Background
Significant fetal hypoxia causing injury or death can occur acutely, such as during labor [
1], 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 [
2]. 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) [
3], biophysical profile (reports the presence/absence of fetal movement, breathing, tone and amniotic fluid volume on ultrasound) [
4] and Doppler waveform velocimetry analysis of fetal vessels [
5]. 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 [
6]. 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% [
7]. 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 [
8]. 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.
Methods
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 <10
th 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.
Sample collection
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.
RNA preparation
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).
Microarray analyses
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.
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.
Discussion
Significant fetal acidemia at birth is strongly associated with perinatal death and adverse perinatal complications, including permanent neurological disability [
6] and cerebral palsy [
22]. 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 [
6]. 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 [
7]. 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) [
1], 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 [
28]. 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 [
29]. 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 [
30], and
Adm is both hypoxic regulated [
31] 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.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
ST conceived the study. ST, CW and SW designed the study. CW, WT, CL, SM and LL collected samples. CW performed the laboratory work. CW analyzed the data, including the microarray bioinformatic analysis. ST, CW and SW wrote the first draft of the paper and all authors provided input and approved the final manuscript.