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The research performed by the principal investigators in the Mother Infant Research Institute at Tufts Medical Center encompasses a broad array of approaches from basic science research, using cells in culture or model animal systems, to translational research, applying state-of-the-art molecular techniques to human material such as blood or saliva samples, to clinical research that involves direct “hands on” interaction with a pregnant woman or her newborn infant. Although in some cases the investigator works at the bedside and not in a laboratory, for the sake of consistency each group is described as a “laboratory.”
The Bianchi Laboratory
The House Laboratory
The Maron Laboratory
The Norwitz Laboratory
The Sen Laboratory
The Bianchi Laboratory
Bi-Directional Trafficking of Cells and Nucleic Acids during Pregnancy
The overall focus of the Bianchi laboratory is the bidirectional trafficking of cells and cell-free nucleic acids between the pregnant woman and her fetus. This translational (T1) work in humans and mice includes: 1) the application of gene expression data from the human fetus and neonate to develop novel diagnostics and therapeutics, and 2) understanding the consequences of the natural acquisition of fetal stem cells by pregnant females to determine if there are long-term beneficial health effects of pregnancy. Currently, most prenatal diagnoses are performed by ultrasound examination or cytogenetic analyses. The former technique addresses fetal structural abnormalities and the latter detects chromosome or DNA abnormalities. Neither approach gives information about fetal functional development.
Transcriptomic Analysis of the Fetus and Newborn
The Bianchi laboratory is particularly interested in using the messenger (m)RNA in amniotic fluid supernatant as a source of information about the growing human fetus (Hui and Bianchi, 2010). We have developed an amniotic fluid transcriptome for normal human fetuses in the second trimester of pregnancy. We have also shown that there are distinctly different gene expressions profiles in fetuses with specific abnormalities. We first investigated mRNA in fetuses with Down syndrome and compared gene expression to normal fetuses matched for gestational age (Slonim et al, 2009). Somewhat surprisingly, our data demonstrated that the fetuses with Down syndrome experience significant oxidative stress even as early as the second trimester of pregnancy. This new information suggests that by treating oxidative stress we may be able to improve outcome. We have recently been awarded a Russo grant from Tufts University to test this hypothesis.
We hypothesize that the use of differentially-regulated gene lists and pathway and network analyses will facilitate the development of inexpensive, noninvasive, point of care, multiplex real-time quantitative polymerase chain reaction amplification assays that could determine fetal or neonatal well-being from a functional (rather than an anatomic) perspective.
Figure 1. Multiplex-reverse transcriptase PCR amplification of cell-free RNA obtained from 10 pregnant women in the third trimester. Each colored line within the box represents a different blood sample. The genes amplified represent fetal sequences that we have previously shown to circulate within the mother (Maron et al, 2007). CAMP= cyclic adenosine monophosphate; NRL=neural retina leucine zipper; S100B=S100 calcium binding protein β; NRP1=neuropilin 1; ROBO4=roundabout homolog 4.
Fetal Cell Microchimerism and Regenerative Medicine
Microchimerism is the presence of two genetically distinct and separately derived populations of cells that coexist within the same organ or individual. Microchimerism is important because as a result of pregnancy, females (both human and murine) acquire populations of fetal cells that survive for years and have the ability to differentiate and proliferate. We are investigating whether these fetal cells have unique properties that will facilitate applications in regenerative medicine. This is an important aspect of gender biology that is frequently overlooked in human adult stem cell research (Bianchi and Fisk, 2007).
In the mouse model, we are exploring the biology of fetal cell microchimerism. We mate wild-type females to syngenic males that are homozygous for the green fluorescent protein (gfp) transgene. All pups inherit one copy of gfp and all of their cells, except for erythrocytes and fur, fluoresce. We are particularly interested in the lung, as our data have demonstrated that the lung is the organ that contains the most fetal cells. We are focusing on gene expression in these cells to inform us as to the type of fetal cells that are in the maternal lung as well as their ability to participate in repair of maternal disease and injury.
Figure 2. Photomicrographs showing examples of fetal mononuclear cells with regular, homogeneous, non-granular GFP positive cytoplasm in maternal lung frozen sections. Upper left panel: 400X magnification, other panels: 1000X magnification.
The House Laboratory
Biomechanics of Cervical Structural Function during Pregnancy
Preterm birth affects 12.3% of all pregnancies in the United States. Although preterm birth is a complex disorder, abnormalities of the cervix are involved in a significant number of preterm deliveries. Cervical shortening and cervical insufficiency are prominent features of preterm birth in many pregnant women. The objective of our research is to study the biomechanical mechanisms of cervical shortening and insufficiency in pregnancy. Our research includes: 1) studies of three-dimensional (3D) anatomic changes associated with cervical shortening and 2) investigation of biochemical and mechanical properties of cervical tissue. This research is valuable for understanding why the cervix stays closed in normal pregnancy but shortens and opens in preterm birth.
Cervical Tissue Engineering for Studying Cervical Remodeling
The mechanical properties of cervical tissue are derived from its extracellular matrix. The cervical extracellular matrix undergoes extensive remodeling in preparation for childbirth. The objective of this work is to use a tissue engineering strategy to develop three-dimensional (3D) engineered tissue suitable for investigating cervical remodeling. We have shown that primary cervical cells synthesize 3D cervical-like tissue with morphology and biochemical content similar to native tissue. We hypothesize that our tissue engineering strategy can help to elucidate the molecular mechanisms responsible for remodeling of the cervix during pregnancy.
Successful accomplishment of this research relies on a collaborative multidisciplinary research team involving investigators with expertise in mechanical engineering, extracellular matrix biochemistry, and maternal fetal medicine.
The Maron Laboratory
The Maron Laboratory focuses its research efforts on exploring neonatal development, physiology, and pathology through salivary gene expression analyses. Saliva is a rich source of genetic information that may be obtained repeatedly and safely from premature infants. Our group has garnered extensive expertise in neonatal salivary gene expression analyses and has developed novel techniques to obtain and process salivary samples from even our tiniest premature infants.
Currently, our laboratory is utilizing important gene expression information found in neonatal saliva to better understand oral feeding maturation in premature infants. Successful oral feeding is one of the most complex neurological tasks of the newborn. In order to safely feed by mouth, an infant must coordinate 26 pairs of muscles, five cranial nerve systems, and thoracic spinal cord segments involved in chest wall movements for coordination of respiration with feeding. These complex interactive mechanisms are poorly understood, particularly in premature neonates who may have multiple co-morbidities limiting their attainment of feeding milestones. Through salivary gene expression analyzes, we have identified key regulatory genes in saliva believed to be involved in oral feeding, including genes associated with satiety, feeding regulation, neurodevelopment, and oral musculature and innervation. We aim to utilize this information to develop a point of care diagnostic platform to accurately and objectively predict successful oral feeding. By objectively identifying infants who are ready to attempt oral feeds, we will limit acute morbidities, such as choking and aspiration, and significantly improve neonatal care and outcomes.
The Norwitz Laboratory
The Norwitz laboratory has a number of complementary research initiatives all focused on a single overall objective: to improve the ability of obstetric care providers to predict and prevent preterm birth. Preterm birth (defined as delivery prior to 37 weeks of gestation) accounts for over 85% of all perinatal morbidity and mortality. It is the leading cause of perinatal death in non-anomalous newborns in the United States. Despite intense efforts, approximately 12.3% (1 in 8) of all deliveries in the United States are preterm, which translates into 500,000 premature infants each year in the United States alone. The long-term goal of our research is to understand in detail the cellular and molecular mechanisms responsible for spontaneous preterm labor and birth with a view towards effectively delaying delivery until term thereby improving perinatal outcome. Individual research project include:
(1) Understanding the role of progesterone-progesterone receptor signaling in spontaneous preterm labor and birth
There is increasing evidence to suggest that withdrawal of progesterone activity at the level of the uterus is a prerequisite for spontaneous labor in humans, both at term and preterm. Moreover, recent studies have shown that progesterone supplementation can prevent preterm birth in some high-risk women. Although this is the first obstetric intervention that has been shown to effectively delay preterm birth in the past 40 years, its mechanism of action is currently not known. Progesterone acts in part by binding to progesterone receptors (PR) and modulating the expression of target genes, although non-genomic pathways are also described (Figure 1). We hypothesize that spontaneous preterm labor and birth results from a genetic predisposition to premature withdrawal of progesterone action at the level of the uterus due to differences in PR gene expression and/or function. To this end, we are investigating:
· The genetics of preterm birth (with particular focus on genetic variations/polymorphisms in progesterone, PR, and PR co-activator/co-repressor genes);
· The effect of progesterone on apoptosis in the fetal membranes (given that one third of preterm birth occurs in the setting of preterm premature rupture of membranes);
· The effect of progesterone supplementation on myometrial contractility both in vivo and in vitro;
· The identity of critical cis-DNA elements and cognate trans-factors that mediate tissue-specific expression of the PR gene as well as regulation by progesterone and other hormones;
· The functional importance of progesterone-PR signaling in preterm labor;
· Identification, characterization, and validation of biomarkers for the prediction of preterm birth (using proteomic / metabolomic analysis of amniotic fluid, cervicovaginal discharge, urine, and serum).
Figure 1: Proposed signaling pathways for progesterone action and the PRs involved
(2) Identification, characterization, and validation of biomarkers of preeclampsia
Preeclampsia (gestational proteinuric hypertension) is a major cause of pregnancy-related maternal death. It is estimated that one woman dies every 6 minutes from complications of preeclampsia somewhere in the world. Preeclampsia is also a major cause of perinatal morbidity and mortality, due primarily to the need to deliver the baby in order to save the mother. This is currently the only effective treatment. As such, prompt diagnosis and optimal timing of delivery is critical. The goal of this research is to better understand the pathogenesis of preeclampsia with a view to improving maternal and perinatal outcome in affected pregnancies. We and others have identified a number of serum and urinary biomarkers for the diagnosis and prediction of preeclampsia, including PP-13, TNFa, and the pro- and anti-angiogenic factors VEGF, PlGF, sFlt1, and sEng. We have recently identified two novel urinary biomarkers for preeclampsia, namely neutrophil gelatinase-associated lipocalin (NGAL) and interleukin-18 (IL-18). We have shown for the first time the presence of NGAL expression at the maternal-fetal interface in vivo (Figure 2) and in purified cytotrophoblast cells in vitro (Figure 3). To this end, we are investigating:
· The genetics of preeclampsia (with particular focus on genetic variations/polymorphisms in candidate biomarker genes followed by GWAS analysis);
· The utility of NGAL and IL-18 in the prediction and diagnosis of preeclampsia, either singly or in combination with each other or with previously described biomarkers (VEGF, PlGF, sFlt1, and sEng) or uterine artery Doppler velocimetry;
· The expression of these putative biomarkers at the maternal-fetal interface in vivo;
· The expression and regulation of these putative biomarkers in purified trophoblast cells under basal conditions and conditions of inflammation and hypoxia.
Figure 2: IHC showing cytokeratin (A,C) (which labels cytotrophoblasts) and NGAL (B,D) expression in placental tissues with / without intraamniotic infection. Negative control is included as an insert.
Figure 3: Fluorescent ICC showing cytokeratin (A,C) and NGAL (B,D) expression in day 1 (cytotropho-blast) cells in vitro with and without IL-1B treatment. DAPI nuclear staining is shown as inser
(3) GnRH-GnRHR signaling at the maternal-fetal interface
The hypothalamic decapeptide, GnRH (GnRH-I), plays a critical role in regulating mammalian reproductive development and function. In the anterior pituitary, GnRH binds to its target, a heptahelical G-protein coupled receptor known as the GnRH receptor (GnRHR) on the cell surface of pituitary gonadotropes. Here it activates intracellular signal transduction pathways to affect the synthesis and release of the gonadotropins, LH and FSH. These hormones enter the systemic circulation to regulate gonadal function, including steroid hormone synthesis and gametogenesis. A second isoform of GnRH (GnRH-II) has been identified, and both GnRH-I and -II are produced also by extra-hypothalamic tissues (including ovary, breast, and placenta), where they exert local autocrine/paracrine functions. We hypothesize that GnRH/GnRHR signaling at the maternal-fetal interface may be critically important in regulating events related to implantation, placentation, and the success of human pregnancy. Specifically, we propose that GnRH may act in an autocrine/paracrine fashion to regulate the production of chemokines, cytokines, and angiogenic factors in trophoblast and/or decidual tissues with important functional implications.
The Sen Laboratory
Maternal obesity has profound consequences on infant and childhood health. The rate of maternal obesity continues to increase and today, the majority of women at reproductive age are either overweight or obese. In addition to the well-established link between maternal obesity and lifelong metabolic dysregulation in their offspring, there have been recent population studies documenting the association between maternal obesity and immune dysregulation in mother and infant. Given the broad current and future impact of maternal obesity, identification of early interventions to prevent intergenerational transfer of these risk factors is of paramount public health importance. From a developmental biology perspective, the detrimental impact of maternal obesity on offspring provides a compelling model to study the broader impact of the in utero environment on offspring phenotype and delineate mechanisms, such as epigenetic programming, governing the transgenerational transfer of non-heritable characteristics. Our laboratory has three main focuses:
Maternal Obesity and Immune Dysregulation (MOOID study)
Obese women are more prone to infectious diseases during pregnancy as compared to lean women. This adversely affects the developing fetus. In addition, children born to obese mothers have a higher incidence and severity of asthma and atopic disease. The MOOID study is a translational study that aims to delineate mechanisms by which obesity in pregnancy dysregulates the maternal and fetal immune system, thus predisposing children to asthma and atopic disease.
Micronutrient Balance in Maternal Obesity
Obesity in pregnancy is associated with inflammation and oxidative stress that create an abnormal in utero milieu in which the embryo and fetus develop. This pro-inflammatory milieu could influence the nascent fetal epigenetic code and lead to changes in offspring. We have previously shown in an animal model that antioxidant supplementation of obese dams ameliorates obesity in offspring. In this pilot study, we will be the first to establish the micronutrient status of obese pregnancy in humans. Current obstetric practice recommends that pregnant women take one prenatal vitamin daily, regardless of their pre-pregnancy body mass index. The results of this study could lead to a trial comparing the maternal and neonatal outcome of a standard prenatal vitamin to a weight adjusted dosage of prenatal vitamins.
Mechanisms by which Maternal Obesity Programs Changes in Offspring
We hypothesize that the abnormal intrauterine milieu of maternal obesity creates early changes in adipocyte determination that lead to a lifelong propensity for obesity and metabolic dysregulation. To test this hypothesis, we will isolate mesenchymal cells from the cord blood of lean and obese mothers and grow them in vitro into adipocytes using well established methods. Using real time PCR and gene expression microarrays, we will measure genes that potentially to participate in adipogenesis, inflammation and insulin resistance, from both the mesenchymal precursors and the cultured adipocytes. This study will be the first to describe how maternal obesity programs fetal adipocytes and could provide preliminary evidence for the mechanisms involved in transgenerational obesity.
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