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Preeclampsia is an increasingly prevalent disorder of placentation that has very limited options for treatment. The disease is characterized by aberrant invasion of placental trophoblasts into the decidualized maternal endometrium. In order to identify pathways of therapeutic interest during placentation, we are focusing on the pathway of the neuropeptide kisspeptin and its receptor KISS1R, both highly expressed in the human placenta. Early functional studies of the ligand-receptor system identified a role for kisspeptin in the inhibition of cancer metastasis. Parallels exist between cancer and placentation, suggesting the possibility of an inhibitory role for kisspeptin during pregnancy as well. Existing functional data supports kisspeptin's inhibitory influence on cellular invasion, but the mechanism remains unknown. Evidence for the localization of the KISS1R receptor in the current literature was established via a nonspecific antibody and requires further investigation. Current literature suggests involvement of the ERK (extracellular signal-regulated kinase) pathway as well. Our work aims to solidify the localization of kisspeptin and KISS1R, avoiding the use of KISS1R antibodies. Using immunohistochemistry for protein localization of kisspeptin and placental fractionation followed by quantitative PCR analysis for gene expression, we provide evidence of kisspeptin's restriction to the syncytiotrophoblast layer of the placenta, and KISS1R gene expression limited to the villous cytotrophoblast layer. This distribution of ligand and receptor suggests a paracrine mechanism for kisspeptin action, with syncytiotrophoblasts secreting kisspeptin to act on its receptor on the villous cytotrophoblast layer, and thus restricting cytotrophoblast invasion. We further attempt to support these data with the use of laser capture microdissection of placental tissue to isolate the different layers, followed by quantitative PCR. This technique introduced a particularly challenging aspect of working with the placenta: maintaining tissue morphology while also preserving RNA integrity. This thesis outlines our troubleshooting process for that technique and introduces alternatives for future work. We also employed Western blot analysis of ERK activation to establish the mechanism of kisspeptin's inhibitory effect on fractionated trophoblasts.

Affecting 6-8% of all pregnancies, preeclampsia is the leading cause of maternal morbidity in the western world and is charactensed by hypertension, proteinuria, edema and platelet aggregation. Despite its prevalence and severity, no comprehensive theory or single factor has been suggested to explain the pathophysiology of this multi system disorder of pregnancy, with the only therapies being bed rest, pharmacological symptom management and if necessary early delivery. Oxidative stress plays an important role in the pathophysiology of preeclampsia, resulting from defective trophoblast invasion, reductions in placental perfusion and placental hypoxia/reoxygenation. The inability of endogenous antioxidant systems up regulated in normal pregnancy, to control increased levels of oxidative stress, is suggested as a possible factor in the feed forward generation of reactive oxygen species and placental oxidative stress. That in turn may stimulate increased syncytiotrophoblast apoptosis, endothelial cell activation and the maternal hyper immune response characteristic of preeclampsia. Analysis of the research literature revealed that previous evaluations of placental oxidation and antioxidant enzyme activity in preeclampsia were by no means comprehensive, and exhibited significant inter-study variations. It was the aim of this thesis to clarify the placental oxidative state and the endogenous antioxidant activity of glutathione peroxidase, thioredoxin reductase, thioredoxin and superoxide dismutase in human placentae in an attempt to determine if variations in antioxidant function were due to changes in gene expression or protein oxidation. The findings reported in this thesis indicate the presence of increased levels of oxidative stress in the preeclamptic placenta, associated with significant reductions in antioxidant enzyme capacity. Quantitative real-time PCR analysis of placental samples revealed that deceases in antioxidant capacity in the placenta are more likely to be related to the significant oxidative burden within the tissue rather than reductions in gene expression. A number of animal models exist to investigate components of preeclampsia pathophysiology, however the ability of these models to mimic the oxidative and antioxidant features of preeclampsia remains unclear. The exposure of pregnant rats to N(G)-nitro-L-arginine methyl ester is a widely used model of endothelial cell dysfunction during preeclampsia. It was the aim of this thesis to determine the biochemical characteristics of this model in an attempt to assess its effectiveness in mimicking oxidative changes in the preeclamptic placenta. Although this model is capable of producing a syndiome in rats similar to the disorder in terms of physiology, this is not manifest in terms of placental biochemistry. The importance of selenium in the synthesis of selenobased antioxidants such as glutathione peroxidase and thioredoxin reductase is well documented. Increasing demand for selenium by the developing fetus may be linked to reductions in selenium status during pregnancy. Considering preeclampsia is associated with significant reductions in selenium status it may be hypothesised that reductions in antioxidant function may be linked to selenium inadequacy. The modulation of dietary selenium in pregnant rats was used to determine the importance of selenium during pregnancy and its effect on antioxidant function and placental oxidative stress. The results of this analysis revealed that selenium deficiency causes a pregnancy specific condition similar to preeclampsia. This condition was found to be associated with increased placental oxidative stress and significant reductions in the systemic activity of selenobased antioxidants that could be modified through selenium supplementation. In summary, data obtained in this thesis indicate that placental oxidative stress and reduced antioxidant enzyme activity play a significant role in the pathogenesis of preeclampsia. These studies support the hypothesis that antioxidant sufficiency is crucial in the maintenance of oxidative balance and that antioxidant dysfunction may result in damage to the placenta and the progression of the disease. These novel data further our understanding of the pathophysiology of preeclampsia and provide new insight into the pathogenesis of clinical complications exhibited in this condition, suggesting antioxidant therapy as a possible means for improving the health outcomes of both mother and baby.

Placenta is a transient association of the fetal and maternal tissues, that develops during pregnancy, in most viviparous animals. The evolution of placenta ensured the development of the fetus inside the womb of the mother, providing a protected environment for the development of the fetus, and preventing the loss of progeny due to unfavorable environmental conditions. Because it is strategically poised at the maternal and fetal interface, the placenta is ideally suited to carry out alimentary, respiratory and excretory functions for the developing fetus. In addition, it serves as an immunological barrier preventing the rejection of the fetal semi-allograft, by the maternal immune system. Furthermore, the placenta elaborates a variety of protein, polypeptide and steroid hormones. These include growth factors, growth factor receptors, neuropeptides, opioids, progesterone and estrogen, whose secretion is dependent on the gestational age of the placenta and its differentiation status. The human placenta, adapts itself remarkably to cater to the changing requirements of the developing fetus. For instance, during the first trimester of pregnancy, the placenta is an actively dividing, a highly invasive and a rapidly differentiating organ; while near term, it represents a fully differentiated and a non-invasive unit. Furthermore, the placenta of the first trimester and that at term differ in their hormone profiles, extents of apoptosis, expression of several transcription factors, etc. This dramatic change in the phenotype of the human placenta can be considered to be the outcome of an intrinsically programmed pattern of differentiation, which may be referred to as the functional differentiation of the placenta. It may be hypothesized therefore, that this functional differentiation could be brought about by the differential expression of genes in the first trimester and the term placenta. The objectives of the present study were: 1. To gain an insight into this process of " functional differentiation” by investigating the differential expression of genes in the two developmentally distinct stages during gestation, viz. during the first trimester and at term. 2. To understand the functional relevance of the differentially expressed genes. A general introduction of the human placenta, describing the importance of differential expression in modulating placental function, is discussed in chapter 1. The functions of the human placenta along with a brief description of its development and differentiation are also briefly described. A Differential Display RT-PCR-based (DD RT-PCR) approach, using total RNA from the first-trimester and term placental villi, was employed to display the differentially expressed genes in the first trimester and the term placenta. The display so generated was used to identify a few differentially expressed cDNAs. This study was aimed at understanding the functional significance of the transcripts which were identified from the display, rather than just concentrate on documenting the differences in the gene expression patterns in the first trimester and the term placental tissue. A detailed description of the methodology adopted for performing DD-PCR using placental tissue, discussing the advantages and disadvantages of using differential display PCR, is described in chapter2. The use of DD-PCR for studying differential gene expression in the human placenta was validated by the finding that one of the cDNAs that was differentially expressed in the first trimester placental tissue, is a fragment of β-hCG cDNA. It is well documented that the differential expression of the β-subunit of hCG (human chorionic gondatrophin) during the first eight weeks of gestation is the rate limiting step in the synthesis and secretion of the functional hormone, which comprises the α and the β-subunits. Furthermore, the use of the model system viz., the first trimester and term placental tissue, was also validated for carrying out DD-PCR by ensuring that all placental samples used for DD analysis were free of endometrial contamination. A detailed description of optimization and validation of DD-PCR in human placental tissues is given in chapter 2. Cloning and sequencing of yet another cDNA from the first trimester differential display revealed that it is T-Plastin. T-Plastin is a member of a family of proteins that are involved in actin-bundling. Northern blot analysis and immunohistochemical studies using an antibody generated to a peptide corresponding to human T-Plastin, confirmed its differential expression and localization in the first trimester placenta. Considering the fact that several carcinomas show enhanced expression of T-Plastin, we tested the hypothesis that its differential expression is correlated with the proliferative potential of the first-trimester placenta It was observed that the first-trimester tissue expressed high levels of beta-actin as compared to the term placental tissue. This is in agreement with the up-regulation of beta-actin following mitogenic stimulation/proliferation and during neoplastic transformation or transformation-associated invasive behaviour of cells, two characteristic features shared by the early placenta with cancerous tissues. Based on our studies and available information in the literature, it is proposed that T-Plastin expression in the first trimester placenta is a growth-associated phenomenon which is partially responsible for the tumor-like phenotype of the first trimester tissue. Studies carried out with the partial T-Plastin cDNA clone that was isolated from the first trimester differential display, are presented in chapter 3. Sequencing of yet another cDNA clone identified from the term placental differential display, T-18 revealed that it had no homology to any known sequence in the nucleotide or est databases. The sequence corresponding to this clone was submitted to the GenBank and was assigned an accession number- AF089811. The differential expression of T-18 was confirmed by Northern blot analysis and RT-PCR analysis. Attempts were made to isolate the full-length cDNA corresponding to T-18 from a commercially available library from Clontech. However, repeated trials to identify the clone corresponding to T-18 did not yield any positive results. However, a genome database search revealed that T-18 was a portion of a large contig contained in chromosome 15. Analysis of the annotated gene sequences in and around the region in which T-18 is located in chromosome 15, revealed that there are very few ests reported in this contig and quite a few repeat sequences reported. Interestingly, it was observed that 6 kb downstream of the region in which T-18 is located, there was an est that had homology to a Bcl-2 precursor protein (an evolutionarily conserved, anti-apoptotic protein, capable of conferring protection against death-inducing signals) and the death adaptor protein, CRADD {Caspase and RIP adapter with death domain). Further updating of the ests in the database might probably be of help in the identification of the full-length cDNA corresponding to T-18 and confirm as to whether T-18 is a part of the gene/gene cluster that comprises the afore-mentioned est. An account of the identification and cloning of T-18 from the term placenta and the attempts to isolate the full-length cDNA clone corresponding to T-18 from a term placental cDNA library, is described in chapter 4. In the absence of any information on the identity of T-18, a study to understand the functional significance of T-18 expression was carried out. Since it was not possible to carry out studies pertaining to the temporal expression of T-18 throughout gestation on the human placenta for ethical reasons, alternate animal/organ models were employed to study T-18 expression. Rat placenta and rat Corpus Luteum (CL) were chosen as alternate models for studying T-18 expression as these two organs/tissues underwent dynamic changes in their function throughout pregnancy. For instance, it is well known that CL is the primary source of progesterone for maintaining pregnancy in the rat and that the progesterone secreting capacity of the luteal cells peak on day 16 of gestation and decline thereafter. Interestingly, a common feature among all the tissues that were chosen for investigating the regulation of T-18 expression, is the fact that they underwent apoptosis with increase in gestational age. The expression of T-18, in tissues exhibiting increased incidence of apoptosis suggested that T-18 maybe an apoptosis-associated gene. Using an explant culture model it was demonstrated that placental villi when cultured in vitro underwent spontaneous apoptosis and that the levels of T-18 message increased, under these conditions. Furthermore, this spontaneous induction of apoptosis in explant cultures could be blocked when villi were cultured in the presence of superoxide dismutase, a free radical scavenging enzyme. In addition, the expression of T-18 was shown to be modulated following treatment with SOD, or in response to oxidative stress. These studies clearly indicate a role for T-18 in placental apoptosis and moreover, implicate the usefulness of explant culture to examine the molecular mechanisms involved in placental apoptosis. Furthermore, the expression of the anti- and pro-apoptotic genes, bcl-x and bax respectively, were investigated, in an attempt to elucidate the signalling pathway(s) that led to the activation of an important downstream protease, caspase-3, in placental apoptosis. The present study revealed that induction of apoptosis in the placenta in vitro involved a bcl/bax independent, caspase-3 dependant pathway. The validation of an explant culture model for studying placental apoptosis and data pertaining to the role of T-18, bcl-x, bax and CPP32 in placental apoptosis, in response to oxidative stress, are presented in chapter 5. The last section titled general discussion summarizes the work carried out in this study and proposes a model for the apoptotic mechanism(s) that may be operating in placenta In conclusion, the present study has led to the identification of two developmentally-regulated factors, T-Plastin and T-18 in the first trimester and term placenta, respectively. The differential expression of these genes, in addition to several other molecular players, is proposed to be responsible for the overall functional differentiation of the placenta through the course of gestation.

Human placental villi are covered by a layer of trophoblast epithelia in direct contact with maternal blood, which exist in a constant steady-state of turnover and renewal ensuring both maternal and fetal health. The process of trophoblast turnover involves proliferation, differentiation and fusion of cytotrophoblast cells to form a terminally differentiated outer syncytiotrophoblast layer which functions as the active transport compartment between mother and fetus. Alterations in the balance between these three processes are thought to diminish both the structural and functional integrity of the syncytiotrophoblast, potentially leading to placental insufficiency associated with severe complications of pregnancy such as pre-eclampsia (PET), intrauterine growth restriction (IUGR) and sudden infant death syndrome (SIDS). Placentas from early (<32 weeks) and late-onset (>33 weeks) pregnancies complicated by PET, IUGR, SIDS and gestational age-matched controls were systematically uniform randomly sampled to assess the morphological basis of placental villous structure and trophoblast turnover (villi, cytotrophoblast, syncytiotrophoblast, apoptotic syncytial knots) using unbiased stereological techniques (volumes and numbers). Villous cytotrophoblast proliferation was assessed using double immunohistochemistry for Ki67 and cytokeratin 7 (CK-7). Severe early-onset IUGR placentas (n=5) were smaller displaying significant reductions in the total number of CT cells, within which the density of proliferating CT was further reduced by 50%. Syncytiotrophoblast volume and number was significantly reduced with an increase in apoptotic syncytial knots. Late-onset IUGR placentas (n=4) also displayed significant reductions in the total number of CT and proliferating CT, but were not associated with changes in the density of proliferating CT. SCT numbers were significantly reduced with an increase in apoptotic knots. Placentas from severe early-onset PET (n=11) were similar to preterm controls, except for a significant increase in apoptotic syncytial knots. However, late-onset PET (n=6) displayed a significant decrease in total CT number, the percentage of which undergoing proliferation was significantly increased for structural villi. There were increased numbers of apoptotic syncytial knots in peripheral villi.

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Thesis (Ph. D.)--University of Hawaii at Manoa, 1995. / Includes bibliographical references (leaves 116-131). / Microfiche. / xvi, 131 leaves, bound ill. (some col.) 29 cm

Insufficient maternal folate during pregnancy increases the risk of the baby being small for gestational age (SGA). Studies in teenagers, a population vulnerable to folate deficiency and SGA birth, have shown that low maternal folate status is associated with impaired placental cell turnover and reduced transport suggesting placental dysfunction underlies SGA in maternal folate deficiency. Mechanisms through which folate-depletion compromises placental function are currently unknown. In non-placental cells, folate modulates microRNAs (miRs), post-transcriptional regulators of cellular functions. Expression of miRs is altered in placentas of SGA compared to normally grown babies but there are no data on differential miR expression or regulation in placentas from folate deficient women. This PhD investigated the hypothesis that placental dysfunction observed in folate deficient women is mediated by altered miR expression. Three placental preparations were compared (villous tissue in explant culture, BeWo choriocarcinoma cells and isolated cytotrophoblast cells) to determine the optimum in vitro system to study the direct effects of folate deficiency. In cytotrophoblast cells, folate deficiency significantly elevated apoptosis and reduced the activity of the system A amino acid transporter, consistent with observations in the placentas of folate-deficient teenagers. The reduction in system A activity by low folate was not associated with altered mRNA expression for the isoforms of system A, implicating an effect of low folate on post-translational regulation of the nutrient transporter. Targeted examination of villous tissue from teenagers with low folate status identified up-regulation of miR-222-3p a folate-sensitive miR. An unbiased miR array identified up-regulation of a further 16 miRs suggesting that maternal folate deficiency in vivo results in aberrant placental miR expression. Bioinformatic analysis of the folate sensitive miRs predicted gene targets known to be altered in placentas from SGA pregnancy that were likely to alter placental function. Two miRs altered in placentas from women with low folate status, miR-30e-3p and miR-34b-5p, were also significantly altered in folate deficient cytotrophoblasts confirming a direct effect of folate on trophoblast miR expression. Inhibition of these miRs in vitro had no effects on placental functions that are altered in vivo in folate-deficient women. Gene array and in silico analysis identified functional endpoints affected by these folate sensitive miRs, including cell signalling for proliferation and survival and oxidative stress, which might contribute to placental dysfunction in folate deplete women. Overall, this study has demonstrated for the first time that folate deficient conditions can directly alter trophoblast system A transport and cell survival and thus could contribute to the increased susceptibility to SGA births in folate deficient women. It has also contributed to the knowledge that miR expression is differentially altered in placentas exposed to folate-deficient versus sufficient conditions in vivo and that miRs are directly altered by folate depletion in vitro. These studies provide the foundation for future research to define the functional consequences of altered expression of folate-sensitive miRs and their target genes to explain how altered miRs could be affecting placental function resulting in development of SGA.

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