Placental OLAH Levels Are Altered in Fetal Growth Restriction, Preeclampsia and Models of Placental Dysfunction

Previously, we identified elevated transcripts for the gene Oleoyl-ACP Hydrolase (OLAH) in the maternal circulation of pregnancies complicated by preterm fetal growth restriction. As placental dysfunction is central to the pathogenesis of both fetal growth restriction and preeclampsia, we aimed to investigate OLAH levels and function in the human placenta. We assessed OLAH mRNA expression (qPCR) throughout pregnancy, finding placental expression increased as gestation progressed. OLAH mRNA and protein levels (Western blot) were elevated in placental tissue from cases of preterm preeclampsia, while OLAH protein levels in placenta from growth-restricted pregnancies were comparatively reduced in the preeclamptic cohort. OLAH expression was also elevated in placental explant tissue, but not isolated primary cytotrophoblast cultured under hypoxic conditions (as models of placental dysfunction). Further, we discovered that silencing cytotrophoblast OLAH reduced the expression of pro- and anti-apoptosis genes, BAX and BCL2, placental growth gene, IGF2, and oxidative stress gene, NOX4. Collectively, these findings suggest OLAH could play a role in placental dysfunction and may be a therapeutic target for mitigating diseases associated with this vital organ. Further research is required to establish the role of OLAH in the placenta, and whether these changes may be a maternal adaptation or consequence of disease.


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.

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.

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.

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.

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% O 2 , 5% CO 2 at 37 • C) or hypoxic (1% O 2 , 5% CO 2 at 37 • C) conditions. Following this, media and cell lysates were collected for subsequent analysis.

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.

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.

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).

Circulating OLAH Transcripts Are Not Significantly Altered between Cases of Normotensive Fetal Growth Restriction, Compared to Cases of Preeclampsia with Growth Restriction
Using next-generation sequencing, we previously identified that OLAH transcripts are elevated in the maternal circulation in pregnancies complicated by fetal growth restriction, approximately half of which had preeclampsia [4]. Here, using data from the same sample set, we sought to evaluate whether those pregnancies that were also complicated by preeclampsia had further altered levels of circulating OLAH transcripts, compared to pregnancies complicated by fetal growth restriction alone (normotensive).
The samples that were used to measure OLAH in the preeclampsia cohort were from patients who had significantly higher BMI and delivered at an earlier gestational age, but were matched for other maternal and perinatal characteristics (Supplementary Table  S1). We found that OLAH mRNA transcripts were not further significantly altered in the maternal circulation when pregnancies were also complicated by preeclampsia (plus growth restriction) compared to normotensive pregnancies with fetal growth restriction ( Figure 1A). Further, we sub-analyzed these cases of fetal growth-restricted pregnancies to assess whether fetal acidemia had an impact on circulating OLAH transcripts, where umbilical artery blood pH < 7.2 indicated acidosis (pH ≥ 7.2 not acidotic). We identified that OLAH transcripts were not altered with fetal acidemia/hypoxia ( Figure 1B). Figure 1. OLAH mRNA transcripts in the maternal circulation of pregnancies complicated by fetal growth restriction with or without preeclampsia or fetal acidemia. Transcript levels were measured via qPCR. OLAH transcripts were not significantly altered in the circulation of cases of preeclampsia with fetal growth restriction, compared to normotensive cases of fetal growth restriction (A). Cases of growth restriction where the fetus was delivered with umbilical cord artery pH < 7.2 did not have significantly altered OLAH transcripts in the maternal circulation compared to those with pH ≥ 7.2 (B). Data presented as relative expression, mean ± SEM. Normotensive n = 44, preeclampsia n = 71, case pH ≥ 7.2 n = 107, case pH < 7.2 n = 18.

OLAH Expression across Gestation
We next investigated whether OLAH was expressed in the human placenta. Placental samples were collected from the first trimester (7-10 weeks' gestation), second trimester (24-28 weeks' gestation), and third trimester (38-39 weeks' gestation) (Supplementary Table S2). OLAH expression was detectable in all placental samples collected across gestation, but the expression was very low in three of the first trimester samples (close to the lower limit of detection). OLAH expression was significantly elevated in second trimester (p = 0.0032) and third trimester (p = 0.0025) placental samples compared to first trimester samples. There was no difference in expression between the second and third trimester placental samples ( Figure 2).  Figure 1. OLAH mRNA transcripts in the maternal circulation of pregnancies complicated by fetal growth restriction with or without preeclampsia or fetal acidemia. Transcript levels were measured via qPCR. OLAH transcripts were not significantly altered in the circulation of cases of preeclampsia with fetal growth restriction, compared to normotensive cases of fetal growth restriction (A). Cases of growth restriction where the fetus was delivered with umbilical cord artery pH < 7.2 did not have significantly altered OLAH transcripts in the maternal circulation compared to those with pH ≥ 7.2 (B). Data presented as relative expression, mean ± SEM. Normotensive n = 44, preeclampsia n = 71, case pH ≥ 7.2 n = 107, case pH < 7.2 n = 18.

OLAH Expression across Gestation
We next investigated whether OLAH was expressed in the human placenta. Placental samples were collected from the first trimester (7-10 weeks' gestation), second trimester (24-28 weeks' gestation), and third trimester (38-39 weeks' gestation) (Supplementary Table S2). OLAH expression was detectable in all placental samples collected across gestation, but the expression was very low in three of the first trimester samples (close to the lower limit of detection). OLAH expression was significantly elevated in second trimester (p = 0.0032) and third trimester (p = 0.0025) placental samples compared to first trimester samples. There was no difference in expression between the second and third trimester placental samples ( Figure 2).

OLAH Expression across Gestation
We next investigated whether OLAH was expressed in the human placenta. Placental samples were collected from the first trimester (7-10 weeks' gestation), second trimester (24-28 weeks' gestation), and third trimester (38-39 weeks' gestation) (Supplementary Table S2). OLAH expression was detectable in all placental samples collected across gestation, but the expression was very low in three of the first trimester samples (close to the lower limit of detection). OLAH expression was significantly elevated in second trimester (p = 0.0032) and third trimester (p = 0.0025) placental samples compared to first trimester samples. There was no difference in expression between the second and third trimester placental samples ( Figure 2).  Figure 2. OLAH gene expression in placental samples collected in first, second, and third trimester. Gene expression was assessed via qPCR. OLAH expression was significantly increased in second and third trimester compared to first trimester. Expression in the second and third trimester was not significantly different. Data presented as relative expression, mean ± SEM. First trimester (7-11 weeks gestation) n = 11, second trimester (24-28 weeks gestation) n = 4, third trimester (38-39 weeks gestation) n = 10 placental samples. ** p < 0.01.

OLAH Levels Are Significantly Altered in Placental Tissue from Cases of Preterm Preeclampsia and Fetal Growth Restriction
Noting that OLAH transcripts are highly increased in the circulation of cases of preeclampsia and growth restriction [4], we next examined OLAH levels in the pathological placenta, as placental dysfunction is central to the pathophysiology of both conditions. We assessed OLAH expression and protein in placental tissue from individuals with preterm preeclampsia and fetal growth restriction, compared to preterm control placental tissue (Supplementary Tables S3 and S4).
Placental mRNA expression of OLAH was significantly increased in preterm preeclampsia (without fetal growth restriction) (p = 0.0004) compared to preterm control, but was not altered in fetal growth restriction alone, or preeclampsia with fetal growth restriction ( Figure 3A). OLAH mRNA expression did not differ significantly between the pathological groups.   Placental OLAH protein production in cases of fetal growth restriction and preeclampsia ± growth restriction was not significantly different from the variable OLAH protein production in the preterm control tissue ( Figure 3B,C; full immunoblots presented in Supplementary Figure S1). However, OLAH protein in the placental tissue from cases of growth restriction was significantly lower than that of the tissue from cases of preeclampsia without growth restriction (p = 0.0042; Figure 3B,C; full immunoblots presented in Supplementary Figure S1). OLAH protein levels were not different in placental tissue from cases of preeclampsia that featured fetal growth restriction compared to the other pathological groups (Figure 3B,C; full immunoblots presented in Supplementary Figure S1).

OLAH Expression Is Upregulated in Placental Explant Tissue Cultured under Hypoxia
To uncover the mechanisms behind altered OLAH levels in the pathological placenta, we next examined OLAH expression in placental tissue explants and isolated cytotrophoblast cells (unique to the placenta) under hypoxia (low oxygen), as a model of placental dysfunction. OLAH expression was detectable in both placental tissue and cytotrophoblast cells under hypoxic (1% O 2 ) and normoxic conditions (8% O 2 ). OLAH expression was significantly increased in placental explant tissue under hypoxia, compared to normoxic conditions (p = 0.0011; Figure 4A). We followed this with western blotting to examine OLAH protein in placental explant tissue, but we could not detect OLAH at the protein concentration used (data not shown). OLAH mRNA expression was not altered in primary cytotrophoblasts cultured under hypoxia ( Figure 4B).

OLAH Expression Is Upregulated in Placental Explant Tissue Cultured under Hypoxia
To uncover the mechanisms behind altered OLAH levels in the pathological placenta, we next examined OLAH expression in placental tissue explants and isolated cytotrophoblast cells (unique to the placenta) under hypoxia (low oxygen), as a model of placental dysfunction. OLAH expression was detectable in both placental tissue and cytotrophoblast cells under hypoxic (1% O2) and normoxic conditions (8% O2). OLAH expression was significantly increased in placental explant tissue under hypoxia, compared to normoxic conditions (p = 0.0011; Figure 4A). We followed this with western blotting to examine OLAH protein in placental explant tissue, but we could not detect OLAH at the protein concentration used (data not shown). OLAH mRNA expression was not altered in primary cytotrophoblasts cultured under hypoxia ( Figure 4B).

Silencing OLAH in Primary Cytotrophoblast Cells Does Not Alter Cell Survival
To elucidate how dysregulated OLAH levels could affect placental function, studies were performed using short interfering (si)RNAs to silence OLAH expression in isolated primary cytotrophoblast cells. Expression was silenced by approximately 90% under both normoxic (p < 0.0001; Figure 5A) and hypoxic conditions (p < 0.0001; Figure 5B), which was determined sufficient knockdown for further experiments. Silencing OLAH did not significantly alter cell survival assessed via MTS assay, under either normoxic or hypoxic conditions (Supplementary Figure S2A,B).

Silencing OLAH in Primary Cytotrophoblast Cells Does Not Alter Cell Survival
To elucidate how dysregulated OLAH levels could affect placental function, studies were performed using short interfering (si)RNAs to silence OLAH expression in isolated primary cytotrophoblast cells. Expression was silenced by approximately 90% under both normoxic (p < 0.0001; Figure 5A) and hypoxic conditions (p < 0.0001; Figure 5B), which was determined sufficient knockdown for further experiments. Silencing OLAH did not significantly alter cell survival assessed via MTS assay, under either normoxic or hypoxic conditions (Supplementary Figure S2A,B).

Silencing Cytotrophoblast OLAH Does Not Alter sFLT1 Secretion
Levels of anti-angiogenic factor sFLT1 are elevated, and angiogenic factor PGF are reduced in the circulation of individuals whose pregnancies are complicated by preeclampsia [14]. Here, we assessed whether OLAH regulates the expression and release of these factors from isolated cytotrophoblast, which would implicate OLAH in the regulation of angiogenesis in preeclampsia. Under hypoxia (mimicking conditions of placental dysfunction akin to that in preeclampsia), silencing cytotrophoblast OLAH did not alter the expression of the two sFLT1 transcript isoforms, sFLT-e15a ( Figure 6A) or sFLT-i13 ( Figure 6B), nor secretion of the functional sFLT1 protein ( Figure 6C). Silencing OLAH under hypoxia significantly reduced PGF mRNA expression (p = 0.0002; Figure 6D), but did not alter PGF secretion ( Figure 6E).

Silencing Cytotrophoblast OLAH Does Not Alter sFLT1 Secretion
Levels of anti-angiogenic factor sFLT1 are elevated, and angiogenic factor PGF are reduced in the circulation of individuals whose pregnancies are complicated by preeclampsia [14]. Here, we assessed whether OLAH regulates the expression and release of these factors from isolated cytotrophoblast, which would implicate OLAH in the regulation of angiogenesis in preeclampsia. Under hypoxia (mimicking conditions of placental dysfunction akin to that in preeclampsia), silencing cytotrophoblast OLAH did not alter the expression of the two sFLT1 transcript isoforms, sFLT-e15a ( Figure 6A) or sFLT-i13 ( Figure 6B), nor secretion of the functional sFLT1 protein ( Figure 6C). Silencing OLAH under hypoxia significantly reduced PGF mRNA expression (p = 0.0002; Figure 6D), but did not alter PGF secretion ( Figure 6E). We also assessed the effects of silencing cytotrophoblast OLAH on sFLT1 and PGF levels in normoxic conditions to evaluate a potential role for OLAH in normal placental function. Here, we found that silencing OLAH did not alter sFLT-e15a expression (Supplementary Figure S3A), but significantly increased sFLT-i13 expression (p = 0.0252; Supplementary Figure S3B), though sFLT1 secretion was not altered (Supplementary Figure  S3C). Silencing OLAH under normoxic conditions did not alter cytotrophoblast PGF expression (Supplementary Figure S3D) or secretion (Supplementary Figure S3E).  We also assessed the effects of silencing cytotrophoblast OLAH on sFLT1 and PGF levels in normoxic conditions to evaluate a potential role for OLAH in normal placental function. Here, we found that silencing OLAH did not alter sFLT-e15a expression (Supplementary Figure S3A), but significantly increased sFLT-i13 expression (p = 0.0252; Supplementary Figure S3B), though sFLT1 secretion was not altered (Supplementary Figure S3C). Silencing OLAH under normoxic conditions did not alter cytotrophoblast PGF expression (Supplementary Figure S3D) or secretion (Supplementary Figure S3E).

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.

Conclusions
Using first trimester, preterm, and term placental tissues and cells from normal and pathological pregnancies, this study identified that OLAH expression is altered over gestation, and may have a role in placental dysfunction. Further investigation is required to establish the precise role of OLAH in the placenta and cytotrophoblast. However, we have identified that this gene could play an important role in the pathogenesis of preeclampsia and fetal growth restriction, whether it is due to its role in fatty acid synthesis is still to be determined.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/antiox11091677/s1, Supplementary Table S1: Patient characteristics of pregnancies complicated with fetal growth restriction with and without preeclampsia (collected as part of the FOX Study); Supplementary  Figure S1: OLAH production in placental tissue collected from preterm pathological pregnancies.; Supplementary Figure S2