In eutherian mammals, proper fetal development depends on a functional placenta. Specialized placental compartments anchor the embryo to the uterus and maintain a steady supply of oxygen and nutrients to the embryo, while other parts manage the disposal of metabolites. Two placental structures facilitate these functions: floating and anchoring villi. The genetic makeup of floating villi has been studied in detail. Little is known about the genetic make-up of anchoring villi, but our cytogenetic studies of fetal cells isolated from anchoring villi or the uterine wall revealed an unexpected large fraction of invading cytotrophoblasts (CTBs) that carry numerical chromosomal aberrations. We postulate that aneuploidy at the fetal-maternal interface is an integral part of the normal placentation program. We hypothesize that in the absence of mutations, genomic changes in CTBs such as gains or losses of chromosomes are normal cellular mechanisms that limit the proliferative as well as the invasive capabilities of CTBs. To test our hypothesis, we propose to investigate the karyotype of CTBs as well as phenotypic markers of cell proliferation or differentiation along an invasive CTB phenotype. In the study proposed here, investigators with complementary expertise in early embryonic development, placental function, molecular cytogenetics and digital image processing will collaborate to localize aneuploid cells in different placental compartments. First, we will determine the types of numerical chromosome abnormalities in the uterine wall and in anchoring as well as floating villi in normal placental specimens and placental tissues carrying a trisomy 21. Next, we will determine the spatial distribution of genomic instability in normal and trisomic second trimester placentas. Techniques used will be fluorescence in situ hybridization (FISH), confocal microscopy and 3D image reconstruction of thick tissue sections to localize aneuploid cells in their histological context. Finally, we will determine the effects of aneuploidy on the proliferative capacity of CTBs as measured by BrdU incorporation and expression of the HLA-G gene, an important marker for CTB differentiation along the invasive pathway. At the end of this project, we will have developed the technology to accurately score all 24 human chromosomes in interphase cells and demonstrated the feasibility to localize aneuploid cells in 3-dimension in thick tissue sections. We will also have detailed information about the frequency, localization and types of aneuploid cells at the fetal-maternal interface and the effects of aneuploidy on CTB proliferation and HLA-G gene expression.
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