Researchers at Stanford University and Chan Zuckerberg Biohub have identified sets of cell-free RNA (cfRNA) biomarkers in the blood of pregnant women that can be used to estimate gestational age and delivery date with the same accuracy as ultrasound and can help to identify mothers who are likely to deliver preterm, up to two months ahead of labor.
David K. Stevenson, M.D., principal investigator at the March of Dimes Prematurity Research Center at Stanford University, describes the PCR-based tests, which can be carried out on a single blood sample, as effectively “eavesdropping on a conversation” among the mother, the fetus, and the placenta. “By measuring cfRNA in the circulation of the mother, we can observe changing patterns of gene activity that happen normally during pregnancy and identify disruptions in the patterns that may signal to doctors that unhealthy circumstances like preterm labor and birth are likely to occur,” he says. “With further study, we might be able to identify specific genes and gene pathways that could reveal some of the underlying causes of preterm birth, and suggest potential targets for interventions to prevent it.”
Stevenson is co-author of the researchers’ published paper in Science, which is entitled “Noninvasive Blood Tests for Fetal Development Predict Gestational Age and Preterm Delivery.”
Approximations of gestational age are typically based on ultrasound imaging and/or the patient’s estimate of her last menstrual period, but neither method is necessarily ideal. Ultrasound is expensive and not always feasible in low-resource settings, while maternal estimates may not be reliable. Current methods also don’t account for the potential for premature birth, which affects 15 million infants worldwide every year, and is “the leading cause of neonatal death and complications later in life,” the authors write. Provisional data for 2017 from the National Center for Health Statistics show that the preterm birth rate in the U.S. has reached 9.93%, up from 9.86% in 2016, the third consecutive annual increase after steady declines over the previous seven years. “To date, no test on the market can reliably predict which pregnant moms will go on to preterm labor,” comments Stacey D. Stewart, president of March of Dimes.
Two thirds of preterm births are spontaneous, but identifying at-risk pregnancies has proven problematic, and “efforts to identify genetic causes and risk factors have had limited success…,” the researchers suggest. Ultrasound measurements and measures of fetal fibronectin (fFN) have similarly had low positive predictive value.
Work by the Stanford-led team had previously shown that the progress of pregnancy could be followed by measuring cfRNA from fetal tissues in maternal blood. The latest research evaluated whether the same method could be developed into blood tests for estimating both gestational age and the risk of preterm birth.
For their initial study, the researchers collected weekly blood samples from 31 healthy pregnant women, all of whom delivered at full term. Each blood sample was analyzed to identify expression levels of genes that were specific to the placenta and to the immune system, or were enriched in fetal liver. The results showed that cfRNA measurements during the course of a pregnancy showed differing time courses, dependent on their tissue of origin. The findings indicated that cfRNA corresponding to a set of placental genes might provide an accurate estimate of fetal development and gestational age throughout pregnancy. Further development and validation of an initial model resulted in the identification of a panel nine placenta-specific RNAs (CGA, CAPN6, CGB, ALPP, CSHL1, PLAC4, PSG7, PAPPA, and LGALS14) in maternal blood that could predict gestational age. “The model’s two most important features, CGA and CGB, encoding chorionic gonadotropin α and β3 subunits of human chorionic gonadotropin (HCG), are known contributors to pregnancy initiation,” the authors point out.
Encouragingly, the cfRNA blood test could predict the gestational age (where the birth occurred within 14 days of the estimated due date) with 45% accuracy, which is in the same range as estimates of gestational age and due date based on ultrasound, at 48%. “Our results are thus generally comparable to ultrasound measurements, can be performed throughout pregnancy, and do not require a priori physiological knowledge such as the woman’s last menstrual period,” the team states.
They next studied two separate cohorts of pregnant women who were known to be at risk of premature birth either because of previous preterm deliveries or because they had experienced premature contractions. Using RNA sequencing technology, the team was able to identify a set of seven cfNRAS (CLCN3, DAPP1, PPBP, MAP3K7CL, MOB1B, RAB27B, and RGS18) that differentiated between the preterm and full-term samples. In validation tests in an independent cohort, they found that “the test accurately classified four of five preterm samples (80%) and misclassified three of 18 full-term samples (17%).
The researchers report that their blood test demonstrated higher mean accuracy than methods based on mass spectroscopic measurements of the ratio of two proteins in blood [sex hormone binding globulin (SHBG) and insulin-like growth factor binding protein 4 (IBP4)], and also exhibited higher predictive value than ultrosonographic measurements of cervical length, or fetal fFN evaluation.
They acknowledge that their studies will need verifiying in much larger, ethnically diverse cohorts, including women who aren’t already known to be at risk of preterm birth. Nevertheless, they suggest that the pilot studies have demonstrated that noninvasive blood tests can be used to predict gestational age and identify women at risk of preterm delivery, using the same blood sample.
“These cfRNA PCR-based tests have two advantages over alternatives: broader applicability and lower cost,” they conclude. “They can be applied across the globe as a complement to or substitute for ultrasound, which can be expensive and inaccurate during the second and third trimester.…These tests hold promise for prenatal care in both the developed and developing worlds.…Conceivably, similar approaches will prove to be useful for identifying and monitoring fetuses with congenital defects that can be treated in utero—a rapidly growing area of fetal medicine.”