Circulating SPINT1 is a biomarker of pregnancies with poor placental function and fetal growth restriction

Abstract

Placental insufficiency can cause fetal growth restriction and stillbirth. There are no reliable screening tests for placental insufficiency, especially near-term gestation when the risk of stillbirth rises. Here we show a strong association between low circulating plasma serine peptidase inhibitor Kunitz type-1 (SPINT1) concentrations at 36 weeks' gestation and low birthweight, an indicator of placental insufficiency. We generate a 4-tier risk model based on SPINT1 concentrations, where the highest risk tier has approximately a 2-5 fold risk of birthing neonates with birthweights under the 3^rd, 5^th, 10^th and 20^th centiles, whereas the lowest risk tier has a 0-0.3 fold risk. Low SPINT1 is associated with antenatal ultrasound and neonatal anthropomorphic indicators of placental insufficiency. We validate the association between low circulating SPINT1 and placental insufficiency in two other cohorts. Low circulating SPINT1 is a marker of placental insufficiency and may identify pregnancies with an elevated risk of stillbirth.

Fig. 1. Plasma concentrations of SPINT1 were significantly reduced preceding the birth of a small for gestational age (SGA) infant.

Scatter plots and receiver operator curves of SPINT1 in two cohorts (a, b; cohort 1, c, d; cohort 2) demonstrate SPINT1 concentrations were significantly reduced (a, p = 2.70 × 10⁻¹³, b, p = 5.24 × 10⁻⁸) in the circulation of women at 36 weeks who subsequently delivered an SGA infant (birthweight < 10th centile). Plasma PlGF concentrations at 36 weeks were also significantly reduced in cohort 2 (e, f, p = 3.81 × 10⁻³). A comparison between receiver operator curves for SPINT1 and PlGF indicated SPINT1 had improved area under the receiver operator curve (g). In a case–control sample set selected from cohort 1, SPINT1 was significantly reduced at 28 weeks’ gestation (h, p = 3.59 × 10⁻⁹). Cases where SPINT1 were low at 28 weeks’ gestation tended to remain low by 36 weeks’ gestation (i). Cohort 1, n = 891 controls, n = 106 SGA. Cohort 2 n = 893 controls, n = 105 SGA, Case control at 28 weeks, n = 100 controls, n = 84 SGA. **p < 0.01, ****p < 0.0001, groups compared using two-tailed Mann–Whitney U tests. AUC—area under the ROC curve, with 95% confidence intervals given in brackets. Data depicted for a, c and e are mean ± s.e.m. Source data included as a Source Data file.

Fig. 2. Plasma SPINT1 at 36 weeks is associated with clinical markers of placental insufficiency.

Plasma SPINT1 concentrations at 36 weeks’ gestation were correlated with uterine artery (UA) Doppler flow resistance (a, n = 327), lean mass of the neonate (b, n = 281), and placental weight (c, n = 305). There was a step-wise reduction in plasma SPINT1 concentration in women whose babies were subsequently born with a birthweight below the 10th centile (d, n = 1803 controls, 5–10th n = 115, p = 9.63 × 10⁻⁸ vs control, 3–5th n = 42, p = 8.57 × 10⁻⁵ vs control, <3rd n = 54, p = 3.06 × 10⁻¹² vs control). Plasma SPINT1 concentrations at 36 weeks were correlated with birth centiles (e). SPINT1 MoMs were combined for both cohort 1 and 2 and grouped according to the birth centile. Graphs d and e depict median +/− interquartile range. Each individual symbol (a–c) represents a patient. ****p < 0·0001 relative to control (two-tailed Mann–Whitney U tests). Source data included as a Source Data file.

Fig. 3. SPINT1 is reduced in an independent high-risk cohort.

Plasma SPINT1 levels were assessed in samples from women who presented at the Manchester Antenatal Vascular Service (MAViS). SPINT1 is reduced in the SGA cohort (a, b, p = 3.31 × 10⁻⁴), with a step-wise reduction when SGA cases were split into 5–10th and <5th centile (c, 5–10th p = 2.83 × 10⁻² vs control, <5th p = 1.10 × 10⁻³ vs control). We also confirm that circulating SPINT1 correlates across all birthweight centiles (d). Uterine artery pulsatility index was assessed at the time of blood sampling, as well as at 22–24 weeks, and for both of these measures we demonstrate a significant correlation with circulating SPINT1 (e, f). We also confirm a significant correlation between circulating SPINT1 and estimated placental surface area at 22–24 weeks. For all panels, each individual symbol represents one patient. a, b: n = 208, control, n = 83 SGA. c: n = 208 control, n = 40 5–10th, n = 43 < 5th. e: n = 221, f: n = 244, g: n = 223. *p < 0.05, **p < 0.01 using two-tailed Mann–Whitney U tests. AUC—area under the ROC curve, with 95% confidence intervals given in brackets. Data depicted for a and c are mean ± s.e.m. Source data included as a Source Data file.

Fig. 4. SPINT1 is reduced in preterm fetal growth restriction.

SPINT1 mRNA (a, n = 19 control, n = 19 fetal growth restriction - FGR) and protein expression (b, n = 22 control, n = 12 FGR) were significantly reduced in placentas from cases of preterm fetal growth restriction (FGR who were delivered <34 weeks’ gestation and had a birthweight <10th centile) relative to gestation matched controls. Plasma SPINT1 concentrations were significantly reduced in women with preterm FGR relative to concentrations in women with healthy pregnancies, where bloods were collected at the same gestations (c, n = 15 control, n = 9 FGR). The area under the receiver operated curve for data shown in c was 0.948 (d). In a mouse model of hypoxia-induced fetal growth restriction, SPINT1 mRNA (e, n = 9 normoxic placentas, n = 9 hypoxic placentas from separate litters) and protein (f, n = 13 normoxic placentas, n = 11 hypoxic placentas from separate litters) expression in the placenta was significantly reduced. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 using two-tailed Mann–Whitney U tests. Individual symbols represent an individual patient, placental isolation or placenta. AUC—area under the ROC curve, with 95% confidence intervals given in brackets. Data depicted for a–c, e, f are mean ± s.e.m. Source data included as a Source Data file.