The Effects of Maternal Periconceptional Ethanol Exposure on the Development of the Pre-Implantation Embryo, Placenta, and the Influence of the Uterine Environment

Abstract

Exposure of the embryo or fetus to perturbations in utero can result in intrauterine growth restriction (IUGR), a primary risk factor for the development of adult disease.One critical window that can result in developmental programming, is the periconceptional period which comprises maturation of the oocyte, conception and the formation of the blastocyst prior to embryo implantation. Alcohol is a common exposure during this period, as it is consumed prior to pregnancy recognition, and often ceases soon after. Maternal periconceptional alcohol exposure (PC-EtOH) in rat dams has been previously shown to cause fetal growth restriction and changes to the late gestation placenta. PC-EtOH also results in the development of adult onset disease, including insulin insensitivity, often in a sexually dimorphic manner. However, the mechanisms by which PC-EtOH can cause programming are relatively unexplored. This thesis aimed to characterise the effects of alcohol on 1. sex-specific pre-implantation development and trophoblast differentiation using in vitro (cell culture) and in vivo (rodent model) techniques, 2. alterations to the early uterine environment and 3. the interactions and communication between the embryo and uterus, and the resultant impacts on placental development across gestation.The direct effect of EtOH on in vitro differentiation was assessed in mouse trophoblast stem cells (Chapter 4). Results demonstrated that doses of 0.2% and 1% EtOH reduced total cell count and expression of trophoblast subtype markers at terminal differentiation (day 6 of culture). Using our in vivo rat model, we also examined basal sex differences in blastocyst and placental development from pre-implantation to lategestation in naturally cycling dams (Chapter 3). Although there were no significant differences between sexes in pre-implantation development (blastocyst cell numbers, trophoblast differentiation), by mid-gestation (E15) placentas from males were heavier and had increased blood space volume and surface area (to both maternal and fetal compartments) than those from females. This likely contributed to the greater fetal body weight in males at E20. This study confirmed the need to examine the effects of PC-EtOH in a sexually dimorphic manner across all of pregnancy.To study the effects of PC-EtOH in vivo, Sprague-Dawley rat dams were given a liquid diet containing either control (0% v/v EtOH) or EtOH (12.5% v/v EtOH) from embryonic (E) day -4 to E4. The following day dams were returned to chow for the remainder of 3 gestation. Dams were sacrificed at E5 to determine cell number of the pre-implantation embryo and its capacity to differentiate into cells required for invasion (Chapter 5).Pre-implantation studies showed PC-EtOH altered inner cell mass count, trophoblast differentiation to trophoblast giant cells (TGCs) and trophoblast behaviour in a sexually dimorphic manner, with females showing the most deleterious outcomes. PC-EtOH females also showed reduced expression of Prl4a1, a gene exclusively expressed by TGCs...