During gonad development and fetal life, the germ cells (GC) undergo a range of different developmental processes necessary for correct postnatal gametogenesis and the production of the next generation. If these fetal events are disrupted by genetic or environmental factors, there could be severe consequences that may not present until adulthood. This is of particular importance in relation to human testicular GC tumours (TGCT), the most common cancer of young men, as TGCT is thought to arise from fetal GCs that have failed to differentiate normally during development and thus persist into adulthood, eventually becoming tumourigenic. TGCT is one of several related disorders of male reproductive health thought to comprise a Testicular Dysgenesis Syndrome (TDS), in which faulty testis development in fetal life may predispose to the development of cryptorchidism, hypospadias, reduced sperm count and TGCT. Currently there is no accepted animal model for TGCT, but some insight into human TDS has been gained through the use of a rat model using in utero Di (n-Butyl) Phthalate (DBP) exposure to induce cryptorchidism, hypospadias, low sperm count and reduced fertility (but not TGCT). However, a previous study suggested that DBP exposure can disrupt GC differentiation, resulting in significantly reduced GC number prior to birth and postnatal consequences. This thesis has been directed at investigating the normal process of GC development in the fetal rat and how this is altered by DBP exposure; such understanding may give insights into the origins of human TGCT by showing how and when disruption of normal fetal GC differentiation can occur. The first objective was to characterise GC development in both the rat testis and ovary to understand the normal events that occur between embryonic day (e)13.5 and e21.5, as most data in the literature is based on the mouse. Analysis by immunohistochemical, stereological and mRNA expression indentified that during this time period, a GC will undergo a dynamic sequence of changes involving migration, proliferation followed by differentiation (manifested by loss of specific protein markers), whilst undergoing germ-line specific remethylation. Whilst whole gonad development is vastly different between testes and ovaries, GC development was broadly the same with only minor differences up to the point where GCs in the ovary enter meiosis. Having established the normal process of GC development in the fetal rat testis, the effects of in utero DBP exposure was then investigated. DBP exposure reduced GC number at all ages investigated even after only 24 hours of exposure and simultaneously prolonged GC proliferation. As apoptosis was unaltered by DBP exposure, the consistent reduction in GC number was suggested to be due to an initial reduction in GC number that does not recover to control levels. GC differentiation was assessed by the expression and localization of specific protein markers (OCT4, DMRT1 and DAZL). The pattern of expression of OCT4 and DMRT1 was altered by DBP exposure. GCs in DBP exposed animals also showed a delay in disaggregation from within the centre of seminiferous cords. These results suggested that a delay in GC differentiation was occurring with DBP exposure. This delay in GC development persisted into early postnatal life, following cessation of DBP exposure. Thus at postnatal day (D)6, GC specific re-expression of DMRT1, GC migration to the basal lamina and resumption of GC proliferation all showed a delay. DBP also induced an increase in the presence of multinucleated gonocytes. DNA methylation in the fetal rat testis was also investigated as a mechanism that could be disrupted by DBP exposure. DNA methylation of GCs increased during the last week of fetal life by global methylation of the GC genome and the increased expression of DNA methyl transferases. No effect of DBP exposure was detected. Inhibition of methylation by 5-aza-2’deoxycytidine was then investigated as a way to block GC differentiation in fetal rat testes and this resulted in a similar transient delay in GC differentiation but was perinatally lethal to the fetus. Bisulphite sequencing of the OCT4 promoter was also performed but proved inconclusive. Methylation patterns may be being altered by DBP exposure, but such changes could not be identified in this thesis. To complement the in vivo DBP exposure studies, an in vitro testis explant system using e14.5 testes was investigated. These in vitro testis explants showed some GC effects with MBP, the active metabolite of DBP, and also suggested a novel role for Hedgehog signalling in GC survival in the fetal rat testis. The studies in this thesis have characterised several aspects of fetal GC development in the rat and identified which of these are affected by DBP exposure, resulting in a delay in GC development. As DBP exposure delays but does not block GC differentiation, this may explain why TGCT is not induced in the DBP exposure rat model for TDS.

Bisphenol A (BPA) and phthalates are chemicals found in many consumer products including water bottles, food packaging and cosmetics. Previous research has shown that there is potential for these compounds to contribute to obesity. In this analysis, the Canadian Health Measures Survey was used to investigate possible associations between urinary concentrations of these compounds and measures of adiposity. BPA urine concentrations were found to decrease with age, and significant associations with BMI and waist circumference were found in linear regression in adults. No associations with measures of adiposity were found in logistic regression for adults and significant negative associations were found in children. A similar discrepancy was found for mono-(2-ethyl-5-hydroxyhexyl) phthalate and mono-(2-ethyl-5-oxohexyl) phthalate, which were significantly associated with obesity in adults, but showed several significant negative associations in children. Overall, this analysis showed that it is unlikely that BPA and phthalates are contributing to adiposity in the Canadian population.

INTRODUCTION Phthalates, a chemical class of plasticizers, are ubiquitous in the environment and recognized as endocrine disrupting compounds (EDCs). Recent data suggest that oxidative stress is a potential mediator of poor male reproductive health associated with phthalate exposure. Mitochondria are implicated in the production of excess oxidative stress and sperm mitochondrial copy number (MtCopy) and deletions (MtDeletion) have been linked with male infertility. However, little is known about the relationship of these mitochondrial biomarkers in sperm with phthalate exposure and oxidative stress. OBJECTIVES To examine associations of urinary phthalate metabolites and isoprostane concentrations on sperm MtCopy and MtDeletions in male partners undergoing assisted reproductive technologies (ART). METHODS A total of (n=97) sperm samples were collected from male partners undergoing ART at Baystate Medical Center, in Springfield, MA from 2014 to 2016 as part of the Sperm Environmental Epigenetics and Development Study (SEEDS). Seventeen urinary phthalate metabolites (n=103) were analyzed by the Centers for Disease Control using tandem mass spectrometry. 15-F2t-Isoprostane (n=101) was measured using a competitive enzyme-linked immonsorbent assay in urine of male individuals. A triplex Taqman probe-based qPCR method was developed for relative quantification of genomic DNA, MtCopy and MtDeletions. Multivariable linear or logistic regression was employed to examine associations with age, BMI, batch and current smoking status with each outcome to determine confounders used for adjustment. RESULTS Quartiles of MtCopy and MtDeletion were positively associated with the odds of male infertility (p for trend < .0001 and 0.007, respectively). Urinary metabolite concentrations of MCNP displayed a positive association with MtCopy (β=1.56; p =0.03). Urinary MEHP concentrations were positively associated with MtDeletion in only infertile individuals (n=30) (β = 0.075; p = 0.006). Urinary isoprostane concentration was not associated with MtCopy or MtDeletion, but was associated with seven phthalate metabolite concentrations (MEOHP, MEHHP, MBzP, MHBP, MiBP, and MHiBP). CONCLUSIONS To our knowledge, this is the first study to investigate the relationship between sperm MtCopy and MtDeletion with oxidative stress and phthalates. These results suggest that certain phthalate metabolites may be associated with a known biomarker of systemic oxidative stress. Sperm mitochondrial function as measured by MtCopy and MtDeletion may be considered biomarkers of male infertility, although no relationship was shown between mitochondrial outcomes and oxidative stress. Future research is investigating these relationships with developmental outcomes including embryo quality.

<p>This work reports the chemical synthesis, characterisation and electrochemical application of titanium dioxide (TiO2) and iron oxide (Fe2O3) nanoparticles in the determination of phthalates. The other part of this work involved electrochemical polymerization of aniline doped with titanium and iron oxide nanoparticles for the sensor platform in the electrolytic determination of phthalates. The TiO2 and Fe2O3 nanoparticles were prepared by sol gel and hydrothermal methods respectively. Particle sizes of 20 nm (TiO2) and 50 nm (Fe2O3) were estimated from transmission electron microscopy (TEM) The other technical methods used in this study for the characterization of the TiO2 and iron oxide Fe2O3 NPs were SEM, XRD and UV- visible spectroscopy. Cyclic voltammetry, square wave voltammetry and electrochemical impedance spectroscopy (EIS) were used to study the electrochemical properties of the nanoparticles. These electrochemical studies of the nanoparticles were performed with a Fe2O3 or TiO2/nafion/glassy carbon membrane electrode in 0.1 M phosphate buffer (pH 7.0) and 0.1 M lithium perchlorate (pH 6.8) under an aerobic condition.</p>

Ph.d.

<p>This work reports the chemical synthesis, characterisation and electrochemical application of titanium dioxide (TiO2) and iron oxide (Fe2O3) nanoparticles in the determination of phthalates. The other part of this work involved electrochemical polymerization of aniline doped with titanium and iron oxide nanoparticles for the sensor platform in the electrolytic determination of phthalates. The TiO2 and Fe2O3 nanoparticles were prepared by sol gel and hydrothermal methods respectively. Particle sizes of 20 nm (TiO2) and 50 nm (Fe2O3) were estimated from transmission electron microscopy (TEM) The other technical methods used in this study for the characterization of the TiO2 and iron oxide Fe2O3 NPs were SEM, XRD and UV- visible spectroscopy. Cyclic voltammetry, square wave voltammetry and electrochemical impedance spectroscopy (EIS) were used to study the electrochemical properties of the nanoparticles. These electrochemical studies of the nanoparticles were performed with a Fe2O3 or TiO2/nafion/glassy carbon membrane electrode in 0.1 M phosphate buffer (pH 7.0) and 0.1 M lithium perchlorate (pH 6.8) under an aerobic condition.</p>

Objective: To determine whether serum phthalate concentrations differ in men and women with infertility compared to those without infertility in Saskatchewan Hypothesis: Serum phthalate concentrations will be greater in men and women with infertility compared to fertile men and women Setting: Patients undergoing assisted reproduction for the treatment of infertility; healthy volunteers recruited from the community Recruitment and sample collection: Infertile couples were recruited prior to in vitro fertilization (IVF) therapy for treatment of unexplained infertility (n=15), polycystic ovarian syndrome (PCOS, n=13), and male factor infertility (n=12); fertile men (n=15) and women (n=15) were recruited using poster advertisements. Blood samples were collected by venipuncture for phthalate analysis. Main outcome measures: Serum phthalates concentrations (ng/mL) Design: Prospective cohort pilot study Methods: In infertile couples, blood samples were collected on the following 3 days of the IVF cycle: early during ovarian stimulation, day of oocyte retrieval and day of embryo transfer. In healthy volunteers, 3 blood samples were collected over a 2 week period. Liquid chromatography and tandem mass spectrometry (LC-MS/MS) was conducted to quantify concentrations of four phthalates: di-n-butyl phthalate (DBP), diethyl phthalate (DEP), di-(2-ethylhexyl) phthalate (DEHP) and diisodecyl phthalate (DIDP). Phthalate concentrations were compared among the four study groups using non-parametric Kruskal-Wallis and Mann-Whitney U post hoc tests. Results: Serum DEHP and DEP concentrations did not differ among control, unexplained, PCOS, and male factor infertility groups in both men and women (p>0.05). DBP in women did not differ among study groups (p=0.205). In contrast, DBP was lesser in men with unexplained, PCOS, and male factor infertility compared to controls (p < 0.05). Similarly, DIDP was lesser in women of couples with unexplained, PCOS and male factor infertility groups compared to fertile women (p < 0.05). Less DIDP was detected in men with unexplained and male factor infertility compared to the control group (p < 0.05) Conclusion: Serum phthalate concentrations in serum were lesser or not different in infertility patients undergoing IVF compared to fertile volunteers. These findings do not support the notion that serum phthalate concentrations are associated with human infertility. Further research is needed to determine whether phthalate concentration in blood cells and adipose tissue differ in infertile versus fertile men and women.

Magister Scientiae - MSc / This work reports the chemical synthesis, characterisation and electrochemical application of titanium dioxide (TiO2) and iron oxide (Fe2O3) nanoparticles in the determination of phthalates. The other part of this work involved electrochemical polymerization of aniline doped with titanium and iron oxide nanoparticles for the sensor platform in the electrolytic determination of phthalates. The TiO2 and Fe2O3 nanoparticles were prepared by sol gel and hydrothermal methods respectively. Particle sizes of 20 nm (TiO2) and 50 nm (Fe2O3) were estimated from transmission electron microscopy (TEM) The other technical methods used in this study for the characterization of the TiO2 and iron oxide Fe2O3 NPs were SEM, XRD and UV- visible spectroscopy. Cyclic voltammetry, square wave voltammetry and electrochemical impedance spectroscopy (EIS) were used to study the electrochemical properties of the nanoparticles. These electrochemical studies of the nanoparticles were performed with a Fe2O3 or TiO2/nafion/glassy carbon membrane electrode in 0.1 M phosphate buffer (pH 7.0) and 0.1 M lithium perchlorate (pH 6.8) under an aerobic condition. / South Africa

Phthalates are endocrine-disrupting chemicals (EDCs) that are known testicular toxicants, used commonly as industrial plasticizers that are found in everyday items. Di-(2-ethylhexyl) phthalate (DEHP) is the most abundant phthalate in the environment, and its primary metabolite mono-(2-ethylhexyl) phthalate (MEHP) is ten-fold more potent. The purpose of this study is to examine the cellular and molecular effects of MEHP in the development of testicular cancer. Proliferation was measured for NT2 cells exposed to 10µM and 100µM MEHP at 24 and 48 hours and for cells under controlled conditions. Methylation-specific PCR (MSP) was used to determine the methylation status of the promoter region of key testicular genes post exposure to MEHP. MEHP caused a dose-dependent negative effect on proliferation and significantly altered methylation levels for key testicular genes following exposure to 10µM MEHP and 100µM, as compared to controls. This suggests that MEHP alters proliferation and methylation of testicular tumour cells in a time- and dose-dependent manner.

Objectives The aims of this thesis were to (1) investigate the association of gestational exposure to environmental phthalates with maternal and perinatal outcomes, and (2) explore phthalate-induced changes to maternal inflammatory responses as potential mediators of possible health effects. Methods A systematic review was performed to summarize existing evidence on the association of gestational exposure to phthalates with obstetrical outcomes, including pre-eclampsia (PE), pregnancy-induced hypertension (PIH), gestational diabetes mellitus (GDM), intrauterine growth restriction (IUGR), birth weight (BW), head circumference (HC), gestational age (GA), preterm birth (PB), and Apgar scores (AS). Additionally, a secondary analysis of data from the MIREC Study was conducted to evaluate the association of phthalate metabolites with clinical outcomes in the mother and infant using multiple linear and logistic regression, and with inflammatory biomarkers using multinomial logistic regression. Results The systematic review identified a total of 24 articles, and observed inconsistent evidence on BW, HC, GA, and PB, a paucity of research on IUGR, PE, GDM, and AS, and a lack of studies on PIH. However, among studies with statistically significant (p<0.05) results, most suggest an association of phthalates with decreased BW and GA, and increased HC and PB. Findings from the MIREC Study indicate a significant (p<0.01) positive association between MBP and HC among female infants; however, null results were identified for BW, GA, PB, AS, and PIH. In relation to the exposure to phthalates, general trends among suggestive associations (p<0.05) for head circumference showed consistent increases in females and decreases in males, and for gestational age displayed decreases in both stratums. Additionally, a significant positive association of MBzP and ∑DEHP was observed with high MMP-2 and low VCAM levels, respectively. Results approaching statistical significance demonstrated a positive association of ∑DEHP with low MCP1 and ICAM levels, MCPP with low GMCSF levels, MBzP with low CRP and high ICAM levels, and MEP with high MMP-7 and IL-2 levels. Conclusion From the systematic review, the effects of phthalates on maternal and perinatal health remain unclear, possibly due to sources of heterogeneity and challenges in exposure assessment. In the MIREC Study cohort, phthalate levels were associated with GA and HC in infants in a sex-specific manner. Phthalates also appear to influence the circulating inflammatory marker levels, possibly explaining the observed adverse effects. Future research is needed to validate these findings.