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Investigating the molecular mechanisms regulated by progesterone governing the ability of the uterus to support pregnancy
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Abstracts / Placenta 45 (2016) 63e133
protein and gene interactions, cellular proliferation and differentiation. Finally, an integration of approaches for the identification of pathological sequelae and vascular changes will be explored using ultrasound, computer assisted tomography and magnetic resonance imaging. (Supported in part by NCS LOI-18, NIAID, NIEHS, NICHHD, NCI, FDA, MCH, NYSDOH, RW and MS Goode Foundation, Merck, Eastman Kodak, Centocor, US/Israel Binational Foundation) KEY.1 ADVANCED ULTRASOUND IMAGING: INSIGHTS FROM PERFUSION AND MOLECULAR IMAGING Jonathan Lindner. Oregon Health & Science University, Portland, USA Novel in vivo ultrasound imaging technologies have been developed for the assessment of tissue perfusion, tissue mechanical properties, and vascular molecular phenotype with site-targeted contrast agents. These technologies are uniquely poised to aid researchers and clinicians by temporally and spatially evaluating tissue and vascular adaptations in health and disease, and the impact of new therapies. This lecture will explore these state-of-the-art methods and their potential applications in assessing the placenta. Contrast-enhanced ultrasound is a technique that is able to spatially quantify tissue perfusion, flow distribution, microvascular blood volume, and microvascular blood flux rate. Accordingly, it can provide information on hierarchical vascular adaptations that govern perfusion and nutrient delivery. Molecular and cellular imaging with ultrasound relies on the detection of site-targeted microbubbles that bear specific targeting ligands on their surface. Because of their intravascular confinement, they can uniquely provide a biologic readout of the expression of specific endothelial cell markers of inflammation, vascular remodeling, oxidative stress, and thrombosis; and the intravascular recruitment/ adhesion of specific blood cell types such as neutrophils, monocytes, and platelets. Finally, shear-wave elastography represents a novel approach for assessing the stiffness of deep tissues which is based on the assessment of tissue motion produced by the pressure of the ultrasound wave in order to calculate elastic modulus. NIH INVESTIGATING THE MOLECULAR MECHANISMS REGULATED BY PROGESTERONE GOVERNING THE ABILITY OF THE UTERUS TO SUPPORT PREGNANCY Francesco DeMayo. Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, USA The ovarian steroid hormone, Progesterone, acting through its cognate receptor, the Progesterone receptor, Pgr, is critical for regulating all processes of the ability of the uterus to support embryo implantation and pregnancy. The Pgr consist of two isoforms , the PgrA and the PgrB. The Pgr isoforms act in all compartments of the uterus. In the preimplantation period the Pgr expressed in the uterine epithelium is critical for enabling the uterine epithelium to become receptive and priming the stroma cells to undergo the decidual reaction to support the implanting embryo. In the stroma compartment the Pgr is necessary for the progression and maintenance of decidual response. Finally in the myometrium the Pgr is critical for maintaining the uterine smooth muscles in a state that will allow the growth of the developing fetus. Utilizing genetically engineered mice in combination with transcriptomic and cistromic analysis we have begun to map the interactions of Pgr with other transcription factors and signaling pathways in the regulation of uterine receptivity, and the ability of the myometrium to support pregnancy. This technology has also been used to investigate the role of PgrA and PgrB in the mouse uterus by specific ablation and overexpression of these isoforms. The pathways identified in the mouse have been validated in primary human samples, primary human endometrial culture cells and human clinical databases to validate their clinical significance. Currently, with the explosion of genome editing tools such as CRISPR/Cas9 the ability to dissect the molecular pathways regulating uterine receptivity will be made simpler and the ability to investigate multiple genetic alterations at once will facilitate the understanding of how the uterus is able to support pregnancy. This work was supported by the NIEHS, and grants from the NICHD, March of Dimes and Burroughs Welcome Fund.
THAN QUO VADIS, TROPHOBLAST? EXPLORING THE NEW WAYS OF AN OLD CELL LINEAGE Sascha Drewlo. Wayne State University School of Medicine, Detroit, USA Traditionally, placental cells are mostly found in close proximity to the implantation site. Some cells get replaced from the conceptus during pregnancy by mechanisms not fully understood. These cells accumulate in the lower cervix and can be safely collected by a simple pap smear. Their presence has been known for many years, but the lack of reliable purification protocols that would provide these cells in numbers and purity necessary for analysis obstructed their assessment for clinical usage and biological function. We recently developed a purification protocol that provides fetal cells with an extravillous trophoblast phenotype in numbers now suitable for various molecular analysis. Here we present an introduction into a novel research field on the applications and usage of trophoblast like cells residing in the cervix. We demonstrate the potential of these cells to investigate the genetic status of the fetus as well as their relationship to placental physiology and disease as early as early as 3 weeks post conception. P1.1 INVOLVEMENT OF INVERTED FORMIN-2 (INF2) IN TROPHOBLAST INVASION AND BIRTH TIMING Katherine Bezold Lamm 1, 2, Emily Holloway 1, Jude McElroy 3, Helen Jones 1, Louis Muglia 1. 1 Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA; 2 University of Cincinnati, Cincinnati, OH, USA; 3 Vanderbilt University, Nashville, TN, USA Objectives: Preterm birth (birth before 37 completed weeks of gestation) is the leading cause of infant mortality worldwide. We identified the inverted formin-2 (INF2) gene through a genome-wide association study of prematurity in infants. INF2 is an important cytoskeletal modulator, highly conserved in both humans and mice. We hypothesize INF2 regulates vascular remodelling by altering trophoblast invasion and, consequently, gestation length. Methods: Inf2-/- timed-matings were performed and gestational length measured from copulatory plug detection (E0.5). Umbilical Doppler was performed at gestational age 18.5 (E18.5) followed by C-section, where litter size, fetal weight, and placental weight were recorded. In additional animals, pregnancies continued to term and birthweight measured at day 1 of life. Matrigel invasion assays were performed using HTR-8/SVneo cells transfected with siRNA against INF2 for 24 hours or treated with LCK inhibitors (PP1 or TX1123) for 7 days. Transwells were fixed, stained with DAPI, and five random fields were counted and normalized to the vehicle control. Data were analysed by Student T-test and a p value of less than 0.05 was deemed significant. Results: Inf2-/- females had significantly longer gestations than wildtype counterparts (19.7±0.08 vs 19.3±0.15 days; p<0.05) with no effect on birthweight or litter size. There was no difference in fetal weight or placental weight at E18.5. Inf2-/- mice demonstrated significant increases in pulsatility index (2.0±0.05 vs 1.9±0.02 a.u.; p<0.05), end diastolic velocity (14.2±0.9 vs 12.0±0.6 mm/s; p < 0.05), and maternal heart rate (604.2±91 vs 424.4±12; p<0.05) at E18.5. Loss of INF2 in vitro significantly reduced trophoblast invasion (14.6±5.9 vs 100±8.3%; p<0.05). Pharmacologic inhibition of Lck, an INF2-dependent tyrosine kinase, significantly reduced trophoblast invasion (31.2±11 PP1 vs 1.3±0.5 TX1123 vs 100±1.3%; p<0.05). Conclusions: These findings suggest INF2 is essential for trophoblast invasion and its loss impacts both vascular function and gestation length. P1.2 ANGIOGENIC FACTORS ARE ASSOCIATED WITH PLACENTAL WEIGHT AND BIRTH WEIGHT IN BANGLADESHI PREGNANCIES Alison Gernand 1, Abdullah Mahmud 2, Eszter Papp 3, Joy Shi 3, Daniel Roth 3. 1 The Pennsylvania State University, University Park, PA, USA; 2 International Center for Diarrhoeal Disease Research, Bangladesh, Dhaka, Bangladesh; 3 Hospital for Sick Children, Toronto, ON, Canada
Abstracts / Placenta 45 (2016) 63e133
protein and gene interactions, cellular proliferation and differentiation. Finally, an integration of approaches for the identification of pathological sequelae and vascular changes will be explored using ultrasound, computer assisted tomography and magnetic resonance imaging. (Supported in part by NCS LOI-18, NIAID, NIEHS, NICHHD, NCI, FDA, MCH, NYSDOH, RW and MS Goode Foundation, Merck, Eastman Kodak, Centocor, US/Israel Binational Foundation) KEY.1 ADVANCED ULTRASOUND IMAGING: INSIGHTS FROM PERFUSION AND MOLECULAR IMAGING Jonathan Lindner. Oregon Health & Science University, Portland, USA Novel in vivo ultrasound imaging technologies have been developed for the assessment of tissue perfusion, tissue mechanical properties, and vascular molecular phenotype with site-targeted contrast agents. These technologies are uniquely poised to aid researchers and clinicians by temporally and spatially evaluating tissue and vascular adaptations in health and disease, and the impact of new therapies. This lecture will explore these state-of-the-art methods and their potential applications in assessing the placenta. Contrast-enhanced ultrasound is a technique that is able to spatially quantify tissue perfusion, flow distribution, microvascular blood volume, and microvascular blood flux rate. Accordingly, it can provide information on hierarchical vascular adaptations that govern perfusion and nutrient delivery. Molecular and cellular imaging with ultrasound relies on the detection of site-targeted microbubbles that bear specific targeting ligands on their surface. Because of their intravascular confinement, they can uniquely provide a biologic readout of the expression of specific endothelial cell markers of inflammation, vascular remodeling, oxidative stress, and thrombosis; and the intravascular recruitment/ adhesion of specific blood cell types such as neutrophils, monocytes, and platelets. Finally, shear-wave elastography represents a novel approach for assessing the stiffness of deep tissues which is based on the assessment of tissue motion produced by the pressure of the ultrasound wave in order to calculate elastic modulus. NIH INVESTIGATING THE MOLECULAR MECHANISMS REGULATED BY PROGESTERONE GOVERNING THE ABILITY OF THE UTERUS TO SUPPORT PREGNANCY Francesco DeMayo. Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, USA The ovarian steroid hormone, Progesterone, acting through its cognate receptor, the Progesterone receptor, Pgr, is critical for regulating all processes of the ability of the uterus to support embryo implantation and pregnancy. The Pgr consist of two isoforms , the PgrA and the PgrB. The Pgr isoforms act in all compartments of the uterus. In the preimplantation period the Pgr expressed in the uterine epithelium is critical for enabling the uterine epithelium to become receptive and priming the stroma cells to undergo the decidual reaction to support the implanting embryo. In the stroma compartment the Pgr is necessary for the progression and maintenance of decidual response. Finally in the myometrium the Pgr is critical for maintaining the uterine smooth muscles in a state that will allow the growth of the developing fetus. Utilizing genetically engineered mice in combination with transcriptomic and cistromic analysis we have begun to map the interactions of Pgr with other transcription factors and signaling pathways in the regulation of uterine receptivity, and the ability of the myometrium to support pregnancy. This technology has also been used to investigate the role of PgrA and PgrB in the mouse uterus by specific ablation and overexpression of these isoforms. The pathways identified in the mouse have been validated in primary human samples, primary human endometrial culture cells and human clinical databases to validate their clinical significance. Currently, with the explosion of genome editing tools such as CRISPR/Cas9 the ability to dissect the molecular pathways regulating uterine receptivity will be made simpler and the ability to investigate multiple genetic alterations at once will facilitate the understanding of how the uterus is able to support pregnancy. This work was supported by the NIEHS, and grants from the NICHD, March of Dimes and Burroughs Welcome Fund.
THAN QUO VADIS, TROPHOBLAST? EXPLORING THE NEW WAYS OF AN OLD CELL LINEAGE Sascha Drewlo. Wayne State University School of Medicine, Detroit, USA Traditionally, placental cells are mostly found in close proximity to the implantation site. Some cells get replaced from the conceptus during pregnancy by mechanisms not fully understood. These cells accumulate in the lower cervix and can be safely collected by a simple pap smear. Their presence has been known for many years, but the lack of reliable purification protocols that would provide these cells in numbers and purity necessary for analysis obstructed their assessment for clinical usage and biological function. We recently developed a purification protocol that provides fetal cells with an extravillous trophoblast phenotype in numbers now suitable for various molecular analysis. Here we present an introduction into a novel research field on the applications and usage of trophoblast like cells residing in the cervix. We demonstrate the potential of these cells to investigate the genetic status of the fetus as well as their relationship to placental physiology and disease as early as early as 3 weeks post conception. P1.1 INVOLVEMENT OF INVERTED FORMIN-2 (INF2) IN TROPHOBLAST INVASION AND BIRTH TIMING Katherine Bezold Lamm 1, 2, Emily Holloway 1, Jude McElroy 3, Helen Jones 1, Louis Muglia 1. 1 Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA; 2 University of Cincinnati, Cincinnati, OH, USA; 3 Vanderbilt University, Nashville, TN, USA Objectives: Preterm birth (birth before 37 completed weeks of gestation) is the leading cause of infant mortality worldwide. We identified the inverted formin-2 (INF2) gene through a genome-wide association study of prematurity in infants. INF2 is an important cytoskeletal modulator, highly conserved in both humans and mice. We hypothesize INF2 regulates vascular remodelling by altering trophoblast invasion and, consequently, gestation length. Methods: Inf2-/- timed-matings were performed and gestational length measured from copulatory plug detection (E0.5). Umbilical Doppler was performed at gestational age 18.5 (E18.5) followed by C-section, where litter size, fetal weight, and placental weight were recorded. In additional animals, pregnancies continued to term and birthweight measured at day 1 of life. Matrigel invasion assays were performed using HTR-8/SVneo cells transfected with siRNA against INF2 for 24 hours or treated with LCK inhibitors (PP1 or TX1123) for 7 days. Transwells were fixed, stained with DAPI, and five random fields were counted and normalized to the vehicle control. Data were analysed by Student T-test and a p value of less than 0.05 was deemed significant. Results: Inf2-/- females had significantly longer gestations than wildtype counterparts (19.7±0.08 vs 19.3±0.15 days; p<0.05) with no effect on birthweight or litter size. There was no difference in fetal weight or placental weight at E18.5. Inf2-/- mice demonstrated significant increases in pulsatility index (2.0±0.05 vs 1.9±0.02 a.u.; p<0.05), end diastolic velocity (14.2±0.9 vs 12.0±0.6 mm/s; p < 0.05), and maternal heart rate (604.2±91 vs 424.4±12; p<0.05) at E18.5. Loss of INF2 in vitro significantly reduced trophoblast invasion (14.6±5.9 vs 100±8.3%; p<0.05). Pharmacologic inhibition of Lck, an INF2-dependent tyrosine kinase, significantly reduced trophoblast invasion (31.2±11 PP1 vs 1.3±0.5 TX1123 vs 100±1.3%; p<0.05). Conclusions: These findings suggest INF2 is essential for trophoblast invasion and its loss impacts both vascular function and gestation length. P1.2 ANGIOGENIC FACTORS ARE ASSOCIATED WITH PLACENTAL WEIGHT AND BIRTH WEIGHT IN BANGLADESHI PREGNANCIES Alison Gernand 1, Abdullah Mahmud 2, Eszter Papp 3, Joy Shi 3, Daniel Roth 3. 1 The Pennsylvania State University, University Park, PA, USA; 2 International Center for Diarrhoeal Disease Research, Bangladesh, Dhaka, Bangladesh; 3 Hospital for Sick Children, Toronto, ON, Canada