1. Introduction
Fetal growth restriction and preeclampsia are serious disorders of pregnancy. Fetal growth restriction is characterized by an estimated fetal weight less than the tenth centile for gestational age, and is associated with an increased risk of stillbirth [
1]. Preeclampsia is a complex condition characterized by new onset hypertension after 20 weeks’ gestation, accompanied by one or more of proteinuria, uteroplacental insufficiency, or maternal organ injury, and can lead to both maternal and perinatal morbidity or mortality [
2]. Unfortunately, these conditions do not have treatments that can reverse disease pathogenesis. Hence, there is an urgent need for novel therapeutic strategies to treat these conditions.
Although preeclampsia and fetal growth restriction are two distinct disorders, they both feature a dysfunctional placenta. The placenta plays a crucial role in pregnancy, acting as the interface between the maternal and fetal systems, facilitating gas exchange, and the transfer of nutrients and waste. Abnormal spiral artery remodeling is a common feature of placental dysfunction, resulting in impaired placental blood supply [
3], which can affect fetal growth. By improving our understanding of placental development and the specific mechanisms behind placental dysfunction, we can identify potential therapeutic targets for prevention and treatment.
Previously, we identified that transcripts for the gene Oleoyl-ACP Hydrolase (
OLAH; also known as S-Acyl fatty acid synthase thioesterase or thioesterase II) were significantly elevated in the maternal circulation in pregnancies complicated by fetal growth restriction (with and without preeclampsia) [
4]. Placenta-derived circulating RNAs are postulated to provide insight into the state of the placenta [
5,
6].
OLAH is an enzyme known for its role in fatty acid synthesis, where it acts alongside fatty acid synthase to preferentially produce shorter, medium chain fatty acids [
7]. OLAH has been identified in mammary gland epithelial cells, particularly in the synthesis of the broad distribution of fatty acids in breast milk [
7,
8], and is elevated in bone marrow-derived mononuclear cells with rheumatoid arthritis [
9]. Intriguingly,
OLAH expression is described to be higher in human placental tissue compared to other species [
10]. This suggests that OLAH may have a unique function in the human placenta and could potentially be involved in the pathogenesis of preeclampsia, a human-specific condition. Although fatty acids play an important role in the growth of the fetus, there is very little published on the role of OLAH in the placenta. Hence, the objective of this study was to examine OLAH levels in the placenta and to determine its role in placental development and disease.
2. Materials and Methods
2.1. FOX Study
As part of the FOX Study, maternal blood was collected from individuals whose pregnancies were complicated by preterm fetal growth restriction and controls with uncomplicated pregnancies, as previously described [
4]. Samples were collected directly into PAXgene Blood RNA tubes (Pre-Analytix, Hombrechtikon, Switzerland) to preserve RNA quality, and processed according to the manufacturer’s instructions. All samples were collected prior to delivery, and after administration of corticosteroids.
Fetal growth restriction was defined as birthweight < 10th centile (
www.gestation.net (accessed on 25 February 2022), Australian parameters) requiring iatrogenic delivery prior to 34 weeks’ gestation with evidence of uteroplacental insufficiency (asymmetrical growth + abnormal artery Doppler velocimetry ± oligohydramnios ± abnormal fetal vessel velocimetry). Fetal growth restriction due to congenital infection, chromosomal or congenital abnormalities, or multiple pregnancies were excluded. For the sub-analysis performed in this manuscript, cases of preterm fetal growth restriction were split into two groups; those where the mother was normotensive (normal blood pressure in pregnancy), and those where the mother had preeclampsia. Fetal hypoxic status was determined by measuring the pH of umbilical artery blood at birth, with fetal hypoxia defined as arterial pH < 7.2. All cases included in sub-analysis received antenatal corticosteroids. Patient characteristics are described in
Supplementary Table S1.
2.2. Placental Tissue Collection
First trimester placental tissue was obtained from conceptus material collected at surgical terminations of singleton pregnancies (7–10 weeks’ gestation) under general anesthesia via curettage, or a combination of aspiration and curettage (according to the surgeon’s preference). Placental tissue was identified and isolated from conceptus material, then washed in phosphate-buffered saline (PBS). Placental tissue was transferred to RNAlater for 48 h, after which the tissue was snap frozen and stored at −80 °C for subsequent analysis. Patient characteristics are described in
Supplementary Table S2.
Placentas were obtained from pregnancies complicated by early onset preeclampsia (requiring delivery ≤ 34 weeks’ gestation). Preeclampsia was defined according to the American College of Obstetricians and Gynecologists guidelines published in 2013 [
11]. Placentas were obtained from cases of preterm fetal growth restriction (delivery ≤ 34 weeks’ gestation), defined as birth weight < 10th centile according to Australian population charts [
12]. Cases associated with congenital infection, chromosomal or congenital abnormalities, multiple pregnancies, or preeclampsia were excluded.
Control healthy, term (delivery 37–40 weeks’ gestation) and preterm (delivery ≤ 34 weeks’ gestation) placentas were collected from normotensive pregnancies where a fetus of normal birth weight centile (>10th centile relative to gestation) was delivered. Placentas with evidence of chorioamnionitis, confirmed by placental histopathology, were excluded.
Term and preterm placental tissue were collected within 30 min of delivery. Preterm delivery of controls was predominantly for iatrogenic conditions including vasa previa, suspected placental abruption, and fetal anemia. Samples from four sites of the placenta were dissected, washed in ice cold PBS and preserved in RNAlater for 48 h, after which the tissue was snap frozen and stored at −80 °C for subsequent analysis. Patient characteristics are described in
Supplementary Tables S2–S4.
Placentas were also obtained from healthy, normotensive term pregnancies (≥37 weeks’ gestation) at the elective cesarean section for explant dissection and cytotrophoblast isolation.
2.3. Collection and Culture of Placental Explants
Placental explants were collected with maternal and fetal surfaces removed by careful dissection. Three pieces of placenta were placed in each well of a 24-well plate (10–15 mg per well), and cultured in Gibco™ Dulbecco’s Modified Eagle Medium (DMEM; ThermoFisher Scientific, Scoresby, VIC, Australia) supplemented with 10% fetal calf serum (FCS; Sigma-Aldrich, St Louis, MO, USA) and 1% Antibiotic Antimycotic (AA; Life Technologies, Carlsbad, CA, USA). Explants were cultured under 8% O2, 5% CO2 at 37 °C overnight (16–18 h). After replacement with fresh media (DMEM/10% FCS/1% AA), explant tissue was cultured for a further 48 h under normoxic (8% O2, 5% CO2 at 37 °C) or hypoxic (1% O2, 5% CO2 at 37 °C) conditions. Following this, explant tissue was snap frozen and stored at −80 °C for subsequent analysis.
2.4. Primary Cytotrophoblast Isolation and Hypoxia Treatment
Primary human cytotrophoblast cells were isolated from healthy, term placentas at an elective cesarean section as previously described [
13]. The cytotrophoblast cells were plated in media (DMEM/10% FCS/1% AA) on fibronectin (10 µg/mL; BD Bioscience, San Jose, CA, USA) coated culture plates. Viable cells were incubated under, 8% O
2, 5% CO
2 at 37 °C overnight to equilibrate and allow adhesion to cell culture plate. After replacement with fresh media (DMEM/10% FCS/1% AA), cytotrophoblasts were either cultured for a further 48 h under normoxic (8% O
2, 5% CO
2 at 37 °C) or hypoxic (1% O
2, 5% CO
2 at 37 °C) conditions or treated as described below. After 48 h, the cells were collected for RNA extraction.
2.5. Silencing OLAH in Primary Cytotrophoblast Cells
Short interfering (si)RNAs designed against OLAH (M-004796-01-0005; Dharmacon, Lafayette, CO, USA) or a pre-tested negative siRNA (Qiagen, Valencia, CA, USA) were combined with lipofectamine (RNAiMax; Invitrogen, Waltham, WA, USA) in Optimem media (ThermoFisher Scientific) at 10 nM, and allowed to complex for 20 min at room temperature. This siRNA complex was then added to overnight equilibrated cytotrophoblasts in fresh media (DMEM/10% FCS) in a dropwise manner. The cells were then incubated for a further 48 h under normoxic (8% O2, 5% CO2 at 37 °C) or hypoxic (1% O2, 5% CO2 at 37 °C) conditions. Following this, media and cell lysates were collected for subsequent analysis.
2.6. MTS Cell Viability Assay
Cell viability was assessed after siRNA treatment using an MTS assay. CellTiter 96-AQueous One Solution (Promega, Madison, WI, USA) was used according to the manufacturer’s instructions. Optical density was measured using a Bio-Rad X-Mark Microplate Spectrophotometer (Hercules, CA, USA) and Bio-Rad Microplate Manager 6 software.
2.7. Real Time Polymerase Chain Reaction (RT-PCR)
Total RNA was extracted from whole blood using the PAXgene
® Blood miRNA Kit (Pre-Analytix) as described previously [
4]. RNA was extracted from placental tissue and cytotrophoblast cells using the Qiagen RNeasy Mini Kit following the manufacturer’s instructions. The RNA was quantified using a Nanodrop 2000 spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA) or LVis Plate for FluoStar Omega Microplate Reader (BMG Labtech, Mornington, VIC, Australia). Extracted RNA was converted to cDNA using the Applied Biosystems
TM High-Capacity cDNA Reverse Transcription Kit, following the manufacturer’s instructions on the iCycler iQ5 (Bio-Rad) or MiniAmp Thermal Cycler (ThermoFisher Scientific). Quantitative Taqman PCR (with primers purchased from Life Technologies) was performed to quantify mRNA expression of
OLAH (Hs00217864_m1),
PGF (Hs00182176_m1),
HMOX1 (Hs01110250_m1),
NOX4 (Hs00418356_m1),
GCLC (Hs00155249_m1),
NLRP3 (Hs00918082_m1),
SPINT1 (Hs00173678_m1),
BAX (Hs00180269_m1),
BCL2 (Hs00608023_m1),
EGFR (Hs01076078_m1),
IGF2 (Hs04188276_m1),
NQO1 (Hs00168547_m1) and
TXN (Hs00828652_m1). The stability of reference genes was confirmed for each sample type and used appropriately; for blood
YWHAZ (Hs01122454_m1),
B2M (Hs00187842_m1) and
GUSB (Hs00939627_m1), for cytotrophoblast
YWHAZ (Hs01122454_m1) and for placental explants and placental tissue:
TOP1 (Hs00243257_m1) and
CYC1 (Hs00357717_m1). Taqman RT-PCR was performed on the CFX384 (Bio-Rad) with the following run conditions: 50 °C for 2 min; 95 °C for 10 min, 95 °C for 15 s, 60 °C for 1 min (40 cycles) or 95 °C for 20 s; 95 °C for 3 s, 60 °C for 30 s (40 cycles; Taqman Fast Advanced Master Mix).
The sFLT1 splice variants sFLT1-i13 and sFLT1-e15a were measured in a SYBR PCR with SYBR Green Master mix (Applied Biosystems) using primers specific for each variant. The primers for sFLT-i13 were 5′-ACAATCAGAGGTGAGCACTGCAA-3′ (forward) 5′-TCCGAGCCTGAAAGTTAGCAA-3′ (reverse), for sFLT-e15a 5-CTCCTGCGAAACCTCAGTG-3′ (forward) 5′-GACGATGGTGACGTTGATGT-3′ (reverse) and for YWHAZ (reference gene) 5′-GAGTCATACAAAGACAGCACGCTA-3′ (forward) 5′-TTCGTCTCCTTGGGTATCCGATGT-3′ (reverse). The SYBR PCR was run on the CFX384 (Bio-Rad), with 40 cycles of 95 °C for 21 s, then 60 °C for 20 min. All data were normalized to the appropriate reference gene as an internal control and calibrated against the average Ct of the control samples (2−ΔΔCt). All cDNA samples were run in duplicate.
2.8. Western Blot Analysis
Protein lysates were extracted from placental tissue collected from pregnancies complicated by preeclampsia, fetal growth restriction, and preterm controls (≤34 weeks), as well as placental explant tissue exposed to hypoxia using RIPA lysis buffer containing proteinase and phosphatase inhibitors (Sigma Aldrich). Protein concentration was determined with Pierce™ BCA Protein Assay Kit (ThermoFisher Scientific, Waltham, MA, USA). Protein lysates (20 µg) were separated on 12% gels before transfer to PVDF membranes (Millipore; Billerica, MA, USA). Membranes were blocked with 1% bovine serum albumin (BSA; Sigma-Aldrich) prior to overnight incubation with the primary anti-OLAH antibody (diluted 1:250 in 2% BSA/TBS-T; HPA037948, Sigma-Aldrich). Following this, membranes were incubated with a secondary anti-rabbit antibody (diluted 1:2500 in 2% BSA; W401, Promega, Madison WI, USA) for 1 h. Bands were visualized using a chemiluminescence detection system (GE Healthcare Life Sciences, Singapore) and ChemiDoc Imaging System (Bio-Rad). Β-actin acted as the loading control, (diluted 1:2500 in 5% skim milk; 51255, Cell Signalling Technology, Danvers, MA, USA). Relative densitometry was measured using Image Lab software 6.0.1 (Bio-Rad).
2.9. Enzyme Linked Immunosorbent Assay (ELISA)
Soluble fms-like tyrosine kinase-1 (sFLT1) and placental growth factor (PGF) secretion were measured in cytotrophoblast conditioned culture media using the DuoSet Human VEGF R1/FLT-1 kit and Human PlGF DuoSet ELISA kit (R&D systems by Bioscience, Waterloo, Australia), respectively, according to manufacturer’s instructions. Optical density was measured using a Bio-Rad X-Mark microplate spectrophotometer and Bio-Rad Microplate Manager 6 software.
2.10. Statistical Analysis
All in vitro experiments were performed with technical triplicates and repeated with a minimum of three different patient samples. Data were tested for normal distribution and for comparisons between two groups either an unpaired t-test or Mann–Whitney test was used as appropriate. For comparisons between three or more groups, a Kruskal–Wallis test was used with Dunn’s correction for multiple comparisons. All data are expressed as mean ± SEM. p values < 0.05 were considered significant. Statistical analysis was performed using GraphPad Prism software 8 (GraphPad Software, Inc.; San Diego, CA, USA).
4. Discussion
In previous studies, our group demonstrated that
OLAH transcripts were highly upregulated in the maternal circulation of individuals whose pregnancies were complicated by fetal growth restriction and preeclampsia [
4]. In this study, we identified that
OLAH is expressed in the placenta throughout gestation, and importantly, its levels are altered in the placenta in cases of fetal growth restriction and preeclampsia (without fetal growth restriction), and under hypoxic conditions. Further, we found that silencing cytotrophoblast
OLAH expression can alter the expression of genes associated with apoptosis, fetal growth, and oxidative stress.
In a sub-analysis of the
OLAH transcripts measured in the maternal circulation of pregnancies complicated by fetal growth restriction, we found no further changes when we split the cases by hypertensive status or fetal hypoxia. Circulating RNAs could give us an insight into the state of the placenta [
5,
6,
16], or act as signaling molecules, taken up by the vasculature to alter function in the maternal system. These findings suggest that dysregulation of OLAH is common to both fetal growth restriction and preeclampsia and may be the result of shared pathophysiological pathways. Further, fetal hypoxia specifically is unlikely to be related to circulating
OLAH levels. If the elevated
OLAH transcripts in these conditions were indeed taken up by the vasculature, exerting their canonical function in fatty acid synthesis could have implications for cardiovascular health. In fact, medium chain fatty acids, which are the products of OLAH-catalyzed reactions, are being considered for their therapeutic benefits on metabolic health in cardiac disease [
17].
We next investigated OLAH in the placenta, as increased OLAH levels in the pathological placenta could contribute to the elevated levels of circulating
OLAH transcripts, especially as both fetal growth restriction and preeclampsia can feature a dysfunctional placenta. We first measured
OLAH expression over gestation, finding generally that its expression appeared at its lowest in the first trimester (with some samples displaying
OLAH expression at the lower limit of detection), but was increased in the second and third trimesters. Placental
OLAH gene expression has previously been demonstrated to be highest at term gestation, albeit in small sample sizes [
18]. This increase in expression as gestation progresses could be associated with the increased circulating lipid levels in pregnancy, especially with rapid lipid lysis that occurs closer to term [
19], as OLAH is required for the preferred synthesis of medium chain fatty acids.
Intriguingly, we found that placentas collected from cases of preeclampsia, but not fetal growth restriction, had significantly elevated OLAH mRNA expression compared to preterm controls. This suggests that OLAH may be regulated distinctly between these two conditions. It is for this reason that we analyzed the samples from cases of preeclampsia featuring growth restriction independently.
Although
OLAH mRNA expression was elevated with preeclampsia, OLAH
protein was not significantly different compared to control. However, we note that while OLAH protein levels in each of the samples from the preeclampsia and growth restriction groups were clustered together, the OLAH protein levels in the preterm controls were quite variable, and thus could have confounded our results. This is a limitation of the preterm control samples, though we have tried to minimize confounding factors within this group, these are not perfectly healthy pregnancies as they have resulted in premature delivery. Nevertheless, we did interestingly find that OLAH protein in growth-restricted placental tissue was significantly lower than that in the placental samples collected from cases of preeclampsia. This again supports the idea that OLAH may be regulated distinctly in these two conditions. Dysregulation of OLAH protein in these two diseases may contribute to differences previously identified in the fatty acid profile of maternal and umbilical cord plasma in pregnancies affected by fetal growth restriction alone versus preeclampsia with fetal growth restriction [
20].
Reduced OLAH in the placenta may contribute to impaired fetal growth. Fatty acids are postulated to be an energy source utilized by the placenta [
21]. Further, the smaller fatty acids are, the more readily they pass through the placenta to enter fetal capillaries [
22]. The implication is that if OLAH levels are reduced, fatty acid synthase will preferentially produce larger chain fatty acids, which move less easily to the fetus, potentially impairing fetal development [
23,
24]. As we have detected low OLAH protein in placentas from cases of fetal growth restriction, we suggest that OLAH could play an important role in disease pathogenesis and could be an important target for improving fetal growth.
In contrast, OLAH levels were high in placenta collected from pregnancies complicated by preterm preeclampsia. Similarly, another study found that cytotrophoblast cells isolated from placentas experiencing preeclampsia had significantly elevated
OLAH mRNA, compared to preterm labor controls [
25]. These findings suggest that preeclampsia is associated with increased placental OLAH.
Preeclampsia is associated with dyslipidemia [
26,
27,
28,
29,
30]. However, most studies focus on circulating and placental levels of long-chain fatty acids, rather than medium-chain fatty acids. Hence, we cannot make a statement on what increased placental OLAH could mean in preeclampsia without further studies, except to suggest that this elevated OLAH may mean increased lipid processing into medium-chain fatty acids, which may move more readily to the fetus for fetal growth. Thus, in preeclampsia, elevated placental OLAH could be beneficial.
In this study, we found that placental hypoxia significantly increased
OLAH mRNA expression in placental explant tissue, similar to our findings in placental tissue from cases of preeclampsia, suggesting that impaired oxygen delivery may cause OLAH dysregulation in preeclampsia. Interestingly, isolated cytotrophoblast cells, which are unique to the placenta, did not have significantly altered
OLAH expression under hypoxia. This suggests that the source of placental OLAH dysregulation may be altered as the trophoblast differentiates to the syncytium, or perhaps is another cell type, such as stromal, vascular, or immune cells that are also part of the placenta. Investigating
OLAH expression in these different cell types could improve understanding of the role of placental OLAH in disease pathology. However, placental hypoxia is just one aspect of the pathology of these conditions. Altered cytotrophoblast
OLAH expression has been demonstrated in cells isolated from preeclamptic placentas [
25], suggesting OLAH may still have an important role in cytotrophoblast cells.
We therefore went on to investigate a potential role for OLAH in placental cytotrophoblasts in the pathogenesis of fetal growth restriction and preeclampsia. Silencing
OLAH in cytotrophoblasts did not alter the expression or secretion of sFLT1, suggesting that it is not involved in the regulation of this anti-angiogenic factor. Under normoxic conditions, silencing cytotrophoblast
OLAH increased the expression of sFLT1 isoform
sFLT-i13, but not isoform
sFLT-e15a. However, this did not affect the secretion of sFLT1 protein, likely because
sFLT-e15a is the predominant isoform [
31,
32]. Under hypoxic conditions, silencing
OLAH significantly increased the mRNA expression of angiogenic
PGF; however, intriguingly did not alter the secretion of PGF protein. This suggests there is post-transcriptional or post-translational regulation of PGF release. Further investigation of intracellular PGF protein would be necessary to elucidate this. Under normoxic conditions, silencing
OLAH did not alter the expression or secretion of PGF. These findings suggest that
OLAH dysregulation in the placenta is unlikely to be associated with changes in these anti-angiogenic and angiogenic factors that are synonymous with preeclampsia, and hence would not be a suitable target for controlling levels of these factors in disease.
However, we did find that silencing
OLAH altered expression of genes associated with apoptosis, fetal growth, and oxidative stress. Under hypoxia, silencing cytotrophoblast
OLAH expression reduced the expression of both
BAX and
BCL2, anti- and pro-apoptotic genes, respectively. The reduction of both means that the balance of these factors may not be altered, and total apoptosis may not be affected [
33]. We also detected a significant reduction in
IGF2; knockdown of IGF2 is associated with growth restriction in mice [
34]. As OLAH protein is low in fetal growth restriction, it is possible that its reduction in disease could decrease IGF2 as well. Silencing
OLAH did not alter
EGFR or
SPINT1 expression, which are also associated with fetal growth. Expression of anti-oxidant genes
HMOX1,
NQO1,
TXN, and
GCLC, and inflammasome gene
NLRP3 were also unaltered, suggesting that cytotrophoblast OLAH does not regulate anti-oxidant pathways or the NLRP3 inflammasome under hypoxia. However,
NOX4, a marker of oxidative stress was significantly reduced—this could be seen as a beneficial effect. However, accompanied by the reduction in
IGF2 expression, it is unclear whether a reduction in OLAH could be protective, or enhance placental dysfunction. Notably, under normoxic conditions, many of these genes were not dysregulated. The only gene we found to be dysregulated was
BCL2, the anti-apoptotic gene, which suggests the loss of cytotrophoblast OLAH under normoxic conditions could be detrimental.
Further studies will evaluate correlations between OLAH levels and placental pathologies in both preterm and term placentas. Assessing pathways altered with OLAH overexpression may give further insight into the implications of OLAH dysregulation in the placenta, especially because placental OLAH is elevated in preeclampsia and with placental hypoxia. Additionally, investigating OLAH protein secretion would help validate whether the low OLAH levels in growth-restricted placentas are truly due to a reduction in OLAH protein production, or increased secretion. An important extension of this work would be to measure specific production of medium chain fatty acids in response to changes in placental OLAH. Assessing the canonical pathways of OLAH and its interactions (e.g.,: with NFκB and PPAR-α pathways) in placental tissue and cytotrophoblast cells would be an important next avenue of investigation.