A subset of patients destined to develop spontaneous preterm labor has an abnormal angiogenic/anti-angiogenic profile in maternal plasma: Evidence in support of pathophysiologic heterogeneity of preterm labor derived from a longitudinal study

Figures & data

Table I.  Clinical characteristics of the study population.

	Normal pregnancy (n = 208)	Preterm labor (n = 52)	p
Age (y)	24 (16–47)	25.5 (16–45)	0.4
Body mass index (kg/m^2)	23.9** (16–39)	23.7^† (17–32)	0.6
Nulliparity	93 (44.7%)	21 (40.4%)	0.6
Previous preterm delivery	1 (0.5%)	10 (19.2%)	<0.001*
Smoking	27 (13.0%)	7 (13.5%)	0.9
GA at first ultrasound	12.3 (6–20)	12.5 (7–23.6)	0.2
GA at delivery (weeks)	39.9 (37–41.9)	35.8 (26.1–36.9)	<0.001*
Birthweight (g)	3475 (2540–4150)	2665 (966–3420)	<0.001*
Delivery ≤32 weeks	0	7(13.5%)	<0.001*
Delivery ≤34 weeks	0	13 (25%)	<0.001*
Amniotic fluid culture positive	0	2/11 (18.2%)	<0.001*

Value expressed as median (range) or number (percent); GA, gestational age; **n = 197; ^†n = 51.

*p < 0.05.

Table II.  Plasma concentrations of angiogenic factors in normal pregnancy and preterm labor who delivered preterm.

	Normal pregnancy	p	Pre-clinical samples preterm labor	p	Clinical samples preterm labor	p^β
1st blood sampling (6–14.9 weeks)
sVEGFR-1 (pg/ml)	1636 (467–5653)	0.2	1428 (758–4755)
PlGF (pg/ml)	33 (0–329)	0.2	27 (0–451)
sEng (ng/ml)	7.1 (3.9–13.9)	0.5	7.2 (4.9–12.0)
sVEGFR-2 (ng/ml)	10.2 (4.7–14.8)	0.5	10.5 (5.9–13.9)
Gestational age (weeks)	12 (6.6–14.9)	0.8	12.3 (8.6–14.9)
	n = 208		n = 48
2nd blood sampling (15–19.9 weeks)
sVEGFR-1 (pg/ml)	1684 (564–6112)	0.2	1551 (388–4374)
PlGF (pg/ml)	114 (0–651)	0.4	102 (32–905)
sEng (ng/ml)	6.8 (3.9–16.9)	0.002*	7.6 (5.3–18.6)
sVEGFR-2 (ng/ml)	10.8 (6.2–19.2)	0.7	10.9 (6.2–19.2)
Gestational age (weeks)	17.3 (15–19.9)	0.8	17.4 (15–19.9)
	n = 208		n = 42
3rd blood sampling (20–24.6 weeks)
sVEGFR-1 (pg/ml)	1470 (388–5723)	0.07	1297 (403–4920)
PlGF (pg/ml)	316 (22–1833)	0.3	274 (78–1087)
sEng (ng/ml)	5.7 (2.5–10.4)	0.03*	6.0 (4.1–16.8)
sVEGFR-2 (ng/ml)	10.8 (2.8–15.7)	0.9	10.9 (6.3–16.1)
Gestational age (weeks)	22.0 (20–24.6)	0.3	21.9 (20–24.6)
	n = 207		n = 52
4th blood sampling (25–27.9 weeks)
sVEGFR-1 (pg/ml)	1659 (518–4962)	0.5	1753 (668–5015)	0.9	1788	0.8
PlGF (pg/ml)	460 (75–4437)	0.4	465 (113–2783)	0.3	255	0.3
sEng (ng/ml)	6.6 (3.0–19.2)	0.01*	7.6 (4.1–12.7)	0.8	8.2	0.3
sVEGFR-2 (ng/ml)	10.9 (5.2–17.1)	0.3	10.3 (6.1–15.4)	0.2	8.8	0.2
Gestational age (weeks)	26.3 (25–27.9)	0.07	26.6 (25–27.9)	0.4	26.0	0.7
	n = 208		n = 38		n = 1
5th blood sampling (28–31.9 weeks)
sVEGFR–1 (pg/ml)	1862 (715–5481)	0.2	2236 (893–7282)	0.9	1885 (1563–3401)	0.6
PlGF (pg/ml)	604 (53–6343)	0.03*	493 (53–1647)	0.06	297 (43–298)	0.02*
sEng (ng/ml)	7.9 (3.4–28.1)	<0.001*	10.8 (5.3–27.8)	0.8	8.5 (7.2–27.6)	0.4
sVEGFR-2 (ng/ml)	10.8 (5.3–17.7)	0.2	10.1 (7.1–14.7)	0.6	12.3 (7.0–14.3)	0.7
Gestational age (weeks)	30.3 (28–31.9)	0.3	30.6 (28.1–31.9)	0.2	28.8 (28.1–30.9)	0.1
	n = 208		n = 36		n = 3
6th blood sampling (32–36.9 weeks)
sVEGFR-1 (pg/ml)	3254 (979–13,163)	0.3	3806 (2012–10,516)	0.2	5614 (1979–14,878)	0.001*
PlGF (pg/ml)	388 (32–4225)	0.3	299 (87–835)	0.2	165 (72–546)	<0.001*
sEng (ng/ml)	11.7 (3.9–36.1)	0.06	17.0 (7.1–49.6)	0.2	17.9 (7.2–46.4)	0.06
sVEGFR-2 (ng/ml)	9.9 (4.9–15.0)	0.9	9.9 (6.7–25.4)	0.2	8.2 (5.8–11.2)	<0.001*
Gestational age (weeks)	35.3 (32.4–36.9)	0.8	35.0 (34.3–36.1)	0.2	35.7 (32.1–36.9)	0.3
	n = 208		n = 10		n = 19

p^β: compared between samples at clinical manifestation of preterm labor and normal pregnancy.

*p < 0.05; value expressed as median (range).

Table III.  Unadjusted and adjusted odds ratio for the identification of spontaneous preterm labor by plasma sEng concentrations above the 3rd quartile for various gestational age intervals in pre-clinical samples.

	Normal pregnancy	Pre-clinical preterm labor	Unadjusted odds ratio (95% CI)	Adjusted oddsratio (95% CI)
1st blood sampling (6–14.9 weeks)
sEng > 8.3 ng/ml (n = 256)	52/208 (25.0%)	10/48 (20.8%)	0.8 (0.4–1.7)	0.6(0.2–1.4)
2nd blood sampling (15–19.9 weeks)
sEng > 7.7 ng/ml (n = 250)	53/208 (25.5%)	19/42 (45.2%)	2.4 (1.2–4.8)	1.9 (0.9–4.4)
3rd blood sampling (20–24.9 weeks)
sEng > 6.6 ng/ml (n = 259)	51/207 (24.6%)	20/52 (38.5%)	1.9 (1.01–3.6)	1.5 (0.7–3.2)
4th blood sampling (25–27.9 weeks)
sEng > 7.7 ng/ml (n = 246)	53/208 (25.5%)	19/38 (50.0%)	2.9 (1.5–5.9)	1.8 (0.8–2.4)
5th blood sampling (28–31.9 weeks)
sEng > 9.7 ng/ml (n = 244)	52/208 (25.0%)	21/36 (58.3%)	4.2 (2.0–8.6)	3.1* (1.3–7.2)
6th blood sampling (32–36.9weeks)
sEng > 16.4 ng/ml (n = 218)	52/208 (25.0%)	6/10 (60.0%)	4.5 (1.3–15.4)	6.6* (1.5–28.6)

Adjusted for previous preterm delivery (yes/no), gestational age at sampling (weeks), duration of sample storage (days), maternal age (years), BMI (Kg/m²), smoking (yes/no), nulliparity (yes/no).

CI, confidence interval; *p < 0.05.

Table IV.  Mixed-effect model comparing the profiles of plasma sEng and sVEGFR-1 concentrations in relation to gestational age between patients with PTL and normal pregnant women adjusting for confounders (see text).

	sEndoglin			sVEGFR-1
	coefficient	p	P^+	coefficient	p	P^+
(Gestational age)^2	0.0012	<0.0001*	<0.0001*	0.0013	<0.0001*	<0.0001*
Preterm labor and delivery (yes/no)	−0.0579	0.0169*	0.0381*	−0.1359	0.0012*	0.0028*
Gestational age (weeks)	−0.0479	<0.0001*	<0.0001*	−0.0521	<0.0001*	<0.0001*
Age (years)	0.0027	0.0233*	0.0442*	−0.0002	0.9180	0.9542
Body mass index (kg/m^2)	−0.0053	0.0003*	0.0009*	−0.0064	0.0181*	0.0384*
Nulliparity (yes/no)	0.0339	0.0212*	0.0423*	0.1026	0.0002*	0.0006*
Previous preterm delivery (yes/no)	0.0279	0.3643	0.4684	−0.0295	0.6035	0.6789
Smoking (yes/no)	0.0045	0.7956	0.8679	−0.0017	0.9568	0.9568
Group × Gestational age (interaction)	0.0045	<0.0001*	<0.0001*	0.0058	<0.0001*	<0.0001*

P⁺, adjusted for false discovery rate; *p < 0.05.

Figure 1.  Profile of plasma soluble endoglin (sEng) concentrations (ng/ml) in relation to GA in normal pregnant women and patients with spontaneous PTL and delivery. Patients destined to developed PTL had a significantly different profile (plasma concentration over time) of plasma sEng concentration from patients with normal pregnancies after adjusting for GA at blood sampling, maternal age, body mass index, nulliparity, a history of preterm delivery, smoking and duration of sample storage (p = 0.04). Plasma sEng concentration was higher in patients destined to develop PTL than in normal pregnant women from 15 to 20 weeks of gestation. The difference became statistical significance at 27.7 weeks and was more pronounce as delivery approached.

Table V.  Mixed-effect model comparing the profiles of plasma PlGF and sVEGFR-2 concentrations in relation to gestational age between patients with PTL and normal pregnant women adjusting for confounders (see text).

	PlGF			sVEGFR-2
	coefficient	p	P^+	coefficient	p	P^+
(Gestational age)^2	−0.0045	<0.0001*	<0.0001*	−0.0002	<0.0001*	<0.0001*
Preterm labor and delivery (yes/no)	0.0889	0.1911	0.2752	0.0265	0.0838	0.1312
Gestational age (weeks)	0.2648	<0.0001*	<0.0001*	0.0096	<0.0001*	<0.0001*
Age (years)	−0.0039	0.2122	0.2938	−0.0016	0.0826	0.1312
Body mass index (kg/m^2)	−0.0035	0.3626	0.4684	0.0010	0.3815	0.4736
Nulliparity (yes/no)	−0.0214	0.5825	0.6764	−0.0206	0.0661	0.1133
Previous preterm delivery (yes/no)	0.0074	0.9277	0.9542	−0.0161	0.4898	0.5878
Smoking (yes/no)	0.0879	0.0562	0.1012	0.0198	0.1327	0.1991
Group* Gestational age (interaction)	−0.0082	0.0005*	0.0015*	−0.0014	0.0009*	0.0022*

P⁺, adjusted for false discovery rate; *p < 0.05.

Figure 2.  Profile of plasma sVEGFR-1 concentrations (pg/ml) in relation to GA in normal pregnant women and patients with spontaneous PTL and delivery. Patients destined to developed PTL had a significantly different profile (plasma concentration over time) of plasma sVEGFR-1 concentration from patients with normal pregnancies after adjusting for GA at blood sampling, maternal age, body mass index, nulliparity, a history of preterm delivery, smoking and duration of sample storage (p = 0.003). Plasma sVEGFR-1 concentrations in patients with PTL were slightly lower than those in normal pregnant women from 10 to 28 weeks of gestation. After this GA, plasma sVEGFR-1 concentrations became higher until delivery.

Figure 3.  Profile of plasma placenta growth factor (PlGF) concentrations (pg/ml) in relation to GA in normal pregnant women and patients with spontaneous PTL and delivery. There was no significant difference in the profile (plasma concentration over time) of plasma PlGF concentration between patients with PTL and normal pregnant women after adjusting for GA at blood sampling, maternal age, body mass index, nulliparity, a history of preterm delivery, smoking and duration of sample storage (p = 0.3).

Figure 4.  Profile of plasma sVEGFR-2 concentrations (ng/ml) in relation to GA in normal pregnant women and patients with spontaneous PTL and delivery. There was no significant difference in the profile (plasma concentration over time) of plasma sVEGFR-2 concentration between patients with PTL and normal pregnant women after adjusting for GA at blood sampling, maternal age, body mass index, nulliparity, a history of preterm delivery, smoking and duration of sample storage (p = 0.1).

Figure 5.  Individual changes in maternal plasma concentration (log 10 (conc+1)) of soluble endoglin (sEng) and soluble vascular endothelial growth factor receptor-2 (sVEGFR-2) in normal pregnant women (control; n = 208) and patients destined to develop PTL and delivery (n = 52) in relation to GA.

Figure 6.  Individual changes in maternal plasma concentration (log10 (conc +1)) of soluble vascular endothelial growth factor receptor-1 (sVEGFR-1) and PlGF in normal pregnant women (control; n = 208) and patients destined to develop PTL and delivery (n = 52) in relation to GA.

Table VI.  Plasma concentrations of angiogenic factors in normal pregnancy and preterm labor who delivered preterm.

	Normal pregnancy	Pre-clinical samples preterm labor	p^β
Interval to delivery 0–1 day
sVEGFR-1 (pg/ml)	2684 (787–8685)	4568 (1563–14,878)	0.001*
PlGF (pg/ml)	459 (68–2432)	184 (43–546)	<0.001*
sEng (ng/ml)	9.9 (3.9–32.2)	14.3 (7.2–46.4)	0.03*
sVEGFR-2 (ng/ml)	9.7 (4.9–15.6)	8.4 (5.8–14.3)	0.003*
Gestational age (weeks)	34.7 (25.1–36.9)	35.4 (26.0–36.9)	0.1
	n = 92	n = 23
Interval to delivery 2–14 days
sVEGFR-1 (pg/ml)	3238 (1392–10,845)	3777 (2012–10,516)	0.3
PlGF (pg/ml)	367 (34–4225)	189 (87–668)	0.054
sEng (ng/ml)	11.4 (3.9–20.4)	17.4 (7.1–49.6)	0.016*
sVEGFR-2 (ng/ml)	9.8 (6.9–13.1)	9.7 (6.7–25.4)	0.8
Gestational age (weeks)	35.2 (29.7–36.6)	35.0 (31.7–36.1)	0.8
Interval to delivery (days)		7 (2–13)
	n = 36	n = 9
Interval to delivery 15–35 days
sVEGFR-1 (pg/ml)	1750 (654–4363)	2772 (797–7282)	0.01*
PlGF (pg/ml)	571 (137–6343)	518 (53–900)	0.08
sEng (ng/ml)	7.5 (3.9–20.3)	11.1(5.6–27.8)	0.002*
sVEGFR-2 (ng/ml)	10.6 (5.3–15.6)	9.5 (7.3–15.4)	0.02*
Gestational age (weeks)	30.1 (22.3–36.0)	30.9 (24.0–34.7)	0.1
Interval to delivery (days)		27 (15–35)
	n = 84	n = 21
Interval to delivery 36–70 days
sVEGFR-1 (pg/ml)	1764 (518–5481)	1791 (605–5015)	0.4
PlGF (pg/ml)	451 (53–4437)	414 (83–2783)	0.3
sEng (ng/ml)	7.0 (3.4–28.1)	8.3 (4.6–21.5)	0.01*
sVEGFR-2 (ng/ml)	10.8 (2.8–17.7)	10.2 (6.1–14.7)	0.2
Gestational age (weeks)	28.0 (17.6–31.9)	27.9 (18.6–31.3)	0.9
Interval to delivery (days)		48 (37–66)
	n = 164	n = 41
Interval to delivery 71–105 days
sVEGFR-1 (pg/ml)	1380 (388–5723)	1204 (403–4920)	0.052
PlGF (pg/ml)	332 (60–1935)	298 (78–1253)	0.8
sEng (ng/ml)	5.9 (2.5–12.6)	6.0 (4.1–16.8)	0.2
sVEGFR-2 (ng/ml)	10.8 (6.2–19.2)	11.1 (6.3–15.8)	0.4
Gestational age (weeks)	22.1 (15.6–27.9)	22.3 (15.6–26.4)	0.9
Interval to delivery (days)		90.5 (71–105)
	n = 168	n = 42
Interval to delivery 106–140 days
sVEGFR-1 (pg/ml)	1563 (465–6112)	1487 (388–4622)	0.2
PlGF (pg/ml)	135 (0–1596)	121 (0–905)	0.6
sEng (ng/ml)	6.3 (2.9–12.9)	7.5 (4.4–18.6)	0.001*
sVEGFR-2 (ng/ml)	10.8 (6.4–15.8)	10.9 (6.2–14.8)	0.8
Gestational age (weeks)	17.9 (8.0–24.0)	18.0 (8.6–21.4)	0.6
Interval to delivery (days)		122 (106–137)
	n = 171	n = 43
Interval to delivery >140 days
sVEGFR-1 (pg/ml)	1676 (467–5653)	1670 (758–4755)	0.4
PlGF (pg/ml)	38.6 (0–396)	34.9 (0–451)	0.8
sEng (ng/ml)	7.1 (3.9–13.9)	7.1 (4.9–12.0)	0.5
sVEGFR-2 (ng/ml)	10.4 (5.3–15.2)	10.6 (6.9–13.9)	0.8
Gestational age (weeks)	12.4 (6.6–19.4)	12.6 (9.3–16.0)	0.8
Interval to delivery (days)		161 (141–183)
	n = 171	n = 43

p^β: compared between samples at clinical manifestation of preterm labor and normal pregnancy; *p < 0.05; value expressed as median (range).

Table VII.  Proportions of PTL patients who had plasma sEng concentrations above the 3rd quartile stratified by placental pathology.

	Pathology consistent with maternal under-perfusion			Pathology consistent with inflammation
	No (n = 26)	Yes (n = 4)	p	No (n = 25)	Yes (n = 5)	p
1st blood sampling (6–14.9 weeks)
sEng > 8.3 ng/ml	2/25 (8.0%)	0/4 (0%)	1.0	2/24 (8.3%)	0/5 (0%)	1.0
2nd blood sampling (15–19.9 weeks)
sEng > 7.7 n/ml	5/23 (21.7%)	3/3 (100%)	0.02*	2/22 (22.7%)	3/4 (75%)	0.07
3rd blood sampling (20–24.9 weeks)
sEng > 6.6 ng/ml	7/26 (26.9%)	3/4 (75.0%)	0.09	9/25 (36%)	1/5 (20%)	0.6
4th blood sampling (25–27.9 weeks)
sEng > 7.7 ng/ml	7/21 (33.3%)	3/4 (75.0%)	0.3	8/20 (40%)	2/5 (40%)	1.0
5th blood sampling (28–31.9 weeks)
sEng > 9.7 ng/ml	9/23 (69.2%)	4/4 (100%)	0.04*	10/23 (43.5%)	3/4 (75%)	0.3
6th blood sampling (32–36.9weeks)
sEng > 16.4 ng/ml	8/17 (47.1%)	2/2 (100%)	0.5	7/15 (46.7%)	3/4(75%)	0.6

Fisher's Exact test; *p < 0.05.