van kraam is volgens departementele protokol uitgevoer. Die primêre uitkomste was veranderinge in fetale hartspoed (variasie, versnellings en verstadigings) pre- en post-toediening van misoprostol. Neonatale hoësorg of intensiewe sorg toelatings was sekondêre uitkomste. Resultate Ons het geen statistiese verskille in gemiddelde fetale hartspoed en basislynvariasie in verhouding tot die tyd na toediening van misoprostol gevind nie. Daar was geen statisties betekenisvolle verskille in die verspreiding van versnellings en verstadigings in verskillende tydsintervalle nie. Daar was meer reaktiewe patrone gedurende alle tydsintervalle na die toediening van misoprostol, maar hierdie verskille was nie statisties betekenisvol nie. In beide studiegroepe was daar geen neonatale komplikasies of intensiewe sorg toelatings nie. Gevolgtrekking In die afwesigheid van kontra-indikasies kan 50 mcg misoprostol aan moeders toegedien word vir induksie van kraam aangesien geen skadelike fetale hartsped abnormaliteite gevind is nie. Groot prospektiewe gerandomiseerde gekontroleerde studies word steeds benodig om effektiwiteit te bevestig en om moederlike en fetale veiligheidskwessies verder te evalueer. Optimale dosis en frekwensie benodig ook robuuste ondersoek. Gebaseer op hierdie tesis kom dit voor of misoprostol waarskynlik nie skadelik vir die fetus onder hierdie omstandighede nie.

Thesis (MMed)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Background Rectovaginal endometriosis accounts for 5-10% of cases of endometriosis and constitutes one of the forms of deep infiltrating endometriosis. . Deep infiltrating endometriosis involving the bowel is most frequently encountered in the rectovaginal septum and is considered to be the most severe form of the disease and the most difficult to treat surgically owing to its invasive nature. There are currently no studies on this topic pertaining to a South African context. Study Objective To document the outcomes in 112 patients undergoing laparoscopic surgery for rectovaginal endometriosis. Methods A retrospective audit of 112 women undergoing laparoscopic surgery for rectovaginal endometriosis at Vincent Pallotti's Aevitas Fertility Clinic was undertaken. Eligibility was established by identifying women from a surgical database based on medical aid coding as well as a review of individual case notes. Patients were telephonically contacted to gather any missing information and to assess further outcomes. Design Classification Study number S11/11/036. This study was approved by the Health Research Ethics Committee at Stellenbosch University and was conducted according to ethical guidelines and principles of The International Declaration of Helsinki, South African Guidelines for Good Clinical Practice and the Medical Research Council (MRC) Ethical Guidelines for Research. Setting Vincent Pallotti’s Aevitas Reproductive Medicine Clinic Patients 112 consecutive patients suffering from rectovaginal endometriosis Interventions: Laparoscopic surgery for treatment of deep infiltrating, namely rectovaginal endometriosis Measurements and Main Results Primary outcome: Complications of laparoscopic surgery for rectovaginal endometriosis included one patient requiring a blood transfusion (0,9%), three cases of rectovaginal fistula (2,7%), two bowel injuries (1,8%)-detected and managed intra-operatively , one ureteric injury (0,9%), one pelvic abscess (0,9%) and the need for three urgent re-operations (2,68%). Secondary outcome: Of the 71 patients desiring fertility 39 (54,9%) fell pregnant of which 27 (69,2%) were spontaneous. Conclusion To our knowledge this is the first study assessing surgical outcomes in the management of deep infiltrating endometriosis from South Africa. These outcomes are in keeping with complication rates quoted in the international literature. Most of the surgery was performed using the shaving technique, in keeping with international trends, whilst fourteen cases required the performance of a segmental resection owing to extensive disease. In trained hands laparoscopic surgery is a valid management option in the management of rectovaginal endometriosis. / AFRIKAANSE OPSOMMING: Agtergrond Vyf tot tien persent van alle endometriose gevalle kan toegeskryf word aan rektovaginale endometriose. Dit word beskou as een van die vorme van diep infiltrerende endometriose. Diep infiltrerende endometriose van die derm kom meestal in die rektovaginale septum voor en word as die ernstigste vorm van die siekte beskou. Dit is die moeilikste om chirurgies te behandel weens sy indringende aard. Daar is tans geen studies beskikbaar oor hierdie onderwerp in die Suid-Afrikaanse konteks nie. Doel van die studie Om die uitkomste te dokumenteer van 112 pasiënte wat laparoskopiese chirurgie vir rektovaginale endometriose ondergaan het. Metodes 'n Retrospektiewe oudit is by Vincent Pallotti se Aevitas Fertiliteitskliniek gedoen van 112 vroue wat laparoskopiese chirurgie vir rektovaginale endometriose ondergaan het. Geskikte pasiënte is geïdentifiseer vanaf 'n chirurgiese databasis gebaseer op mediese kodering, sowel as vanaf 'n oorsig van pasiënt notas. Pasiënte is telefonies genader om ontbrekende inligting in te samel en verdere uitkomste te evalueer. Klassifikasie Ontwerp Studie nommer S11/11/036. Hierdie studie is deur die Gesondheids Navorsing Etiese Komitee van die Universiteit van Stellenbosch goedgekeur en uitgevoer volgens die etiese riglyne en beginsels van die Internasionale Verklaring van Helsinki, Suid-Afrikaanse Riglyne vir Goeie Kliniese Praktyk en die Mediese Navorsingsraad (MNR) se Etiese Riglyne vir Navorsing. Instelling Vincent Pallotti se Aevitas Reproduktiewe Medisyne Kliniek Pasiënte 112 agtereenvolgende pasiënte met rektovaginale endometriose. Ingrepe: Laparoskopiese chirurgie vir die behandeling van diep infiltrende, rektovaginale endometriose. Resultate Primêre uitkoms: Komplikasies van laparoskopiese chirurgie vir rektovaginale endometriose het ingesluit: een pasiënt wat 'n bloedoortapping benodig het (0,9%), drie gevalle van rektovaginale fistels (2,7%), twee dermbeserings (1,8%) - intraoperatief gediagnoseer en herstel, een ureter besering (0,9%), een bekkenabses (0,9%) en drie dringende herhaal operasies (2,68%). Sekondêre uitkoms: Van die 71 pasiënte wat fertiliteit verlang het: 39 (54,9%) het swanger geraak, waarvan 27 (69,2%) spontaan was. Gevolgtrekking Sover ons kennis strek, is dit die eerste Suid-Afrikaanse studie waar daar na die chirurgiese uitkomste in die behandeling van diep infiltrerende endometriose gekyk is. Hierdie uitkomste stem ooreen met internasionale literatuur in terme van komplikasie syfers. Die meeste van die operasies is uitgevoer met behulp van die skeer-tegniek, in ooreenstemming met internasionale tendense, terwyl veertien gevalle segmentele reseksies vereis het weens uitgebreide siekte. In goed opgeleide hande is die laparoskopiese behandeling van rektovaginale endometriose ‘n geldige behandelings opsie.

Thesis (MMed)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Introduction Pregnancies in women with a previous unexplained stillbirth may be jeopardized by increased antenatal surveillance and higher rates of induction of labour and caesarean delivery without clear evidence of benefit. Despite the fact that there have been no studies that adequately tested fetal benefit in routine induction of labour for a previous stillbirth, a policy of routine induction of labour at 38 weeks, with all the associated maternal, fetal and health-care associated costs, was in practice at Tygerberg Hospital for the past 30 years. This study aimed to investigate the safety of continuation of these pregnancies until term (≥39 weeks). Aims and Objectives To assess the clinical outcome and impact on the health service in a pregnancy with a previous unexplained intra-uterine demise (IUD) by routine induction of labour at term instead of at 38 weeks. Methodology This was a prospective observational study on the safety of a new hospital protocol which was introduced in 2012. The protocol extended the gestation for induction after a previous IUD from 38 weeks to term. The study population included all pregnant patients with a current singleton pregnancy, and a previous unexplained or unexplored (no data available) singleton fetal demise ≥24 weeks/500grams. All patients with a previous stillbirth in the metropolitan drainage area of Tygerberg Hospital are referred to Tygerberg for further care; and all referrals during 2012 were recruited for the study. Patients with known or recurrent risks for intra-uterine death were managed according to the relevant clinical condition and were excluded from the study. Results During the audit period, 306 patients with a previous intra-uterine fetal death were referred for further management. Of these, 161 had a clear indication for either earlier intervention or no intervention and were excluded from the protocol. Of the remaining 145 patients, 9 met exclusion criteria and there were 2 patients who defaulted. Forty-two of the study patients (with no known previous medical problems) developed complications during their antenatal course that necessitated a change in clinical management and earlier (<39 weeks) delivery. Of the remaining 92 patients in the audit, 47 (51%) went into spontaneous labour before their induction date. There were no intra-uterine deaths prior to delivery. Conclusions Careful follow up at a high risk clinic identifies new or concealed maternal or fetal complications in 29% of patients with a previous IUD and no obvious maternal or fetal disease in the index pregnancy. When all risks are excluded and the pregnancy allowed to progress to 39 weeks before an induction is offered, 51% will go into spontaneous labour. / AFRIKAANSE OPSOMMING: Inleiding Swangerskappe in vroue met vorige onverklaarbare stilgeboorte mag in gevaar gestel word deur meer intense voorgeboorte sorg en ‘n groter hoeveelheid induksies van kraam en keisersnitte sonder duidelike bewyse dat dit tot voordeel strek. Ten spyte van die feit dat daar geen studies is wat bewys het dat roetine induksie van kraam vir ‘n vorige stilgeboorte op 38 weke tot voordeel van die baba was nie, was ‘n beleid van roetine induksie van kraam op 38 weke, met al die geassosieërde moederlike en fetale risikos daaraan verbonde; asook die hoë gesondheidskostes, roetine praktyk in Tygerberg Hospitaal vir die afgelope 30 jaar. Hierdie studie het ten doel gehad om die veiligheid van voortsetting van hierdie swangerskappe tot voltyd (≥39 weke) te ondersoek. Doelwitte Om die kliniese uitkoms; asook die impak op gesondheidsdienste te evalueer in ‘n swanger vrou met n vorige onverklaarbare intra-uteriene sterfte; deur roetine induksie van kraam aan te bied op voltyd in plaas van 38 weke. Metodologie Hierdie was n prospektiewe kohort studie om die veiligheid van ‘n nuwe hospitaal protokol wat in 2012 geïmplimenteer is, te bepaal. Hierdie protokol het die gestasie tydperk van induksie van kraam van alle swanger pasiënte na ‘n vorige onverklaarbare stilgeboorte van 38 weke na voltyd verleng. Die studiepopulasie het alle swanger pasiënte met ‘n huidige enkelswangerskap en ‘n vorige onverklaarbare of onbekende (geen data beskikbaar) enkelvoudige fetale sterfte ≥24 weke/500gram, ingesluit. Alle pasiënte in die metropolitaanse dreineringsarea van Tygerberg Hospitaal met ‘n vorige stilgeboorte word na Tygerberg verwys vir verdere hantering, en alle verwysings gedurende 2012 was gewerf vir die studie. Pasiënte met bekende of herhalende risikofaktore vir ‘n intra-uteriene sterfte was hanteer volgens die relevante kliniese inligting en was uitgesluit by die studie. Resultate Drie-honderd-en-ses pasiënte met ‘n vorige intra-uteriene fetale sterfte was gedurende die oudit periode verwys vir verdere hantering. In 161 pasiënte was daar ‘n duidelike indikasie vir of vroeër intervensie of geen intervensie nie; en hulle was uitgesluit van die protokol. Van die oorblywende 145 pasiënte is 9 pasiënte uitgesluit as gevolg van die uitsluitingskriteria en daar was 2 pasiënte wat versuim het om op te volg. Twee-en-veertig pasiënte (met geen bekende vorige mediese probleme nie) het komplikasies gedurende hulle voorgeboorte verloop ontwikkel wat gelei het tot verandering in kliniese hantering en vroeëre verlossing (≤39 weke) genoodsaak het. Van die oorblywende 92 pasiënte in die oudit, het 47 (51%) in spontane kraam gegaan voor hulle induksiedatum. Daar was geen intra-uteriene sterftes voor verlossing nie. Gevolgtrekkings Noukeurige opvolg by ‘n hoërisiko kliniek identifiseer nuwe of versteekte moederlike en fetale komplikasies in 29% van pasiënte met ‘n vorige intra-uteriene sterfte sonder enige duidelike moederlike of fetale siekte in die indeks swangerskap. Wanneer alle risikos uitgesluit word en die swangerskap toegelaat word om voort te gaan tot 39 weke voor ‘n induksie aangebied word, sal 51% van pasiënte spontaan in kraam gaan.

Thesis (D.Phil.)--Stellenbosch University, 2000. / ENGLISH ABSTRACT: Chapter 1 provides literature based background information on the clinical importance of sperm morphology as recorded by strict criteria during the diagnostic approach of the infertile couple. Furthermore, the use of a sequential diagnostic schedule for couples in an assisted reproductive programme is emphasized. The author revisited the literature on chromatin packaging of spermatozoa and addresses this issue as an additional semen parameter providing information relating to DNA damaged spermatozoa. The chapter also includes evidence underlining the growing need for the implementation of the acrosome reaction as an important contribution to the assisted reproductive programme. Chapter 2 provides detailed descriptions of the material and methods used during the study. Chapter 3 is sub-divided into 5 sections, each of which represents a separate study that was prepared as a scientific paper. The study included 338 couples consulting for infertility treatment at various gynaecologists in Pretoria and Johannesburg. The diagnostic assisted reproductive laboratory support was provided by the Andrology laboratory of Drs du Buisson and partners from Pretoria. In the first study the role of chromatin packaging as an indicator of in vitro fertilization rates, the semen samples from 72 men were used to record their chromatin packaging quality as well as their sperm morphology classification. Significant different percentages CMA3staining (mean±SE) were recorded among the 2 morphology groups, namely 65.9%±3.5 and 44.5%±1.7 (p=0.001). Using cut off values for chromatin packaging established during the first study, the second study utilized semen from 140 men in the in vitro fertilization (IVF) and intracytoplasmic sperm injection programme (ICSI) to analyze for sperm concentration, motility, morphology and chromatin packaging (CMA3).IVF and ICSI data were stratified using 3 basic cut off values for CMA3staining, namely <44%, >44-60% and >60%. The study concluded that results on the chromatin packaging quality of spermatozoa could be used as an additional parameter of sperm quality since it could provide valuable information on decondensation status of a given sperm population. The third study aimed to establish zona pellucida induced acrosome reaction response (ZIAR) among 35 couples with normal and G-pattern sperm morphology and repeated poor fertilization results during assisted reproduction treatment. Interactive dot diagrams, divided patients into 2 groups i.e. ZIAR<15% and ZIAR>15% with mean fertilization rates of 49% and 79%, respectively. The study concluded that the ZIAR test has diagnostic potential, since it can assist the clinician to identify couples that will benefit from ICSI therapy. The forth study revisited the importance of micro-assay for acrosome reaction determinations in a diagnostic andrology laboratory. The micro-assay not only allows the use of a single zona pellucida, but also facilitates the future possibility of using recombinant zona pellucida proteins in a diagnostic test system. The final study in Chapter 3 includes results obtained from 49 couples (172 oocytes) and aimed to evaluate the role of chromatin packaging and sperm morphology during sperm-zona binding, sperm decondensation and the presence of polar bodies among 170 oocytes that failed in vitro fertilization (IVF). Odds ratio analyses indicated that being in the a group with elevated CMA3 staining i.e. >60%, the risk of decondensation failure increases 15.6 fold relative to normal CMA3 staining <44%. Chapter 4 underlines the validity of the sequential diagnostic approach and summarizes the results and value of a multistep diagnostic scheme. The chapter concludes with the recommendation that both chromatin packaging quality and zona pellucida mediation of the acrosome reaction should be part of the diagnostic tools in the assisted reproductive programme. / AFRIKAANSE OPSOMMING: Die literatuuroorsig in Hoofstuk 1 konsentreer in hoofsaak op die kliniese belang van sperm morfologie en die uitbreiding van die diagnostiese toetse en hantering van die egpaar in die reproduktiewe ondersteuningsprogram. Die kromatien pakkingskwaliteit van die spermsel word onderskryf as In belangrike toevoeging tot die diganostiese program, aangesien ONS skade dikwels saam met kromatiendefekte aangetref word. Die rol van die akrosoomreaksie word ook in detail literatuuroorsigtelik beklemtoon. Hoofstuk 2 bevat volledige inligting omtrent materiaal en metodes wat in die studie gebruik is. Hoofstuk 3 bevat die eksperimentele gegewens wat in 5 afsonderlike sub-afdelings as wetenskaplike publikasies aangebied word. Die studies bestaan uit data van 338 pasiënte, wat deur verskillende ginekoloë van Pretoria en Johannesburg gekonsulteer is waartydens drs. du Boisson en vennote van Pretoria die diagnostiese reproduktiewe laboratoriumdienste verskaf het. Die eerste studie stel dit ten doel om die belang en korrelasie van die spermsel kromatienpakkingskwaliteit van 72 mans te vergelyk met die morfologiese bou van sie sel. Aangesien morfologie reeds gevertig is as 'n kliniese voorspeller van bevrugting was dit nodig om hierdie parameter te vergelyk met die kromatienpakking van die sel. Twee afsnypunte word vir die normo-en teratozoospermiese mans identifiseer naamlik, 44.5%±1.7 en 65.9%±3.5 (p=O.001),respektiewelik. Die tweede studie gebruik die afsnypunte 44% en 66% om die in vitro bevrugting en intrasellulêre sperm inspuiting (ICSI) data te ontleed. Die resultate dui aan dat kromatienpakking In waardevolle bydrae tot die diagnostiek van die pasiënte lewer. Die derde studie stel dit ten doelom die waarde van die zona pellucida geinduseerde akrosoomreaksie (ZIAR) te bepaal. Die studie sluit die data van 35 egpare in wat almal normale of G-patroon morfologie het en verder onverklaarde swak bevrugtings resultate tydens in vitro bevrugtingsterapie. Interaktiewe punt diagram (interactive dot diagrams) verdeel die data in twee groepe naamlik, ZIAR<15% en ZIAR>15% met gemiddelde bevrugtingssyfers van 49% en 79%, respektiewelik. Die studie sluit af met die gedagte dat die ZIAR toets 'n groep pasiënte identifiseer met 'n besondere fisiologiese afwyking d.i. subnormale akrosoom respons op zona pellucida blootstelling. Die vierde afdeling van die hoofstuk onderstreep die belang van die mikro-tegniek vir die bepaling van die akrosoom reaksie, wat tydens die projek gebruik is Die vyfde afdeling van Hoofstuk 3 stel dit ten doelom 170 onbevrugde eierselle van 49 pasiënte te ontleed vir moontlike oorsake vir die mislukte bevrugting. Ondersoeke sluit in die kromatienpakking, sperm-zona binding, sperm dekondensasie en die teenwoordigheid van polêre liggaampies. Statisties blyk dit dat indien 'n kromtienpakking nie normaal is nie (>66%) het die spermsel 'n 15 keer groter kans om nie te dekondenseer nie. Hoofstuk 4 bespreek die noodsaaklikheid van die diagnostiese skedule by die hantering van die onvrugbare egpaar in.

Thesis (DSc)--Stellenbosch University, 2012. / Please refer to full text for abstract.

Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: CHAPTER 1 In this chapter the aim is to outline the different chapters under section A. Against this background, we will conduct a literature review of relevant studies performed, and evaluate their comments regarding identifying embryo markers which can be utilized to improve overall ART outcome. We will evaluate the embryo marker sHLA-G in detail, using a prospective randomized study as well as a retrospective multi-centre study. The role of the morphology and genetic profile of an oocyte, zygote and embryo and subsequent blastocyst formation will be evaluated with the help of WGA/CGH. The work will then be summarized and conclusions will be made as well as possible suggestions for future directions will be indicated. In section B the methodology of the studies explaining the role of the candidate is illustrated. CHAPTER 2 In this chapter the impact of the oocyte/zygote and the embryo on implantation/pregnancy rate was discussed. The morphologic characteristics of the oocyte, the cumulus–oocyte-complex (COC), the zona pellucida, the perivitelline space, cytoplasm and meiotic spindle and the polar body and its appearance were discussed in detail. The morphologic characteristics of embryo fragmentation and its effect on embryo development, ploidy and blastocyst formation were also studied. Embryo markers to predict pregnancy outcome were researched based on the international literature. The pronuclear morphology and early cleavage were highlighted as non-invasive embryo markers to predict outcome. A non-invasive biochemical marker, soluble Human Leucocyte-Antigen-G (sHLA-G), that is expressed by developing embryos was researched. The value of blastocyst transfer and the improved ongoing pregnancy rate compared to cleavage stage embryos were highlighted based on a recent meta-analysis. A detailed discussion on sHLA-G as well as Array-CGH and the future of these tests followed. CHAPTER 3 In this chapter the aim was to compare pregnancy and implantation rates when embryos are selected based on a single Day 3 (D 3) morphology score vs. a GES score plus sHLA-G expression. This was a prospective randomized study (n=214) undergoing fresh ICSI cycles. Embryos were selected for transfer based on either Day 3 morphology score (Group A) or GES-scoring plus sHLA-G expression (Group B). The following results were reported: Clinical [35/107 (33%) vs. 52/107 (49%)] and ongoing pregnancy [20/107 (19%) vs. 52/107 (49%)] rates were significantly different between Group A and Group B (p<0.05). Implantation rates were not significantly different between Group A [52/353 (15%)] and Group B [73/417 (18%)] (p<0.05). The number of pregnancies lost during the first trimester was nearly 12 times higher in Group A [25/52 (48%)]. It was concluded that the miscarriage rate was significantly lower in Group B than Group A and the pregnancy results were superior when embryos were selected based on GES plus sHLA-G expression. CHAPTER 4 Several studies have reported an association between the presence of soluble human leukocyte antigen-G (sHLA-G) in human embryo culture supernatants (ES) with implantation and pregnancy outcome in vitro. However, the actual presence role during implantation and effect on implantation and pregnancy outcome are still controversial. A retrospective multi-centre study was performed on 2040 ICSI patients in six different centers. All embryos were individually cultured and a chemiluminescence enzyme-linked immunosorbent assay (ELISA) was used to detect the presence of sHLA-G in culture medium surrounding embryos. In all centers, a positive sHLA-G result was associated with an increase in odds of multiple clinical implantations (OR: 1.48, 95% CI: 1.07 to 2.05, p-value: 0.0170), and an increased odds of multiple on-going pregnancies (OR: 1.66, 95% CI: 1.10, 2.51, p-value: 0.0170). Data from this multi-centre study emphasize that sHLA-G expression is a valuable non-invasive embryo marker to assist in improving pregnancy outcome with the theoretical potential to reduce multiple pregnancies. A combination of sHLA-G expression and extended embryo culture to the blastocyst stage might provide future tools by which to select single embryos for transfer and reduce the risk of multiple gestational, without compromising their pregnancy rates. CHAPTER 5 In this chapter the ploidy status of first and second polar bodies and Day 3 blastomere, embryo morphology and biochemical (sHLA-G) characteristics were correlated with blastocyst development and subsequent pregnancy outcome. All oocytes/zygotes and embryos were individually cultured to the blastocyst stage. PB-I, PB-II and blastomeres underwent whole genome amplification (WGA) and comparative genome hybridization (CGH) and complete karyotyping. Each embryo‟s culture medium supernatant was collected and analyzed for sHLA-G expression on Day 2. The following results were reported: Fifty seven mature (MII) donor oocytes were obtained, 33/57 (57.9%) were aneuploid, 21/57 (36.8%) were euploid and 3/57 (5%) were “inconclusive”. No correlation was found between CGH status of PB-I, PB-II and the GES-score. Furthermore, no correlation was established between PB-I CGH results and blastocyst morphology grade. There was a significant correlation between PB-I CGH and blastomere CGH results. Euploid and aneuploid PB-I developed into 58% and 67% blastocysts, respectively. Kappa statistics (>0.7) revealed a positive correlation between the ploidy of PB-I, PB-II and the blastomeres. It was concluded that following ICSI and sequential genetic karyotyping of the oocyte/zygote and subsequent blastomeres, the majority of oocytes fertilized and subsequent zygotes developed into blastocysts, despite their ploidy status. We therefore conclude that blastocyst development is not associated with ploidy. CHAPTER 6 Identifying a developmentally competent embryo to transfer that has the highest probability to develop into a live baby has been an issue of debate and continues research. The aim of this chapter is to discuss the morphological, biochemical and genetic features of an embryo that has been shown to be predictive of implantation and pregnancy outcome in ART using most current evidence. A literature search was performed looking at the correlation between pronuclear morphology, early cleavage, cleavage stage embryos, blastocyst development, the presence of sHLA-G, CGH, embryo development and implantation/pregnancy rates in ART. Based on the available literature, a combination of observations could assist the scientist with embryo selection. The pronuclear stage morphology, the early embryo division, cleavage embryo stage and quality of the day 3 embryos provides limited guidance. However, choosing a blastocyst with a positive sHLA-G result on Day 5 is the optimal combination to make the final selection before embryo transfer or freezing. This non-invasive approach should improve pregnancy outcome and reduce multiple pregnancy rates. As far as the use of the more invasive technology such as aCGH is concerned, more research on pregnancy outcome is needed. CHAPTER 7 A combination of observations for embryo selection, starting with oocyte grading, pronuclear stage morphology, early zygote cleaving and cleavage-stage embryo morphology/quality on Day-3, however, ultimately using extended embryo culture and choosing a blastocyst on Day 5 with positive sHLA-G values available, will assist the scientist in making the final decision before selecting an embryo for transfer or cryopreservation. The use of aCGH (for chromosomal analysis) is invasive and is still considered experimental. Finally we conclude that despite all the above mentioned parameters to select an embryo for transfer that will develop into a live baby, more extensive research and international corroboration is needed in order to improve and standardize embryo selection criteria. / AFRIKAANSE OPSOMMING: HOOFSTUK 1 Die doel in hierdie hoofstuk is om die verskillende hoofstukke onder Afdeling A uiteen te sit. Daar word beplan om „n literatuur oorsig te doen van toepaslike studies rakend embriomerkers wat swangerskap-uitkoms in in vitro bevrugting kan verbeter. Verder sal die embriomerker sHLA-G deeglike bestudeer word met behulp van „n prospektiewe gerandomiseerde studie, asook „n retrospektiewe multisentrum studie. Die rol van embrio morfologie en die genetiese profiel van die ovum, sigoot asook die embrio en die daaropvolgende blastosist vorming sal geëvalueer word met behulp van WGA/CGH. Alle bevindings sal daarna opgesom word, gevolg deur „n sinvolle gevolgtrekking en laastens sal voorstelle gemaak word vir toekomstige navorsing op die gebied. In Afdeling B sal die metodiek van die studies verduidelik word, asook „n beskrywing gegee word van die kandidaat se rol gedurende die navorsings projekte in hierdie tesis. HOOFSTUK 2 In hierdie hoofstuk word die impak van die oösiet en die embrio op die inplanting/swangerskap-koers bespreek. Die morfologiese eienskappe van die oösiet, die kumulus-oösiet kompleks, die sona pellucida, die perivitelline spasie, sitoplasma en meiotiese spoel, die poolliggaam en die se voorkoms word breedvoerig bespreek. Die morfologiese eienskappe van die embrio, fragmentasie en die invloed daarvan op die embrio, ploïdie, en blastosistvorming word bespreek. Embriomerkers om swangerskapsuitkoms te voorspel, gebaseer op internasionale literatuur, is ook nagevors. Die pronukleêre morfologie en vroeë deling word as nie-indringende embriomerkers uitgelig om swangerskapsuitkoms te voorspel. „n Biochemiese, nie-indringende merker wat deur ontwikkelende embrios uitgedruk word, oplosbare menslike leukosiet antigeen-G (sHLA-G), word bespreek. Die waarde van blastosist oordrag en die verbeterde koers van voortgaande swangerskappe in vergelyking met verdelende embrios, is ook uitgelig, gebaseer op „n onlangse metanalise. „n Breedvoerige bespreking van sHLA-G asook “Array-CGH” en die toekoms van hierdie toetse word behandel. HOOFSTUK 3 Die doel van hierdie hoofstuk is om swangerskap en inplantingskoerse te vergelyk wanneer embrios geselekteer word op „n enkel Dag 3 (D 3) morfologie beoordeling, teenoor „n kumulatiewe GES-telling plus sHLA-G uitdrukking. Hierdie was „n prospektiewe ewekansige studie (n=214) waar pasiënte ICSI-siklusse ondergaan het. Embrios is geselekteer vir terugplasing gebaseer op óf Dag 3 morfologie telling (Groep A), óf „n kumulatiewe GES-telling plus sHLA-G uitdrukking (Groep B). Die volgende resultate is gerapporteer: kliniese swangerskappe [35/107 (33%) vs 52/107 (49%)] en voortgaande swangerskappe [20/107 (19%) vs. 52/107 (49%)] se sukses koerse is beduidend verskillend tussen Groep A en Groep B (p<0.05). Inplantingskoerse is nie beduidend verskillend tussen Groep A [52/353 (15%)] en Groep B [73/417 (18%)] (p<0.05) nie. Die aantal swangerskappe wat tot niet gegaan het tydens die eerste trimester was bykans 12 keer hoër in Groep A [25/52 (48%)]. Die slotsom was dat die miskraamsyfer beduidend laer in Groep B as in Groep A is en die swangerskap syfer betekenisvol beter was wanneer die selektering van embrios op GES plus sHLA-G gebaseer is. HOOFSTUK 4 Verskeie studies het „n assosiasie getoon tussen die teenwoordigheid van oplosbare menslike leukosiet antigeen-G (sHLA-G) in menslike embrio kultuur en swangerskaps uitkoms in vitro. „n Retrospektiewe studie is op 2040 ICSI pasiënte by 6 verskillende sentra gedoen om die effek van s-HLAG verder te bestudeer. Alle embrios is individueel gekweek om die teenwoordigheid van sHLA-G in „n kultuurmedium rondom die embrios te identifiseer. In alle sentra is „n positiewe sHLA-G uitslag met „n toename in die waarskynlikheid van veelvuldige inplantings geassosieer (OR: 1.48, 95% CI: 1.07 tot 2.05, p-waarde: 0.0170), asook „n toename in waarskynlikheid van meervoudige swangerskappe wat voortduur (OR: 1.66, 95% CI: 1.10, 2.51, p-waarde: 0.0170). Data uit die multisentriese studie beklemtoon dat sHLA-G uitdrukking „n waardevolle nie-indringende embriomerker is om by te dra tot die verbetering van swangerskapsuitkoms, asook die teoretiese potensiaal om meervoudige swangerskappe te verminder. „n Kombinasie van sHLA-G uitdrukking en verlengde embrio kultuur tot die blastosist stadium mag moontlik „n toekomstige hulpmiddel wees waardeur enkele embrios vir terugplasing geselekteer kan word. Daardeur kan die risiko van meervoudige swangerskappe beperk word sonder om die swangerskapkoerse in gevaar te stel. HOOFSTUK 5 In dié hoofstuk word die ploïdie status van die eerste en tweede poolliggaampies en Dag 3 blastomere, embrio morfologie en biochemiese (sHLA-G) eienskappe gekorrelleer met blastosist ontwikkelling en uiteindelike swangerskapsuitkoms. Alle oösiete/sigote en embrios is individueel tot die blastosist stadium gevolg. PB-I, PB-II en blastomere het “volledige kariotipering ondergaan deur gebruik te maak van die toets “comparative genome hybridization (CGH)”. Elke embrio se kultuurmedium supernatant is versamel en ontleed vir sHLA-G uitdrukking op Dag 2. Die volgende uitslae is gerapporteer: Sewe-en-vyftig mature (MII) donor oösiete is verkry; 33/57 (57.9%) is aneuploïd, 21/57 (36.8%) is euploïd en 3/57 (5%) is onbeslis. Geen verwantskap is gevind tussen CGH status van PB-I, PB-II en die GES-telling. Geen verwantskap is gevind tussen CGH status van sHLA-G. Verder was daar geen verwantskap gevind tussen PB-I CGH uitslae en blastosist morfologie graad nie. Daar was „n beduidende korrelasie tussen PB-I CGH en blastomeer CGH uitslae. Euploïde en aneuploïde PB-I het onderskeidelik in 58% en 67% blastosiste ontwikkel. Daar is „n positiewe verwantskap tussen die ploïdie van PB-I, PB-II en die blastomere aangetoon [Kappa (>0.7)]. Dit is afgelei dat na ICSI en sekwensiële genetiese kariotipering van die oösiet/sigoot en daaropvolgende blastomere, die meerderheid oösiete bevrug is en die daaropvolgende sigote ontwikkel het tot blastosiste, ongeag hul ploïdie status. Ons afleiding is dus dat blastosist ontwikkelling nie aan ploïdie verwant is nie. HOOFSTUK 6 In hierdie hoofstuk bespreek ons waarnemings wat betref seleksie kriteria om die beste embrios te kies vir terugplasing wat uiteindelik tot „n suksesvolle swangeskap sal lei. Morfologiese, biochemiese en genetiese faktore is ondersoek. „n Onderskeiding is gemaak tussen nie-indringende (mikroskopiese en biochemiese) en indringende (embrio biopsie, aCGH) tegnieke. 'n Kombinasie van nie-indringende observasies, wat insluit pronukliere mofologie, vroee sigoot verdeling en vroeë embrio morfologie/kwalitieit op Dag-3 het beperkte inligting verskaf wat betref swangerskapkans. Verlengde embrio kweking tot die blastosist stadium (Dag-5) plus „n positiewe sHLA-G resultaat gee egter veel meer voordelige inligting aan die embrioloog met die embrio seleksie proses, voor embrio terugplasing of bevriesing. Laasgenoemde inligting sal die swangerskap syfer bevoordeel en die meervoudige swangerskap kans verlaag. Wat die indringende tegniek (aCGH) betref, word veel meer data benodig rakend die potensiele voor- en nadele wat betref swangerskap uitkoms, voordat „n sinvolle gevolgtrekking gemaak kan word. HOOFSTUK 7 „n Volledige literatuur oorsig dui daarop dat alle beskikbare riglyne om embrios te kies vir terugplasing, ingespan moet word. In die studie is daar gekyk na „n kombinasie van hierdie voorstelle. Daar is begin met die morfologie van die pronukliere stadium, gevolg deur vroeë sigoot-verdeling, asook beoordeling van embrios se morfologie/kwaliteit op Dag-3 van ontwikkeling. Daar word voorgestel dat die keuse van „n blastosist op Dag 5, gekombineerd met „n positiewe oplosbare menslike leukosiet antigeen G (shla-G) die embrioloog van hulp kan wees om die beste embrio te kies vir terugplasing of bevriesing. Hierdie nie-indringende riglyn behoort swangerskap-uitkoms te verbeter asook meervoudige swangerskappe te verminder. Indringende tegnieke soos ACGH benodig verdere in diepte navorsing en data verkryging om die waarde van hierdie toets te kan beoordeel.

Thesis (MA)--Stellenbosch University, 1999. / ENGLISH ABSTRACT: There is no standardised instrument available in South Africa to measure patient satisfaction with antenatal care. The measurement of patient satisfaction is especially important after the implementation of a free antenatal care service in the South African health system. The purpose of this study was to develop and validate an appropriate scale to measure patient satisfaction. Several methods to measure patient satisfaction are described in the literature. A questionnaire was developed for the Tygerberg Hospital patients. This questionnaire was tested in 200 antenatal patients through a structured interview. The importance of cross-cultural research is emphasised in the validation of the measuring instrument. Factor analysis was used to validate the instrument. This showed that a single factor accounted for most of the total variance. All the items had to do with the process of antenatal care. The findings of this survey showed the following: • One cannot use overseas measuring instruments without adjusting for cross-cultural differences. • The patient satisfaction score is negatively skewed with a high mean. • Social desirability response sets may play an important role in these questionnaires. • There is a statistically significant difference in patient satisfaction with antenatal care between the different antenatal clinics, even after controlling for socio-demographic differences. • That the satisfaction score is a reflection of the service rendered to the patient and not of the socio-demographic differences. This research identified the difficulties of developing a standardised instrument to measure patient satisfaction with antenatal care and opens the way for future research into patient satisfaction with medical services. / AFRIKAANSE OPSOMMING: Daar is geen gestandaardiseerde meetinstrument om pasiente se tevredenheid met voorgeboortesorg in Suid Afrika te bepaal nie. Die noodsaaklikheid van die bepaling van tevredenheid met voorgeboortesorg het nou belangriker geword nadat 'n stelsel van gratis voorgeboortesorg in Suid-Afrika gei"mplementeer is. Die doel van hierdie navorsing was om 'n skaal te ontwikkel om pasiente se tevredenheid met voorgeboortesorg te bepaal en om die geldigheid van hierdie meetinstrument plaaslik te toets. In die literatuur is daar verskeie metodes om pasiente se tevredenheid te bepaal. 'n Vraelys is ontwikkel vir Tygerberg Hospitaal se voorgeboorte pasiente. Hierdie vraelys is getoets by 200 pasiente in die voorgeboorte klinieke in Tygerberg Hospitaal deur middel van 'n gestruktureerde onderhoud. In die geldigheidsbepaling van die meetinstrument 1s die belangrikheid van kruiskulturele navorsing beklemtoon. Faktoranalise is gebruik vir die bepaling van geldigheid. Met faktoranalise is aangetoon dat een onderliggende faktor, naamlik die voorgeboortesorgsisteem, pasiente se tevredenheid verklaar. Die bevindings in hierdie ondersoek het die volgende getoon: • Dat aile meetinstrumente nie summier transkultureel toegepas kan word nie . • Dat die tevredenheidsmeting van voorgeboortesorg 'n negatiewe skewe verspreiding het, met 'n hoe gemiddelde telling. • Sosiaal-aanvaarbare antwoorde speel waarskynlik 'n groat rol in hierdie vraelyste. • Dat daar 'n statistiese betekenisvolle verskil is in die tevredenheidsgraad van pasiente met voorgeboortesorg tussen sommige klinieke; selfs nadat gekontroleer is vir sosiodemografiese verskille tussen pasiente. • Dat die tevredenheidsmeting 'n weerspieeling is van die diens gelewer aan die pasient, en nie net 'n weerspieeling is van die pasient se sosio-demografiese verskille nie. Hierdie navorsmg identifiseer die probleme met die opstel van 'n gestandaardiseerde meetinstrument vir die bepaling van pasente se tevredenheid met voorgeboortesorg en baan die weg vir verdere navorsing oor pasiente se tevredenheid met mediese dienste.

Thesis (D.Phil.)--Stellenbosch University, 2000. / ENGLISH ABSTRACT: Chapter 1 provides literature based background information on the clinical importance of sperm morphology as recorded by strict criteria during the diagnostic approach of the infertile couple. Furthermore, the use of a sequential diagnostic schedule for couples in an assisted reproductive programme is emphasized. The author revisited the literature on chromatin packaging of spermatozoa and addresses this issue as an additional semen parameter providing information relating to DNA damaged spermatozoa. The chapter also includes evidence underlining the growing need for the implementation of the acrosome reaction as an important contribution to the assisted reproductive programme. Chapter 2 provides detailed descriptions of the material and methods used during the study. Chapter 3 is sub-divided into 5 sections, each of which represents a separate study that was prepared as a scientific paper. The study included 338 couples consulting for infertility treatment at various gynaecologists in Pretoria and Johannesburg. The diagnostic assisted reproductive laboratory support was provided by the Andrology laboratory of Drs du Buisson and partners from Pretoria. In the first study the role of chromatin packaging as an indicator of in vitro fertilization rates, the semen samples from 72 men were used to record their chromatin packaging quality as well as their sperm morphology classification. Significant different percentages CMA3staining (mean±SE) were recorded among the 2 morphology groups, namely 65.9%±3.5 and 44.5%±1.7 (p=0.001). Using cut off values for chromatin packaging established during the first study, the second study utilized semen from 140 men in the in vitro fertilization (IVF) and intracytoplasmic sperm injection programme (ICSI) to analyze for sperm concentration, motility, morphology and chromatin packaging (CMA3).IVF and ICSI data were stratified using 3 basic cut off values for CMA3staining, namely <44%, >44-60% and >60%. The study concluded that results on the chromatin packaging quality of spermatozoa could be used as an additional parameter of sperm quality since it could provide valuable information on decondensation status of a given sperm population. The third study aimed to establish zona pellucida induced acrosome reaction response (ZIAR) among 35 couples with normal and G-pattern sperm morphology and repeated poor fertilization results during assisted reproduction treatment. Interactive dot diagrams, divided patients into 2 groups i.e. ZIAR<15% and ZIAR>15% with mean fertilization rates of 49% and 79%, respectively. The study concluded that the ZIAR test has diagnostic potential, since it can assist the clinician to identify couples that will benefit from ICSI therapy. The forth study revisited the importance of micro-assay for acrosome reaction determinations in a diagnostic andrology laboratory. The micro-assay not only allows the use of a single zona pellucida, but also facilitates the future possibility of using recombinant zona pellucida proteins in a diagnostic test system. The final study in Chapter 3 includes results obtained from 49 couples (172 oocytes) and aimed to evaluate the role of chromatin packaging and sperm morphology during sperm-zona binding, sperm decondensation and the presence of polar bodies among 170 oocytes that failed in vitro fertilization (IVF). Odds ratio analyses indicated that being in the a group with elevated CMA3 staining i.e. >60%, the risk of decondensation failure increases 15.6 fold relative to normal CMA3 staining <44%. Chapter 4 underlines the validity of the sequential diagnostic approach and summarizes the results and value of a multistep diagnostic scheme. The chapter concludes with the recommendation that both chromatin packaging quality and zona pellucida mediation of the acrosome reaction should be part of the diagnostic tools in the assisted reproductive programme. / AFRIKAANSE OPSOMMING: Die literatuuroorsig in Hoofstuk 1 konsentreer in hoofsaak op die kliniese belang van sperm morfologie en die uitbreiding van die diagnostiese toetse en hantering van die egpaar in die reproduktiewe ondersteuningsprogram. Die kromatien pakkingskwaliteit van die spermsel word onderskryf as In belangrike toevoeging tot die diganostiese program, aangesien ONS skade dikwels saam met kromatiendefekte aangetref word. Die rol van die akrosoomreaksie word ook in detail literatuuroorsigtelik beklemtoon. Hoofstuk 2 bevat volledige inligting omtrent materiaal en metodes wat in die studie gebruik is. Hoofstuk 3 bevat die eksperimentele gegewens wat in 5 afsonderlike sub-afdelings as wetenskaplike publikasies aangebied word. Die studies bestaan uit data van 338 pasiënte, wat deur verskillende ginekoloë van Pretoria en Johannesburg gekonsulteer is waartydens drs. du Boisson en vennote van Pretoria die diagnostiese reproduktiewe laboratoriumdienste verskaf het. Die eerste studie stel dit ten doel om die belang en korrelasie van die spermsel kromatienpakkingskwaliteit van 72 mans te vergelyk met die morfologiese bou van sie sel. Aangesien morfologie reeds gevertig is as 'n kliniese voorspeller van bevrugting was dit nodig om hierdie parameter te vergelyk met die kromatienpakking van die sel. Twee afsnypunte word vir die normo-en teratozoospermiese mans identifiseer naamlik, 44.5%±1.7 en 65.9%±3.5 (p=O.001),respektiewelik. Die tweede studie gebruik die afsnypunte 44% en 66% om die in vitro bevrugting en intrasellulêre sperm inspuiting (ICSI) data te ontleed. Die resultate dui aan dat kromatienpakking In waardevolle bydrae tot die diagnostiek van die pasiënte lewer. Die derde studie stel dit ten doelom die waarde van die zona pellucida geinduseerde akrosoomreaksie (ZIAR) te bepaal. Die studie sluit die data van 35 egpare in wat almal normale of G-patroon morfologie het en verder onverklaarde swak bevrugtings resultate tydens in vitro bevrugtingsterapie. Interaktiewe punt diagram (interactive dot diagrams) verdeel die data in twee groepe naamlik, ZIAR<15% en ZIAR>15% met gemiddelde bevrugtingssyfers van 49% en 79%, respektiewelik. Die studie sluit af met die gedagte dat die ZIAR toets 'n groep pasiënte identifiseer met 'n besondere fisiologiese afwyking d.i. subnormale akrosoom respons op zona pellucida blootstelling. Die vierde afdeling van die hoofstuk onderstreep die belang van die mikro-tegniek vir die bepaling van die akrosoom reaksie, wat tydens die projek gebruik is Die vyfde afdeling van Hoofstuk 3 stel dit ten doelom 170 onbevrugde eierselle van 49 pasiënte te ontleed vir moontlike oorsake vir die mislukte bevrugting. Ondersoeke sluit in die kromatienpakking, sperm-zona binding, sperm dekondensasie en die teenwoordigheid van polêre liggaampies. Statisties blyk dit dat indien 'n kromtienpakking nie normaal is nie (>66%) het die spermsel 'n 15 keer groter kans om nie te dekondenseer nie. Hoofstuk 4 bespreek die noodsaaklikheid van die diagnostiese skedule by die hantering van die onvrugbare egpaar in.

Introduction: Neuroactive steroids represent a class of both synthetic and naturally occurring steroids that have an effect on neural function. In addition to classical genomic mechanism by the hormones progesterone, deoxycorticosterone and testosterone 3α-OH metabolites of these hormones enhance GABAA receptor through rapid non-genomic mechanism. The site(s) of action of these neuroactive steroids namely 3α-OH-5α-pregnan-20 one, (3α,5α)-3,21-deoxycorticosterone(3α5α-THDOC) and 5α androstane-3α,17β-diol on GABAA receptor are distinct from that of benzodiazepines and barbiturate binding sites. The modulation site(s) has a well-defined structure activity relationship with a 3α-hydroxy and a 20-ketone configuration in the pregnane molecule required for agonistic action. Pregnenolone sulfate is a noncompetitive GABAA receptor antagonist and inhibit GABA activated Cl- current in an activation dependant manner. 3β-hydroxy A-ring reduced pregnane steroids are also GABAA receptor antagonist and inhibit GABAA receptor function and its potentiation induced by their 3α-diesteromers in a noncompetitive manner. Aim: The aim was to investigate if the effect of GABA, pentobarbital antagonism by bicuculline and if the effect of GABA-agonist and antagonist neuroactive steroids including pregnenolone sulfate is dependant on the α-subunits of GABAA receptor. Furthermore, the studies aimed at investigating the binding site of pregnenolone sulfate and if its effect is dependent on γ-subunit. In addition, the inhibitory effect of pregnenolone sulfate and 3β-hydroxy steroids has been characterized. We also wanted to investigate if the neuroactive steroids effect vary between the human and rat recombinant α1β2γ2L receptors and between the long (L) and short (S) variants of γ2-subunit. Method: Experiments were performed by the two electrodes voltage-clamp technique using oocytes of Xenopus laevis expressed with recombinant GABAA receptors containing α1, α4 or α5, β2, γ2L and γ2S-subunits. Results: There was no difference between the α1, α4 and α5-containing subunits regarding GABA and pentobarbital inhibition by bicuculline. GABA-activated current in the binary αβ was potent than that of ternary αβγ receptor. Unlike Zn2+ effect, inhibition by pregnenolone sulfate on the GABAA receptor is not dependant on the γ-subunit. It is likely that the 2’ residue closest to the N-terminus of the protein at M2 helix on both α1 and β2 subunit are critical to the inhibitory actions of PS and the function of Cl- channels. Point mutation at M2 helix of the β2-subunit (b2A252S) can dramatically reduce the inhibitory effect of PS on the GABAA receptors without affecting the inhibitory properties of 3β-hydroxysteroids. Agonist and antagonist steroids also varied in their efficacy between the human and rat α1β2γ2L receptor. Neuroactive steroids also showed difference between human γ2L and γ2S-containing receptor. Conclusions: GABA and pentobarbital antagonism by bicuculline is not dependant on α-subunit. Pregnenolone sulfate binding site is different from that of Zn2+. 3β-hydroxysteroids and pregnenolone sulfate inhibit GABAA receptor through different mechanisms. Neuroactive steroids also differ between species and between the long and short variant of γ- subunit.

Regulation of the growth and differentiation of trophoblast cells is critical for successful embryo implantation and placentation. Cytokines are key players in these processes, as well as modulating the maternal immune response to prevent rejection of the conceptus. This thesis focused on the investigation of the cytokine interleukin (IL) - 33 and its receptor, ST2. ST2 has two isoforms, a functional cell surface receptor (ST2L) and a soluble decoy receptor (sST2). Previous work in this laboratory had shown that the human placenta expresses both IL-33 and sST2 at term. The aim of this thesis was to investigate IL-33 and ST2 in early pregnancy, the time when trophoblast is at its most active, with a view to better understanding their role. IL-33 and ST2 mRNA and protein were examined in 14 first trimester placentas from 6-12 weeks of gestation. IL-33 was localized to cells in the villous stroma, whereas ST2 was present in the syncytiotrophoblast, villous cytotrophoblast and the invasive extravillous cytotrophoblast of the cell columns. Secretion of sST2, but not IL-33, by the placenta was found. Investigation of pre-implantation embryos showed the presence of ST2, but not IL-33 protein. Decidualized endometrium was investigated as a potential source of IL-33 and sST2 at the maternal-fetal interface and, although mRNA for both was present, no protein could be found. The key finding was that sST2, rather than ST2L, was the predominant isoform in the placenta. This led us to reconsider the hypothesis that IL-33/ST2 interactions in the placenta are important for successful pregnancy and raised the possibility that they may have independent roles. Using trophoblast cell lines as a model, it was shown that sST2 binds to trophoblast cells, significantly inhibits their proliferation and stimulates their invasion in vitro. This is the first report of this novel role for sST2 in pregnancy. Thus these studies have shown that sST2 may play an important role in implantation and placentation through controlling trophoblast invasion.