Quantifying circulating hypoxia-induced RNA transcripts in maternal blood to determine in uterofetal hypoxic status
© Whitehead et al.; licensee BioMed Central Ltd. 2013
Received: 8 July 2013
Accepted: 11 November 2013
Published: 9 December 2013
Hypoxia in utero can lead to stillbirth and severe perinatal injury. While current prenatal tests can identify fetuses that are hypoxic, none can determine the severity of hypoxia/acidemia. We hypothesized a hypoxic/acidemic fetus would up-regulate and release hypoxia-induced mRNA from the fetoplacental unit into the maternal circulation, where they can be sampled and quantified. Furthermore, we hypothesized the abundance of hypoxia induced mRNA in the maternal circulation would correlate with severity of fetal hypoxia/acidemia in utero. We therefore examined whether abundance of hypoxia-induced mRNA in the maternal circulation correlates with the degree of fetal hypoxia in utero.
We performed a prospective study of two cohorts: 1) longitudinal study of pregnant women undergoing an induction of labor (labor induces acute fetal hypoxia) and 2) pregnancies complicated by severe preterm growth restriction (chronic fetal hypoxia). For each cohort, we correlated hypoxia induced mRNA in the maternal blood with degree of fetal hypoxia during its final moments in utero, evidenced by umbilical artery pH or lactate levels obtained at birth. Gestational tissues and maternal bloods were sampled and mRNAs quantified by microarray and RT-PCR.
Hypoxia-induced mRNAs in maternal blood rose across labor, an event that induces acute fetal hypoxia. They exhibited a precipitous increase across the second stage of labor, a particularly hypoxic event. Importantly, a hypoxia gene score (sum of the relative expression of four hypoxia-induced genes) strongly correlated with fetal acidemia at birth. Hypoxia-induced mRNAs were also increased in the blood of women carrying severely growth restricted preterm fetuses, a condition of chronic fetal hypoxia. The hypoxia gene score correlated with the severity of ultrasound Doppler velocimetry abnormalities in fetal vessels. Importantly, the hypoxia gene score (derived from mRNA abundance in maternal blood) was significantly correlated with the degree of fetal acidemia at birth in this growth restriction cohort.
Abundance of mRNAs coding hypoxia-induced genes circulating in maternal blood strongly correlates with degree of fetal hypoxia/acidemia. Measuring hypoxia-induced mRNA in maternal blood may form the basis of a novel non-invasive test to clinically determine the degree of fetal hypoxia/acidemia while in utero.
KeywordsCirculating mRNA Pregnancy Fetal growth restriction Fetal hypoxia Biomarker Diagnostics
Significant fetal hypoxia causing injury or death can occur acutely, such as during labor , or it can occur chronically as a result of poor placental function (placental insufficiency). Chronic hypoxia arising from placental insufficiency can also cause severe fetal growth restriction (FGR).
When FGR occurs at significantly preterm gestations, the risks of stillbirth are high and clinicians must judge the optimal time to deliver the fetus. They are required to balance the probability of stillbirth, neonatal death or permanent disability (caused by severe fetal acidemia) if the pregnancy is left to continue versus the risks of iatrogenic prematurity if the preterm fetus is delivered unnecessarily early in gestation . To help make these decisions, tests of fetal well-being are used to determine the likelihood that the fetus is significantly hypoxic. These include the cardiotocograph (reports fetal heart rate patterns) , biophysical profile (reports the presence/absence of fetal movement, breathing, tone and amniotic fluid volume on ultrasound)  and Doppler waveform velocimetry analysis of fetal vessels . While access to these tests has improved perinatal outcomes, FGR fetuses are still lost to stillbirth or neonatal demise or they suffer significant perinatal injury . In a large study of 604 live-born cases of preterm FGR (<33 weeks gestation), major morbidity occurred in 35.9% of cases and mortality was 21.5% . As such, there is scope for significant improvements in clinical outcomes.
A potential limitation of existing tests is that they observe fetal physiological responses to hypoxia . As such, significant heterogeneity may be expected, where the threshold of hypoxia/acidemia required to provoke specific physiological responses captured by these tests will vary between fetuses. In addition, current tests only provide a likelihood that significant hypoxia is likely to be present. Importantly, none are validated to provide a quantitative estimate of the fetal blood pH/lactate concentrations (fetal acidemia). An approach measuring fetal hypoxia at a biochemical/molecular level may be a more direct strategy to determine the degree of fetal hypoxia/acidemia in utero.
The discovery that fetoplacentally derived mRNA is constantly released into the maternal blood from early pregnancy until delivery raises the possibility of a new way to monitor for the presence of fetal hypoxia/acidemia [9, 10]. We hypothesized a hypoxic fetus would up-regulate and release hypoxia-induced mRNA into maternal blood. Furthermore, the relative abundance of such transcripts may quantitatively correlate with the degree of fetal acidemia. Thus, measuring hypoxia-induced mRNA abundance in the maternal circulation might form the basis of a non-invasive test to estimate in utero fetal blood pH concentrations. We therefore investigated whether hypoxia-induced mRNA abundance in maternal blood correlated with the degree of fetal hypoxia/acidemia in utero.
Study participants and specimens
Participants were recruited from two tertiary hospitals in Melbourne (Monash Medical Centre and Mercy Hospital for Women). We obtained approval from The Mercy Hospital for Women Human Research Ethics Committee (MHW R10/02) and The Southern Health Research Ethics Committee B (MMC 09154B). All women provided written informed consent.
To examine acute hypoxia, a prospective study was undertaken in the birth suite. Maternal whole blood was collected from women undergoing induction of labor at term. An intravenous cannula was inserted at recruitment reserved for sample collection for the study. Serial blood samples were collected: prior to induction and commencement of uterine contractions; at commencement of the second stage of labor (full dilatation) and at delivery. Fetal umbilical artery blood samples and placental biopsies were collected immediately after delivery. Fetal hypoxic/acidemic status was determined by measuring umbilical artery blood lactate levels. Thirty women with documented fetal umbilical artery blood lactate levels at delivery and complete sampling were matched according to gestation, parity and maternal characteristics to identify a normoxic (fetal umbilical cord lactate <6 mmol/L) and hypoxic cohort (fetal umbilical lactate >6 mmol/L).
To examine chronic hypoxia, serial maternal whole blood samples were collected from 20 women carrying severely growth restricted preterm fetuses and 30 controls. Severe FGR was defined as a customized birthweight <10th centile (http://www.gestation.net, Australian parameters) requiring iatrogenic delivery prior to 34 weeks’ gestation with uteroplacental insufficiency (asymmetrical growth + abnormal umbilical artery Doppler velocimetry +/- oligo-hydramnios). Women with superimposed preeclampsia were included. Control blood samples (n = 30) were collected from women carrying an appropriately grown fetus (matched for gestation, parity and maternal characteristics) and subsequently delivered at term without complications.
Preterm placental samples (n = 8) were collected from women delivering preterm an appropriate grown fetus without hypertensive disease or histological evidence of chorioamnionitis. We only included those in the FGR cohort who delivered by caesarean to avoid the potential bias caused by acute hypoxia of labor. For the FGR cohort, fetal hypoxic status at delivery was determined by measuring fetal blood pH levels obtained from the umbilical artery at birth.
A total of 2.5 mls of either maternal peripheral whole blood and/or fetal umbilical cord blood samples were collected in PAXgene whole blood RNA tubes (PreAnalytix, Hombrechtikon, Switzerland). According to the manufacturer’s instructions, they were stored at room temperature for 24 hours, then at -80C until processing.
Placental biopsies were obtained immediately after delivery from the maternal side of the placenta avoiding the decidua and fetal membranes (placental samples from the FGR cohort were all after caesarean section). Biopsies were washed in sterile phosphate buffered saline, snap frozen and stored at -80°C until processing.
Total RNA was extracted using the Paxgene miRNA system (PreAnalytix/BD) according to the manufacturer’s instructions. Total RNA was extracted from placental tissue using the mirVana isolation kit (Ambion, Austin, TX, USA) according to the manufacturer’s instructions. Genomic DNA was removed using DNAse treatment. RNA concentration and purity was measured using a NanoDrop ND100 spectrophotometer (Thermo Scientific, Pittsburgh, PA, USA). Microarray sample quality were evaluated further by the BioAnalyser 2100 system (Agilent, Santa Clara, CA, USA).
RNA samples were hybridized to Illumina Human Ref-8 Expression Beadchips for the labor ward study and the Illumina HumanHT-12 Expression Beadchips (Illumina Inc., San Diego, CA, USA) for the FGR study. Beadchip processing was performed by the Australian Genome Research Facility (Melbourne, VIC, Australia) according to the manufacturer’s instructions. Scanned images were analyzed using Illumina GenomeStudio. Gene expression analysis was performed using BioConductor in R (http://www.r-project.org) after quality control, preprocessing, background subtraction and normalization was performed. Linear modeling was performed using the Limma package (http://www.bioinf.wehi.edu.au/limma/) and fold change calculated using the t-test adjusted for multiple comparisons using the Benjamini and Hochberg methodology for false discovery rate. Unsupervised hierarchical clustering and principal component analysis were performed to illustrate how well the patient groups could be separated on the basis of the different molecular signatures. GSEA software (http://www.broadinstitute.org/gsea) was used to investigate over-representation of biological pathways, comparing published biological pathways and the gene-set developed in this study.
Microarray data are available in the ArrayExpress database (http://www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-2054.
Validation with quantitative real-time reverse-transcription polymerase chain reaction analysis
Real-time reverse-transcription polymerase chain reaction (RT-PCR) validation of the 48 gene molecular signature was performed using the Taqman Hypoxia TLDA (Applied Biosystems, Carlsbad, CA, USA) and specific Taqman primers and probes for the candidate four gene signature (Applied Biosystems). Reverse transcription of 200 ng of total RNA was performed using Superscript Vilo or Superscript III (Invitrogen, Carlsbad, CA, USA). All RT-PCRs were performed in triplicate using multiple negative controls including RT and no-template controls. We chose the combined expression of Gapdh, B2m and Gusb as our internal control having validated that their expression was not altered by hypoxia in these samples. The comparative CT method was used to determine relative expression. Non-parametric statistical tests were used for comparison of gene expression and the Bonferroni correction for multiple comparisons was used where appropriate.
Expression of hypoxia-induced mRNA in fetal blood and placenta sampled from fetuses acutely hypoxic during labor
During labor, each uterine contraction abrogates maternal blood flow within the myometrium, decreasing placental oxygenation . Fetuses are rendered progressively hypoxic as labor advances . Therefore, labor is effectively an in vivo functional ‘model’ of acute human fetal hypoxia.
We first examined whether hypoxia-induced mRNA transcripts are increased in gestational tissues (fetal blood and placenta) in the presence of acute fetal hypoxia caused by labor. Blood lactate concentrations in the umbilical artery (from the placenta) at birth are measured by clinicians to retrospectively determine whether the fetus was genuinely hypoxic during its final moments in utero, where levels >6 mmol/L are considered elevated . Therefore, we grouped our cohort according to whether umbilical artery lactate concentrations were high (>6 mmol/L; hypoxia cohort) or not (<6 mmol/L; controls).
Expression of hypoxia-induced mRNA in maternal blood sampled at the moment of delivery in cases where the fetus was significantly hypoxic at birth
Next, we investigated whether hypoxia-induced transcripts were increased in maternal blood sampled at the moment of birth, comparing the hypoxic cohort (umbilical artery lactate concentrations >6 mmol/L) with controls. We performed a prospective labor ward study, recruiting women undergoing an induction of labor. We placed a second intravenous cannula reserved for collecting samples for the study (Additional file 1: Table S1 lists clinical details). The ‘moment of birth’ sample was taken when the head was on view at the perineum (vaginal opening) and delivery was imminent.
Expression of hypoxia-induced mRNA in maternal blood sampled longitudinally across labor
To exclude the possibility of a non-specific global rise in mRNA transcripts in maternal blood across labor, we assessed five transcripts coding growth-related genes by PCR. None of these significantly increased across labor (Additional file 1: Figure S1).
The second stage of labor (full cervical dilatation until birth) is shorter in duration than the first stage (from onset of contractions until full cervical dilatation), but particularly hypoxic for fetuses where rapid decreases in fetal arterial pO2, base excess and pH occur [1, 12–14]. We measured Hif1α, Hif2α, Adm and LdhA expression in maternal blood obtained from samples straddling the first stage and second stage of labor (mean (± SD) 486 (± 242) minutes between sample collection) and compared the relative increase to paired samples straddling the second stage of labor (mean (± SD) 44 (± 55) minutes between sample collection). There were only minimal increases in gene expression across the first stage of labor but far steeper increases across the second stage (Figure 3B). Thus, hypoxia-induced transcripts do not gradually increase across labor linearly with time (this might be expected to occur if these transcripts were released primarily in response to general inflammation that occurs during labor  rather than fetal hypoxia). Instead, they rose far more steeply during the much shorter period of the second stage of labor. We suggest the likely explanation is that fetuses are more hypoxic during the second stage [1, 12–14].
Correlation between hypoxia-induced mRNA in maternal blood sampled at the moment of birth with fetal hypoxic status at birth
Expression of hypoxia-induced mRNA in maternal blood sampled from women with severe preterm growth restricted fetuses
Patient characteristics for fetal growth restriction and control cohorts
FGR (n = 20)
Control (n = 30)
Maternal age (yrs)a
Parity (% primiparous)
Gestational age at delivery (weeks)a
29 + 5 (21)
40 + 1 (10)
Gestational age at sampling (weeks)a
29 + 5 (21)
29 + 0 (18)
Mode of birth (%)
Five-minute Apgar scorea
Neonatal admission (%)
Perinatal death (%)
Gestational hypertension/pre-eclampsia (%)
Pre-gestational diabetes (%)
Umbilical artery Doppler USS waveforms (%)
Expression of hypoxia induced mRNA in maternal blood in the FGR cohort, split according to whether preeclampsia was also present
To examine the possibility that co-existent preeclampsia may affect expression of hypoxia-induced mRNA in maternal blood (and thus, be a confounder), we split our FGR cohort according to whether there was concurrent preeclampsia (n = 8) or not (n = 12). mRNA expression of Hif1α, Hif2α, Adm and LdhA were all significantly elevated in both FGR cohorts (that is, FGR with concurrent preeclampsia, and FGR without preeclampsia; see Additional file 1: Figure S3) compared to gestationally-matched controls (healthy pregnancies that progressed to delivery at term of an infant with a normal birth weight). Importantly, mRNA expression levels of all four genes were no different between the FGR cohort with concurrent preeclampsia and FGR without preeclampsia. These data suggest mRNA coding hypoxia induced genes are increased in the presence of severe FGR, irrespective of the presence of preeclampsia.
Correlation between abundance of hypoxia-induced RNA in maternal blood with severity of Doppler velocimetry abnormalities
Correlation between dynamic changes in hypoxia-induced mRNA in maternal circulation and acute changes in umbilical artery Doppler waveforms
Corticosteroids are often administered via intramuscular injection to the mother to accelerate fetal lung maturation, in preparation for preterm birth [18, 19]. They can induce acute, but transient improvements of the umbilical artery Doppler waveforms over 24 to 72 hours (for example, from AEDF to raised SDR) , while in some cases, waveforms worsen . Paired maternal blood samples were taken just prior to, and 24 hours after, corticosteroid administration. Decreased expression of Hif1α (Figure 6B) was observed when the Doppler waveforms improved and conversely, increased expression of Hif1α (Figure 6C) was observed when waveforms deteriorated after corticosteroid administration. This suggests circulating hypoxia-induced transcripts in maternal blood promptly change in parallel with very acute alterations in presumed fetal hypoxic status.
Correlation between hypoxia-induced mRNA in maternal blood sampled on the day of delivery with acidemic status of FGR infants at the moment of birth
Significant fetal acidemia at birth is strongly associated with perinatal death and adverse perinatal complications, including permanent neurological disability  and cerebral palsy . In a study of 60 preterm fetuses delivered at ≤28 weeks gestation, an umbilical artery blood pH of ≤7.15 was strongly associated with severe adverse neurological outcomes (sensitivity 30% at 98% specificity) compared with higher pH levels . In another study of 604 neonates delivered at ≤33 weeks gestation, an umbilical cord pH of ≤7.20 was associated with a 4.2 likelihood ratio of fetal death . Therefore, a non-invasive test that can estimate fetal acidemic status could help clinicians’ better time delivery. While current non-invasive antenatal tests can identify fetuses at higher risk of being acidemic, none have been validated to accurately estimate the degree of fetal acidemia in utero.
Here we have presented evidence to suggest quantifying hypoxia-induced mRNA in the maternal circulation may be a novel approach to determining in-utero fetal hypoxic status. Hypoxia-induced transcripts in the maternal circulation appear tightly correlated with expression in human gestational tissues, and they dynamically change with acute alterations in presumed fetal hypoxic status. Furthermore, we generated a hypoxia gene expression score that sums the relative abundance of mRNA in the maternal circulation that code four hypoxia-induced genes. This score appeared to be highly correlated with acute (labor cohort) and chronic (FGR cohort) fetal hypoxia.
While the measurement of free mRNA in the maternal circulation has been studied previously, we believe our study represents a significant conceptual advance. Previous studies have proposed the use of free mRNA as a ‘static’ tool, where levels are measured once in order to either diagnose [23, 24] or predict pregnancy complications [25–27]. Here we propose serial measurements to observe dynamic changes within the same patient, monitoring hypoxic status over time and delivering when significant acidemia is predicted.
The cardiotocograph is the mainstay of monitoring to identify hypoxia during labor. While it performs well in identifying the presence of fetal hypoxia (85% sensitivity) , its specificity is notoriously poor because heart rate decelerations, including late decelerations, can either be caused by hypoxia or be induced by mechanical reflex autonomic responses unrelated to hypoxia. As a result, use of the cardiotocograph results in unnecessary interventions . Ours may be the first ‘theoretical’ non-invasive test for women in labor that can determine the degree of in utero fetal acidemia. The speed of current PCR technologies means such a test is not feasible as a clinical tool to make decisions during labor but improvements in nucleic acid detection technologies might make such a test possible in the future.
We have also presented evidence suggesting hypoxia-induced mRNA in the maternal circulation correlates with acidemic status of FGR fetuses’ in utero. It is conceivable that day-to-day clinical decisions regarding timing of an FGR fetus can await the results of a PCR result performed using machines available today. Therefore, our test may have a role in situations where current tests of fetal well-being are equivocal and the clinician is left unsure whether the fetus should be delivered. This occurs quite frequently. A prospective study examining a preterm FGR cohort found biophysical profile results were discordant with the umbilical artery Doppler findings in 55% of cases . Thus, a test that can provide a reliable estimate of in utero fetal blood pH levels in such situations may help clinicians decide whether immediate delivery is warranted.
A limitation of our study is that we have not decisively proven the hypoxia-induced mRNA we are measuring in the maternal blood originates from the fetoplacental unit. This may be possible with the use of next-generation sequencing technologies where sequence information could be used to identify the origin of mRNA transcripts (maternal or fetal). However, we have presented strong circumstantial evidence to suggest the hypoxia induced mRNA are of fetoplacental origin: 1) they increase with situations of likely severe acute and chronic fetal hypoxia, 2) they correlate with an increase of hypoxic mRNA transcripts in gestational tissues, and 3) their relative abundance displays a highly significant and tight correlation with fetal acidemic status at birth. Ultimately, if hypoxia-induced transcripts in maternal blood were validated to reflect fetal acidemic status, it would not be absolutely essential to establish their origin, although a fetoplacental source seems the most likely.
To translate our potential test to the monitoring of fetuses with severe FGR, our test requires validation with a study of larger numbers. Such a validation study could also help determine whether clinical factors, such as smoking and maternal obesity, alter hypoxia induced mRNA levels in maternal blood. We are currently undertaking such a large prospective validation study.
Furthermore, in this proof of concept study, we summed the relative expression of mRNA in maternal blood that codes Hif1α, Hif2α, LdhA and Adm to generate a gene hypoxia score. These genes were chosen on the basis of their biology; the former three have central roles in the hypoxic response , and Adm is both hypoxic regulated  and very highly expressed in placenta. Future studies should bioinformatically screen other hypoxia-induced genes to develop the most accurate test to determine degree of in utero fetal acidemia. Finally, it may be more optimal to develop a clinical test that expresses mRNA abundance by copy number rather than relative expression.
In conclusion, we have presented evidence to show measuring circulating hypoxia-induced transcripts in maternal blood may be a promising approach to clinically assess fetal hypoxic status in utero. It may be useful to help clinicians’ time delivery, especially in cases of severe preterm FGR, potentially improving perinatal outcomes and decreasing rates of stillbirth.
Abundance of mRNAs coding hypoxia-induced genes circulating in maternal blood strongly correlates with the degree of fetal hypoxia/acidemia, and they dynamically change with acute alterations in presumed fetal hypoxic status. Furthermore, a hypoxia gene expression score that sums the relative abundance of mRNAs in the maternal circulation was highly correlated with acute (labor ward cohort) and chronic (FGR cohort) fetal hypoxia/acidemia. Therefore, measuring hypoxia-induced mRNA in maternal blood may form the basis of a novel non-invasive test to clinically determine the degree of fetal hypoxia/acidemia while in utero.
Absent end diastolic flow
Fetal growth restriction
Hypoxia inducible factor 1α
Hypoxia inducible factor 2α
Lactate dehydrogenase A
Message Ribonucleic acid
Polymerase chain reaction
Reversed end diastolic flow
Raised systolic-diastolic ratio.
We thank study participants for donating tissues and clinical midwives for helping with sample collection. This research was supported by The National Health and Medical Research Council (#1028521 #1050765), The Viertel Charitable Foundation, Royal Australian and New Zealand College of Obstetricians and Gynecologists (Arthur Wilson Fellowship, Luke Proposch Perinatal Scholarship) and ANZ Trustees.
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