Doctoral Degrees (Obstetrics and Gynaecology)

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Evaluation of gamete dysfunction as a cause of failed human in vitro fertilization
(Stellenbosch : Stellenbosch University, 2000-12) Esterhuizen, Aletta Dorothea; Franken, D. R.; Stellenbosch University. Faculty of Medicine & Health Sciences. Dept. of Medicine.
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.
Human gamete micromanipulation and intracytoplasmic sperm injection (ICI) : its impact on severe male infertility
(Stellenbosch : Stellenbosch University, 2000-12) Windt, Marie-Lena; Kruger, T. F.; Stellenbosch University. Faculty of Medicine & Health Sciences. Dept. of Obstetrics & Gynaecology.
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.
The interaction between human spermatozoa and its homologous zona pellucida : scientific advances and clinical significance
(Stellenbosch : Stellenbosch University, 2002-12) Oehninger, Sergio C.; Franken, D. R.; Stellenbosch University. Faculty of Medicine & Health Sciences . Dept. of Obstetrics & Gynaecology.
ENGLISH ABSTRACT: Infertility is a very common problem worldwide. Recent data have shown that disorders of the male represent the most common single defined cause of infertility. This proposal examines the clinical significance and fundamental physiological aspects of human gamete interaction. These studies are focused on the assessment of the cellular-molecular mechanisms involved in human sperm binding to its homologous zona pellucida resulting in the physiologic induction of the acrosome reaction. We have developed and validated in vitro bioassays that assess specific steps of the fertilization process that are critical for early embryo development. The results of our translational research have already had a significant impact on the overall evaluation of male infertility and on the clinical management of the infertile man in the assisted reproduction arena. Furthermore, the unveiling of the basic mechanisms involved in human gamete interaction will ultimately allow for both (i) the development of new male reproductive diagnostic capabilities and (ii) the design of improved and safer therapies aiding conception in childless couples suffering from male infertility.
Molecular-genetic investigation into host susceptibility and variability to HIV/AIDS in the South African population
(Stellenbosch : Stellenbosch University, 2003-12) Pretorius, Gideon Stephan; Kotze, Maritha J.; De Jong, Geertje; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Obstetrics and Gynaecology .
ENGLISH ABSTRACT: The risk of human immunodeficiency virus type-1 (HIV-1) infection and rate of progression towards development of the acquired immunodeficiency syndrome (AIDS) is determined by a combination of viral characteristics, immune function and host genetic variation. Although mutations of the chemokine and chemokine co-receptor genes and allelic variation of the major histocompatibility complex (MHC) have been studied extensively, variation in these host genetic factors does not explain the differences in HIV/AIDS susceptibility in all cases. This study represents the first analysis of new candidate genes implicated in iron metabolism and immune function in relation to HIV-1 disease in the African context. Both case-control association studies and genotype-phenotype correlations were performed to determine the potential functional significance of genetic variants that may be involved, either directly or indirectly, in susceptibility to HIV-1 disease in the South African population. Genotyping was performed to identify potentially important polymorphisms in the solute carrier family 11 member 1 (SLC11A 1), haemochromatosis (HFE) and protein-tyrosine phosphatase receptor-type C (PTPRC/CD45) genes in HIV-seropositive versus HIVseronegative individuals. This was followed by HLA-B27 genotyping in HIV-1 infected individuals with known disease status to determine the potential impact of combined genotypes for different mutations identified in the same study cohort. Preferential association with any of the mutations screened for in the CCR5, SLC 11A1, HFE or CD45 genes were not detected in HLA-B27 positive individuals identified. These findings were in accordance with the independent protective role of HLA-B27 in relation to disease progression in HIV-1 infected individuals. Although differences in allelic distribution were not significant between the study groups, an apparently African-specific mutation 32A~G, identified in an exonic splicing silencer element (ESS-1) of the CD45 gene, appeared to predominate in HIV-1 infected subjects with WHO Class I disease status and slow progression to AIDS. This mutation was present in 35.7% (5/14) of HIV-seropositive individuals with WHO Class I disease status, whilst absent in 22 HIV-seropositive patients with rapid disease progression. This finding may be related to differences in proportions of both CD4+ and CD8+ subsets observed following flow cytometry (FACs) analys.s in two HIV-seropositive individuals with mutation 32A~G, compared with an HIV-seropositive individual without this mutation. Analysis of the iron-related SLC11A1 and HFE genes did not reveal significant associations with modified risk of HIV-1 infection or progression to AIDS in our predominantly African study population. However, the effect of the virus on iron metabolism was demonstrated for the first time at the DNA level. Haemoglobin levels were significantly reduced in both HIV-seropositive (P=O.004) and HIV-seronegative (P=O.02) Black Africans with mutation IVS3-48c~g in the HFE gene, compared with mutationnegative individuals in both groups. Since this effect was more pronounced in HIV-infected individuals compared with controls, presence of the HFE mutation seems to result in an even stronger effect on haemoglobin levels, which may be related to the acute phase response following virus infection. This effect possibly results from genetic variation in a nearby gene involved in innate immunity, most likely in the HLA region on chromosome 6. It therefore seems possible that genetic variation in any of the host molecules involved in response to infection could contribute to clinical outcome. The significance of the multitude of host genetic factors investigated in this study, or previously implicated in susceptibility to HIV-1 infection and disease progression, revealed a complex interrelationship between the host and HIV-1. In some instances the disease process following HIV-1 infection depends on combined effects of different mutations occurring in the same individual, while independent effects of specific genes in conjunction with environmental influences may explain diverse clinical outcomes in others.
Sperm DNA fragmentation : implications in assisted reproductive technologies
(Stellenbosch : Stellenbosch University, 2007-12) Hoogendijk, Christiaan F. (Christiaan Frederik); Kruger, T. F.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Obstetrics and Gynaecology.
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.

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