Theses and Dissertations (Obstetrics and Gynaecology)

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The identification of microrna signatures associated with glycaemic control and pregnancy outcomes in pregnancies complicated by Type 1, Type 2, and gestational diabetes mellitus
(University of Pretoria, 2024-04-02) Pheiffer, Carmen; Adam, Sumaiya; Dias, Stephanie; u12174506@tuks.co.za; Malaza, Nompumelelo L.
Background. Diabetes in pregnancy (DIP) is associated with short- and long-term adverse pregnancy outcomes for both mother and child. However, pregestational type 1 (T1DM) and type 2 (T2DM) are associated with more common and severe pregnancy outcomes compared to gestational diabetes (GDM). Maternal biochemical and epigenetic markers and knowledge of diabetes have been associated with glycaemic control and adverse pregnancy outcomes in women with DIP, therefore they offer potential to serve as markers. This may aid in reducing adverse outcomes and improve mother and child health. Aim. The aim of this study was to compare adverse pregnancy outcomes by DIP type and explore the candidacy of adiponectin, leptin, sex hormone binding globulin (SHBG), microRNAs (miRNAs) and diabetes knowledge to serve as markers of glycaemic control during pregnancy and perinatal outcomes in pregnancies complicated by T1DM, T2DM and GDM. Methods. A prospective study was conducted at the high-risk antenatal clinic at Steve Biko Academic Hospital, Pretoria, South Africa between May 2017 and April 2023. The study population consisted of 232 pregnant women with pregestational T1DM (n=27) or T2DM (n=78), GDM (n=58), and normoglycaemia (n=69). Maternal serum adiponectin, leptin and SHBG levels were measured using enzyme linked immunosorbent assays (ELISAs). Maternal serum miRNAs were measured using quantitative real-time PCR. The diabetes knowledge and perceptions questionnaire was developed in three phases and content validity was tested in 20 women with GDM using researcher-administered interviews. Results. Pregestational T1DM and T2DM were associated with an increased risk of preterm birth (p=0.002). Obesity was associated with a higher frequency of GDM (p=0.036), while body weight ≥ 80 kg was associated with caesarean section before the onset of labour (p<0.05). Lower maternal leptin levels were associated with large for gestational age (LGA; p=0.036), macrosomia (birthweight more than 4 kg; p=0.060) and preterm birth (PTB; p=0.004). Lower levels of maternal SHBG were associated with macrosomia (p=0.025) and levels were negatively correlated with neonatal birthweight (r=-0.263, p=001). No association between maternal adiponectin levels and neonatal birth outcomes was observed. Four miRNAs (miR-124-3p, miR-128-3p, miR-20a-5p and miR-210-3p) were associated with small for gestational age (SGA) and were able to predict SGA, miR-124-3p (AUC=0.815), miR-128-3p (AUC=0.760), miR-20a-5p (AUC=0.841) and miR-210-3p (AUC=0.779). MiR-210-3p was associated with macrosomia and demonstrated good predictive ability (AUC=0.779). MiR-222-3p was increased in women with good glycaemic control compared to women with poor glycaemic control during pregnancy. A comprehensive questionnaire for evaluating diabetes knowledge in South African pregnant women with GDM was developed, which performed well in terms of content validity and was able to assess knowledge of diabetes in pregnant women with GDM. Conclusion. To our knowledge, this is the first study to investigate the association between maternal adiponectin, leptin, SHBG and miRNAs with neonatal birth outcomes in South Africa. Our findings suggest that maternal leptin, SHBG and miRNAs may offer potential as biomarkers of neonatal birth outcomes and glycaemic control. Additionally, this is the first study to develop a questionnaire to evaluate diabetes knowledge in women with GDM in South Africa. The developed questionnaire may aid in identifying knowledge gaps in pregnant women with GDM, thereby enhancing education programs and developing interventions to improve glucose management and pregnancy outcomes in women with GDM. However, further studies in larger and multi-ethnic populations are warranted to explore the candidacy of biochemical and miRNA biomarkers for glycaemic control and neonatal birth outcomes. Additionally, the developed questionnaire requires a comprehensive validation process to evaluate construct validity, internal consistency, and test-retest reliability in future.
Development of a screening algorithm for the cervical cancer prevention programme in Botswana
(University of Pretoria, 2023) Dreyer, Greta; Hofmeyr, Justus; Morroni, Chelsea; doreen.masire@gmail.com; Ramogola-Masire, Doreen
In this thesis, Development of a screening algorithm for the cervical cancer prevention programme in Botswana, the candidate utilised a new human papillomavirus (HPV) deoxyribonucleic acid (DNA) assay called Antila AmpFire to test for high-risk HPV in a semi-urban area in Botswana. This prospective cohort study evaluated several triage methods for high-risk HPV screen-positive women. The methods were visual inspection after acetic acid, colposcopy, and various partial HPV genotypes. Performance of the various triage methods were calculated and compared to that of the primary hrHPV screening. Restricted 8-hrPHV (16,18,31,33,35,45,52,58) maintained the highest sensitivity of all the triage methods. Although our finding supported the 8-hrHPV triage method as the most effective strategy, additional research is needed to guide further management as the relatively poor specificity of this method would still lead to overtreatment of women with no established precancer disease.
Investigating Molecular Biomarkers During Gestational Diabetes Mellitus
(University of Pretoria, 2019) Pheiffer, Carmen; Adam, Sumaiya; Rheeder, Paul; Stephanie.Dias@mrc.ac.za; Dias, Stephanie Charmaine
Introduction: Gestational diabetes mellitus (GDM) is a significant public health concern, due to its association with short- and long-term complications in both mothers and offspring. DNA methylation and single nucleotide polymorphisms (SNPs) offer potential to serve as molecular biomarkers, which may lead to improved detection of GDM with positive effects on health outcomes. Aim: The aim of this study was to investigate whether DNA methylation and SNPs are associated with GDM and may offer potential as molecular biomarkers for GDM in South Africa (SA). Methods: This study followed a two-pronged approach. Firstly, literature searches were conducted to collate and synthesise all published articles reporting on the prevalence of GDM in SA, the screening and diagnostic strategies used, and the current status of DNA methylation and SNPs as biomarkers for GDM. Secondly, we conducted experiments to investigate global (n=201), genome-wide (n=24) and gene-specific DNA methylation (n=286) of the adiponectin gene (ADIPOQ) in whole blood of women with and without GDM, using an Enzyme-Linked Immunosorbent Assay, a methylationEPIC BeadChip Array and pyrosequencing, respectively. In addition, genotype and allele frequencies of ADIPOQ rs266729 and rs17300539, and methylenetetrahydrofolate reductase (MTHFR) rs1801133 were determined, using quantitative real-time PCR (n=449) and DNA sequencing for validation. Results: The literature search showed that the prevalence of GDM in SA has increased over the years. Furthermore, it showed that the lack of uniformity in screening and diagnosis between and within countries hamper the accurate detection of GDM. Lastly, the literature search identified several studies that support the use of DNA methylation and SNPs as potential biomarkers for GDM. Experimentally, we showed no differences in global DNA methylation between GDM and non-GDM groups. Interestingly, global DNA methylation levels were 18% (p=0.012) higher in obese compared to non-obese pregnant women. Genome-wide methylation analysis identified 1046 differentially methylated CpG sites (associated with 939 genes) (Cut-off threshold: M>0.06 and p<0.01). Among the top five CpG sites identified, one CpG mapped to the calmodulin-binding transcription activator 1 (CAMTA1) gene, which has been shown to regulate insulin production and secretion. Two CpG sites (-3410: p=0.048 and -3400: p=0.004) in the ADIPOQ promoter were hypomethylated during GDM in HIV negative, but not in HIV positive women. Lastly, no association between the ADIPOQ and MTHFR polymorphisms and GDM was observed in our population. Conclusion: To our knowledge, this is the first study to investigate the association between DNA methylation or ADIPOQ (rs266729 and rs17300539) and MTHFR (rs1801133) polymorphisms and GDM in SA. Findings suggest that gene-specific, but not global methylation nor SNPs rs266729, rs17300539 and rs1801133, may offer potential as molecular biomarkers of GDM in this population. Future longitudinal studies in larger samples that include both HIV negative and positive pregnant women are warranted to explore the candidacy of DNA methylation as molecular biomarkers for GDM.
Optimization of techniques required for somatic cell nuclear transfer using a mouse model
(University of Pretoria, 2018) Pepper, Michael Sean; Huyser, Carin; u29005010@tuks.co.za; Gouveia, Chantel
Introduction: Somatic cell nuclear transfer (SCNT) is the removal (enucleation) of chromosomes from a metaphase II oocyte, followed by the transfer and fusion of a donor somatic cell to the enucleated oocyte (cytoplast). The reconstructed oocyte is then activated to induce embryonic development. One of the main purposes of creating SCNT blastocysts is the derivation of embryonic stem cells (ESCs) for therapeutic cloning. The consensus for the low SCNT blastulation rate (12-15% in mice and 10% in humans) is epigenetic reprogramming failure of the donor somatic cell nucleus by the oocyte. The optimization and technical efficiency of SCNT was investigated in this study. Methods: Female B6D2F1 mice were used as oocyte and cumulus cell (somatic cell) donors. In study 1, non-invasive spindle imaging by Hoffman modulation contrast microscopy was used to identify the spindle within 484 oocytes. Once located, the spindles were enucleated. Successful enucleation was confirmed through artificial activation (317 cytoplasts) using calcium ionophores and kinase or protein synthesis inhibitors that caused fragmentation of the cytoplasts; and Hoechst DNA staining (167 cytoplasts) to microscopically confirm the absence of chromosomes. Appropriate controls for the techniques were included. In study 2, enucleation of 564 oocytes was followed by the transfer a single cumulus cell exposed to a membrane fusogen into close contact with the oolemma of the cytoplast. Post fusion, the reconstructed oocytes were artificially activated and subsequently cultured to the blastocyst stage, with the addition of a histone deacetylase inhibitor to aid epigenetic reprogramming. The reconstructed oocytes (80-90%) were expected to survive nuclear transfer; with 70-80% surviving activation, 60-70% pseudo-pronucleus formation; 50-60% cleavage to the 2-cell stage after 24 hours of nuclear transfer; and 30-50% development to the morula/blastocyst stage 72-96 hours post nuclear transfer. Results: The first enucleation confirmation technique was the analysis of cytoplast fragmentation between 16-18 hours after activation, which indicated that 85% of cytoplasts were effectively enucleated. A cohort of non-enucleated control oocytes confirmed the efficiency of the activation protocol by showing a pseudo-pronucleus formation rate of 95.4%. The second confirmation technique revealed an enucleation efficiency of 97.5%, which was confirmed by the absence of chromosomes in stained cytoplasts. The staining protocol was verified using a group of non-enucleated control oocytes, resulting in 100% of the oocytes presenting with stained and visible chromosomes. In study 2, enucleation was performed with a survival rate of 99.1%. The cytoplasts then underwent cumulus cell nuclear transfer with 100% survival. Subsequently, a fusion rate of 72.3% and an activation rate of 81.7% was achieved in this study. Blastocyst formation by SCNT was significantly lower than that of the control group (5.4% vs. 55.1%), and more poor-quality blastocysts were produced by SCNT (63.6%). Therefore, according to statistical analyses, the chance of forming a blastocyst by SCNT in this project was 0.041-fold that of the control group. Discussion: Results revealed that the rates of enucleation and nuclear transfer survival, fusion efficiency, activation survival, pseudo-pronucleus formation, cell division, compaction and morula development of the SCNT embryos were as good as those reported. However, the formation of SCNT blastocysts was below the published average, which may be a consequence of epigenetic reprogramming failure. Experimental adaptations such as medium supplementation or nuclear reprogramming strategies can be applied to improve epigenetic reprogramming by SCNT, which could be evidenced by a greater number of embryos progressing to good-quality blastocysts. In humans, use of SCNT as a tool to generate specific ESCs from the somatic cells of an individual could ultimately lead to the analysis of disease mechanisms, as well as improve the efficiency of cell-based therapies with a negligible risk of immune rejection in the treatment of degenerative diseases. SCNT is a cutting-edge technique that can offer innovative clinical applications in the field of assisted reproduction such as preventing the transmission of mitochondrial DNA diseases from mother to child, as well as the treatment of ooplasm pathologies.
Maternal health after having survived a potentially life-threatening complication in pregnancy
(University of Pretoria, 2017) Pattinson, Robert Clive; priya.somapillay@up.ac.za; Soma-Pillay, Priya
OBJECTIVES Maternal near-miss audits were initially introduced to evaluate the quality of obstetric care at the time of the event. One of the principle advantages of studying near-misses is that these women are able to adequately report the obstacles and delays they face to assure their continued survival. The primary objective of the study was to compare the long-term health of women who suffered a serious acute morbidity event in pregnancy to a control group of low risk women who have had a normal pregnancy outcome. We wanted to determine whether the insult associated with maternal near-misses makes women more vulnerable to further organ dysfunction. The secondary aim was to determine the spectrum of morbidity in the Pretoria Academic Complex (PAC) and the barriers to obstetric care. METHODS AND MATERIALS This study consists of 6 sub-studies to address the research problem. The first study investigated the epidemiology of the spectrum of morbidity in the PAC. Delivery data related to demographic and reproductive characteristics, pregnancy and childbirth complications were collected on a daily basis from hospitals in the PAC: Steve Biko Academic Hospital, Kalafong Provincial Tertiary Hospital, Tshwane District Hospital, Pretoria West and Mamelodi Hospitals and from Stanza Bopape and Eersterust Midwife Obstetric Units. Daily audit meetings were held at the 2 tertiary hospitals to identify women with potentially life-threatening conditions. We further investigated the delays/barriers in providing obstetric care to women who were classified as a maternal near miss. The “three delays model” was used to identify the phases of delay in the health system. Three studies investigated the long-term complications in women with severe pre-eclampsia in pregnancy. Magnetic resonance imaging, echocardiography, and digital photos of the eye were taken at delivery and 1-year post-partum. In the last study, we compared the quality of life of women classified as a maternal near miss to a group of women with an uncomplicated low-risk pregnancy using the World Health Organisation Quality of Life questionnaire. RESULTS About one in 20 pregnant women in the PAC had a potentially life-threatening condition and 0.5% a life-threatening condition in pregnancy. One or more factors causing a delay in accessing care were identified in 83% of near-miss cases. Near miss women have a poorer quality of life 1-year after delivery than women with uncomplicated pregnancies. Severe pre-eclampsia in pregnancy was found to have the following long-term effects on maternal health: ? Cerebral white matter lesions were demonstrated in 48% of women with severe pre-eclampsia at 1 year postpartum ? Women with early onset pre-eclampsia requiring delivery prior to 34 weeks had an increased risk of cardiac diastolic dysfunction ? Retinal artery and venular calibers in women with pre-eclampsia are smaller than those of normotensive women at 1-year postpartum CONCLUSION Women who experience a severe acute morbidity event in pregnancy must be recognised as a vulnerable group who are require increased postpartum care and surveillance. Ideally all tertiary centres should have near-miss clinics for postpartum care. Any risk factors for future disease should be identified and modified to promote long-term health.
Efficacy of the sentinel lymph node biopsy algorithm and PET/CT scan in assessing regional lymph node status in women with early stage endometrial and cervical cancer in a South African population
(University of Pretoria, 2017) Lindeque, B. Gerhard; Sathekge, Mike Machaba; Abu-Rustum, Nadeem; leon.snyman@up.ac.za; Snyman, Leon Cornelius
Abstract Introduction Knowledge about the oncologic status of pelvic lymph nodes forms an essential and integral part in the management of women with uterine cancer. Lymph node status is part of endometrial cancer staging and plays an important role in primary treatment and adjuvant treatment planning and prognosis in women with cervical cancer. Current practice in the management of uterine cancers involves systematic full pelvic lymphadenectomy, mainly to determine the oncological status of the nodes, as there is no high-quality evidence suggesting a therapeutic effect attributable to lymphadenectomy. Imaging in the form of computed tomography (CT) scans and magnetic resonance (MRI) scan is not accurate to determine pelvic lymph node status in women with uterine cancer. Functional scans such as 18Fluoro-deoxy-glucose positron emission/computed tomography (FDG-PET/CT) scan might provide better access in this setting. Sentinel lymph node biopsy (SLNB) procedures, specifically the SLNB algorithm, have been proposed as a safe and accurate alternative procedure to full systematic lymphadenectomy in women with uterine cancers. It has also been proposed as a better alternative than complete omission of lymphadenectomy in women with presumed low risk early stage endometrial cancer. SLNB procedures might also be able to detect higher rates of lymph node metastases with the detection of micro metastases following pathological ultrastaging The presence or absence of high risk human papilloma virus (hrHPV) DNA in sentinel lymph nodes of women with cervical cancer has also been suggested to be a useful adjunct to frozen section examination (FSE) in assisting with determination of the status of the non-sentinel nodes. Some data suggest the combination of negative FSE and absence of hrHPV accurately predict the absence of metastases. South African women have high prevalence of human immunodeficiency virus infection, tuberculosis (TB) and pelvic inflammatory disease (PID). All these infections involve the lymphatic system. Data on SLNB procedures are form well-developed countries with different disease burdens and socioeconomic profiles, and there is no data from women living in low-resource settings. Aims This study aimed to determine the efficacy of and performance of FDG-PET/CT scan and SLNB and SLNB algorithm in accurately predicting the regional lymph node status of the pelvis in women with early stage cervical cancer and presumed early stage endometrial cancer. It also aimed to investigate the usefulness of HPV DNA testing of sentinel nodes in women with cervical cancer. Population and setting This was a prospective observational study performed in the Gynaecologic Oncology Unit at the Kalafong Provincial Tertiary Hospital and Steve Biko Academic Hospital. Patients aged 18 years and older, with operable stages cervical cancer and presumed early stage endometrial cancer willing and able to provide informed consent were eligible for inclusion. Materials and methods Sentinel node mapping was done using methylene blue (MB) and indocyanine green (ICG) injected into the cervix after induction of anaesthesia at the time of primary surgery. 99Technetium nanocolloid (99Tc) was administered one day pre-operatively followed by lymphoscintigram. FDG-PET/CT scans were performed prior to surgery. Following mapping and removal, FSE, HPV DNA typing, haematoxylin and eosin (H&E) examination with ultrastaging on H&E negative specimens were performed on the SLNs. All patients underwent systematic full pelvic lymphadenectomy and appropriate cancer surgery. Results One hundred patients were prospectively recruited to the study and results of 94 patients were available for analysis. SNL detection rate of the whole group was 60.6% with bilateral detection 29.2%. Twenty-four patients (25.5%) had pelvic metastases. Sixty-five percent of women with cervical cancer in this study were HIV positive, and the SLN detection rate in this group was 65% with bilateral detection rate of 30%. The detection rate was significantly higher in women without nodal metastases, those with stage IA2 – IB2 disease, with tumour less than 2 cm and women with BMI less than 25 kg/m2. HIV status, history of TB, PID and the presence of adhesions did not influence the SLN detection rate. The sentinel lymph node biopsy algorithm has a sensitivity of 100%, NPV of 100% and a false negative rate of 0% in this study. The SLNB procedure identified two women with only micro metastases (15.4%). These women would not have been identified with systematic lymphadenectomy and H&E examination. Indocyanine green and the combination of methylene blue and 99Technetium nanocolloid had significantly better sentinel node detection rates compared to methylene blue alone FDG-PET/CT scan was performed in 28 women. The sensitivity, specificity, positive and negative predictive values of FDG-PET/CT scans to accurately predict nodal status, were 66.67%, 82%, 30.77% and 95.38% respectively. The false negative rate of FDG-PET/CT scans was 33.3%. The sensitivity, specificity, PPV and NPV for FSE in this cohort was 66.67%, 100%, 100% and 96.05% respectively. The FNR for FSE was 23.1%. Thirty-two patients with cervical cancer had tumour and SLN hrHPV DNA data. The sensitivity, specificity, PPV and NPV of sentinel lymph node HPV DNA to predict metastases was 50%, 69.6%, 30 and 84.2% respectively with a false negative rate of 42.8%. Conclusions Although the SLN detection rate was lower compared to the published literature, the SLNB algorithm performed excellently in this group of patients of which the majority were HIV-infected. The SLNB procedure can be considered as a treatment option in selected cases in the management of women with early stage endometrial and cervical cancer. PET/CT should not be used as part of the primary diagnosis and staging investigations in women with uterine cancer, and is recommended only in selected cases for initial staging of locally advanced cervical cancer being considered for radical chemoradiation therapy. In this study, testing for the presence of hrHPV DNA in the sentinel lymph nodes was not useful as a predictor of pelvic lymph node status. The combination of negative FSE and negative hrHPV in the SLNs did not have a reliable negative predictive value for the absence of pelvic nodal metastases.
Comparing Screening Strategies for Gestational Diabetes in a South African Population
(University of Pretoria, 2017) Rheeder, Paul; u04332555@up.ac.za; Adam, Sumaiya
Globally, there is an alarming increase in the incidence of Type II diabetes mellitus (T2DM). It is well recognized that women who develop gestational diabetes (GDM) in their pregnancies are at increased risk of T2DM in later life. In addition, poor glycaemic control in pregnancy impacts adversely on the neonatal outcome, as well as the long term disease risks of that child. The risk of these outcomes increases continuously as maternal fasting plasma glucose levels increases. Several adverse outcomes have been associated with DM during pregnancy. These include pre-eclampsia, polyhydramnios, fetal macrosomia, fetal hepatomegaly and cardiomegaly, birth trauma, operative delivery, perinatal mortality and neonatal respiratory problems and metabolic complications such as hypoglycaemia, hyperbilirubinaemia, hypocalcaemia and polycythaemia. Despite five decades of research there is little consensus regarding the optimal approach to screening for GDM. Recently most international organisations have recommended that all women should be screened for GDM. South Africa is a diverse multi-racial society with an increasing burden of non-communicable diseases. The health system is already overburdened, and the optimal approach to screening for GDM remains unclear. A prospective cohort observational study was conducted at the Eyethu Yarona clinic (Lion Park Clinic), in Johannesburg, South Africa (SA). One thousand (1000) consecutive non-diabetic women who were less than 26 weeks pregnant were recruited. At recruitment the women completed a demographic questionnaire, and had a random glucose and glycated haemoglobin (HbA1c) drawn. A fasting blood glucose was assessed within 2 weeks, and a serum specimen was frozen at -40°C for further testing at a later stage. Patients had a 75 g 2-hour oral glucose tolerance test (OGTT) and HbA1c between 24 – 28 weeks gestation. All glucose measurements were done at the laboratory using standardized tests (venous blood) and on a Roche Accuchek Active® glucometer (Roche Diagnostics, Mannheim, Germany) (capillary blood). GDM was diagnosed according to the International Federation of Gynecology and Obstetrics (FIGO) criteria, i.e. any one abnormal reading was diagnostic of GDM: 0-hour ≥5.1 mmol/l, 1-hour ≥10 mmol/l, or 2-hour ≥8.5 mmol/l. Thereafter a nested cohort study of HIV negative patients was conducted to investigate the association between the concentrations of biomarkers associated with glucose homeostasis and GDM in a South African population. C-reactive protein (CRP), adiponectin, and fasting insulin were measured on the stored serum samples. The Insulin Sensitivity Index (HOMA-IR = fasting insulin (microU/L) x fasting glucose (mmol/L) / 22.5), and Quantitative Insulin Sensitivity Check Index (QUICKI = 1 / [log (I0) + log (G0)]) were calculated for further evaluation of markers of insulin sensitivity. The significance of this research was to assess the burden of disease of GDM in a South African population. The different diagnostic criteria were also compared, as well as the universal versus the traditional risk-factor based screening approach to GDM. Screening methods were compared so as to propose a simple, effective, cost efficient screening and diagnostic tool that may be implemented at primary health care level, which will in turn identify those pregnant women who warrant referral to a high care obstetric unit, thus improving both maternal and neonatal outcomes in our population.
Evaluation of pelvic floor morphology in South African females
(University of Pretoria, 2017) Lindeque, B. Gerhard; Dietz, Hans Peter; zeelha.abdool@up.ac.za; Abdool, Zeelha
Pelvic floor dysfunction in the form of pelvic organ prolapse (POP) is a common gynaecological condition, especially in the elderly. Although the aetiology is poorly understood, several risk factors such as vaginal childbirth, chronically raised intra-abdominal pressure (such as asthma and chronic constipation), ageing, previous hysterectomy and connective tissue disorders are thought to play a role in the pathophysiology of POP. Studies have shown that vaginal childbirth can result in both gross and micro-architectural distortion/alteration of the pelvic floor musculature and is thus considered to play a major role in the development of POP. Although ethnicity has been proposed as a risk factor, there are limited studies on this subject. Recently, transperineal ultrasound (TPUS) has been used to study the structural integrity and the dynamic interaction between the pelvic organs and pelvic floor musculature. Using a specified methodology we intended to determine and compare pelvic floor morphology, namely pelvic organ descent and levator hiatal distensibility in a multi-ethnic South African population (Asian, Caucasian and Black) in both asymptomatic nulliparous and symptomatic multiparous women. Secondly we also intended to study the association between prolapse symptoms and functional anatomy of the pelvic floor, and finally to determine the impact of vaginal childbirth on the pelvic floor morphology 3-6 month postpartum. For all the studies women were recruited from the local nursing school, general gynaecology and tertiary urogynaecology clinic. Pregnant women were recruited from the district antenatal clinic. This cohort included only Black pregnant women. After informed consent all ultrasound volumes were acquired at rest, maximal pelvic floor contraction and Valsalva maneuver. Volumes were deindentified and analysed 6-8 weeks later using GE Kretz 4D View (GE Kretztechnik Gmbh, Zipf, Austria). In the nulliparous cohort, we found that Black South African women had greater pelvic organ descent on ultrasound and clinically and greater distensibility compared to South Asian and Caucasian women. Multivariate modelling revealed that Black 2 ethnicity remained a significant factor for pelvic organ mobility on clinical examination, (P=0.024). In women with symptomatic POP, there was significant variation in clinical prolapse stage, levator distensibility and pelvic organ descent in this racially diverse population presenting with pelvic organ prolapse, with South Asians having a lower avulsion rate than the other two ethnic groups (P= 0.014). As regards the association between prolapse symptoms and functional anatomy of the pelvic floor we found a significant association between awareness, visualization and/or feeling of a vaginal lump and abnormal pelvic floor functional anatomy, that is, hiatal ballooning and levator avulsion (all P< 0.05). The fourth part of the study included eighty four women who returned at a mean of 4.8 months postpartum. We found significant alteration in pelvic organ support and levator hiatal distensibility after vaginal delivery i.e. a significant increase in mean values from ante to postpartum measurements, more so for the vaginal delivery group. 15% of Black primiparous women sustained levator trauma after their first vaginal delivery. In conclusion, to the author‘s knowledge this is the first study on pelvic floor morphology in South African women. Contrary to previous publications inferring that Black women rarely develop PFD, we have shown that this particular ethnic group had significantly different pelvic floor dynamics than Caucasian and South Asian women for both nulliparous and multiparous symptomatic women. Levator trauma occurs in 15% of Black women after vaginal childbirth.
Investigating a novel in vitro embryo culture system – The Walking Egg Affordable Assisted Reproductive Technology
(University of Pretoria, 2017) Huyser, Carin; Ombelet, Willem; boshoffgm@gmail.com; Boshoff, Gerhardus Marthinus
Introduction: The desire to have a biological child transcends race, religion and socio-economic status. However for those faced with infertility, the financial resources needed to conceive are often not available. Current research in assisted reproduction has gravitated towards cost reduction to address restricting financial factors, without compromising quality of treatment. One such initiative is the development of a low-cost embryo culture method by The Walking Egg foundation. This method utilizes a standard chemical reaction and simple equipment to equilibrate culture media pH and to regulate temperature; both aspects were investigated in this study. An exploration into the insemination concentration to achieve oocyte fertilization was also undertaken. Methods: Quality control of temperature regulation on six different heating devices, including a comparison of inter- and intra-variations was carried out. The utilization of citric acid and bicarbonate of soda for carbon dioxide production, which subsequently facilitate setting of pH values, was tested by injecting increasing citric acid volumes (1.2 ml – 3.0 ml in 0.2 ml increments) into set volumes of bicarbonate of soda. Further investigation evaluated gas production at various temperatures (37°C, 25°C and 15°C), at increasing intervals (16 – 30 hours) of equilibration and these were compared by measuring pH of the culture media. The influence of altitude on pH was explored by repeating the chemical reaction experiment at five different locations in South Africa. Furthermore, the addition of water to citric acid before gas generation was explored. The minimal insemination concentration needed for fertilization was determined by the addition of decreasing numbers of spermatozoa to non-fertilized bisected oocytes. The experiment was repeated with a selected sperm insemination number in 1 ml or 50 μl culture media to compare the tested culture system with conventional culture. Spermatozoa bound to the hemi-zonae were counted with the aid of an inverted phase contrast microscope. Hemi-zonae with bound sperm were also stained with ethidium homodimer and evaluated using a confocal laser-scanning microscopy system. After removal of hemi-zonae, the spermatozoa in culture were isolated for deoxyribonucleic acid fragmentation analyses and reactive oxygen species presence in the culture media was measured. Additionally, reactive oxygen species generation in simulated culture was measured over time. Results: All the equipment tested bar one, the warming oven, proved useable with the simplified Walking Egg in vitro fertilization culture system. By decreasing the citric acid volumes, it was indicated that 1.8 ml citric acid, diluted with 1.2 ml water, is the optimal volume to facilitate the required culture media pH. Omitting the water dilution from citric acid volumes affected the culture media pH adversely, however reducing the temperature during gas equilibration did not. A change in altitude had no effect on culture media pH. Lower insemination numbers resulted in decreased sperm binding, with 2 x 103 motile sperm insemination providing the lowest number to still obtain sufficient sperm–zona binding (≥20 sperm bound). Incubation in 1 ml vs. 200 μl culture media indicated decrease in sperm bound. Sperm deoxyribonucleic acid fragmentation and the presence of reactive oxygen species in the culture media were similar in both the test and control groups. A comparison over time revealed less reactive oxygen species in 1 ml culture media, from the simplified Walking Egg in vitro fertilization culture system after three days of culture, than 200 μl culture media drops under oil, from conventional culture after 18 hours, however the results were not statistically significant. Discussion: Purpose-made heating devices provide superior stabilization of culture media temperature. When selecting a heating device, intra-variations should be considered. Culture media can be manipulated to the required pH by carbon dioxide production, with meticulous attention paid to the citric acid volumes used. However, if gas generation is performed at room temperature, equilibration time must be increased. In conventional culture, the minimum insemination number can be reduced to 2 x 103 motile sperm. Due to lower binding of sperm in large volumes of culture media, 2 – 5 x 103 motile sperm should be considered for the simplified culture system, depending on a holistic consideration of all sperm parameters. Extended culture for at least three days with the simplified culture system can be performed without increasing reactive oxygen species present in culture media. Further research of this novel culture method should include the application of the culture method in a South African environment.
Cervical neoplasia in women with and without HIV-related immune depletion : epidemiology and pathogenesis related to HPV types
(University of Pretoria, 2016) Dreyer, Greta; mc@vanaardt.net; Van Aardt, Cornelis
Objectives: Despite current comprehension of HPV epidemiology, the question of how HPV type distribution in the South African population differs from that in the rest of the world remains largely unanswered for both prevalence and oncogenic potential. The primary objective of this study was to investigate the causal relationship between oncogenic or high-risk HPV (HrHPV) types and disease of the cervix, ranging from healthy women to pre-neoplastic and malignant disease. The secondary objective was to investigate the potential differences in the importance of oncogenic HPV types between HIV-infected and HIV-non-infected women. This information is crucial to design country-specific primary and secondary prevention programmes. Methods and materials: This study consists of five smaller studies to address the research problem. Firstly, we investigated the prevalence and distribution of HPV types in sample South African women representative of the general population and subset women with AIDS, both with normal cytology. Secondly, we assessed HPV types present in patients with biopsy-confirmed CIN II/III and to compare HPV type distribution between HIV-infected- and -non-infected women. DNA typing was done on the surface as well as in tissue samples of the dysplastic lesion, in an attempt to identify the lesion-causing virus. Immunohistochemical markers was utilised to insure accurate histological diagnosis and reduce inter- and intra-observer variability. Lastly, we investigated type-specific prevalence in women with invasive cervical cancer (ICC) with and without HIV co-infection. All the above data was then collated to determine the importance of HPV types in cervical oncogenesis in South African women with and without HIV. Results: High-risk HPV DNA was detected from 45% of women with normal cytology, 93% of CIN II/III and 88% of ICC. The four most prevalent HrHPV types found in women without cytological abnormalities were HPV 16, 51, 58 and 45; among women with CIN II/III HPV 16, 52, 35 and 18 were the most common single types; and in ICC samples HPV 16, 18, 45 and 35 were most common. HPV 16, 18, 31, 33, 35, 51, 52 and 56 were all found to be important causes of cervical dysplasia. HPV 16, 18 and 35 were more common in ICC than in women with normal cytology, while HPV 16, 18 and 45 were more common in ICC than preinvasive disease. Infection with HPV and with multiple HPV types was more common among HIVpositive women in all disease groups of the study. Among HIV-positive women HPV 18, 35, 45 and 56 seem to be more important in CIN II/III, while HPV 18, 33, 45 and 58 may be more important causes of ICC. Only HPV 45 was statistically significantly more common among HIV-positive women. Conclusion: The studied population of South African women differs significantly from published data. We also described potential differences in the oncogenic importance of specific HPV types among immune depleted women never discussed before. It is recommended that efforts for both vaccination and screening should be focused only on HPV alpha-9 and alpha-7 groups and firstly only on HPV 16, 18, 45 and 35.
Evaluating the feasibility of low cost sperm preparation methods within a prospective intrauterine insemination program in Gabon
(University of Pretoria, 2015) Huyser, Carin; u12232310@tuks.co.za; Moungala, Lionel Wildy
Introduction: Intrauterine insemination (IUI) is frequently used as the first-line treatment for infertility, due to the relative simplicity and cost-effectiveness of the procedure. Even though, several sperm processing techniques are used for IUI, semen samples need to be washed using a single and appropriate method prior to insemination. Implementing low-cost sperm preparation methods for IUI would benefit those seeking help in developing countries such as Gabon, where treatment for infertility is currently virtually non-existent. The first section of this research was aimed towards the evaluation of a sperm swim-up method (SW-10) while in the second section the feasibility of establishing an IUI programme in Gabon was explored. Methods: The initial section involved three sets of experiments. Semen samples were obtained from patients and donors participating in the Reproductive Biology Laboratory?s research donor registry programme. For experiment (i), semen samples (n=25) were divided into 3 equal aliquots and processed using three syringes with volumes of 5 ml (SW-5),10 ml (SW-10) and 20 ml (SW-20), respectively. During experiment (ii), semen samples (n=20) were split into 2 equal volumes and processed using either the SEP-D kit or the SW-10 method. In experiment (iii) (n=20) two sperm preparation methods i.e. the single layer centrifugation (SLC) and the density gradient centrifugation (DGC) were compared. For post-processing analyses, concentration, motility and total motile sperm count (experiments (i), (ii) and (iii)), as well as morphology, viability and DNA integrity (experiment (ii)) were evaluated. Information on the feasibility of establishing an IUI programme in Gabon in the second section was obtained from a questionnaire completed by gynaecologists practising in Gabon. Results: In the first section, experiment (i) indicated a significant increase in motility and concentrations in spermatozoa processed using the SW-10 method when compared to SW-5 and SW-20 (p<0.05). Experiment (ii) revealed that the SW-10 method yielded spermatozoa with significant superior motility and concentration when compared to that obtained using the SEP-D kit (p<0.05). The SW-10 yielded a statistically significant larger number of spermatozoa with intact plasma membranes, DNA content and normal morphology (p<0.05). In experiment (iii), semen processed using SLC resulted in spermatozoa with statistically significant higher concentrations. However spermatozoa obtained from the DGC had superior motility. The second section comprised a gynaecological survey conducted in Gabon. Seventeen (85%) of the 20 registered gynaecologists participated in the survey. Gynaecologists were particularly interested in a basic infertility treatment and training programme, as well as in the establishment of an ART unit in Libreville. All participants were in agreement that ART services would improve both diagnostic and therapeutic patient services in Gabon. Discussion and conclusion: This study indicated that spermatozoa recovered from the simplified sperm swim-up method had statistically significantly higher sperm parameters when compared to those from the SEP-D kit. Processing semen using SLC resulted in significantly higher sperm concentration when compared to that of the DGC, which yielded a higher percentage of progressively motile spermatozoa. The need to establish an IUI programme in Gabon was proved in the second section of this study. In conclusion, the simplified swim-up method can possibly be an effective low-cost alternative in the preparation of semen samples for IUI procedures. The implementation of low-cost sperm preparation methods can be important in developing countries such as Gabon.
Investigating alternative sperm preservation methods for assisted reproductive technologies
(University of Pretoria, 2013) Huyser, Carin; Du Plessis, S.S.; marisa.slabbert@gmail.com; Slabbert, Marisa
Introduction: Cryopreservation of human sperm is considered a routine practice in assisted reproduction laboratories. Semen samples are mainly cryopreserved as a back-up for procedures, donor sperm, and validation of samples from human immunodeficiency virus-positive patients. Human immunodeficiency virus semen samples generally result in a low yield of purified spermatozoa after decontamination. These samples need to be cryopreserved for later use. Unlike conventional cryopreservation, vitrification does not use harmful cryoprotectants, thereby potentially reducing sperm damage. Vitrification is not yet common practice for sperm cryopreservation in assisted reproduction. The aim of this study was to establish the feasibility of utilising vitrification as an alternative to current conventional cryopreservation of spermatozoa. Methods: Semen samples were collected from human immunodeficiency virus-negative patients seeking diagnostic assistance from the unit. All samples were processed according to the unit’s standard protocol. For Study 1A (n=10) washed samples were divided and cryopreserved using three different cryopreservation media, and two different freezing protocols. In Study 1B (n=15), washed samples were divided and preserved using cryoprotectant-free vitrification in 100 μl, 300 μl and 500 μl volumes. For Study 2 (n=35) washed samples were split and cryopreserved using cryoprotectant-free vitrification (utilizing the volume that resulted in the highest quality spermatozoa in Study 1B) and conventional slow freezing (using the medium and protocol that resulted in superior quality spermatozoa in Study 1A). Post thawing, motility and kinetic parameters (Studies 1 and 2), viability (Study 1), mitochondrial membrane potential (Study 2), and DNA fragmentation (Study 2) of the two groups were compared. vi Results: Study 1A indicated that cryopreserving spermatozoa using Freezing Medium resulted in the highest quality spermatozoa with regards to motility and viability (p<0.05). Comparing the two preservation protocols, no conclusion could be reached on which protocol yielded superior results (p>0.05). The RBL freezing method is shorter, simpler and requires less equipment, and was therefore deemed the preferred method. Study 1B showed that the larger vitrification volumes (300 μl and 500 μl) yielded better spermatozoa in terms of motility and viability (p<0.05). No significant difference was observed with respect to the 300 μl and 500 μl vitrification volume groups. For practical reasons, 300 μl volumes will provide sufficient sperm for any procedure and, the intermediate volume ensures that more than one straw can be preserved. Study 2 found that cryoprotectant-free vitrification resulted in spermatozoa with significantly higher mitochondrial membrane potential and significantly lower apoptosis post thawing (p<0.05). Discussion: Conventional cryopreservation methods may compromise various sperm parameters and final yield. In this study, cryopreservation and cryoprotectant-free vitrification had equivalent outcomes with respect to sperm motility. However, the latter method yielded superior results in terms of ΔΨ and DNA sperm fragmentation. In conclusion, vitrification is an easy, rapid and more affordable technique that requires no special equipment. Using vitrification for purified sperm samples of patients could potentially result in a better post thaw quality for ART procedures.
Assisted reproduction services : accessible screening and semen profiling of HIV-positive males
(University of Pretoria, 2013) Huyser, Carin; Richter, Karin Louise; ; Stander, Melissa
Introduction International guidelines endorse the screening of patients for human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV) and Chlamydia trachomatis before assisted reproductive techniques (ART). At present no such guidelines exists in South Africa. At the Reproductive and Endocrine Unit (referred to as “the Unit”) of Steve Biko Academic Hospital, all patients with unknown HIV status are counselled and a blood sample is collected during the initial visit for automated laboratory based HIV screening. These HIV results are not available before semen samples are processed. Furthermore, patients are not screened for HBV, HCV and Chlamydia trachomatis. Couples attending the Unit are of a low to middle socio-economic status and experience financial constraints. Moreover, automated laboratory based assays are expensive to perform. Rapid testing is a cost effective and practical method from screening patients, with a 20–30 minute result turnover time. Until screening at the Unit is improved, the possible identification of semen characteristics that could indicate HIV infection would be a useful tool. Materials and Methods The following rapid point-of-care assays were evaluated: Determine® HIV-1/2 combo test (n=100), Determine® HBsAg test (n=100), DIAQUICK HCV kit (n=74), and the DIAQUICK Chlamydia trachomatis kit (n=30). For profiling, parameters from a basic semen analysis of HIV-positive males (n=60) were compared with HIV-negative males (n=60). Information pertaining to CD4 count, antiretroviral treatment and plasma viral load of HIV-positive males were analysed. Results From all patients included in the study, 8% tested positive for HIV. The risk of a female being HIV-positive was 3.73 times higher than for males. In the pilot study to explore rapid testing for HBV and HCV, 1% and 1.4% of patients tested positive respectively. When testing for Chlamydia trachomatis 31.3% of females, but no males tested positive. Comparing semen profiles, no significant differences were found between samples from HIV positive and negative males or between HIV positive males categorised by CD4 cell count (p>0.05). For the HIV-positive group with a detectable plasma HIV viral load (>40 copies/ml), a significant difference was observed in the semen viscosity (p=0.0460). Significant differences were noted in the sperm motility (immotile sperm p=0.0456, progressive sperm p=0.0192) of patients receiving antiretroviral (ARV) therapy. Discussion and Conclusion The use of rapid testing is an acceptable and feasible option for improving current screening protocols at the Unit. The absence of definite alterations in the semen characteristics of HIV-positive men further motivates the need for a simpler, point-of-care screening protocol. The prevalence of HBV was lower than that reported in the general population of South Africa and further investigation is needed. Although the sample size was small, HCV prevalence was similar to that of the general population. One third of females tested positive for Chlamydia trachomatis. The methodology used was possibly not appropriate for males. This study highlighted the need for guidelines that address the specialised needs of ART clinics in resource-limited and developing countries with a high HIV prevalence.
Semen decontamination for the elimination of seminal pathogens
(University of Pretoria, 2013) Huyser, Carin; Loskutoff, Naida; jozef.fourie@up.ac.za; Fourie, Jozef Markus
The presence of pathogens in semen can compromise the outcome of assisted reproductive treatment, together with the possibility of the female partner or offspring becoming infected. This is cause for concern, especially in South Africa with a high prevalence of HIV-1. Most of these infected individuals are in their reproductive years with the desire to have their own genetically related children. Therefore, assisted reproductive treatment with effective risk reduction procedures, such as semen processing for the elimination of these pathogens is crucial. However, during sperm preparation by standard discontinuous density gradient centrifugation, the supernatant is aspirated to allow access to the purified sperm pellet. Pathogens from the upper layers can adhere to the inside surface of the test tube and flow down to re-infect the purified sperm sample. The use of a centrifuge tube insert may prevent the re-contamination of sperm samples after discontinuous density gradient centrifugation. Furthermore, seminal pathogens can bind specifically or non-specifically to spermatozoa, rendering semen decontamination procedures ineffective. Serine proteases, such as trypsin, have been demonstrated to effectively inactivate viruses and to break pathogen-sperm bonds. However, the addition of a protease to density gradient layers during semen processing could have a negative impact on sperm parameters. This research was therefore aimed towards the determination of: i) The effect of semen processing with trypsin and trypsin inhibitor on sperm parameters. ii) The prevalence of various bacteria in semen samples from men attending the Reproductive and Endocrine Unit at Steve Biko Academic Hospital. iii) The effectiveness of semen processing by discontinuous density gradient centrifugation with a centrifuge tube insert, for the elimination of some of the most prevalent bacteria, white blood cells and in vivo derived HIV-1. Evaluation of sperm parameters after semen processing indicated that trypsin and trypsin inhibitor did not have an impact on sperm mitochondrial membrane potential, vitality, motility and zona binding potential, or acrosin activity, respectively. Seminal bacteria were highly prevalent in patients wishing to participate in the Unit’s assisted reproductive program, with 49.5% of semen samples presenting with positive bacterial cultures. Semen processing by means of discontinuous density gradient centrifugation with the tube insert, eliminated significantly more in vitro derived (spiked) bacteria and white blood cells from semen compared to processing without the insert. Furthermore, the semen decontamination procedure was effective in removing HIV-1 RNA from 100% of samples and proviral DNA from 98.1% of semen samples from HIV-1 sero-positive patients. The effectiveness of discontinuous density gradient centrifugation for the elimination of seminal pathogens could, therefore, be improved by the addition of trypsin to the upper density layer, without supplementing the bottom layer with trypsin inhibitor. Additionally, semen decontamination efficiency could also be improved by the prevention of re-contamination of processed sperm samples by the utilization of a tube insert during discontinuous density gradient centrifugation.
An investigation cervical cancer, human papillomavirus (HPV) infection and steroid contraception
(University of Pretoria, 2011-10-25) Lindeque, B. Gerhard; moodleym29@ukzn.ac.za; Moodley, Manivasan
PROJECT ONE Introduction HPV is detected in about 99.7% of cervical cancers. However, the HPV type distribution in South African women is unknown. Objectives To determine HPV-type distribution among women with cervical dysplasia in relation to oral contraceptive usage. Methods Prospective cross-sectional study of four groups of patients according to oral contraceptive usage: non-users, users of less than five years duration, users of between five years and ten years and users of more than ten years duration. Swabs of the cervix were analysed for HPV DNA using polymerase chain reaction method. Results A total of 124 women were recruited for the study. There were 75 HIV-infected patients (seroprevalence 61%). Of the 102(82%) HPV-positive patients, 79 patients had high-risk HPV DNA (78%). In terms of the four oral contraceptive groups, high-risk HPV DNA was detected in 70% (n=21), 79% (n=22), 90% (n=21) and 71% (n=15) of patients, respectively. The odds of having HPV DNA was six times higher for the combination of contraceptive users of less than 5 years duration/non-users (OR 5.9, 95% CI: 1.87 - 18.77). There was no change when adjustment was made for age (OR 6.1, 95% CI: 1.9 - 19.4). HPV DNA types 16 and or 18 was present in a total of 21 patients (49%) (non-contraceptive users and users < 5years duration) versus 15 patients (42%) who used oral contraceptives of more than 5 years duration (p=0.524). HPV type 16 was the commonest HPV type detected (20.2%) and HPV type 58 was the next commonest high-risk HPV type (16.1%). HPV types 58 and 33 was detected in a much greater percentage of our population and HPV 16 in a much smaller percentage of our population compared with a non-South African population. Conclusion The findings of this study demonstrate an interesting distribution of HPV types in a South African population. PROJECT TWO Introduction Various risk factors have been implicated in the causation of cervical cancer including human papillomavirus (HPV), the early genes (E6 and E7) of which encode the main transforming proteins. Studies have suggested that steroid hormones may enhance the expression of these genes leading to loss of p53 gene-mediated cell apoptosis. Methods A total of 120 cervical tissue samples were obtained from patients with proven cervical cancer. Patients who used depo-medroxyprogesterone acetate steroid contraception were recruited as part of the study arm. Only HPV DNA type 16 samples were used for the study. Controls included three cell lines (CaSki, SiHa,&C33A) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal housekeeping gene. Of 120 patients, there were 111 patients with HPV type 16 identified. Of this number, RNA was present in 63 samples. There were 30 women (30/63) who used steroid contraception. In relation to patients who used contraception, HPV 16 E6 gene expression was present in 79% (n = 23) and 88% (n = 30) of steroid users compared to nonusers, respectively. In total there were 25 patients (40%) with expression of the HPV 16 E6*I gene and 30 patients with expression of the E6*II gene. There were 57% of steroid users (n = 17) who had expression of the E6*I/E6*II gene, compared to 52% (n = 17) of nonusers (P = 0.800). Conclusion From a molecular level, this study reflects almost similar distribution of the HPV 16 E6/E6*1 and E6*11 and does not confirm the role of injectable progesterones in cervical carcinogenesis. Further studies with larger patient numbers are needed.
The role of the tumour suppressor gene PTEN in the etiology of cancers of the female genital tract
(University of Pretoria, 2011-10-24) Lindeque, B. Gerhard; Jansen van Rensburg, Elizabeth; gretadreyer@mweb.co.za; Dreyer, Greta
The phosphorylation and dephosphorylation of the tyrosine amino-acids in proteins play an important role in the regulation of many cellular processes in all eukariotic organisms, including the regulation of cell cycle control, growth control, cellular differentiation and gene and synaptic transmission. The involvement of the phosphatase genes in human carcinogenesis was long-suspected, but PTEN is the first important phosphatase gene proven to be a true tumour suppressor. The basic function of normal PTEN is the dephosphorylation of the kinases and inhibition of the integrin and growth factor mediated kinase signalling pathways. The central hypothesis of this study is that PTEN plays an important role in tumours of the upper female genital tract. The involvement of aberrations in the coding regions of this gene was studied in specific gynaecologic tumours and tissues using polymerase chain reaction based mutation analysis. The research model was to study both the malignant tumour and the closest available pre-malignant or benign counterpart to demonstrate different levels of involvement of PTEN in the evolving steps. The PTEN gene was found to be intimately involved in endometrial carcinogenesis. Involvement was demonstrated in hyperplasia and was common in endometroid carcinoma (54%). Pathogenic PTEN mutations were much more common in cancer than in hyperplasia (10%). Multiple mutations were found in some late stage tumours, suggesting that the already malignant tumour cells accumulate more genetic mutations over time. All tumours with more than one pathogenic mutation occurred in African patients. The latter twofindings are unique to the current study. Selective involvement of the PTEN gene was demonstrated in uterine soft tissue tumours. PTEN involvement was neither found in benign soft tissue tumours nor significantly in leiomyosarcoma or endometrial stromal sarcoma. However, PTEN plays a significant role in uterine carcinosarcoma (13%) and specifically in tumours with an endometroid epithelial component, where mutations were found in 17%. This finding is a highly significant and unique research result which supports the hypothesis of the endometrial origin of these tumours. It also supports the observation of a strong link between this gene and endometroid differentiation, with morphology strongly linked to cellular genetics. PTEN gene mutation was demonstrated in ovarian endometroid carcinoma in ~29% of cases investigated. This finding confirms PTEN involvement in carcinogenesis in this tumour type. The finding suggests that PTEN involvement is linked to endometroid epithelial morphology. We could not sufficiently test the involvement of the gene in benign or pre-malignant ovarian endometroid lesions and thus cannot comment on the chronology of mutations in this tissue type. When all tumour types were included, there was a tendency towards a lower frequency ofPTEN mutations in African women. PTEN mutations correlated with endometroid histology. In combination, these results confirm the racial disparity in tumour type distribution or morphology. In summary this study demonstrated significant though highly selective PTEN gene involvement and a strong and interesting association between genotype and histological phenotype was confirmed. The findings enhance our understanding of carcinogenesis and should lead to translational research into new anti-neoplastic drugs. AFRIKAANS: Fosforilering en defosforilering van die tirosien aminosure in proteine speel ‘n belangrike rol in die regulering van sellulêre prosesse in alle eukariotiese organismes. Dit sluit die regulering van selsikluskontrole, groeikontrole, sellulêre differensiasie sowel as genetiese en sinaptiese oordrag in. Dit word lank reeds gespekuleer dat die fosfatase-gene betrokke is in menslike karsinogenese, maar die PTEN geen is die eerste fosfatase geen wat bewys word om ‘n ware tumoronderdrukker geen te wees. As basiese funksie defosforileer normale PTEN die kinases en inhibeer dit die kinase sinjaal kontrolepaaie wat deur integrien en groeifaktor beheer word. Die sentrale hipotese van hierdie studie is dat PTEN ‘n belangrike rol speel in tumore van die boonste genitale traktus. Die frekwensie van abnormaliteite in die koderingsareas van hierdie geen is bestudeer in spesifieke ginekologiese tumore en weefsels met die gebruik van polimerase kettingreaksie gebaseerde mutasie-analise. Die maligne tumore sowel as die mees verwante pre-maligne of benigne weefsel- of tumortipes wat beskikbaar was, is gebruik as navorsingsmateriaal om sodoende die verskillende vlakke van PTEN betrokkenheid in die ontwikkeling van neoplasie te demonstreer.Intieme betrokkenheid van die PTEN geen is gevind in endometriële karsinogenese. PTEN mutasies is in hiperplasie gevind en dit was algemeen in endometroiede karsinoom (54%). Patogene mutasies was baie meer algemeen in kanker as in hiperplasie (10%). Veelvuldige mutasies is in sommige laat stadium tumore aangetoon, wat suggereer dat reeds maligne selle meer genetiese mutasies oor tyd verkry. Alle tumore waar meer as een patogeniese mutasie gevind is het voorgekom by swart pasiënte. Die laaste twee bevindinge is uniek tot hierdie studie.Selektiewe betrokkenheid van die PTEN geen is gevind in die ontwikkeling van sagte weefsel tumore van die uterus. PTEN mutasies is nie in benigne sagte weefsel tumore gevind nie en geen betekenisvolle betrokkenheid is in leiomiosarkome of endometriële stromale sarkome aangetoon nie. PTEN was egter betekenisvol betrokke in karsinosarkome van die uterus (13%) en veral in tumore met ‘n endometrioiede epiteelkomponent waar mutasies in 17% gevind is. Hierdie bevinding is ‘n hoogs betekenisvolle en unieke navorsingsbevinding wat die hipotese ondersteun dat hierdie tumore uit die endometrium ontstaan. Dit onderskryf ook die indruk dat ‘n sterk band bestaan tussen hierdie geen en endometroiede differensiasie, met morfologie sterk gekoppel aan sellulêre genetika.Mutasie in die PTEN geen is aangetoon in ovariële endometroiede karsinoom in ~29% van gevalle wat ondersoek is. Die bevinding bevestig PTEN betrokkenheid in karsinogenese in hierdie tumortipe. Weereens toon die resultaat dat PTEN betrokkenheid gekoppel is aan endometroiede morfologie. Die ondersoek van benign of pre-maligne letsels in hierdie orgaan was nie voldoende om kommentaar oor die tydsberekening van mutasie te kan lewer nie.Met alle tumortipes in ag genome, is daar ‘n tendens aangetoon van minder PTEN mutasies in swart vroue. PTEN mutasies korreleer met endometroiede histologie. In kombinasie bevestig hierdie resultaat ‘n rasse-diskrepansie in die distribusie van tumourtipe of morfologie. In opsomming is die bevinding van hierdie studie dat daar betekenisvolle dog hoogs selektiewe PTEN geen betrokkenheid in boonste genitale traktus tumore is. ‘n Sterk en interessante verband is bevestig tussen genotipe en histologiese fenotipe. Hierdie resultate verbeter die begrip van karsinogenese en behoort ‘n bydrae te lewer in die soeke na nuwe anti-neoplastiese middels.
The effect of non thermal 900 MHZ mobile phone radiation on human spermatozoa
(University of Pretoria, 2008-06-02) Huyser, Carin; Franken, Daniel R.; Leszczynski, Dariusz; upetd@up.ac.za; Falzone, Nadia
Several studies have highlighted the possibility that radio-frequency electromagnetic fields (RF-EMF) used in mobile phone technology could influence DNA integrity of male germ cells as well as sperm motility. Current knowledge concerning the influence of RF-EMF on male germ cells is extremely limited. In the present study the hypothesis that 900 MHz GSM radiation could induce the activation of stress response in human spermatozoa was investigated. Ejaculated, density purified, human spermatozoa from donors were exposed to 900 MHz GSM mobile phone radiation at specific absorption rate (SAR) levels of 2.0 and 5.7 W/kg and examined at various time points post exposure. Sperm motility and morphology were evaluated by computer-aided sperm analysis (CASA). The ability of RF-EMF exposed sperm to undergo the acrosome reaction was evaluated by flow cytometry. Sperm binding to the zona pellucida of human oocytes was determined by the hemi-zona (HZA) assay. Apoptotic markers, phosphatidylserine (PS) externalization, change in mitochondrial membrane potential (Δψm), reactive oxygen species (ROS) generation, caspase activation and DNA fragmentation were analysed using flow cytometry. Heat shock protein (Hsp) 27 and 70 expression and activity were analyzed using specific antibodies with flow cytometry and Western blot methods. Stress fibre stabilization (F-actin polymerization) was visualized using fluorescent dye labelled phalloidin. No effect was seen on kinematic parameters assessed at SAR 2.0 W/kg, however straight line velocity (VSL) and beat cross frequency (BCF) were significantly altered after exposure at SAR 5.7 W/kg. Sperm shrinkage (decrease in surface area) was observed at both exposure levels. RF-EMF did not influence exposed spermatozoa’s ability to undergo the acrosome reaction. A significant decrease in sperm-zona binding was observed at both exposure levels. RF radiation did not have an effect on any apoptotic markers. ROS generation increased significantly with an increase in SAR (5.7 W/kg). RF-EMF did not induce a stress response in exposed sperm (no activation of Hsp70 and 27 activity). These results cannot be ascribed to heating, as the temperature did not increase by more than 0.2 - 0.3ºC during exposure. The decrease in sperm-zona binding is the result of an alternative non-stress inducible pathway. This study should be replicated at lower SAR levels that would simulate the radiation absorption from carrying the cell phone in a pocket close to the testes.

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