- Email: [email protected]
Immune protection against placental cytomegalovirus transmission in nonhuman primate models
70
Abstracts / Placenta 45 (2016) 63e133
The syncytiotrophoblast is a complex, multinucleated component of the placenta that is formed and maintained by the fusion of cytotrophoblast cells. During this dynamic cell fusion process, the specific cascade of epigenetic changes is currently unknown. To elucidate these mechanisms we conducted a cytotrophoblast fusion experiment and profiled the chromatin landscape at a series of time points. Chromatin accessibility is an informative property of the epigenome and is highly correlated with transcriptional regulation, histone modifications, DNA methylation, and the three dimensional conformation of DNA within the nucleus. In most cell types profiled to date, between 1-4% of the genome is present in regions of open, accessible chromatin, which are predominantly active promoter and enhancer elements. To assess this property we implemented ATAC-seq (Assay for Transposase Accessible Chromatin), which allows for the rapid production of chromatin accessibility sequencing libraries from limited numbers of cells, including a recent advancement that allows for the simultaneous production of thousands of single cell chromatin accessibility libraries from a single sample. Since ATAC-seq can be performed on isolated nuclei, we are able to avoid many of the challenges imposed by a multinucleated cell type. We can then utilize these data in computational models to produce a high resolution map of epigenetic remodeling during the fusion process which may reveal valuable insights into the mechanisms and pathways that drive the formation and maintenance of a healthy syncytiotrophoblast. O2.01 TROPHOBLAST STEM CELLS FROM MURINE FIBROBLASTS e CAN THE MOUSE SERVE AS BLUEPRINT FOR THE HUMAN SITUATION? Hubert Schorle. University of Bonn, Bonn, Germany Trophoblast stem cells represent the stem cell population of the extraembryonic lineage and arise as result of the first cell fate decision. From blastocyst stage onwards, a distinct epigenetic lineage barrier strictly separates mouse embryonic and extra-embryonic lineages. This epigenetic barrier cannot be fully overcome as the expression of TS-determining factors in embryonic stem cells lead to incomplete transdifferentiation. We recently demonstrated that transient expression of Tfap2c, Gata3, Eomes and Ets2 in fibroblasts suffices to generate cells, which resemble trophoblast stem cells in terms of morphology, expression and methylation pattern. The iTSC (induced trophoblast stem cells) display transgene independent self-renewal, differentiate along the extra-embryonic lineage and chimerize the placenta upon blastocyst injection. Our findings provide insights into the transcription factor networks governing trophoblast stem cell identity and raise the question whether such an approach would work in the human situation, where the hTSC is still evasive. O2.02 HUMAN TROPHOBLAST STEM CELLS: REAL OR NOT REAL? Mana Parast. University of California, San Diego, USA Mouse trophoblast stem cells (TSC) were derived over 15 years ago from preimplantation blastocysts, and can be maintained in a proliferative, multipotent state using a combination of FGF and activin signaling. Studies with transgenic mice have identified a number of transcription factors which are involved in specification of the trophoblast and maintenance of the TSC state. At the same time, little is known about similar cells in the human blastocyst and placenta. Many investigators, both within and outside this field, assume that what is true in mice must be true in human. Nevertheless, recent studies point to major differences in both pre- and early post-implantation events, markers associated with the trophoblast progenitor state, and signaling pathways involved in trophoblast lineage determination, in mice and human. For example, our studies have identified p63, a marker of stem cells in stratified epithelia, as a marker of proliferative cytotrophoblast (CTB), only in the human placenta. More recently, we have identified a subgroup of early post-implantation CTB which co-express p63 and CDX2, a marker of mouse TSC. This p63+/CDX2+ CTB population is lost by the end of the first trimester, co-incident with a decrease in CTB proliferation and bipotential differentiation. We therefore hypothesize that this population may define TSC in the human placenta. Our current efforts include further characterization of CTB in the early
post-implantation period using a combination of single cell RNAseq, flow cytometric analysis of cell surface markers, and measurement of differentiation potential. Simultaneously, we are performing chemical library screens for identification of small molecules which could potentially maintain such TSCs. Our goal is to establish a reproducible TSC model, which can recapitulate early events in human placental development, and which could be widely disseminated to researchers for the study of placenta-based pregnancy complications, such as pre-eclampsia and fetal growth restriction. O2.03 CONTROLLING MULTI-STAGE TROPHOBLAST DEVELOPMENT: ORCHESTRATION OF PROGENITOR AND DIFFERENTIATED CELL FATE BY TRANSCRIPTION FACTORS Soumen Paul. University of Kansas Medical Center, USA Mammalian reproduction is critically dependent on trophoblast cells, which assure embryo implantation and placentation. Development of trophoblast cell lineages is a multi-step process and relies upon proper spatial and temporal regulation of gene expression during (i) trophectoderm-development in preimplantation embryo (ii) maintenance of selfrenewal within trophoblast stem and progenitor cells (TSPCs) of an early postimplantation embryo; and (iii) subsequent differentiation of trophoblast progenitors to specialized trophoblast subtypes of a matured placenta. However, we have a poor understanding of transcriptional mechanisms that regulate distinct stages of trophoblast development. Here, we studied developmental stage-specific transcriptional mechanisms with inducible gene knockout mouse models of transcription factors GATA2, GATA3 and TEAD4. We show that GATA factors occupy both transcriptionally active and silent genes to orchestrate developmental stagespecific gene expression thereby establishing both stem/progenitor and differentiated cell-fate. In contrast, TEAD4 function is critical to maintain self-renewal of TSPCs. We also show that expression patterns of these key transcription factors are conserved during human trophoblast lineage development and they mediate largely conserved transcriptional program in human trophoblast progenitors. Our study reveals how key transcription factors fine-tune gene expression in extra-embryonic trophoblast lineage to ensure early progression of mammalian development. O3.01 IMMUNE PROTECTION AGAINST PLACENTAL CYTOMEGALOVIRUS TRANSMISSION IN NONHUMAN PRIMATE MODELS Sallie Permar. Duke University Medical Center, USA Cytomegalovirus (CMV) is the most common congenital infection worldwide, and a significant cause of infant hearing loss and neurodevelopmental delay. One potential therapeutic intervention to eliminate this disease is a maternal vaccine that blocks placental CMV transmission. Previously, we developed a nonhuman primate model of placental CMV infection in rhesus monkeys and demonstrated a role for maternal immunity in protection against severe congenital CMV disease. In our model, CD4+ T cell depleted dams, who also demonstrate a delayed neutralizing antibody response, experienced high rates of transmission (100%) and fetal loss (75%) shortly after rhesus CMV (rhCMV) challenge. To further investigate the role of maternal antibodies in protection against congenital CMV, we designed a study to determine if passive antibody infusion of CMV-seronegative, CD4+ T cell depleted dams with rhCMV hyperimmune globulin could protect against placental rhCMV transmission or placental and fetal disease, and whether the presence of CMV-specific antibody alone could impact in vivo CMV evolution. 6 rhCMV-seronegative rhesus macaque dams in the second trimester of pregnancy were CD4+ depleted and given a single or multiple dose regimen of hyperimmune globulin (HIG) 1 hour prior to intravenous (IV) challenge with a mixture of fibroblast-adapted (180.92) and epithelial-tropic (UCD52/UCD59) rhCMV strains. Passive infusion of hyperimmune globulin (HIG) prior to IV rhCMV challenge of CD4+ depleted dams provided complete protection against fetal loss (0/6 dams aborted; p<0.01). Furthermore, 2 doses of a highlyneutralizing HIG product achieved protection from placental transmission of rhCMV (0/3 transmitted), while a single dose of low-potency HIG
Abstracts / Placenta 45 (2016) 63e133
product did not (2/3 dams transmitted). Moreover, targeted deep sequencing of rhCMV glycoprotein L (gL) and glycoprotein B (gB) from maternal plasma revealed that infused HIG created a genetic bottleneck, decreasing the diversity of haplotypes at both loci. Finally, transcriptome analysis of the placenta of HIG-treated compared to untreated dam revealed dysregulation in the genes within pathways of angiogenesis, inflammation, and in particular, the fatty acid binding protein 4, a placental factor previously associated with preeclampsia. Our data suggest that the presence of highly-neutralizing antibodies at the time rhCMV acquisition can prevent congenital infection, perhaps by influencing early systemic and intrauterine viral replication and eventual placental dysregulation. A better understanding of how antibody-mediated viral selection and impairment impacts placental rhCMV transmission and fetal outcome will inform future vaccine development efforts that may be able to focus on the elicitation of this response. O3.02 ROLE OF POLYMICROBIAL INFECTION ON VIRAL-INDUCED TERATOGENIC EFFECTS Gil Mor. Yale University School of Medicine, CT, USA Perinatal outcomes from viral infections during pregnancy can range from no effect to pregnancy loss by spontaneous abortion to fetal infection with resulting congenital viral syndromes. Prenatal care currently holds no true standard for antenatal management of viral infections during pregnancy aside from those known as TORCH infections (toxoplasmosis, “other”, rubella, CMV, and HSV). And while these guidelines allow for a diagnosis of infection no treatment or preventative strategy is available to prevent adverse pregnancy outcomes. The placenta is generally an effective barrier preventing microorganisms from the uterus or maternal circulation from accessing the developing fetus. Consequently, for a microorganism to reach the fetal cavity it needs to overcome the defense mechanism provided by trophoblast cells. We have shown that a viral infection of the placenta disrupts the normal protective mechanisms of the placental-fetal unit, leading to preterm birth in response to sub-lethal/sub-clinical doses of bacterial products. Furthermore, we demonstrated that the Zika virus is able to infect first trimester trophoblast cells, inhibit Type I interferon expression and function and trigger trophoblast apoptosis. We will discuss our findings showing that placental IFNb is a major immune modulatory factor at the implantation site by preventing viruses from reaching the developing embryo/fetus while inhibiting uncontrolled “cytokine storm” and immune cell activation that could have a detrimental effect on the pregnancy. However, due to their potent anti-viral properties, interferons are major viral targets for neutralization and consequently affecting the protective effect of the placenta. AGA UNDERSTANDING THE MOLECULAR UNDERPINNINGS OF DISTINCT SUBCLASSES OF PREECLAMPSIA Shannon Bainbridge. University of Ottawa, Ottawa, Canada Preeclampsia (PE) is a hypertensive disorder affecting 3-5% of all pregnancies and remains a leading cause of fetal and maternal morbidity and mortality. Consensus has been reached that PE is not a single disease with common pathophysiology. In support, we have recently used a systems biology approach to identified molecular subclasses of PE with distinct pathophysiology. Specifically our findings indicated that PE can result from maternal maladaptation to pregnancy (‘maternal PE’), placental malperfusion (‘canonical PE’), or maternal-fetal incompatibility and heightened immune activation (‘immune-driven PE’). These findings open the door to individualized therapeutic approaches for defined subclasses of PE. Most recently we have turned our attention to better understanding the molecular underpinnings of the ‘immune-driven PE’ subclass, as this group of women are under-represented in our current descriptions of PE pathophysiology. Interestingly, several members of the Poly ADP-ribose polymerase (PARP) family of proteins are specifically overexpressed within the placentae of women with ‘immune-driven PE’. Excess PARP activity plays a pivotal role in chronic inflammatory disease states through depletion of its enzymatic cofactor NAD+. NAD+ is a key regulator of energy metabolism
71
within a cell, modulating the enzymatic activities of various sirtuin proteins to sustain mitochondrial health. We therefore hypothesized that placental dysfunction observed in ‘immune-driven PE’ is dependent on placental NAD+ depletion. Using a combined investigation of human placental pathology biopsies, in vitro human trophoblast culture and a rat model of ‘immune-driven PE’, we have collected evidence to support the speculation that a pro-inflammatory environment at the maternal-fetal interface triggers excess placental PARP activity and subsequent NAD+ depletion in the immune-driven subclass of PE. Reduced placental NAD+ stores would lead to energy deficiency within the placenta, causing damage to this vital organ of pregnancy. This provides the rationale to replenish cellular NAD+ stores to improve the prognosis of immune activated forms of PE. HPP THE HUMAN PLACENTA PROJECT: CURRENT PROGRESS AND FUTURE DIRECTIONS David H. Weinberg, Caroline Signore, Catherine Y. Spong. The Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, USA Abnormalities of placental development and function are known to underlie many major pathologies of pregnancy. However, the assessment of the placenta across pregnancy presents special challenges due to the need to avoid risk to the mother and developing fetus. The Human Placenta Project (HPP) is an initiative aimed at revolutionizing understanding of the human placenta. The development and application of innovative technologies that can be used safely in pregnant women should allow researchers to produce a new dynamic picture of placental structure and function in real time, one that assesses key developmental trajectories of placental formation and functional cues critical for successful human pregnancy. The application of these new tools and technologies should ultimately lead to new ways to treat, cure, and even prevent placental dysfunction disorders. It is likely that the methods developed will also be applicable to assessment of other organs; thus, the impact may be farreaching. To achieve these goals will require the collaborative efforts of clinicians, placental biologists, and technology experts from diverse research areas, utilizing novel technologies, including some developed for other research areas, and applying them to the placenta. To be successful, this must be a global effort. NICHD has invested more than $50M to support the HPP, awarding 19 grants to date to researchers inside and outside the U.S. The current awards support a wide range of imaging and nonimaging approaches. NICHD has also published 2 additional funding opportunities aimed at placental assessment across pregnancy; one focused on omics, and the other on the use of existing data sets. A yearly HPP meeting allows the scientific community to be active participants in the development of the project roadmap. This talk will outline progress made on the HPP to date, and discuss plans for continuing to move the project forward. IFPA IFPA SENIOR AWARD LECTURE: PLACENTA TOXICOLOGY e SUSCEPTIBILITY, ASSESSMENT AND CONTRIBUTIONS TO REPRODUCTIVE AND FETAL/NEONATAL OUTCOME Richard Miller. University of Rochester, Rochester, USA The placenta and associated membranes have often been considered the conduit for exchange not only for nutrients and waste products but also for the entry of therapeutic and environmental/infectious/occupational contaminants. Whether defined as a nutrient, contaminant, infectious agent or waste product, the placenta itself can be a site for the production of reproductive and developmental toxicants as well as the site for toxic action. A variety of in utero (human and animal) and in vitro studies have been conducted to unravel the kinetics and actions for a diversity of molecules. An range of different research approaches will be examined using examples of nutrients (amino acids, Vitamins A and B12), infectious agents, medications (abciximab, anti-HIV, diethylstilbestrol, gadolinium, retinoids) and contaminants (bisphenol A, cadmium, flame retardants, phthalates) with functional placental endpoints of energy metabolism,
Abstracts / Placenta 45 (2016) 63e133
The syncytiotrophoblast is a complex, multinucleated component of the placenta that is formed and maintained by the fusion of cytotrophoblast cells. During this dynamic cell fusion process, the specific cascade of epigenetic changes is currently unknown. To elucidate these mechanisms we conducted a cytotrophoblast fusion experiment and profiled the chromatin landscape at a series of time points. Chromatin accessibility is an informative property of the epigenome and is highly correlated with transcriptional regulation, histone modifications, DNA methylation, and the three dimensional conformation of DNA within the nucleus. In most cell types profiled to date, between 1-4% of the genome is present in regions of open, accessible chromatin, which are predominantly active promoter and enhancer elements. To assess this property we implemented ATAC-seq (Assay for Transposase Accessible Chromatin), which allows for the rapid production of chromatin accessibility sequencing libraries from limited numbers of cells, including a recent advancement that allows for the simultaneous production of thousands of single cell chromatin accessibility libraries from a single sample. Since ATAC-seq can be performed on isolated nuclei, we are able to avoid many of the challenges imposed by a multinucleated cell type. We can then utilize these data in computational models to produce a high resolution map of epigenetic remodeling during the fusion process which may reveal valuable insights into the mechanisms and pathways that drive the formation and maintenance of a healthy syncytiotrophoblast. O2.01 TROPHOBLAST STEM CELLS FROM MURINE FIBROBLASTS e CAN THE MOUSE SERVE AS BLUEPRINT FOR THE HUMAN SITUATION? Hubert Schorle. University of Bonn, Bonn, Germany Trophoblast stem cells represent the stem cell population of the extraembryonic lineage and arise as result of the first cell fate decision. From blastocyst stage onwards, a distinct epigenetic lineage barrier strictly separates mouse embryonic and extra-embryonic lineages. This epigenetic barrier cannot be fully overcome as the expression of TS-determining factors in embryonic stem cells lead to incomplete transdifferentiation. We recently demonstrated that transient expression of Tfap2c, Gata3, Eomes and Ets2 in fibroblasts suffices to generate cells, which resemble trophoblast stem cells in terms of morphology, expression and methylation pattern. The iTSC (induced trophoblast stem cells) display transgene independent self-renewal, differentiate along the extra-embryonic lineage and chimerize the placenta upon blastocyst injection. Our findings provide insights into the transcription factor networks governing trophoblast stem cell identity and raise the question whether such an approach would work in the human situation, where the hTSC is still evasive. O2.02 HUMAN TROPHOBLAST STEM CELLS: REAL OR NOT REAL? Mana Parast. University of California, San Diego, USA Mouse trophoblast stem cells (TSC) were derived over 15 years ago from preimplantation blastocysts, and can be maintained in a proliferative, multipotent state using a combination of FGF and activin signaling. Studies with transgenic mice have identified a number of transcription factors which are involved in specification of the trophoblast and maintenance of the TSC state. At the same time, little is known about similar cells in the human blastocyst and placenta. Many investigators, both within and outside this field, assume that what is true in mice must be true in human. Nevertheless, recent studies point to major differences in both pre- and early post-implantation events, markers associated with the trophoblast progenitor state, and signaling pathways involved in trophoblast lineage determination, in mice and human. For example, our studies have identified p63, a marker of stem cells in stratified epithelia, as a marker of proliferative cytotrophoblast (CTB), only in the human placenta. More recently, we have identified a subgroup of early post-implantation CTB which co-express p63 and CDX2, a marker of mouse TSC. This p63+/CDX2+ CTB population is lost by the end of the first trimester, co-incident with a decrease in CTB proliferation and bipotential differentiation. We therefore hypothesize that this population may define TSC in the human placenta. Our current efforts include further characterization of CTB in the early
post-implantation period using a combination of single cell RNAseq, flow cytometric analysis of cell surface markers, and measurement of differentiation potential. Simultaneously, we are performing chemical library screens for identification of small molecules which could potentially maintain such TSCs. Our goal is to establish a reproducible TSC model, which can recapitulate early events in human placental development, and which could be widely disseminated to researchers for the study of placenta-based pregnancy complications, such as pre-eclampsia and fetal growth restriction. O2.03 CONTROLLING MULTI-STAGE TROPHOBLAST DEVELOPMENT: ORCHESTRATION OF PROGENITOR AND DIFFERENTIATED CELL FATE BY TRANSCRIPTION FACTORS Soumen Paul. University of Kansas Medical Center, USA Mammalian reproduction is critically dependent on trophoblast cells, which assure embryo implantation and placentation. Development of trophoblast cell lineages is a multi-step process and relies upon proper spatial and temporal regulation of gene expression during (i) trophectoderm-development in preimplantation embryo (ii) maintenance of selfrenewal within trophoblast stem and progenitor cells (TSPCs) of an early postimplantation embryo; and (iii) subsequent differentiation of trophoblast progenitors to specialized trophoblast subtypes of a matured placenta. However, we have a poor understanding of transcriptional mechanisms that regulate distinct stages of trophoblast development. Here, we studied developmental stage-specific transcriptional mechanisms with inducible gene knockout mouse models of transcription factors GATA2, GATA3 and TEAD4. We show that GATA factors occupy both transcriptionally active and silent genes to orchestrate developmental stagespecific gene expression thereby establishing both stem/progenitor and differentiated cell-fate. In contrast, TEAD4 function is critical to maintain self-renewal of TSPCs. We also show that expression patterns of these key transcription factors are conserved during human trophoblast lineage development and they mediate largely conserved transcriptional program in human trophoblast progenitors. Our study reveals how key transcription factors fine-tune gene expression in extra-embryonic trophoblast lineage to ensure early progression of mammalian development. O3.01 IMMUNE PROTECTION AGAINST PLACENTAL CYTOMEGALOVIRUS TRANSMISSION IN NONHUMAN PRIMATE MODELS Sallie Permar. Duke University Medical Center, USA Cytomegalovirus (CMV) is the most common congenital infection worldwide, and a significant cause of infant hearing loss and neurodevelopmental delay. One potential therapeutic intervention to eliminate this disease is a maternal vaccine that blocks placental CMV transmission. Previously, we developed a nonhuman primate model of placental CMV infection in rhesus monkeys and demonstrated a role for maternal immunity in protection against severe congenital CMV disease. In our model, CD4+ T cell depleted dams, who also demonstrate a delayed neutralizing antibody response, experienced high rates of transmission (100%) and fetal loss (75%) shortly after rhesus CMV (rhCMV) challenge. To further investigate the role of maternal antibodies in protection against congenital CMV, we designed a study to determine if passive antibody infusion of CMV-seronegative, CD4+ T cell depleted dams with rhCMV hyperimmune globulin could protect against placental rhCMV transmission or placental and fetal disease, and whether the presence of CMV-specific antibody alone could impact in vivo CMV evolution. 6 rhCMV-seronegative rhesus macaque dams in the second trimester of pregnancy were CD4+ depleted and given a single or multiple dose regimen of hyperimmune globulin (HIG) 1 hour prior to intravenous (IV) challenge with a mixture of fibroblast-adapted (180.92) and epithelial-tropic (UCD52/UCD59) rhCMV strains. Passive infusion of hyperimmune globulin (HIG) prior to IV rhCMV challenge of CD4+ depleted dams provided complete protection against fetal loss (0/6 dams aborted; p<0.01). Furthermore, 2 doses of a highlyneutralizing HIG product achieved protection from placental transmission of rhCMV (0/3 transmitted), while a single dose of low-potency HIG
Abstracts / Placenta 45 (2016) 63e133
product did not (2/3 dams transmitted). Moreover, targeted deep sequencing of rhCMV glycoprotein L (gL) and glycoprotein B (gB) from maternal plasma revealed that infused HIG created a genetic bottleneck, decreasing the diversity of haplotypes at both loci. Finally, transcriptome analysis of the placenta of HIG-treated compared to untreated dam revealed dysregulation in the genes within pathways of angiogenesis, inflammation, and in particular, the fatty acid binding protein 4, a placental factor previously associated with preeclampsia. Our data suggest that the presence of highly-neutralizing antibodies at the time rhCMV acquisition can prevent congenital infection, perhaps by influencing early systemic and intrauterine viral replication and eventual placental dysregulation. A better understanding of how antibody-mediated viral selection and impairment impacts placental rhCMV transmission and fetal outcome will inform future vaccine development efforts that may be able to focus on the elicitation of this response. O3.02 ROLE OF POLYMICROBIAL INFECTION ON VIRAL-INDUCED TERATOGENIC EFFECTS Gil Mor. Yale University School of Medicine, CT, USA Perinatal outcomes from viral infections during pregnancy can range from no effect to pregnancy loss by spontaneous abortion to fetal infection with resulting congenital viral syndromes. Prenatal care currently holds no true standard for antenatal management of viral infections during pregnancy aside from those known as TORCH infections (toxoplasmosis, “other”, rubella, CMV, and HSV). And while these guidelines allow for a diagnosis of infection no treatment or preventative strategy is available to prevent adverse pregnancy outcomes. The placenta is generally an effective barrier preventing microorganisms from the uterus or maternal circulation from accessing the developing fetus. Consequently, for a microorganism to reach the fetal cavity it needs to overcome the defense mechanism provided by trophoblast cells. We have shown that a viral infection of the placenta disrupts the normal protective mechanisms of the placental-fetal unit, leading to preterm birth in response to sub-lethal/sub-clinical doses of bacterial products. Furthermore, we demonstrated that the Zika virus is able to infect first trimester trophoblast cells, inhibit Type I interferon expression and function and trigger trophoblast apoptosis. We will discuss our findings showing that placental IFNb is a major immune modulatory factor at the implantation site by preventing viruses from reaching the developing embryo/fetus while inhibiting uncontrolled “cytokine storm” and immune cell activation that could have a detrimental effect on the pregnancy. However, due to their potent anti-viral properties, interferons are major viral targets for neutralization and consequently affecting the protective effect of the placenta. AGA UNDERSTANDING THE MOLECULAR UNDERPINNINGS OF DISTINCT SUBCLASSES OF PREECLAMPSIA Shannon Bainbridge. University of Ottawa, Ottawa, Canada Preeclampsia (PE) is a hypertensive disorder affecting 3-5% of all pregnancies and remains a leading cause of fetal and maternal morbidity and mortality. Consensus has been reached that PE is not a single disease with common pathophysiology. In support, we have recently used a systems biology approach to identified molecular subclasses of PE with distinct pathophysiology. Specifically our findings indicated that PE can result from maternal maladaptation to pregnancy (‘maternal PE’), placental malperfusion (‘canonical PE’), or maternal-fetal incompatibility and heightened immune activation (‘immune-driven PE’). These findings open the door to individualized therapeutic approaches for defined subclasses of PE. Most recently we have turned our attention to better understanding the molecular underpinnings of the ‘immune-driven PE’ subclass, as this group of women are under-represented in our current descriptions of PE pathophysiology. Interestingly, several members of the Poly ADP-ribose polymerase (PARP) family of proteins are specifically overexpressed within the placentae of women with ‘immune-driven PE’. Excess PARP activity plays a pivotal role in chronic inflammatory disease states through depletion of its enzymatic cofactor NAD+. NAD+ is a key regulator of energy metabolism
71
within a cell, modulating the enzymatic activities of various sirtuin proteins to sustain mitochondrial health. We therefore hypothesized that placental dysfunction observed in ‘immune-driven PE’ is dependent on placental NAD+ depletion. Using a combined investigation of human placental pathology biopsies, in vitro human trophoblast culture and a rat model of ‘immune-driven PE’, we have collected evidence to support the speculation that a pro-inflammatory environment at the maternal-fetal interface triggers excess placental PARP activity and subsequent NAD+ depletion in the immune-driven subclass of PE. Reduced placental NAD+ stores would lead to energy deficiency within the placenta, causing damage to this vital organ of pregnancy. This provides the rationale to replenish cellular NAD+ stores to improve the prognosis of immune activated forms of PE. HPP THE HUMAN PLACENTA PROJECT: CURRENT PROGRESS AND FUTURE DIRECTIONS David H. Weinberg, Caroline Signore, Catherine Y. Spong. The Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, USA Abnormalities of placental development and function are known to underlie many major pathologies of pregnancy. However, the assessment of the placenta across pregnancy presents special challenges due to the need to avoid risk to the mother and developing fetus. The Human Placenta Project (HPP) is an initiative aimed at revolutionizing understanding of the human placenta. The development and application of innovative technologies that can be used safely in pregnant women should allow researchers to produce a new dynamic picture of placental structure and function in real time, one that assesses key developmental trajectories of placental formation and functional cues critical for successful human pregnancy. The application of these new tools and technologies should ultimately lead to new ways to treat, cure, and even prevent placental dysfunction disorders. It is likely that the methods developed will also be applicable to assessment of other organs; thus, the impact may be farreaching. To achieve these goals will require the collaborative efforts of clinicians, placental biologists, and technology experts from diverse research areas, utilizing novel technologies, including some developed for other research areas, and applying them to the placenta. To be successful, this must be a global effort. NICHD has invested more than $50M to support the HPP, awarding 19 grants to date to researchers inside and outside the U.S. The current awards support a wide range of imaging and nonimaging approaches. NICHD has also published 2 additional funding opportunities aimed at placental assessment across pregnancy; one focused on omics, and the other on the use of existing data sets. A yearly HPP meeting allows the scientific community to be active participants in the development of the project roadmap. This talk will outline progress made on the HPP to date, and discuss plans for continuing to move the project forward. IFPA IFPA SENIOR AWARD LECTURE: PLACENTA TOXICOLOGY e SUSCEPTIBILITY, ASSESSMENT AND CONTRIBUTIONS TO REPRODUCTIVE AND FETAL/NEONATAL OUTCOME Richard Miller. University of Rochester, Rochester, USA The placenta and associated membranes have often been considered the conduit for exchange not only for nutrients and waste products but also for the entry of therapeutic and environmental/infectious/occupational contaminants. Whether defined as a nutrient, contaminant, infectious agent or waste product, the placenta itself can be a site for the production of reproductive and developmental toxicants as well as the site for toxic action. A variety of in utero (human and animal) and in vitro studies have been conducted to unravel the kinetics and actions for a diversity of molecules. An range of different research approaches will be examined using examples of nutrients (amino acids, Vitamins A and B12), infectious agents, medications (abciximab, anti-HIV, diethylstilbestrol, gadolinium, retinoids) and contaminants (bisphenol A, cadmium, flame retardants, phthalates) with functional placental endpoints of energy metabolism,