CARBON MONOXIDE AND PREGNANCY: A SEARCH FOR A POSSIBLE THERAPEUTIC IN THE TREATMENT OF PRE-ECLAMPSIA

Pre-eclampsia (PE) is a pregnancy disorder that affects roughly 5-7% of all pregnancies and is a leading cause of both maternal and fetal/neonatal morbidity and mortality. With no present cure for the disease, researchers are interested in the lower incidence of PE observed among the cigarette smoking pregnant population. However, women who use smokeless tobacco do not experience the same decreased incidence of PE, leading to hypothesis of protection against PE from the largest combustible product of cigarette smoke, carbon monoxide (CO). Studies evaluated levels of CO in PE women and found that they were statistically lower than those of healthy pregnancy. Researchers have found CO to possess many cytoprotective and regulatory properties and specifically within the placenta, it has been found to increase perfusion pressure, decrease oxidative stress, decreases ischemia/reperfusion induced apoptosis and maintain endothelial functioning. The idea for use of CO as a possible therapeutic for PE has thus become a real possibility. This study determined CO levels in pregnant women ± smoking as well as in PE women±smoking, as to discover a possible therapeutic range for future treatments. The best correlated automated CO measurement device with blood CO levels was determined, for use in future clinical studies. This thesis also sought a possible CO delivery concentration, in order to achieve the CO levels observed in the human correlation study. A threshold level of maternal CO exposure in a murine animal model was found, for which fetal and maternal negative toxicities were not observed. The results of this thesis lend a few more pieces to the complicated puzzle involving CO and PE and offer another step toward the possibility of a therapeutic treatment/prevention using this gaseous molecule.

Mouse Uterine Natural Killer Cell Functions During Early Pregnancy

Early pregnancy is characterized by complex interactions between blood vessels, leukocytes, and conceptus-derived trophoblasts within the gestational uterus. Uterine Natural Killer (uNK) cells become the most abundant leukocyte during decidualization and produce a wide array of angiogenic factors, yet little is known regarding their early pregnancy functions. To characterize the role(s) of uNK cells, whole mount in situ immunohistochemistry of live early implant sites was performed. A timecourse examination of murine early pregnancy (virgin, and gd4.5-9.5) implantation sites was performed. Comparison of Gd6.5, 8.5 and 9.5 implant sites from BALB/c+/+ controls (BALB/c) and BALB/c-Rag2-/-Il2rg-/- (alymphoid) identified anomalies that result from the absence of lymphocytes. In alymphoid decidua basalis, mesometrial angiogenesis was widespread but pruning of nascent vessels within alymphoid decidua basalis was deficient. As early gestation progressed, vessels of alymphoid decidua basalis showed no evidence for remodeling. Alymphoid implantation sites showed ~24h delay in uterine lumen closure and embryonic development. To determine if uNK cells would normalize the anomalies observed in alymphoid implantation sites, adoptive cell transfer of NK+ B- T- marrow to alymphoid mice was performed. All of the above anomalies were reversed by adoptive transfer of NK+B-T- marrow. My results suggest that uNK cells support vascular growth and development which ensures the decidua can support the growing conceptus early in pregnancy prior to formation and function of the placenta. Human decidual NK cells may fill similar roles and be important targets for strategies designed to correct intra-uterine growth restriction.

Relationship between abnormal maternal inflammation and insulin resistance during pregnancy

Abnormal maternal inflammation during pregnancy is linked to complications such as preeclampsia and fetal growth restriction. There is growing evidence that insulin resistance is also associated with a heightened inflammatory state, and is linked to pregnancy complications such as gestational diabetes. This study tested the hypothesis that abnormal inflammation during pregnancy is causally linked to elevations in blood glucose and insulin resistance. To induce a state of abnormal systemic inflammation, bacterial lipopolysaccharide (LPS) was administered to pregnant rats on gestational days (GD) 13.5-16.5. Dams treated with LPS exhibited an abnormal immune response characterized by an elevation in white blood cells, which was linked to reduced fetal weight and increased glucose levels over pregnancy. Abnormal inflammation is characterized by increased levels of circulating pro-inflammatory cytokines such as tumour necrosis factor alpha (TNF) and interleukin-6, which contribute to insulin resistance by inhibiting the insulin signalling pathway. TNF in particular induces a serine phosphorylation (pSer307) of insulin receptor substrate 1 (IRS-1). In our model, insulin resistance was assessed by measuring the extent of pSer307 of IRS-1 and total IRS-1 expression in skeletal muscle, as well as changes in metabolic parameters and pancreas tissue morphology associated with insulin resistance. LPS-treated dams exhibited a significant reduction in IRS-1 expression, elevation in fasting glucose levels, and reduction in insulin sensitivity indices. There were also biologically relevant increases in fasting plasma insulin levels and insulin resistance indices, but not pSer307 of IRS-1 and pancreatic islet size. To determine whether inflammation plays a role in reducing insulin signalling and the other changes associated with LPS administration, etanercept, a TNF antagonist, was administered on GDs 13.5 and 15.5 prior to LPS injections. With the exception of IRS-1 expression, in rats treated with etanercept all of the measured parameters remained at the levels observed in saline controls, indicating a link between abnormal inflammation and insulin resistance. The results of this study support the practice of monitoring the inflammatory conditions of the mother prior to and during pregnancy, and support further investigation into the potential use of anti-inflammatory agents during pregnancy in women at risk of insulin resistance and gestational diabetes.