Reduced circulating NrCAM as a biomarker for fetal growth restriction

Abstract

Background: Placental insufficiency underpins pregnancy complications, fetal growth restriction (FGR) and preeclampsia, yet predictive biomarkers are limited. Neuronal Cell Adhesion Molecule (NrCAM) may be a promising biomarker of placental dysfunction. This study investigated whether NrCAM can predict diseases of placental insufficiency.

Methods: Circulating NrCAM was measured across independent cohorts. Plasma NrCAM was assessed at 36 weeks' gestation in women who later delivered FGR infants (<3rd centile birthweight), or developed preeclampsia at term. Circulating NrCAM was also measured in international cohorts: a UK high-risk cohort of women presenting with reduced fetal movements and delivered an FGR infant; a high-risk cohort from South Africa diagnosed with preeclampsia or eclampsia. NrCAM was also assessed in pregnancies with preterm FGR or preeclampsia (<34 weeks gestation). The effect of hypoxia on NrCAM expression was measured in trophoblast stem cells, primary trophoblasts, and a murine FGR model.

Findings: Circulating NrCAM was reduced at 36 weeks' gestation in women who later delivered FGR infants (p = 4.75 x 10^-6, AUC = 0.76, n = 26 FGR, n = 957 controls). In the UK cohort, reduced NrCAM levels were associated with FGR (p = 9.34 × 10^-3, AUC = 0.72, n = 12 FGR, n = 235 control). In the South Africa cohort, circulating NrCAM was reduced with preeclampsia (p = 0.03, AUC = 0.70, n = 27 preeclampsia, n = 15 control). Placental NrCAM expression was lower in FGR (p = 0.0003, n = 23 FGR) and preeclampsia (p = 0.0003, n = 41 preeclampsia, n = 20 controls). Hypoxia reduced NrCAM expression in human trophoblast stem cells (p < 0.01) primary trophoblasts (p < 0.0001) and in a murine FGR model (p < 0.01, n = 9 per group).

Interpretation: Reductions in plasma and placental NrCAM are strongly associated with FGR and may be driven by hypoxia.

Funding: This study was funded by a grant from National Health and Medical Research Council.

Keywords: Biomarker; FGR; Hypoxia; NrCAM; Placenta; Preeclampsia.

Fig. 1

Bar graphs and receiver operator curves of Neuronal Cell Adhesion Molecule (NrCAM) in two large cohorts (a-d, Cohort 1, e-h, Cohort 2). NrCAM concentrations significantly reduced (a, p = 4.75 x 10⁻⁶) in the circulation of women at 36 weeks' gestation who subsequently delivered an FGR infant (birthweight <3rd centile). The discriminatory power of NrCAM is shown as a receiver operating characteristic (AUROC) curve with an area of the curve of 0.76 (b). Plasma Placental Growth Factor (c, d; PlGF) concentrations at 36 weeks were also significantly reduced in the same cohort (c, p = 8.15 × 10⁻⁷). The AUROC of PlGF is 0.77 (d). Cohort, n = 957 controls, n = 26 FGR. In a high-risk international cohort (FEMINA, Manchester, UK) (e, f) show NrCAM concentrations reduced (e, p = 9.34 x 10⁻³) in the circulation of women who presented at the clinic with reduced fetal movements who subsequently delivered an FGR infant (birthweight <3rd centile), AUROC of 0.72 (f). Plasma PlGF (g, h) concentrations in the same cohort remain unchanged (g, p = 0.13). The AUROC of PlGF is 0.63 (h). Cohort, n = 235 controls, n = 12 FGR. groups compared using Mann–Whitney U tests. AUROC, area under the AUROC curve, with 95% confidence intervals presented in brackets. Data depicted for a, c, e and g are mean ± SEM. ∗∗p < 0.01, ∗∗∗∗p < 0.0001.

Fig. 2

Circulating Neuronal Cell Adhesion Molecule (NrCAM) (a, b) concentrations were not altered (a, p = 0.09) in the circulation of 24 women at 36 weeks' gestation who subsequently developed preeclampsia (PE) at term, compared with 936 controls. The discriminatory power of NrCAM is shown as a receiver operating characteristic curve with a modest area of the curve (AUROC) of 0.60 (b). Plasma Placental Growth Factor (c, d; PlGF) concentration is significantly reduced in participants who develop preeclampsia at term (c, p = 5.45 x 10⁻⁸). The AUROC of PlGF is 0.84 (d). Controls, n = 959 controls, n = 24 PE. In samples from a case control cohort (PROVE, South Africa), circulating NrCAM was significantly reduced (e, p = 0.03) in participants with severe preeclampsia. The AUROC of 0.70 (f). NrCAM is not altered in participants with eclampsia (g), with a modest AUROC of 0.59 (h). Control, n = 15 controls, n = 27 preeclampsia (PE), n = 29 eclampsia. Groups compared using Mann–Whitney U tests. AUROC, area under the ROC curve, with 95% confidence intervals presented in brackets. Data depicted for a, c, e and f are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001.

Fig. 3

Circulating Neuronal Cell Adhesion Molecule (NrCAM) concentrations were significantly reduced in a case control of participants with preterm fetal growth restriction (FGR, <34 weeks' gestation), compared to gestation-matched controls (a, p = 0.0003). Gestation-matched controls, n = 20, FGR, n = 23. The discriminatory power of NrCAM is shown as a receiver operating characteristic (AUROC) of 0.82 (c). Circulating NrCAM is reduced in participants with preterm preeclampsia (<34 weeks' gestation), compared to gestation-matched controls (b, p = 0.0003). Gestation-matched controls, n = 20, preeclampsia, n = 41. The AUROC is 0.79 (d). NrCAM protein concentrations are reduced in placenta lysates from participants who delivered an FGR infant (e, p = 0.005). Gestation-matched controls, n = 19, FGR, n = 43. NrCAM protein concentrations are reduced in placenta lysates from participants who are diagnosed with preeclampsia (f, p = 0.0002), compared to gestation-matched controls. Gestation-matched controls, n = 21, preeclampsia, n = 27. NRCAM mRNA expression is not altered in placenta from participants who delivered an FGR infant (g) or diagnosed with preeclampsia (h) compared with preterm controls. Gestation-matched controls, n = 17, FGR, n = 63, preeclampsia, n = 78. NFASC mRNA expression is significantly reduced in the placenta from participants who delivered an FGR infant (i, p = 0.03) or developed preeclampsia at term (j, p = 0.003), compared with gestation-matched controls. Gestation-matched controls, n = 18, FGR, n = 30, preeclampsia, n = 78. Groups compared using Mann–Whitney U tests. Data presented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Fig. 4

NRCAM mRNA expression is reduced following differentiation of cytotrophoblast stem cells to syncytiotrophoblast and extravillous trophoblast (EVT) cells. hTSCs were differentiated into syncytiotrophoblast and EVT cells from 0 h to 96 h. To confirm EVT differentiation, reduction in cytotrophoblast marker TEAD4 (a, p = 0.006 72 h, p = 0.001 96 h), and increase in EVT marker HLAG (b, p = 0.02 72 h, p = 0.0002 96 h) mRNA expression was observed. To confirm syncytiotrophoblast differentiation, reduction in cytotrophoblast marker TEAD4 (d), and increase in syncytiotrophoblast marker SDC1 (e) mRNA expression was observed. NRCAM mRNA expression was reduced in EVT (c) and syncytiotrophoblast (f) following differentiation. NRCAM mRNA expression was reduced when cytotrophoblast hTSC cells (g), but not syncytiotrophoblast cells (h) exposed to hypoxia (1% O₂), compared to control physiological conditions (normoxia; 8% O₂). NRCAM mRNA expression was reduced in primary trophoblast cells (i) exposed to hypoxia (1% O₂) compared to normoxia (8% O₂). mRNA expression was normalized to the geometric mean of housekeeper genes. All experiments were repeated 5 times in triplicate. In primary term human trophoblast cells, NRCAM mRNA expression was significantly reduced in cells exposed to hypoxia (1% O₂), compared to normoxic (8% O₂) controls (e, normoxia, n = 6, hypoxia n = 6). For data with two groups, unpaired t-test or a Mann–Whitney (non-parametric) test was used. For 3 or more groups, a one-way ANOVA (parametric) or a Kruskal Wallis (nonparametric) test was used. For tests using multiple comparison tests, data was compared to the control group. Data was presented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Fig. 5

In a mouse model of hypoxia-induced fetal growth restriction, fetal weight (a) is significantly reduced in hypoxia treated mothers (10% inspired O₂), compared to normoxia controls (21% inspired O₂). Placental weight remained unchanged (b), whilst placenta-to-body ratio is significantly increased in maternal hypoxia treated group, compared with normoxic controls (c). NrCAM mRNA expression is significantly decreased in placentas from the maternal hypoxia group, compared with normoxic controls (d, n = 9 normoxic placentas, n = 9 hypoxic placentas from separate litters, n = 9 mice in each group). Data was presented as mean ± SEM. ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.