Michael Coughtrie

The precise coordination of inflammatory signaling, oxidative stress responses, steroidogenesis, and steroid metabolism are required in all aspects of pregnancy, from fertilization and implantation to parturition. Dysfunction in any of these processes can lead to unfavourable pregnancy outcomes and complications requiring obstetric interventions. As the use of assisted reproductive technologies (ART) has increased in recent decades, it has become evident that these pregnancies are associated with increased risks compared to unassisted pregnancies. However, the underlying reason for these increased risks is unknown. Preliminary studies from our laboratory using a mouse model of ART provide evidence that inflammatory, antioxidant, or steroid pathways could be dysregulated in ART. In this thesis, we investigated the levels inflammatory mediators, measures of antioxidant defense, and steroid hormones in pregnancy to determine any associations with pregnancy outcome. Beginning with an investigation of non- steroidal anti-inflammatory drug (NSAID) use in pregnancy (Chapter 2), we determined the prevalence of NSAID use close to labour. Next, to investigate the differences between ART and unassisted pregnancies, we used a cohort of placentas from ART pregnancies and unassisted pregnancies matched for gestational age, maternal age, ethnicity, and singleton/twin pregnancy. In this cohort, we evaluated the levels of cytokines and vascular endothelial growth factors and identified associations with pregnancy outcome using the accompanying clinical chart profiles (Chapter 3). Further, the activity of antioxidant enzymes and levels of antioxidants were measured in these placentas (Chapter 4). In Chapter 5, we measured the levels of steroid hormones and steroid sulfates in maternal, placental, and fetal compartments, again comparing these results between ART and unassisted pregnancy and identifying associations with pregnancy outcome. No widespread dysregulation of these processes was found between ART and unassisted pregnancies. However, when stratified for pregnancy outcome, two significant associations were found. In pregnancies complicated by chorioamnionitis and in twin pregnancy, there was dysregulation across all three signalling processes. These signalling pathways are complex and interconnected, but the data generated here adds novel findings to the ART field, and identifies potential pathways for future investigation to resolve the etiology of increased pregnancy complications in ART.

View record

PURPOSE Uridine diphosphate glucuronosyltransferases (UGTs) are Phase II conjugation enzymes that are critical for vertebrate detoxication. They catalyze the glucuronidation biotransformations of numerous endo- and xenobiotics to facilitate their clearance and elimination and are important for hormonal regulation. Glucuronidation-related disorders include congenital syndromes, excessive chemical toxicities, adverse drug reactions, and dysregulated hormonal status. Since the 1980s it has been known that UGT proteins can be expressed but not fully active, yet little is known about how the UGT protein structure regulates its activity and function, because of the lack of a complete structure. We hypothesized that translational and post-translational mechanisms may play a role in regulating the activity and function of human UGTs. Aim 1 and 2 focused on UGT isoform 1A6, one of the most important isoforms in endo- and xenobiotic metabolism. Aim 3 studied the co-expression of major human hepatic UGT isoforms to gain an insight on their co-regulation in vivo.METHODSN-glycosylation variants of human UGT1A6 were established to study the influence of N-glycosylation on their expression, activity, cellular function and localization. HEK293 expressed UGT1A6-(His)6 and Sf9-insect expressed soluble UGT1A6 were purified and their activity and latency were characterized. The co-expression between 7 UGTs was investigated based on a western blot database collected from healthy liver donors, aged fetal (20 weeks) – 87 years.GENERAL CONCLUSIONSN-glycosylation is an important regulator of human UGT1A6 in expression, activity, and cellular function. Different forms of N-glycosylation were observed for UGT1A6 between HEK293 cells and human liver microsomes, showing a caveat of using recombinant cell lines to study UGT structure/function. Sf9-insect expressed soluble UGT1A6 is a good candidate for structure analysis due to sufficient yield and activity after purification. The influence of membranes on UGTs may be different from traditional beliefs as the data suggested alamethicin and Brij 58 can directly activate the enzyme. Lastly, expression of major human hepatic UGTs was significantly correlated and correlations were affected by age. The UGTs may be co-regulated in the human liver so single-isoform systems may have limitations on reflecting UGT activity in vivo.

View record