Amniotic fluid cell-free RNA: A novel source of information about human development

Amniotic fluid (AF) is a complex, dynamic solution that surrounds the fetus and contains cell-free fetal (cff) RNA. Study aims The overall objective of this thesis was to investigate AF cff RNA as a source of fetal gene expression information in normal and abnormal pregnancies. The specific aims were: • To develop an understanding of the mid-trimester AF transcriptome, including the putative tissues sources and biological functions of AF cell-free RNA • To investigate the changes that occur in AF cff RNA with increasing gestational age • To explore AF cff RNA from aneuploid fetuses for gene expression information about abnormal neurodevelopment • To better understand the pathophysiology involved in twin-twin transfusion syndrome by performing global gene expression analysis of AF cff RNA from pregnancies with active disease Methods RNA was isolated from AF supernatants (AFS), amplified and hybridized to whole genome microarrays. Statistically significantly differentially-regulated and/ or “present” genes were identified. Functional analyses were performed with bioinformatics software tools including Ingenuity Pathway Analysis™ (IPA) and the Database for Annotation, Visualization and Integrated Discovery (DAVID). Results • Cell-free RNA in AF is derived from a variety of tissues and provides data on multiple fetal organ systems, including the nervous system. • The tissue expression patterns and enriched biological pathways in AF cff RNA vary with gestational age and, at term, reflect fetal maturation processes. • Mid-trimester AFS from aneuploid fetuses is enriched for nervous system gene expression and neurological disease pathways. • Functional genomic analysis of AFS from TTTS recipient fetuses reveals dysregulation of cardiovascular and nervous system gene expression that precedes intervention with laser therapy. Conclusions AF cff RNA is a s! ource of meaningful biological information about the fetus. Functional genomic analysis of AF cff RNA can reveal novel molecular data about the impact of chromosomal or structural abnormalities on human development. These findings advance the concept of AFS as a gene expression “summary fluid” of the live human fetus.