Objective
To examine whether mRNA circulating in maternal blood coding genes regulating fetal growth are differentially expressed in (1) severe preterm fetal growth restriction (FGR) and (2) at 28 weeks’ gestation in pregnancies destined to develop FGR at term.
Study Design
mRNA coding growth genes were measured in 2 independent cohorts. The first was women diagnosed with severe preterm FGR (<34 weeks’ gestation; n = 20) and gestation matched controls (n = 15), where the mRNA was measured in both maternal blood and placenta. The second cohort was a prospective longitudinal study (n = 52) of women whom had serial ultrasound assessments of fetal growth. mRNA coding growth genes in maternal blood were measured at 28 and 36 weeks in pregnancies with declining growth trajectories (ending up with term FGR; n = 10 among the 52 recruited) and controls who maintained normal growth trajectory (n = 15).
Results
In women with severe preterm FGR, there was increased expression of placental growth hormone (6.3-fold), insulin-like growth factors (IGF1, 3.4-fold; IGF2, 5.0-fold), IGF receptors (2.1-fold) and IGF binding proteins (3.0-fold), and reduced expression of ADAM12 (0.5-fold) in maternal blood (and similar trends in placenta) compared with controls ( P < .05). Notably, at 28 weeks’ gestation there was increased IGF2 (3.9-fold), placental growth hormone (2.7-fold), and IGF BP2 (2.1-fold) expression in maternal blood in women destined to develop FGR at term ( P < .05).
Conclusion
Measuring mRNA coding growth genes in maternal blood may detect unsuspected severe preterm FGR already present in utero, and predict term FGR when measured at 28 weeks’ gestation.
Fetal growth restriction (FGR) is a leading cause of stillbirth and strongly associated with increased perinatal morbidity. There are no effective intrauterine treatments, and therefore the management relies on early detection and timely delivery.
Unfortunately, clinical detection of FGR is suboptimal and many cases remain undiagnosed. It is thought that unrecognized FGR represents an important cause of stillbirth and therefore a reliable screening test for FGR could significantly improve clinical outcomes. There are 2 situations where a noninvasive biomarker test for FGR may be useful: (1) to identify cases of unsuspected preterm FGR that are already present in utero, or (2) predict cases of normally grown fetuses at risk of developing FGR.
The discovery that nucleic acids circulate in the maternal blood where they can be quantified offers a novel avenue to identify biomarkers of placental function. RNA can be extracted reliably and subjected to molecular analyses with precision. In contrast, the detection of proteins relies on the existence of high quality monoclonal antibodies. Importantly, it is possible circulating mRNA in the maternal circulation may be derived from the placenta and reflected trends in differential transcript expression in the distant placental transcriptome. If true, then this approach could also provide unique insights into placental pathology previously not attainable while the fetus remains in utero.
The insulin-like growth factor (IGF) system is essential for fetal and placental growth and development. It includes IGF1 and IGF2, which can bind to 1 of 6 IGF binding proteins (IGFBPs) to modulate their bioavailability. The IGFs exert their metabolic actions by interacting with cell surface tyrosine kinase receptors (IGF1R, IGF2R), which selectively bind the IGFs and insulin. Notably, these IGF ligands, receptors, and binding proteins are expressed in the placenta, and the IGFs are released into the maternal circulation from early pregnancy. Placental growth hormone (PGH) and ADAM12 are recently discovered placental-derived growth factors that also modulate their effects via the IGF system. There is mounting evidence that abnormalities in the IGF system play a crucial role in the pathogenesis of FGR. However, the literature has been conflicting as to whether FGR is associated with changes in circulating IGF proteins in maternal blood.
Therefore in this study, we investigated whether mRNA of IGF1, IGF2, IGFBP-2, IGF1R, PGH, and ADAM12 were dysregulated in the maternal blood in pregnancies affected by severe preterm FGR, and whether the circulating mRNA might reflect placental gene expression. We also studied a second cohort where we prospectively collected serial maternal samples from 28 weeks’ gestation in a low-risk population and followed fetal growth until delivery. We identified cases with well-grown fetuses at recruitment (as determined by ultrasound) but who exhibited a decline in growth trajectory, developing FGR at term. We measured mRNA coding these 6 growth factors in maternal blood from this cohort at 28 and 36 weeks’ gestation to determine their potential to predict term FGR.
Materials and Methods
Clinical cohorts and recruitment of participants
Subjects were recruited from Mercy Hospital for Women and Monash Medical Centre, Melbourne between 2008-2011. Written informed consent was obtained and the study protocol was approved by both institutions’ Human Research and Ethics Committees.
The 2 cohorts include a case-control study investigating severe preterm FGR, and a prospective longitudinal study investigating term FGR.
In the first cohort, cases of severe preterm FGR were defined as FGR (customized birthweight 10th percentile) requiring iatrogenic delivery <34 weeks’, with evidence of uteroplacental insufficiency (asymmetrical growth + abnormal umbilical artery Doppler velocimetry, ± oligohydramnios or abnormal fetal vessel velocimetry). FGR because of infection, chromosomal, or congenital abnormalities, and multiple pregnancies were excluded. Both maternal blood and placenta was collected (n = 20) and those with or without preeclampsia (American Congress of Obstetricians and Gynecologists guidelines ) were included.
Preterm control blood samples were collected from women (n = 20) with an appropriately grown fetus, at a gestation matched to the preterm FGR cases, and who subsequently delivered appropriately grown fetuses at term without obstetric complications (birthweight 20-80th percentile). Preterm placental samples (n = 8) were collected from women delivering preterm (<34 weeks) an appropriate grown fetus, in the absence of hypertensive diseases of pregnancy or chorioamnionitis. Term placental samples (n = 8) were collected from uncomplicated, appropriately grown (birthweight 20-80th percentile) singleton pregnancies at >37 weeks’. All placental samples were collected at prelabor cesarean section.
In the second cohort, low-risk healthy pregnant women were recruited at 28 weeks’ gestation. At the time of recruitment, none of the participants had obstetric, medical, or surgical complications. Ultrasound assessment of fetal growth was performed at 28 weeks’ gestation, and only those with a well-grown fetus were included. Maternal blood samples were collected at 28 and 36 weeks’ gestation. Ultrasound assessment of fetal growth was repeated at 36 weeks’ gestation and final birthweight recorded. After birth the subjects were stratified into 3 cohorts.
- (1)
Appropriate for gestational age fetuses (AGA, n = 15), defined as fetuses with an estimated fetal weight 20-95th percentile at 28 weeks who maintained their growth trajectory to term (change in centiles between the 28 weeks and birth <30%).
- (2)
Term FGR (n = 10), defined as fetuses appropriately grown at 28 weeks (20-95th percentile) who subsequently exhibited a fall in growth trajectory resulting in term FGR (serial fall in estimated weight percentiles from 28 weeks’ to 36 weeks’ gestation, with birthweight <10th percentile).
- (3)
The remainder of the pregnancies (n = 27), had variable growth trajectories and could not be clearly classified into either of the above groups. Samples from this cohort were not analyzed.
Estimated fetal weight was calculated using the Hadlock equation. The customized percentiles for estimated fetal weight and birthweight were generated using the Australian dataset of the GROW software ( www.gestation.net ), which takes into consideration maternal height, weight, ethnicity, parity, and fetal sex. All ultrasounds were performed by 1 operator, trained, and experienced in fetal biometry.
Sample collection and laboratory analysis: maternal peripheral whole blood samples (2.5 mL) were collected in PAXgene blood RNA tubes (PreAnalytix, Hombrechtikon, Switzerland) and processed as per manufacturer’s instructions and stored at −80°C until processing.
Placental biopsies were obtained immediately after delivery by cesarean section. Placental biopsies were taken from the maternal side of the placenta, to a depth of 2/3 of the placenta, avoiding the decidua. The biopsies were clear of obvious infarction or calcification. Placental biopsies were washed in sterile PBS to remove contamination by maternal blood, snap frozen, and stored at −80°C until processing.
RNA was extracted from peripheral whole maternal blood using the PAXgene blood miRNA kit (PreAnalytix) according to manufacturer’s instructions, as previously described. Placental RNA was extracted using the mirVana Isolation Kit (Ambion, Austin, TX) according to manufacturer’s instructions. Genomic DNA was removed using DNAse treatment, and total RNA eluted and stored at −80°C if not used immediately. RNA concentration and purity were measured using a NanoDrop ND1000 spectrophotometer (Thermo Scientific, Pittsburgh, PA).
Real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis was performed after reverse transcription of 200 ng RNA using Superscript Vilo (Invitrogen, Carlsbad, CA) according to manufacturers’ instructions. Quantitative gene expression analyses were performed with commercially available Taqman Gene Expression Assays (Applied Biosystems, Carlsbad, CA) for insulin-like growth factor (IGF)1, IGF2, IGF1R, IGFBP-2, PGH, and ADAM12. The RT-PCR was performed in triplicate, with multiple negative controls (including no template and no RT controls), on the CFX 384 (BioRad, Foster City, CA), with the following cycling conditions: 50°C for 2 minutes, 95°C for 10 minutes, and 40 cycles of 95°C for 15 seconds, 60°C for 1 minute, and 72°C for 30 seconds.
Relative quantification was determined by the comparative CT method. Gene expression was normalized against the mean expression of glyceraldehyde-3-phosphate dehydrogenase, GUSB, and B2M to increase the stringency of the comparison, and calibrated against the mean expression level of the preterm control group.
Statistical analysis
All data was statistically analyzed using Graphpad Prism v 5 (GraphPad Software Inc, San Diego, CA). Differences in RT-PCR gene expression was assessed using Mann-Whitney U or Kruskal-Wallis test, or where the data was considered normally distributed the t test or analysis of variance. Patient characteristics were compared using χ 2 where appropriate. Data was presented as mean ± SEM. Significance was defined as P < .05.
Results
mRNA coding growth genes are differentially expressed in placenta from cases of severe preterm FGR
We first examined the expression of IGF1, IGF2, IGF1R, IGFBP2, PGH, and ADAM12 in the placenta in severe preterm FGR (n = 20), and compared them with preterm (n = 8) and term (n = 8) controls. The clinical characteristics are described in Table 1 .
| Characteristic | FGR blood and placenta (n = 20) | Preterm blood (n = 20) | Preterm placenta (n = 8) | Term placenta (n = 8) |
|---|---|---|---|---|
| Maternal age, y | 30.4 (5.9) | 31.1 (1.5) | 31.8 (1.7) | 31.9 (1.1) |
| Parity (% primiparous) | 65 | 60 | 12.5 | 25 |
| Gestational age at delivery, wk | 29+5 (3) | 40+2 (1.4) | 29+4 (1) | 38+3 (0.6) |
| Gestational age at sampling, wk | 29+5 (3) | 30+1 (2.1) | 29+4 (1) | 38 +3 (0.6) |
| Birthweight, g | 885 (273) | 3565 (35) | 1492 (203) | 3400 (122) |
| Customized birth percentile, n (%) | 3 (3) | 48 (6) | 66 (7.5) | 69 (4) |
| Perinatal death, % | 13 | 0 | 0 | 0 |
| Preeclampsia, % | 40 | 0 | 0 | 0 |
| Umbilical artery Doppler velocimetry waveforms, % | ||||
| REDF | 30 | 0 | 0 | 0 |
| AEDF | 40 | 0 | 0 | 0 |
| ↑PI | 30 | 0 | 0 | 0 |
| Normal | 0 | 100 | 100 | 100 |
In preterm FGR, there was a substantial increase in placental mRNA expression of the following compared with preterm controls ( Figure 1 , A): IGF1 (2.3-fold, P < .05), IGF2 (3.0-fold, P < .01), IGF1R (2.6-fold, P < .01), IGFBP2 (3.0-fold, P < .05), and PGH (5.6-fold, P < .05). In contrast, ADAM12 was significantly down-regulated in preterm FGR ( P < .001). Interestingly, only IGFBP2 significantly increased across gestation in the controls, with a 3-fold increase in term controls compared with preterm controls ( P < .05).
mRNA coding growth genes in maternal blood are differentially expressed in cases of severe preterm FGR, and mirror trends seen in the placenta
To investigate whether circulating mRNA in the maternal circulation might reflect changes in the placental transcriptome, the expression of these 6 genes were measured in maternal blood ( Figure 1 , B). The expression of all 6 genes in maternal blood with severe preterm FGR closely correlated with the trends in the placental transcriptome (compared with Figure 1 , A). Interestingly, the degree of fold change was generally higher in maternal blood compared with placenta. Thus, in preterm FGR there was a 3.4-fold increase in IGF1 ( P < .001), 5.0-fold increase in IGF2 ( P <.01), 2.1-fold increase in IGF1R ( P < .05), 6.3-fold increase in PGH ( P < .01), and 3.0-fold increase in IGFBP2. Like placenta, ADAM12 expression was down-regulated in the maternal blood in preterm FGR compared with controls ( P < .05).
Expression mRNA coding growth genes in maternal blood correlates with the severity of disease, in severe preterm FGR
The severity of preterm FGR is characterized by increased placental resistance, which can be assessed by Doppler ultrasound of placental and fetal vessels. The mildest abnormality is reflected by an increase in the umbilical artery resistance (pulsatility index), whereas absent and reversal of end diastolic flow in the umbilical artery (AREDF) is associated around 30-70% destruction of the placental villous tree, and strongly correlated with fetal hypoxia/academia. We therefore examined expression of mRNA coding growth genes in maternal circulation was further dysregulated with increasing severity of FGR.
Figure 2 demonstrates increased expression in both the placenta ( Figure 2 , A) and maternal blood ( Figure 2 , B) for IGF1, IGF2, and PGH with AREDF compared with increased resistance but positive end diastolic flow (increased pulsatility index; P < .05). Again, the fold changes in the maternal blood were far greater than that seen in placenta. There was a 2-fold increase in IGF1 in the placenta compared with a 4.8-fold increase in the maternal blood with AREDF. However, there was no difference in expression of IGFBP2, IGF1R, or ADAM2 in either the placenta or maternal blood in FGR, stratified according to the severity of umbilical artery Doppler resistance.
Stay updated, free articles. Join our Telegram channel
Full access? Get Clinical Tree
