Abstract 17β-Hydroxysteroid dehydrogenases (17HSDs)/17-ketosteroid reductases (17KSRs) modulate the biological activity of certain estrogens and androgens by catalyzing dehydrogenase and reductase reactions between 17β-hydroxy and 17-ketosteroids. In the present study, cDNAs encoding mouse and rat 17HSD/KSR1 were cloned in order to study the role of rodent type 1 enzyme in ovarian estradiol (E2) biosynthesis and its enzymatic characteristics. Both rat and mouse 17HSD/KSR1 were expressed in granulosa cells of developing follicles, where diethylstilbestrol and follicle-stimulating hormone stimulated follicular maturation and up-regulated the expression of 17HSD/KSR1, whereas human chorionic gonadotropin caused luteinization of follicles and down-regulation of the enzyme. In line with this, the rodent type 1 enzymes are not expressed in the corpus luteum (CL). Mouse 17HSD/KSR1 showed substrate specificity different from that of the human counterpart. The mouse type 1 enzyme catalyzed the reaction from androstenedione to testosterone at least as efficiently as estrone (E1) to E2, while human 17HSD/KSR1 clearly preferred the E1 to E2 reaction. A mouse mammary epithelial cell line was found to possess strong estrogenic 17KSR activity. A novel type of 17HSD/KSR responsible for this activity was expression-cloned on the basis of its ability to convert E1 to E2 and it was chronologically named 17HSD/KSR7. Interestingly, it showed 89 % identity with a rat protein called prolactin receptor-associated protein (PRAP), which is expressed in the CL. Enzymatic characterization showed that both mouse 17HSD/KSR7 and PRAP efficiently catalyzed the reaction from E1 to E2. The mouse type 7 enzyme was most abundantly expressed in the ovary and placenta. Similar primary structure, enzymatic characteristics, and tissue distribution of mouse 17HSD/KSR7 and PRAP suggest that PRAP is rat 17HSD/KSR7. Further studies showed that in rat ovaries 17HSD/KSR7 is primarily expressed in the middle and second half of pregnancy, in parallel with E2 secretion from the CL. Using in situ hybridization, cell-specific expression of 17HSD/KSR7 was studied in the mouse ovary, uterus and placenta. In the mouse ovary, the enzyme was expressed exclusively in the CL. In the uterus on day 5 post coitum (p.c.), the type 7 enzyme was expressed in the decidua, mostly in the inner zone of antimesometrial decidua. Between day 8 and 9 p.c. the enzyme was abundant in decidua capsularis of the developing placenta, after which expression moved to the basal zone. On days 12 and 14 p.c., mouse type 7 enzyme was abundantly expressed in the spongiotrophoblasts, where expression decreased towards parturition. Altogether, rodent 17HSD/KSR7 is a new 17HSD/KSR which is involved in the biosynthesis of E2 in the ovaries. In addition, E2 produced locally in the decidua and placenta by the type 7 enzyme may have a role in decidualization and/or implantation and placentation.

Fetal growth restriction (FGR) is a serious complication of human pregnancy where the fetus fails to reach its genetically pre-determined growth potential. It is a common condition, affecting 5 -15% of all pregnancies (Gardosi 2009) and is linked to a third of all antepartum deaths (CEMACH 2008). An ongoing problem for obstetricians is the difficulty in diagnosing and predicting FGR and those at highest risk of poor outcomes. Placental insufficiency is a major cause of FGR and specific abnormalities in placental morphology and function occur in this condition; constituting an abnormal FGR placental phenotype (Sibley, Turner et al. 2005). Magnetic Resonance Imaging (MRI) is a powerful tool that allows quantitative analysis of several indices relating to tissue structure and function and, therefore, is of potential use in identifying this phenotype. We hypothesised that a range of MR indices would be feasible in the placenta at 1.5 T, that these indices would be altered in FGR and that there would be correlations with relevant parameters of placental morphology. Ultimately, we aimed to assess whether these indices could be used in the assessment of FGR in utero.Using MRI we estimated placental volume, widths, length and depths in groups of women with normal and FGR pregnancies. We also measured placental relaxation times, T1 and T2, which relate to tissue composition and assessed parameters relating to blood flow using Intra-Voxel Incoherent Motion (IVIM) and Arterial Spin Labelling (ASL). We demonstrated an FGR placental phenotype that was reduced in volume but increased in depth, by around 10mm, with a shorter T2 relaxation time and lower values of D (the diffusion coefficient) measured by IVIM. A trend for reduced perfusion measured by ASL was observed in pregnancies with birthweights less than 10th centile (Gardosi, Chang et al. 1992). T2 and D also correlated with stereological indices of placental morphology.In conclusion, the studies in this thesis illustrate these MRI indices show great potential asbiomarkers for identifying the FGR placenta

An essential function of the placenta is the exchange of nutrients, and wasteproducts of fetal metabolism, between mother and fetus. The placenta thereforeplays a key role in determining fetal growth and size at birth. Fetal growthrestriction (FGR) is a complication affecting around 5% of pregnancies. Thereare several possible causes of FGR but the most common in the Western world isplacental dysfunction. The FGR baby is at much greater risk of stillbirth andneurodevelopmental morbidities than the normally grown baby. Furthermore, thesmaller baby per se has an increased risk of a range of morbidities as an adult.The thrust of the work covered in this thesis was to improve understanding of theabnormalities in placental exchange physiology associated with FGR. The goalwas (and is) to develop new placental diagnostic biomarkers for the disease andnew treatments based on improving placental function.The first tranche of work described showed that there are specific changes intransporter activities in the placenta in FGR. My colleagues and I showed thatSystem A amino acid transporter activity in the microvillous plasma membrane(MVM) of the syncytiotrophoblast (transporting epithelium of the placenta) isreduced, per mg membrane protein, and that this reduction is related to theseverity of the disease. This contrasted with our observation in normal pregnancythat MVM System A activity, per mg protein, is inversely related to size of the babyat birth, and first suggested the concept of placental adaptation to fetal growthdemand. We, and others, went on to show that a number of other transporters inthe syncytiotrophoblast are altered in FGR. However, not all transporters areaffected and at least one is upregulated. This led me to hypothesise that some ofthese changes are causal to FGR and some are responses, or adaptations, toabnormal fetal growth. The direct causes of transporter activity changes are notknown but our work, and that of others, suggests that glucocorticoids play a role.We also showed that transporter activities in the placenta are affected in othercomplications where fetal growth is aberrant. Furthermore, we provided evidencethat denuded areas of the syncytiotrophoblast might be the morphologicalcorrelate of a route of passive transfer of hydrophilic solutes across the placenta.Our studies in a mouse model of FGR suggest that abnormalities in such aparacellular route may be part of the placental dysfunction in the disease.In the final group of publications of this thesis I describe work showing gestationalchanges in placental transporter activities. I suggest that these are primarilyregulated to maintain fetal growth trajectory, but may also provide for specificnutrient demands at particular times in gestation. This explanation was supportedby work with genetically modified mice showing experimentally that placentaltransporter activity is regulated, or adapted, in relation to fetal growth demand. Itappears from several studies described here that there is a matching of fetalgrowth demand and placental nutrient supply. However, other work shows that thematernal nutritional environment does modify this matching.The studies described here have led to three ongoing lines of investigation: (1)applying knowledge of placental phenotypes of FGR to assist in early diagnosis ofwomen at risk; (2) using mouse models of FGR to test drugs for treating thedisease in humans; (3) investigations into the nature of the fetal nutrient demandsignal(s) to the placenta, and whether these signals are altered in FGR.

The use of assisted reproductive technologies (ART) in Canada has increased rapidly over the past decade, with a 235% increase in reported ART cycle starts and a 190% increase in resultant clinical pregnancies from 2008-2018, outpacing our understanding of potential adverse perinatal outcomes. This dissertation project recognizes that the use of ART will continue to increase in both scale and scope, and that it will continue to be a valued means of overcoming infertility and involuntary childlessness. The balance of risks and benefits of these rapid technological innovations for human health is uncertain. Therefore, this project seeks to produce evidence about both the risks associated with ART as well as component treatment factors amenable to modification to make its use safer. While available evidence indicates that medically assisted pregnancies are at greater risk of placental complications, data are scarce, and estimates of specific effects are varied and not reported in the context of service provision for Canada. Linking data from the Canadian Assisted Reproductive Technologies Register to the Better Outcomes Registry & Network Ontario data, this project commenced with a foundational retrospective cohort study to assess the relationship between ART (in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI)) and placental and placentally-mediated complications by comparing assisted conceptions from four years of ART cycle starts to non-assisted conceptions. Building on the findings from the foundational study, two additional studies were performed to investigate the contributions of two treatment factors, day or stage of embryo at transfer and cryopreservation, to the observed patterns of risk. Compared to non-assisted pregnancies, we find that assisted pregnancies are associated with an increased risk of placental and placentally-mediated complications. Within assisted pregnancies, risk profiles are best achieved by stratifying the analysis by both treatment type (IVF or ICSI) and embryo transfer type (fresh or frozen), in keeping with clinical practice. Although we did not find the day or stage of embryo at transfer to be a primary independent contributor to the observed increase in risk of these complication, patterns observed in the relationship between method of cryopreservation and these outcomes warrant further investigation. This thesis concludes by arguing for the pursuit of a hierarchy of risk approach to the use of ART, which is supported by the findings of this project, as it may be the most effective method to guide technological innovation and policy implementation to mitigate the risks associated with ART while not being overly restrictive of the options available to those seeking to build a family.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Veronika Váchalová Supervisor: Prof. PharmDr. František Štaud, Ph.D. Consultant: Mgr. Rona Karahoda Title of diploma thesis: The influence of antidepressants on serotonin homeostasis in placenta Depression is a serious mental disorder affecting 10-20% of women during pregnancy. Up to 10% of these pregnant women are prescribed antidepressants (ADs), most frequently from the class of selective serotonin (5-HT) reuptake inhibitors (SSRIs). While the safety of this treatment is questionable due to reported impaired pregnancy/fetal outcomes, understanding of potential mechanistic causes is still lacking. During pregnancy, 5-HT is important for normal placental function and proper fetal development and programming. 5-HT homeostasis in the placenta is maintained via the 5-HT transporter (SERT/SLC6A4) on the apical side and the recently characterized organic cation transporter 3 (OCT3/SLC22A3) on the basal side of trophoblast. These transporters take up 5-HT from the maternal and fetal circulations, respectively into the syncytiotrophoblast (STB) where it is degraded by monoamine oxidase-A (MAO-A). As all ADs interfere with the 5-HT system it is important to study their potential interactions in the...

After the discovery that Zika virus (ZIKV) infection in pregnant women may result in severe adverse outcomes such as fetal microcephaly, ZIKV must be added to an ever-expanding list of teratogenic viruses. As only a small minority of newborns will display congenital abnormalities after maternal primary infection, innate immune mechanisms must exist in the placenta to prevent viral transmission to the fetus. Understanding the innate antiviral defenses of the placenta is critical to improving diagnosis, treatment, and prevention of adverse pregnancy outcomes stemming from viral infections. We hypothesized that RLRs are expressed in either one or both of the outer cell layers of the chorionic villi, either the syncytiotrophoblast (STB, outermost layer) or in the villous cytotrophoblast (CTB, inner layer), and that expression of these receptors will increase with advancing gestational age.  In order to determine the expression of RLRs in placental tissue (6-32 weeks and full-term), we used immunohistochemistry to stain for the RIG-I-like receptors (RLRs) RIG-I, DHX58/LGP2, and MDA5, as well as the endosomal Toll-like receptor TLR7, that serve as antiviral innate immune receptors involved in detecting microbial ligands and cytoplasmic viral nucleic acids. Hofbauer (HB) cells stained positive for all receptors and served as a positive internal control. TLR7 was not present in either the STB or CTB throughout gestation. MDA5 was localized to the STB cytoplasm up to 13 weeks. After 13 weeks, MDA5 was localized to the CTB cytoplasm. DHX58/LGP2 was localized to the STB cytoplasm at 6 weeks of gestation, the STB apical and basal membranes in addition to cytoplasm at 7 weeks of gestation, and also CTB cytoplasm after 7 weeks of gestation. Lastly, RIG-I was localized to the CTB cytoplasm throughout gestation. The differential expression of these RLRs suggest an innate immune defense system unique to the placenta that is responsible for protecting the conceptus from viral attack.  These findings will complement ongoing work in characterizing replication of teratogenic viruses in the placenta.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Veronika Kučerová Supervisor: doc. PharmDr. Martina Čečková, Ph.D. Consultant: PharmDr. Lenka Ťupová, Ph.D. Title of diploma thesis: Study on expression of selected ABC transporters in placenta The placenta is a temporary organ through which the fetus is supplied with nutrients and oxygen from the mother's blood and conversely waste substances are transferred into the mother's blood during pregnancy. Substance transfer through the placenta is a complex process controlled by a number of physiological mechanisms, including passive diffusion, facilitated diffusion or active transport, which is realized by activity of membrane transporters with energy consumption. Presence of active ABCs efflux transporters in the placenta has been known for a long time and their function is associated primarily with limiting the entry of harmful substances into the placenta and further into the fetus, thus contributing to its protection. Among the best described transporters belong P-glycoprotein (MDR1/ABCB1), breast cancer resistance protein (BCRP/ABCG2) and multidrug resistance protein 2 (MRP2/ABCC2), whose expression has been confirmed in the apical membrane of the placental syncitiotrophoblast facing...

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The high incidence of pregnancy loss is a major issue facing the cattle industry. Use of in vitro fertilized (IVF) bovine embryos has become increasingly popular to help alleviate several of these reproductive issues and provide a means to enhance genetic gain for production traits. An uterine paracrine factor cocktail containing epidermal growth factor (EGF), fibroblast growth factor 2 (FGF2), and insulin-like growth factor 1 (IGF1) (collectively termed EFI) was recently identified as a means for improving in vitro derived bovine embryo development and trophectoderm cell numbers. The objectives of this work were to determine if EFI treatment during in vitro bovine embryo culture improves transferable embryo quality and post-transfer placental and fetal development. For each replicate (3 total), slaughterhouse-derived bovine oocytes were matured and fertilized in vitro. At day 4 post-fertilization, ≥8 cell embryos were harvested, pooled, and exposed to either the EFI treatment (10ng/ml EGF, 10ng/ml FGF2, 50ng/ml IGF1) or carrier only (1% Bovine Serum Albumin). At day 7, individual embryos were transferred to estrous synchronized beef cattle. Artificial insemination (AI) was completed on a subset of cows. The EFI treatment increased (P<0.05) the percentage of transferable embryos. Pregnancy rate at day 28 post-estrus was similar among treatments. Circulating concentrations of pregnancy-associated glycoproteins (PAGs) were determined from plasma harvested at day 28, 42 and 56. Transrectal ultrasonography was used to measure fetal crown-rump length (CRL) at day 42 and 56 and to determine fetal sex at day 60. There were no main effect differences observed across days for PAG concentration. Fetus sex by ET/AI group interactions were absent at day 28 but existed at days 42 and 56 (P<0.05). At both days, this interaction reflected fetus sex-dependent changes within the ET control group, where PAG concentrations were greater (P<0.05) in male fetuses than female fetuses. No CRL differences or interactions existed among fetal sex and pregnancy group. In summary, addition of the EFI cocktail during bovine embryo culture improved the quality of transferable embryos, but did not affect placental function or embryonic/fetal development. Increasing the numbers of transferable embryos is of value given the cost of in vitro embryo production, but no apparent increases in embryo or placental competency were detected. The EFI treatment increased (P<0.05) the percentage of transferable embryos. / Master of Science / The high incidence of pregnancy loss is a major issue facing the cattle industry. Use of in vitro fertilized (IVF) bovine embryos has become increasingly popular to help alleviate several of these reproductive issues and provide a means to enhance genetic gain for production traits. An uterine paracrine factor cocktail containing epidermal growth factor (EGF), fibroblast growth factor 2 (FGF2), and insulin-like growth factor 1 (IGF1) (collectively termed EFI) was recently identified as a means for improving in vitro derived bovine embryo development and trophectoderm cell numbers. The objectives of this work were to determine if EFI treatment during in vitro bovine embryo culture improves transferable embryo quality and post-transfer placental and fetal development. For each replicate, slaughterhouse-derived bovine oocytes were matured and fertilized in vitro and at day 4 post-fertilization, embryos were exposed to either the EFI treatment (10ng/ml EGF, 10ng/ml FGF2, 50ng/ml IGF1) or carrier only (1% Bovine Serum Albumin). Artificial insemination (AI) was completed on a subset of cows and the remaining cattle receive embryos at day 7. The EFI treatment increased (P<0.05) the percentage of transferable embryos. Pregnancy rate at day 28 post-estrus was similar among treatments. Circulating concentrations of pregnancy-associated glycoproteins (PAGs) were determined from plasma harvested at day 28, 42 and 56. Transrectal ultrasonography was used to measure fetal crown-rump length (CRL) at day 42 and 56 and to determine fetal sex at day 60. There were no main effect differences observed across days for PAG concentration. Fetus sex by ET/AI group interactions were absent at day 28 but existed at days 42 and 56 (P<0.05). At both days, this interaction reflected fetus sex-dependent changes within the ET control group, where PAG concentrations were greater (P<0.05) in male fetuses than female fetuses. No CRL differences or interactions existed among fetal sex and pregnancy group. In summary, addition of the EFI cocktail during bovine embryo culture improved the quality of transferable embryos, but did not affect placental function or embryonic/fetal development. Increasing the numbers of transferable embryos is of value given the cost of in vitro embryo production, but no apparent increases in embryo or placental competency were detected. The EFI treatment increased (P<0.05) the percentage of transferable embryos.