Abstract
Background: Preeclampsia is a severe complication of pregnancy. Chemerin is an adipokine secreted from adipose tissue and highly expressed in placenta. This study evaluated the biomarker potential of circulating chemerin to predict preeclampsia.
Methods: Maternal plasma and placenta were collected from women with early-onset preeclampsia (<34 weeks), with preeclampsia and eclampsia, or before preeclampsia diagnosis (36 weeks). Human trophoblast stem cells were differentiated into syncytiotrophoblast or extravillous trophoblasts across 96 hours. Cells were cultured in 1% O2 (hypoxia) or 5% O2 (normoxia). Chemerin was measured by enzyme-linked immunosorbent assay (ELISA) and RARRES2 (gene coding chemerin) by reverse transcription-quantitative polymerase chain reaction.
Results: Circulating chemerin was increased in 46 women with early-onset preeclampsia (<34 weeks) compared to 17 controls (P < .0006). Chemerin was increased in placenta from 43 women with early-onset preeclampsia compared to 24 controls (P < .0001). RARRES2 was reduced in placenta from 43 women with early-onset preeclampsia vs 24 controls (P < .0001).
Chemerin was increased in plasma from 26 women with established preeclampsia (P = .006), vs 15 controls. Circulating chemerin was increased in 23 women who later developed preeclampsia vs 182 who did not (P = 3.23 × 10−6).
RARRES2 was reduced in syncytiotrophoblast (P = .005) or extravillous trophoblasts (P < .0001). Hypoxia increased RARRES2 expression in syncytiotrophoblast (P = .01) but not cytotrophoblast cells.
Conclusions: Circulating chemerin was elevated in women with early-onset preeclampsia, established preeclampsia, and preceding preeclampsia diagnosis of preeclampsia. RARRES2 was dysregulated in placenta complicated by preeclampsia and may be regulated through hypoxia. Chemerin may have potential as a biomarker for preeclampsia but would need to be combined with other biomarkers.
Keywords: chemerin, adipokine, placenta, hypoxia, preeclampsia, biomarker
Preeclampsia is a complication of pregnancy that affects between 2% and 8% of pregnancies and remains a leading contributor to maternal and perinatal morbidity and mortality worldwide (1).
Protein biomarkers found in the maternal circulation offers the potential to detect preeclampsia early. Clinical biomarkers such as soluble fms-like tyrosine kinase-1 (sFlt-1) and placental growth factor (PlGF) are proteins used to assist in detection of women with preeclampsia (2). Although these markers perform well, they are useful as a “rule out” test and do not describe patients that will develop preeclampsia (3). Unfortunately, there are still no biomarkers that can effectively “rule in” and predict those who will develop preeclampsia (3). Our laboratory focused on investigating placental proteins that are released into the maternal circulation (4-7). Such placental enriched proteins could be useful for identifying placental dysfunction, a hallmark of preeclampsia.
Chemerin is an adipokine that is secreted by white adipose tissue and skin (8). Chemerin likely plays an important role in obesity and metabolic diseases (9). In pregnancy, elevated chemerin serum levels have been identified among women with gestational diabetes (10) and preeclampsia (11). As this molecule is highly expressed in the placenta and is elevated in blood samples obtained from individuals with inflammatory diseases, it may be a molecule used to predict preeclampsia. Although other studies have identified elevated chemerin levels in preeclampsia, the predictive potential of this protein remains to be clarified.
This study assessed chemerin levels in both the placenta and maternal circulation from several large prospective cohorts. This includes 2 cohorts from Australia and a large biobank obtained from women with severe preeclampsia and eclampsia collected from South Africa (12). We further characterized chemerin and the possible role it plays in the pathogenesis of preeclampsia through in vitro assays using human first-trimester cytotrophoblast stem cells.
Materials and Methods
Early-Onset Preeclampsia <34 Weeks' Gestation
The established cohort is a prospective study conducted at Mercy Hospital for Women where samples were collected from 46 participants with established early-onset preeclampsia (<34 weeks' gestation) and 17 gestation matched controls with no complications. Early-onset preeclampsia was defined according to the American College of Obstetricians and Gynaecologists (ACOG) guidelines (13). Ethics approval was granted by Mercy Health Human Research Ethics Committee (R11/34), and participants presenting to the Mercy Hospital for Women gave informed, written consent for sample collection. No evidence of subclinical infection was identified. Clinical characteristics are provided in Table 1. Maternal blood was collected in a 9 mL EDTA tube, samples were centrifuged, and plasma was stored at −80°C until further analysis.
Table 1.
Maternal clinical characteristics for <34 weeks plasma samples
| Controls (n = 23) | Preeclampsia (n = 52) | P-value | |
|---|---|---|---|
| Maternal age (years) Mean ± SEM | 32.63 ± 0.95 | 30.83 ± 0.84 | .3304 |
| Gestation at blood collection (weeks) Mean ± SEM | 26.38 ± 0.98 | 29.65 ± 0.39 | .0063 |
| Gestation at delivery (weeks) Mean ± SEM | 37.38 ± 1.64 | 29.95 ± 0.43 | <.0001 |
| Parity no. (%) | |||
| 0 | 11 (45.83) | 33 (71.74) | |
| 1 | 8 (33.33) | 9 (19.56) | .0943 |
| ≥2 | 5 (20.84) | 4 (8.70) | |
| SBP at delivery (mmHg) Median (IQR) | 120.00 (125.00-110.00) | 173.50 (180.00-165.00) | <.0001 |
| DBP at delivery (mmHg) Median (IQR) | 75.00 (80.00-70.00) | 100.00 (110.00-99.75) | <.0001 |
| BMI (kg/m2) Median (IQR) | 24.25 (28.93-20.55) | 28.50 (35.20-26.00) | .0046 |
| Birth weight (g) Median (IQR) | 3405 (3823-3180) | 1277 (1625-777.50) | <.0001 |
| Male no. (%) | 12 (50.00) | 19 (41.30) | .4869 |
Mann-Whitney test was used for nonparametric data and chi-square tests for categorical variables. BMI data missing for 3/46 PE samples. SBP and DBP data missing for 1/24 control samples.
Abbreviations: BMI, body mass index; IQR, interquartile range; SBP, systolic blood pressure; DBP, diastolic blood pressure.
Placental tissue was collected from 43 women with early-onset preeclampsia and 24 controls matched for gestational age who delivered via caesarean section. Control samples did not have fetal growth restriction or hypertensive disorders and delivered preterm due to rupture of membranes or placenta previa. Ethics approval was granted by Mercy Health Human Research Ethics Committee (R11/34), and participants presenting to the Mercy Hospital for Women gave informed, written consent for sample collection. Patient characteristics are provided in Table 2.
Table 2.
Maternal clinical characteristics for <34 weeks placental protein samples
| Controls (n = 21) | Preeclampsia (n = 43) | P-value | |
|---|---|---|---|
| Maternal age (years) Mean ± SEM |
30.14 ± 1.61 | 31.51 ± 0.83 | .4058 |
| Gestation at delivery (weeks) Mean ± SEM |
30.11 ± 0.54 | 30.20 ± 0.36 | .8995 |
| BMI (kg/m2) Median (IQR) |
28.20 (34.85-24.25) | 27.00 (35.60-25.00) | .9958 |
| Parity no. (%) | |||
| 0 | 5 (23.81) | 32 (74.42) | .0006 |
| 1 | 11 (52.38) | 7 (16.28) | |
| ≥2 | 5 (23.81) | 4 (9.30) | |
| SBP at delivery (mmHg) Median (IQR) |
120 (130-111) | 170 (185-160) | <.0001 |
| DBP at delivery (mmHg) Median (IQR) |
70 (80.00-67.50) | 100 (110.00-95.00) | <.0001 |
| Birth weight (g) Median (IQR) |
1585 (1943-1278) | 1253 (1479-866.8) | .0128 |
| Male no. (%) | 11 (52.38) | 23 (53.49) | .9336 |
Unpaired t-test was used for normally distributed data, Mann-Whitney U tests for nonparametric data, and chi-square tests for categorical variables. BMI data missing for 5/21 control samples and 10/43 PE samples. Birth weight data missing for 1/43 PE samples.
Abbreviations: BMI, body mass index; DBP, diastolic blood pressure; IQR, interquartile range; SBP, systolic blood pressure.
Placental samples were collected immediately after delivery. Tissue was randomly sampled from 4 separate sites on the maternal side of the placenta, washed with ice-cold phosphate buffered saline, homogenized together, and aliquoted and preserved in RNAlater stabilization solution (Thermo Fisher Scientific). Samples were stored at −8 °C for isolation of RNA and protein.
BUMPS Cohort
The biomarker and ultrasound measures for preventable stillbirth (BUMPS) study is a large prospective study conducted at the Mercy Hospital for Women, Melbourne, Australia, which involved the collection of blood at 36 weeks' gestation (35+0-37+0) (6, 14). From the first 1000 BUMPS participants, a case cohort of 205 samples were selected for further analysis: 23 who developed preeclampsia and 182 randomly selected controls (representing the “cohort,” women who did not develop preeclampsia). Preeclampsia was diagnosed according to the ACOG guidelines (15). No evidence of subclinical infection was identified. Ethical approval was obtained from the Mercy Health and Human Research Ethics Committee (approval number 2019-012), and participants gave informed, written consent. Clinical characteristics are provided in Table 3. Whole blood was collected in 9 mL EDTA tubes. Tubes were centrifuged, and plasma supernatant was obtained and stored at −80°C until sample analysis.
Table 3.
Maternal characteristics and pregnancy outcomes for the BUMPS cohort
| BUMPs Cohort Clinical Characteristics | ||||
|---|---|---|---|---|
| Control | Preeclampsia | P-value | ||
| Gestation at collection (weeks) Median (IQR) |
36.14 (35.71-36.57) | 36.28 (35.57-36.57) | .89 | |
| Birth weight (grams) Median (IQR) |
3490 (3185-3820) | 3150 (2550-3450) | .0002 | |
| Crown-heel length (cm) Median (IQR) |
51 (50-52) | 51 (47-52) | .2152 | |
| Fetal sex No. (%) |
Male | 91 (50) | 91 (50) | .84 |
| Female | 12 (52) | 11 (48) | ||
| Maternal age (years) Median (IQR) |
32 (29.75-35) | 34 (32-37) | .02 | |
| BMI (kg/m2) Median (IQR) |
24.55 (22.29-28.12) | 27.88 (24.14-30.66) | .02 | |
| Parity no. (%) | 0 | 89 (49) | 19 (82.6) | .007 |
| 1 | 72 (39) | 4 (17.4) | ||
| ≥2 | 21 (12) | 0 (0) | ||
| Smoking no. (%) | Nonsmoker | 170 (93.4) | 21 (91.3) | .71 |
| Former smoker | 6 (0.03) | 1 (0.04) | ||
| Smoker | 6 (0.03) | 1 (0.04) | ||
| GDM no. (%) | None | 160 (92) | 17 (74) | .01 |
| Diet-controlled | 14 (8) | 6 (26) | ||
| Insulin-controlled | 0 (0) | 0 (0) | ||
Data presented as median (25th-75th percentile) and as number (%) if categorical. Kruskal-Wallis tests were used for continuous data. Fisher's exact tests were used for categorical variables. Small for gestational age is defined as an infant birthed <10th birth weight centile. Significant difference is identified when P < .05.
Abbreviations: BMI, body mass index, BUMPS, biomarker and ultrasound measures for preventable stillbirth; GDM, gestational diabetes mellitus.
PROVE Cohort
The Preeclampsia Obstetric Adverse Events (PROVE) cohort is a collaborative study being conducted in Tygerberg Hospital (Stellenbosch University, Cape Town, South Africa) (12). We examined plasma samples from 26 women in the PROVE cohort who had preeclampsia and significant end-organ complications (pulmonary oedema, raised liver enzymes, haemolysis, or renal failure), 34 with eclampsia and 15 normotensive pregnancies. Clinical characteristics have been previously published (5), and diagnosis was described according to the ACOG guidelines (13). No evidence of subclinical infection was identified. All participants gave informed, written consent, and ethical approval was obtained from Stellenbosch University (approval number N17/05/048). Whole blood was collected in 9 mL EDTA tubes, samples were centrifuged, and plasma was stored at −80°C until sample analysis.
Human Trophoblast Stem Cells
Human trophoblast stem cells (hTSCs) are cytotrophoblast stem cell lines that were imported from the RIKEN BRC through the National BioResource Project of the MEXT/AMED, Japan. Cells were cultured and differentiated into either syncytiotrophoblast or extravillous trophoblast (EVT) cells according to Okae et al (16). See Tables 4-6 for cell media reagents.
Table 4.
Reagent information for cytotrophoblast media
| TS medium – 50 mL (stable for 2 weeks at 4°C) | |
|---|---|
| TS basal medium | 50 mL |
| DMEM/F12 (Life Technologies) | 48.45 mL |
| KSR (Life Technologies) | 4% |
| ITS-X supplement (Novachem Pty Ltd) | 1% |
| Penicillin-streptomycin (Life Technologies) | 0.5% |
| Bovine serum albumin (Novachem Pty Ltd) | 0.3% |
| 200 mM L-Ascorbic acid (Novachem Pty Ltd) | 1.5µg/mL |
| 10 mM A83-01 (Novachem Pty Ltd) | 25 μL |
| 4 mM CHIR99021 (Novachem Pty Ltd) | 25 μL |
| 10 mM Y27632 (Novachem Pty Ltd) | 12.5 μL |
| 1005 µg/mL EGF (Novachem Pty Ltd) | 12.5 μL |
| VPA (Valporic acid) (Novachem Pty Ltd) | 6.25 μL |
Table 5.
Reagent information for syncytiotrophoblast media
| ST(2D) medium – 50 mL (prepare fresh before use) | |
|---|---|
| Differentiating basal medium | 50 mL |
| DMEM/F12 (Life Technologies) | 48.75 mL |
| ITS-X supplement (Novachem Pty Ltd) | 1% |
| Penicillin-streptomycin (Life Technologies) | 0.5% |
| Bovine serum albumin (Novachem Pty Ltd) | 0.3% |
| KSR (Life Technologies) | 2 mL |
| 5 mM Forskolin (Novachem Pty Ltd) | 20 μL |
| 10 mM Y27632 (Novachem Pty Ltd) | 12.5 μL |
Table 6.
Reagent information for EVT media
| EVT medium – 50 mL (prepare fresh before use) | |
|---|---|
| Differentiating basal medium | 50 mL |
| DMEM/F12 (Life Technologies) | 48.75 mL |
| ITS-X supplement (Novachem Pty Ltd) | 1% |
| Penicillin-streptomycin (Life Technologies) | 0.5% |
| Bovine serum albumin (Novachem Pty Ltd) | 0.3% |
| KSR (Life Technologies) | 2 mL |
| 10 mM A83-01 (Novachem Pty Ltd) | 37.5 μL |
| 200µg/mL NRG1 (Cell Signalling Technology, Danvers, MA, USA) | 25 μL |
| 10 nM Y27632 (Novachem Pty Ltd) | 12.5 μL |
Abbreviations: EVT, extravillous trophoblast.
hTSCs Cultured Under Hypoxic Conditions
Cells were seeded at 60 000 cells per well in a 24-well cell culture plate or 15 000 cells per well in a 96-well tissue culture place with syncytial (ST[2D]) media. Cells were incubated at 37°C with 8% O2, and 5% O2 (physiological normoxic conditions) for 96 hours to allow syncytilization to occur. Cells were transferred to culture in 1% O2 for hypoxic conditions or remained under 8% O2 for normoxic conditions for an additional 48 hours (treated in triplicate, n = 5). Following treatment, cell lysates were collected for RNA extraction to perform reverse transcription-quantitative polymerase chain reaction (qRT-PCR) and confirm cell differentiation and experiment.
hTSCs Exposed to Tumor Necrosis Factor α and Interleukin 6
Cells were seeded and incubated at 3 °C with 8% O2 and 5% O2 for 72 hours to allow for syncytilization to occur. Cells were then treated with 0, 0.1 and 1 ng/mL of tumor necrosis factor α (17) or interleukin 6 (18) for 24 hours (treated in triplicate, n = 5). Following treatment, cell lysates were collected for RNA extraction to perform qRT-PCR.
ELISA Measurement of Circulating Chemerin
Chemerin was measured using the R&D Systems Human Chemerin DuoSet ELISA kit following the manufacturer's instructions (R and D Systems catalog no. DY2324, RRID: AB_2928995).
Quantitative RT-PCR to Measure Chemerin Gene Expression
RNA was extracted from placental samples, cytotrophoblast, syncytiotrophoblast, and EVT cells using the GenElute Mammalian Total RNA Miniprep Kit (Sigma-Aldrich), according to the manufacturer's instructions. RNA was quantified using the Nanodrop ND 100 spectrophotometer (Nanodrop Technologies Inc). A total of 1 μg (placental) or 100 ng (cellular) RNA was reverse transcribed to cDNA using the Applied Biosystems high-capacity cDNA Reverse Transcriptase Kit (Life Technologies, Carlsbad, CA, USA) following the manufacturer's instructions. Taqman fast advanced Master Mix (Applied Biosystems) and specific fluorescein amidite-labeled Taqman Gene expression Assays (Life Technologies) were used to measure the gene expression of RARRES2 (Retinoic Acid Receptor Responder 2, Assay ID: Hs01123775_m1). To confirm differentiation into different cell types, cell morphology was confirmed. In addition, we measured well-known EVT markers, TEAD4 (TEA Domain Transcription Factor 4, Assay ID: Hs01125032_m1), HLAG (Human Leukocyte Antigen G, Assay ID: Hs03045108_m1). To confirm differentiation into syncytiotrophoblast, we measured CDH2 (Cadherin-2, Assay ID: Hs00983056_m1), syncytiotrophoblast SDC1 (Syndecan 1, Assay ID: Hs00896423_m1). These genes are used to characterize these cell types in Okae et al (16). qRT-PCR was performed on the CFX384 (Bio-Rad) with thermocycling parameters: 95°C for 20 seconds, 40 cycles of denaturation for 3 seconds at 95°C, and 60°C for 30 seconds. No product was detected in the nontemplate control and gene expression was normalized to the geometric mean of CYC1 (Cytochrome C1, Assay ID: Hs00357717_m1) and TOP1 (DNA Topoisomerase I, Assay ID: Hs00243257_m1) housekeepers (run in duplicate). Housekeepers were optimized and expression was stable across cell types and pathologies of pregnancy (19). Samples were run in duplicate, and an average threshold (Ct) value was used. Gene expression was normalized to the Ct mean of each control group and the 2−ΔΔCt method of the mean was used and expressed as fold change relative to controls.
Statistical Analysis
Maternal characteristics were compared for women diagnosed with early-onset preeclampsia, before diagnosis, or after clinical diagnosis of preeclampsia (13), or eclampsia compared to normotensive, gestation-matched controls using a Mann-Whitney U test for continuous data and Fisher's exact test for categorical data. All data generated was tested for normality using the Anderson-Darling test, D’Agostino and Pearson test, Shapiro-Wilk test, and Kolmogorov-Smirnov test. When 2 groups were analyzed for an unpaired dataset, either an unpaired t-test or a Mann-Whitney (nonparametric) test was used. For the analysis of paired data (hTSCs exposed to hypoxia), either a paired t-test (parametric) or a Wilcoxon ranked (nonparametric) test was used. For 3 or more groups, a one-way analysis of variance (parametric) or a Kruskal-Wallis (nonparametric) test was used. Predictive performance was measured using area under receiver operating characteristic curve (AUC). All data was represented as mean ± SEM, median [interquartile range (IQR) or fold change (95% CI)]. P < .05 was considered significant. All analysis was performed using GraphPad Prism 9.3.1 (GraphPad Software, LLC).
Results
Chemerin Protein Expression is Increased in Maternal Circulation From Women Diagnosed With Preeclampsia
Circulating chemerin was measured in 46 women with early-onset preeclampsia and compared to 24 controls. Circulating chemerin was increased 1.34-fold in women with early-onset preeclampsia (Fig. 1A, P < .0006; preeclampsia median: 4.7 × 105 pg/mL; IQR, 3.8 × 105-6.1 × 105 pg/mL; control median: 3.5 × 105 pg/mL; IQR, 2.6 × 105-4.3 × 105 pg/mL). AUC was 0.75 (Fig. 1B).
Figure 1.
Circulating and placental chemerin was increased in patients with preeclampsia, and preeclampsia and eclampsia with significant end-organ failure, whereas gene expression of RARRES2 was reduced. Circulating chemerin was increased in 46 women with early-onset preeclampsia compared to 17 gestation matched controls (A). The discriminatory power of chemerin is shown as a receiver operating characteristic curve with an area under the curve of 0.75 (B). Chemerin protein expression was increased in placental samples from 43 women with early onset preeclampsia, compared to 21 term controls (C). Chemerin gene RARRES2 was significantly reduced in placenta from 61 women with early-onset preeclampsia compared to 18 controls (D). Circulating chemerin was the measure in PROVE cohort. Circulating chemerin was increased in the PROVE cohort where 15 women had severe preeclampsia and 35 with eclampsia (E). Data was presented as median ± interquartile range with each dot representing an individual patient. *P < .05, **P < .01, ****P < .0001.
Abbreviations: PROVE, Preeclampsia Obstetric Adverse Events.
Next, chemerin was assessed in placenta from women with early-onset preeclampsia and normotensive controls. Chemerin protein expression was increased in placenta from 43 women with preeclampsia compared to 21 controls (Fig. 1C; P < .02). mRNA expression of RARRES2 (chemerin gene) was reduced in placentas from 61 women with early-onset preeclampsia compared to 21 controls (Fig. 1D; P < .0001).
To validate the findings that chemerin was increased in preeclampsia, we measured circulating chemerin in the PROVE cohort from South Africa. Women from the PROVE cohort were diagnosed with preeclampsia with severe features, including significant end-organ dysfunction or eclampsia. In the PROVE cohort, circulating chemerin was increased 1.62-fold in 26 women with preeclampsia (P = .006; median = 1.93 × 105 pg/mL; IQR, 1.2 × 105 pg/mL-2.4 × 105 pg/mL) and 1.38-fold in 34 women with eclampsia (P = .054; median: 1.6 × 105 pg/mL; IQR 1.0 × 105 pg/mL-2.2 × 105 pg/mL) compared with 15 controls (Fig. 2C; median: 1.1 × 105 pg/mL; IQR, 8.5 × 104 pg/mL-1.4 × 105 pg/mL).
Figure 2.
Circulating chemerin is increased in plasma collected from women diagnosed with either preeclampsia or eclampsia. Circulating chemerin was measured in the BUMPS cohort. Circulating chemerin was increased in 23 women who developed preeclampsia in the BUMPS cohort compared to 182 controls (A). Area under the receiver operator curve of 0.80 (B). Data is presented as median ± interquartile range with each dot representing an individual sample. *P < .05, ****P < .0001.
Abbreviations: BUMPS, biomarker and ultrasound measures for preventable stillbirth.
Circulating Chemerin is Increased in Women Later Diagnosed With Preeclampsia, Established Preeclampsia or Eclampsia
To validate findings from the early onset preeclampsia cohort and the PROVE cohort, we assessed circulating chemerin in a predictive cohort (BUMPS). The BUMPS cohort allows us to assess the biomarker potential of chemerin at 36 weeks' gestation, collected before disease onset, which can give insight into its predictive power. Next, chemerin levels were assessed among 36-week blood samples obtained from the BUMPS study. We observed a 1.29-fold increase in plasma chemerin levels from 23 women with preeclampsia compared with 182 controls (Fig. 2A, P = 3.23 × 10−6). Preeclampsia median was 1.1 × 105 pg/mL; IQR, 9.3 × 104 pg/mL-1.3 × 105 pg/mL; control median was 8.5 × 104 pg/mL; IQR, 7.3 × 104 pg/mL-1.0 × 105 pg/mL). AUC was 0.80 (Fig. 2B).
Chemerin gene RARRES2 was measured following cellular differentiation of cytotrophoblast stem cells into syncytiotrophoblast or EVT cells. We confirmed differentiation to syncytiotrophoblast cells through a reduction in cell border marker CDH2 (Fig. 3A; P < .001) and increase in syncytiotrophoblast gene SDC1 (Fig. 3C; P < .0001). In ST, RARRES2 was significantly decreased at 48 hours (P = .005) and 96 hours (Fig. 3E; P = .03). We confirmed cell differentiated into EVTs by a reduction in cytotrophoblast marker TEAD4 (Fig. 3B; P < .0001) and increased expression of EVT marker HLAG (Fig. 3D; P = .03). RARRES2 expression was reduced in EVT cells 48 hours (Fig. 3F; P < .0001) and 96 hours (P < .0001) after differentiation.
Figure 3.
RARRES2 gene expression is reduced following differentiation of cytotrophoblast stem cells to syncytiotrophoblast and EVT cells. hTSCs were differentiated into syncytiotrophoblast and EVT cells from 0 hours (0 hours) to 96 hours (96 hours). To confirm cytotrophoblast differentiation, reduction in cytotrophoblast CDH2 (A), reduction in TEAD4 (B), increase in syncytiotrophoblast marker SDC1 (C), and increase in EVT marker HLAG (D) mRNA expression were observed. RARRES2 mRNA expression was measured throughout differentiation from cytotrophoblast (0 hours) to either syncytiotrophoblast (E) or EVT (F) differentiation (96 hours). RARRES2 expression was reduced in syncytiotrophoblast (E) and EVT cells (F) following differentiation. mRNA expression was normalized to the geometric mean of housekeeper genes. All experiments were repeated 5 times in triplicate. Data was presented as mean ± SEM. *P < .05, **P < .01, ***P < .001, ****P < .0001.
Abbreviations: EVT, extravillous trophoblast; hTSC, human trophoblast stem cell.
Preeclampsia is associated with poor perfusion and placental hypoxia. To further investigate RARRES2 in a model of preeclampsia, we measured expression of RARRES2 in cytotrophoblast and syncytiotrophoblast cells following exposure to hypoxia (1% O2) and normoxia (8% O2). RARRES2 mRNA expression was not altered in cytotrophoblast cells (Fig. 4A; P = .68) but was increased in syncytiotrophoblast cells when cultured in hypoxia (Fig. 4B; P = .01).
Figure 4.
The effect of hypoxia on RARRES2 expression in cytotrophoblast and syncytiotrophoblast placental cells. Culture under hypoxia (1% O2) did not alter RARRES2 expression in cytotrophoblast cells (A) but increased expression in syncytiotrophoblast cells (B) compared to control physiological conditions (normoxia; 8% O2). Differentiation of hTSCs into EVT and ST cells observed a decrease in RARRES2 mRNA expression (C). After culture in hypoxic conditions, RARRES2 mRNA expression was not altered in cytotrophoblast cells but increased in syncytiotrophoblast cells. mRNA expression was normalized to the geometric mean of housekeeper genes (D). Experiments were repeated 5 times in triplicate. Data was presented as mean ± SEM. Created with BioRender.com. *P < .05, **P < .01, ***P < .001, ****P < .0001.
Abbreviations: EVT, extravillous trophoblast; hTSC, human trophoblast stem cell; ST, syncytiotrophoblast.
Overall, in vitro studies found RARRES2 mRNA expression was reduced following cellular differentiation from cytotrophoblast to syncytiotrophoblast and EVT cells (Fig. 4C). Once exposed to hypoxia, RARRES2 mRNA expression was increased in syncytiotrophoblast but not cytotrophoblast cells (Fig. 4D).
Discussion
This study identified increased circulating chemerin in plasma collected before and after diagnosis of preeclampsia. The in vitro gene studies showed a reduction in RARRES2 when trophoblast cells were differentiated into syncytiotrophoblast and EVT cells. Following culture in hypoxic conditions, RARRES2 expression was increased in syncytiotrophoblast but not cytotrophoblast cells.
Chemerin is a proinflammatory chemokine that influences vascular and metabolic changes in chronic conditions, such as obesity (20). It is highly expressed in white adipose tissue, lung, liver, and placental tissue. Our study was unique as we measured circulating chemerin in 3 separate cohorts. We found elevated circulating chemerin in a cohort of women diagnosed with early-onset preeclampsia. This was supported by our second cohort, which found increased chemerin levels in plasma from women with severe preeclampsia (with severe features) and eclampsia (cohort from South Africa). The increase in circulating chemerin from women with preeclampsia has been reported in the literature (11, 21, 22).
In addition to our initial analysis, we corrected circulating chemerin for maternal body mass index (BMI) (an established risk factor for preeclampsia; data not shown). Interestingly, a positive correlation between circulating chemerin and BMI in the early-onset cohort was identified, but this was not the case in the BUMPs or PROVE cohorts. Patients with early-onset preeclampsia typically display more severe symptoms compared to those who are diagnosed with late-onset preeclampsia. Although clinical characteristics of preeclampsia are the same, recent studies highlight differences in molecular pathways between both subtypes (23). Differences in molecular pathways associated with chemerin signaling should be assessed in early- and late-onset preeclampsia in future studies.
A significant limitation of these studies includes the small number of samples used in the analyses. Our study was unique as we also measured the biomarker potential of chemerin in women before their diagnosis of preeclampsia (collected at 36 weeks' gestation). We found increased circulating chemerin in women who later developed preeclampsia, which increases the potential of chemerin as a biomarker. As placental molecules are known to leak into the maternal circulation, we hypothesized that the increased levels within the plasma are mainly sourced from the placenta; however, further studies would need to be undertaken to confirm this.
Whilst this study found associations with increased circulating chemerin and incidence of preeclampsia, we also discovered elevated chemerin levels in placental lysates from women with early-onset preeclampsia (<34 weeks' gestation). Interestingly, we also identified a reduction in chemerin (RARRES2) mRNA expression in the placenta. In our study, reduced RARRES2 mRNA expression was opposite to the increase in chemerin protein expression in the placenta. Other studies have also shown discrepancies in chemerin mRNA and protein levels. Dobrzyn et al found increased mRNA but decreased protein expression of chemerin receptor gene and protein levels in endometrial tissue explants when treated with prostaglandins in early gestation (24). Our study may indeed reflect more increased translation of chemerin in the placenta, although additional studies are required to confirm this.
In the PROVE cohort, we found significant increase of chemerin in plasma from women in South Africa with severe preeclampsia and eclampsia. Samples from the PROVE cohort were collected from women with severe preeclampsia (some with end-organ damage) and eclampsia when high blood pressure results in increased neurological imbalances, leading to seizures. Patients from this cohort demonstrate end-organ dysfunction, which is different to the other cohorts in this study. The changes in chemerin concentration in the PROVE cohort (1.54 vs 1.34-fold change) may reflect severity of preeclampsia cases compared to the other cohorts in which we measured chemerin. Of note however, the PROVE cohort is a smaller cohort with a wide range in gestational age at sampling. Thus, further validation is important.
Our study also identified high circulating chemerin in patients preceding the diagnosis of preeclampsia, the BUMPS cohort. Current literature suggests that chemerin acts as a proinflammatory cytokine when inflammation is induced. As inflammation is implicated in the onset of preeclampsia, chemerin might be a contributor to the inflammatory response (25). Following receiver operating characteristic analysis, the AUC for chemerin in this cohort was 0.80. This is comparable to the sFlt/PlGF ratio measured in the fetal longitudinal assessment of growth study, another cohort collected at 36 weeks before diagnosis of preeclampsia (AUC 0.79) (5). We suggest that future studies combine sFLT1/PlGF and chemerin to potentially improve disease prediction.
Our in vitro studies found RARRES2 is highly expressed in cytotrophoblast cells of the placenta. As placental hypoxia is a major characteristic of preeclampsia, we exposed mononuclear cytotrophoblasts and multinuclear syncytiotrophoblast cells to 1% O2. Increased RARRES2 mRNA was measured in syncytiotrophoblast cells following exposure to hypoxia. Chua et al has also observed increased RARRES2 in human coronary artery endothelial cells after exposure to hypoxia (26). Therefore, our data suggests that placental hypoxia may abhorrently increase RARRES2 expression in the syncytiotrophoblast. Given there is now strong evidence that preeclampsia is associated with intermittent hypoxia or hypoxia and reoxygenation injury, mimicking this condition in vitro may produce further increases in cellular RARRES2 or chemerin protein. Although we attempted to measure chemerin in the media from hypoxia-exposed syncytiotrophoblast, we were unable to detect its presence.
Overall, circulating chemerin levels were increased in 2 cohorts of women with established preeclampsia and eclampsia and in a cohort of women who later developed preeclampsia. We identified dysregulated RARRES2 in placenta of patients diagnosed with preeclampsia and suggest hypoxia as a possible contributor. While chemerin is significantly higher at 36 weeks' gestation in those destined to deliver with term preeclampsia, it may have potential as a biomarker for preeclampsia as a rule in test but would need to be combined with other biomarkers, such as PlGF and sFLT.
Acknowledgments
We thank Alison Abboud, Danica Idzes, and Valerie Kyritsis for recruitment and collection of blood samples. We also thank Gabrielle Pell, Rachel Murdoch, and Genevieve Christophers for their assistance in recruiting participants and blood samples. We also thank the pathology, health information services, and antenatal clinic staff at the Mercy Hospital for Women for their assistance in conducting this research. First trimester cytotrophoblast stem cell line was obtained from the RIKEN BRC through the National BioResource Project of the MEXT/AMED, Japan.
Contributor Information
Lucy A Bartho, Translational Obstetrics Group, The Department of Obstetrics and Gynaecology, Mercy Hospital for Women, University of Melbourne, Heidelberg, Victoria 3084, Australia; Mercy Perinatal, Mercy Hospital for Women, Heidelberg, Victoria 3084, Australia.
Manju Kandel, Translational Obstetrics Group, The Department of Obstetrics and Gynaecology, Mercy Hospital for Women, University of Melbourne, Heidelberg, Victoria 3084, Australia; Mercy Perinatal, Mercy Hospital for Women, Heidelberg, Victoria 3084, Australia.
Susan P Walker, Translational Obstetrics Group, The Department of Obstetrics and Gynaecology, Mercy Hospital for Women, University of Melbourne, Heidelberg, Victoria 3084, Australia; Mercy Perinatal, Mercy Hospital for Women, Heidelberg, Victoria 3084, Australia.
Catherine A Cluver, Department of Obstetrics and Gynecology, Stellenbosch University, Cape Town 7505, South Africa.
Roxanne Hastie, Translational Obstetrics Group, The Department of Obstetrics and Gynaecology, Mercy Hospital for Women, University of Melbourne, Heidelberg, Victoria 3084, Australia; Mercy Perinatal, Mercy Hospital for Women, Heidelberg, Victoria 3084, Australia; Department of Obstetrics and Gynecology, Stellenbosch University, Cape Town 7505, South Africa.
Lina Bergman, Department of Obstetrics and Gynecology, Stellenbosch University, Cape Town 7505, South Africa; Department of Women's and Children's Health, Uppsala University, Uppsala 751 85, Sweden; Department of Obstetrics and Gynecology, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Gothenburg 405 30, Sweden.
Natasha Pritchard, Translational Obstetrics Group, The Department of Obstetrics and Gynaecology, Mercy Hospital for Women, University of Melbourne, Heidelberg, Victoria 3084, Australia; Mercy Perinatal, Mercy Hospital for Women, Heidelberg, Victoria 3084, Australia.
Ping Cannon, Translational Obstetrics Group, The Department of Obstetrics and Gynaecology, Mercy Hospital for Women, University of Melbourne, Heidelberg, Victoria 3084, Australia; Mercy Perinatal, Mercy Hospital for Women, Heidelberg, Victoria 3084, Australia.
Tuong-Vi Nguyen, Translational Obstetrics Group, The Department of Obstetrics and Gynaecology, Mercy Hospital for Women, University of Melbourne, Heidelberg, Victoria 3084, Australia; Mercy Perinatal, Mercy Hospital for Women, Heidelberg, Victoria 3084, Australia.
Georgia P Wong, Translational Obstetrics Group, The Department of Obstetrics and Gynaecology, Mercy Hospital for Women, University of Melbourne, Heidelberg, Victoria 3084, Australia; Mercy Perinatal, Mercy Hospital for Women, Heidelberg, Victoria 3084, Australia.
Teresa M MacDonald, Translational Obstetrics Group, The Department of Obstetrics and Gynaecology, Mercy Hospital for Women, University of Melbourne, Heidelberg, Victoria 3084, Australia; Mercy Perinatal, Mercy Hospital for Women, Heidelberg, Victoria 3084, Australia.
Emerson Keenan, Translational Obstetrics Group, The Department of Obstetrics and Gynaecology, Mercy Hospital for Women, University of Melbourne, Heidelberg, Victoria 3084, Australia; Mercy Perinatal, Mercy Hospital for Women, Heidelberg, Victoria 3084, Australia.
Natalie J Hannan, Translational Obstetrics Group, The Department of Obstetrics and Gynaecology, Mercy Hospital for Women, University of Melbourne, Heidelberg, Victoria 3084, Australia; Mercy Perinatal, Mercy Hospital for Women, Heidelberg, Victoria 3084, Australia.
Stephen Tong, Translational Obstetrics Group, The Department of Obstetrics and Gynaecology, Mercy Hospital for Women, University of Melbourne, Heidelberg, Victoria 3084, Australia; Mercy Perinatal, Mercy Hospital for Women, Heidelberg, Victoria 3084, Australia.
Tu’uhevaha J Kaitu’u-Lino, Translational Obstetrics Group, The Department of Obstetrics and Gynaecology, Mercy Hospital for Women, University of Melbourne, Heidelberg, Victoria 3084, Australia; Mercy Perinatal, Mercy Hospital for Women, Heidelberg, Victoria 3084, Australia.
Funding
Funding for this work was provided by: National Health and Medical Research Council (#1065854, 2000732). Additional sources of funding for the cohort from South Africa was received from the Swedish Medical Society, Märta Lundqvist Foundation, Swedish Foundation for International Cooperation in Research and Higher Education, Jane and Dan Olssons Foundation, Mercy Perinatal (Australia), the Swedish Research Council (Vetenskapsrådet), Sweden, and the Center for Clinical Research Dalarna, Sweden. Salary support was received from the National Health and Medical Research Council Fellowships to T.K.L. (#1159261), S.T. (#1136418) N.J.H. (#1146128), R.H. (#1176922). The funders had no role in the study.
Disclosure
The authors have nothing to disclose.
Data Availability
Some or all datasets generated during and/or analyzed during the current study are not publicly available but are available from the corresponding author on reasonable request.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
Some or all datasets generated during and/or analyzed during the current study are not publicly available but are available from the corresponding author on reasonable request.
