Wendy Robinson

The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.

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DNA methylation (DNAm) is an epigenetic mark that can control or reflect gene expression in a highly cell-specific manner. Placental DNAm has been studied in various contexts, however, several sources of variation remain uncharacterized. Before we are able to understand how placental DNAm contributes to important health-relevant contexts, we must develop an understanding of the underlying normal variation that occurs in the placental methylome. For example, a high proportion of variation in DNAm can vary by ethnicity and genotype. DNAm is also highly cell-specific, and the placenta is a heterogeneous tissue comprised of several distinct cell populations. However, due to difficulty of isolating cell populations, most placental DNAm research is conducted on whole placental chorionic villi tissue. Isolating placental tissue without contamination from maternal tissue, such as decidua and blood, can be challenging, especially in earlier gestation samples where sampling enough tissue is difficult.In this thesis, I hypothesize that a large proportion of the variation in placental DNAm can attributed to ethnicity, genetic ancestry, cell composition, cell-specific effects, and the presence of maternal cells. Using high-density DNAm microarray profiling, and open access genomic data repositories, I assessed these factors in placental samples with various phenotypes. I found that ethnicity and genetic ancestry are associated with placental DNAm variation in samples containing self-reports of White/Caucasian, East Asian/Asian, and Black/African American ethnicity. Further, I found that it is possible to predict ethnicity and genetic ancestry with high accuracy and reliability from placental DNAm. Another major source of variation is cell-specific DNAm, which I characterized from placental samples of first trimester and term pregnancies. I found that trophoblast and Hofbauer cells are highly epigenetically distinct, and many placental epigenetic features are conserved in trophoblasts but not always in other placental cells. I developed a reference to estimate cell composition from placental chorionic villi DNAm. Lastly, I developed an approach to estimate maternal cells present in chorionic villi samples, using DNAm, and found several previously published placental DNAm studies to contain maternally-contaminated samples. Overall, I contributed to our understanding of placental DNAm, and have provided bioinformatic tools for future placental DNAm research.

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Fetal growth restriction (FGR) is a common pregnancy complication in which the fetus does not grow to its genetic potential due to a pathological cause, which puts it at greater risk for morbidity and mortality in the perinatal period and poor health outcomes in childhood and adulthood. Although the etiology of FGR is diverse, insufficient function of the placenta underlies many cases, as the placenta is a crucial organ to support fetal growth and development and a healthy pregnancy. One of the few established genetic contributors to placental insufficiency and non-syndromic FGR is trisomy confined to the placenta. Beyond this, the contribution of smaller genomic imbalances (copy number variants) or common single nucleotide variants and their impact on gene regulation in the placenta, for example through DNA methylation, remains largely unexplored.In this thesis, I hypothesized that placental genomic imbalances, including aneuploidy and copy number variants (CNVs), and candidate single nucleotide variants in a gene relevant to DNA methylation (DNAme) are associated with poor fetal growth and/or altered placental DNAme. Using molecular-cytogenetic and microarray techniques, I assessed aneuploidy and CNVs in placentas from infants born small-for-gestational age (SGA) and adequately-grown controls. I found that confined placental mosaicism of autosomal aneuploidies or rare candidate CNVs involving genes related to placental function or growth were present in about 18% of SGA cases, and that CNV load was not associated with SGA. I also characterized a novel case of eight 2-4 Mb duplications confined to the placenta of an infant with FGR, in which the CNVs arose de novo in a cell in the trophoblast lineage. Finally, I studied two candidate single nucleotide polymorphisms in MTHFR, involved in the metabolic pathway that produces one-carbon units for methylation reactions and purine synthesis. I found that these variants were not associated with altered placental DNAme, and that there was only a trend for increased risk of placental insufficiency complications of FGR and/or preeclampsia. Through these studies, I contributed to our understanding of genetic variation in the placenta and its association with FGR and placental insufficiency, and provided a foundation from which future studies can build.

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Acute chorioamnionitis (aCA), a preterm birth (PTB) associated inflammatory condition, can have adverse effects on the health of the baby. This condition is characterized by inflammatory lesions in the fetal membranes and can also involve the chorionic plate in the placenta. Histologic examination of the placenta is the gold standard for diagnosing aCA, but is only possible after delivery; thus, this method is not suitable for prenatal diagnosis of aCA. This necessitates the development of non-invasive biomarkers to allow effective management of the disease and hence, reduce the incidence of PTB. Additionally, genetic variation in immune-system genes may contribute to the placenta’s inflammatory responses, thus influencing susceptibility to aCA. The overarching objective of this dissertation is to understand how genetic, epigenetic, and miRNA variation in the placenta is associated with the disruption of immune balance in aCA. To achieve this, I first examined the association of single nucleotide polymorphisms (SNPs) in innate immune system genes and aCA status. I observed that differences in IL6 (rs1800796) placental allele frequencies were associated with the presence of aCA. Further, I showed the IL6 SNP may regulate IL6 gene expression and DNA methylation (DNAme) in the placenta, and alter disease risk to aCA. Secondly, using the Illumina HumanMethylation850 BeadChip, I characterized epigenetic variation associated with aCA in placenta and fetal membranes. Specifically, I observed that aCA-affected placentas showed a unique DNAme profile that may reflect an increase in immune cell number as a response to inflammation and/or represent activation of the innate immune response in the placenta. Lastly, I investigated whether altered miRNA profiles were associated with aCA-affected placentas. Expression was quantified for six inflammation-related miRNAs using quantitative real-time PCR. I observed that expression of miR-518b and miR-338-3p were differentially expressed in aCA-affected placentas. I also showed that miR-518b expression in placenta was associated with IL6 (rs1800796) genotype, where carriers of the C allele exhibited decreased miR-518b expression compared to the carriers of the G allele. In summary, this research uniquely investigated genetic alterations, DNAme, and miRNA expression patterns in aCA-affected placentas, adding insights into the processes likely impacting immune function during aCA.

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Preeclampsia (PE), characterized by maternal hypertension and proteinuria, is the leading cause of maternal and perinatal morbidity and mortality worldwide. PE can be subdivided into early-onset PE (EOPE), diagnosis
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High-throughput methods have resulted in a large volume of studies measuring genome-wide DNA methylation (DNAm) in association with human health and disease. Understanding of DNAm patterns may be translated, for example, into predicting children at risk for illness or identifying etiological subtypes within a heterogeneous disease. Addressing biological and technical factors affecting measurement of genome-wide DNAm is essential to reduce false discovery in such studies. This dissertation develops principles for analyzing genome-wide DNAm, with the aim of improving collection and analysis of human developmental data. To this end, I present four studies employing several techniques to measure genome-wide DNAm: DNAm of L1 and Alu repetitive elements in addition to Illumina 27k and 450k DNAm microarrays. In the first of these studies, I found that tissue type, gestational age, technical platform and CpG density contribute to variable measurement of genome-wide DNAm. Subsequent studies primarily used the 450k array to measure genome-wide DNAm, a technology targeting 485,577 sites in the genome. A detailed annotation of the 450k array was created and tested, to enhance this platform’s utility. Array probes targeting sites containing SNPs (4.3%) and non-specific probes (8.6%) were identified, and I examined how these compromised probes may result in spurious discoveries. A pilot study in placental tissue identified batch effects in 450k data. A computational tool was applied to reduce the batch signal, but I demonstrated that when applied to a problematic study design, false biological signal may be introduced. The workflow for processing and analyzing genome-wide DNAm data was finally applied to profile five tissues ascertained from second trimester neural tube defect (NTD)-affect pregnancies. Despite research, medical interventions and public health changes, NTDs remain the second most common congenital abnormality in many parts of the world, and the etiology of these cases is unknown. Using the 450k array, I found 3,342 differentially methylated sites in the kidneys of spina bifida cases compared to gestational-age matched controls, but little alteration in genome-wide DNAm in other NTD tissues. This dissertation contributes methodologies and analytical tools that will help manage bias, improve reproducibility and reduce false discoveries in studies of genome-wide DNAm.

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Recurrent miscarriage (RM), defined as 3 or more consecutive spontaneous losses of pregnancy before 20 weeks gestation, affects 1-2% of couples and has a complex etiology. Half of miscarriages from RM cases are caused by chromosomal abnormalities in the embryo and while there are several associated maternal factors, underlying causes and clinically relevant biomarkers have been elusive. I hypothesized that genetic and/or epigenetic factors associated with maternal meiotic non-disjunction, reproductive aging and endocrinological profile, or placental functioning will contribute to the etiology of RM. In these case-control studies, I investigated the association between RM and 1) maternal mutations in synaptonemal complex protein 3 (SYCP3), 2) maternal telomere lengths, 3) maternal polymorphisms in genes in the hypothalamus-pituitary-ovarian (HPO) axis and 4) placental DNA methylation patterns. The findings suggest that maternal mutations in SYCP3 and polymorphisms in HPO axis genes may not contribute significantly to risk for RM. No mutations in SYCP3 were identified in women with RM with at least one trisomic conception. While associations between polymorphisms within the estrogen receptor β, activin receptor 1, prolactin receptor and glucocorticoid receptor genes and RM were identified, these were not significant after correction for multiple comparisons. Aspects of chromosomal biology may be important factors in the etiology of RM. Women with RM had significantly shorter telomeres compared to controls, suggesting altered rates of biological aging. In the placental villi of RM samples, there were few differences in DNA methylation at targeted sites when compared to isolated miscarriages and elective terminations. However, gene ontology analysis showed that imprinted genes and immune response pathways were overrepresented among those sites differentially methylated between RM and elective termination placentas. The RM group additionally had an increase in the number of outlier cases at a select number of imprinted loci. Furthermore, several placental samples from both cases and controls showed aberrant DNA methylation profiles at many loci investigated, suggesting these samples may have global dysregulation of DNA methylation and/or differences in placental composition/functioning. These studies have improved our understanding of mechanisms involved in RM and will contribute to the direction of future research.

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Dysregulation of placental and fetal epigenetics can affect gene expression patterns, including the parent-of-origin dependent expression in imprinted genes. While defects of imprinted genes have been implicated in some adverse pregnancy outcomes, little is currently known about the role of epigenetics in regulating normal or pathological human pregnancy and development. The objective of this thesis is to provide fundamental DNA methylation profiles of human fetal and placental development so as to offer insights into the etiology of human disease and adverse pregnancy outcomes. Taking advantage of the unbalanced parental genomic constitutions in triploidies, 45 novel imprinted genes were identified by comparing the genome-wide DNA methylation profiles between 10 diandries and 10 digynies. A comparison of DNA methylation profiles between placentas of different gestations and other somatic tissues showed tissue-specific and gestational age-specific DNA methylation changes in many imprinted genes. To gain insight into the genomic pattern of tissue-specific methylation, DNA methylation profile was evaluated in 5 somatic tissues (brain, kidney, lung, muscle and skin) from eight normal second-trimester fetuses. Tissue-specific differentially methylated regions (tDMRs) were identified in 195 loci, suggesting that tissue-specific methylation is established early in the second trimester. Importantly, only 17% of the identified fetal tDMRs were found to maintain this same tissue-specific methylation in adult tissues, implicating an extensive epigenetic reprogramming between fetus and adult. Besides intra-individual differences, there is also substantial DNA methylation variation between individuals. While many sites show a continuous pattern of DNA methylation variation between different placentas, WNT2, TUSC3 and EPHB4 were identified to have epipolymorphisms at their promoter region. The methylation status at the TUSC3 promoter showed an association with preeclampsia, suggesting a role of DNA methylation change in adverse pregnancy outcomes. A further investigation of DNA methylation profiles in 26 placentas from preeclampsia, IUGR and control subjects showed 34 loci were hypomethylated in the early-onset preeclamptic placentas, with TIMP3 having a potential of being a biomarker for the disorder. These results provided comprehensive DNA methylation profiles for both normal and abnormal fetal and placental tissues, which contribute to the biological and clinical aspects of the pathogenesis of fetal and placental disorders.

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