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Transcriptomic Analysis of Amniotic Fluid Uncovers Gene Expression Changes Linked to Fetal Maturity

NEW YORK (GenomeWeb) – By comparing amniotic fluid transcriptome profiles, Cincinnati Children's Hospital Medical Center researchers have uncovered genes whose expression could reflect fetal maturity and potentially guide preterm delivery decisions.

First author Beena Kamath-Rayne from Cincinnati Children's and her colleagues isolated and sequenced RNA from the amniotic fluids of 16 women at different time points in their pregnancies. As they reported today in BMC Medical Genomics, a number of transcripts were uniquely expressed at different stages of pregnancy and were associated with certain neonatal co-morbidities.

"This study demonstrates the feasibility of testing amniotic fluid to identify biomarkers for fetal organ maturation to better enable obstetricians to make delivery planning decisions for preterm births," Kamath­Rayne said in a statement. "This will allow pediatricians and neonatologists to prepare for the various neonatal morbidities these preterm infants may face, and allow obstetricians to better weigh risks to the baby when making decisions about preterm delivery."

Kamath­Rayne and her colleagues obtained four second trimester amniotic fluid samples (18 weeks to 24 weeks), six late preterm samples (34 weeks to 36 weeks), and six full-term samples (39 weeks to 40 weeks) for transcriptome analysis. RNA sequencing yielded between 25 million and 54 million reads per sample that the researchers then aligned to the human reference genome and transcriptome.

Hierarchical clustering found that the expressed genes grouped by gestational age and that some genes were induced over time as others were suppressed, the researchers reported.

The genes that were induced over time were typically involved in signaling transduction pathways, lipid and surfactant homeostasis, mediated cell immune response, and response to growth factors. Mutations or deletions in these genes, the researchers said, are known to affect lipid homeostasis, surfactant physiology, and adipose and liver morphology.

In contrast, those genes that were suppressed over time were enriched for cell cycle, protein targeting to ER, cell proliferation, embryogenesis and development functions — and mutations or deletions in these genes have been associated with embryonic lethality in mice, abnormal embryogenesis or development, abnormal prenatal growth or body size, and embryonic growth arrest.

Kamath­Rayne and her colleagues further reported that genes associated with surfactant physiology and VEGF signaling — like SFTPA1, SFTPD, VEGFA, and AKT1, among others — were significantly increased in the full-term amniotic fluid samples, noting that genes that regulate pulmonary function are critical for lung function at birth.

By contrast, the expression of genes involved in the Wnt and Hippo signaling pathways — such as FZD1, WNT2, and CCND2 — decreased with gestational age. These two pathways, the researchers said, are important for morphogenesis, tissue growth, and organ size.

Overall, some 257 genes were differentially expressed in late preterm versus full-term fetuses, the researchers reported.

Of these, 111 genes were more highly expressed in term fetuses. Those genes were enriched for biological processes like immune response, protein transport, apoptosis, and response to stress, growth factors, and lipids, according to a Gene Ontology analysis.

Meanwhile, 146 genes whose expression was higher in late pre-term fetuses were enriched for biological processes like cilium morphogenesis, mRNA processing, cell cycle, and protein catabolism. Mutations or deletions in these genes have been associated with small lung, decreased lean body mass, and prenatal growth retardation, the researchers noted.

Kamath­Rayne and her colleagues also mapped these differentially expressed RNAs to the GNF Gene Expression Atlas to gauge their expression across 66 different tissues. They were able to trace RNAs from their samples to the respiratory tract, skin, and other major organ systems. Using the GO­Elite pathway analysis software package, they conducted a cell and tissue marker enrichment analysis to find that RNAs associated with neutrophils, lung, tongue, salivary gland, oral mucosa, adipocytes, oligodendrocyte progenitors, and CD14+ cells, among others, are not as highly expressed in late preterm fetuses as they are in term fetuses.

Preterm birth is associated with a number of co-morbidities like the need for respiratory support, gavage feeding, and thermoregulation difficulties, the researchers noted.

In this sample set, they found that preterm infants requiring gavage feeding expressed fewer salivary gland, oral mucosa, pharyngeal mucosa, and tongue markers than term infants did. Meanwhile, they reported that infants that needed respiratory support expressed a similar percent of markers for salivary gland, oral mucosa, pharyngeal mucosa, and tongue, though they did exhibit a decrease in markers for adipose tissue and fetal lungs.

This, Kamath­Rayne and her colleagues said, suggests that amniotic fluid markers could serve as indicators of organ system maturity.

After additional studies, such with a larger cohort, they said these markers could be turned into a less invasive tool that relies on, for instance, maternal blood or urine samples rather than amniotic fluid, to assess fetal maturity.