Alexander Guillermo Beristain

The metzincin family of metalloproteases coordinates cell and tissue developmental processes through regulation of growth factor availability, receptor signaling potential, and cell-cell/cell-matrix adhesion. During placental development, metzincin proteases are crucial for successful trophoblast differentiation and invasion. While distinct roles for metzincin proteases in controlling specific trophoblast functions have been described, a comprehensive assessment of metzincins in trophoblasts during discrete stages of differentiation has yet to be performed.In this thesis, I investigate the function of metzincin proteases in the novel human trophoblast stem cell lines with the use of the broad protease inhibitor Batimastat. I show that Batimastat leads to decreased size and invasive outgrowth of organoids cultured in regenerative and extravillous differentiation media respectively. I further undertake a comprehensive single cell transcriptomic assessment of metzincin protease expression in diverse states of human trophoblasts from 11 first trimester placentas. In the 8 distinct trophoblasts states categorized [four progenitor cytotrophoblast (CTB), one syncytiotrophoblast precursor (SCTp), two column CTB (cCTB), and one extravillous trophoblast (EVT) state], I identify 24 metzincin proteases, including 12 adamalysins, 2 pappalysins, 3 astacins and 7 matrixins. Our follow-up cell trajectory modeling shows that most (19/24) metzincins increase along the EVT pathway, though multiple proteases also increase along the villous pathway as CTB fuse into syncytiotrophoblast. Within the CTB niche, single-cell velocity ordering identifies 11 metzincin proteases (ADAM10, -17, MMP14, -15, -19, -23B, ADAMTS1, -6, -19, TLL-1, -2) expressed in progenitors proximal to the predicted origin. To elucidate factors governing CTB maintenance, I analyze metzincin-substrate interactions within the CTB niche. This reveals ~150 substrates and binding partners, including FBN2 as an ADAMTS6-specific substrate preferentially expressed in trophoblast progenitors. Lastly, Iundertake a single cell RNA sequencing analysis of metzincin protease dynamics in the human trophoblast stem cell derived organoids which reveal a high degree of overlap with our in vivo dataset.Together, this work characterizes the metzincin transcriptomic landscape in human first trimester trophoblasts and establishes insight into the roles specific proteases perform within distinct trophoblast niches and during differentiation. This thesis serves as a guide for future investigations into the roles of metzincin proteases in placental development.

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Infertility, the inability to achieve a natural pregnancy within twelve months, affects roughly 10% of couples worldwide. To address this difficulty, many people use assisted reproductive technologies (ART) such as in vitro fertilization, intracytoplasmic sperm injection, and intrauterine insemination. There is some concern over the safety of these techniques, however, as some adverse birth outcomes such as growth restriction, low fetoplacental weight ratio, and preterm birth, as well as certain developmental disorders known as imprinting disorders have been reported at higher rates in children conceived via ART. In this thesis, I investigate a number of birth outcomes and placental metrics as they relate to different factors of ART procedures. I also evaluate expression levels and DNA methylation (DNAme) profiles of imprinted genes in the context of births that display some of the adverse outcomes associated with ART. Birth weight, gestational age at birth, and APGAR scores at one and five minutes were not seen to be significantly different between ART and spontaneously conceived (SC) children. Placental metrics (weight, diameter, thickness), however, were seen to be significantly larger in ART children. These differences were most closely associated with which ART technique was used and whether or not it included in vitro embryo culturing. DNAme and expression profiles of imprinted genes associated with imprinting disorders did not differ significantly between births with low and normal fetoplacental weight ratios, despite previous studies finding significant differences between ART and SC in the same dataset. When only considering births that were preterm, growth-restricted, or of abnormal birth weight for their gestational age, expression of imprinted genes was not seen to be significantly different between ART and SC. The findings of these studies suggest that an increase in placental size is rather consistent among ART births, while the altered expression and DNAme profiles sometimes reported for ART are less common and more stochastic. There is also some evidence to suggest that the increased placental size is largely attributable to the in vitro culturing and ART techniques themselves, while the changes in DNAme and gene expression sometimes seen may be more attributable to the underlying infertility.

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Pre-pregnancy obesity associates with adverse reproductive outcomes that impact maternal and fetal health. While obesity-driven mechanisms underlying adverse pregnancy outcomes remain unclear, local uterine immune cells are strong but poorly studied candidates. Uterine immune cells, particularly uterine natural killer cells (uNK), play central roles in orchestrating developmental events in pregnancy. However, the effect of obesity on uNK biology is poorly understood. Using an obesogenic high fat/high sugar diet (HFD) mouse model, I set out to examine the effects of maternal obesity on uNK composition and establishment of the maternal-fetal interface. HFD exposure resulted in weight gain-dependent increases in systemic inflammation and rates of fetal resorption. While HFD did not affect total uNK proprotions, HFD exposure led to an increase in natural cytotoxicity triggering receptor-1 (Ncr1) expressing uNKs as well as overall uNK activity. Importantly, HFD-associated changes in uNK coincided with impairments in uterine artery remodeling in mid-pregnancy, but these vascular alterations were no longer observed by late pregnancy. Comparison of uNK mRNA transcripts from control and HFD mice identified HFD-directed transcriptional changes in genes that play roles in promoting activity/cytotoxicity and vascular biology. Together, this work provides new insight into how obesity may impact uNK-processes central to the establishment of the maternal-fetal interface in early and mid-pregnancy. Moreover, these findings shed light on the cellular processes affected by maternal obesity that may relate to overall pregnancy health.

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Oxygen tension is thought to regulate many of the cellular and molecular processes that contribute to placenta development. An area of recent focus has been on the importance of a low-oxygen environment in controlling progenitor trophoblast differentiation along the invasive extravillous pathway. However, previously published research on the role of oxygen in trophoblast differentiation is conflicting. In this thesis, the effects of low (1%), physiologically normal (5%) and high (20%) oxygen conditions on extravillous trophoblast differentiation and column formation are examined using a human placental explant model. I show that culture in low oxygen conditions enhances column outgrowth and promotes the expression of pro-extravillous genes and gene pathways. By contrast, culture in high oxygen conditions promotes trophoblast proliferation, reduces column outgrowth and stalls differentiation along the extravillous pathway. I show that both low and physiologically normal oxygen conditions increase expression of the lysyl oxidase gene, and that this gene plays an important role in promoting extravillous column outgrowth. Together, these findings support hypoxia as an important factor driving trophoblast differentiation along the extravillous pathway. Additionally, this work provides new insight into specific molecular processes, regulated by oxygen tension, that may play an important role in early placentation.

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Assisted Reproductive Technology (ART) has been used for nearly four decades to help infertile couples achieve pregnancy. Such pregnancies are at a greater risk of restricted fetal growth and preterm birth, though whether these risks arise from environmental factors imposed on embryos cultured in vitro, or from genetic or epigenetic factors related to underlying infertility of the parents, remains unclear. Embryos generated using ART are frequently mosaic – having at least two genetically distinct cell populations within the same embryo. Mosaicism that persists in the cells that form the placenta can be observed as confined placental mosaicism (CPM), wherein genetic imbalances that may be fatal for a fetus are tolerated in the placenta, albeit with consequences such as restricted fetal growth, premature birth, and fetal demise. To investigate whether genetic imbalances and mosaicism occur more frequently for ART conceptions, term placentas from in-vitro fertilization (IVF) conceptions were analyzed at three distinct sites for large variations in chromosomal copy number. The incidence of CPM (4/82, 4.9%) was not significantly greater than what has previously been observed in term placentas from spontaneous conceptions (8/528, 1.5%, P=0.064, Fisher’s Test).The presence of constitutional genetic abnormalities and mosaicism were also investigated by karyotyping live-born infants from ART and natural conception pregnancies, and abnormalities were detected at similar rates between IVF (3/220, 1.36%), intracytoplasmic sperm injection (ICSI) (4/255, 1.57%), and naturally conceived cases (5/336, 1.49%).My results support observations that ART pregnancies are not at significantly greater risk of chromosomal abnormality or mosaicism than spontaneous pregnancies, and suggest that the poorer perinatal outcomes observed for ART conceptions may be attributable to other factors, such as small copy number variation or epigenetics.

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INTRODUCTION: In Canada, nearly half of women of child-bearing age are overweight (BMI 25-29.9 kg/m²) or obese (BMI ≥30 kg/m²). Current guidelines recommend moderate exercise for all pregnant women. However, little is known about the effect of exercise during pregnancy on the developing offspring. Rodent studies demonstrate that offspring from dams fed a high-fat diet have vessel-specific impairment in endothelium-dependent vascular function, greater adiposity, and impairments in glucose homeostasis. Further, offspring from exercised dams have improved glucose homeostasis. OBJECTIVE: In this thesis, I determined if maternal exercise during pregnancy mitigates the adverse effects of maternal obesity on offspring adiposity, glucose homeostasis, and vascular health in female offspring.METHODS: Dams (C57BL/6) were fed a control (10% energy from fat) or western diet (45% energy from fat) from weaning for 13 weeks and through breeding, pregnancy, and lactation. Just prior to breeding, dams were put into cages with or without a running wheel for voluntary exercise through breeding, pregnancy, and lactation. Offspring were weaned onto the control or western diet and maintained on the diet for 20 weeks; only female offspring were studied. Lean and fat mass were quantified by ¹H-NMR. Intraperitoneal insulin tolerance (IPITT), glucose tolerance (IPGTT) and insulin secretion tests (IPIST) were performed prior to the end of the feeding period. Vascular endothelial-dependent and independent dilatation were assessed ex vivo by isometric force measurements of aortic rings in response to phenylephrine, acetylcholine, and sodium nitroprusside.RESULTS: Weaning weights were greater in offspring from western-fed dams than control-fed dams. Control-fed offspring from western-fed dams had higher heart and kidney weights than those from control-fed dams. Control-fed offspring from exercised dams had higher blood glucose concentrations at IPGTT₃₀minutes than those from sedentary dams. Western-fed offspring from exercised dams had greater body weights, retroperitoneal fat weights, and Il10 mRNA in retroperitoneal adipose tissue, and lower blood glucose concentrations at IPGTT₉₀minutes than those from sedentary dams. Endothelium-dependent vasorelaxation of thoracic aortae was likely unaffected by maternal western diet or maternal exercise in control-fed and western-fed offspring.CONCLUSION: These findings suggest maternal exercise is beneficial in improving glucose homeostasis and adiposity in western-fed female offspring.

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Human placental development is a complex process dependent on continuous cross talk between fetal and maternal uterine compartments. Trophoblasts, placental cells of fetal epithelial lineage, invade the maternal uterine lining, forming the maternal-fetal interface. This interface is vital for controlling nutrient, gas, and waste exchange between maternal and fetal circulatory systems. The differentiation of progenitor trophoblasts into highly-specific cell subsets important for placental function is a highly-regulated developmental process. Trophoblast differentiation into invasive subtypes is essential for enhancing optimal uterine artery physiology, maternal-fetal nutrient exchange, and immunotolerance in pregnancy. Trophoblast invasion is controlled by cell-cell and cell-matrix interactions, as well as through production of various growth factors, cytokines, and hormones. Insufficient trophoblast invasion results in insufficient arterial remodeling that underlies cellular events responsible for the development of pregnancy disorders such as preeclampsia and fetal growth restriction. A Disintegrin and Metalloproteinase 28 (ADAM28) is a multi-functional protein belonging to the metzincin superfamily of metalloproteinases. ADAM28 exists as two alternative splice variants: a full-length transmembrane form (ADAM28-L) and a truncated secreted variant (ADAM28-S). Recently, ADAM28 has been shown to be differentially expressed in two distinct trophoblast subtypes ex vivo; however despite this knowledge, little is known about the role of ADAM28 in placental development or trophoblast biology. My research has demonstrated that both ADAM28 isoforms are highly expressed in invasive trophoblasts of distal regions in placental columns. Utilizing a loss-of-function strategy, I demonstrate that ADAM28 promotes trophoblast column outgrowth, and directs trophoblast cell migration and survival. Collectively, these findings describe a novel role for ADAM28 in regulating the differentiation of progenitor trophoblasts into extravillous trophoblast subsets and highlight ADAM28 as a key protease in human placental development.

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Placental development is a highly regulated process requiring signals from both fetal and maternal uterine compartments. Within this complex system, trophoblasts, placental cells of epithelial lineage, form the maternal-fetal interface controlling nutrient, gas and waste exchange. The commitment of progenitor villous cytotrophoblasts to differentiate into diverse trophoblast subsets is a fundamental process in placental development. Differentiation of trophoblasts into invasive stromal- and vascular- remodeling subtypes is essential for uterine arterial remodeling and placental function. Inadequate placentation, characterized by defects in trophoblast differentiation, may underlie the earliest cellular events driving pregnancy disorders such as preeclampsia and fetal growth restriction. Molecularly, invasive trophoblasts acquire characteristics defined by profound alterations in cell-cell and cell-matrix adhesion, cytoskeletal reorganization and production of proteolytic factors. A Disintegrin and Metalloproteinase 12 (ADAM12) is a multifunctional protein belonging to the ADAM family of metalloproteinases. ADAM12 exists as two alternatively spliced isoforms: ADAM12L (a transmembrane form) and ADAM12S (a truncated secreted form). ADAM12 is highly expressed in the human placenta and promotes matrix remodelling, cell proliferation and invasion in tumorigenesis. Importantly, low ADAM12 serum levels have been associated with pregnancies linked to poor pregnancy outcome. In spite of this knowledge, the importance of ADAM12 in directing trophoblast biology in early placentation is poorly understood. In this thesis, I have determined that both ADAM12 isoforms are expressed in distal ends of trophoblast columns, highly-invasive matrix-degrading trophoblasts and the invasive HTR8/SVneo trophoblastic cell line. Utilizing loss-of and gain-of-function strategies, I have demonstrated that ADAM12S plays a central role in promoting trophoblast invasion, as well as extravillous cytotrophoblast (EVT) column outgrowth through a mechanism requiring its intrinsic proteolytic activity. Moreover, I have identified cAMP signalling as the upstream regulator of ADAM12 expression and function in trophoblasts. Collectively, these findings describe a novel role for ADAM12 in directing an invasive phenotype in trophoblasts, and highlights ADAM12 as a key protease in human placental development.

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