Medicine, Faculty of
Obstetrics and Gynaecology, Department of
DSpace
UBCV
Ahmed, Faten
Doctor of Philosophy - PhD
University of British Columbia
Placental insufficiency disorders, including preeclampsia (PET), intrauterine growth restriction (IUGR) and preterm labor (PTL), are major obstetric complications that can have devastating effects on both the mother and the fetus. These syndromes share a common phenomenon of poor placental trophoblast cell invasion into the uterine tissue. To date there are no effective treatments for these illnesses. Placental invasion is controlled by many hormones and growth factors, among which the transforming growth factor (TGF)-β superfamily is a well-known regulator. Myostatin (MSTN) is a TGF-β superfamily member recognized for its important role in muscle growth control. Recently, MSTN was shown to be secreted and functioning in the placenta. In addition, MSTN serum and/or placental levels were found to be upregulated in common pregnancy complications including PET and IUGR. Yet, the role of MSTN in placental biology is poorly understood. In this study, I confirmed the positive effect of MSTN treatment on human trophoblast invasion and performed mRNA sequencing on control and MSTN-treated primary trophoblast cells (n=5). This analysis identified 610 differentially expressed genes (DEGs) with a false discovery rate <0.05, of which 380 were upregulated and 230 genes were downregulated. DEGs were highly enriched in epithelial mesenchymal transition (EMT) genes, among which serine protease inhibitor E2 (SERPINE2), plasminogen activator inhibitor-1 (PAI-1), SRY-Box Transcription Factor 4 (SOX4) and the cell adhesion molecule N-cadherin (N-Cad) were validated using RT-qPCR and Western blot. I then demonstrated that each of those genes was involved in MSTN-induced trophoblast cell invasion. Finally, I investigated the upstream signaling and found that MSTN induced upregulation of SERPINE2, PAI-1 and N-Cad via the canonical ALK4/5-SMAD2/3/4 signaling pathway. iv However, SMAD2 does not appear to be involved in MSTN-induced PAI-1 upregulation. In addition, SOX4 was involved in MSTN-induced SERPINE2 upregulation. Collectively, the current study deciphers the underlying molecular mechanisms involved in MSTN-induced human trophoblast cell invasion and provides insight into the functional consequences of its dysregulation in placental insufficiency disorders.
ROLE OF MYOSTATIN SIGNALING IN HUMAN TROPHOBLAST CELL INVASION by Faten Ahmed MD Assiut School of Medicine, Assiut University, Egypt 2000 Doctoral degree in Obstetrics and Gynecology, Assiut University, Egypt 2014 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (REPRODUCTIVE AND DEVELOPMENTAL SCIENCES) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) December 2022 © Faten Ahmed, 2022 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: Role of Myostatin Signaling in Human Trophoblast Cell Invasion submitted by Faten Ahmed in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Reproductive and Developmental Sciences Examining Committee: Dr. Peter C. K. Leung, Department of Obstetrics & Gynecology, UBC Supervisor Dr. Christian Klausen, Department of Obstetrics & Gynecology, UBC Supervisory Committee Member Dr. Michael Anglesio, Department of Obstetrics & Gynecology, UBC Supervisory Committee Member Dr. Michael Kobor, Department of Medical Genetics, UBC University Examiner Dr. Anthony Cheung, Department of Obstetrics & Gynecology, UBC University Examiner iii Abstract Placental insufficiency disorders, including preeclampsia (PET), intrauterine growth restriction (IUGR) and preterm labor (PTL), are major obstetric complications that can have devastating effects on both the mother and the fetus. These syndromes share a common phenomenon of poor placental trophoblast cell invasion into the uterine tissue. To date there are no effective treatments for these illnesses. Placental invasion is controlled by many hormones and growth factors, among which the transforming growth factor (TGF)-β superfamily is a well-known regulator. Myostatin (MSTN) is a TGF-β superfamily member recognized for its important role in muscle growth control. Recently, MSTN was shown to be secreted and functioning in the placenta. In addition, MSTN serum and/or placental levels were found to be upregulated in common pregnancy complications including PET and IUGR. Yet, the role of MSTN in placental biology is poorly understood. In this study, I confirmed the positive effect of MSTN treatment on human trophoblast invasion and performed mRNA sequencing on control and MSTN-treated primary trophoblast cells (n=5). This analysis identified 610 differentially expressed genes (DEGs) with a false discovery rate <0.05, of which 380 were upregulated and 230 genes were downregulated. DEGs were highly enriched in epithelial mesenchymal transition (EMT) genes, among which serine protease inhibitor E2 (SERPINE2), plasminogen activator inhibitor-1 (PAI-1), SRY-Box Transcription Factor 4 (SOX4) and the cell adhesion molecule N-cadherin (N-Cad) were validated using RT-qPCR and Western blot. I then demonstrated that each of those genes was involved in MSTN-induced trophoblast cell invasion. Finally, I investigated the upstream signaling and found that MSTN induced upregulation of SERPINE2, PAI-1 and N-Cad via the canonical ALK4/5-SMAD2/3/4 signaling pathway. iv However, SMAD2 does not appear to be involved in MSTN-induced PAI-1 upregulation. In addition, SOX4 was involved in MSTN-induced SERPINE2 upregulation. Collectively, the current study deciphers the underlying molecular mechanisms involved in MSTN-induced human trophoblast cell invasion and provides insight into the functional consequences of its dysregulation in placental insufficiency disorders. v Lay Summary The placenta is very important for pregnancy and needs to invade into the womb. Poor invasion may result in pregnancy complications including hypertensive preeclampsia as well as poor intrauterine fetal growth. Placental invasion is regulated by many factors including members of the transforming growth factor-β superfamily, of which myostatin was recently found to be expressed in the placenta and dysregulated in pregnancies with preeclampsia and poor intrauterine growth. I performed this study to examine the effect of myostatin on placental invasion and investigate the underlying genes by which it carries out this function. Using human placental cells, I showed that myostatin increases placental invasion via a specific group of genes. I also studied the pathway used by myostatin to affect some of those genes. My study illustrates a role for myostatin in pregnancy and helps in understanding the effects of its dysregulation in pregnancy complications. vi Preface This study was approved by the Research Ethics Board of the University of British Columbia (certificate #H07-01149). The research in this dissertation was conducted at the BC Children’s Hospital Research Institute by Faten Ahmed under the supervision of Dr. Peter C.K. Leung. Dr. Peter C.K. Leung and I generated the hypothesis and experimental design for this work. The bioinformatic analysis of the RNA seq data in Chapter 4 was performed by Yuyin Yi, a previous PhD candidate in Dr. Peter Leung`s lab at the BC Children`s Hospital Research Institute. The remaining experiments and data analyses were carried out by Faten Ahmed. A version of Chapters 4 and 6 has been published [(Myostatin Increases Human Trophoblast Cell Invasion by Upregulating N-cadherin via SMAD2/3-SMAD4 Signaling) Faten Ahmed, Christian Klausen, Hua Zhu, and Peter C.K. Leung. Biol Reprod. 2022 Jun 13;106(6), PMID: 35020826]. Most of this manuscript was written by Faten Ahmed, with critical edits from the other authors. A version of Chapters 4 (RNA sequencing data) and 5 is being prepared for journal submission. [(Upregulation of Serine Protease Inhibitors E1 and E2 Contributes to Myostatin-Induced Human Trophoblast Cell Invasion) Faten Ahmed, Christian Klausen, Yuyin Yi, Hua Zhu, and Peter C.K. Leung]. vii Table of Contents Abstract .................................................................................................................................. iii Lay Summary .......................................................................................................................... v Preface .................................................................................................................................... vi Table of Contents .................................................................................................................. vii List of Tables ........................................................................................................................... x List of Figures ........................................................................................................................ xi List of Abbreviations ........................................................................................................... xiv Acknowledgments .............................................................................................................. xviii Dedication .............................................................................................................................. xx Chapter 1: Introduction ..................................................................................................... 1 1.1 The Human Placenta ............................................................................................. 1 1.2 Epithelial-Mesenchymal Transition (EMT) ......................................................... 8 1.3 TGF-β Superfamily and Placenta ....................................................................... 12 1.4 Myostatin (MSTN) ............................................................................................. 15 1.5 Placenta-Mediated Diseases ............................................................................... 24 1.6 Models for the Study of Placental Development and Function .......................... 28 Chapter2: Rationale, Hypothesis and Objectives. ......................................................... 40 2.1 Rationale and Research Question ....................................................................... 40 2.2 Overall Hypothesis ............................................................................................. 40 2.3 The Objectives of the Study ............................................................................... 41 viii Chapter 3: Materials and Methods ................................................................................. 42 3.1 Antibodies and Reagents .................................................................................... 42 3.2 Primary Human Trophoblast Isolation and Culture............................................ 42 3.3 Transcriptomic Analysis Using RNA-Sequencing ............................................. 44 3.4 Gene Set Enrichment Analysis (GSEA) ............................................................. 45 3.5 Culture of HTR8/Svneo Immortalized Human Trophoblast Cells ..................... 45 3.6 Matrigel-Coated Transwell Invasion Assay ....................................................... 45 3.7 Reverse Transcription Quantitative Real-Time PCR (RT-qPCR) ...................... 46 3.8 Western Blot ....................................................................................................... 47 3.9 Small Interfering RNA (siRNA) Transfection ................................................... 48 3.10 MTT Assay ......................................................................................................... 48 3.11 Statistical Analysis.............................................................................................. 49 Chapter 4: Myostatin Increases Cell Invasion and Regulates Several Core EMT Genes in Primary Human Trophoblast Cells ................................................................. 50 4.1 Introduction......................................................................................................... 50 4.2 Results................................................................................................................. 52 4.3 Discussion ........................................................................................................... 56 Chapter 5: Upregulation of Serine Protease Inhibitors E1 and E2 Contributes to Myostatin-Induced Human Trophoblast Cell Invasion ................................................ 73 5.1 Introduction......................................................................................................... 73 5.2 Results................................................................................................................. 76 5.3 Discussion ........................................................................................................... 80 ix Chapter 6: Myostatin Increases Human Trophoblast Cell Invasion by Upregulating N-cadherin via SMAD2/3-SMAD4 Signaling ................................................................. 95 6.1 Introduction......................................................................................................... 95 6.2 Results................................................................................................................. 97 6.3 Discussion ........................................................................................................... 99 Chapter7: Conclusions ................................................................................................... 107 7.1 Summary of Findings ....................................................................................... 107 7.2 Significance and translational potential ............................................................ 111 7.3 Strengths of the study ....................................................................................... 113 7.4 Limitations ........................................................................................................ 114 7.5 Future Directions .............................................................................................. 116 References ............................................................................................................................ 118 Appendices........................................................................................................................... 147 Appendix A List of the DEGs in human first-trimester primary trophoblasts (MSTN vs Control) ............................................................................................................................. 147 Appendix B The hallmark genes enriched among the DEGs in MSTN-treated primary trophoblast cells as analyzed by GSEA. ........................................................................... 162 Appendix C The hallmark EMT genes differentially expressed in MSTN-treated primary trophoblast cells ................................................................................................................ 163 Appendix D List of the DEG in MSTN-treated primary trophoblast cells based on a FC of 1.3 ..................................................................................................................................... 165 x List of Tables Table 3. 1 Antibodies and Reagents Used for the Study ........................................................ 42 Table 3. 2 Primer Sequences Used for RT-qPCR Studies. ..................................................... 47 Table 4. 1 Top Significant KEGG Pathways Enriched Among the DEG with a 1.3 FC cut-off in MSTN Treated Human Primary Trophoblasts ........................................................... 60 Table 4. 2 Top Significant MSigDB Hallmark Terms Enriched Among the DEG (with a 1.3 FC cut-off) in MSTN-Treated Human Primary Trophoblasts ........................................ 61 Table 4. 3 Top Significant GO Biological Processes Enriched Among the DEG (with a 1.3 FC cut-off) in MSTN-Treated Human Primary Trophoblasts ........................................ 62 Table 4. 4 Top Significant GO Cellular Components Enriched Among the DEG (with a 1.3 FC cut-off) in MSTN-Treated Primary Trophoblasts ..................................................... 63 xi List of Figures Figure 1. 1 The journey of the human embryo. ........................................................................ 2 Figure 1. 2 Development of the human placenta during the first three weeks of gestation. .... 3 Figure 1. 3 Schematic diagram of trophoblast differentiation and spiral artery remodeling. ... 5 Figure 1. 4 Cellular modifications associated with EMT. ........................................................ 9 Figure 1. 5 MSTN precursor protein and its cleavage. ........................................................... 16 Figure 1. 6 Canonical MSTN signaling .................................................................................. 17 Figure 1. 7 Localization of MSTN in human primary isolated EVTs. ................................... 22 Figure 1. 8 Localization of MSTN in third-trimester placentae. ............................................ 23 Figure 1. 9 Placental study models ......................................................................................... 28 Figure 1. 10 Summary of human trophoblast invasion study models. ................................... 39 Figure 4. 1 MSTN increases cell invasion in HTR8/SVneo and primary human trophoblast cells. ................................................................................................................................ 64 Figure 4. 2 MSTN activates the canonical SMAD2/3 signaling pathway. ............................. 65 Figure 4. 3 SMAD2/3-SMAD4 signaling mediates the MSTN-induced trophoblast invasion. ........................................................................................................................................ 66 Figure 4. 4 MSTN-induced trophoblast cell invasion is mediated by activin/TGF-β type 1 receptors. ......................................................................................................................... 67 Figure 4. 5 MSTN regulates the gene profile expression in primary trophoblasts. ................ 68 Figure 4. 6 MSTN-treated primary trophoblasts were highly enriched in hallmark EMT genes as shown using GSEA and GO analyses. ....................................................................... 69 Figure 4. 7 Visualization of DEG in human primary trophoblasts treated with or without MSTN. ............................................................................................................................ 70 xii Figure 4. 8 KEGG pathway and GO analyses of DEG in human primary trophoblasts treated with or without MSTN using a fold change cut off. ....................................................... 71 Figure 4. 9 Comparison of DEGs in human primary trophoblasts treated with or without MSTN. ............................................................................................................................ 72 Figure 5. 1 MSTN upregulates SERPINE2 and PAI-1 mRNA levels in human trophoblast cells. ................................................................................................................................ 85 Figure 5. 2 MSTN upregulates SERPINE2 and PAI-1 protein levels in human trophoblast cells. ................................................................................................................................ 86 Figure 5. 3 SERPINE2 and PAI-1 are efficiently knocked down in human trophoblast cells as shown by RT-qPCR. ....................................................................................................... 87 Figure 5. 4 SERPINE2 and PAI-1 are efficiently knocked down in trophoblast cells as shown by Western blot. .............................................................................................................. 88 Figure 5. 5 SERPINE2 and PAI-1 are involved in MSTN-induced trophoblast invasion. .... 89 Figure 5. 6 MSTN-induced SERPINE2 and PAI-1 upregulations are mediated by activin/TGF-β type 1 receptors as shown by RT-qPCR. ................................................ 90 Figure 5. 7 MSTN-induced SERPINE2 and PAI-1 up-regulations are mediated by activin/TGF-β type 1 receptors as shown by Western blot. ........................................... 91 Figure 5. 8 MSTN upregulated SERPINE2 and PAI-1levels via canonical SMAD signaling pathway. .......................................................................................................................... 92 Figure 5. 9 MSTN induces trophoblast invasion through SOX4 upregulation. ..................... 93 Figure 5. 10 Role of SOX4 in MSTN-induced SERPINE2 and PAI-1 upregulation. ............ 94 Figure 6. 1 MSTN increases N-Cad production in HTR8/SVneo and primary human trophoblast cells. ........................................................................................................... 102 xiii Figure 6. 2 N-Cad is efficiently knocked down in human trophoblast cells using si-RNA. 103 Figure 6. 3 Knockdown of N-Cad attenuates MSTN-induced trophoblast cell invasion. .... 104 Figure 6. 4 MSTN-induced N-Cad upregulation and trophoblast cell invasion are mediated by activin/TGF-β type1receptors. ................................................................................. 105 Figure 6. 5 SMAD2/3-SMAD4 signaling mediates the upregulation of N-Cad by MSTN. 106 Figure 7. 1 Potential molecular mechanisms regulating MSTN-induced human trophoblast invasion. ........................................................................................................................ 110 xiv List of Abbreviations -Tub alpha tubulin °C degree Celsius 2D two dimensional 3D three dimensional ACVR2A activin A receptor type 2A ACVR2B activin A receptor type 2B ALK activin receptor-like kinase AMH anti-Mullerian hormone AMHR anti-Mullerian hormone receptor ANOVA analysis of variance BAMBI BMP and activin membrane bound inhibitor BMP bone morphogenetic protein BMPR bone morphogenetic protein receptor BP blood pressure BSA bovine serum albumin cDNA complimentary DNA CTB cytotrophoblast Ctrl Control DEG differentially expressed gene DMEM Dulbecco’s modified eagle medium DMSO dimethyl sulfoxide DOHaD developmental origins of health and disease xv ECM extracellular matrix eEVT endovascular extravillous trophoblast EGF epidermal growth factor EGFR Epidermal growth factor receptor EMT epithelial-mesenchymal transition EVT extravillous trophoblast FBS fetal bovine serum FC fold change FDR false discovery rate FGF fibroblast growth factor FPKM fragments per kilobase of transcript per million mapped reads FSTL3 follistatin-like 3 GAPDH glyceraldehyde 3-phosphate dehydrogenase GDF growth differentiation factor GDM gestational diabetes mellitus GO gene ontology GSEA gene set enrichment analysis GW gestational weeks HCG human chorionic gonadotropin HDAC histone deacetylase HELLP hemolysis, elevated liver enzymes and low platelet count hESC human embryonic stem cell HLA human leukocyte antigen xvi hPSC human pluripotent stem cell hrs hours hTSC human trophoblast stem cell ICM inner cell mass iEVT interstitial extravillous trophoblast IL interleukin iPSC induced pluripotent stem cell IUGR intrauterine growth restriction IVF in vitro fertilization KRT7 Cytokeratin-7 LRP LDL receptor related protein MAPK mitogen-activated protein kinase MMP matrix metalloprotease MSigDB molecular signal database MSTN myostatin MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide N-Cad N-cadherin PAI-1 plasminogen activator inhibitor-1 PBS phosphate buffered saline PCOS polycystic ovarian syndrome PET preeclamptic toxemia PI3K Phosphoinositide 3-kinase PS primitive syncytium xvii PTL preterm labor RNA-seq RNA sequencing RT-qPCR reverse transcription quantitative real-time polymerase chain reaction SDS-PAGE sodium dodecyl-sulfate polyacrylamide gel electrophoresis SEM standard error of mean SERPIN serine protease inhibitor siRNA small interfering ribonucleic acid, SMAD suppressor of mothers against decapentaplegic SOX4 SRY-Box transcription factor 4 STB syncytiotrophoblast t-PA tissue plasminogen activator TBS tris-buffered saline TCF-4 T-cell factor 4 TE trophectoderm TGF-β transforming growth factor-β TGF-βR transforming growth factor-β receptor u-PA urokinase type plasminogen activator uNK uterine natural killer cells xviii Acknowledgments This work wouldn’t have been achieved without the contribution of many personnel who supported and encouraged me throughout my PhD journey. I would like to thank Dr Peter Leung for his continuous support through all my PhD years. His lab offered me a friendly and a supportive working environment. He has been very encouraging and understanding throughout my PhD. My sincere gratitude goes to Dr Christian Klausen who has been always there whenever I had questions, confusions, or misunderstandings. His valuable advice and meticulous thorough revisions for this thesis and the work published out of it are remarkable. Moreover, he has been always available for me and my fellow lab mates anytime we needed help or guidance Also, I would like to offer my appreciation to my supervisory committee members; Dr Dan Rurak and Dr Michael Anglesio for their remarkable and valuable critiques for the current work. Dr Anglesio`s great experience in genomics and bioinformatics has been invaluable to accomplish this work. I am very grateful for our lab manager Dr Hua Zhu for her continuous technical and scientific support. Dr Zhu was the first person in the lab to teach me not only how to perform the experiments but also their rational and scientific basis. xix I would also like to thank Dr Yuyin Yi, who used to be my lab mate and who graduated earlier this year. Her help and support in analysing the RNA sequencing data has been crucial and vital in finishing this work. My gratitude is extended to all my previous and current lab mates in Dr Leung`s lab for their help and support during my PhD years. Additionally, I would like to extend my gratitude to my PhD colleagues; Nikita Telkar and Amy Inkster in Dr Robinson`s lab at the BC Children Hospital Research Institute for their valuable advice in RNA sequencing data presentation and analysis. This work would not have been done without the cooperation of the patients, doctors, and nurses at CARE program. The placental tissues provided by them have been crucial for performing the experiments included in this thesis. Moreover, the current work was supported by a CIHR grant and a graduate studentship award from Healthy Starts program at the BC Children Hospital Research Institute. Finally, I would like to thank my family for their continuous patience, support, and encouragement throughout my PhD years. This work wouldn’t have come to light if it wasn’t for them. xx Dedication To My Parents My always supportive husband, Mohamed My lovely kids; Yomna, Mariam Abdullah and Ali 1 Chapter 1: Introduction Placenta-mediated pregnancy complications account for a significant number of poor reproductive outcomes and can have negative impacts not only on maternal and fetal health [1], but also on well-being throughout childhood and into adulthood [2]. The human placenta is a highly invasive organ that must invade maternal tissues and vasculature to guarantee sufficient blood supply to the developing fetus. In addition to infertility, pregnancy loss and preterm labor (PTL) related to implantation defects, aberrant invasion of placenta-forming trophoblast cells and inadequate remodeling of the uterine vasculature are associated with early-onset preeclampsia (PET) and/or fetal intrauterine growth restriction (IUGR) [3, 4]. Conversely, excessive placental invasion can result in placenta accreta spectrum disorders, severe postpartum hemorrhage or metastatic malignant choriocarcinoma [4, 5]. To date, the molecular mechanisms that regulate placental development and invasion have yet to be fully deciphered. 1.1 The Human Placenta 1.1.1 Placental Development: It all starts when a sperm succeeds in penetrating the oocyte membrane and fertilizes it in the distal third of the fallopian tube to form the one-cell embryo, the zygote. This cell then undergoes several divisions while travelling from the tube to the uterine body. At around day 5/6 the dividing embryo, now called the blastocyst, arrives at the uterus where it will settle down (Fig.1.1). 2 The blastocyst is formed of two cellular compartments: the inner cell mass (ICM) and the outer trophectoderm (TE) layer. To anchor itself to the uterus, the trophectoderm attaches to the uterine lining which has undergone decidualization in preparation for implantation. The interaction between the polar TE (near the ICM) and the decidua starts the implantation process 6-7 days post-fertilization [7]. This process occurs in three successive steps: apposition, adhesion (attachment) and penetration (invasion) [8]. The ICM divides into the epiblast and hypoblast which form the embryo and the yolk sac, respectively. Moreover, both layers contribute to the formation of the extraembryonic mesoderm which in turn gives rise to the mesenchyme of chorionic villi as well as the umbilical cord [9]. After implantation, TE stem cells give rise to mononuclear cytotrophoblasts as well as the multinuclear primitive syncytiotrophoblast (PS) [10, 11]. The PS is the first invasive cell type that grows into the decidua and invades into the maternal uterine capillaries. At the time of PS expansion, rows of proliferative cytotrophoblasts break into the syncytium forming the primary villi which invade into the uterine vessels and glands. Extraembryonic mesoderm then migrates into the primary villi transforming them into secondary villi which become tertiary villi a few days later when hemangiogenic foci appear in the mesenchyme and initiate formation of the vascular network connecting the fetal and placental vessels [12]. The epithelial layer of the villi branches and expands by proliferation of villous Figure 1. 1 The journey of the human embryo. After ovulation, the oocyte is picked up by the fimbria of the fallopian tube where it meets the spermatozoa that will fertilize it in the distal third. The resultant zygote starts dividing while moving towards the upper part of the uterine body. It arrives there as a blastocyst around day5/6 post-fertilization and starts the implantation process (reproduced from [6]). 3 cytotrophoblasts which fuse into a multinuclear layer called the syncytiotrophoblast (Fig.1.2). Figure 1. 2 Development of the human placenta during the first three weeks of gestation. A, Human blastocyst implanting into the pregnant uterus. B, Development of the first placental structures and the embryonic disc. C, Formation of primary villi and yolk sac. D, Development of tertiary villi and the embryonic germ layers. AC amniotic cavity, CS connecting stalk, ChC chorionic cavity, CTB cytotrophoblast, DSC decidual stromal cell, Ec ectoderm, En endoderm, Ep epiblast, EVT extravillous trophoblast, ExC exocoelomic cyst, ExM extraembryonic mesoderm, Hy hypoblast, ICM inner cell mass, L lacunae system, LY lymphatic vessel, Md mesoderm, MS maternal blood sinusoid, pEC placental endothelial cell, PS primitive syncytium, pSC placental stromal cell, PV primary villi, PYo primitive yolk sac, SA spiral artery, TBS trophoblastic shell, TV tertiary villi, UC uterine capillary, UG uterine gland, ULE uterine luminal epithelium, VE venous vessel, vCTB villous CTB, Yo yolk sac (reproduced from [7]). 4 There are two types of mature villi; floating villi which move freely in the maternal blood and anchoring villi that anchor the developing fetus to the uterine wall. Cytotrophoblast cells at the distal ends of mature anchoring villi proliferate to form rows of proximal cell columns. These proximal column trophoblasts give rise to the distal cell column trophoblasts which stop undergoing mitosis and start invading into the decidual stroma as invasive extravillous trophoblasts (EVTs) [7]. Interestingly, these cells do not exit the cell cycle but, rather, undergo a process of endoreduplicative cycles culminating in the formation of giant cells at the junction between the inner third and outer two thirds of the myometrium [13]. These placental bed giant cells remain until term with a possible endocrine function [14]. The proliferating cells of the proximal cell columns are considered the pool of progenitor cells for the invasive EVTs. EVTs differentiate into 2 types of cells: interstitial and endovascular EVTs [15, 16]. Interstitial EVTs (iEVTs) invade into the uterine glands [17] as well as the lymphatics and veins [18]. Glandular EVTs are essential for the early histotrophic nutrition of the developing embryo, whereas invasion of decidual lymphatics and veins may facilitate drainage of blood and lymph fluid from the decidua and the placenta [19]. The endovascular EVTs (eEVTs) invade the spiral arteries and participate in the spiral artery remodelling process (Fig.1.3). 5 Uterine natural killer (uNK) cells and macrophages recruit iEVTs to invade the decidual stroma and participate in spiral artery remodelling that extends to the inner third of the myometrium (junctional zone myometrium) [21, 22]. The recruited iEVTs secrete cytokines such as IL-6 and IL-8 which in turn induce the secretion of vascular endothelial factors that attract the uNK cells into the vascular smooth muscle layer [21, 23]. These cells initiate the breakdown of vascular extracellular matrix (ECM) and are joined later by the Figure 1. 3 Schematic diagram of trophoblast differentiation and spiral artery remodeling. Mononuclear villous cytotrophoblasts differentiate into multinucleated syncytiotrophoblast or invasive EVTs. As trophoblasts differentiate along the invasive pathway, they migrate into and remodel decidual stroma and arterioles. This trophoblast-mediated remodeling of maternal stroma and vasculature ultimately increases the blood flow towards the placenta and fetus. vCT, villous cytotrophoblast; synCT, syncytiotrophoblast; cEVT, columnar extravillous cyto-trophoblast; eEVT, endovascular extravillous cytotrophoblast; iEVT, interstitial extravillous cytotrophoblast; mSA, maternal spiral artery; EC, endothelial cell; EG, endometrial gland; uNK, uterine Natural Killer cell; MF, macrophages; TGC, trophoblast giant cell (reproduced from [20]). 6 iEVTs [24, 25] which invade into the spiral arteries and acquire the vascular phenotype of eEVTs. These differentiated eEVTs induce apoptosis of endothelial cells and replace them. At the same time iEVTs stimulate apoptosis of the smooth muscle and break down the basal lamina, which is replaced by a fibrinoid matrix [22]. This process transforms spiral arteries from narrow high-resistance vessels into wide vasomotor-irresponsive vascular conduits of low-resistance that allows the flow of large volumes of blood at low speed, thus ensuring maximum perfusion and preventing injuries resulting from high blood flow [15, 16]. Interestingly, complete blood flow through spiral arteries does not commence until 12-13 gestational weeks (GW) [26] when the placenta develops its antioxidant defence in the form of free radical scavengers like superoxide dismutase and catalase [27]. eEVTs form trophoblast plugs that prevent blood flow during early pregnancy to create a hypoxic environment that protects the developing fetal organs from oxidative stress and promotes placental development as well as vasculogenesis and angiogenesis [7]. Before the establishment of hemochorial circulation, the developing embryo depends on maternal glandular secretions referred to as histotrophs [28]. 1.1.2 Placental Function The placenta is vital to fetal development as it anchors the fetus to the mother and performs multiple functions that are important for the development of a healthy pregnancy. The syncytiotrophoblasts secrete many factors and hormones that help the mother adapt to pregnancy and help the fetus grow. Some examples of these factors include human chorionic gonadotropin (HCG), estrogen, progesterone, placental growth factor and placental lactogen, among others [29]. Moreover, the placenta is a critical transport conduit for nutrients and 7 waste products between the mother and the fetus. It also acts as the fetal lung, being the site of oxygen and carbon dioxide exchange [30]. Furthermore, the placenta is a metabolically active organ that synthesizes substances like glycogen, fatty acids and cholesterol, and stores others such as iron, vitamins, and glycogen [30]. Another important function of the placenta is immunotolerance, which prevents the mother’s body from attacking the fetus who has a different genetic makeup from her. EVTs interact with maternal decidual stromal cells, uNK cells and macrophages to allow for immunological acceptance of the placental-fetal allograft [31, 32]. Similarly, the placenta acts as a protective barrier to many harmful substances and organisms that might be present in the maternal blood [30]. 8 1.2 Epithelial-Mesenchymal Transition (EMT) For the placenta to properly carry out its functions, it needs to undergo a tightly controlled process of invasion into the uterine wall. One of the mechanisms used by distal column trophoblasts to migrate and invade into the decidua and inner myometrium is called EMT [33]. 1.2.1 Definition of EMT EMT is a process where epithelial cells undergo several biochemical changes to acquire characteristics of mesenchymal cells. These changes involve aspects of cell shape, polarity, adhesion, and motility [34]. Epithelial cells have apical-basal polarity and are attached to the basal lamina and neighbouring cells via inter-cellular adhesion complexes, including adherens junctions, tight junctions, gap junctions and desmosomes [35]. On the other hand, mesenchymal cells are flat, spindle shaped and have antero-posterior polarity that facilitates their motility. The main features marking EMT are the loss of cell-cell adhesion and changes in attachment to extracellular matrix (ECM), especially the loss of cell attachment to the basal lamina [36] (Fig.1.4). 9 1.2.2 Types of EMT There are three main types of EMT [38]. Type 1 occurs during embryonic development and involves gastrulation and neural crest formation [39]. Type 2 is found in wound healing, tissue regeneration and organ fibrosis [40]. Type 3 is involved in cancer formation and metastasis [41]. The EMT process used during CTB/EVT differentiation is unique and does not belong to any of the previous three types [42]. Some researchers refer to Figure 1. 4 Cellular modifications associated with EMT. After activation of the EMT program, epithelial cells switch off the expression of epithelial markers, such as E-cadherin and cytokeratin, and turn on expression of mesenchymal markers, including N-cadherin and fibronectin. Conversely, mesenchymal cells can re-differentiate into epithelial cells by mesenchymal-epithelial transition. Depending on the tissue and signaling context, epithelial cells may lose only some characteristics or may show both epithelial and mesenchymal properties; this can be considered a partial EMT. EMT, epithelial-mesenchymal transition. (Reproduced from [37]). 10 it as type 0 as it occurs very early during fetal development and precedes all the other types [42]. During the very first weeks of pregnancy, the EVTs change their epithelial phenotype and acquire some mesenchymal characteristics that enable them to migrate and invade into the maternal decidua and vessels [33]. To date, the molecular mechanisms of placental trophoblast differentiation and invasion are still not completely understood. But given the similarity between the placenta and malignant cells, one may extrapolate some of these mechanisms from cancer biology. Both placental and cancer cells are highly proliferative, migratory, invasive and can escape the immune system [43, 44]. However, EVTs are under tight control by the uterine environment [44] to limit their invasion beyond the inner third of the myometrium [15]. Also, EVTs undergo a “transient EMT” whereby the EMT process slows down and EVTs become immotile and non-invasive by the end of the second trimester [45]. Interestingly, third trimester EVTs isolated from abnormally invasive placentae exhibit EMT characteristics similar to their first trimester counterparts [46]. Indeed, EVTs undergo terminal differentiation into trophoblast giant cells, a process that appears to add an extra layer of protection against deeper invasion [7]. 1.2.3 Molecular Regulation of EMT For EMT to occur a well-orchestrated series of molecular signals is required to activate specific transcription factors and signaling pathways that end in regulation of downstream target genes which directly affect cell adhesion profiles, motility, and polarity [47]. The transforming growth factor-β (TGF-β) superfamily is a well-known molecular regulator of EMT [48]. Epidermal growth factor (EGF) and its receptors are other strong 11 inducers of EMT during placental development [47]. Also, SNAIL, SLUG, TWIST, TCF-4/ β catenin complex and SOX4 are among the important EMT transcription factors [47, 49]. The resultant downstream target genes that induce the phenotypic changes characteristic of the EMT process include, among others, adhesion molecules, proteases, and integrins [47]. During EMT, E-cadherin (E-Cad) is down regulated while N-cadherin (N-Cad) is upregulated. This is often referred to as the cadherin switch and is one of the hallmarks of the EMT process [38]. EMT in trophoblast differentiation may be considered “partial” as the main epithelial marker cytokeratin-7 (KRT7) is not downregulated in EVTs [47]. Also, the mesenchymal marker vimentin is not induced in EVT cells [47]. Unsurprisingly, abnormal trophoblast invasion can arise if the EMT molecular machinery is dysregulated ending in placenta-mediated diseases. Since the TGF-β superfamily is a well-known EMT inducer, investigating the effects of its members on placental development and function may help in understanding the complex molecular mechanisms underlying placenta-mediated diseases. 12 1.3 TGF-β Superfamily and Placenta 1.3.1 Members and Signaling of TGF-β Superfamily The TGF-β superfamily is a diverse group of more than 40 growth factors that play vital roles in different biological processes and are mainly characterized by the presence of the cystine knot growth factor domain [50]. The superfamily has several subfamilies including, TGF-β, bone morphogenetic proteins (BMPs), activins and inhibins, growth differentiation factors (GDFs), anti-Mullerian hormone (AMH) and nodal [50]. To execute their functions, each factor binds a type-2 transmembrane serine threonine kinase receptor which in turn phosphorylates and activates a type-1 transmembrane serine threonine kinase receptor [51]. There are five type-2 receptors: TGF-β receptor 2 (TGFβR2), activin receptor type-2A (ACVR2A), activin receptor type-2B (ACVR2B), BMP receptor type-2 (BMPR2) and AMH receptor type 2 (AMHR2). There are seven type-1 receptors designated as activin-like kinase (ALK)1-7 [51]. Interestingly, most of the TGF-β ligands and their receptors are located at the maternal-fetal interface, suggesting important roles in the placentation process [52]. Phosphorylated type-1 receptors phosphorylate and activate receptor-regulated small mothers against decapentaplegic (R-SMADs), including SMAD1/5/8 and SMAD2/3, which in-turn form a complex with common SMAD4 and translocate to the nucleus to act as transcription factors regulating the expression of target genes [53]. Differential effects among TGF-β ligands occurs, in part, via binding to different receptors and R-SMADs. For example, TGF-βs, activins, and nodal bind to ALK4/5/7 in combination with type 2 receptors ACVR2A, ACVR2B or TGFβR2, and activate SMAD2/3-mediated signaling. In contrast, BMPs, GDFs [except for myostatin (MSTN) and GDF11], and AMH bind to ALK2/3/6 in 13 combination with type 2 receptors ACVR2A, ACVR2B, BMPR2 or AMHR2, and activate SMAD1/5/8-mediated signaling [54]. SMAD-dependant signaling is the canonical pathway for TGF-β superfamily members; however, they can also signal through SMAD-independent pathways involving mitogen activated protein kinases (MAPKs), phosphatidylinositol 3-kinase (PI3K)/AKT, and RAS /Rho GTPase [55]. In addition, the quantitative and qualitative effects of TGF-β superfamily ligands are regulated by type-3 receptors (co-receptors) like betaglycan, endoglin, and cripto [50]. Other regulators that have been shown to neutralize the function of TGF-β ligands include antagonists like follistatin, follistatin-like protein 3 (FSTL3), noggin, BMP and activin membrane binding inhibitor (BAMBI), chordin, and gremlin [50]. 1.3.2 Role of TGF-β Superfamily in the Placenta In humans TGF-β superfamily members regulate many cellular differentiation and developmental processes including placentation [56]. The TGF-β subfamily comprises three isoforms (TGF-β1, TGF-β2, TGF-β3) which are all expressed by the placenta and endometrium [56]. TGF-β subfamily ligands were shown to supress trophoblast proliferation and promote syncytialization [57, 58]. However, reports regarding their role in EVT invasion are conflicting, as some studies show that TGF-β subfamily inhibits EVT invasion [59] while others have shown the opposite effect [60, 61]. Similarly, three isoforms of activin (activin A, B and C) are expressed in the placenta and endometrium [62] and have been shown to promote trophoblast syncytialization [63] and invasion [64-66]. In contrast, inhibin A and inhibin B promote syncytialization [63] but suppress trophoblast invasion [67]. 14 Nodal has been reported to suppress trophoblast proliferation [68] and EVT invasion [69]. Moreover, nodal along with fibroblast growth factor 4 (FGF-4) creates a stemness promoting microenvironment for trophoblast stem cells by inhibiting their precocious differentiation [70]. The BMP subfamily contains about 20 isoforms [71] of which, only BMP2 and BMP4 have been shown to play a role in placentation [56]. BMP2 induces trophectoderm differentiation into trophoblasts [72] and promotes trophoblast invasion [73], while BMP4 stimulates syncytialization of cytotrophoblast cells [74]. Other members such as MSTN have been shown to increase trophoblast proliferation [75], migration [75] and invasion [76]. Given the important roles of TGF-β superfamily signaling in placental development, it is not surprising that serum and/or placental levels of TGF-β1 [77], TGF-β2 [78], TGF-β3 [79], nodal [69], activin A [80, 81], inhibin A [82], MSTN [83], GDF15 [84], and AMH [85] are dysregulated in PET. Similarly, serum/ placental levels of TGF-β3 [86], activin A [87], inhibin A [87], inhibin B [88] and MSTN [83] are abnormal in cases of IUGR. Women who have a miscarriage have also been shown to have dysregulated serum/placental levels of TGF-β1 [89], TGF-β3 [90], and inhibin A [91]. 15 1.4 Myostatin (MSTN) 1.4.1 Structure of MSTN MSTN is a member of the TGF-β superfamily belonging to the activin/inhibin subfamily [92] and shares 90% sequence identity with GDF11[92]. It was discovered by McPherron et al. in 1997 when they inactivated the myostatin gene (MSTN), giving rise to a hyper-muscular mouse [93]. MSTN is made up of two exons and three introns that encode 375 protein residues [93]. Although MSTN is a highly conserved secretory protein [94], a number of naturally occurring mutations have been reported in a variety of species including humans [95-97]. MSTN inhibits both the growth and differentiation of skeletal muscles, hence the name “myostatin.” High levels of MSTN are linked to muscle wasting and cachexia associated with old age, liver disease, AIDS, malignancy, and other debilitating disorders [98]. MSTN is synthesized as a pro-protein precursor consisting of a signal peptide, a large N-terminal pro-peptide, and a smaller C-terminal mature peptide. The immature precursor undergoes a proteolytic cleavage via a furin to remove the signal peptide. In general, MSTN exists in an inactive dimer state non-covalently bound to the pro-peptide. A second proteolytic cleavage occurs by a BMP-1/Tolloid family enzyme to free the mature peptide dimer [99, 100] (Fig. 1.5). 16 MSTN activity is controlled at both the transcriptional and post-translational levels. After its cleavage from pro-MSTN, mature MSTN is stored in the extracellular space as a latent complex bound to its pro-peptide that prevents it from interacting with its receptors [101, 102]. Besides the pro-peptide, other proteins like follistatin and follistatin-like proteins are known to bind and inactivate MSTN [103]. However, these proteins are not MSTN-specific and can bind to and antagonize other TGF-β superfamily members like activin, GDF11 and members of the TGF-β subfamily [104]. Several factors activate the release of active MSTN from its latent complex including proteases, free radicals, low pH, and hypoxia [105-107]. Interestingly, the latent MSTN complex is not completely inert and has been shown by luciferase reporter assay to have some activity which can be blocked by WFIKKN1 (GASP2) and WFIKKN2 (GASP1) [108]. 1.4.2 Signaling and Function of MSTN MSTN functions in both autocrine and paracrine manners [93, 109]. The active dimer binds to two type-2 transmembrane serine-threonine kinase receptors, mainly ACVR2B [110]. Which then phosphorylates and activates two type-1 receptors, mainly ALK5 and to a lesser extent ALK4 [111]. ALK7 is not usually involved in MSTN signaling [111]. Activation of ALK4/5 leads to the downstream phosphorylation of SMAD2 and Figure 1. 5 MSTN precursor protein and its cleavage. The MSTN precursor protein consists of 3 parts: signal peptide, N-terminal pro-peptide and C-terminal mature peptide. The precursor is hydrolyzed twice to produce the active mature peptide dimer. The first is via a furin enzyme family to remove the signal peptide. The second cleavage is by a BMP-1/Tolloid family enzyme to remove the pro-peptide. S-S, disulphide bond (original picture created in PowerPoint). 17 SMAD3 (R-SMADs). Phosphorylated SMAD2/3 then binds with common SMAD4 to form a heterotrimeric complex that translocates to the nucleus as a transcription factor to modulate gene expression. This SMAD-dependent signaling is the canonical signaling pathway for MSTN (Fig.1.6), however there is evidence that MSTN can also signal in a SMAD-independent fashion via ERK, p-38 MAPK, and JNK pathways [112-114]. MSTN is mainly synthesized and expressed in skeletal muscles [98] and plays an important role in negatively regulating their growth and development [109]. The increase in muscle size associated with MSTN knockout is due to both hyperplasia and hypertrophy of muscle fibers [115]. MSTN is also expressed in other tissues such as brain [94], mammary Figure 1. 6 Canonical MSTN signaling Active MSTN dimer binds to two ACVR2B receptors which in turn phosphorylate two ALK4/5 type1 receptors that subsequently activates SMAD2/3. Phosphorylated SMAD2/3 form a complex with the common SMAD4. The SMAD2/3/4 complex enters the nucleus and acts as a transcription factor to regulate the expression of the MSTN target genes. (original picture created in BioRender). 18 [116], adipose [117], cardiac [118], endometrial [119], ovarian [120], as well as placental [121] tissues. MSTN regulates muscles during development and adulthood periods. During development, MSTN inhibits myogenesis by suppressing myoblast growth and differentiation [122, 123]. However, in adults, MSTN inhibits the activation and self-renewal of muscle stem cells, known as satellite cells [124]. MSTN serum or muscle levels are elevated in catabolic conditions associated with muscle wasting, including cancer [125], AIDS [126], cardiac failure [127], renal failure [128], chronic obstructive pulmonary disease [129], aging [130] and prolonged bed rest [131]. MSTN also plays a role in metabolic regulation [132, 133], obesity [134] and insulin resistance [135]. Thus, it is considered by some researchers as the holy grail of muscle, bone, and fat [136]. Indeed, MSTN knockout mice are hyper-muscular with less adipose tissue, improved metabolic status, and higher bone mineral density [93, 117, 137, 138]. Accordingly, MSTN inhibition is currently being tested in many clinical trials to study its effectiveness in treating different musculoskeletal and metabolic diseases [139]. 1.4.3 Human MSTN Gene Mutations Genetic studies have shown several mis-sense mutations in the coding region of human MSTN including E164K, I225T, P198A, A55T, and K153R [140]. The last two mutations are polymorphisms in the general population [140]. The K153R mutation is the most frequent [141] and consequential, resulting in low muscle strength, obesity, and extreme longevity [142]. Its effects have been attributed to higher proteolysis and activation of pro-myostatin by furin, without any changes in cleavage of latent MSTN by BMP-1/Tolloid enzyme, leading to an overall increase in the amount of latent MSTN. This leads to 19 an overall increase in MSTN effects such as low muscle strength and obesity, since latent MSTN is shown to have a significant activity [108], leading to higher overall levels of active MSTN [143]. 1.4.4 Myostatin and the Female Reproductive Organs MSTN is expressed in the female reproductive system, including granulosa and theca cells [143], follicular fluid [120], endometrial cells [119], myometrial cells [144], and trophoblasts [75]. Moreover, it has been shown to be dysregulated in several reproductive disorders such as polycystic ovary syndrome (PCOS), leiomyoma, endometriosis, adenomyosis, PET, IUGR and gestational diabetes mellitus (GDM). PCOS affects 6% of adolescent girls and 8-13% of reproductive age women, making it the most common endocrine reproductive disorder. PCOS is characterized by androgen excess, irregular menstruation, and infertility [145]. PCOS patients are more prone to obesity, diabetes, anxiety, and depression [146]. PCOS patients tend to display higher serum MSTN levels [147], however smaller studies have reported no differences between women with PCOS and controls [148]. MSTN is expressed in human granulosa cells and follicular fluid [120]. Granulosa and theca cells of large antral follicles of PCOS patients express higher levels of MSTN and its receptors (ACVR2A, ACVR2B and ALK5) compared to those of normal ovaries [143]. Moreover, MSTN levels in follicular fluid of PCOS patients are higher than those of control women [149]. These levels are even higher in PCOS patients with insulin resistance compared to those without insulin resistance [150]. Interestingly MSTN is found to inhibit glucose uptake of granulosa lutein cells and thus may play a role in metabolic dysregulation in PCOS [150]. 20 Follicular fluid MSTN levels are inversely correlated to progesterone levels suggesting a role for MSTN in inhibiting premature luteinization [151]. MSTN may also play a role in ECM regulation and proliferative activity of granulosa cells by up regulating lysyl oxidase and connective tissue growth factor [152, 153]. Accordingly, some have suggested that during in-vitro fertilization (IVF) cycles a higher MSTN level before HCG administration is important to keep progesterone levels low and prevent premature luteinization. In contrast, lower MSTN levels after HCG administration may be essential for implantation and the continuation of pregnancy by maintaining higher progesterone levels [154]. Indeed, serum levels of MSTN are higher in poor ovarian response patients undergoing controlled ovarian stimulation, underlining the role of MSTN in ovarian response in IVF cycles and suggesting its potential role as a predictor for IVF response [155]. In summary, MSTN regulates follicular development and ovulation. Serum MSTN is elevated in PCOS and poor ovarian response patients. Similarly, MSTN levels are upregulated in follicular fluid and follicular cells of PCOS patients. MSTN may play an important role in metabolic dysregulation in PCOS patients. Leiomyoma is a very common benign tumour arising from the myometrium. It affects 20-40% of reproductive age women, usually presents with heavy uterine bleeding, abdominal mass, and may be associated with infertility, recurrent miscarriages and PTL [156, 157]. Expression of MSTN, but not its receptors, is higher in leiomyoma compared to normal adjacent myometrium [144]. Like skeletal muscles [158] healthy myometrial cells respond to MSTN treatment by upregulating SMAD7 [144] and decreasing cell proliferation via a SMAD signaling [159]. However, this is not the case with leiomyoma due to higher levels of FSTL3 in leiomyoma cells that antagonizes MSTN actions. [144]. Moreover, 21 MSTN levels in leiomyoma cells are positively correlated with MMP14 levels and both significantly correlate with the intensity of dysmenorrhea (pain during menstruation) [160]. Estrogen treatment significantly decreases MSTN expression in human myometrium [144]. In summary, MSTN plays a role in the pathogenesis of leiomyoma and dysmenorrhea. Endometriosis is a relatively common chronic inflammatory condition characterized by the presence of endometrial tissue outside the uterus and is classified into three types: superficial peritoneal lesions, ovarian endometrioma, and deep infiltrating endometriosis [161]. Women with endometriosis may present with chronic pelvic pain, dysmenorrhea, pain during intercourse or micturition and infertility [162]. Interestingly, MSTN and its receptors are highly expressed by ovarian endometrioma and deep infiltrating endometriosis, suggesting potential pro-invasive effects of MSTN [163]. Moreover, endometrial adenocarcinoma has also been shown to express higher levels of MSTN and its receptors compared to healthy endometrium [163]. In summary, MSTN might play a role in upregulating endometrial cell invasion as implied from its higher expression levels in deep infiltrating endometriosis and endometrial adenocarcinoma. Adenomyosis is a benign gynecological condition characterized by the presence of endometrial glands among the myometrial cells and usually presents with chronic pelvic pain, dysmenorrhea, heavy menstruation, and infertility [164, 165]. A widely accepted mechanism for the development of adenomyosis is that endometrial glandular cells undergo EMT and invade the underlying myometrium [166]. Interestingly, adenomyotic tissue has been shown to express high levels of MSTN, follistatin as well as ACVR2A and ACVR2B [167]. 22 MSTN mRNA and protein are expressed in primary placental tissues throughout gestation [121] as well as in trophoblast cell lines (HTR8/SVneo, BeWo, JAR, and JEG3) [168]. Also, MSTN receptor ACVR2B is expressed in human third trimester placenta as well as primary and immortalized EVTs [75]. Furthermore, MSTN protein is localized to EVTs of first trimester (Fig. 1.7) and term human placentas (Fig. 1.8) [75]. Figure 1. 7 Localization of MSTN in human primary isolated EVTs. A, EVTs isolated from first-trimester placental villous tissues positive for MSTN. B, HLA-G-positive EVTs. C, KRT7-positive trophoblast cells. D, Negative staining of EVTs for vimentin. E and F, Negative staining for isotype controls for the rabbit and mouse antibodies used in immunocytochemistry and secondary antibody interactions (G and H). Staining was viewed using a Nikon Eclipse 80i microscope with a Nikon D5-Ri1 camera and NIS-Elements imaging software (Nikon) and presented at 10x magnification (and 40x magnification for MSTN in A). MSTN, myostatin; HLA-G, human leukocyte antigen; KRT7, cytokeratin7; EVT, extravillous trophoblast (reproduced from [75]). 23 Placental MSTN expression is negatively correlated with gestational age, being more abundant in early pregnancy [121], suggesting a potential role in placental development. Furthermore, MSTN has been shown to increase human trophoblast migration [75], invasion [76, 169] and glucose uptake [121]. Importantly, placental and/or blood levels of MSTN are dysregulated in common pregnancy complications including PET [83, 170], IUGR [83] and GDM [171]. Moreover, MSTN has been suggested as a potential biomarker for GDM because its early second trimester serum levels are higher in pregnancies that eventually develop the disease [172]. Figure 1. 8 Localization of MSTN in third-trimester placentae. A and B, Localization of MSTN in EVT of third-trimester placentae. C, HLA-G positive EVT. D, KRT7-positive trophoblast cells. E, Negative staining of EVT for vimentin. F and G, Negative staining for isotype controls for the rabbit and mouse antibodies used in immunocytochemistry and secondary antibody interactions (H and I). Human muscle tissue positive for MSTN (J) is shown. Sections were imaged with Aperio ScanScope XT digital scanner at 40x magnification. MSTN, myostatin; HLA-G, human leukocyte antigen; KRT7, cytokeratin7; EVT, extravillous trophoblast (reproduced from [75]). 24 1.5 Placenta-Mediated Diseases 1.5.1 Definition of Placenta-Mediated Diseases A heterogenous group of diseases that are linked to abnormal placental development usually related to trophoblast invasion and affect about 5% of pregnancies [1]. 1.5.2 Aetiology and Pathophysiology of Placenta-Mediated Diseases The underlying pathophysiology of placenta-mediated diseases is related to the process of trophoblast invasion. Excessive placental invasion results in placenta accreta spectrum of abnormally adherent placentae that are usually complicated by severe maternal haemorrhage and maternal mortality [173]. While uncontrolled invasion causes a malignant type of trophoblast disease called choriocarcinoma [174]. On the other hand, poor trophoblast invasion results in insufficient spiral artery remodeling and placental insufficiency. This leads to pregnancy complications including PET, IUGR, PTL and still birth [1]. These diseases are commonly termed the “Great Obstetric Syndromes” which refers to conditions that occur in pregnancy and share the following criteria: 1) multiple aetiologies, 2) with fetal involvement, 3) have a long pre-clinical period, 4) result from a complex interaction between mother, fetus, and the environment, and 5) are adaptive in nature [175]. Importantly, these complications can extend far beyond the intra-uterine and neonatal periods to affect childhood and adulthood as well, according to the theory of “Developmental Origin of Health and Disease” (DOHaD). This theory hypothesizes that the origin of some non-communicable childhood and adulthood disorders such as obesity, cardiovascular diseases, metabolic syndrome, asthma, psychiatric illnesses, and diabetes is rooted in the intra-uterine and neonatal periods [176-178]. 25 Defective deep placentation, which is a common feature in placenta-mediated diseases, is defined as absent or incomplete remodeling of the junctional zone myometrial spiral arteries [179]. Placental blood flow is regulated by two main factors, the size of the placental bed which is determined by the number of spiral arteries, and the depth of the spiral artery remodeling [179]. Accordingly, defective deep placentation can be classified according to the extent of the junctional zone myometrial spiral artery remodelling into 1) partial transformation as in cases of PTL and IUGR without hypertension, 2) absent transformation as in PET without IUGR, 3) absent transformation with obstructive lesions as in cases of PET with IUGR [179]. PET is defined as new onset of hypertension (systolic BP ≥140 and/or diastolic BP ≥90) and proteinuria or new onset of hypertension and significant end organ dysfunction with or without proteinuria after 20 GW or postpartum [180]. It affects 4.6% of pregnancies on a global scale [181]. Eclampsia is the occurrence of convulsions complicating 2% of cases of PET [182]. PET/eclampsia are leading causes of maternal morbidity and mortality worldwide [183] leading to 60,000 maternal deaths / year [184, 185]. Moreover, approximately 10% of perinatal deaths occur in pregnancies complicated by PET [186]. Moreover, preeclamptic/eclamptic mothers and their babies who survive may suffer many negative consequences. In the mother, PET may result in renal failure, HELLP (hemolysis, elevated liver enzymes, and low platelet counts), liver failure and seizures. While fetal complications include IUGR, intrauterine fetal death, and iatrogenic prematurity [187]. Moreover, PET can cause an increased risk for long-term maternal and fetal cardiovascular abnormalities [188, 189]. 26 In 1991, Rodman introduced the two-stage model to explain the underlying pathological mechanism of PET [190]. The first stage of this model is the pre-clinical stage and is caused by defective spiral artery remodelling resulting in placental ischemia and consequent release of harmful placental products into the maternal circulation. The second stage is the clinically overt maternal illness and is a consequence of the placental ischemia stage [190]. In 1993, Roberts and Redman suggested that endothelial dysfunction is the link between the 2 stages of the model [191]. In 1996, Ness and Roberts introduced the terms placental and maternal types of PET [192]. Placental PET refers to early-onset disease that occurs before 34 weeks, accounts for around 10% of cases, and is usually severe and complicated. It is due to an abnormality in placental circulation and is commonly associated with IUGR. In contrast, the maternal type defines the late-onset PET that occurs at or after 34 weeks, accounts for most cases, and is usually less severe. According to this classification, the underlying pathology of late-onset PET is not related to the placenta but to pre-pregnancy maternal inflammatory diseases that cause her vascular system to react abnormally to pregnancy stressors [192]. The original 2-stage PET model [190] can explain early-onset type PET but not the maternal (late-onset type) PET. In 2019, this model was updated such that placenta and maternal factors come to contribute to the occurrence of both early- and late-onset PET [193]. Placental compression around term, due to uteroplacental malperfusion secondary to placental outgrowth beyond the uterine capacity, results in placental hypoxia and the release of factors contributing to the systemic vascular inflammation associated with late-onset PET. According to the updated 2-stage model, there is no maternal form of PET without involvement of the placenta [193]. Thus, PET is a result of a stressed placenta which secretes 27 factors that cause maternal systemic vascular pathology or supress factors that stabilize vascular and immune interfaces [194]. It is very important to differentiate between early and late-onset PET as their underlying pathology and molecular changes are completely different. A recent RNA sequencing analysis shows that early-onset severe PET has 2,977 DEGs, while late-onset severe and mild PET had only 375 and 42 DEGs respectively. This attests to the different underlying molecular mechanisms between both types and implies less involvement of genetic factors in the pathogenesis of mild PET [185]. IUGR, another common placenta-mediated disorder, is commonly defined as an impaired fetal growth and development with an estimated fetal weight below the 10th percentile and is usually associated with placental insufficiency [195]. It affects 5-10% of pregnancies and commonly ends in PTL, intrauterine fetal death and perinatal mortality [196]. IUGR is due to fetal causes in 70-80% of cases [197]. Maternal or placental causes account for 20-30% of IUGR cases [197]. Like PET, there are two types of IUGR depending on the time of onset (early and late). Early-onset IUGR occurs before 32 weeks and is associated with extensive placental insufficiency compared to the late-onset type [196]. Although early onset IUGR is discovered in the late second or beginning of third trimester, the underpinning pathology starts earlier during first trimester [198]. The placenta is vital for fetal growth being responsible for nutrient and oxygen transport to the fetus. Early IUGR in high income countries is usually due to placental insufficiency while malnutrition during pregnancy is the main cause in low-income countries [197]. Placental dysfunction in early IUGR includes malformed placental villi and defective trophoblast proliferation and invasion [197]. 28 1.6 Models for the Study of Placental Development and Function Given the important role played by the placenta during pregnancy and in the development of pregnancy complications, it is important to understand the underlying molecular mechanisms controlling placental development. However, this process is an early pregnancy event (first 10-12 GW) and thus using easily accessible term placenta obtained after birth is not optimal. Therefore, it is of utmost importance to develop various systems that allow us to study trophoblast cell development and function. These models can be generally divided into in vivo, ex vivo, in vitro and in silico (Fig.1.9). Figure 1. 9 Placental study models Placental models can be classified according to the site into in vivo, ex vivo, in silico and in vitro. In vitro systems can be categorized according to the type of studied cells or the type of the culture system. TB, Trophoblast; hTSCs, human trophoblast stem cells; hESCs, human embryonic stem cells; hiPSCs, human induced pluripotent stem cells; TE, trophectoderm; CTB, cytotrophoblasts from first trimester placenta; 2D, 2-dimensional; 3D, 3dimentional. 29 1.6.1 In Vivo Animal Models In vivo models refer to those where placental development and function are investigated inside a living organism. Different animal models are used including mouse, rat, guinea pig, rabbit, sheep, and non-human primates. Despite the numerous merits to the use of those animals, there are many shortcomings related to inter-species differences in the gestation period as well as the placental structure and function. The mouse is a popular and commonly used model given the low cost of breeding, high litter number, and feasibility of producing genetically edited strains [199]. Moreover, it has a hemochorial placenta like humans where the fetal trophoblasts are in direct contact with the maternal blood. However, the additional presence of a yolk sac placenta functioning throughout pregnancy, the short gestation period (19-21 days), and the shallow placental invasion (not surpassing the decidua basalis) are among the features that limit the utility of mouse models for studying human placental development [199, 200]. Other models include guinea pigs, sheep, and non-human primates. Guinea pigs have a longer gestation period (65-70 days) and a deeply invasive placenta that traverses the myometrium and reaches the serosa. Sheep are expensive to breed and have a small litter size and a non-invasive placenta [200]. Although non-human primates can be good models for studying placental development, their use is very limited due to ethical considerations and high cost of breading. Another in vivo placental model is the human placental xenograft [201, 202] where human placental tissue is grafted into nude mice. Interestingly, the grafted tissue maintains its immunohistochemical structure and hormonal secretory function [201, 202]. However, the environment in which placental tissue is grafted is very different from the unique maternal uterine milieu. 30 Consequently, there is no existing animal model that can completely recapitulate the distinctive aspects of human placental development and function [199]. In addition, ethical and financial restrictions regarding the use of animals in research has led to the recent implementation of the 3R movement, which proposes the reduction, replacement, and refinement of animal use in scientific research [203]. Hence, there has been a drive to develop alternative models derived from human cells to study the distinctive structure and function of the human placenta. 1.6.2 Ex Vivo Models A placental perfusion model is established where placental structure is kept intact and perfused while in culture [204]. This model gives insight into placental transport and metabolism of nutrients and new drugs of unknown effects on pregnancy [205] and is still considered the gold standard in such studies [206] especially due to absence of any ethical restriction. However, placental perfusion model is not without limitations. It is short lived and cannot be maintained more than 24hrs in culture [203]. Moreover, this model is usually used with term placental tissue which doesn’t truly reflect placental transport in early pregnancy when it is very important to explore effects of fetal exposure to potential teratogenic factors. Additionally, placental perfusion system doesn’t account for non-placental factors such as fetal metabolism and clearance [207]. 1.6.3 In Vitro Models In vitro models can be classified by either the type of cultured cells or the nature of the culture system (Fig. 1.9). 31 1.6.3.1 Types of Cultured Cells 1.6.3.1.1 Placental tissue explants and primary trophoblasts Like ex vivo systems, tissue explants maintain the relationship between maternal and fetal cells while having the potential to be cultured for a relatively longer period (10-12 days) [208]. Villous explants are used to study trophoblast proliferation and differentiation. Moreover, explants isolated from first trimester placentas may be used to study EVT invasion. However, the use of explants is limited by the availability of fresh placental tissue. Nevertheless, advances in cryopreservation and thawing technology can overcome this limitation [209]. Also, villous explant outgrowth can be interpreted differently by researchers as trophoblast proliferation or trophoblast migration [32]. Another limitation of villous explants is the difficulty to attribute effects to a specific cell type because the explant is a cocktail of different cell types. In this case, isolation of primary trophoblasts from villous explants [210, 211] may solve this problem. Two-step isolation methods have been developed which involve trypsinization of placental villi followed by different purification techniques to isolate specific cell types [212]. Another method entails culturing mechanically isolated first trimester placental villi and using outgrowing trophoblasts in downstream studies after their characterization [57, 210]. There are four criteria that if used in combination can define a mononuclear trophoblast. These include methylation of the ELF5 promoter and the expression of the chromosome 19 mi-RNA cluster (C19MC) as well as a panel of protein markers [213]. For a mononuclear cell to be a trophoblast, it needs to have a hypomethylated ELF5 promoter and to express C19MC, KRT7, TFAP2C, and GATA3. If all the above criteria are present, then the cell is either an EVT or a villous trophoblast. EVTs are positive for human leukocyte 32 antigen (HLA)-C, -E and -G, whereas villous trophoblasts are negative for the pan HLA class I molecules including HLA-G [213]. Yet, a major limitation with the use of primary trophoblasts is their scarcity and limited life span in culture. 1.6.3.1. Trophoblastic Cell Lines: To overcome the limitations of primary cell models, trophoblastic cell lines have been developed and are widely used in the field of placental research. Trophoblast cell lines are stable throughout multiple passages and each cell line has the same cell progeny, facilitating the reproducibility of data [209]. Trophoblast cell lines are classified according to the International Federation of Placenta Associations (IFPA) into three types: 1) cell lines derived from malignant tissue (e.g., BeWo, JAR, JEG3), 2) cell lines generated from normal tissue (e.g., HTR8, HTR8/SVneo, SGHPL), and 3) cell lines generated from embryonal carcinoma with evidence of trophoblast differentiation (e.g. H9, HT-H) [214]. Choriocarcinoma cell lines are commonly used to study the placental barrier [215]. In addition, BeWo cells are used as a model for syncytiotrophoblasts to study syncytialization and hormone secretion [216]. Choriocarcinoma cell lines are not appropriate to study trophoblast proliferation and invasion due to their malignant nature. However, HTR8/SVneo cells are often used as an EVT cell model to investigate trophoblast proliferation, migration, and invasion [217, 218]. These cells are derived from first trimester trophoblasts transfected with the simian virus 40 large T antigen [219]. The suitability of using HTR8/SVneo cell lines is debatable due to the presence of a mixed population of epithelial and mesenchymal cells [220]. However, the presence of mesenchymal cells is likely due to an EMT process that occurs in HTR8/SVneo cells while in culture rather than an initial contamination [217]. Still, methods that prolong 33 the life span of trophoblast cell lines, including transfection with immortalization factors and spontaneous transformation, can affect the function and genetic make-up of these trophoblastic cell lines [221]. 1.6.3.1.3 Human Stem Cells: Inadequacies of the above-mentioned models have necessitated the development of better systems to be used in placental research. Stem cells are unspecialized and have the potential to differentiate to different cell types. They are present both in embryo and adult cells. Stem cells are classified according to this potential into totipotent, pluripotent, multipotent, oligopotent, and unipotent [222]. Human totipotent stem cells can develop into all cell types and are only present in the zygote. Human embryonic stem cells (hESCs) are a type of human pluripotent stem cells (hPSCs) derived from the ICM. Another example of hPSCs is induced pluripotent stem cells (iPSCs) derived from the somatic adult cell that can be reprogrammed back into an embryonic-like pluripotent state and can differentiate into different cell types [222]. The spectrum of differentiation narrows with multipotent and oligopotent stem cells until it becomes unipotent and can only differentiate into one type of specialized cell [222]. Human trophoblast stem cells (hTSCs) are multipotent cells that refer to a placental niche that can differentiate into different types of trophoblasts [223]. Trophoblasts can be derived from hESCs [224], iPSCs [225], TE of blastocyst [226] and pure villous cytotrophoblasts isolated from early first trimester placenta [226]. hESCs can produce trophoblast-like cells if cultured in media containing BMP4 [224]. However, this media results in a mixed lineage of mesoderm and trophoblasts rather than a pure trophoblast one [227]. The media can be further optimized to include 34 activin/nodal inhibitor and FGF receptor inhibitor [228]. hESCs develop into TP63 positive cytotrophoblast progenitor cells before their final differentiation into syncytiotrophoblast and EVTs [229]. Krendel et al. identified cells positive for the transcription factors GATA2, GATA3, TFAP2A, and TFAP2C upstream of the TP63 positive cells. These transcription factors are expressed by TE cells and known to inhibit pluripotency and induce trophoblast lineage genes [230]. Recently, Horri et al. obtained a pure cytotrophoblast progenitor lineage when culturing hESCs in media containing BMP4 and a wingless-related integration site (Wnt) inhibitor [231]. Importantly, hESCs and iPSCs allow the opportunity to study not only trophoblast development and differentiation but also trophoblast lineage [232]. Nevertheless, these models are not without limitations and trophoblasts derived from hESCs do not perfectly replicate primary trophoblasts in terms of expression of characterizing markers [233]. One example is the absence or low expression of ELF5 in trophoblasts derived from hESCs [234]. Accordingly, researchers felt a need to develop a model which closely resembles the human trophoblast cell. Recently, Okae et al. succeeded in deriving hTSCs from TE of blastocysts and cytotrophoblasts of first trimester placenta [226]. These hTSCs express the pan-trophoblast marker (CK7), cytotrophoblast markers (TP63 and TEAD4) and the mononuclear trophoblast marker (GATA3) along with a hypomethylated ELF5 promoter [226]. These cells can self-renew and propagate in culture using EGF, Wnt activator and inhibitors for TGF-β, Rho associated protein kinase (ROCK) and histone deacetylase (HDAC). In addition, they can be cryopreserved for a later use. These hTSCs can differentiate into EVTs when cultured on 35 Matrigel in the presence of neuregulin-1and TGF-β inhibitor. Also adding the cAMP agonist forskolin, allows the differentiation of hTSCs into syncytiotrophoblasts. 1.6.3.2 Types of Culture System Trophoblast cells can be cultured in isolation (monotypic) or in co-culture with other cell types (complex). 1.6.3.2.1 Monotypic Culture System This is the commonly used system for placental research. It contains only one cell type and is suitable for studying the effect of different factors on isolated trophoblasts (e.g., effects of adding or removing specific growth factors on trophoblast function). Examples of this system include the culture of isolated primary EVTs and trophoblastic cell lines. 1.6.3.2.2 Complex Culture System This system contains more than one type of differentiated trophoblasts cultured with or without other cell types such as decidual (stromal, uNK and macrophages) and endothelial cells. This system is ideal for studying interactions between different cell types and is a better representation of in vivo conditions where different trophoblast cells are not present in isolation but with other cells that affect their ultimate function and response to investigated factors. Cells in monotypic and co-culture systems are cultured in either a 2D or a 3D format. 2D Culture Systems This is the commonly used system in which cells attach to the culture vessel substrate, lose their original spherical architecture, and acquire a flat 2D shape. Trophoblast culture on plastic petri dishes is an example of monotypic 2D culture system. While the transwell membrane system which is commonly used for co-culture of trophoblasts and decidual cells to study the effects of paracrine decidual secretions on trophoblast function is 36 an example of the 2D complex culture system [235]. Similarly, the transwell co-culture system can be used to simulate the placental barrier for transport studies where trophoblast cells and endothelial cells are cultured in the upper and lower sides of the porous membrane, respectively [236]. hTSCs are also cultured in a defined culture media using this 2D system where they propagate and differentiate into EVTs and syncytiotrophoblasts [226]. 3D Culture Systems These systems are developed to maintain the 3D structure of cultured cells and tissues to create more anatomically and physiologically relevant culture conditions. Examples of this system include spheroid, organoid, and microfluidic systems. Spheroid systems were originally developed to study tumor progression and consist of clusters of single cells or cell aggregates grown without ECM on low attachment plates [203]. The system is also adapted for use with placental cells [237, 238]. Co-culture of trophoblast spheroids and decidual cells are used to study the effects of decidual factors on trophoblast expansion [239]. Cells also keep their 3D structure when cultured embedded in an ECM. An organoid is a 3D organ-like structure originating from human stem cells embedded in ECM. Using a defined culture media, stem cells propagate and differentiate into different organ specific cell types that self-organize to resemble their natural equivalent structurally and functionally [240]. Placental organoids constitute a major advance and are gaining much popularity lately. Placental organoids can originate from iPSCs [225] or pure villous cytotrophoblast (CTB-organoid) [241-243]. CTB-organoids develop from pure villous CTBs isolated from early first trimester placentas (6-7 GW) and embedded in Matrigel in the presence of a defined culture media containing Wnt activator, EGF, and TGF-β as well as BMP-signaling inhibitors [241]. 37 Villous CTB proliferate into spheroid-like structures in which cell proliferation decreases toward the centre where cell fusion occurs forming multi-nuclear STB-like cells. Thus, the CTB-organoids have an inverse organization compared to that of the in vivo placental villous epithelium where cell fusion occurs towards the outer part of the villi [241, 243]. Moreover, CTB towards the periphery of the organoid can differentiate into EVT like cells by the removal of Wnt activator. This shows the bipotentiality of hTSCs as they can differentiate into both types of cells; STB and EVTs. Interestingly CTB-organoids can self-propagate in culture for >1year and are of close anatomical, genetic, and functional resemblance to placental villi in vivo [241, 243]. CTB-organoids offer a great opportunity to biobank TSCs from patients with different placental mediated diseases [244]. This will enable us to study and understand the underlying molecular mechanisms of such diseases. The application of microfluidic technology has gained attention from placenta researchers recently and led to the development of the placenta on a chip system. It provides a microscale structure of the placenta in vivo. Moreover, it mimics in vivo conditions by allowing continuous perfusion of the culture and enabling the creation of chemical gradients [245]. This system is invaluable for the study of drug transport across the placental barrier [246-248]. The placental barrier is also modeled in a 3D microfluidic device using a 3D printing technique called 2 photon polymerizations [249]. Immortalized trophoblasts and endothelial cell lines are commonly used in these systems however, given the shortcomings of these cell lines, combining stem cell and microfluidic technologies to develop patient specific chips will be invaluable for individualized precision medicine [203]. 38 1.6.4 In Silico Models In silico models are computer-based mathematical replicas developed to study and predict placental development under physiological and pathological scenarios [250]. These models combine the advantages of in vivo and in vitro systems and can circumvent ethical- and cost-related concerns [251]. Importantly they allow for the simultaneous study of different parameters, thus better simulating the complexity of the in vivo human placenta [203]. 1.6.5 Models of Trophoblast Invasion The transwell invasion assay is commonly used to study trophoblast invasion. Trophoblasts are cultured on Matrigel-coated transwell inserts with a chemoattractant in the lower well [66, 252, 253]. Matrigel is mainly composed of laminin, collagen IV, entactin and heparin sulphate proteoglycan [254] which are among the natural components of the placental bed along with fibronectin [255, 256]. When 3D spheroids of EVTs are included, they allowed for a more complex cell-cell interaction. Indeed, EVTs migrate from the spheres and invade through Matrigel only when endometrial stromal cells are plated in the lower well [257]. Bioprinting can incorporate different biomaterials and growth factors to more closely mimic the in vivo invasion process [258, 259]. In addition, microfluidics is used to design models of invasion where stable gradients can be created on a chip [260, 261]. Fig.1.10 summarizes the different models used to study trophoblast invasion. 39 Figure 1. 10 Summary of human trophoblast invasion study models. (a) Diagram of trophoblast invasion of the endometrium in vivo. (b) A transwell model with a monolayer of trophoblasts invading a gel towards the chemoattractant in the well. (c) A spheroid model in a well plate with trophoblasts invading the underlying gel towards a monolayer of cells (d) A bio-printed model consisting of concentric rings of a gel, with trophoblasts in the outermost layer and a chemoattractant in the innermost layer (e) A microfluidic device with trophoblasts suspended in a gel in the center channel with medium flowing through the channels on either side. The chemoattractant is included in only one media channel in this design (reproduced from [218]. 1.6.6 Shortcomings of Current Models: Biomechanical cues such as tissue elasticity and stiffness are important in placental development [262] and these cues are poorly represented in available models [218]. The absence of immune cells especially Hoffbauer cells from current models is another hurdle [218]. Although a perfusable vasculature culture system for placental cells is still lacking [218], a vascularized microfluidic placental model perfused by blood serum obtained from women with placenta-mediated disease is currently under study [218]. Such models will be invaluable in placental research and clinical fields. 40 Chapter2: Rationale, Hypothesis and Objectives. 2.1 Rationale and Research Question Being an indispensable organ for a healthy pregnancy, the placenta needs to invade through the uterine wall to perform its different vital functions. This process is tightly and precisely regulated [3, 4]. The TGF-β superfamily members are among the important regulators of placental invasion [52]. MSTN, a relatively newly discovered member of this superfamily [93] is abundantly expressed in skeletal muscles [98] but was also found in other organs including the ovary [120], uterus [119] and placenta [121]. MSTN mRNA and protein are expressed in primary placental tissues throughout gestation [121] as well as in commonly used cell lines (HTR8/SVneo, BeWo, JAR, and JEG3) [168]. It is localized to EVTs in first trimester and term human placentas [75]. Placental MSTN expression is negatively correlated with gestational age, being more abundant in early pregnancy [121]. Compelling evidence suggests that placental and/or blood levels of MSTN are altered in pregnancies complicated with PET [83, 170], IUGR [83] and gestational diabetes [171]; however, the functions of MSTN in placental biology remain poorly studied. MSTN was found to increase human trophoblast migration [75] and invasion [76, 169], however little is known about the molecular mechanisms underlying these effects in trophoblast cells. To this end, my research was designed to delineate the role of MSTN in human trophoblast invasion and clarify the molecular mechanisms regulating this process. 2.2 Overall Hypothesis MSTN increases human trophoblast cell invasiveness by regulating genes involved in the EMT process through the canonical ALK4/5-SMAD2/3/4 signaling pathway. 41 2.3 The Objectives of the Study Objective 1: To study the effects of MSTN on human trophoblast cell invasion and identify target genes essential for those effects. Objective 2: To investigate the function and signaling pathway of serine protease inhibitors (SERPINs) E1 and E2 in MSTN-induced trophoblast cell invasion. Objective 3: To investigate the involvement of N-Cad and its signaling pathway in MSTN-induced human trophoblast cell invasion. 42 Chapter 3: Materials and Methods 3.1 Antibodies and Reagents Table 3. 1 Antibodies and Reagents Used for the Study Reagent Species Company Catalogue# Concentration Myostatin Mouse myeloma cell-derived recombinant human myostatin R&D systems 788-G8-010 5-100ng/mL SB431542 NA Sigma Aldrich S4317 5-10μM Anti-cytokeratin 7 Mouse monoclonal Santa Cruz Biotechnology sc-23876 1:100 Anti-HLA-G Mouse monoclonal ExBio, 11-499-C100 1:100 Anti-SERPINE2/Nexus Mouse monoclonal Life Span Biosciences LS-C173926 0.5 μg/mL Anti-α-Tubulin Mouse monoclonal Santa Cruz Biotechnology sc-23948 0.01 μg/mL Anti-PAI-1 Mouse monoclonal BD Transduction laboratories 612024 0.1 μg/mL Anti-SMAD2 Mouse monoclonal Cell Signaling Technology 3103 1 μL/mL Anti-phosphoSMAD2 Rabbit monoclonal Cell Signaling Technology 3108 1 μL/mL Anti SMAD3 Rabbit monoclonal Cell Signaling Technology 9523 1 μL/mL Anti phosphoSMAD3 Rabbit monoclonal Cell Signaling Technology 9520 1 μL/mL Anti-SMAD4 Rabbit polyclonal Cell Signaling Technology 9515 1 μL/mL Anti-SOX4 Mouse monoclonal Santa Cruz Biotechnology sc-518016 0.5 μg/mL Anti-N-Cad Mouse monoclonal BD Biosciences 610920 0.001 μg/mL Anti-mouse 2° antibody Horseradish peroxidase-conjugated goat Bio Rad 170-6516 0.2 μL/mL Anti-rabbit 2° antibody Horseradish peroxidase-conjugated goat Bio Rad 170-6515 0.25 μL/mL 3.2 Primary Human Trophoblast Isolation and Culture This study was approved by the Research Ethics Board of the University of British Columbia (certificate #H07-01149) and all patients provided informed written consents. First 43 trimester (6-10 GW) human placental tissues were collected from patients undergoing elective pregnancy termination. Primary human trophoblast cells were isolated from chorionic villous explants as previously reported [211]. Briefly, chorionic villous tips were washed in cold Dulbecco’s modified phosphate buffered saline (PBS; Gibco, Cat# 14190144), minced into small pieces, and cultured in Dulbecco's Modified Eagle's medium (DMEM; Gibco, Cat# 11965092) containing 10% (vol/vol) fetal bovine serum (FBS; Gibco, Cat# 12483020), 100 U/mL penicillin and 100 µg/mL streptomycin (Gibco, Cat# 15140122). Cultures were incubated in flasks at 37°C in a humidified 5% CO2/air atmosphere for 3–4 days after which non-attached pieces were washed away. Attached villous tissue fragments were cultured for an additional 10-14 days to allow for outgrowth of trophoblast cells, which were subsequently separated from attached fragments by trypsinization. Trophoblasts were used from passages 2-5 and each experiment performed with primary trophoblast cells was replicated using cells isolated from placentas of at least 3 separate patients. Vehicle and MSTN treatments were conducted in DMEM supplemented with 0.1% FBS following a period of 18-24 hr starvation in DMEM supplemented with 0.1%FBS. Immunofluorescence Primary trophoblast cells were cultured on chamber slides, fixed with 4% paraformaldehyde for 10 min at room temperature, and permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) for 5 min. Slides were blocked with serum-free protein block (X0909, Dako) for 1 hr at room temperature and then incubated with anti-HLA-G and anti-KRT7 overnight at 4°C (see Table 3.1 for details on all antibodies). Slides were then washed with PBS and incubated for 1 h at room temperature with Alexa Fluor 594-conjugated goat anti-mouse secondary antibody (see Table 3.1 for details on all antibodies) and mounted using 44 ProLong Gold mounting media containing 4′,6-diamidino-2-phenylindole (DAPI; Life Technologies; Cat# P36931). 3.3 Transcriptomic Analysis Using RNA-Sequencing Primary trophoblasts from 5 patients were cultured with or without 50 ng/mL MSTN for 24 hrs and total RNA was extracted using the RNeasy Mini Kit. Sample quality control was performed with a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, California, United States) and qualifying samples (RNA integrity number > 9) were prepared following the standard protocol for Next Ultra II Stranded mRNA (New England Biolabs, Ipswich, Massachusetts, United States). RNA sequencing was performed on an Illumina NextSeq 500 with paired end 42bp x 42bp reads at the Biomedical Research Centre (University of British Columbia, Canada). Reads were aligned with STAR to the UCSC Homo sapiens hg19 (GRCh37) reference sequence [263]. Fragments per kilobase of transcripts per million reads (FPKMs) and differential expression of reference transcripts were calculated using Cufflinks2 [264]. Identified DEGs satisfying the significance parameters were expressed as q-values representing false discovery rate (FDR)-adjusted P value < 0.05. DESeq2 package in R was used for analysis of DEGs, accounting for both patients and treatments. Genes ≥10 FPKM with adjusted q-values of less than 0.05 (P<0.05) and fold change (FC) ≥ ±1.5 were considered significantly differentially expressed. Gene Ontology (GO) enrichment and pathway analysis were performed using the Enricher online tool (https://maayanlab.cloud/Enrichr/). 45 3.4 Gene Set Enrichment Analysis (GSEA) Differentially expressed sequences with a Benjamini-Hochberg adjusted p-value < 0.05 were considered significant. Sequences were ranked in ascending order of FC to generate the ranked list format (.rnk) required by the GSEA software (v4.2.2) [265] downloaded from the Molecular Signatures Database (http://www.gsea-msigdb.org/gsea/msigdb/index.jsp). GSEA was used to test for enrichment using default parameters (nperm = 1000, enrichment statistic = weighted) with the Human HGNC_ID as the chip platform (MSigDB v7.4) and with the ‘no collapse to gene symbols’ option. The hallmark gene sets from the database were chosen to run the enrichment analysis against the DEG. Gene sets were considered significant if the FDR<25%. 3.5 Culture of HTR8/SVneo Immortalized Human Trophoblast Cells The HTR8/SVneo simian virus 40 large T antigen-immortalized human trophoblast cell line was generously provided by Dr. P. K. Lala (Western University, Canada) [219]. Cells were cultured in DMEM supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin, and 10% (vol/vol) FBS. Cultures were incubated at 37°C in a humidified 5% CO2 air atmosphere and the medium was changed every other day. Each experiment was replicated using at least 3 different passages. Vehicle and MSTN treatments were conducted as described above for primary human trophoblast cells. 3.6 Matrigel-Coated Transwell Invasion Assay Cell invasiveness was measured using 24-well transwell inserts (8 µm pore size; BD Biosciences) coated with 40 µL of growth factor-reduced Matrigel (BD Biosciences) at a concentration of 1 mg/mL. Inserts were seeded with 5 x 104 cells in 250 µL DMEM/F12 46 medium supplemented with 0.1% (vol/vol) FBS, and 750 µL medium with 10% (vol/vol) FBS was added to the lower chamber. Cells were incubated at 37°C for 48 hours (hrs), after which non-invading cells were wiped from the upper side of the membrane and cells on the lower side were fixed with cold methanol (-20°C) and air dried. Cell nuclei were stained with Hoechst 33258 (Sigma-Aldrich), imaged with a fluorescent microscope, and analyzed using Image J software (National Institutes of Health). Duplicate inserts were used for each individual experiment, and five random microscopic fields were counted per insert. 3.7 Reverse Transcription Quantitative Real-Time PCR (RT-qPCR) Cells were washed with cold PBS and total RNA was extracted with Trizol Reagent (Life Technologies) according to the manufacturer’s instructions. Reverse transcription was performed using 2 µg of total RNA, random primers, and Moloney murine leukemia virus reverse transcriptase (Promega, Madison, Wisconsin, United States; Cat# M170A) in a final volume of 20 µL. RT-qPCR was performed on an Applied Biosystems 7300 or Quant Studio 3 real-time PCR system equipped with 96-well optical reaction plates. Each 20 µL RT-qPCR reaction contained 1xSYBR Green PCR Master Mix (Applied Biosystems), 12 ng cDNA, and 250 nM of each specific primer. The primers used are summarized in Table 3.2 and the specificity of each assay was confirmed by dissociation curve analysis. At least three separate experiments were performed, and each sample was assayed in triplicates. A mean value was used for the determination of mRNA levels by the comparative Cq method with Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the reference gene and using the formula 2-ΔΔCq. 47 Table 3. 2 Primer Sequences Used for RT-qPCR Studies. Target Forward 5` to 3` Reverse 5` to 3` SERPINE2 TGGTGATGAGATACGGCGTAA GTTAGCCACTGTCACAATGTCTT PAI-1 GAGAAACCCAGCAGCAGATT TGGTGCTGATCTCATCCTTG F2RL1 TGGATGAGTTTTCTGCATCTGTCC CGTGATGTTCAGGGCAGGAATG N-Cad GGA CAG TTC CTG AGG GAT CA GGA TTG CCT TCC ATG TCT GT SOX4 AGCGACAAGATCCCTTTCATTC CGTTGCCGGACTTCACCTT SMAD2 GCC TTT ACA GCT TCT CTG AAC AA ATG TGG CAA TCC TTT TCG AT SMAD3 CCC CAG CAC ATA ATA ACT TGG AGG AGA TGG AGC ACC AGA AG SMAD4 TGG CCC AGG ATC AGT AGG T CAT CAA CAC CAA TTC CAG CA GAPDH GAG TCA ACG GAT TTG GTC GT GAC AAG CTT CCC GTT CTC AG 3.8 Western Blot Cells were twice washed with cold PBS and lysed in ice-cold lysis buffer (Cell Signaling Technology, Danvers, Massachusetts, United States; Cat# 9803) with added protease inhibitor cocktail (Sigma-Aldrich, Cat# P8340). Extracts were centrifuged at 13,000 rpm (8000 g) for 15 minutes at 4°C, and supernatant protein concentrations were determined using the DC protein assay kit (Bio-Rad Laboratories, Mississauga, Ontario, Canada; Cat# #5000111) with bovine serum albumin (BSA) as the standard. Equal amounts of protein (30 µg) were separated by SDS-PAGE and electro-transferred to polyvinylidene fluoride membrane (Bio-Rad Laboratories). Membranes were blocked with Tris-buffered saline containing 5% (wt/vol) non-fat dry milk or BSA for 1 hr and incubated overnight at 4°C with 48 primary antibodies diluted in Tris-buffered saline with 5% (wt/vol) non-fat dried milk or BSA and 0.1% (vol/vol) Tween-20. After incubation with horseradish peroxidase-conjugated secondary antibody at room temperature for 1 hr, bound antibodies were visualized using enhanced chemiluminescent (CL) or Super Signal West Femto CL substrates (Thermo Fisher) and CL-Exposure film (Thermo Fisher). Membranes were stripped with stripping buffer [50 mM Tris-HCl, (pH 7.6), 10 mM β-mercaptoethanol, and 1% (wt/vol) SDS] at 50°C for 50 min and re-probed with anti-α-tubulin, SMAD2 or SMAD3 antibodies as loading controls. Densitometric quantification was performed using Image-J software with α-tubulin, SMAD2 or SMAD3 as internal controls for normalization. 3.9 Small Interfering RNA (siRNA) Transfection Fifty percent confluent cells were transfected for 48 hrs with 20 nM ON-TARGETplus NON-TARGETING pool control or 20 nM ON-TARGETplus SMARTpool siRNA targeting SERPINE2, PAI-1, SOX4, SMAD2, SMAD3, SMAD4, and N-Cad (Dharmacon) using Lipofectamine RNAiMAX and Opti-MEM (Life Technologies) according to the manufacturer’s instructions. Knockdown efficiency was assessed by RT-qPCR and Western Blot analyses. 3.10 MTT Assay MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma] assay was used to assess cell viability. HTR8/SVneo cells were seeded in 24-well plates (1 x 104 cells/well in 1 mL of medium) and treated every 24 hrs with vehicle or MSTN in medium containing 0.1% (vol/vol) FBS for a total of 72 hrs. Cells were incubated with 0.5 mg/mL 49 MTT for 4 hrs, the medium was replaced with 1 mL dimethylsulfoxide (DMSO), and absorbances were measured at 490 nm using a microplate reader. 3.11 Statistical Analysis Results are presented as the mean ± standard error of mean (SEM) of at least three independent experiments and were analyzed by either unpaired Student T test or one-way ANOVA followed by Newman-Keuls test for multiple comparisons of means using PRISM 9.2 software (GraphPad Software Inc.). Means were considered significantly different if P <0.05 and were indicated by different letters. 50 Chapter 4: Myostatin Increases Cell Invasion and Regulates Several Core EMT Genes in Primary Human Trophoblast Cells 4.1 Introduction Despite being a temporary organ, the placenta performs many functions that are indispensable for a healthy pregnancy [1, 2]. Improper placental development leads to a variety of common pregnancy complications including PET, IUGR, and preterm labor [3, 4]. In addition, poor placental function may result in childhood and late adulthood illnesses such as cardiovascular, metabolic, and mental health disorders [2]. To function properly, the placenta invades into the uterine wall traversing the decidua as well as the inner third of the myometrium [7]. If this invasion is too shallow it can lead to placental insufficiency resulting in placenta-mediated pregnancy complications. Placental invasion is controlled by different hormones, cytokines, and growth factors [266]. TGF-β superfamily growth factors are known to play various roles in regulating trophoblast invasion [52]. MSTN is a member of the TGF-β superfamily and has recently been shown to be expressed by the human placenta (8-10). Moreover, MSTN serum/placental levels are dysregulated in PET [83, 170], IUGR [83] and gestational diabetes [171]. Functionally, MSTN has been shown to increase trophoblast proliferation [75], migration [75] and Matrigel invasion [76, 169]. However, very little is known regarding its mechanisms of action in trophoblast cells. Placental transcript profiling by various means, including RNA sequencing, provides a great opportunity to explore the underlying molecular mechanisms governing placental development and function in normal and pathological pregnancies [267]. Classical bulk RNA sequencing is used to study gene expression at the level of the whole sample, 51 while single cell RNA sequencing explores gene profiling at the single cell level [268]. Both bulk [269-275] and single cell [276-278] RNA sequencing have been invaluable in providing insight on physiological placental development. Moreover bulk RNA sequencing has been specially useful in studying the underlying molecular changes in common pregnancy complications including PET [279, 280] and IUGR [281-283]. Likewise, transcriptomic profiling of in vitro cultured primary trophoblast cells and trophoblast cell lines have been performed to study the effects of a given treatment while controlling other experimental conditions (reviewed in [267])[284]. The current study was conducted to confirm MSTN-induced trophoblast invasion and to shed light on the molecular mechanisms of MSTN-induced trophoblast invasion by profiling the MSTN-regulated transcriptome in primary human trophoblast cells. I tested the effect of MSTN treatment on trophoblast invasion using primary and HTR8/SVneo trophoblast cells via the transwell invasion assay. I then used RNA sequencing to compare MSTN treated primary trophoblasts to controls. 52 4.2 Results 4.2.1 MSTN increases invasion of immortalized and primary human trophoblast cells: As shown in Fig. 4.1A, treatment of HTR8/SVneo cells with varying concentrations of MSTN (0-100 ng/mL) significantly increased cell invasiveness at all the concentrations tested. I then decided to go with the 50-ng/mL dose which I thought was high enough to result in observable effects but still was a biologically relevant concentration. Moreover, treatment with 50 ng/mL MSTN had no effect on HTR8/SVneo cell viability for up to 72 hrs (Fig. 4.1B). Importantly, the pro-invasive effects of MSTN were confirmed in primary human trophoblast cells treated with 50 ng/mL MSTN (Fig. 4.1C). 4.2.2 MSTN-induced trophoblast cell invasion is mediated by SMAD2/3-SMAD4 signaling: To investigate the role of canonical SMAD2/3-SMAD4 signaling in MSTN-induced trophoblast invasion, I first used Western blot to measure MSTN-induced changes in SMAD2/3 phosphorylation/activation. As shown in Fig. 4.2, treatment with MSTN increased the phosphorylation of both SMAD2 and SMAD3 in a time-dependent manner (15-60 min) in both HTR8/SVneo and primary human trophoblast cells. To investigate the role of SMAD2/3-SMAD4 signaling in MSTN-induced trophoblast invasion, I next performed a series of knockdown experiments with siRNAs targeting receptor-regulated SMAD2+3 as well as common SMAD4. As shown in Fig. 4.3A and 4.3B, pre-treatment with each of the siRNAs alone or in combination significantly reduced the respective mRNA and protein levels of each target both in the presence or absence of MSTN. Importantly, MSTN-induced trophoblast cell invasion was abolished by combined knockdown of SMAD2+3 or depletion of common SMAD4 (Fig. 4.3C). 53 4.2.3 MSTN-induced trophoblast cell invasion is mediated by activin/TGF-β type 1 receptors To investigate whether activin/TGF-β type 1 receptors mediate the effects of MSTN on cell invasion, I employed a pharmacological approach with the ALK4/5/7 type 1 receptor inhibitor SB431542. Western blot results showed that MSTN-induced SMAD2/3 phosphorylation was abolished by pre-treatment with SB431542 in both HTR8/SVneo and primary human trophoblast cells (Fig.4.4A). Next, I examined the effects of SB431542 pre-treatment on MSTN-induced trophoblast invasion. As shown in Fig.4.4B, pre-treatment of HTR8/SVneo and primary trophoblast cells with SB431542 significantly attenuated MSTN-induced trophoblast invasion as well as the basal invasion of primary trophoblasts. 4.2.4 MSTN-regulated gene profile in primary human trophoblast cells RNA sequencing analysis was conducted with primary human trophoblast cells treated for 24 hrs with vehicle control or 50 ng/mL MSTN (n=5). The purity of trophoblast cultures was assessed immunocytochemically and showed >90% positive staining for KRT7 (epithelial marker) and HLA-G (EVT marker) (Fig.4.5A). Principal components analysis (PCA) revealed that differences between placentas from the 5 patients were stronger than differences due to treatment (Fig.4.5B). Therefore, we used DESeq2 analysis with a paired sample design including sample information to identify DEGs between MSTN-treated and control groups. This accounted for differences between the samples while estimating treatment-induced effects. In total 610 of the 16482 genes with ≥ 10 FPKM were differentially expressed with an Benjamini-Hochberg adjusted P value < 0.05; 380 were upregulated and 230 were downregulated in MSTN-treated trophoblasts compared to controls (Appendix A). 54 4.2.5 MSTN-treated primary trophoblasts were highly enriched in hallmark EMT genes 4.2.5.1 GSEA, KEGG and GO analyses of DEG (with q-value<0.05) in MSTN treated primary trophoblasts When GSEA was performed with the 610 pre-ranked (ranked based on FC) DEGs, we observed a positive enrichment in 15 and a negative enrichment in 4 of the 50 hallmark gene sets (Appendix B). However, only the hallmark EMT gene set (Appendix C) reached significance with an FDR < 25% (Fig.4.6A). KEGG pathway analysis with the pre-ranked DEGs demonstrated a significant enrichment in genes involved in focal adhesion and ECM receptor interaction, including integrins, fibronectin, collagen, and laminin. Other enriched pathways were those involved in cancer and in TGF-β, PI3K-AKT and Hippo signaling (Fig. 4.6B). Interestingly, the GO of the biological processes enriched in the DEGs was related to ECM organization and structure, cell migration and motility as well as cell proliferation (Fig. 4.6C). Moreover, there was an enrichment in genes located in ECM, cytoskeleton, and cell junctions (Fig. 4.6D). 4.2.5.2 KEGG and GO analyses of DEG (using a FC cut-off) in MSTN treated primary trophoblasts We identified 25 DEGs using a 1.5 FC cut-off (Fig.4.7A), however given the small sample size and inter-sample variability, we conducted further analyses with a 1.3 FC cut-off. This analysis identified 96 DEGs between control and MSTN-treated cells; 81 upregulated and 15 downregulated (adjusted P-value < 0.05) (Appendix D). Hierarchical clustering of these 96 DEGs is shown in Fig.4.7B. 55 KEGG pathway analysis revealed an enrichment in genes regulating ECM receptor interactions (TNXB, ITGB3, ITGA1, TNC, LAMC2) (Table 4.1, Fig.4.8A). The MSigDB Hallmark 2020 analysis showed an enrichment in genes regulating the EMT process including (IL32, LRRC15, SERPINE2, IGFBP3, NTM, ITGB3, PAI-1, TNC, HTRA1, FZD8, LAMC2, INHBA, PTHLH, SFRP1, N-Cad, LOX, COL7A1, ADAM12, PMEPA1) (Table 4.2, Fig.4.8B). Moreover, MSTN-treated trophoblasts were enriched in genes regulating ECM structure and organization, including ITGB3, ITGA1, PAI-1, TNC, HTRA1, LAMC2 MMP10, ADAM19, ADAMTS4, LOX, COL7A1 ADAM12, HAS2 and ADAMTS6. We also observed enrichment of genes involved in cell motility and migration (LRRC15, SFRP1, SERPINE2, IGFBP5, SEMA3C, PAI-1, HAS2, F2RL1, LAMC2, ADRA2A) (Table 4.3, Fig.4.8C). GO analysis of cellular processes showed that most of the DEGs were enriched in the ECM, plasma membrane and focal adhesion areas (Table 4.4, Fig. 4.8D). RNA sequencing data for some of the DEGs involved in the EMT process are shown in Fig.4.9A and were validated by RT-qPCR (Fig.4.9B). 56 4.3 Discussion In the current study, I showed that MSTN increases the invasion of primary and immortalized human trophoblast cells. Moreover, I found that the pro-invasive effects of MSTN can be observed at concentrations as low as 5 ng/mL, which is well within the reported range for serum MSTN levels (3.2- 40 ng/mL) [83, 148, 285]. Furthermore, I profiled the MSTN-regulated transcriptome in primary human trophoblast cells and demonstrated that MSTN regulates many genes involved in ECM structure and organization as well as in cell migration and motility. To my knowledge, this is the first study to investigate the effects of MSTN on the genetic machinery of trophoblast cells. MSTN RNA and protein have been shown to be expressed in the human placenta throughout gestation, with higher protein levels in the first and second trimesters [121]. MSTN has also been shown to be localized in EVTs of the human placenta [75]. Higher MSTN levels in early gestation may suggest a role in placental development which mainly takes place during the first trimester. Previous studies have demonstrated effects of MSTN on several trophoblast functions, including proliferation [75], migration [75] and invasion [76]. Effects of MSTN on cell proliferation/viability are somewhat debatable as initial reports with real-time cell imaging of HTR8/SVneo cells suggested pro-proliferative effects over 40 hrs at concentrations ranging from 5-1000 ng/mL [75]. However, subsequent studies found no effects of MSTN (1000 ng/mL) on the viability of placental explants over a 24-hrs period [83]. Similarly, the current study found that treatment with MSTN (50 ng/mL) did not affect HTR8/SVneo cell viability over a period of 72 hrs. These discrepancies in effects on proliferation/viability may be due to differences in the assays used (e.g., cell imaging vs. MTT), trophoblast models, HTR8/SVneo passage numbers, and/or sources of recombinant 57 human MSTN. Moreover, proliferation is mainly a function of column cytotrophoblasts which give rise to EVTs that migrate and invade the maternal tissues [30]. Therefore, proliferative effects of MSTN on human trophoblasts need to be further investigated using a more appropriate cytotrophoblast model. Reports on the effects of MSTN on trophoblast cell motility and invasiveness are more consistent. Peiris et al. demonstrated stimulatory effects of MSTN on HTR8/SVneo and primary trophoblast cell migration using the scratch wound assay [75]. My findings agree with two recent studies showing that MSTN enhances trophoblast invasiveness in the Matrigel-coated transwell invasion assay [76]. Interestingly, deep infiltrating endometriosis and endometrial adenocarcinoma were shown to have higher MSTN mRNA and protein levels compared to normal endometrium [163], suggesting pro-invasive effects of MSTN in endometriotic cells. MSTN exerts its functions by binding to activin type 2 receptors which in turn activate type 1 receptors, primarily ALK4 and ALK5 (Fig.1.6) [286]. Using siRNA knockdown of ALK4 and ALK5, my lab has shown that MSTN-induced FSTL3 production is mediated by ALK5, and not ALK4, in human trophoblast cells [76]. In the current study, I used an ALK4/5/7 inhibitor (SB431542) to confirm that MSTN-induced trophoblast invasion is mediated by similar activin type 1 receptors. SMAD signaling is one of the important pathways regulating trophoblast invasion [287]. Downstream MSTN signaling involves phosphorylation of receptor regulated SMAD2/3 and subsequent binding to common SMAD4 to form a complex that translocates into the nucleus and modulates gene expression. In the present study, combined knockdown of SMAD2 and SMAD3 suppressed MSTN-induced trophoblast invasion. Similarly, other TGF-β superfamily members like activin [66] 58 and BMP2 [73] increased trophoblast cell invasion via SMAD2/3-SMAD4 signaling. However, the involvement of SMAD-independent signaling for MSTN-induced trophoblast invasion remains to be investigated. The process of EMT contributes to EVT formation and is characterized by the differentiation of immotile polar epithelial cells to EVTs with mesenchymal characteristics such as motility and the ability to invade maternal tissues [288]. Acquisition of these characteristics requires changes in cell shape, apical-basolateral polarity, and cell-cell/ECM adhesion [38]. Beside its role in placental development, EMT is a cornerstone in embryonic development and cancer metastasis [34]. Interestingly, profiling the transcriptome of MSTN-treated trophoblasts showed a significant enrichment of EMT genes. TGF-β signaling is a well-known pathway to induce EMT [48, 289, 290] by upregulating key EMT transcription factors, including Snail [291], Slug [292] and Twist [293]. Indeed, TGF-β superfamily members such as TGF-β, activins, BMP2, MSTN and nodal were shown to be important players in the process of placental invasion (reviewed in [52]). MSTN has been found to upregulate trophoblast cell migration and invasion. Similar changes have also been reported in ovarian cancer cells [294], non-small cell lung cancer cells [295], keratinocytes [296] and chick dermatomes [295]. Indeed, many of the DEGs in my analysis are regulated during the EMT process (ADAM12, N-Cad, COL7A1, CTGF, FZD8, HAS2, HTRA1, IGFBP3, IL32, ITGB3, INHBA, LRRC15, LAMC2, LOX, NTM, PTHLH, PMEPA1, PAI-1, SERPINE2, SFRP1, TNC) [297]. Moreover, some of these DEGs have previously been shown to regulate trophoblast cell invasion (ADAM12, N-Cad, INHBA, IGFBP3, SERPINE2, ITGB3, HTRA1, LOX) [66, 298-304]. Similarly, profiling the transcriptome of BMP2-treated primary human trophoblasts 59 revealed that the top DEGs were related to cell adhesion and ECM molecules [284]. This might explain the molecular mechanism of BMP2-induced trophoblast cell invasion [73, 305-307] and it would be of interest to compare it to that of MSTN in future studies. RNA sequencing analysis of MSTN-treated primary trophoblasts resulted in a relatively small number of DEGs with ≥ 1.5-fold change. This might be mainly related to the technical and inter-patient variabilities as well as the small sample size, and the duration of MSTN treatment. A major contributor to inter-patient variability is the inability to predict pregnancy outcome of the first trimester samples used for trophoblast isolation, which could yield a mix of normal and diseased placentas [267]. Placental sex could also be a factor where two (#33 and #35) out of the five analyzed samples were males based on expression of the DDX3Y gene [308]. Interestingly these two samples were grouped together as shown in Fig.4.4B and 4.7B. Sexual dimorphism has been shown to affect placental gene expression [274]. Accordingly, I performed a paired analysis to mitigate some of these inter-sample differences. Another explanation for the relatively small number of DEGs could be that I analyzed only a single time-point (24 hrs). Future studies examining MSTN-regulated transcriptome at multiple time points (e.g., 1, 3, 6 and 12 hrs) might reveal more DEGs and yield greater insight into the transcriptional programs governed by MSTN in human trophoblasts. My findings provide insight into some of the genetic changes that may contribute to the pro-invasive effects of MSTN on human trophoblast cells and can pave the road towards understanding the role of MSTN in the human placenta and pregnancy complications. 60 Table 4. 1 Top Significant KEGG Pathways Enriched Among the DEG with a 1.3 FC cut-off in MSTN Treated Human Primary Trophoblasts Term q-value Overlapped genes ECM-receptor interaction 0.007675 [TNXB, ITGB3, ITGA1, TNC, LAMC2] p53 signaling pathway 0.020120 [CDK6, TP53I3, IGFBP3, PAI-1] Arrhythmogenic right ventricular cardiomyopathy 0.020120 [N-Cad, ITGB3, ITGA1, SLC8A1] Human papillomavirus infection 0.031595 [CDK6, TNXB, ITGB3, ITGA1, TNC, FZD8, LAMC2] PI3K-Akt signaling pathway 0.037164 [CDK6, TNXB, IRS1, ITGB3, ITGA1, TNC, LAMC2] 61 Table 4. 2 Top Significant MSigDB Hallmark Terms Enriched Among the DEG (with a 1.3 FC cut-off) in MSTN-Treated Human Primary Trophoblasts Term q-value Overlapped genes Epithelial-Mesenchymal Transition 3.967852e-18 [IL32, LRRC15, SERPINE2, IGFBP3, NTM, ITGB3, PAI-1, TNC, HTRA1, FZD8, LAMC2, INHBA, PTHLH, SFRP1, N-Cad, LOX, COL7A1, ADAM12, PMEPA1] TNF-alpha Signaling via NF-kB 8.348441e-04 [PAI-1, TNC, F2RL1, PMEPA1, SIK1, INHBA, IFIT2] KRAS Signaling Up 4.482770e-03 [IGFBP3, F2RL1, INHBA, ANGPTL4, ETV4, MMP10] UV Response Dn 5.380145e-03 [IGFBP5, IRS1, ITGB3, PAI-1, HAS2] Inflammatory Response 1.838938e-02 [TNFSF15, ITGB3, PAI-1, HAS2, INHBA] Coagulation 2.428499e-02 [ITGB3, PAI-1, HTRA1, MMP10] 62 Table 4. 3 Top Significant GO Biological Processes Enriched Among the DEG (with a 1.3 FC cut-off) in MSTN-Treated Human Primary Trophoblasts Term q-value Overlapped genes Extracellular matrix organization (GO:0030198) 1.925614e-07 [ITGB3, ITGA1, PAI-1, TNC, HTRA1, LAMC2, MMP10, ADAM19, ADAMTS4, LOX, COL7A1, ADAM12, HAS2, ADAMTS6] Extracellular structure organization (GO:0043062) 2.062854e-07 [ADAM19, ADAMTS4, LOX, COL7A1, ITGB3, ITGA1, PAI-1, ADAM12, TNC, LAMC2, MMP10, ADAMTS6] External encapsulating structure organization (GO:0045229) 2.062854e-07 [ADAM19, ADAMTS4, LOX, COL7A1, ITGB3, ITGA1, PAI-1, ADAM12, TNC, LAMC2, MMP10, ADAMTS6] Negative regulation of smooth muscle cell migration (GO:0014912) 1.351156e-04 [NDRG4, IGFBP5, IGFBP3, PAI-1] Regulation of cell population proliferation (GO:0042127) 1.507899e-04 [IL11, MYOCD, IRS1, IGFBP3, ITGA1, AKR1C3, LAMC2, INHBA, ADRA2A, PTHLH, SFRP1, CDK6, HAS2, NOX4, SOX4, KCNH1] Negative regulation of cell population proliferation (GO:0008285) 3.655449e-04 [MYOCD, NDRG4, SFRP1, CDK6, IGFBP5, IGFBP3, ITGA1, NOX4, INHBA, PTHLH, SOX4] Regulation of smooth muscle cell migration (GO:0014910) 3.655449e-04 [NDRG4, IGFBP5, IGFBP3, PAI-1] Negative regulation of cellular process (GO:0048523) 2.289188e-03 [MYOCD, SFRP1, CDK6, IGFBP3, ITGA1, LPXN, NR1H3, NOX4, OSGIN2, INHBA, PTHLH, SOX4] Negative regulation of smooth muscle cell proliferation (GO:0048662) 2.289188e-03 [CNN1, NDRG4, IGFBP5, IGFBP3] Regulation of cell migration (GO:0030334) 2.974167e-03 [LRRC15, SFRP1, SERPINE2, IGFBP5, SEMA3C, PAI-1, HAS2, F2RL1, LAMC2, ADRA2A] 63 Table 4. 4 Top Significant GO Cellular Components Enriched Among the DEG (with a 1.3 FC cut-off) in MSTN-Treated Primary Trophoblasts Term q-value Overlapped genes Collagen-containing extracellular matrix (GO:0062023) 3.220479e-09 [LRRC15, TNXB, SERPINE2, PAI-1, TNC, HTRA1, LAMC2, COCH, ADAM19, ADAMTS4, SFRP1, N-Cad, COL7A1, NCAM1, HMCN1, ANGPTL4] Microvillus membrane (GO:0031528) 4.441764e-02 [SLC7A5, ITGB3] Integral component of plasma membrane (GO:0005887) 4.441764e-02 [SEMA3C, TNFSF15, IRS1, PCDH10, ITGB3, NPY1R, GRIK2, SHISA9, PCDH19, ADRA2A, SLC8A1, N-Cad, HAS2, NOX4, F2RL1, KCNH1] Endoplasmic reticulum lumen (GO:0005788) 4.441764e-02 [N-Cad, IGFBP5, COL7A1, IGFBP3, TNC, ARSI] Focal adhesion (GO:0005925) 4.441764e-02 [ARHGAP22, CNN1, N-Cad, ITGB3, ITGA1, TNC, LPXN] Cell-substrate junction (GO:0030055) 4.441764e-02 [ARHGAP22, CNN1, N-Cad, ITGB3, ITGA1, TNC, LPXN] 64 Figure 4. 1 MSTN increases cell invasion in HTR8/SVneo and primary human trophoblast cells. A, HTR8/SVneo cells were treated with varying concentrations of MSTN (0, 5, 10, 25, 50 or 100 ng/mL) and cell invasiveness was examined by Matrigel-coated transwell assay. Representative images of the transwell membrane of the invasion assay (upper left panels; scale bars 100 µm) and summarized quantitative results (right panel) are shown separately. B, Treatment with 50 ng/mL MSTN had no effect on HTR8/SVneo cell viability for up to 72 hrs. C, Primary human trophoblast cells were treated with vehicle control or 50 ng/mL MSTN and cell invasiveness was examined by Matrigel-coated transwell assay. Representative images of the invasion assay (left panel; scale bars 100 µm) and combined quantitative results (right panel) are shown separately. MSTN, myostatin; Ctrl, control; hrs, hours. 65 Figure 4. 2 MSTN activates the canonical SMAD2/3 signaling pathway. HTR8/SVneo cells (A) and primary human trophoblast (B) cells were treated with vehicle control or 50 ng/mL MSTN for different lengths of time (15-60 min). Levels of phosphorylated SMAD2 (P-SMAD2) and SMAD3 (P-SMAD3) were examined by Western blot with corresponding phospho-specific antibodies. Membranes were stripped and re-probed with antibodies for total SMAD2 and SMAD3. Phosphorylation levels were quantified on the right side of each panel Ctrl, control; MSTN, myostatin; min, minutes. 66 Figure 4. 3 SMAD2/3-SMAD4 signaling mediates the MSTN-induced trophoblast invasion. A-C, HTR8/SVneo cells were transfected for 48 hrs with non-targeting control siRNA (si-Ctrl) or siRNAs targeting SMAD2 (si-2), SMAD3 (si-3), SMAD2+3 (si-2+3) or SMAD4 (si-4) prior to treatment with or without 50 ng/mL MSTN for 24 hrs. SMAD2, SMAD3 and SMAD4 mRNA (A) and protein (B) levels were analysed by RT-qPCR and Western blot. Representative Western blot images (upper panels) and summarized quantitative results (lower panels) are shown separately. C, Matrigel-coated transwell invasion assays were used to examine the effects of SMAD2+3 and SMAD4 knockdown on MSTN-induced invasion in HTR8/SVneo cells. Representative images of the invasion assay (left panels; scale bars 100 µm) and combined quantitative results (right panel) are shown separately. Ctrl, control; MSTN, myostatin; α-Tub, α-Tubulin. 67 Figure 4. 4 MSTN-induced trophoblast cell invasion is mediated by activin/TGF-β type 1 receptors. A, HTR8/SVneo cells (left panels) and primary human trophoblast cells (right panels) were pre-treated for 1 hr with vehicle control (DMSO) or 10 µM SB431542 (ALK4/5/7 inhibitor) prior to treatment for 60 min with vehicle control or 50 ng/mL MSTN. SMAD2/3 phosphorylation was measured by Western blot with corresponding phospho-specific and total antibodies. B, Matrigel-coated transwell invasion assays were used to examine the effects of SB431542 pre-treatment on myostatin-induced invasion in HTR8/SVneo cells (left panels) and primary trophoblast cells (right panels). Results are displayed as the mean ± SEM of at least three independent experiments and values without common letters are significantly different (P < 0.05). Scale bars 100 µm. Ctrl, control; MSTN, myostatin; hr, hour; SB, SB431542. 68 Figure 4. 5 MSTN regulates the gene profile expression in primary trophoblasts. A, Purified primary human trophoblasts were stained with immunofluorescence antibodies directed against KRT7 and HLA-G, and nuclei were stained with DAPI (200 x magnification, scale bars = 100 μm). B, The principal component analysis revealed that the differences between placentas from the 5 included patients were stronger than the differences due to treatment. KRT7, cytokeratin7; HLA-G, human leukocyte antigen G; Ctrl, control; MSTN, myostatin; PCA, principal component analysis; VST, variance stabilizing transformation. AFigure 4.5 MSTN regulates the gene profile expression in primary trophoblasts.A, Purified primary human trophoblasts were stained with immunofluorescence antibodiesdirected against KRT7 and HLA-G , and nuclei were stained with DAPI (200 x magnification, scale bars = 100 μm). B, The principal component analysis revealed that the differences between placentas from the 5 included patients were stronger than the differences due to treatment. KRT7, cytokeratin7; HLA-G, human leukocyte antigen G; Ctrl, control; MSTN, myostatin; PCA, principal component analysis.BDAPIDAPIDAPIKRT7HLA-GIgGMergeMergeMergeCtrlMSTNGroup 69 Figure 4. 6 MSTN-treated primary trophoblasts were highly enriched in hallmark EMT genes as shown using GSEA and GO analyses. In total 610 genes were differentially expressed with an adjusted P<0.05; 380 DEG were upregulated while 230 DEG were downregulated. A, GSEA of the DEG s revealed a significant enrichment in the Hallmark EMT genes. B, KEGG analysis showed an enrichment of pathways regulating focal adhesion, and ECM receptor interaction. C, GO of biological processes identified an enrichment of genes involved in ECM organization and structure, as well as in cell motility and migration. D, Among the DEGs, there was an enrichment in genes located in ECM, focal adhesion, and cell-substrate junctions. an asterisk (*) next to a P-value indicates the term also has a significant adjusted P-value (<0.05). MST, myostatin; DEG, differentially expressed gene; GSEA, gene set enrichment analysis; GO, gene ontology; EMT, epithelial-mesenchymal transition; ECM, extracellular matrix. A BC DKEGG 2021 HumanGO Biological process 2021 GO Cellular Component 2021Figure 4.6 MSTN-treated primary trophoblasts were highly enriched in hallmark EMT genes as shown using GSEA and GO analysis.In total 610 genes were differentially expressed with an adjusted P<0.05; 380 DEG were upregulated while 230 DEG were downregulated. A, GSEA of the DEG s revealed a significant enrichment in the Hallmark EMT genes. B, KEGG analysis showed an enrichment of pathways regulating focal adhesion, and ECM receptor interaction. C, GO of biological processes identified an richment of genes involved in ECM rganization and structure, as well as in cell motility and migrat on. D, Among the DEGs , there was an enrichment in genes located in EC , focal adhesion and cell-substrate junctions. a asterisk (*) n xt to a P-value indicates the term also has a significant adjusted P-value (<0.05). MST, myostatin; DEG, differentially expressed gene; GSEA, gene set enrichment analysis; GO, gene ontology; EMT, epithelial mesenchymal transition; ECM, extracellular matrix. 70 Figure 4. 7 Visualization of DEG in human primary trophoblasts treated with or without MSTN. A, A volcano plot of DEG according to fold change B, A hierarchical clustering of Ctrl and MSTN-treated trophoblasts using the 96 DEGs from the RNA sequencing analysis. Ctrl, control; MSTN, myostatin; DEG, differentially expressed gene. ABFigure 4.7 Visualization of DEG in human primary trophoblasts treated with or without MSTN.A, A scatter plot of DEG according to fold change B, A hierarchical clustering of Ctrl and MSTN-treated trophoblasts using the 96 DEGs from the RNA sequencing analysis. Ctrl, control; MSTN, myostatin; DEG, differentially expressed gene.SampleCtrlMSTN 71 Figure 4. 8 KEGG pathway and GO analyses of DEG in human primary trophoblasts treated with or without MSTN using a fold change cut off. A, KEGG 2021 human pathway analysis of 96 DEG showing an enrichment of genes involved in ECM-receptor interaction. B, MSigDB Hallmark 2020 pathway analysis of 96 DEG presenting an enrichment of genes involved in EMT. C, Gene Ontology biological process of 96 DEG showing an enrichment of genes involved in ECM organization and cell migration D, There was an enrichment in genes located in ECM and plasma membranes as well as focal adhesion areas, an asterisk (*) next to a P-value indicates the term also has a significant adjusted P-value (<0.05). MSTN, myostatin; GO, gene ontology; DEG, differentially expressed gene; EMT, epithelial-mesenchymal transition; ECM, extracellular matrix. A BC DKEGG 2021 Human MSigDB Hallmark 2020GO Biological process 2021 GO Cellular Component 2021Figure 4.8 KEGG pathway and GO analysis of DEG in hu an primary trophoblasts treated with or without MSTN using a fold change cut off.A, KEGG 2021 human pathway analysis of 96 DEG showing an enrichment of genes involved in ECM-receptor interaction. B, MSigDB Hallmark 2020 pathway analysis of 96 DEG presenting an enrichment of genes involved in EMT. C, Gene Ontology biological process of 96 DEG showing an enrichment of genes involved in ECM organization and cell migration D, There was an enrichment in genes located in ECM and plasma membra es as well as focal adhes on ar as, an asterisk (*) next to a P-value indicates the term also has a significant adjusted P-value (<0.05). MSTN, myostatin; GO, gene ontology; DEG, differentially expressed gene; EMT, epithelial mesenchymal transition; ECM, extracellular matrix.-log10 (p values) -log10 (p values) 72 Figure 4. 9 Comparison of DEGs in human primary trophoblasts treated with or without MSTN. A, RNA-seq data of differentially expressed MSTN signaling targets in primary trophoblasts treated with or without 50ng/mL MSTN. B, RT-qPCR validation of the differential expression of MSTN signaling target genes in primary trophoblasts treated with or without 50ng/mL MSTN, Data were normalized to GAPDH, and values represent the mean ± SEM. *P< 0.05, **P < 0.005, using multiple unpaired Student's t test with correction of multiple comparison using Holm-Sidak method. MSTN, myostatin; N-Cad, N-cadherin; FPKM, fragments per kilobase of exon per million reads; PAI-1, plasminogen activator inhibitor-1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. 73 Chapter 5: Upregulation of Serine Protease Inhibitors E1 and E2 Contributes to Myostatin-Induced Human Trophoblast Cell Invasion 5.1 Introduction Placental trophoblast cells undergo a process of EMT to invade into the maternal decidua and the inner third of the myometrium. To invade, cytotrophoblast cells leave the cell columns and invade into the decidual ECM. Serine, cysteine and matrix metalloproteinases are the three main families of proteases involved in the implantation process [309]. Serine proteases constitute over one third of all known proteolytic enzymes [310] and are involved in many physiological and pathological processes, including blood coagulation, protein metabolism, apoptosis, immunity development and fertilization [310, 311]. Tissue and urokinase plasminogen activators (t-PA and u-PA respectively) are examples of serine proteases that are expressed in the human placenta [312, 313]. They are considered the major plasminogen activators catalyzing its conversion into plasmin [314]. Plasmin is an important serine protease involved in the EMT process [315]. Moreover, plasmin activates some matrix metalloproteinases (MMPs) which, in turn, degrade collagen [316]. However, pregnancy is a hypercoagulability state to safeguard against potential severe intra-partum bleeding [317]. Thus, fibrinolysis needs to be regulated via serine protease inhibitors (SERPINs) [318]. Serine protease inhibitors constitute the largest protease inhibitor superfamily and are widely distributed within the body [319]. Each of the 36 SERPINs expressed in humans performs different functions including protease inhibition, hormone transport, and tumour suppression [319]. Placenta-mediated disorders such as PET and IUGR are associated with 74 aberrant expression of several placental SERPINs including SERPINA3, SERPINA5, SERPINA8, SERPINB2 (PAI-2), SERPINB5, SERPINB7, SERPINE1 (PAI-1), SERPINE2 and SERPING1 [320]. SERPINE2 or protease nexus-1 is a broad-spectrum inhibitor that acts on thrombin, u-PA, plasmin, trypsin and prostasin, among others [321]. It is expressed in different body systems including the nervous, respiratory, vascular, renal, and reproductive systems [321]. However, its expression is the highest in the placenta, especially near term [301]. In the placenta, SERPINE2 is localized in syncytio-, columnar- and extravillous- trophoblasts as well as decidual cells [301]. Additionally, SERPINE2 knockdown decreases villous outgrowth as well as trophoblast migration, invasion, and capillary-like tube formation [301]. SERPINE1 or plasminogen activator inhibitor-1 (PAI-1) is a paralogue of SERPINE2 and the main inhibitor of u-PA and t-PA [322]. PAI-1 can also inhibit plasmin directly and is considered the main plasmin regulator in vivo [323]. PAI-1 is considered an immunocytochemical marker of invading trophoblasts [324]. Interestingly, SERPINE2 and PAI-1 were shown to be induced by hypoxia and their placental levels were dysregulated in PET [325]. Moreover, a recent systemic review reports a positive association between high plasma levels of PAI-1 and PET [326]. MSTN is a member of the TGF-β superfamily [92]. MSTN mRNA and protein are expressed in primary placental tissues throughout gestation [121] as well as in placental cell lines (HTR8/SVneo, BeWo, JAR, and JEG3) [168]. It is localized to EVTs of the first trimester and term human placentas [75]. Placental and/or blood levels of MSTN are dysregulated in common pregnancy complications including PET [83, 170], IUGR [83] and 75 gestational diabetes [171]. In human trophoblast cells, MSTN has been shown to exert pro-migratory [75] and pro-invasive [76, 169] effects. Importantly, SERPINE2 and PAI-1 are among the top DEGs in the MSTN-treated primary trophoblasts RNA seq analysis as shown in Chapter 4. To this end the current study was performed to investigate the role of SERPINE2 and its paralog PAI-1 in the MSTN-induced trophoblast invasion and to explore the underlying molecular mechanisms. The effects of MSTN treatment on SERPINE2 and PAI-1 levels were investigated using RT-qPCR and Western blot. Moreover, the role played by those SERPINs in trophoblast invasion was studied using the transwell invasion assay, small molecule inhibitors and siRNA knockdown. 76 5.2 Results 5.2.1 MSTN upregulates SERPINE2 and PAI-1 in human trophoblast cells: HTR8/SVneo and primary trophoblast cells were treated for varying times (3-24 hrs) with 50 ng/mL MSTN. SERPINE2 and PAI-1 mRNA and protein levels were measured by RT-qPCR and Western blot, respectively. MSTN treatment significantly increased SERPINE2 mRNA levels beginning at 6 hrs in HTR8/SVneo (Fig.5.1A) and primary trophoblast (Fig.5.1B) cells. On the other hand, MSTN significantly increased PAI-1 mRNA levels beginning at an earlier time point (3hrs) and gradually attenuating over time in both HTR8/SVneo (Fig.5.1C) and primary trophoblast (Fig.5.1D) cells. When trophoblast cells were treated with 50 ng/mL MSTN for 24 and 48 hrs, SERPINE2 protein levels increased at both time points in HTR8/SVneo (Fig.5.2A) and primary trophoblast cells (Fig.5.2B). Similarly, MSTN upregulated PAI-1 protein levels in both cell types (Fig.5.2C-D). 5.2.2 MSTN-induced trophoblast cell invasion is mediated by SERPINE2 and PAI-1: To determine if SERPINE2 and PAI-1 are involved in MSTN-induced trophoblast cell invasion, siRNA was used to deplete SERPINE2 and PAI-1 prior to MSTN treatment and analysis of cell invasion. Knockdown of SERPINE2 and PAI-1 abolished basal and MSTN-induced SERPINE2 and PAI-1 mRNA (Fig.5.3A-D) and protein levels (Fig.5.4A-B) in HTR8/SVneo and primary human trophoblast cells. Importantly, SERPINE2 knockdown suppressed MSTN-induced but not basal trophoblast cell invasion in HTR8/SVneo cells (Fig.5.5A). However, SERPINE2 knockdown suppressed both basal and MSTN-induced trophoblast cell invasion in primary human trophoblast cells (Fig.5.5B). On the other hand, PAI-1 knockdown increased basal invasion but completely blocked the pro-invasive effects of MSTN in HTR8/SVneo cells (Fig.5.5C). Meanwhile, depletion of PAI-1 significantly 77 reduced both basal and MSTN-induced invasion in primary human trophoblast cells (Fig.5.5D). 5.2.3 MSTN-induced SERPINE2 and PAI-1 upregulation are mediated by activin/TGF-β type 1 receptors: To confirm that the effects of MSTN on SERPINE2 and PAI-1 production are mediated by activin/TGF-β type 1 receptor, we pretreated cells with the ALK4/5/7 type 1 receptor inhibitor SB431542. Treatment with SB431542 eliminated the stimulatory effects of MSTN on SERPINE2 (Fig.5.6A-B) and PAI-1 (Fig.5.6C-D) mRNA levels in both HTR8/SVneo and primary human trophoblast cells. Similar effects of SB431542 were observed on MSTN-induced SERPINE2 (Fig.5.7A-B) and PAI-1 (Fig.5.7C-D) protein levels. 5.2.4 The role of SMAD signaling in MSTN-induced SERPINE2 and PAI-1 upregulation: SMAD signaling is the canonical signaling pathway via which MSTN induces its effects. Downstream MSTN signaling involves phosphorylation of SMAD2 and SMAD3 which form a complex with common SMAD4 and enters the nucleus as a transcription factor to regulate MSTN target gene expression (Fig1.6). siRNA-mediated knockdown was used to investigate the role of canonical SMAD2/3-SMAD4 signaling in MSTN-induced SERPINE2 and PAI-1 upregulation. At the mRNA level, MSTN-induced SERPINE2 was abolished by knockdown of SMAD3, SMAD2+3 and SMAD4, but not SMAD2 (Fig. 5.8A). However, MSTN-induced SERPINE2 protein levels were partially inhibited by SMAD2 knockdown and completely abolished by SMAD3, SMAD2+3 and SMAD4 downregulation (Fig.5.8B). Interestingly, knockdown of SMAD2 alone had no effect on 78 MSTN-induced upregulation of PAI-1 mRNA (Fig.5.8C) or protein (Fig.5.8D) levels which were completely abolished by SMAD3, SMAD2+3 and SMAD4 downregulation (Fig.5.8C-D). 5.2.5 SOX4 contributes to MSTN-induced trophoblast cell invasion: SOX4 is a member of the sex determining region Y (SRY)-related high mobility-group-box (HMG box) transcription factors and is crucial for embryonic development and organogenesis [327, 328]. Moreover, SOX4 is considered a master regulator of the EMT process [329]. My lab has recently shown the pro-invasive role of SOX4 in first trimester human trophoblast cells [330]. To investigate its role in MSTN-induced trophoblast invasion, primary trophoblast cells were treated with SOX4 siRNA before adding 50 ng/mL MSTN. MSTN treatment upregulated SOX4 mRNA (Fig.5.9A) and protein (Fig.5.9B) levels when measured at 24hrs. Moreover, after confirming the efficiency of SOX4 siRNA knockdown (Fig.5.9A-B), SOX4 siRNA was used to investigate role of SOX4 in MSTN-induced trophoblast invasion. Interestingly, SOX4 siRNA treatment reduced basal and abolished MSTN-induced trophoblast cell invasion (Fig.5.9C). 5.2.6 Upregulation of transcription factor SOX4 contributes to MSTN-induced SERPINE2 but not PAI-1 production: Recently, my lab has showed that SOX4 is involved in BMP2-induced SERPINE2 upregulation [330]. Similarly, knockdown of SOX4 eliminated MSTN-induced upregulation of SERPINE2 mRNA (Fig.5.10A) and protein (Fig.5.10B) levels in primary human trophoblast cells. In contrast, depletion of SOX4 in primary human trophoblast cells enhanced the stimulatory effects of MSTN on PAI-1 mRNA (Fig.5.10C) and protein 79 (Fig.5.10D) levels, suggesting that SOX4 is not involved and/or antagonizes MSTN-induced PAI-1 production. 80 5.3 Discussion The present study demonstrates for the first time the involvement of the serine protease inhibitors SERPINE2 and PAI-1 in MSTN-induced trophoblast cell invasion. Moreover, the upregulation of both SERPINs is dependent on activin/TGF-B type 1 receptors (ALK4/5) and canonical SMAD2/3-SMAD4 signaling. Previous studies have also shown that MSTN increases trophoblast invasion by upregulating FSTL3 [76] and MMP2 [169] via ALK5 receptor and SMAD2/3-SMAD4 signaling. Interestingly, I identified differences in the involvement of SMAD2 and transcription factor SOX4 between MSTN-induced SERPINE2 and PAI-1 production. The role of SERPINE2 in placental invasion is poorly understood, however SERPINE2 knockdown has been shown to decrease explant outgrowth as well as migration and invasion of immortalized 3A-sub-E trophoblast cells [301]. However, Chern et al. [301] mistakenly described 3A-sub-E cell line as a derivative of first trimester placenta when it actually represents term villous trophoblasts [331]. Indeed, it has been shown that the EMT changes regulating the invasive potential of term EVTs is much less than that of first trimester EVTs [46]. In the current study, SERPINE2 knockdown attenuated basal invasion in primary trophoblasts but not HTR8/SVneo cells. Nevertheless, SERPINE2 knockdown completely abolished MSTN-induced invasion in both primary and immortalized human trophoblast cells. The pro-invasive effect of SERPINE2 on trophoblast cells may seem at odds with the notion that a protease inhibitor is expected to inhibit invasion as opposed to facilitating it. Intriguingly, SERPINE2 is known to exert both anti- and pro-invasive effects in various types of cancer [332]. For example, SERPINE2 plays an anti-invasive role in prostate carcinoma [333] and C6 glioma cells [334]. In contrast, SERPINE2 promotes 81 migration/invasion of pancreatic [335], breast [336], and melanoma cancer cells [337]. SERPINE2 is also overexpressed in squamous oral and colorectal carcinomas [338, 339]. The pro-invasive effect of SERPINE2 on breast cancer cells has been partly attributed to SERPINE2 binding to LRP1 receptor which activates ERK signaling that in turn upregulates MMP9 production [340]. Also, SERPINE2 has also been shown to increase local invasion of pancreatic cancer cells by upregulating extracellular matrix production [335]. The underlying mechanism by which SERPINE2 exerts its pro-invasive effects on trophoblast cells warrants further investigation. PAI-1 also appears to exert somewhat paradoxical effects as a serine protease inhibitor; an anti-invasive factor in the human placenta [341, 342], and a pro-migratory factor in breast cancer cells [343]. In the current study, PAI-1 had an anti-invasive effect on HTR8/SVneo basal invasion compared to a pro-invasive one on that of primary trophoblasts. This might reflect the differences between transformed and primary cells. Meanwhile PAI-1 had a pro-invasive effect on MSTN-induced invasion in both study models. The opposite effects of PAI-I on basal and MSTN-induced invasion of HTR8/SVneo cells deserve further investigation. PAI-1 seems to have pro- or anti-migratory effects on vascular cells depending on its concentration; low doses stimulate vascular cell migration, angiogenesis and tumour growth whereas high doses have the opposite effect [344]. PAI-1 promigratory/invasive effects may be attributed to its binding to non-proteinase ligands such as the cell adhesion protein vitronectin [345], which in turn becomes activated to support cell adhesion [346] and vascular cell migration [347] by binding to integrins. Moreover, PAI-1 has been shown to contribute to EMT-like processes in gastric [348], breast [349] and lung [350] cancer cells. 82 Human [351] as well as mouse and rat [352] placentas express high levels of SERPINE2. In addition, SERPINE2 protein is highly expressed in the uterine endometrium around implantation in humans [353], mice [352] and rats [354], implying a role for SERPINE2 in the implantation process. Likewise, PAI-1 is essential for the remodelling of maternal tissues during implantation [309, 355]. Interestingly, SERPINE2 mRNA and protein levels are downregulated in placental tissues from patients with severe PET complicated with HELLP syndrome [320]. Whether this is related to the pro-invasive effects of SERPINE2 on trophoblast cells warrants further investigation. On the other hand, placental expression of PAI-1 is higher in cases of poor placental invasion like PET/IUGR [356] and was among the hub genes in placentas of early onset PET patients along with epidermal growth factor receptor (EGFR), collagen-α-1 (I) chain (COL1A1), secreted phosphoprotein 1 (SPP1), leptin (LEP), collagen-α-2 (I) chain (COL1A2), Thy-1 membrane glycoprotein (THY1), BMP4, vascular cell adhesion protein 1 (VCAM1) and matrix metallopeptidase 1 (MMP1) [357]. However, although a positive association between high PAI-1 levels and PET has been reported [326], whether the increase is a cause or result of PET could not be determined given that PAI-1 levels are usually measured later in pregnancy after the development of PET [326]. Being a master regulator of EMT [358], SOX4 is considered a cancer signature gene as its levels are upregulated in a wide range of human cancers [359]. Indeed, SOX4 is known to regulate a variety of cancer-related phenotypes including cell migration and invasion [328]. Interestingly, SOX4 is required for TGF-β-induced EMT in mammary cells [360]. The role of SOX4 in pregnancy has not been the subject of much investigation, however, it was shown to play a role in the process of decidualization [361]. Placental SOX4 is mainly 83 localized in EVTs as well as decidual cells suggesting a role in placental invasion and implantation [330]. My lab has recently shown that SOX4 directly regulates SERPINE2 expression in human trophoblast cells by binding to regulatory sequences in the SERPINE2 promoter and enhancer areas [330]. SOX4 was also found to be involved in BMP2-induced SERPINE2 upregulation and BMP2- induced trophoblast cell invasion [330]. Likewise, in the current study, SOX4 mediates MSTN-induced SERPINE2 upregulation as well as MSTN-induced invasion in human trophoblast cells. Interestingly, SOX4 does not mediate the stimulatory effects of MSTN on PAI-1 production, suggesting that SOX4 mediates a subset of the effects of MSTN on human trophoblast cells. MSTN has previously been shown to induce the phosphorylation of receptor regulated SMAD2/3 in human trophoblast cells [76, 169]. Moreover, we [76] and others [169] have shown that the invasive effects of MSTN on human trophoblast cells are mediated by ALK5 and not ALK4. Previous studies have characterized cell-type specific differential involvement of ALK4/5 in MSTN-dependent functions; MSTN preferentially binds to ALK4 in myoblast cells as opposed to binding ALK5 in non-myogenic tissues [362]. Furthermore, the current study identifies differences in the involvement of SMAD2/3 in MSTN-induced SERPINE2 and PAI-1. Notably, the effects of MSTN on PAI-1 were mediated by SMAD3-SMAD4, which agrees with studies showing that the human PAI-1 promoter contains binding sites for SMAD3 and SMAD4, but not SMAD2 [363]. In contrast, MSTN-induced SERPINE2 was mediated by SMAD2/3-SMAD4 signaling, which is in agreement with identification of binding sequences for all three of these SMADs in the human SERPINE2 gene [364]. In addition, Chip-seq analyses have demonstrated that SOX4 and SMAD3 co-exist at multiple genetic loci, and that SOX4 acts as a cofactor for SMAD3 in regulating the 84 expression of TGF-β-induced EMT-related genes in breast cancer cells [365]. Future studies are required to determine whether SOX4 regulates MSTN-induced SERPINE2 upregulation directly or via interactions with receptor regulated SMADs. My study demonstrates for the first time the role of serine protease inhibitors SERPINE2 and PAI-1 in MSTN-induced trophoblast cell invasion. I also clarify the differential roles of canonical SMAD2/3-SMAD4 signaling as well as transcription factor SOX4 in MSTN-induced SERPINE2 and PAI-1 upregulation. My results provide insight into the potential functional consequences of dysregulated serum and placental MSTN levels in normal and pathological (PET and IUGR) placental development. 85 Figure 5. 1 MSTN upregulates SERPINE2 and PAI-1 mRNA levels in human trophoblast cells. HTR8/SVneo cells (left panel) and primary trophoblast cells (right panel) were treated for 3-24 hours with or without 50 ng/mL MSTN. SERPINE2 (upper panel) and PAI-1 (lower panel) mRNA levels were examined by RT-qPCR with GAPDH as the reference gene. MSTN upregulates SERPINE2 (A-B) and PAI-1 (C-D) mRNA levels in immortalized HTR8/Svneo and in primary trophoblast cells. Results are displayed as the mean ± SEM of at least three independent experiments and values without common letters are significantly different (P < 0.05). MSTN, myostatin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; h, hours. 86 Figure 5. 2 MSTN upregulates SERPINE2 and PAI-1 protein levels in human trophoblast cells. HTR8/SVneo cells (left panels) and primary trophoblast cells (right panels) were treated with or without 50 ng/mL MSTN for 24 and 48 hours. SERPINE2 (upper panel) and PAI-1(lower panel) protein levels were analyzed using Western blot and normalized to α-tubulin. Representative Western blot images and summarized quantitative results are shown separately. MSTN upregulates SERPINE2 (A-B) and PAI-1 (C-D) protein levels in immortalized HTR8/SVneo and primary trophoblast cells. Results are displayed as the mean ± SEM of at least three independent experiments and values without common letters are significantly different (P < 0.05). Ctrl, control; MSTN, myostatin; α-tub, α-tubulin; h, hours. 87 Figure 5. 3 SERPINE2 and PAI-1 are efficiently knocked down in human trophoblast cells as shown by RT-qPCR. HTR8/SVneo cells (left panels) or primary human trophoblast cells (right panels) were transfected for 48 hours with non-targeting control siRNA (si-Ctrl) or siRNA targeting SERPINE2 and PAI-1 prior to treatment with vehicle control or 50 ng/mL MSTN. SERPINE2 (upper panel) and PAI-1 (lower panel) mRNA levels were examined by RT-qPCR with GAPDH as the reference gene. SERPINE2 (A-B) and PAI-1(C-D) were efficiently knocked down in HTR8/SVneo and primary trophoblasts. Results are displayed as the mean ± SEM of at least three independent experiments and values without common letters are significantly different (P < 0.05). Ctrl, control; MSTN, myostatin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. 88 Figure 5. 4 SERPINE2 and PAI-1 are efficiently knocked down in trophoblast cells as shown by Western blot. SERPINE2 (upper panel) and PAI-1 (lower panel) protein levels were analyzed in HTR8/SVneo (left panel) and primary trophoblasts (right panel) by Western Blot and normalized to α-tubulin. Representative Western blot images and summarized quantitative results are shown separately. SERPINE2 (A-B) and PAI-1 (C-D) were efficiently knocked down in HTR8/SVneo and primary trophoblasts as verified by protein levels. Results are displayed as the mean ± SEM of at least three independent experiments and values without common letters are significantly different (P < 0.05). Ctrl, control; MSTN, myostatin; α-tub, α-tubulin. 89 Figure 5. 5 SERPINE2 and PAI-1 are involved in MSTN-induced trophoblast invasion. Matrigel-coated transwell invasion assay was used to examine the effects of SERPINE2 and PAI-1knockdown on MSTN-induced invasion in HTR8/SVneo (left panels) and primary trophoblast (right panels) cells. Representative images of the invasion assay (scale bars 100 µm) and combined quantitative results are shown separately. SERPINE2 knockdown (si-SERPINE2) abolished MSTN-induced trophoblast invasion of HTR8/Svneo (A) and primary trophoblast cells (B). It also decreased basal invasion of primary trophoblast cells (B) but not of HTR8/Svneo cell line (A). PAI-1 knockdown (si-PAI-1) abolished MSTN-induced trophoblast invasion of HTR8/Svneo (C) as well as primary trophoblast (D) cells. Moreover, it decreased basal invasion of primary cells (D). However, PAI-1 knockdown increased basal invasion of HTR8/SVneo cells (C). Results are displayed as the mean ± SEM of at least three independent experiments and values without common letters are significantly different (P < 0.05). Ctrl, control; MSTN, myostatin. 90 Figure 5. 6 MSTN-induced SERPINE2 and PAI-1 upregulations are mediated by activin/TGF-β type 1 receptors as shown by RT-qPCR. HTR8/SVneo cells (left panel) or primary trophoblast cells (right panel) were pre-treated for 1 hour with vehicle control (DMSO) or 10 µM SB431542 (ALK4/5/7 inhibitor) prior to treatment for 24 hours with vehicle control or 50 ng/mL MSTN. SERPINE2 (upper panel) and PAI-1 (lower panel) mRNA levels were examined by RT-qPCR with GAPDH as the reference gene. Pre-treating the trophoblast cells with the ALK4/5/7 type I receptor inhibitor SB431542 abolished the MSTN-induced upregulation of SERPINE2 (A-B) and PAI-1 (C-D) mRNA in HTR8/SVneo and primary trophoblasts. Results are displayed as the mean ± SEM of at least three independent experiments and values without common letters are significantly different (P < 0.05). Ctrl, control; MSTN, myostatin. 91 Figure 5. 7 MSTN-induced SERPINE2 and PAI-1 up-regulations are mediated by activin/TGF-β type 1 receptors as shown by Western blot. SERPINE2 (upper panel) and PAI-1 (lower panel) protein levels in HTR8/SVneo (left panel) and primary trophoblasts (right panel) were analyzed by Western Blot and normalized to α-tubulin. Representative Western blot images and summarized quantitative results are shown separately. Pre-treating the trophoblast cells with the ALK4/5/7 type I receptor inhibitor SB431542 abolished the MSTN-induced upregulation of SERPINE2 (A-B) and PAI-1 (C-D) protein levels in HTR8/SVneo and primary trophoblasts. Results are displayed as the mean ± SEM of at least three independent experiments and values without common letters are significantly different (P < 0.05). Ctrl, control; MSTN, myostatin; α-tub, α-tubulin. 92 Figure 5. 8 MSTN upregulated SERPINE2 and PAI-1levels via canonical SMAD signaling pathway. HTR8/SVneo cells were transfected for 48 hours with non-targeting control siRNA (si-Ctrl) or siRNAs targeting SMAD2 (si-2), SMAD3 (si-3), SMAD2+3 (si-2+3) or SMAD4 (si-4) prior to treatment with or without 50 ng/mL MSTN for 24 h SERPINE2 and PAI-1 mRNA (left panel) and protein (right panel) levels were measured by RT-qPCR and Western blot, respectively. Representative Western blot images and summarized quantitative results are shown separately. SMAD2 knockdown didn't abolish MSTN-induced SERPINE2 (A) or PAI-1 (C) mRNA upregulation. MSTN-induced SERPINE2 protein upregulation is mediated through SMAD2/3-SMAD4 signalling (B). MSTN-induced PAI-1 protein upregulation is mediated through SMAD3-SMAD4 signalling (D). Results are displayed as the mean ± SEM of at least three independent experiments and values without common letters are significantly different (P < 0.05). Ctrl, control; MSTN, myostatin; α-tub, α-tubulin. 93 Figure 5. 9 MSTN induces trophoblast invasion through SOX4 upregulation. Primary trophoblast cells were transfected for 48 hours with non-targeting control siRNA (si-Ctrl) or siRNAs targeting SOX4 (si-SOX4) prior to treatment for 24 hours with vehicle control or 50 ng/mL MSTN. SOX4 mRNA levels (upper left panel) were examined by RT-qPCR with GAPDH as the reference gene. SOX4 protein levels (upper right panel) were analyzed by Western blot and normalized to α-tubulin. Representative Western blot images and summarized quantitative results are shown separately. Matrigel-coated transwell invasion assays were used to examine the effects of SOX4 knockdown on MSTN-induced invasion in primary trophoblast cells. Representative images of the invasion assay (lower left panels; scale bars 100 µm) and combined quantitative results (lower right panel) are shown separately. A-B, Primary trophoblast cells treated with 50ng/mL MSTN showed an increase in SOX4 mRNA (A) and protein (B) levels, and this increase was abolished when SOX4 was knocked down using si-RNA. C, When SOX4 was knocked down in primary trophoblasts before MSTN treatment, cells invaded in lower numbers compared to controls. Results are displayed as the mean ± SEM of at least three independent experiments and values without common letters are significantly different (P < 0.05). Ctrl, control; MSTN, myostatin; α-tub, α-tubulin. 94 Figure 5. 10 Role of SOX4 in MSTN-induced SERPINE2 and PAI-1 upregulation. Primary trophoblast cells were transfected for 48 hours with non-targeting control siRNA (si-Ctrl) or siRNAs targeting SOX4 (si-SOX4) prior to treatment for 24 hours with vehicle control or 50 ng/mL MSTN. SERPINE2 and PAI-1 mRNA levels (left panels) were examined by RT-qPCR with GAPDH as the reference gene. SERPINE2 and PAI-1 protein levels (right panels) were analyzed by Western blot and normalized to α-tubulin. Representative Western blot images and summarized quantitative results are shown separately. A-B, Primary trophoblast cells treated with 50ng/mL MSTN showed an increase in SERPINE2 mRNA (A) and protein (B) levels, and this increase was abolished when SOX4 was knocked down using si-RNA. C-D, MSTN-induced increase in the expression levels of PAI-1 mRNA (C) and protein (D) was not removed when primary trophoblast cells were pre-treated with si-SOX4. Results are displayed as the mean ± SEM of at least three independent experiments and values without common letters are significantly different (P < 0.05). Ctrl, control; MSTN, myostatin; α-tub, α-tubulin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. 95 Chapter 6: Myostatin Increases Human Trophoblast Cell Invasion by Upregulating N-cadherin via SMAD2/3-SMAD4 Signaling 6.1 Introduction Placenta-mediated pregnancy complications account for a significant number of poor reproductive outcomes and can have negative impacts on maternal and fetal health [1]. Most of these diseases are related to the process of placental invasion. To properly perform its functions, the human placenta invades into the maternal uterine wall as deep as the inner third of the myometrium. In cases where the invasion is poor and superficial, pregnancy complications such as early onset PET, IUGR and PTL may occur. Placental cells that perform the invasion function are called EVT. The process underlying EVT formation is thought to involve a form of EMT [288]. EMT is a process where epithelial cells are reprogramed to become less adhesive and more migratory and/or invasive [366]. This process is regulated by a variety of different proteins, among which are a unique family called the cadherins. Cadherins are a group of transmembrane glycoproteins that regulate cell adhesion and migration in a calcium dependant manner [367]. The cadherin family comprises 114 members classified into classical, desmosomal, proto-cadherins and atypical [368]. Classical cadherins are characterized by the presence of 5 extracellular cadherin repeats for cadherin-cadherin interaction, a transmembrane domain, and a cytoplasmic domain that connects the cytoskeleton to the cell membrane via β- and α-catenin [369, 370]. Classical cadherins come in several varieties, of which type I are comprised of E-Cad, N-Cad, placental cadherin (CDH3), retinal cadherin (CDH4) and muscle cadherin (CDH15) [370]. E-Cad maintains epithelial tissue integrity [371] whereas N-Cad promotes heterotypic 96 cell-cell adhesion and collective/aggregative cell movement [372]. This means that it encourages the adhesion between epithelial and stromal/endothelial cells. N-Cad also interacts directly with other membrane proteins including fibroblast growth factor receptor (FGFR) [373]. Studies have shown that this interaction can inhibit FGFR internalization and cause persistent MAPK-ERK signaling and MMP9 expression [374]. The EMT process is often associated with a switch from E-Cad to N-Cad, thus facilitating cell migration and invasion [366, 375]. β-catenin plays an important role in cell adhesion and migration as it connects transmembrane type I cadherins to actin cytoskeleton [376]. E-Cad forms a complex with β-catenin and prevent Wnt signaling [377].Thus, the loss of E-Cad frees β-catenin and facilitates EMT [377]. N-Cad is expressed in invasive EVTs and HTR8/SVneo cells at both the mRNA and protein levels [378]. Interestingly, results from my transcriptomic analysis of MSTN-treated primary trophoblasts suggest an upregulation of N-Cad. However, the role of N-Cad in MSTN-induced trophoblast invasion is unknown. Studies have shown that the migratory and invasive effects of N-Cad can vary by cell type. Although N-Cad is upregulated and exerts a pro-invasive effect in most cancers, it is neither altered nor downregulated in other malignancies like osteosarcoma, where it has anti-invasive effects (reviewed in [379]). To this end, I decided to investigate the role of N-Cad in MSTN-induced trophoblast invasion. The effects of MSTN treatment on N-Cad levels was investigated using RT-qPCR and Western blot. Moreover, the role played by N-Cad in trophoblast invasion was studied via the transwell invasion assay, small molecule inhibitors and siRNA knockdown. 97 6.2 Results 6.2.1 Myostatin treatment upregulates N-Cad in trophoblast cells To investigate the molecular mechanisms of MSTN-induced human trophoblast cell invasion, we first examined the effects of MSTN treatment on the mesenchymal adhesion molecule N-Cad. HTR8/SVneo and primary human trophoblast cells were treated for varying times (3-24 hrs) with 50 ng/mL MSTN, and N-Cad mRNA levels were measured by RT-qPCR. As shown in Fig. 6.1A, treatment with MSTN significantly increased N-Cad mRNA levels beginning at 6 hrs in HTR8/SVneo cells and 12 hrs in primary trophoblast cells. Western blot analysis confirmed that MSTN treatment significantly increased N-Cad protein levels in HTR8/SVneo and primary trophoblasts cells treated with MSTN for 24 and 48 hrs (Fig. 6.1B). 6.2.2 N-Cad upregulation contributes to MSTN-induced trophoblast cell invasion To investigate if N-Cad upregulation contributes to MSTN-induced trophoblast cell invasion, N-Cad siRNA was used to deplete N-Cad prior to treatment with MSTN. Pre-treatment of HTR8/SVneo and primary human trophoblast cells with N-Cad siRNA abolished both basal and MSTN-induced N-Cad mRNA (Fig.6.2A) and protein (Fig.6.2B) levels. Importantly, N-Cad knockdown suppressed MSTN-induced trophoblast cell invasion in both HTR8/SVneo (Fig.6.3A) and primary trophoblast cells (Fig.6.3B). 6.2.3 MSTN-induced N-Cad upregulation is mediated by activin/TGF-β type 1 receptors To investigate whether activin/TGF-β type 1 receptors mediate the effects of MSTN on N-Cad production and cell invasion, we employed a pharmacological approach with the ALK4/5/7 type 1 receptor inhibitor SB431542. RT-qPCR (Fig.6.4A) and Western blot 98 (Fig.6.4B) results showed that pre-treatment of HTR8/SVneo and primary trophoblast cells with SB431542 blocked the stimulatory effects of MSTN on N-Cad mRNA and protein levels, respectively. 6.2.4 MSTN-induced N-Cad production is mediated by SMAD2/3-SMAD4 signaling To investigate the role of canonical SMAD2/3-SMAD4 signaling in MSTN-induced N-Cad upregulation, we performed a series of knockdown experiments with siRNAs targeting receptor-regulated SMAD2, SMAD3, SMAD2+3 as well as common SMAD4. Interestingly, although knockdown of SMAD2 or SMAD3 alone did not alter the effects of MSTN on N-Cad mRNA levels, combined knockdown of SMAD2+3 or depletion of common SMAD4 abolished MSTN-induced N-Cad mRNA upregulation (Fig.6.5A). Likewise, only knockdown of SMAD2+3 or SMAD4 was able to suppress MSTN-induced increases in N-Cad protein levels (Fig.6.5B). 99 6.3 Discussion Using both primary and immortalized human trophoblast cells, I show for the first time that MSTN increases trophoblast cell invasion by upregulating N-Cad production via canonical SMAD2/3-SMAD4 signaling. Peiris et al. demonstrated stimulatory effects of MSTN on HTR8/SVneo and primary trophoblast cell migration using the scratch wound assay [75]. Similarly, the current as well as the recent results of my lab [76] show that MSTN enhances trophoblast invasiveness in the Matrigel-coated transwell invasion assay. To migrate from the chorionic villi and invade into the decidua, EVTs lose their epithelial characteristics and acquire mesenchymal traits in a process similar to EMT [33]. Several TGF-β superfamily members are well-known inducers of EMT during embryogenesis, cancer progression, and fibrosis [48, 289, 290]. Indeed, my lab and others have reported MSTN-induced EMT-like effects in various systems such as chick dermomyotome, keratinocytes, non-small cell lung cancer cells, and ovarian cancer cells [294-296]. These EMT-like changes often involve a cadherin switch from E-Cad to N-Cad. My results suggest that MSTN-induced trophoblast invasion involves elements of EMT, including upregulation of N-Cad. Likewise, my lab has previously shown that other TGF-β superfamily members (activins and BMP2) increased human trophoblast cell invasion by upregulating N-Cad [66, 73]. However, I was not able to investigate the effects of MSTN on E-Cad in the present study because E-Cad is markedly reduced or absent in columnar and extravillous trophoblasts [49, 380, 381] as well as in HTR8/SVneo cells [381]. MSTN exerts its functions by binding to activin type 2 receptors which in turn activate type 1 receptors, primarily ALK4 and ALK5 [286]. Using siRNA knockdown of 100 ALK4 and ALK5, my lab has recently shown that MSTN-induced FSTL3 production is mediated by ALK5, and not ALK4, in human trophoblast cells [76]. In this study, I used an ALK4/5/7 inhibitor (SB431542) to confirm that MSTN-induced N-Cad production is mediated by similar type 1 receptors. However, receptor involvement may be context dependent because in ovarian cancer cells the effects of MSTN on E-Cad are mediated by both ALK4 and ALK5 [294]. Downstream MSTN signaling involves phosphorylation of receptor regulated SMAD2/3 and subsequent binding to common SMAD4 to form a complex that translocates into the nucleus and modulates gene expression. In the present study, combined knockdown of SMAD2 and SMAD3 was required to suppress MSTN-induced N-Cad production, suggesting each SMAD can compensate for the other. In contrast, we have recently shown that individual knockdown of SMAD2 or SMAD3 is sufficient to abolish MSTN-induced FSTL3 in trophoblast cells [76]. Thus, even within a given cell type, SMAD2 and SMAD3 can act redundantly or cooperatively depending on the molecular context. Other TGF-β superfamily members such as activin [66] and BMP2 [73] have also been shown to increase trophoblast cell invasion by upregulating N-Cad via SMAD2/3-SMAD4 signaling. In contrast, MSTN treatment did not alter N-Cad levels despite inducing SMAD2/3 phosphorylation and E-Cad downregulation in ovarian cancer cells [294]. Overall, the effects of MSTN and other TGF-β superfamily members on N-Cad expression cannot be explained simply by activation of SMAD2/3-SMAD4 signaling and suggest other important regulatory mechanisms, whether at the level of the promoter or upstream. Studies in pancreatic ductal epithelial and non-small cell lung cancer cells have demonstrated recruitment of SMAD4 and SMAD3/4 complexes to SMAD binding elements in the N-Cad promoter region [382, 383]. 101 However, SMAD2/3 and SMAD4 have also been shown to form a complex with Notch1 intracellular domain and the transcription factor recombination signal binding protein for immunoglobulin kappa J region (RBP-J) that is recruited to an RBP-J binding site in the N-Cad promoter in cerebrovascular endothelial cells [384]. In addition to context-dependent interactions with various transcriptional regulators, SMAD protein functions can also be modulated by other signaling pathways (e.g. ERK and p38 MAPK) that phosphorylate an internal linker region that is unique from the region phosphorylated by activin/TGF-β type I receptors [385]. Future studies will be required to further clarify the molecular determinants of MSTN-induced N-Cad production in human trophoblast cells. 102 Figure 6. 1 MSTN increases N-Cad production in HTR8/SVneo and primary human trophoblast cells. A, HTR8/SVneo cells (left panels) or primary trophoblast cells (right panels) were treated for 3-24 hours with or without 50 ng/mL MSTN, and N-Cad mRNA levels were examined by RT-qPCR with GAPDH as the reference gene. B, HTR8/SVneo cells (left panels) or primary trophoblast cells (right panels) were treated with or without 50 ng/mL MSTN for 24 and 48 hours, and N-Cad protein levels were analyzed by Western blot and normalized to α-tubulin. Representative Western blot images (upper panels) and summarized quantitative results (lower panel) are shown separately. Results are displayed as the mean ± SEM of at least three independent experiments and values without common letters are significantly different (P < 0.05). Ctrl, control; MSTN, myostatin; N-Cad, N-cadherin; α-tub, α-tubulin; h, hours. 103 Figure 6. 2 N-Cad is efficiently knocked down in human trophoblast cells using si-RNA. HTR8/SVneo cells (left panels) or primary human trophoblast cells (right panels) were transfected for 48 hours with non-targeting control siRNA (si-Ctrl) or siRNA targeting N-cadherin (si-N-Cad) prior to treatment with vehicle control or 50 ng/mL MSTN. N-Cad mRNA (A) and protein (B) levels were analyzed by RT-qPCR and Western blot (normalized to α-tubulin) respectively. Representative images (upper panels) and summarized quantitative results (lower panel) are shown separately. Ctrl, control; MSTN, myostatin; N-Cad, N-cadherin; α-tub, α-tubulin. 104 Figure 6. 3 Knockdown of N-Cad attenuates MSTN-induced trophoblast cell invasion. HTR8/SVneo cells (A) or primary human trophoblast cells (B) were transfected for 48 hours with non-targeting control siRNA (si-Ctrl) or siRNA targeting N-cadherin (si-N-Cad) prior to treatment with vehicle control or 50 ng/mL MSTN. Matrigel-coated transwell invasion assays were used to examine the effects of N-Cad knockdown on MSTN-induced invasion. Representative images of the invasion assay (left panels; scale bars 100 µm) and combined quantitative results (right panels) are shown separately. Results are displayed as the mean ± SEM of at least three independent experiments and values without common letters are significantly different (P < 0.05). Ctrl, control; MSTN, myostatin; N-Cad, N-cadherin; α-tub, α-tubulin. A0.00.51.01.52.02.5 CtrlCell invasionFold change relative to controlabc cMSTNsi-Ctrl si-N-CadCtrl0.00.51.01.52.02.5si-Ctrl si-N-CadCell invasionFold change relative to controla abaMSTNasi-Ctrlsi-N-Cad+MSTNsi-Ctrl+MSTNsi-N-Cadsi-Ctrl si-Ctrl+MSTNsi-N-cad si-N-cad+MSTNFigure 6.3 Knockdown of N-Cad attenuates MSTN-induced trophoblast cell invasion.HTR8/SVneo cells (A) or primary human trophoblast cells (B) were transfected for 48 hours with non-targeting control siRNA (si-Ctrl) or siRNA targeting N-cadherin (si-N-Cad) prior to treatment with vehicle control or 50 ng/mL MSTN. Matrigel-coated transwell invasion assays were used to examine th effects of N-Cad knockdown on MSTN-induced invasion. Representativ images of the invasion assay (left panels; scale bars 100 µm) and combined quantitative results (right panels) are shown separately. Results are displayed as the mean ±SEM of at least three independent experiments and values without common letters are significantly different (P < 0.05). Ctrl, control; MSTN, myostatin; N-Cad, N-cadherin; α-tub, α-tubulin.HTR8/SVneo cellsPrimary trophoblast cellsB 105 Figure 6. 4 MSTN-induced N-Cad upregulation and trophoblast cell invasion are mediated by activin/TGF-β type1receptors. HTR8/SVneo cells (left panels) and primary human trophoblast cells (right panels) were pre-treated for 1 hour with vehicle control (DMSO) or 10 µM SB431542 (ALK4/5/7 inhibitor) prior to treatment for 60 min with vehicle control or 50 ng/mL MSTN. N-Cad mRNA (A) and protein (B) levels were analyzed by RT-qPCR and Western blot respectively. Representative Western blot images (upper panels) and summarized quantitative results (lower panels) are shown separately. Ctrl, control; MSTN, myostatin; N-Cad, N-cadherin; α-tub, α-tubulin. 106 Figure 6. 5 SMAD2/3-SMAD4 signaling mediates the upregulation of N-Cad by MSTN. HTR8/SVneo cells were transfected for 48 hours with non-targeting control siRNA (si-Ctrl) or siRNAs targeting SMAD2 (si-2), SMAD3 (si-3), SMAD2+3 (si-2+3) or SMAD4 (si-4) prior to treatment with or without 50 ng/mL MSTN for 24 h. N-Cad mRNA (A) and protein (B) levels were likewise measured by RT-qPCR and Western blot. Representative Western blot images (upper panels) and summarized quantitative results (lower panels) are shown separately. Results are displayed as the mean ± SEM of at least three independent experiments and values without common letters are significantly different (P < 0.05). Ctrl, control; MSTN, myostatin; N-Cad, N-cadherin; α-tub, α-tubulin. 107 Chapter 7: Conclusions 7.1 Summary of Findings The placenta is the first fetal organ to develop. This reflects its extreme importance and vital role in pregnancy. Interestingly, its effect is not limited to the nine months of pregnancy but rather extends to childhood and adulthood periods according to the DOHaD theory [386]. This theory hypothesizes that the causes of many non-infectious childhood and adulthood diseases are rooted during the in-utero and early neonatal periods due to epigenetic effects of environmental stressors [176]. To properly performs its functions, the placenta needs to invade into the uterine wall. This invasion is tightly regulated by many growth factors and cytokines secreted by decidual stromal and immune cells as well as by placental trophoblasts [32]. Any dysregulation may result in either excessive or insufficient invasion leading to pregnancy complications. Placental over-invasion causes placenta accreta spectrum which can end in fatal post-partum bleeding [42]. On the other hand, poor placental invasion results in several pregnancy complications including early onset-PET, IUGR, PTL and fetal loss [179]. The process of placental invasion is carried by a specialized type of cells called EVTs. These cells invade into the decidual interstitial tissues and into the spiral arteries reaching the inner third of the myometrium [244]. Together with uNKs, they remodel the spiral arteries into wide low pressure blood conduits to allow the smooth delivery of large blood volumes under low pressure [22]. To invade, distal column villous trophoblasts, undergo a transition process from epithelial to mesenchymal cells called EMT [33]. Through this process, column trophoblasts become migratory and leave the cell column as EVTs. A popular group of growth factors called TGF- β superfamily is a well-known player in EMT. 108 A member of this family, MSTN, well known as a negative muscle regulator [93] was shown to be expressed by EVTs [75] and to upregulate their migration [75] and invasion [169] More importantly MSTN serum levels and/or placental expression were upregulated in a number of placenta-mediated disorders including PET [83, 170] and IUGR [83]. However, its role and regulating molecular mechanisms in placental biology is not yet clear. The main aim of the current study was to investigate the pro-invasive role of MSTN in human trophoblast cells and to decipher the transcriptomic changes of MSTN-treated EVTs to understand the underlying molecular mechanisms orchestrating MSTN-induced trophoblast invasion. I hypothesized that MSTN induces trophoblast invasion via ALK4/5 receptors and through the canonical SMAD2/3-SMAD4 pathway to regulate the expression of genes involved in trophoblast invasion. To achieve those aims, I used two models; immortalized HTR8/SVneo cell line and primary human trophoblast cells isolated from first trimester placentas obtained from patients who underwent elective pregnancy termination. Matrigel transwell invasion assay was used to study the effect of MSTN and genes of interest on trophoblast invasion. I also performed an RNA sequencing analysis to profile the transcriptome of MSTN-treated primary trophoblast cells. RT-qPCR and Western blot were used to measure mRNA and protein levels, respectively. Small molecule inhibitor and siRNA transfection were used to block ALK4/5/7 receptor and to knockdown SMADs and other genes of interest, respectively. The current study found that MSTN exerts its pro-invasive effect on human trophoblast cells via ALK4/5 receptors and the canonical SMAD2/3-SMAD4 signaling pathway. Furthermore, the RNA sequencing analysis of MSTN-treated primary trophoblasts showed that MSTN differentially expressed (FDR<0.05) 610 genes; 380 were upregulated 109 while 230 were downregulated. GSEA analysis of the 610 DEGs revealed a significant positive enrichment in the EMT genes involved in cell adhesion, cell migration, and ECM assembly. I then investigated the role and molecular regulation of four of the DEGs, namely, SERPINE2, PAI-1, SOX4 and N-Cad in MSTN-induced trophoblast invasion. I chose SERPINE2 because it is among the top DEGs in my RNA-seq analysis [Appendix A]. I was also intrigued to know the function of PAI-1which is a SERPINE2 paralogue, in MSTN-induced trophoblast invasion. Interestingly, SERPINE2 and PAI-1 are highly expressed in human placenta [301, 324] and their levels are dysregulated in cases of PET [320, 356]. The transcription factor SOX4 is also a top DEG in my RNA-seq analysis [Appendix A] and is a master EMT transcription factor [358]. Furthermore, SOX4 was shown recently by our lab to play a role in BMP2-induced trophoblast invasion and SERPINE2 upregulation [330]. Additionally, N-Cad was chosen because it is a hallmark gene in the EMT process [387] and was previously shown by our lab to be involved in activin- [66] and BMP2- [73]induced trophoblast invasion. In the current study, MSTN upregulated the mRNA and protein levels of SERPINE2, PAI-1, and N-Cad through ALK4/5 receptor and canonical SMAD signaling. Interestingly MSTN used the canonical SMAD2/3-SMAD4 to upregulate SERPINE2 and N-Cad. However, SMAD2 didn’t seem to be involved in MSTN-induced PAI-1 upregulation which was mediated by SMAD3-SMAD4 signaling. Using siRNA knockdown, I showed that SERPINE2, PAI-1, SOX4 and N-Cad were involved in the MSTN-induced trophoblast invasion. Moreover, I found the transcription regulator factor SOX4 to be involved in the SERPINE2 upregulation in primary trophoblast cells. A graphic summary of the results is shown in Fig.7.1. 110 Figure 7. 1 Potential molecular mechanisms regulating MSTN-induced human trophoblast invasion. MSTN binds to human EVT cell and starts a signaling cascade that ends in changes in transcription of EMT genes that eventually induces trophoblast invasion. MSTN binding to ACVR2B receptors on human EVT activates ALK4/5 which in turn results in phosphorylation of receptor-regulated SMAD2/3. Phosphorylated SMAD2/3 form a complex with the common SMAD4 and enters the nucleus as a transcription factor to upregulate transcription of SERPINE2 and N-Cad. Phosphorylated SMAD3 alone forms a complex with the common SMAD4 and enters the nucleus to induce the transcription of PAI-1. MSTN also upregulates the expression of the transcription factor SOX4 which in turn induces the transcription of SERPINE2. SERPINE2, PAI-1, N-Cad and SOX4 proteins play a pro-invasive role in human trophoblast and induces its invasion into the decidual interstitial tissue till inner third of the myometrium to support the developing fetus (Original figure created in Biorender.com). 111 7.2 Significance and translational potential The current thesis focuses on the role of MSTN on placental invasion which is related to several pregnancy complications including early onset PET, IUGR, PTL and fetal loss [179]. These diseases are commonly referred to as placenta-mediated [1] because insufficient placental invasion is among the underpinning aetiologies. Poor placental invasion and insufficiency are thought to be major contributing factors in early-onset PET (< 34 GW), whereas the late-onset subtype is mainly related to maternal, and not placental factors [388]. Early-onset PET accounts for only 10% of PET cases [389], however it is the most severe type and results in serious maternal [390] and fetal complications [391]. In addition to PET, IUGR is a common complication of placental insufficiency resulting from poor placental invasion [391]. Indeed, the dysregulation of MSTN in PET and IUGR [83, 170] was shown in previous work by other groups [83, 170]. First [83] and third [170] trimester serum MSTN levels were elevated in patients who later developed PET and those diagnosed with PET, respectively. Moreover, placental MSTN mRNA [170] and protein [83, 170] levels were higher in placental tissue from PET pregnancies. Similarly, placental but not serum MSTN levels were elevated in cases of IUGR [83]. Elevated serum and placental MSTN levels suggest that the placenta may be the source of MSTN in PET. On the other hand, second trimester serum MSTN levels were not different in women who developed PET later in pregnancy compared to controls [392]. However, it is important to keep in mind that none of the above referenced groups clearly differentiated between early- and late-onset PET despite the pathophysiological and molecular [185, 393] differences between the two types. 112 Impaired trophoblast invasion results in insufficient spiral artery remodeling which can generate an ischemic placental environment [394], resulting in secretion of harmful factors into the systemic circulation that can induce endovascular and organ damage [395]. Our findings of a positive role for MSTN in trophoblast cell invasion may appear contradictory to the findings of elevated serum and/or placental MSTN in PET and IUGR. The increase in MSTN levels in PET and IUGR may be a compensatory mechanism to increase trophoblast invasion which is commonly impaired in such cases. Similarly, placental expression of FSTL3 was found to be higher in cases of PET [170] and IUGR [396]. FSTL3 antagonizes MSTN [99] and thus may weaken its pro-invasive effects. Another role that MSTN may have in PET can be through its effect on cytokine production. PET is associated with an imbalance between pro-inflammatory T cells and anti-inflammatory T-reg cells resulting in higher levels of pro-inflammatory cytokines and lower levels of anti-inflammatory cytokines [397]. Interestingly, MSTN was shown to lower the levels of pro-inflammatory cytokines including tumor necrosis factor alpha, interferon gamma and interleukin (IL)-8 and increases the levels of anti-inflammatory cytokine IL-4 in placental explant cultures [83]. It can be suggested that increased placental MSTN in cases of PET and IUGR might be a compensatory mechanism to increase trophoblast invasion and anti-inflammatory cytokines. However, high levels of FSTL3 binds to MSTN and prevent its action. Alternatively, MSTN might play different roles in different trophoblast cell populations. My study investigated its functions in EVTs, where it positively regulates trophoblast invasion. However, MSTN might have different roles in other placental cell types, such as villous trophoblasts, syncytiotrophoblast, and decidual stromal cells. Future 113 studies investigating MSTN functions in these and other placental cells will be of great interest. The current study gives insight into the role played by MSTN in placental biology. Furthermore, it provides a functional context for dysregulated MSTN levels in placenta-mediated pregnancy complications, mainly PET and IUGR. However, whether MSTN can have a therapeutic role in those complications warrants further investigations. 7.3 Strengths of the study To my knowledge, this is the first study to profile the transcriptome of MSTN-treated human trophoblasts. This transcriptome might pave the road for other investigators to deepen our understanding of genomic regulation by MSTN in human trophoblast cells. The enrichment of the EMT genes, including those involved in cell adhesion and ECM structure and organization, in the transcriptome of MSTN-treated trophoblasts shed some light on the molecular determinants of MSTN-induced trophoblast invasion. Although the pro-invasive effect of 25ng/mL MSTN was previously shown [76, 169], the current study demonstrated that MSTN -induced trophoblast invasion can be achieved at more physiologically relevant doses as low as 5ng/mL. Moreover, the present study showed for the first time, that MSTN performed its pro-invasive effects through the serine protease inhibitors; SERPINE2, PAI-1, SOX4 as well as the cell adhesion molecule N-Cad. The current study also investigated the signaling pathway adopted by MSTN to exert its effects on the above-mentioned target genes. Notably, I also showed for the first time that MSTN upregulated the transcription factor SOX4 which also contributed to MSTN-induced SERPINE2 upregulation. 114 7.4 Limitations Gene expression in vivo is very complex and studying a few molecules can only tell a small part of the story [398]. Moreover, how trophoblasts react in culture might differ from their behavior in vivo, where they are in contact with other placental and decidual cells. Also, in vivo trophoblasts cells are under the influence of the interplay of different hormones, growth factors, and cytokines which can synergize, neutralize, or even oppose MSTN pro-invasive effects on human trophoblast cells. Furthermore, the 3D architecture of tissues is completely lost in the culture dish, adding to the potential differences between in vivo and in vitro MSTN effects on trophoblast cells. Consequently, the in vivo effects of MSTN may or may not closely match our findings using in vitro models. 7.4.1 Study model I chose the HTR8/SVneo cell line as a model to study trophoblast invasion because it was commonly used as an EVT study model. Also, immortalized cell lines are easy to obtain and maintain in culture for many passages, allowing for enhanced reproducibility of results. However, the use of HTR8/SVneo cells has been questioned by some researchers because they do not meet all the trophoblast criteria proposed by Lee et al. [213]. A trophoblast is characterized by unmethylated ELF5 promoter, expression of miRNA on chromosome 19, expression of certain protein markers (KRT7, TFAP2C and GATA3) and either the absence of HLA class I molecule for villous trophoblast or expression of HLA-C, G and E in case of EVTs [213]. In contrast, HTR8/SVneo cells have a methylated ELF5 promoter and an HLA expression that doesn’t match with villous or extravillous trophoblasts [213]. Moreover, it has been reported that the HTR8/SVneo cell line contains a mixed population of epithelial and 115 mesenchymal cells [220]. However, the presence of mesenchymal cells is suggested to be a result of an EMT process while in culture, rather than being a part of the progenitor cells of the cell line [217]. Yet to date, HTR8/SVneo cells remain a commonly used human EVT cell model. Importantly, my results were replicated using primary trophoblasts isolated from first trimester placentas. 7.4.2 RNA sequencing I used primary trophoblast cells isolated from only 5 first trimester placentas to study MSTN-induced transcriptomic changes. Unfortunately, those placental samples didn’t have any associated clinical data, including maternal age, parity, reason for pregnancy termination or history of placenta-mediated diseases. Miscarriages or pregnancies terminated for medical reasons might be genetically abnormal [399]. Even pregnancies terminated for social reasons can’t be guaranteed to have developed normally if pregnancy to be continued [399]. Thus, I ended up with placentas that displayed some differences in basal gene expression, but I mitigated this problem by using analysis with a paired sample design to identify DEGs between MSTN-treated and control groups. Another limitation is studying MSTN effect at a single time point only. I studied the effect of a 24 hr MSTN treatment on trophoblast transcriptome. Examining the effect at multiple earlier time points might have resulted in more differentially expressed genes especially those for which transcription occurs shortly after treatment (e.g., 3-6hrs). 7.4.3 Invasion assay The Matrigel transwell invasion assay is very popular in trophoblast invasion studies. However, it is very simplistic and cannot reflect the complexity of in vivo invasion. It is an endpoint assay that doesn’t give any information regarding the velocity and direction 116 of invasion. The Roche xCELLigence System is an expensive adaptation of the transwell invasion assay that uses microelectrodes to quantify cell invasion in real-time [400] Moreover, trophoblast invasion is essential for a successful implantation process and is under the control of multiple types of maternal cells including decidual stromal, endothelial and immune cells [401]. Thus, the transwell invasion assay doesn’t recapitulate the heterogeneity of the maternal fetal interface as well as the 3D in vivo cellular environment [402]. A better alternative that can more accurately reflect the in vivo implantation environment is an implantation on a chip device which is a microfluidic system in which fetal (trophoblast) and decidual (stromal, endothelial, and natural killer) cells are cultured separated by an ECM in a 3D system [403]. This system can measure not only trophoblast invasion through an ECM but also through a spiral artery model. Moreover, it allows the study of the effect of the different maternal decidual cells on trophoblast invasion [403]. However, this is a brand new and custom manufactured system and is not yet commercially available. 7.5 Future Directions Due to the limited sample size (n=5) of the current RNA sequencing analysis, it was difficult to study the effect of fetal sex on MSTN-induced placental transcriptomic changes. Indeed, fetal gender has a prominent effect on the placental transcriptome [274] and pregnancy complications including PET [404, 405] and PTL [406, 407]. In the future performing an RNA sequencing analysis using a larger sample size will give us a deeper understanding of the effect of sex on MSTN-induced placental transcriptomic effects. Future studies will also need to take in consideration the complexity of the in vivo environment regarding cell architecture and interaction between different types of fetal and maternal cells. 117 A 3D co-culture system that includes fetal trophoblasts as well as decidual stromal, immune, and endothelial cells will hopefully paint a better picture regarding MSTN effects on trophoblast invasion. Future studies could also explore potential roles for non-canonical MSTN signaling via ERK1/2, p-38 MAPK, and JNK pathways [112-114]. Furthermore, whether MSTN plays a role in other placental functions including hormone secretions and placental transport (other than glucose) needs to be explored. In addition, more clinical studies are needed to deepen our understanding of the role of MSTN in different pregnancy complications. The studies need to clearly differentiate between early- and late-onset PET. Also, serum MSTN needs to be measured at multiple time points during pregnancy to span the three trimesters and then correlated to the pregnancy outcome. This would allow us to determine if MSTN can be used as a serum biomarker to predict different pregnancy complications. The studies will also clarify if MSTN can be of any therapeutic benefit in placental-mediated diseases. Also, to my knowledge, MSTN was not investigated in pregnancy complications with excessive trophoblast invasion including placenta accreta spectrum. It would be interesting to know if MSTN serum and placental levels are dysregulated in such conditions. 118 References 1. Duffett, L. and M. Rodger, LMWH to prevent placenta-mediated pregnancy complications: an update. Br J Haematol, 2015. 168(5): p. 619-38. 2. Lu, H.Q. and R. Hu, Lasting Effects of Intrauterine Exposure to Preeclampsia on Offspring and the Underlying Mechanism. AJP Rep, 2019. 9(3): p. e275-e291. 3. 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Early Hum Dev, 2004. 76(1): p. 47-54. 147 Appendices Appendix A List of the DEGs in human first-trimester primary trophoblasts (MSTN vs Control) ID log2 Fold Change Fold Change P-value Adjusted P-value CST1 1.0726 2.1032 0.0000 0.0000 CALB2 0.8766 1.8361 0.0000 0.0000 F2RL1 0.8693 1.8267 0.0000 0.0000 TNC 0.8631 1.8190 0.0000 0.0000 KCNH1 0.8562 1.8103 0.0000 0.0000 TNFSF15 0.8316 1.7796 0.0000 0.0000 SERPINE2 0.8217 1.7674 0.0000 0.0000 PMEPA1 0.7859 1.7241 0.0000 0.0000 DIRC3 0.7564 1.6893 0.0000 0.0000 KCNG1 0.7535 1.6859 0.0000 0.0006 LRRC15 0.7482 1.6798 0.0000 0.0000 SEMA3C 0.7469 1.6782 0.0000 0.0000 NTM 0.7211 1.6485 0.0000 0.0000 PTHLH 0.7198 1.6469 0.0001 0.0018 INHBA 0.6321 1.5499 0.0000 0.0000 SHISA9 0.6272 1.5445 0.0000 0.0001 SPON2 0.6148 1.5314 0.0000 0.0000 MEX3A 0.6130 1.5295 0.0000 0.0000 PLCXD3 0.6084 1.5246 0.0000 0.0001 NOX4 0.6070 1.5231 0.0001 0.0030 SIK1 0.5935 1.5089 0.0000 0.0000 SOX4 0.5882 1.5033 0.0000 0.0000 ANGPTL4 0.5786 1.4933 0.0001 0.0020 HAS2 0.5738 1.4884 0.0000 0.0007 MMP10 0.5617 1.4761 0.0000 0.0000 CTGF 0.5588 1.4730 0.0000 0.0000 HMCN1 0.5544 1.4686 0.0000 0.0000 COCH 0.5463 1.4604 0.0001 0.0036 NCAM1 0.5441 1.4581 0.0000 0.0000 HUNK 0.5411 1.4550 0.0001 0.0028 DACT1 0.5375 1.4514 0.0000 0.0001 SLC22A23 0.5305 1.4444 0.0000 0.0000 ADAM19 0.5275 1.4415 0.0000 0.0000 DIO2 0.5240 1.4379 0.0000 0.0000 KRT7 0.5205 1.4345 0.0000 0.0000 CNN1 0.5185 1.4324 0.0000 0.0000 148 ID log2 Fold Change Fold Change P-value Adjusted P-value DGKI 0.5046 1.4187 0.0000 0.0000 ZSWIM5 0.4994 1.4136 0.0000 0.0000 MYOCD 0.4901 1.4045 0.0000 0.0013 OSGIN2 0.4833 1.3980 0.0000 0.0000 HTRA1 0.4804 1.3952 0.0000 0.0000 TMEM2 0.4764 1.3913 0.0000 0.0000 ITGA1 0.4596 1.3752 0.0000 0.0000 GRIK2 0.4580 1.3736 0.0000 0.0000 LOX 0.4555 1.3712 0.0000 0.0000 PCDH10 0.4533 1.3692 0.0000 0.0000 LRRC2 0.4529 1.3687 0.0017 0.0335 ETV4 0.4440 1.3604 0.0000 0.0000 NREP 0.4411 1.3577 0.0005 0.0113 TP53I3 0.4393 1.3559 0.0000 0.0000 ANOS1 0.4369 1.3537 0.0000 0.0008 RASSF2 0.4319 1.3490 0.0003 0.0087 IGFBP3 0.4277 1.3451 0.0000 0.0000 ITGB3 0.4277 1.3451 0.0000 0.0000 ADAMTS6 0.4257 1.3432 0.0000 0.0002 SLC8A1 0.4254 1.3429 0.0000 0.0000 KIAA1024 0.4174 1.3355 0.0001 0.0029 LPXN 0.4145 1.3329 0.0004 0.0113 FAM196B 0.4138 1.3322 0.0000 0.0002 COL7A1 0.4123 1.3308 0.0002 0.0064 ARSI 0.4119 1.3305 0.0002 0.0057 LAMC2 0.4114 1.3300 0.0000 0.0000 IL32 0.4091 1.3278 0.0000 0.0001 PCDH19 0.4080 1.3268 0.0011 0.0234 ENC1 0.4073 1.3262 0.0000 0.0000 FZD8 0.4038 1.3230 0.0000 0.0001 ADAM12 0.4020 1.3213 0.0000 0.0000 N-Cad 0.3993 1.3189 0.0000 0.0000 MEOX1 0.3992 1.3188 0.0001 0.0036 PAI-1 0.3970 1.3168 0.0000 0.0000 ADAMTS4 0.3938 1.3138 0.0001 0.0038 CDK6 0.3937 1.3137 0.0000 0.0000 DSE 0.3930 1.3131 0.0000 0.0000 SLC7A5 0.3900 1.3104 0.0000 0.0000 PGM2L1 0.3889 1.3094 0.0000 0.0001 NDRG4 0.3883 1.3089 0.0000 0.0012 IGFBP5 0.3856 1.3064 0.0000 0.0000 FAM26E 0.3824 1.3035 0.0000 0.0003 149 ID log2 Fold Change Fold Change P-value Adjusted P-value ARHGAP22 0.3821 1.3032 0.0000 0.0000 IRS1 0.3801 1.3014 0.0000 0.0000 LRIG1 0.3783 1.2998 0.0000 0.0000 LIMS1 0.3621 1.2853 0.0000 0.0000 P4HA3 0.3621 1.2853 0.0006 0.0156 ZSWIM4 0.3616 1.2848 0.0002 0.0052 RGMB 0.3604 1.2837 0.0000 0.0000 TUBA1A 0.3568 1.2806 0.0000 0.0000 DYSF 0.3532 1.2774 0.0000 0.0002 RUNX1 0.3528 1.2771 0.0000 0.0000 HK2 0.3461 1.2711 0.0000 0.0000 AMPD3 0.3454 1.2705 0.0000 0.0000 VDR 0.3433 1.2687 0.0000 0.0000 PFKFB3 0.3432 1.2686 0.0000 0.0000 NEGR1 0.3401 1.2658 0.0000 0.0000 RFTN2 0.3361 1.2624 0.0009 0.0206 SLC16A2 0.3339 1.2604 0.0000 0.0000 CASK 0.3290 1.2561 0.0000 0.0000 TNFSF4 0.3286 1.2558 0.0000 0.0000 STK38L 0.3261 1.2536 0.0000 0.0000 UPP1 0.3259 1.2535 0.0000 0.0000 MFAP2 0.3248 1.2525 0.0000 0.0001 CD82 0.3248 1.2525 0.0000 0.0000 COL4A1 0.3238 1.2516 0.0000 0.0000 TPM1 0.3227 1.2507 0.0000 0.0000 PRR5L 0.3224 1.2504 0.0000 0.0002 NAV1 0.3202 1.2485 0.0000 0.0000 PDCD1LG2 0.3183 1.2469 0.0000 0.0000 MEGF6 0.3180 1.2466 0.0014 0.0283 MET 0.3157 1.2447 0.0000 0.0000 YOD1 0.3149 1.2439 0.0001 0.0032 IER3 0.3144 1.2435 0.0000 0.0000 CSF2RB 0.3102 1.2399 0.0016 0.0322 POU2F2 0.3059 1.2362 0.0000 0.0000 COTL1 0.3049 1.2353 0.0000 0.0000 IL7R 0.3044 1.2349 0.0000 0.0000 ABLIM3 0.3033 1.2340 0.0000 0.0000 KIF13B 0.3021 1.2329 0.0000 0.0005 MT2A 0.3018 1.2327 0.0000 0.0000 MMP12 0.2986 1.2299 0.0000 0.0000 DSP 0.2976 1.2291 0.0000 0.0000 FN1 0.2967 1.2283 0.0000 0.0000 150 ID log2 Fold Change Fold Change P-value Adjusted P-value PLBD1 0.2954 1.2272 0.0000 0.0000 FAM46C 0.2951 1.2269 0.0000 0.0012 PAPPA 0.2948 1.2268 0.0000 0.0000 BCAT1 0.2947 1.2266 0.0000 0.0000 ACTG2 0.2940 1.2260 0.0002 0.0061 LUZP1 0.2924 1.2247 0.0000 0.0000 PALM2 0.2890 1.2218 0.0015 0.0314 SMURF2 0.2889 1.2217 0.0000 0.0000 FSTL3 0.2857 1.2190 0.0001 0.0020 ARNTL2 0.2813 1.2153 0.0001 0.0035 COL5A1 0.2811 1.2151 0.0000 0.0000 LRRC17 0.2803 1.2144 0.0000 0.0001 WNT5B 0.2795 1.2138 0.0000 0.0000 PLAU 0.2791 1.2135 0.0000 0.0000 WNT5A 0.2781 1.2126 0.0000 0.0000 PLEKHO1 0.2778 1.2124 0.0000 0.0000 COL1A1 0.2774 1.2120 0.0000 0.0006 ENAH 0.2771 1.2118 0.0000 0.0000 SCUBE3 0.2768 1.2115 0.0000 0.0000 MEX3B 0.2757 1.2106 0.0015 0.0298 DCBLD1 0.2730 1.2084 0.0000 0.0000 PLOD2 0.2728 1.2081 0.0000 0.0000 NEDD9 0.2703 1.2060 0.0000 0.0002 TIPARP 0.2698 1.2056 0.0000 0.0003 FBXO32 0.2676 1.2038 0.0000 0.0007 SMAD7 0.2668 1.2031 0.0002 0.0067 SEMA7A 0.2660 1.2024 0.0000 0.0010 PKIA 0.2657 1.2022 0.0025 0.0467 MXRA5 0.2648 1.2014 0.0000 0.0000 TXNDC5 0.2633 1.2002 0.0001 0.0033 UBASH3B 0.2626 1.1996 0.0000 0.0000 SYNE1 0.2599 1.1974 0.0000 0.0001 FRMD6 0.2597 1.1972 0.0000 0.0000 P4HA2 0.2588 1.1965 0.0000 0.0000 ARHGEF28 0.2583 1.1961 0.0001 0.0043 DOPEY2 0.2582 1.1960 0.0010 0.0216 DPYSL3 0.2568 1.1949 0.0000 0.0000 TANC2 0.2559 1.1941 0.0000 0.0007 MAP3K7CL 0.2530 1.1917 0.0001 0.0032 SPOCD1 0.2511 1.1901 0.0000 0.0006 MARCH4 0.2506 1.1897 0.0000 0.0006 SNCAIP 0.2496 1.1889 0.0022 0.0424 151 ID log2 Fold Change Fold Change P-value Adjusted P-value MARCKS 0.2491 1.1885 0.0000 0.0000 TMEM158 0.2488 1.1882 0.0000 0.0001 CADM1 0.2486 1.1880 0.0000 0.0004 LTBP2 0.2481 1.1876 0.0000 0.0000 SKIL 0.2461 1.1860 0.0000 0.0000 TGFB2 0.2453 1.1853 0.0000 0.0006 COL4A2 0.2449 1.1850 0.0000 0.0000 NRP2 0.2433 1.1837 0.0000 0.0000 EFHD1 0.2428 1.1833 0.0024 0.0448 THBS1 0.2424 1.1829 0.0000 0.0000 YPEL5 0.2421 1.1828 0.0000 0.0000 VCAN 0.2418 1.1825 0.0000 0.0000 TES 0.2414 1.1822 0.0000 0.0000 CDKN2B 0.2403 1.1812 0.0000 0.0001 COL16A1 0.2402 1.1812 0.0000 0.0000 LPCAT2 0.2390 1.1802 0.0000 0.0000 KCTD1 0.2390 1.1801 0.0002 0.0046 FBN1 0.2360 1.1777 0.0000 0.0000 PSMD2 0.2355 1.1773 0.0000 0.0000 SEPT11 0.2345 1.1765 0.0000 0.0000 RHOBTB1 0.2341 1.1762 0.0003 0.0087 SPDL1 0.2336 1.1758 0.0002 0.0067 RALA 0.2336 1.1758 0.0000 0.0000 MMP2 0.2316 1.1741 0.0000 0.0000 SLC1A4 0.2306 1.1733 0.0003 0.0070 CD55 0.2304 1.1732 0.0000 0.0000 PPFIBP1 0.2279 1.1712 0.0000 0.0000 VAC14 0.2261 1.1697 0.0000 0.0001 TUBB3 0.2258 1.1694 0.0000 0.0000 SMTN 0.2249 1.1687 0.0000 0.0000 BGN 0.2227 1.1669 0.0000 0.0004 PSD3 0.2225 1.1668 0.0000 0.0001 VEGFC 0.2221 1.1665 0.0000 0.0000 NT5DC2 0.2214 1.1658 0.0000 0.0000 UCK2 0.2211 1.1656 0.0000 0.0008 NET1 0.2205 1.1651 0.0002 0.0059 NUAK1 0.2203 1.1650 0.0000 0.0002 MLLT11 0.2197 1.1645 0.0000 0.0000 PDPN 0.2169 1.1623 0.0000 0.0000 RAB3B 0.2160 1.1615 0.0000 0.0000 SAT1 0.2148 1.1606 0.0000 0.0004 MYO1D 0.2146 1.1604 0.0000 0.0001 152 ID log2 Fold Change Fold Change P-value Adjusted P-value TNFRSF12A 0.2142 1.1600 0.0000 0.0004 MICAL2 0.2137 1.1597 0.0000 0.0000 PODXL 0.2129 1.1590 0.0000 0.0000 HHAT 0.2127 1.1589 0.0014 0.0293 TMEM132A 0.2121 1.1584 0.0000 0.0000 GADD45A 0.2111 1.1576 0.0000 0.0000 BNC2 0.2109 1.1574 0.0012 0.0250 RBP1 0.2092 1.1560 0.0016 0.0324 MAPK8 0.2086 1.1556 0.0000 0.0007 CXCL8 0.2076 1.1548 0.0000 0.0000 CALM2 0.2074 1.1546 0.0000 0.0000 BACE2 0.2071 1.1543 0.0001 0.0020 PSME4 0.2060 1.1535 0.0000 0.0001 SRGAP1 0.2054 1.1530 0.0004 0.0092 SPSB1 0.2044 1.1522 0.0002 0.0052 PFKP 0.2032 1.1513 0.0000 0.0000 SH3RF1 0.2026 1.1508 0.0004 0.0097 SGK1 0.2026 1.1507 0.0000 0.0002 LOXL2 0.2023 1.1506 0.0000 0.0000 CTHRC1 0.2022 1.1505 0.0001 0.0023 FAM214B 0.2022 1.1505 0.0001 0.0025 HYOU1 0.2015 1.1499 0.0000 0.0000 MMP3 0.2013 1.1498 0.0000 0.0000 PPP1R14B 0.2013 1.1497 0.0000 0.0002 SH3PXD2B 0.2010 1.1495 0.0000 0.0001 TBX3 0.1999 1.1486 0.0000 0.0000 MARCKSL1 0.1999 1.1486 0.0000 0.0014 SPECC1 0.1996 1.1483 0.0000 0.0012 FAT4 0.1988 1.1477 0.0004 0.0096 DAP 0.1985 1.1475 0.0000 0.0000 SIPA1L3 0.1967 1.1461 0.0006 0.0137 ITGB5 0.1958 1.1453 0.0000 0.0000 PPP2R3A 0.1950 1.1447 0.0009 0.0199 SH3D19 0.1949 1.1446 0.0000 0.0003 RAP2B 0.1946 1.1444 0.0002 0.0045 MTHFD1L 0.1939 1.1439 0.0002 0.0048 BICC1 0.1927 1.1429 0.0000 0.0016 LCP1 0.1914 1.1419 0.0000 0.0000 PLXNB3 0.1899 1.1407 0.0026 0.0484 COL1A2 0.1895 1.1404 0.0000 0.0000 TMEM57 0.1894 1.1403 0.0009 0.0194 CLSTN2 0.1883 1.1394 0.0005 0.0126 153 ID log2 Fold Change Fold Change P-value Adjusted P-value AMIGO2 0.1879 1.1391 0.0001 0.0019 TAGLN2 0.1872 1.1385 0.0000 0.0001 CSPG4 0.1856 1.1373 0.0002 0.0066 TAGLN 0.1848 1.1366 0.0000 0.0000 GALNT10 0.1838 1.1359 0.0000 0.0009 SYTL5 0.1838 1.1359 0.0000 0.0005 ST3GAL2 0.1828 1.1351 0.0000 0.0012 PIK3CD 0.1819 1.1344 0.0003 0.0075 NAV3 0.1817 1.1342 0.0000 0.0004 ATP9A 0.1817 1.1342 0.0003 0.0087 PLAUR 0.1809 1.1336 0.0003 0.0076 TMEM237 0.1787 1.1318 0.0001 0.0035 OTUD7B 0.1780 1.1313 0.0008 0.0189 GREM1 0.1780 1.1313 0.0000 0.0000 SPHK1 0.1778 1.1312 0.0002 0.0043 HIVEP1 0.1776 1.1310 0.0016 0.0317 B4GALT1 0.1774 1.1308 0.0000 0.0000 MMP15 0.1767 1.1303 0.0007 0.0172 FGF2 0.1760 1.1297 0.0000 0.0001 SMARCC1 0.1743 1.1284 0.0000 0.0004 HMGA2 0.1741 1.1283 0.0007 0.0167 CDK14 0.1738 1.1280 0.0008 0.0183 ADAMTS7 0.1737 1.1279 0.0010 0.0219 TGFBR1 0.1737 1.1279 0.0012 0.0252 TNFRSF10D 0.1733 1.1276 0.0001 0.0031 PDLIM7 0.1728 1.1273 0.0001 0.0019 IVNS1ABP 0.1726 1.1271 0.0001 0.0024 CEBPB 0.1723 1.1269 0.0001 0.0017 COL8A1 0.1722 1.1268 0.0000 0.0008 PELO 0.1708 1.1256 0.0000 0.0007 KCNMA1 0.1690 1.1243 0.0000 0.0010 FRMD4A 0.1687 1.1241 0.0001 0.0028 ITGA2 0.1677 1.1233 0.0000 0.0003 MANF 0.1675 1.1231 0.0001 0.0029 DCBLD2 0.1675 1.1231 0.0000 0.0000 COL12A1 0.1662 1.1221 0.0000 0.0000 ZNF827 0.1658 1.1218 0.0024 0.0461 CSRP2 0.1648 1.1210 0.0006 0.0139 ST5 0.1641 1.1205 0.0000 0.0002 MYH9 0.1629 1.1195 0.0000 0.0000 PCGF3 0.1625 1.1193 0.0003 0.0088 MAFF 0.1591 1.1166 0.0006 0.0148 154 ID log2 Fold Change Fold Change P-value Adjusted P-value RIC1 0.1590 1.1165 0.0004 0.0112 CTPS1 0.1581 1.1159 0.0014 0.0282 TSPAN13 0.1565 1.1146 0.0014 0.0288 STMN1 0.1563 1.1144 0.0000 0.0016 FBLN5 0.1562 1.1143 0.0000 0.0000 SGPL1 0.1553 1.1137 0.0009 0.0208 DLC1 0.1552 1.1136 0.0000 0.0010 SLFN5 0.1552 1.1136 0.0000 0.0012 TRIM32 0.1541 1.1128 0.0008 0.0183 PTPRF 0.1539 1.1126 0.0001 0.0031 ITGA5 0.1536 1.1123 0.0000 0.0000 CAV1 0.1534 1.1122 0.0000 0.0001 TLN2 0.1532 1.1120 0.0005 0.0128 TPBG 0.1496 1.1092 0.0000 0.0008 MOB1B 0.1484 1.1084 0.0016 0.0326 LRRC8A 0.1477 1.1078 0.0000 0.0010 FOXN3 0.1475 1.1077 0.0009 0.0207 MSN 0.1465 1.1069 0.0000 0.0000 IARS 0.1453 1.1059 0.0000 0.0003 ITPRIP 0.1451 1.1058 0.0008 0.0186 LRP12 0.1449 1.1056 0.0012 0.0250 PXDN 0.1448 1.1056 0.0000 0.0000 ELK3 0.1443 1.1052 0.0008 0.0180 MLXIP 0.1442 1.1051 0.0010 0.0226 KLF7 0.1435 1.1046 0.0023 0.0434 TBC1D16 0.1430 1.1042 0.0004 0.0100 TRIO 0.1430 1.1042 0.0000 0.0012 SH3BP5L 0.1424 1.1038 0.0022 0.0425 IGF1R 0.1424 1.1037 0.0024 0.0450 TGFBI 0.1413 1.1029 0.0001 0.0025 TNIP1 0.1389 1.1011 0.0001 0.0032 AKAP9 0.1388 1.1010 0.0017 0.0334 BIRC2 0.1376 1.1001 0.0003 0.0079 SSR3 0.1373 1.0998 0.0000 0.0002 AFAP1 0.1369 1.0995 0.0001 0.0042 SLC4A7 0.1368 1.0995 0.0021 0.0411 TUBB 0.1366 1.0993 0.0000 0.0001 CEP170 0.1357 1.0987 0.0008 0.0192 ITGB1 0.1356 1.0985 0.0000 0.0000 NEK7 0.1339 1.0972 0.0007 0.0163 MAP1B 0.1335 1.0969 0.0012 0.0261 JUN 0.1320 1.0958 0.0004 0.0101 155 ID log2 Fold Change Fold Change P-value Adjusted P-value TXNIP 0.1314 1.0954 0.0009 0.0208 HSPA5 0.1312 1.0952 0.0000 0.0004 PBXIP1 0.1306 1.0948 0.0002 0.0061 PDIA4 0.1291 1.0936 0.0000 0.0008 MAPKAPK2 0.1282 1.0929 0.0003 0.0069 PXDC1 0.1281 1.0929 0.0025 0.0462 ADAMTS5 0.1281 1.0928 0.0009 0.0201 TTYH3 0.1261 1.0913 0.0004 0.0091 SRPX2 0.1260 1.0913 0.0004 0.0102 PTK7 0.1254 1.0908 0.0000 0.0012 RBPJ 0.1254 1.0908 0.0006 0.0140 P3H1 0.1243 1.0900 0.0001 0.0041 DIP2B 0.1242 1.0899 0.0026 0.0479 PTPN12 0.1233 1.0892 0.0015 0.0309 NOTCH2 0.1233 1.0892 0.0003 0.0087 EDEM1 0.1224 1.0885 0.0010 0.0227 RCN3 0.1198 1.0866 0.0010 0.0211 SPARC 0.1194 1.0863 0.0000 0.0013 PYCR1 0.1184 1.0855 0.0024 0.0450 SSH1 0.1175 1.0849 0.0010 0.0221 HN1 0.1164 1.0840 0.0019 0.0377 AXL 0.1156 1.0834 0.0001 0.0031 ARHGAP1 0.1134 1.0818 0.0017 0.0344 NF2 0.1130 1.0815 0.0020 0.0399 EMP1 0.1120 1.0807 0.0007 0.0157 MMP14 0.1118 1.0806 0.0002 0.0066 STC2 0.1110 1.0799 0.0006 0.0137 SPOCK1 0.1109 1.0799 0.0009 0.0200 P4HA1 0.1108 1.0798 0.0009 0.0202 FNDC3B 0.1103 1.0795 0.0002 0.0051 THBS2 0.1096 1.0789 0.0006 0.0140 PALLD 0.1067 1.0768 0.0001 0.0028 TUBA1B 0.1049 1.0754 0.0004 0.0097 PHC2 0.1048 1.0753 0.0022 0.0428 BASP1 0.1042 1.0749 0.0021 0.0409 FKBP1A 0.1040 1.0747 0.0005 0.0134 SEMA5A 0.1033 1.0742 0.0022 0.0430 FAP 0.1016 1.0730 0.0010 0.0226 ANTXR1 0.1011 1.0726 0.0006 0.0137 USP22 0.1006 1.0722 0.0010 0.0214 SERPINH1 0.1002 1.0719 0.0004 0.0098 ITGA11 0.0990 1.0710 0.0021 0.0404 156 ID log2 Fold Change Fold Change P-value Adjusted P-value EXT1 0.0983 1.0705 0.0008 0.0182 IGFBP7 0.0980 1.0703 0.0001 0.0035 FTH1 0.0956 1.0685 0.0012 0.0249 VIM 0.0907 1.0649 0.0008 0.0191 CANX 0.0897 1.0641 0.0007 0.0159 CALR 0.0883 1.0631 0.0009 0.0194 FSTL1 0.0828 1.0590 0.0014 0.0294 CKAP4 0.0787 1.0561 0.0022 0.0428 ATP1B3 -0.0932 0.9 0.0016 0.0323 LSS -0.0941 0.9 0.0012 0.0250 ACLY -0.0974 0.9 0.0010 0.0217 QPRT -0.1028 0.9 0.0025 0.0478 PAPSS2 -0.1035 0.9 0.0006 0.0155 NQO1 -0.1036 0.9 0.0007 0.0157 PGRMC1 -0.1050 0.9 0.0014 0.0291 USP53 -0.1065 0.9 0.0013 0.0270 LDHA -0.1101 0.9 0.0012 0.0252 S100A6 -0.1107 0.9 0.0008 0.0176 CD9 -0.1108 0.9 0.0026 0.0479 MGAT5 -0.1120 0.9 0.0017 0.0335 FJX1 -0.1125 0.9 0.0024 0.0447 HSPH1 -0.1134 0.9 0.0014 0.0282 F2RL2 -0.1140 0.9 0.0009 0.0204 ARL6IP5 -0.1152 0.9 0.0005 0.0124 PDGFRB -0.1169 0.9 0.0006 0.0140 MKLN1 -0.1181 0.9 0.0016 0.0326 FAM213A -0.1216 0.9 0.0006 0.0144 SMAD3 -0.1226 0.9 0.0012 0.0246 CCND3 -0.1238 0.9 0.0020 0.0397 FASN -0.1247 0.9 0.0000 0.0011 PPT1 -0.1263 0.9 0.0012 0.0249 LAMA4 -0.1267 0.9 0.0011 0.0232 LPIN1 -0.1267 0.9 0.0001 0.0042 EVA1B -0.1274 0.9 0.0010 0.0224 G6PD -0.1282 0.9 0.0000 0.0012 SLC44A1 -0.1292 0.9 0.0019 0.0371 ARHGAP23 -0.1346 0.9 0.0003 0.0070 PRRX1 -0.1353 0.9 0.0000 0.0006 EPAS1 -0.1362 0.9 0.0000 0.0006 TOB1 -0.1367 0.9 0.0026 0.0483 SULF1 -0.1386 0.9 0.0000 0.0006 RASSF8 -0.1396 0.9 0.0005 0.0125 157 ID log2 Fold Change Fold Change P-value Adjusted P-value PCDH18 -0.1417 0.9 0.0001 0.0030 PCYT2 -0.1430 0.9 0.0002 0.0053 MOSPD2 -0.1430 0.9 0.0018 0.0359 GBP1 -0.1439 0.9 0.0023 0.0447 MRPS6 -0.1449 0.9 0.0005 0.0114 SYNPO -0.1457 0.9 0.0019 0.0369 TBL1X -0.1470 0.9 0.0009 0.0197 PDE5A -0.1475 0.9 0.0009 0.0199 TSC22D4 -0.1475 0.9 0.0002 0.0067 LITAF -0.1478 0.9 0.0000 0.0009 TKT -0.1504 0.9 0.0000 0.0002 CHST2 -0.1508 0.9 0.0001 0.0024 PRRG1 -0.1520 0.9 0.0010 0.0226 KRT19 -0.1533 0.9 0.0000 0.0000 CFH -0.1534 0.9 0.0007 0.0167 IGFBP6 -0.1537 0.9 0.0000 0.0009 CCL2 -0.1550 0.9 0.0012 0.0250 GLUL -0.1582 0.9 0.0001 0.0041 FMN2 -0.1609 0.9 0.0016 0.0314 CITED2 -0.1666 0.9 0.0000 0.0000 PKIG -0.1671 0.9 0.0000 0.0009 TFAP2C -0.1674 0.9 0.0011 0.0245 TNFRSF1A -0.1685 0.9 0.0002 0.0057 DUSP10 -0.1701 0.9 0.0014 0.0282 CYBRD1 -0.1706 0.9 0.0000 0.0004 S1PR3 -0.1720 0.9 0.0000 0.0000 FAM105A -0.1745 0.9 0.0007 0.0158 PCED1A -0.1748 0.9 0.0023 0.0435 GATA2 -0.1774 0.9 0.0000 0.0010 NID2 -0.1789 0.9 0.0001 0.0039 CDC25B -0.1791 0.9 0.0001 0.0029 DMXL2 -0.1791 0.9 0.0004 0.0096 CLDN11 -0.1792 0.9 0.0000 0.0016 TNFAIP2 -0.1793 0.9 0.0000 0.0001 CPT1A -0.1794 0.9 0.0002 0.0052 BCL6 -0.1803 0.9 0.0008 0.0191 DRAM1 -0.1841 0.9 0.0000 0.0000 ATP1B1 -0.1874 0.9 0.0008 0.0186 STAMBPL1 -0.1890 0.9 0.0002 0.0053 TBC1D2 -0.1890 0.9 0.0000 0.0004 ANXA11 -0.1893 0.9 0.0000 0.0001 KITLG -0.1893 0.9 0.0004 0.0096 158 ID log2 Fold Change Fold Change P-value Adjusted P-value S100A10 -0.1893 0.9 0.0001 0.0017 SETDB2 -0.1898 0.9 0.0019 0.0377 GLRX -0.1900 0.9 0.0000 0.0000 TMEM119 -0.1904 0.9 0.0001 0.0020 KAT2B -0.1911 0.9 0.0023 0.0444 BMP4 -0.1923 0.9 0.0013 0.0269 SLC38A2 -0.1931 0.9 0.0000 0.0000 TNFRSF11B -0.1935 0.9 0.0007 0.0157 CARHSP1 -0.1935 0.9 0.0000 0.0012 CYTH3 -0.1960 0.9 0.0001 0.0029 PPA1 -0.1969 0.9 0.0000 0.0003 HEG1 -0.1975 0.9 0.0000 0.0003 CEMIP -0.1979 0.9 0.0012 0.0249 ST6GALNAC6 -0.2020 0.9 0.0000 0.0013 FHL1 -0.2037 0.9 0.0000 0.0012 STXBP5 -0.2071 0.9 0.0000 0.0002 FLOT1 -0.2084 0.9 0.0000 0.0001 STON1 -0.2090 0.9 0.0015 0.0308 SYNM -0.2116 0.9 0.0011 0.0245 PTGR1 -0.2119 0.9 0.0000 0.0000 ID3 -0.2125 0.9 0.0015 0.0298 PDE8B -0.2164 0.9 0.0019 0.0377 HACD4 -0.2164 0.9 0.0021 0.0414 HAND2 -0.2178 0.9 0.0000 0.0001 CDC42EP4 -0.2180 0.9 0.0001 0.0022 NABP1 -0.2183 0.9 0.0000 0.0000 AMOT -0.2188 0.9 0.0011 0.0232 SOCS2 -0.2205 0.9 0.0017 0.0342 DLL4 -0.2210 0.9 0.0026 0.0481 LOXL4 -0.2221 0.9 0.0000 0.0015 BRIX1 -0.2227 0.9 0.0001 0.0021 ETV1 -0.2249 0.9 0.0004 0.0102 GJA1 -0.2252 0.9 0.0000 0.0000 NMT2 -0.2256 0.9 0.0000 0.0012 LIMA1 -0.2258 0.9 0.0000 0.0000 GBP2 -0.2269 0.9 0.0000 0.0001 MAP3K5 -0.2286 0.9 0.0000 0.0000 S100A4 -0.2303 0.9 0.0000 0.0000 PDGFRA -0.2307 0.9 0.0000 0.0000 PCDH7 -0.2312 0.9 0.0000 0.0000 GPRC5A -0.2316 0.9 0.0000 0.0000 CYB5B -0.2347 0.8 0.0000 0.0000 159 ID log2 Fold Change Fold Change P-value Adjusted P-value MASP1 -0.2392 0.8 0.0000 0.0002 SDC4 -0.2414 0.8 0.0000 0.0000 KCNMB4 -0.2417 0.8 0.0002 0.0052 EHD3 -0.2419 0.8 0.0000 0.0000 LBH -0.2437 0.8 0.0000 0.0000 NME3 -0.2446 0.8 0.0001 0.0035 DPYD -0.2453 0.8 0.0005 0.0130 PYGL -0.2455 0.8 0.0000 0.0009 TNFRSF21 -0.2456 0.8 0.0000 0.0000 HSPA1B -0.2484 0.8 0.0002 0.0051 ADD3 -0.2493 0.8 0.0000 0.0000 RGCC -0.2514 0.8 0.0000 0.0012 SSX2IP -0.2515 0.8 0.0002 0.0066 ID1 -0.2524 0.8 0.0008 0.0176 ETS2 -0.2551 0.8 0.0000 0.0000 LIPA -0.2553 0.8 0.0000 0.0000 ADGRG6 -0.2562 0.8 0.0000 0.0001 TNS2 -0.2572 0.8 0.0000 0.0001 GPRC5B -0.2596 0.8 0.0000 0.0002 IFITM2 -0.2601 0.8 0.0000 0.0001 ABCA7 -0.2619 0.8 0.0004 0.0100 MEST -0.2643 0.8 0.0000 0.0000 WWC3 -0.2646 0.8 0.0000 0.0000 CPZ -0.2654 0.8 0.0022 0.0425 VCAM1 -0.2687 0.8 0.0019 0.0369 MBOAT1 -0.2701 0.8 0.0008 0.0178 PC -0.2711 0.8 0.0002 0.0050 GNAI1 -0.2721 0.8 0.0000 0.0000 HNMT -0.2726 0.8 0.0000 0.0011 APOL1 -0.2751 0.8 0.0001 0.0017 AASS -0.2780 0.8 0.0000 0.0000 MRGPRF -0.2797 0.8 0.0000 0.0000 SKP2 -0.2818 0.8 0.0000 0.0003 CTNNAL1 -0.2824 0.8 0.0000 0.0000 MTUS1 -0.2832 0.8 0.0003 0.0087 SDC2 -0.2840 0.8 0.0000 0.0000 IL1R1 -0.2848 0.8 0.0000 0.0000 TNFRSF19 -0.2884 0.8 0.0000 0.0000 ACSS2 -0.2887 0.8 0.0000 0.0000 TWIST1 -0.2907 0.8 0.0007 0.0167 SOX13 -0.2950 0.8 0.0000 0.0005 MTMR10 -0.2978 0.8 0.0000 0.0002 160 ID log2 Fold Change Fold Change P-value Adjusted P-value LPAR6 -0.2987 0.8 0.0001 0.0019 SYPL2 -0.3013 0.8 0.0000 0.0013 STOM -0.3017 0.8 0.0000 0.0000 FBLN1 -0.3107 0.8 0.0000 0.0000 HSBP1L1 -0.3112 0.8 0.0001 0.0033 SLC40A1 -0.3128 0.8 0.0004 0.0108 ISYNA1 -0.3130 0.8 0.0000 0.0000 A2M -0.3137 0.8 0.0012 0.0249 CPA4 -0.3157 0.8 0.0000 0.0003 SLC5A3 -0.3213 0.8 0.0000 0.0000 CFB -0.3263 0.8 0.0012 0.0249 GREM2 -0.3285 0.8 0.0000 0.0010 TMEM173 -0.3344 0.8 0.0000 0.0000 PRKAR2B -0.3363 0.8 0.0004 0.0098 COL14A1 -0.3366 0.8 0.0000 0.0000 CSF1 -0.3391 0.8 0.0000 0.0000 TBC1D8 -0.3413 0.8 0.0000 0.0011 LAG3 -0.3422 0.8 0.0007 0.0167 CRABP2 -0.3425 0.8 0.0001 0.0024 FAM124A -0.3434 0.8 0.0000 0.0002 RAB27B -0.3444 0.8 0.0000 0.0000 CYB5A -0.3458 0.8 0.0000 0.0000 LEPR -0.3479 0.8 0.0000 0.0000 PTX3 -0.3501 0.8 0.0000 0.0000 CYGB -0.3560 0.8 0.0000 0.0008 PLA2G4A -0.3575 0.8 0.0000 0.0001 AGFG2 -0.3608 0.8 0.0000 0.0000 ERMP1 -0.3691 0.8 0.0000 0.0000 LRRC8C -0.3748 0.8 0.0000 0.0000 ISM1 -0.3752 0.8 0.0011 0.0244 CEBPD -0.3759 0.8 0.0000 0.0000 OSR2 -0.3763 0.8 0.0002 0.0046 GBP4 -0.3772 0.8 0.0000 0.0000 NDRG1 -0.3790 0.8 0.0000 0.0000 RSPO3 -0.3849 0.8 0.0000 0.0010 TGFBR3 -0.3896 0.8 0.0002 0.0056 SPRY1 -0.3924 0.8 0.0007 0.0159 TMTC1 -0.4037 0.8 0.0000 0.0000 ADAMTS9 -0.4197 0.7 0.0003 0.0086 ADRA2C -0.4202 0.7 0.0018 0.0354 SEMA6A -0.4219 0.7 0.0000 0.0001 C10orf54 -0.4249 0.7 0.0000 0.0001 161 ID log2 Fold Change Fold Change P-value Adjusted P-value AGPAT9 -0.4275 0.7 0.0000 0.0012 TIMP3 -0.4346 0.7 0.0000 0.0001 PLSCR4 -0.4348 0.7 0.0000 0.0000 ITGA6 -0.4387 0.7 0.0000 0.0000 TMEM26 -0.4401 0.7 0.0000 0.0006 IL6R -0.4450 0.7 0.0000 0.0000 LSAMP -0.4501 0.7 0.0017 0.0342 PRELP -0.4505 0.7 0.0003 0.0085 FAM167B -0.4532 0.7 0.0005 0.0115 SLCO4A1 -0.4632 0.7 0.0000 0.0005 HSD17B2 -0.4725 0.7 0.0000 0.0000 KRT18 -0.4987 0.7 0.0000 0.0000 RASGRP2 -0.5115 0.7 0.0010 0.0227 AKR1C3 -0.5153 0.7 0.0012 0.0246 SFRP1 -0.5292 0.7 0.0000 0.0000 IFIT2 -0.5693 0.7 0.0006 0.0142 HSPA2 -0.5999 0.7 0.0000 0.0000 NPY1R -0.6262 0.6 0.0000 0.0000 SECTM1 -0.6524 0.6 0.0000 0.0000 NR1H3 -0.6558 0.6 0.0000 0.0000 PTGES -0.6952 0.6 0.0000 0.0000 COLEC12 -0.7255 0.6 0.0001 0.0025 FAM65B -0.7480 0.6 0.0000 0.0000 ADRA2A -0.9337 0.5 0.0000 0.0000 ALDH1L1 -0.9626 0.5 0.0000 0.0000 TNXB -1.0731 0.5 0.0000 0.0000 ALDH1A1 -1.1196 0.5 0.0000 0.0000 CBLN2 -1.2655 0.4 0.0000 0.0000 162 Appendix B The hallmark genes enriched among the DEGs in MSTN-treated primary trophoblast cells as analyzed by GSEA. Hallmark genes Size FDR q-value Enrichment Epithelial mesenchymal transition 81 0.035 Positive Myogenesis 22 1.000 Positive Hypoxia 37 0.844 Positive Inflammatory response 23 0.655 Positive KRAS signaling up 23 0.568 Positive Apical junction 23 0.502 Positive TNF-_signaling via NFKB 37 0.681 Positive Coagulation 22 0.740 Positive UV response 29 0.675 Positive MTORC1_signaling 22 0.886 Positive Complement 15 0.859 Positive IL2_STAT 5_signaling 21 0.924 Positive Glycolysis 27 0.880 Positive p53_pathway 19 0.819 Positive Estrogen response early 21 0.959 Positive Interferon gamma response 15 0.425 Negative Xenobiotic metabolism 21 0.318 Negative Apoptosis 22 0.437 Negative Estrogen response late 17 0.523 Negative 163 Appendix C The hallmark EMT genes differentially expressed in MSTN-treated primary trophoblast cells Gene Rank in gene list Rank metric score Core enrichment TNC 3 1.819 Yes SERPINE2 6 1.767 Yes PMEPA1 7 1.724 Yes LRRC15 10 1.680 Yes NTM 12 1.648 Yes PTHLH 13 1.647 Yes INHBA 14 1.550 Yes HTRA1 40 1.395 Yes LOX 44 1.371 Yes IGFBP3 52 1.345 Yes ITGB3 53 1.345 Yes COL7A1 59 1.331 Yes LAMC2 61 1.330 Yes IL32 62 1.328 Yes FZD8 65 1.323 Yes ADAM12 66 1.321 Yes N-Cad 67 1.319 Yes PAI-1 69 1.317 Yes COL4A1 101 1.252 Yes TPM1 102 1.251 Yes FN1 119 1.228 Yes FSTL3 128 1.219 Yes COL5A1 130 1.215 Yes WNT5A 134 1.213 Yes COL1A1 136 1.212 Yes PLOD2 141 1.208 Yes MXRA5 148 1.201 Yes DPYSL3 156 1.195 Yes CADM1 164 1.188 Yes COL4A2 168 1.185 Yes THBS1 171 1.183 Yes VCAN 173 1.182 Yes COL16A1 176 1.181 Yes FBN1 179 1.178 Yes MMP2 185 1.174 Yes BGN 192 1.167 Yes VEGFC 194 1.166 Yes SAT1 202 1.161 Yes TNFRSF12A 204 1.160 Yes 164 Gene Rank in gene list Rank metric score Core enrichment GADD45A 209 1.158 Yes CXCL8 213 1.155 Yes LOXL2 222 1.151 Yes CTHRC1 223 1.150 Yes MMP3 226 1.150 Yes ITGB5 235 1.145 Yes COL1A2 243 1.140 Yes TAGLN 249 1.137 Yes PLAUR 256 1.134 Yes GREM1 259 1.131 Yes FGF2 264 1.130 Yes ITGA2 278 1.123 Yes COL12A1 281 1.122 Yes FBLN5 292 1.114 Yes ITGA5 298 1.112 Yes TGFBI 317 1.103 Yes ITGB1 326 1.099 Yes JUN 329 1.096 Yes P3H1 341 1.090 Yes NOTCH2 344 1.089 Yes SPARC 347 1.086 Yes MMP14 355 1.081 Yes SPOCK1 357 1.080 Yes THBS2 360 1.079 Yes BASP1 364 1.075 Yes FAP 367 1.073 Yes SERPINH1 370 1.072 Yes VIM 375 1.065 Yes FSTL1 378 1.059 Yes PDGFRB 396 0.922 No PRRX1 409 0.910 No NID2 443 0.883 No TNFRSF11B 463 0.875 No GJA1 488 0.855 No SDC4 499 0.846 No MEST 519 0.833 No VCAM1 522 0.830 No FBLN1 543 0.806 No PTX3 563 0.785 No TGFBR3 575 0.763 No TIMP3 583 0.740 No SFRP1 596 0.693 No 165 Appendix D List of the DEG in MSTN-treated primary trophoblast cells based on a FC of 1.3 ID Log 2-FC FC Adjusted P value CST1 1.0726 2.1032 0.0000 CALB2 0.8766 1.8361 0.0000 F2RL1 0.8693 1.8267 0.0000 TNC 0.8631 1.8190 0.0000 KCNH1 0.8562 1.8103 0.0000 TNFSF15 0.8316 1.7796 0.0000 SERPINE2 0.8217 1.7674 0.0000 PMEPA1 0.7859 1.7241 0.0000 DIRC3 0.7564 1.6893 0.0000 KCNG1 0.7535 1.6859 0.0006 LRRC15 0.7482 1.6798 0.0000 SEMA3C 0.7469 1.6782 0.0000 NTM 0.7211 1.6485 0.0000 PTHLH 0.7198 1.6469 0.0018 INHBA 0.6321 1.5499 0.0000 SHISA9 0.6272 1.5445 0.0001 SPON2 0.6148 1.5314 0.0000 MEX3A 0.6130 1.5295 0.0000 PLCXD3 0.6084 1.5246 0.0001 NOX4 0.6070 1.5231 0.0030 SIK1 0.5935 1.5089 0.0000 SOX4 0.5882 1.5033 0.0000 ANGPTL4 0.5786 1.4933 0.0020 HAS2 0.5738 1.4884 0.0007 MMP10 0.5617 1.4761 0.0000 CTGF 0.5588 1.4730 0.0000 HMCN1 0.5544 1.4686 0.0000 COCH 0.5463 1.4604 0.0036 NCAM1 0.5441 1.4581 0.0000 HUNK 0.5411 1.4550 0.0028 DACT1 0.5375 1.4514 0.0001 SLC22A23 0.5305 1.4444 0.0000 ADAM19 0.5275 1.4415 0.0000 DIO2 0.5240 1.4379 0.0000 KRT7 0.5205 1.4345 0.0000 CNN1 0.5185 1.4324 0.0000 DGKI 0.5046 1.4187 0.0000 ZSWIM5 0.4994 1.4136 0.0000 MYOCD 0.4901 1.4045 0.0013 166 ID Log 2-FC FC Adjusted P value OSGIN2 0.4833 1.3980 0.0000 HTRA1 0.4804 1.3952 0.0000 TMEM2 0.4764 1.3913 0.0000 ITGA1 0.4596 1.3752 0.0000 GRIK2 0.4580 1.3736 0.0000 LOX 0.4555 1.3712 0.0000 PCDH10 0.4533 1.3692 0.0000 LRRC2 0.4529 1.3687 0.0335 ETV4 0.4440 1.3604 0.0000 NREP 0.4411 1.3577 0.0113 TP53I3 0.4393 1.3559 0.0000 ANOS1 0.4369 1.3537 0.0008 RASSF2 0.4319 1.3490 0.0087 IGFBP3 0.4277 1.3451 0.0000 ITGB3 0.4277 1.3451 0.0000 ADAMTS6 0.4257 1.3432 0.0002 SLC8A1 0.4254 1.3429 0.0000 IL11 0.4207 1.3386 0.0535 KIAA1024 0.4174 1.3355 0.0029 LPXN 0.4145 1.3329 0.0113 FAM196B 0.4138 1.3322 0.0002 COL7A1 0.4123 1.3308 0.0064 ARSI 0.4119 1.3305 0.0057 LAMC2 0.4114 1.3300 0.0000 IL32 0.4091 1.3278 0.0001 PCDH19 0.4080 1.3268 0.0234 ENC1 0.4073 1.3262 0.0000 FZD8 0.4038 1.3230 0.0001 ADAM12 0.4020 1.3213 0.0000 N-Cad 0.3993 1.3189 0.0000 MEOX1 0.3992 1.3188 0.0036 PAI-1 0.3970 1.3168 0.0000 ADAMTS4 0.3938 1.3138 0.0038 CDK6 0.3937 1.3137 0.0000 DSE 0.3930 1.3131 0.0000 SLC7A5 0.3900 1.3104 0.0000 PGM2L1 0.3889 1.3094 0.0001 NDRG4 0.3883 1.3089 0.0012 IGFBP5 0.3856 1.3064 0.0000 FAM26E 0.3824 1.3035 0.0003 ARHGAP22 0.3821 1.3032 0.0000 IRS1 0.3801 1.3014 0.0000 AKR1C3 -0.5153 0.6996 0.0246 167 ID Log 2-FC FC Adjusted P value SFRP1 -0.5292 0.6929 0.0000 IFIT2 -0.5693 0.6739 0.0142 HSPA2 -0.5999 0.6598 0.0000 NPY1R -0.6262 0.6479 0.0000 SECTM1 -0.6524 0.6362 0.0000 NR1H3 -0.6558 0.6347 0.0000 PTGES -0.6952 0.6176 0.0000 COLEC12 -0.7255 0.6048 0.0025 FAM65B -0.7480 0.5954 0.0000 ADRA2A -0.9337 0.5235 0.0000 ALDH1L1 -0.9626 0.5131 0.0000 TNXB -1.0731 0.4753 0.0000 ALDH1A1 -1.1196 0.4602 0.0000 CBLN2 -1.2655 0.4159 0.0000
Thesis/Dissertation
eng
Reproductive and Developmental Sciences
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Leung, P. C. K.
Role of myostatin signaling in human trophoblast cell invasion
Text