NR4A2 expression is not altered in placentas from cases of growth restriction or preeclampsia, but is reduced in hypoxic cytotrophoblast

Nuclear Receptor Subfamily 4 Group A Member 2 (NR4A2) transcripts are elevated in the circulation of individuals whose pregnancies are complicated by preterm fetal growth restriction (FGR). In this paper, we show that the cases with preeclampsia (PE) have increased circulating NR4A2 transcripts compared to those with normotensive FGR. We aimed to establish whether the dysfunctional placenta mirrors the increase in NR4A2 transcripts and further, to uncover the function of placental NR4A2. NR4A2 expression was detected in preterm and term placental tissue; expressed higher at term. NR4A2 mRNA expression and protein were not altered in placentas from preterm FGR or PE pregnancies. Hypoxia (1% O2 compared to 8% O2) significantly reduced cytotrophoblast NR4A2 mRNA expression, but not placental explant NR4A2 expression. Silencing cytotrophoblast NR4A2 expression under hypoxia (via short interfering (si)RNAs) did not alter angiogenic Placental Growth Factor, nor anti-angiogenic sFlt-1 mRNA expression or protein secretion, but increased expression of cellular antioxidant, oxidative stress, inflammatory, and growth genes. NR4A2 expression was also not altered in a model of tumour necrosis factor-α-induced endothelial dysfunction, or with pravastatin treatment. Further studies are required to identify the origin of the circulating transcripts in pathological pregnancies, and investigate the function of placental NR4A2.


Results
Circulating NR4A2 mRNA levels are increased in individuals with coexistent preeclampsia and fetal growth restriction. Using next generation sequencing, we previously reported increased NR4A2 transcripts in the circulation of women whose pregnancies were complicated by preterm fetal growth restriction (< 34 weeks gestation) 13 . In the current study, we performed a sub-analysis, directly comparing cases with preterm preeclampsia and growth restriction to cases of normotensive growth restriction. We found significantly increased circulating NR4A2 transcripts in the growth restricted cases with preeclampsia, compared to normotensive cases (Fig. 1).
Placental NR4A2 expression is increased at term. NR4A2 expression was detected in all placental tissues collected, preterm (24-36 weeks) and term (37-41 weeks). NR4A2 expression was higher in term placental tissue compared to preterm placental tissue (Fig. 2).
NR4A2 expression is not altered in preterm pathological placental tissue. To elucidate whether NR4A2 has a role in placental dysfunction, we investigated NR4A2 expression in placenta collected from pregnancies complicated by preterm preeclampsia and fetal growth restriction (≤ 34 weeks gestation), conditions that Circulating NR4A2 mRNA in cases of normotensive preterm fetal growth restriction and preeclampsia with growth restriction (< 34 weeks gestation). RNA levels were assessed by qPCR. Cases with preeclampsia and growth restriction had significantly higher circulating NR4A2 mRNA compared to normotensive cases of growth restriction. Data presented as relative change from normotensive levels; mean ± SEM. **p < 0.01. Normotensive, n = 45; preeclampsia, n = 71.
Scientific Reports | (2021) 11:20670 | https://doi.org/10.1038/s41598-021-00192-y www.nature.com/scientificreports/ feature impaired placental development and dysfunction. There was no significant difference in NR4A2 expression in placental tissue from pregnancies complicated by either preterm fetal growth restriction or preeclampsia (≤ 34 weeks gestation) compared to preterm control tissue (Fig. 3a). NR4A2 protein levels were also not altered in the preterm pathological tissue compared to preterm controls (Fig. 3b,c; Supplementary Fig. S1). NR4A2 mRNA expression and protein levels were not significantly altered by fetal sex in preterm pathological placental tissue or controls (data not shown).
NR4A2 expression is decreased in cytotrophoblast, but not placental explants under hypoxia. To further elucidate the role of NR4A2 in the placenta, its expression was assessed in placental explants and cytotrophoblast, cells unique to the placenta. These cells and tissues were exposed to hypoxia to simulate a low oxygen environment akin to that in placental insufficiency. NR4A2 expression was detectable in both placental explant tissue and primary cytotrophoblast. There was no difference in NR4A2 expression in placental explants under hypoxia compared to normoxic conditions (Fig. 4a). In cytotrophoblast, NR4A2 expression was significantly reduced under hypoxia (p < 0.0001; Fig. 4b). However, there was no significant change in cytotrophoblast NR4A2 protein levels in hypoxic compared to normoxic conditions ( Fig. 4c; Supplementary  Fig. S2). Silencing NR4A2 in cytotrophoblasts does not alter expression or secretion of sFLT-1 or PGF. We next assessed the effects of NR4A2 knockdown on sFlt-1, an anti-angiogenic factor increased in preeclampsia, and placental growth factor (PGF), an angiogenic factor decreased in preeclampsia. Silencing NR4A2 in cytotrophoblasts under hypoxia had no significant effect on the expression of the sFlt-1 isoforms, sFlt-1-e15a and sFlt-1-i13 (Fig. 5a,b) or sFlt-1 secretion (Fig. 5c). Silencing NR4A2 under hypoxia had no significant effect on PGF expression (Fig. 5d). Silencing NR4A2 under normoxic conditions did not alter sFlt-1 and PGF expression or sFlt-1 secretion ( Supplementary Fig. S4).
Under hypoxia, silencing NR4A2 did not alter expression of pro-apoptotic gene: BCL2 Associated X (BAX; Supplementary Fig. S5a), pro-survival gene: B-cell lymphoma 2 (BCL2; Supplementary Fig. S5b)  www.nature.com/scientificreports/ growth and proliferation genes: epidermal growth factor receptor (EGFR) and insulin-like growth factor 2 (IGF2) (Supplementary Fig. S5c and S5d). Gene expression was also assessed in cytotrophoblasts with silenced NR4A2 under normoxic conditions. In these cells we found significantly decreased expression of BAX (p = 0.0123; Supplementary  NR4A2 is not altered with TNF-α-induced endothelial dysfunction or pravastatin treatment. Given there was no change in NR4A2 expression in the pathological preterm placenta, we assessed whether the increased circulating levels of NR4A2 transcripts in pregnancies complicated by fetal growth restriction and preeclampsia might originate in the vasculature. We assessed this in endothelial cells, which form the inner lining of blood vessels. NR4A2 expression was detectable in Human Umbilical Vein Endothelial cells (HUVECs). Moreover, as preeclampsia is associated with endothelial dysfunction 37 , we assessed whether dysfunction may alter NR4A2 expression. Tumor necrosis factor (TNF)-α, an inflammatory mediator increased in the circulation of women with preeclampsia 38 , was used to induce endothelial dysfunction in HUVECs. Our model of endothelial dysfunction revealed that TNF-α-induced endothelial dysfunction did not alter NR4A2 expression (Fig. 7). Furthermore, treatment with 200 µM pravastatin with TNF-α, a candidate drug for prevention of preeclampsia 39 , did not significantly alter NR4A2 expression (Fig. 7).

Discussion
Circulating nucleic acid transcripts involved in fetal-maternal signalling 40 may provide a means for early identification of conditions involving placental dysfunction, such as preeclampsia and fetal growth restriction 12,13 .
In this study, we extended the findings reported previously 13 , identifying that preeclampsia further increased circulating NR4A2 transcript levels in pregnancies complicated by preterm fetal growth restriction (< 34 weeks gestation). However, this finding is not mirrored in placental tissue, where we observed no difference in NR4A2 expression in placentas from cases of preterm growth restriction or preeclampsia (≤ 34 weeks gestation). We aimed to elucidate the role of NR4A2 in the placenta, finding that NR4A2 expression is higher in term placenta compared to preterm tissue, and its expression is significantly decreased in cytotrophoblast under hypoxia. Furthermore, we identified that decreased cytotrophoblast expression of NR4A2 is associated with the dysregulation of genes involved in oxidative stress, inflammation, and growth and development.
A key aim of this paper was to identify whether expression of NR4A2 in the pathological placenta mirrored the increased NR4A2 transcripts demonstrated in the maternal circulation of cases of preterm preeclampsia and growth restriction (< 34 weeks gestation)-if so, supporting the concept that they may originate in the dysfunctional placenta. However, we did not find any significant change in NR4A2 expression or protein in placentas from pregnancies complicated by preterm fetal growth restriction or preeclampsia. This contrasts the findings and hypoxic (1% O 2 ) conditions. NR4A2 expression was unaltered in placental explant tissue with hypoxia (a). In primary cytotrophoblasts, NR4A2 expression was significantly decreased under hypoxic conditions (b). There was no change in NR4A2 protein production with hypoxia (c). Data presented as fold change from control ± SEM. qPCR: n = 4-5 experimental replicates, each sample from a different patient. Each sample was run in triplicate. Western blot: n = 4 experimental replicates, with each sample from a different patient. Each experiment was run in triplicate and replicate lysates were pooled. β-actin acted as the loading control. ****p < 0.0001.  41 . However, their study included both preterm and term samples. As we've shown that NR4A2 expression is altered between preterm and term gestations, we suggest that this disparity is likely due to gestational differences. Further, early-and late-onset preeclampsia have distinct molecular processes, with the early-onset cases associated with increased severity 42,43 . Our study overcomes this potential confounder by assessing the harder to source clinical samples exclusively from cases of preterm preeclampsia delivering ≤ 34 weeks. Choosing early-onset cases also allows us to compare levels of placental mRNA to circulating transcripts in the FOX blood samples. Our findings suggest that the growth restricted or preeclamptic placenta is not the origin of the altered circulating mRNA transcripts. Furthermore, we identified that silencing NR4A2 did not alter the expression or secretion of sFlt-1 or PGF, key factors central to the pathogenesis of preeclampsia, suggesting that placental NR4A2 is not involved in driving the release of anti-angiogenic factors from the dysfunctional placenta, a key process in the pathogenesis of preeclampsia. However, we have yet to distinguish whether placental NR4A2 may have a role in late-onset preeclampsia, as we only assessed NR4A2 levels in samples collected from early-onset cases of preeclampsia with preterm delivery (≤ 34 weeks). Though NR4A2 expression has been previously detected in the placenta 20,21,41 , alterations in placental expression across gestation had not yet been determined. We found that NR4A2 expression was higher at term compared to preterm gestation. Upregulation in placental NR4A2 has been hypothesised to be associated with inflammatory processes 21,44 . To further understand these changes across gestation, it would be beneficial to examine NR4A2 in first trimester placental tissue, where, if NR4A2 is involved in inflammation as hypothesised, there may be increased NR4A2 expression given many inflammatory pathways are integral in driving successful extravllous cytotrophoblast invasion and critical arteriole remodelling 20 .

Scientific Reports
Although NR4A2 was not significantly altered in the preterm pathological placenta, there is potential it could play a role in the pathophysiology underpinning placental dysfunction and disease. Our in vitro models simulate conditions that drive placental dysfunction, offering insight into whether NR4A2 has a role in placental development, and the development of placental disease. Placental hypoxia is a feature of placental dysfunction, where impaired uteroplacental blood supply can cause periods of abnormally low oxygen tension 8 . Simulating these  www.nature.com/scientificreports/ This study identified several potential actions that NR4A2 expression may regulate in the placenta specific cytotrophoblast cells under dysfunctional conditions. Silencing NR4A2 under hypoxia increased expression of the cytoprotective antioxidant genes HMOX-1 and GCLC. Indeed, given placental dysfunction is associated with oxidative stress 45 , this suggested that reducing NR4A2 expression could be beneficial. Intriguingly, silencing NR4A2 under hypoxia also increased the expression of SPINT1. Our team has recently reported the important finding that low expression of SPINT1 in the human placenta and maternal circulation is associated with placental insufficiency and growth restriction 9 . Again, this result suggests that therapeutic interventions that reduce NR4A2 may be advantageous.
However, we also found that silencing NR4A2 increased the expression of NLRP3, which is a key regulator of the inflammatory response and has been implicated in preeclampsia pathogenesis 46 . NR4A2 has been previously identified as a NLRP3 inflammasome activation-responsive gene in a human monocyte cell line, with the suggestion that its induction acts in a negative feedback loop to prevent sustained inflammasome activation 47 . This implies, in contrast to our first suggestion, that loss of NR4A2 may result in persistent inflammation, detrimental to the placenta. Furthermore, silencing NR4A2 also increased expression of NOX4, a marker of oxidative stress. Enhancement of oxidative stress could be harmful to the already stressed placenta. Thus, these findings reveal that a more complex regulation may be at play and further studies are required to gain a better understanding of compensation and causation.
As NR4A2 acts as a transcription factor 48 , it was not surprising that it was involved in the regulation of many different pathways in cytotrophoblast cells. These results have allowed us to identify the pathways associated with NR4A2, but the conflicting responses mean we cannot clearly conclude whether reduction of NR4A2 in cytotrophoblast cells is a harmful consequence of hypoxic stress or a beneficial adaptation to mediate hypoxic damage in the placenta. Advanced protein assessment could be undertaken in the future to validate these findings.
Although this work predominantly aimed to assess NR4A2 expression and function in the dysfunctional placenta, we were also able to look at the function of NR4A2 at a physiologically normal oxygen tension. We identified several important genes to be differentially expressed in normoxic conditions with NR4A2 suppression, not altered under hypoxia. Silencing NR4A2 beneficially decreased cytotrophoblast levels of BAX, a proapoptotic gene, but also adversely decreased expression IGF2 and EGFR, which are markers of cell proliferation and growth. These findings indicate that NR4A2 responds variably under different oxygen tensions. However, once again it is unclear whether reduction of NR4A2 expression may be beneficial.
Given these results suggest that the placenta is unlikely to be the source of increased circulating NR4A2 transcripts in individuals with pregnancies complicated by fetal growth restriction and preeclampsia, we examined whether the vasculature may be the source. Though we could detect NR4A2 expression in endothelial cells, we did not find increased expression under TNF-α-induced dysfunction. This suggests that the endothelium is also unlikely to be the origin of the increased circulating NR4A2 transcripts-but additional studies using additional endothelial cell types beyond HUVECs, and different inducers of endothelial dysfunction are required before this can be confirmed. An important consideration is that the circulating transcripts may also originate from other organs in the maternal system that we did not assess here. Additionally, treatment with the therapeutic pravastatin, a drug currently in trial to prevent preeclampsia, had no effect on NR4A2 expression under endothelial dysfunction, suggesting that pravastatin treatment does not affect regulation of this transcription factor.

Conclusion
The origin of elevated circulating NR4A2 transcripts in patients with preterm fetal growth restriction and preeclampsia remains unknown, but is unlikely to be from the placenta. Although we found that placental NR4A2 expression is not altered with preterm preeclampsia or fetal growth restriction, the gene does regulate several important intracellular pathways associated with oxidative stress, fetal growth, and inflammation under the dysfunctional condition of hypoxia. More research is required to confirm the role of NR4A2 in the placenta, Figure 7. NR4A2 expression in human umbilical vein endothelial cells with TNF-α induced dysfunction, and pravastatin treatment. There was no significant change in NR4A2 mRNA expression with the addition of TNFα. Pravastatin treatment did not alter NR4A2 expression from TNF-α only levels. The control group was not exposed to TNF-α. Data presented as fold change from TNF-α treatment ± SEM. n = 3 experimental replicates, with each sample from a different patient. Each experiment was run in duplicate.

Methods
Fetal oxygenation (FOX) study. Maternal blood was collected from women whose pregnancies were complicated by preterm fetal growth restriction across six tertiary hospitals in Australia and New Zealand, as previously described 13 . The blood was directly collected (after corticosteroid administration, prior to delivery) into PAXgene ® Blood RNA tubes (Pre-Analytix, Hombrechtikon, Switzerland) and processed according to manufacturer's instructions. Preterm fetal growth restriction was defined as a birthweight < 10th centile (www. gesta tion. net, Australian parameters) requiring iatrogenic delivery prior to 34 weeks' gestation with uteroplacental insufficiency (asymmetrical growth + abnormal artery Doppler velocimetry +/− oligohydramnios +/− abnormal fetal vessel velocimetry). Fetal growth restriction due to infection, chromosomal or congenital abnormalities, and multiple pregnancy was excluded. For our sub-analysis in the current study, the cases of growth restriction were split into preeclamptic and normotensive groups. Patient clinical characteristics are presented in Table 1.

Placenta and umbilical cord collection. Ethical approval was obtained from the Mercy Health Human
Research Ethics Committee (R11/34). Women presenting to the Mercy Hospital for Women (Heidelberg, Victoria) gave informed, written consent for the collection of their placenta and umbilical cord. Experiments were performed following institutional guidelines and regulations.
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 49 . Placentas were also obtained from cases of preterm fetal growth restriction (requiring delivery ≤ 34 weeks gestation) defined as a birthweight < 10th centile, according to Australian population charts 50 . Placental tissue from cases associated with congenital infection, chromosomal or congenital abnormalities and multiple pregnancies were excluded.
Term (delivery 37-41 weeks' gestation) and preterm placentas (delivery 24-36 weeks' gestation) were also collected from normotensive pregnancies where a fetus of normal birthweight percentile (> 10th centile relative to gestation), and no clinical evidence of growth restriction, was delivered. Preterm deliveries in this group were predominantly for iatrogenic reasons (including vasa/placenta previa and suspected placental abruption) or premature rupture of membranes. Cases with hypertensive disease or evidence of chorioamnionitis (confirmed by placental histopathology) were excluded.
Placental tissue was collected within 30 min of delivery. For the groups detailed above, tissue was cut from four sites of the placenta and washed in cold phosphate buffered saline (PBS; 137 mM NaCl, 10 mM Na 2 HPO 4 , Table 1. Patient characteristics of pregnancies complicated with fetal growth restriction with and without preeclampsia. Data are n (%),or median (IQR). Missing BMI data for n = 3 normotensive samples and n = 6 preeclamptic samples, and umbilical artery pH for n = 1 preeclamptic sample. Estimated fetal weight not available for n = 4 normotensive and n = 5 preeclamptic samples. *p < 0.05, **p < 0.01.  Tables 2, 3 and 4. Placentas were also obtained from healthy, normotensive term pregnancies (≥ 37 weeks' gestation) at elective caesarean section for explant dissection and cytotrophoblast isolation. Umbilical cords were collected for the isolation of Human Umbilical Vein Endothelial Cells (HUVECs).
Primary cytotrophoblast isolation and hypoxia treatment. Human primary cytotrophoblast were isolated from healthy, term placentas from elective caesarean section as previously described 54 . The cells were Table 2. Patient characteristics for placental tissue used to assess NR4A2 expression between preterm and term gestation. Maternal age unavailable for n = 1 term sample. BMI data unavailable for n = 6 preterm samples. ****p < 0.0001.  Table 3. Patient characteristics of cases with preeclampsia, fetal growth restriction and control samples for gene (mRNA) expression studies. BMI data unavailable for n = 3 preterm controls, n = 1 fetal growth restriction samples. Birthweight data unavailable for n = 1 preeclamptic sample. Statistical analysis compared the preeclamptic or fetal growth restricted samples to the preterm controls. *p < 0.05, **p < 0.01, ****p < 0.0001. Endothelial cell isolation and culture. HUVECs were isolated from the umbilical cord of normotensive pregnancies as previously described 55 . The HUVECs were cultured in M199 media (Life Technologies, California, USA) containing 20% newborn calf serum, 1% endothelial cell growth factor, 1% heparin (Sigma-Aldrich) and 1% AA and used between passages 1-3. Cells were plated in 24-well plates containing M199 media supplemented with 10% FCS, 1% endothelial cell growth factor, 1% heparin and 1% AA.

Endothelial dysfunction and statin treatment.
To induce endothelial dysfunction, the HUVECs were pre-treated with 10 ng/mL tumour necrosis factor (TNF)-α, and incubated at 37 °C, 20% O 2 and 5% CO 2 for 2 h as previous 53,56 . Following this, 200 µM pravastatin (candidate drug for the treatment of preeclampsia) (Sigma-Aldrich) was added and cells incubated for 24 h. Cell lysates were collected for subsequent analysis.
Enzyme linked immunosorbent assay (ELISA). Soluble fms-like tyrosine kinase-1 (sFlt-1) secretion was measured in cytotrophoblast conditioned culture media using the DuoSet Human VEGF R1/FLT-1 kit (R&D systems by Bioscience, Waterloo, Australia) 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 duplicates or triplicates and repeated with a minimum of three different patient samples. Data were tested for normal distribution and statistically tested as appropriate. Either an unpaired t test or Mann-Whitney test was used. 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).

Data availability
The datasets generated during and analysed during the current study are available from the corresponding author on reasonable request.