Global Gene Expression Analysis of the Living Human Fetus Using Cell-Free Messenger RNA in Amniotic Fluid

Abstract

ECAUSE OF THE INHERENT DIF-ficulties with research on human fetuses, the analysis of fetal gene expression has in large part been limited to the examination of tissue from human abortuses and the assessment of animal models for genes and developmental pathways that are conserved across species. Fetal monitoring in vivo is limited to noninvasive methods such as the measurement of uterine size or anatomic evaluation by fetal sonography. In addition, genetic analysis can be performed on amniotic fluid components, including amniocytes, which typically require time-consuming expansion in vitro before use, and cellfree proteins in the amniotic fluid, such as ␣-fetoprotein, which can serve as biomarkers for genetic anomalies. The cellfree component of the amniotic fluid is discarded after these analyses and is therefore available for research and future clinical applications.Cell-free fetal DNA in the serum and plasma of pregnant women was first described by Lo et al 1 in 1997 after others demonstrated the presence of circulating tumor-specific DNA sequences in cancer patients. [2][3][4] After it was shown that cell-free fetal DNA is also present in the urine of pregnant women, 5 we hypothesized that amniotic fluid, as a reservoir of fetal urine, would contain fetal DNA. In a preliminary study, we demonstrated that much larger quantities of fetal DNA are present in amniotic fluid than in maternal serum (100-to 200-fold difference). 6

Embryonic stem cell-derived microvesicles reprogram hematopoietic progenitors: evidence for horizontal transfer of mRNA and protein delivery

“…A similar mechanism may also explain why vesicles derived from apoptotic cells are also highly enriched in mRNA. 27,28,37,38 In support of the concept of MV (e.g., ES-MV) playing a role in horizontal exchange of molecules between cells, it has been demonstrated that eucaryotic cells possess highly sophisticated membrane trafficking pathways that define specific membrane domains and provide a means for moving vesicles between them. Recently, it was demonstrated, for example, that cells cultured in vitro may exchange MV in so-called tunneling nanotubules.…”

The Amniotic Fluid Transcriptome as a Guide to Understanding Fetal Disease

“…3). The RNA was extracted, amplified, labeled and hybridized to either the Affymetrix U133A expression microarray (Larrabee et al 2005) or the Affymetrix GeneChip Human Genome U133 Plus 2.0 expression microarray (Slonim et al 2009;Koide et al 2011;Hui et al 2012aHui et al ,b, 2013aEdlow et al 2014;Massingham et al 2014). Despite the availability of more sophisticated expression microarrays, the U133 Plus 2.0 was chosen because of the need for compatibility with an essential downstream in silico tool, the Connectivity Map (Lamb et al 2006).…”

“…Cell-free fetal RNA in amniotic fluid supernatant provides an opportunity to study fetal gene expression in real-time. questions about the effects of sex, GA, and disease on the transcripts present in AFS (Larrabee et al 2005). AF was obtained from four pregnancies in which each woman underwent therapeutic amnioreduction for polyhydramnios caused by twin -twin transfusion syndrome (TTTS) or fetal hydrops.…”

“…A long-standing question within the field has been whether these morbidities arise from the disease itself, or are complications resulting from the laser ablation treatment. As discussed above, preliminary examination of fetal gene expression in the context of TTTS suggested differential expression of the water-transport gene AQP1 in the recipient twin (Larrabee et al 2005). In 2013, Hui et al revisited this disease, making a focused study of transcriptome-wide changes to fetal gene expression of the recipient twin in the second trimester, comparing gene expression between unaffected fetuses and fetuses with stage III TTTS, and comparing expression for fetuses with stages II and III TTTS.…”

“…It is a complex biofluid, containing both membrane-bound fetal cells (amniocytes) and myriad cell-free components, including placenta-derived microparticles, proteins, cffDNA and cffRNA from the fetus (Fig. 1) (Larrabee et al 2005). Amniocytes are suspended in the AF, and sometimes undergo programmed cell-death, releasing cell-free transcripts.…”