Could alterations in maternal plasma visfatin concentration participate in the phenotypes definition of preeclampsia and SGA?

Abstract

Objective

Women with preeclampsia and those who delivered a small-for-gestational-age (SGA) neonate share several mechanisms of disease, including chronic uteroplacental ischemia and failure of physiologic transformation of the spiral arteries. However, the clinical manifestation of these obstetrical syndromes is remarkably different. It has been proposed that an altered maternal metabolic state, as well as a unique circulating cytokines milieu, predispose women to develop either preeclampsia or SGA. Compelling evidence suggests that adipose tissue orchestrates both metabolic pathways and immunological responses via the production of adipokines. Visfatin is a novel adipocytokine with metabolic and immunomodulating properties. The objective of this study was to determine whether preeclampsia and SGA are associated with alterations in maternal circulating visfatin concentrations.

Methods

This cross-secitonal study included pregnant women in the following groups: (1) normal pregnancy (n = 158); (2) patients with preeclampsia (n = 43) of which 32 had an AGA and 11 had an SGA neonate; (3) patients without preeclampsia who delivered an SGA neonate (n = 55). Maternal plasma visfatin concentrations were measured by ELISA. Nonparametric tests and multiple linear regression analysis were used.

Results

(1) Women who delivered an SGA neonate had a higher median maternal plasma visfatin concentration than those with a normal pregnancy (20.0 ng/ml, interquartile range: 17.2-24.6 vs. 15.2 ng/ml, 12.1-19.2, respectively; P < 0.001) than those with preeclampsia (14.5 ng/ml, 12.5-18.7; P < 0.001); (2) the median maternal plasma visfatin concentration did not differ significantly between patients with preeclampsia and those with a normal pregnancy (P = 0.8); (3) among patients with preeclampsia, there was no significant difference in the median maternal plasma visfatin concentration between those with or without an SGA neonate (P = 0.5); (4) in a linear regression model, delivery of an SGA neonate and pregestational body mass index were independently associated with increased visfatin concentration after adjustment for confounding factors (maternal age, smoking, gestational age at blood collection and the presence of preeclampsia or SGA).

Conclusion

(1) Patients with SGA, but not those with preeclampsia, had a higher maternal plasma visfatin concentration than those with a normal pregnancy; (2) this finding suggests differential involvement of visfatin in SGA and preeclampsia; (3) we propose that changes in circulating maternal visfatin concentration may be implicated in the phenotypic definitions and distinction of preeclampsia and SGA.

INTRODUCTION

Preeclampsia and small-for-gestational-age (SGA) neonates are two of the most prevalent and important ‘great obstetrical syndromes’ [1]. Consistent with their syndromic nature, and in accordance with the related risk factors [2-11], several common mechanisms of disease have been proposed for both entities, including defective physiologic transformation of the spiral arteries [12-14], an antiangiogenic state [15-35], endothelial cell dysfunction [36-41], chronic uteroplacental ischemia [42,43] and an increased maternal intravascular inflammatory response [32,44-54].

Despite these similarities, the clinical manifestation of preeclampsia and SGA is different. Preeclampsia is characterized by hypertension, proteinuria and multiple organ involvement; in 10-25% of patients, concomitant fetal growth restriction will be diagnosed [55]. SGA is usually defined as a birthweight below the 10^th percentile for gestational age at birth according to the birthweight distribution of a particular population [56]. The clinical features of hypertension, proteinuria and multiple organ involvement are not present in patients with isolated SGA. This apparent paradox can be articulated by the following question: why do some patients develop preeclampsia while others develop SGA, in the presence of similar risk factors and underlying mechanisms of disease?

Ness and SIbai [37] have proposed that underlying maternal metabolic derangements are the cause for the distinct clinical manifestations of these complications of pregnancy. Specifically, this concept holds that patients with increased adiposity, insulin resistance/hyperglycemia or dyslipidemia will develop preeclampsia, whereas in those without these metabolic complications, SGA will be the clinical manifestation.

Adipose tissue is now recognized as a highly active endocrine organ [57-60] that can exert its pleiotropic effects via the production and secretion of adipokines. Moreover, Dysregulation of adipokines has been implicated in the pathophysiology of insulin resistance [57,61-63], obesity [64], dyslipidemia and the metabolic syndrome [65]. Consistent with these findings, adipokine have been implicated in metabolic adaptations to normal gestation [57,66-73], as well as in preeclampsia [74-94], SGA [95-97] and other complications of pregnancy [69,93,98-109].

Visfatin, a newly discovered 52 kDa adipokine, has been implicated in regulation of glucose hemostasis [110,111]. Indeed, high circulating concentrations of visfatin are associated with hyperglycemia [112], Type-2 diabetes mellitus (Type-2 DM) [113,114] and gestational diabetes mellitus (GDM) [99,115-118]. Thus, the objective of this study was to determine if preeclampsia and SGA are associated with alterations in maternal circulating visfatin concentrations.

MATERIALS AND METHODS

Study population

A cross-sectional study was conducted by searching our clinical database and bank of biological samples, and included pregnant women in the following groups: (1) normal pregnant women who delivered an appropriate for gestational age (AGA) newborn (n = 158); (2) patients with preeclampsia (n = 43) of which 32 had an AGA and 11 had an SGA neonate; (3) patients without preeclampsia who delivered an SGA neonate (n = 55).

Samples and data were retrieved from our bank of biological samples and clinical databases. Many of these samples have previously been employed to study the biology of inflammation, hemostasis, angiogenesis regulation and growth factor concentrations in normal pregnant women and those with pregnancy complications.

All participating women provided written informed consent prior to enrollment and the collection of blood samples. The collection and use of blood for research purposes was approved by the Institutional Review Boards of the Sotero del Rio Hospital (Santiago, Chile) and the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NIH, DHHS, Bethesda, MD).

Definitions

The definition of normal pregnancy included all of the following: (1) no medical, obstetrical or surgical complications; (2) intact membranes; (3) delivery of a term neonate (> 37 weeks) with a birth weight above the 10^th percentile [119]; (4) a normal oral 75-g oral glucose tolerance test (OGTT) between 24 and 28 weeks of gestation based on World Health Organization (WHO) criteria [120].

Clinical definitions

The inclusion criteria for normal pregnant women were as follows: singleton gestation, no prior diabetes mellitus, no maternal or fetal complications during pregnancy, normal plasma glucose concentration in the first trimester, normal oral glucose challenges test [121] and delivery at term of a healthy neonate with a birthweight above the 10^th percentile for gestational age [122].

Preeclampsia was defined as the presence of hypertension (systolic blood pressure ≥ 140 mmHg or diastolic blood pressure ≥ 90 mmHg on at least two occasions, 4 h to 1 week apart) occurring after 20 weeks of gestation in women with previously normal blood pressure, and proteinuria (≥ 300 mg in a 24-h urine collection or at least one dipstick measurement ≥ 1+) [123]. Severe preeclampsia was defined as severe hypertension (diastolic blood pressure ≥ 110 mmHg) plus mild proteinuria, or mild hypertension and sever proteinuria (a 24-h urine sample containing ≥ 3.5 g of protein or a urine specimen ≥ 3+ protein by dipstick measurement on at least two occasions) or severe hypertension plus severe proteinuria [124]. Patients with abnormal liver function test (aspartate aminotransferase > 70 IUI/1) and thrombocytopenia (platelet count < 100,000/cm³) were also classified as having severe preeclampsia [125]. The diagnosis of SGA was based on ultrasonographic estimated fetal weight and confirmed by a birth weight below the 10^th percentile for gestational age [56,122].

The body mass index (BMI) was calculated according to the formula: weight (kg)/height (m²). Normal weight women were defined as those with a BMI of 18.5-24.9 kg/m² according to the definition of the WHO [126].

Sample collection and human visfatin C-terminal immunoassay

Maternal blood samples were collected at clinical visit. Blood was centrifuged at 1300g for 10 min at 4 °C. The plasma obtained was stored at -80 °C until analysis. Concentrations of visfatin in maternal plasma were determined using specific and sensitive enzyme immunoassays (Phoenix Pharmaceuticals, Belmont, CA). An initial assay validation was performed in our laboratory prior to the conduction of this study. Detailed description of the assay has been previously published [73,99,103,107]. The calculated inter and intraassay coefficients of variation for visfatin C-terminal immunoassays in our laboratory were 5.3% and 2.4%, respectively. The sensitivity was calculated to be 0.04 ng/ml.

Statistical analysis

Normality of the data was tested using the Kolmogorov-Smirnov test. Because maternal plasma visfatin concentrations were not normally distributed, Kruskal-Wallis tests with post-hoc analysis by Mann-Whitney U-tests were used for comparisons of continuous variables between the different groups. Comparison of proportions was performed by Chi-square or Fisher’s exact tests. Multiple linear regression analysis was performed to determine which factors were significantly and independently associated with maternal plasma visfatin concentrations; because of skewed distribution, logarithmic (log) transformation was employed in the latter analysis. The following parameters were included in the model: maternal age, maternal pregestational BMI (as continuous variable), gestational age at blood sampling, smoking status and the presence of preeclampsia of SGA. A P-value of < 0.05 was considered statistically significant. Analysis was performed with SPSS< version 14 (SPSS, Chicago, IL).

RESULTS

Demographic and clinical characteristics of the study groups are presented in Table I. The median gestational age at blood sampling was significantly higher in patients with an SGA neonate than in those with either normal pregnancy or preeclampsia. The median gestational age at delivery of patients with preeclampsia was significantly lower than those with either normal pregnancy or SGA (Table I).

Table I.

Clinical and demographic characteristics of the study population.

	Normal pregnancy (n = 158)	P *	Preeclampsia (n = 43)	P χ	SGA (n = 55)	P ‡
Maternal age (years)	26 (22-31)	0.8	26 (21-30)	0.1	22 (19-29)	0.35
Parity	1 (0-2)	0.1	1 (0-1)	0.1	1 (0-1)	0.5
Pre-gestational BMI (kg/m2)	23.5 (21.9-29.9)	0.62	24.5 (21.1-28.5)	0.2	23.2 (21.1-25.0)	0.24
GA at sampling (weeks)	33.0 (28.0-38.7)	0.22	31.7 (29.7-33.8)	< 0.001	39.0 (38.0-39.7)	< 0.001
GA at delivery (weeks)	39.7 (38.7-40.4)	< 0.001	32.4 (30.4-34.1)	0.003	38.5 (38.0-39.8)	< 0.001
Birth weight (g)	3430 (3190-3690)	< 0.001	1460 (1130-2010)	< 0.001	2600 (2330-2777)	< 0.001

Values expressed as median (interquartile range).

GA, gestational age; BMI, body mass index.

^*P: comparison between normal pregnancy and preeclampsia groups.

^χP: comparison between normal pregnancy and SGA groups.

^‡P: comparison between preeclampsia and SGA groups.

Visfatin concentrations in patients with preeclampsia and those with an SGA neonate

The median maternal plasma concentration of visfatin was significantly higher in patients with an SGA neonate than in those with either normal pregnancy (20.0 ng/ml, interquartile range [IQR]: 17.2-24.6 vs. 15.2 ng/ml, IQR: 12.1-19.2; P < 0.001, Figure 1) or those with preeclampsia (14.5 ng/ml, IQR: 12.5-18.7; P < 0.001, Figure 1). The median maternal plasma visfatin concentration did not differ significantly between patients with preeclampsia and those with a normal pregnancy (P = 0.8, Figure 1).

Figure 1.

Maternal plasma visfatin concentration in normal pregnant women, patients with preeclampsia and patients who delivered an SGA neonate. The median maternal plasma concentration of visfatin was significantly higher in patients with an SGA neonate than in those with either a normal pregnancy (20.0 ng/ml, interquartile range [IQR]: 17.2-24.6 vs. 15.2 ng/ml, IQR: 12.1-19.2, respectively; P < 0.001). The median maternal plasma visfatin concentration did not differ significantly between patients with preeclampsia and those with a normal pregnancy (P = 0.8)

Visfatin concentrations in patients with preeclampsia with and without SGA

Among patients with preeclampsia, there was no significant difference in the median maternal plasma visfatin concentration between those with and without an SGA neonate (14.1 ng/ml, IQR: 11.8-17.5 vs. 14.9 ng/ml, IQR 12.8-19.0, respectively; P = 0.5, Figure 2). The median maternal plasma concentration of visfatin was significantly higher in patients with an isolated SGA neonate than in those with preeclampsia either with or without an SGA neonate (P < 0.001 for both comparisons, Figure 2).

Figure 2.

Maternal plasma visfatin concentration in patients with preeclampsia who delivered an SGA neonate, patients with preeclampsia who delivered and AGA neonate and patients without preeclampsia who delivered an SGA neonate. Among patients with preeclampsia, there was no significant difference in the median maternal plasma visfatin concentration between those with and without an SGA neonate (14.1 ng/ml, IQR: 11.8-17.5 vs. 14.9 ng/ml, IQR: 12.8-19.0, respectively; P = 0.5). The median maternal plasma concentrations of visfatin were significantly higher in patients with an isolated SGA neonate than in those with preeclampsia either with or without an SGA neonate (P , 0.001 for both comparisons).

Visfatin concentrations in patients with mild vs. severe preeclampsia

Among patients with preeclampsia, there was no significant difference in the median maternal plasma visfatin concentration between those with mild and severe preeclampsia (14.0 ng/ml, IQR: 9.9-21.7 vs. 14.5 ng/ml, IQR: 12.6-18.3, respectively; P = 0.8). The median maternal plasma concentrations of visfatin were significantly higher in patients with an isolated SGA neonate than in those with either mild or severe preeclampsia (P < 0.001 for both comparisons).

Linear regression analysis was used to examine the association between the presence of SGA or preeclampsia and maternal plasma visfatin concentration while adjusting for maternal age, pregestational BMI (as a continuous variable), gestational age at blood sampling and smoking status. The final regression model suggested that the presence of SGA and pregestational BMI were independently associated with maternal plasma concentrations visfatin (P < 0.001 and P = 0.04, respectively).

DISCUSSION

Principal findings of the study

(1) Women who delivered an SGA neonate had a higher median maternal plasma visfatin concentration than those either with normal pregnancy or with preeclampsia; (2) the median maternal plasma visfatin concentration did not differ significantly between patients with preeclampsia and those with normal pregnancy; (3) among patients with preeclampsia, there was no significant difference in the median maternal plasma visfatin concentration between those with or without an SGA neonate.

The physiological role of visfatin

Visfatin, a newly discovered 52 kDa adipokine, was originally identified as a growth factor for early B cell, and thus was termed Pre-B cell colony-enhancing factor (PBEF) [127]. Visfatin/PBEF is preferentially produced by visceral fat depot [111]. However, expression of visfatin is not limited to adipose tissue and it can be expressed in placenta, fetal membranes [128-135], myometrium [136], bone marrow, liver, muscle [127], heart, lung, kidney [127], macrophages [137] and neutrophils [127,138].

Visfatin has been implicated in regulation of glucose homeostasis, as well as in inflammatory response. Several lines of evidence support the role of this adipokine in metabolic regulation: (1) adipocytes secrete visfatin in response to treatment with glucose [112]; (2) visfatin can exert insulin-mimicking effects [110,111] through the activation of an insulin receptor; (3) visfatin deficient mice have an impaired glucose tolerance [139]; (4) a visfatin promoter polymorphism is associated with a susceptibility to Type-2 DM [140].

A compelling body of evidence suggests a role for visfatin as a mediator of the inflammatory response. The following findings characterized visfatin as an immunomodulator: (1) it synergizes with IL-7 and stem cell factors to promote the growth of B-cell precursors [67]; (2) treatment of human monocytes with visfatin results in an increased secretion of IL-6, TNF-α and IL-1β in a dose dependent manner [141]; (3) the expression of visfatin is increased following exposure to TNF-α (in monocytes [137], macrophages [142] and neutrophils [138]), IL-6 (in synovial [143] and amniotic epithelial cells), IL-8 and granulocyte/macrophage colony stimulating factor (in neutrophils [138]); (4) chronic inflammatory disorders such as inflammatory bowel disease [141] and rheumatoid arthritis [144] are associated with higher circulating visfatin concentrations than normal subjects.

Visfatin in normal gestation and in complications of pregnancy

Alterations in circulating adipokines have been associated with adaptations to gestation, as well as in complications of pregnancy [66-72,93,95,98,100-104,106,107]. Consistent with this view, normal pregnancy is associated with high maternal circulating visfatin concentrations [73,145-149]. In addition, gestational diabetes mellitus is associated with altered maternal concentrations of this adipokine than nondiabetic pregnant women [99,115,117,118,150]. Recently, we have reported that intra-amniotic infection/inflammation is associated with higher amniotic fluid concentrations of visfatin than the absence of infection [107]. This finding is in agreement with previous reports in which visfatin expression in fetal membranes increased after exposure to inflammatory stimuli [135,151].

Dysregulation of circulating maternal visfatin concentrations is a feature of patients with an SGA neonate but not of preeclampsia

Reports regarding maternal circulating visfatin concentrations in patients with preeclampsia are scarce and inconsistent. Fasshauer et al. [152] and subsequently Adali et al. [153] reported higher maternal concentrations of circulating visfatin in patients with preeclampsia than in normotensive pregnant women during eh third trimester, while Hu et al. [149] found significantly lower maternal concentrations of this adipokine in preeclampsia than in the control group. Moreover, in the latter study, patients with severe preeclampsia had a significantly lower serum visfatin concentration than those with mild preeclampsia [149]. In contrast to the aforementioned studies, we found no significant differences in maternal circulating visfatin concentrations in pregnant women with and without preeclampsia. Differences in study population and study design may account for these apparent discrepancies. Specifically, the sample size, ethnic origin, gestational age at enrollment, differences in BMI and neonatal birth weights differ among the studies.

Only two studies by Fasshauer et al. [154] and Malamitsi-Puchner et al. [155] reported the results of comparison of maternal visfatin concentrations between the patients with an SGA neonate and those with a normal pregnancy [154,155]. The results reported herein are in agreement with these studies; however, our findings extend the aforementioned reports by demonstrating that patients with an SGA neonate have higher circulating maternal visfatin concentrations than those with preeclampsia. Of note, this novel finding did not change after adjusting for maternal age, maternal pregestational BMI, gestational age at sampling and smoking. In addition, we were able to report that the presence of an SGA neonate in patients with preeclampsia was not accompanied by significant alterations in maternal circulating visfatin concentrations suggesting that the effect of the presence of an SGA neonate on maternal plasma visfatin concentration is overwhelmed by preeclampsia.

Why is visfatin differentially regulated in patients with preeclampsia and in those with SGA neonates?

A lingering conundrum in obstetrics is why preeclampsia and pregnancy complicated by an SGA neonate have profoundly different clinical manifestation, despite their common risk factors and similar mechanisms of disease. Several explanations have been proposed to account for the divergence between preeclampsia and SGA including exposure to infection during pregnancy [156-158], differences in the profile of angiogenic and antiangiogenic response to intrauterine insults [30,35], altered activity of the coagulation system [159,160] and altered concentrations of placental growth hormone [161] and pro-inflammatory chemokines such as CSCL10/IP-10 [32].

Ness and Sibai [37] have proposed that underlying maternal metabolic derangements are the cause for the distinct clinical manifestations of these complications of pregnancy. Specifically, increased maternal adiposity, insulin resistance/hyperglycemia or dyslipidemia are associated with preeclampsia, whereas absence of these metabolic complications is associated with SGA. Indeed, obesity [3,162-164] and insulin resistance [165-168] are independent risk factors for preeclampsia and the latter is associated with dyslipidemia [169,170] as well as with metabolic syndrome-related morbidity [171,172] and mortality [173] later in life. In contrast, maternal overweight/obesity has a protective effect for the development of SGA fetuses [162,174].

The cross-sectional nature of this study limited our ability to infer a causal relationship between visfatin and SGA. However, several explanations can account for the differences in maternal circulating visfatin concentrations between patients with preeclampsia and those with an SGA neonate:

1. High circulating maternal visfatin concentration confers a beneficiary metabolic effect and thus protects the mother from preeclampsia; Compelling evidence suggests that visfatin may have beneficiary metabolic effects. Indeed, injection of visfatin in mice lowered plasma glucose, and visfatin deficient mice have an impaired glucose tolerance suggesting that high concentrations of visfatin may be associated with insulin sensitivity. Thus, it can be postulated that in the presence of common risk factors for preeclampsia and SGA, elevated visfatin concentration will result in increased insulin sensitivity that is associated with a decreased risk for preeclampsia. In contrast, the failure to increase maternal visfatin concentration may be associated with preeclampsia.

2. Transport from the fetal to maternal compartment: Ibáñez et al. [175] reported that cord blood visfatin concentration in SGA neonates is higher than AGA neonates. Thus, it can be hypothesized that a transport from fetal to maternal circulation can account for the higher concentrations of this adipokine in patients with an SGA neonate. Nevertheless, the relatively high molecular weight of visfatin (52 kDa) and lack of evidence for a specific mechanism of cross-placental transport do not support this explanation. Furthermore, in a study conducted by Malamitsi-Puchner [155], cord blood visfatin concentration did not differ between SGA and AGA neonates.

In summary, this study is the first to compare circulating maternal visfatin concentrations between patients with an SGA neonate and those with preeclampsia. The novel findings reported herein suggest that changes in circulating maternal visfatin concentration participate in the phenotype definition of preeclampsia and SGA. This observation is in line with and lends credence to the report by Ness and Sibai as well as to the growing body of evidence concerning the role of adipokines in complications of pregnancy.