Maternal plasma fetuin-A concentration is lower in patients who subsequently developed preterm preeclampsia than in uncomplicated pregnancy: a longitudinal study^*

Abstract

Objective: Fetuin-A is a negative acute phase protein reactant that acts as a mediator for lipotoxicity, leading to insulin resistance. Intravascular inflammation and insulin resistance have been implicated in the mechanisms of disease responsible for preeclampsia (PE). Maternal plasma concentrations of fetuin-A at the time of diagnosis of preterm PE are lower than in control patients with a normal pregnancy outcome. However, it is unknown if the changes in maternal plasma fetuin-A concentrations precede the clinical diagnosis of the disease. We conducted a longitudinal study to determine whether patients who subsequently developed PE had a different profile of maternal plasma concentrations of fetuin-A as a function of gestational age (GA) than those with uncomplicated pregnancies.

Methods: A longitudinal case–control study was performed and included 200 singleton pregnancies in the following groups: (1) patients with uncomplicated pregnancies who delivered appropriate for gestational age (AGA) neonates (n = 160); and (2) patients who subsequently developed PE (n = 40). Longitudinal samples were collected at each prenatal visit and scheduled at 4-week intervals from the first or early second trimester until delivery. Plasma fetuin-A concentrations were determined by ELISA. Analysis was performed using mixed-effects models.

Results: The profiles of maternal plasma concentrations of fetuin-A differ between PE and uncomplicated pregnancies. Forward analysis indicated that the rate of increase of plasma fetuin-A concentration in patients who subsequently developed PE was lower at the beginning of pregnancy (p = 0.001), yet increased faster mid-pregnancy (p = 0.0017) and reached the same concentration level as controls by 26 weeks. The rate of decrease was higher towards the end of pregnancy in patients with PE than in uncomplicated pregnancies (p = 0.002). The mean maternal plasma fetuin-A concentration was significantly lower in patients with preterm PE at the time of clinical diagnosis than in women with uncomplicated pregnancies (p < 0.05). In contrast, there were no significant differences in maternal plasma fetuin-A concentration in patients who developed PE at term.

Conclusions: (1) The profile of maternal plasma concentrations of fetuin-A over time (GA) in patients who develop PE is different from that of normal pregnant women; (2) the rate of change of maternal plasma concentrations of fetuin-A is positive (increases over time) in the midtrimester of normal pregnancy, and negative (decreases over time) in patients who subsequently develop PE; (3) at the time of diagnosis, the maternal plasma fetuin-A concentration is lower in patients with preterm PE than in those with a normal pregnancy outcome; however, such differences were not demonstrable in patients with term PE.

• α₂-Heremans–Schmid glycoprotein
• hypertensive disorders in pregnancy
• insulin resistance
• intravascular inflammation
• negative acute phase protein reactant

Introduction

Preeclampsia (PE), one of the “great obstetrical syndromes” [1–4], is a leading cause of maternal [5–19] and neonatal morbidity [20–27]. Several mechanisms of disease have been implicated in the genesis of the syndrome, including: (1) failure of physiologic transformation of the spiral arteries [28–34]; (2) an anti-angiogenic state [35–69]; (3) systemic intravascular inflammation [70–77]; (4) endothelial dysfunction [78–84]; (5) oxidative stress [84–89]; (6) endoplasmic reticulum stress [88,90]; (7) platelet [91–95] and thrombin activation [96–103]; (8) anti-angiotensin-II antibodies [104–113]; and (9) insulin resistance [114–137].

Fetuin-A, a negative acute phase protein reactant (plasma protein whose concentration decreases during an inflammatory state [138–142]), has been implicated in the pathophysiology of PE [143–148]. This fetal protein was first described by Kai Pedersen [149]. It has a very high concentration in the neonatal calf, is mainly produced by the liver and decreases with time; therefore, he proposed that the protein be called “Fetuin” (from the Latin “Foetus”) [149]. In 1990, homology between human α₂-Heremans-Schmid glycoprotein (AHSG) and fetuin in cattle was demonstrated [150]. Fetuin-A can inhibit LPS- or IFN-γ-induced high mobility group box-1 proteins (HMGB-1) released in macrophages [142,151]. Moreover, fetuin-A may influence the resolution of inflammation by acting as a bacterial opsonin, thereby facilitating macrophage-mediated ingestion and the elimination of apoptotic neutrophils [152–154]. The administration of fetuin-A can protect against death during the course of endotoxemia and experimental sepsis induced by cecal puncture ligation [151]. Fetuin-A is also a hepatokine with metabolic effects [155–160]. Specifically, this protein has been identified as a mediator of lipotoxicity through the toll-like receptor-4 pathway, and can lead to insulin resistance [161].

We have reported that the maternal plasma concentrations of fetuin-A are significantly lower in women with preterm PE at the time of diagnosis than in normal pregnant women [162]. Moreover, such concentrations are correlated with the concentrations of other negative acute phase reactants, such as albumin and transferrin, in patients with PE. There is no information on whether the changes in fetuin-A occur prior to the development of PE. This longitudinal study was performed to address this question.

Materials and methods

Study design and participants

This retrospective, longitudinal, nested case–control study included 200 singleton pregnancies in the following groups: (1) women with uncomplicated pregnancies who delivered an appropriate for gestational age (AGA) neonate (controls; n = 160); and 2) patients who had PE (n = 40). Multiple gestations, pregnancies with fetal congenital anomalies and pregnancies complicated by chronic hypertension, diabetes mellitus, and/or renal disease were excluded.

Plasma samples were obtained serially during pregnancy. All patients had a minimum of three samples during pregnancy (range: three to seven samples). Plasma samples were selected once from each patient at the following seven intervals: 6–14.9; 15–19.9; 20–24.9; 25–27.9; 28–31.9; 32–36.9; 37–41.1 weeks of gestation. The earliest sample for each interval was used if multiple samples had been collected in a particular window. Samples collected after the clinical diagnosis of PE were excluded.

Clinical definition

PE was diagnosed in the presence of systolic blood pressure >140 mmHg or diastolic blood pressure >90 mmHg on at least two occasions, 4 h to 1 week apart, and proteinuria >300 mg in 24-h urine collection or one dipstick with >2+ urine protein [163,164]. Pregnant women were considered “normal” if they had no medical, obstetrical or surgical complications, and delivered a normal term (>37 weeks) infant whose birthweight was AGA (10th–90th percentile) [165,166].

The collection and utilization of the samples was approved by both the Human Investigation Committee of the Sótero del Rió Hospital, Santiago, Chile (a major affiliate for the Catholic University of Santiago) and the Institutional Review Board of the Eunice Kennedy Shriver National Institute of Child Health and Human Development (National Institutes of Health, Department of Health and Human Services). Many of these samples were used in previous studies.

Sample collection and fetuin-A immunoassays

Blood samples were collected into tubes containing EDTA, then centrifuged for 10 min at 4 °C and stored at −70 °C. Laboratory personnel were blinded to clinical diagnosis. Maternal plasma concentrations of fetuin-A were determined using sensitive and specific immunoassays (BioVender LLC, Candler, NC). The immunoassay was performed in duplicate and utilized a sandwich enzyme-based technique and had been validated for plasma determinations of the analytes. The inter- and intra-assay coefficients of variation were 4.7% and 2.9%, respectively. The sensitivity of the assays was 4.1 ng/mL.

Statistical analysis

Cross-sectional analysis of demographic and clinical characteristic data

The Kolmogorov–Smirnov test was used to assess the distribution of the data. Since the data were not normally distributed, we used the Kruskal–Wallis test for comparisons among groups and the Mann–Whitney U test for comparisons between groups for continuous variables. Chi-square or Fisher’s exact tests were used for comparisons of categorical variables. Statistical analysis was performed using SPSS 19 (IBM Corp, Armonk, NY) and SAS 9.3 (Cary, NC). A p value <0.05 was considered statistically significant.

Longitudinal analysis of plasma fetuin-A concentration

The data collected in this study contain serial measurements from each individual belonging to two groups (PE and controls). We used linear mixed effects models for analysis, which included fixed and random effects [167,168]. Using this approach, each subject has its own baseline response (fetuin-A concentration; random intercept) but is assumed to follow the same (fixed) profile over time (GA). The fixed effects included the diagnostic group (PE versus controls), polynomial terms of the GA at venipuncture up to the third degree, body mass index (BMI) (kg/m²), and the duration of sample storage (years). Parity and maternal age were tested but did not improve the model fit as determined by a likelihood ratio test. In addition, interaction terms between polynomial components of GA and the diagnostic group were also considered fixed effects. The effect of the group (difference in the mean fetuin-A concentration between women with PE and controls) was assessed at various GA (10–41 weeks) using the linear mixed effects model.

The lmer function from the lme4 package under the R statistical environment (www.r-project.org) was used for mixed-effects model fitting. Significance of the fixed effects in the linear model was determined using the ANOVA method in the lme4 package, which performs a likelihood ratio test between the model fit with and without the fixed effects of interest. A p value <0.05 was considered significant.

Backward longitudinal analysis

Each patient with PE was matched with up to four controls based on GA at sampling. Samples from matched controls collected after delivery of patients with PE were excluded from the analysis. The GA at the time of diagnosis of patients with PE and their matched controls was subtracted from the GA at sampling to compute the number of weeks before the diagnosis. The same mixed effects model used in the forward analysis was also applied to test for differences in the plasma concentrations of fetuin-A as a function of the time to diagnosis.

Results

Demographic and clinical characteristics of patients across the study groups

This study included a total of 1328 samples (1101 from uncomplicated pregnancies and 227 from patients with PE). The demographic and clinical characteristics of the study groups are displayed in Table 1. Patients who developed PE had a significantly lower median maternal age but higher median pre-pregnancy BMI and proportion of nulliparous women than those with uncomplicated pregnancies. There was no significant difference in the median GA at enrollment between patients who subsequently developed PE and those with uncomplicated pregnancies. The median GA at delivery and birthweight were lower in patients with PE than in the uncomplicated pregnancy group (p < 0.001 both; Table 1). Twenty-seven percent (11/40) of patients with PE delivered a small-for-gestational-age (SGA) neonate, and 37.5% (15/40) delivered before 37 weeks of gestation.

Table 1. Demographic and clinical characteristics of the study groups.

	Uncomplicated pregnancy (n = 160)	Preeclampsia (n = 40)	p
Maternal age (years)	24 (20–30)	20.5 (19–25)	0.02
Pre-pregnancy body mass index (BMI) (kg/m^2)	23.9 (21.6–26.8)	25.7 (23.5–29.2)	0.005
Smoking	23 (14.4)	2 (5)	0.55
Nulliparity	71 (44.4)	26 (65)	0.02
Gestational age at enrollment (weeks)	9.4 (8–11.4)	10 (8.7–11.4)	0.3
Gestational age at delivery (weeks)	40 (39.2–40.7)	38 (34.8–39.3)	<0.001
Birthweight (g)	3415 (3190–3560)	2980 (1990–3537)	<0.001
Birthweight <10th percentile	0	11 (27.5)	<0.001
Delivery <37 weeks	0	15 (37.5)	<0.001

Data expressed as median (interquartile range) and number (percentage).

Maternal plasma concentrations of fetuin-A in preeclampsia (forward longitudinal analysis)

The average profile of maternal plasma fetuin-A concentration as a function of GA was different between women who developed PE and the control group [p < 0.05 for the interaction terms between PE and polynomial components of the GA (“PE × GA”, “PE × GA²” and “PE × GA³”) of the mixed effects model (Table 2).

Table 2. Longitudinal analysis of the association between fetuin-A and preeclampsia after adjusting for confounding factors.

	All preeclampsia versus uncomplicated pregnancy
	Estimate	Standard error	t-value	p value
Intercept	265.7	5.3	49.89	< 0.001
(GA-41)	−4.9	1.2	−4.25	< 0.001
(GA-41)^2	−0.198	0.088	−2.24	0.01
(GA-41)^3	−0.0014	0.0019	−0.77	0.22
Group	−43.2	15.1	−2.87	0.002
BMI-25	1.63	0.72	2.27	0.01
Storage-8.13	−27.0	4.6	−5.82	<0.001
(GA-41) × PE	−10.3	3.4	−3.06	0.001
(GA-41)^2 × PE	−0.700	0.238	−2.94	0.002
(GA-41)^3 × PE	−0.014	0.005	−2.86	0.002

GA, gestational age; BMI, body mass index; PE, preeclampsia; (GA-41) × PE, interaction term between diagnosis and GA; (GA-41)² × PE, interaction between diagnosis and GA², (GA-41)³ × PE, interaction between diagnosis and GA³.

The changes in plasma concentrations of fetuin-A in patients who subsequently developed PE and those who had uncomplicated pregnancies across all GA are displayed in Figure 1. The curves in the figures represent a polynomial fit of the analyte concentration as a function of GA among uncomplicated pregnant women and those who developed PE (after adjusting for BMI and storage time).

Figure 1. Maternal plasma concentrations of fetuin-A in women with uncomplicated pregnancy (black dot) and patients who subsequently developed preeclampsia (PE) (red dot). The gestational age dependence of the fetuin-A concentration in uncomplicated pregnant women (black line) and those in preeclampsia group (red line) was estimated using linear mixed-effects using a third degree polynomial function. The vertical lines on the preeclampsia curve denote statistical significance of the difference between the two groups at the corresponding gestational age according to a linear mixed-effects model adjusting for covariates [body mass index (BMI) (kg/m²) and the duration of sample storage (years)].

The rate of the increase in plasma fetuin-A concentration in patients who subsequently developed PE was lower at the beginning of pregnancy (p = 0.001), yet increased faster in the midtrimester of pregnancy (p = 0.0017) to reach a similar concentration as uncomplicated pregnancies by 26 weeks. Subsequently, the rate of decrease was greater towards the end of gestation in women who developed PE than in those who had uncomplicated pregnancies (p = 0.002; Figure 1).

Maternal plasma fetuin-A concentrations before the diagnosis of preeclampsia

While the change over time in maternal plasma concentration of fetuin-A is different in women who subsequently developed PE from women who have a normal pregnancy outcome, an important clinical question is whether the absolute concentrations are different at any given time before diagnosis – this could have implications for the prediction of the syndrome. Therefore, we conducted a backward analysis to address this specific question. The mean maternal plasma fetuin-A concentration was significantly lower in patients with preterm PE at the time of diagnosis than in those with uncomplicated pregnancies (p < 0.05; Table 3), but not before. However, there was no significant difference in the mean maternal plasma fetuin-A concentration before or at the time of clinical diagnosis between patients with term PE and those with uncomplicated pregnancies (Table 4).

Table 3. The difference in plasma fetuin-A concentration and the statistical differences (p value) according to weeks before clinical diagnosis between patients with preterm preeclampsia and uncomplicated pregnancies.

Weeks before clinical diagnosis	Difference in plasma fetuin-A concentration between patients with preterm preeclampsia and uncomplicated pregnancies	p value
0	−31.46	0.03
−1	−20.10	0.14
−2	−11.00	0.40
−3	−3.98	0.76
−4	1.12	0.93
−5	4.49	0.74
−6	6.29	0.65
−7	6.69	0.63
−8	5.88	0.66
−9	4.02	0.76
−10	1.28	0.92
−11	−2.15	0.87
−12	−6.11	0.64
−13	−10.43	0.42
−14	−14.93	0.26
−15	−19.43	0.15

Table 4. The difference in plasma fetuin-A concentration and the statistical differences (p value) according to weeks before clinical diagnosis between patients with term preeclampsia and uncomplicated pregnancies.

Weeks before clinical diagnosis	Difference in plasma fetuin-A concentration between patients with term preeclampsia and uncomplicated pregnancies	p value
0	−10.84	0.42
−1	−4.97	0.69
−2	−0.20	0.99
−3	3.56	0.76
−4	6.40	0.59
−5	8.40	0.48
−6	9.66	0.42
−7	10.26	0.40
−8	10.29	0.39
−9	9.84	0.41
−10	8.99	0.45
−11	7.84	0.51
−12	6.47	0.58
−13	4.97	0.67
−14	3.42	0.77
−15	1.93	0.87

Discussion

Principal findings

(1) This is the first longitudinal study reporting a change in plasma fetuin-A concentrations in patients who subsequently developed PE; (2) the rate of change of maternal plasma concentrations of fetuin-A is positive (increases over time) in the midtrimester of normal pregnancy, and negative (decreases over time) in patients who subsequently developed PE; and (3) the mean maternal plasma fetuin-A concentration was significantly lower in patients with preterm PE than in those with uncomplicated pregnancies at the time of clinical diagnosis.

Maternal plasma fetuin-A concentration and preeclampsia

Previous studies examining serum concentrations of fetuin-A in PE yield conflicting results. Gomez et al. [147] and Park et al. [146] reported that the serum concentration of this protein was increased in patients with PE, while Movalec et al. [143,144] reported that it was lower in women with PE than in those with an uncomplicated pregnancy.

Our findings demonstrate that the profile (concentration over time) of maternal plasma fetuin-A among patients who subsequently developed PE throughout pregnancy differs from that of uncomplicated pregnancies. The mean fetuin-A maternal plasma concentration was lower in preterm PE at the time of diagnosis: these observations are consistent with a previous cross-sectional study at the time of diagnosis of PE performed in two different populations (Hispanics and African-Americans) [162] and with those of Molvarec et al. [143,144] who reported that median serum fetuin-A concentrations were lower in women with PE and patients with HELLP syndrome compared to women with uncomplicated pregnancies. Moreover, fetuin-A concentrations showed significant inverse correlations with serum C-reactive protein concentration [143] while having a significant positive correlation with plasma albumin and transferrin [162] in women with PE, supporting the role of fetuin-A as an acute negative phase protein reactant.

Fetuin-A: a negative acute-phase reactant protein

A likely explanation for the lower concentration of fetuin-A in PE is that this protein is a negative acute-phase reactant. PE is characterized by intravascular inflammation [70–77], demonstrated with flow cytometry studies [73] as well as the determination of cytokines/chemokines [169–181], complement split products [182], etc. Moreover, maternal peripheral blood mononuclear cells from patients with PE produced higher concentrations of proinflammatory cytokines than those with uncomplicated pregnancies [173,174,178,183–187].

Evidence that fetuin-A acts as a negative acute phase protein reactant [139–141] includes: (1) in patients with bacterial infection, serum fetuin-A concentrations decreased, and had a positive correlation with acute negative positive phase protein reactants (e.g. alpha anti-trypsin, orosomucoid and haptoglobin) and a positive correlation with acute negative phase reactants (e.g. albumin and transferrin) [139]; (2) the administration of recombinant human interleukin-6 and interleukin-1β decreases the synthesis of fetuin in human hepatoma HepG2 cells [140]; and (3) circulating fetuin-A concentrations decrease at approximately 24–48 h, returning toward the base-line approximately 72 h after onset of endotoxemia or sepsis in a mouse model of lethal systemic inflammation (induced by endotoxemia or cecal puncture ligation) [151].

In a previous study [162], we reported that patients with two pregnancy complications (PE and acute pyelonephritis) in which there is intravascular inflammation [73,188] have lower concentrations of fetuin-A than normal pregnant women. Moreover, we demonstrated that there was a significant correlation between the concentrations of fetuin-A and two other acute negative-phase reactants – albumin and transferrin – particularly among women with PE [162]. One potential explanation for the lack of demonstrable difference in the concentration of fetuin-A between patients with PE at term and normal pregnancy may be that intravascular inflammation is less severe in term than in preterm PE [189,190].

Another possible mechanism for the decreased plasma fetuin-A concentrations in PE is explained by the protective role of vascular calcification of fetuin-A [191–196]. Fetuin-A knock-out mice developed severe calcification in multiple organs [192], and the administration of fetuin-A to cultured bovine vascular smooth muscle cell resulted in stiffness of mineralization [193]. In humans, plasma fetuin-A concentration had an inverse correlation with the arterial stiffness determined by flow-mediated dilatation in both healthy subjects [194] and those with chronic kidney disease [195–198]. Endothelial dysfunction and increased arterial stiffness have been detected in patients with PE [199–201].

Strengths and limitations of this study

This is the first longitudinal study that evaluated the changes of the maternal plasma concentration of fetuin-A in patients with PE from the first trimester of pregnancy. Due to substantial overlapping in absolute concentrations, we believe it is unlikely that fetuin-A concentrations could be a useful biomarker for the prediction of PE.

Conclusion

The profile of maternal plasma fetuin-A concentration (over time) differs between patients who subsequently developed PE and those with uncomplicated pregnancies. The most likely explanation for the lower concentration of fetuin-A in maternal plasma in patients with preterm PE at the time of diagnosis is that this protein is a negative acute phase reactant, and therefore, its concentration may reflect the presence of intravascular inflammation.

Declaration of interest

The authors report no declarations of interest. This research was supported, in part, by the Perinatology Research Branch, Division of Intramural Research, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Department of Health and Human Services (NICHD/NIH); and, in part, with Federal funds from NICHD, NIH under Contract No. HHSN275201300006C.

Notes

* Presented as an oral presentation at the World Congress of Perinatal Medicine, 19–22 June 2013, Moscow, Russia.