Molecular Regulators of Embryo Implantation and Pregnancy
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MOLECULAR REGULATORS OF EMBRYO IMPLANTATION AND PREGNANCY Brooke Campbell Matson A dissertation submitted to the faculty at the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Cell Biology and Physiology in the School of Medicine. Chapel Hill 2017 Approved by: Kathleen Caron Keith Burridge Richard Cheney Robert Tarran Steven Young © 2017 Brooke Campbell Matson ALL RIGHTS RESERVED ii ABSTRACT Brooke Campbell Matson: Molecular Regulators of Embryo Implantation and Pregnancy (Under the direction of Kathleen Caron) Embryo implantation is one of the first steps in the establishment of pregnancy and sets the stage for proper embryonic and placental development. We are beginning to appreciate the complexity of implantation and its demand for synchrony between embryo and endometrium. Over the years, many groups have identified and studied both embryonic and endometrial factors affecting implantation, but there is still much to learn. Here, we focus on the endocrine peptide adrenomedullin (Adm, AM) and its association with female reproductive physiology and disease in both mice and humans. In Part I, we elaborate on the subfertility phenotype of Adm heterozygous female mice by testing whether AM supplementation before implantation can improve fertility and pregnancy outcomes. We find that administration of AM directly to the mouse uterus before blastocyst transfer enhances implantation success and inter-embryonic spacing. Intra- uterine delivery of AM also confers morphological and cellular changes to the uterine epithelium and stroma that are associated with endometrial receptivity and believed to be beneficial for implantation. We also test mid-regional pro-adrenomedullin (MR-proADM), a stable surrogate for AM peptide, as a biomarker for endometriosis, which is comorbid with infertility. Our studies identify novel mechanisms of action of AM in the endometrium in support of implantation and the establishment of pregnancy, providing a foundational basis for the use of AM as a clinical therapeutic for infertility. In Part II, we address the role of AM in the maintenance of a healthy pregnancy by expanding upon previous studies in mouse models with clinical samples from women with severe preeclampsia, a placental vascular disorder characterized by hypertension and iii proteinuria. Because Adm-/- placentas exhibit a preeclampsia-like phenotype, we test the hypothesis that MR-proADM concentrations are decreased in plasma from women with severe preeclampsia. Indeed, we find that MR-proADM levels are blunted in women with severe preeclampsia and that MR-proADM is similarly effective as other established biomarkers of preeclampsia at distinguishing between cases and controls. Altogether, our studies in both mice and humans point to diagnostic and therapeutic applications for AM in the context of female reproductive disease. iv ACKNOWLEDGEMENTS For the studies in Chapter 2, we thank the UNC Animal Models Core, specifically Dale Cowley, Kim Kluckman, and Eric Holle, for their expertise and technical assistance concerning blastocyst transfer. We also thank the Animal Histopathology Core and the Histology Research Core Facility at UNC for tissue embedding and sectioning. Finally, we thank the Microscopy Services Laboratory at UNC, specifically Vicky Madden and Kristen White, for scanning electron microscopy expertise. This work was supported by U.S. National Institutes of Health grants R01 HD060860 (KMC), R01 HD067721 (SLY), F30 HD085652 (BCM), and T32 GM008719 and by Ferring Research Institute, Inc. For the studies in Chapter 3, we thank Angela Houwing of Greenville Health System and Jana Phillips of the University of North Carolina at Chapel Hill for clinical research support and acquisition of clinical characteristics of study participants donating plasma for MR-proADM analysis. We also acknowledge Imani Sweatt for experimental efforts toward identifying a link between STAT3 and Adm. The work in Chapter 3 was supported by U.S. National Institutes of Health grants R01 HD060860 (KMC), R01 HD067721 (BAL and SLY), F30 HD085652 (BCM), and T32 GM008719 and by the Triangle Community Foundation’s Gertrude B. Elion Mentored Medical Student Research Award. For the studies in Chapter 7, we thank Brian Antczak and Kristin Weaver of Duke University for collection and processing of patient samples. This study was supported by the Department of Obstetrics and Gynecology at Duke University and by U.S. National Institutes of Health grant R01 HD060860 (KMC). Chapters 5 and 6 were funded by U.S. National Institutes of Health grants R01 HD060860 (KMC) and T32 GM008719, and Chapters 4 and 8 were funded by U.S. National v Institutes of Health grants R01 HD060860 (KMC), F30 HD085652 (BCM), and T32 GM008719 and by the Triangle Community Foundation’s Gertrude B. Elion Mentored Medical Student Research Award. Finally, for all chapters, I would like to thank Dr. Kathleen Caron and all past and present members of the Caron laboratory for their technical advice and assistance as well as for many thoughtful discussions and insights into this work over the years. vi TABLE OF CONTENTS LIST OF TABLES .................................................................................................................... x LIST OF FIGURES ................................................................................................................. xi LIST OF ABBREVIATIONS .................................................................................................... xii PART I – ESTABLISHMENT OF PREGNANCY Chapter 1: Introduction – Embryo Implantation and Adrenomedullin .................................... 1 Embryo Implantation and Endometrial Receptivity ...................................................... 1 Adrenomedullin and the Establishment of Pregnancy ................................................. 2 References .................................................................................................................. 6 Chapter 2: Adrenomedullin Improves Fertility and Promotes Pinopodes and Cell Junctions in the Peri-Implantation Endometrium ........................................ 10 Overview ................................................................................................................... 10 Introduction ................................................................................................................ 11 Materials and Methods .............................................................................................. 13 Results ...................................................................................................................... 19 Discussion ................................................................................................................. 25 Figures ...................................................................................................................... 30 References ................................................................................................................ 40 Chapter 3: MR-proADM is a Potential Biomarker of Endometriosis, an Infertility-Associated Disease ............................................................................. 45 Overview ................................................................................................................... 45 vii Introduction ................................................................................................................ 46 Materials and Methods .............................................................................................. 47 Results ...................................................................................................................... 50 Discussion ................................................................................................................. 52 Tables ........................................................................................................................ 55 Figures ...................................................................................................................... 56 References ................................................................................................................ 60 Chapter 4: Conclusions and Future Directions ..................................................................... 64 Summary of Results .................................................................................................. 64 Future Directions ....................................................................................................... 65 Concluding Remarks ................................................................................................. 72 References ................................................................................................................ 74 PART II – MAINTENANCE OF PREGNANCY Chapter 5: Uterine Natural Killer Cells as Modulators of the Maternal-Fetal Vasculature ........................................................................................................ 78 Overview ................................................................................................................... 78 Introduction ................................................................................................................ 78 uNK Cell Differentiation ............................................................................................. 80 uNK-Trophoblast