Elevated Circulating and Placental SPINT2 Is Associated with Placental Dysfunction

Abstract

Biomarkers for placental dysfunction are currently lacking. We recently identified SPINT1 as a novel biomarker; SPINT2 is a functionally related placental protease inhibitor. This study aimed to characterise SPINT2 expression in placental insufficiency. Circulating SPINT2 was assessed in three prospective cohorts, collected at the following: (1) term delivery (n = 227), (2) 36 weeks (n = 364), and (3) 24-34 weeks' (n = 294) gestation. SPINT2 was also measured in the plasma and placentas of women with established placental disease at preterm (<34 weeks) delivery. Using first-trimester human trophoblast stem cells, SPINT2 expression was assessed in hypoxia/normoxia (1% vs. 8% O2), and following inflammatory cytokine treatment (TNFα, IL-6). Placental SPINT2 mRNA was measured in a rat model of late-gestational foetal growth restriction. At 36 weeks, circulating SPINT2 was elevated in patients who later developed preeclampsia (p = 0.028; median = 2233 pg/mL vs. controls, median = 1644 pg/mL), or delivered a small-for-gestational-age infant (p = 0.002; median = 2109 pg/mL vs. controls, median = 1614 pg/mL). SPINT2 was elevated in the placentas of patients who required delivery for preterm preeclampsia (p = 0.025). Though inflammatory cytokines had no effect, hypoxia increased SPINT2 in cytotrophoblast stem cells, and its expression was elevated in the placental labyrinth of growth-restricted rats. These findings suggest elevated SPINT2 is associated with placental insufficiency.

Keywords: SPINT2/HAI-2; foetal growth restriction; intrauterine growth restriction; placental insufficiency; preeclampsia; small for gestational age.

Figure 1

SPINT2 expression in placentas of patients with established placental disease. Compared to preterm controls, SPINT2 mRNA expression (a) was not altered in placentas from normotensive pregnancies affected by foetal growth restriction (FGR), but it was significantly decreased in those from pregnancies compromised by concurrent preeclampsia (PE) and FGR, and increased in preeclamptic placentas of AGA infants. SPINT2 protein expression (b) in these same placentas was also not changed in FGR-affected normotensive pregnancies, but was significantly elevated in all PE cases (with and without FGR). Each data point represents an individual patient sample; data are expressed as median ± IQR; * p < 0.05, ** p < 0.01.

Figure 2

Circulating SPINT2 levels preceding preeclampsia development or birth of a small-for-gestational-age infant. In the blood of women on the day of delivery (a), SPINT2 protein expression was increased (approaching statistical significance, p = 0.051) in those who delivered an SGA infant, compared to AGA controls. This association was stronger at 36 weeks’ gestation, in which there was a significant elevation of SPINT2 levels among women who later delivered a small-for-gestational-age (SGA) infant (b) as well as in those women who subsequently developed PE (d), although the significance of the latter was lost when accounting for outliers. Earlier in the pregnancy, however, at 24–34 weeks, there was no association between circulating SPINT2 and SGA (c) nor PE (e) cases in samples from women with underlying vascular disease. In this cohort, SPINT2 did not fluctuate across gestation in controls nor PE; however, there was an apparent increase in SPINT2 across gestation in those women destined to birth an SGA infant. In the plasma collected on the day of delivery from women with diagnosed placental insufficiency (f), circulating SPINT2 was unchanged in cases, relative to controls. Each data point represents an individual patient sample; data are expressed as median ± IQR; linear regression showing 95% confidence intervals; * p < 0.05, ** p < 0.01.

Figure 3

The effect of hypoxia on SPINT2 expression in placental cells. SPINT2 mRNA and protein secretion was measured in the following three types of trophoblast cultures: primary trophoblasts isolated from term placentas, and first-trimester cytotrophoblast and syncytiotrophoblast from a stem cell line. In the term primary trophoblasts (a), SPINT2 transcripts were significantly increased in response to hypoxia, while secreted protein levels (b) were not changed. Hypoxic conditions caused no alteration to the first-trimester cytotrophoblast stem cell SPINT2 mRNA (c), but did significantly increase the levels of SPINT2 secretion (d) compared to normoxic controls. The syncytialised first-trimester stem cells had no change in mRNA (e), although they did demonstrate decreased SPINT2 secretion (f). Experiments were repeated n = 3–5 times; data are expressed as mean ± SEM. In placentas from a rat model of uteroplacental insufficiency, changes were identified in rat SPINT2 mRNA (rSpint2) expression in both the basalis (g) and labyrinth (h) zones, being depressed and elevated, respectively. Each data point represents an individual rat placenta; data are expressed as median ± IQR; * p < 0.05, ** p < 0.01.

Figure 4

Inflammatory cytokine regulation of SPINT2 in placental cells. First-trimester cytotrophoblasts (a–d) and syncytiotrophoblasts (e–h) were treated with inflammatory cytokines, TNF or IL-6, at various doses. No significant changes were observed in SPINT2 mRNA (a,c,e,g) expression, nor in secreted SPINT2 (b,d,f); with the exception of IL-6–treated syncytiotrophoblasts (h), which stimulated a modest, but significant, decrease in SPINT2 secretion at lower doses. Experiments were repeated n = 5 times; data are expressed as mean ± SEM; ** p < 0.01.