Hypertension in Pregnancy
ISSN: 1064-1955 (Print) 1525-6065 (Online) Journal homepage: http://www.tandfonline.com/loi/ihip20
Maternal Serum Resistin Is Reduced in First Trimester Preeclampsia Pregnancies and Is a Marker of Clinical Severity Michael Christiansen, Paula L. Hedley, Sophie Placing, Karen R. Wøjdemann, Anting L. Carlsen, Jennifer M. Jørgensen, Anne-Cathrine Gjerris, AnneCathrine Shalmi, Line Rode, Karin Sundberg & Ann Tabor To cite this article: Michael Christiansen, Paula L. Hedley, Sophie Placing, Karen R. Wøjdemann, Anting L. Carlsen, Jennifer M. Jørgensen, Anne-Cathrine Gjerris, Anne-Cathrine Shalmi, Line Rode, Karin Sundberg & Ann Tabor (2015) Maternal Serum Resistin Is Reduced in First Trimester Preeclampsia Pregnancies and Is a Marker of Clinical Severity, Hypertension in Pregnancy, 34:4, 422-433, DOI: 10.3109/10641955.2014.913615 To link to this article: http://dx.doi.org/10.3109/10641955.2014.913615
Published online: 04 Dec 2015.
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Hypertens Pregnancy, 2015; 34(4): 422–433 ! Informa Healthcare USA, Inc. ISSN: 1064-1955 print / 1525-6065 online DOI: 10.3109/10641955.2014.913615
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Maternal Serum Resistin Is Reduced in First Trimester Preeclampsia Pregnancies and Is a Marker of Clinical Severity Michael Christiansen,1 Paula L. Hedley,1,2 Sophie Placing,1 Karen R. Wøjdemann,3,4 Anting L. Carlsen,1 Jennifer M. Jørgensen,1 Anne-Cathrine Gjerris,3,5 Anne-Cathrine Shalmi,3,5 Line Rode,3 Karin Sundberg,3 and Ann Tabor3 1
Department of Clinical Biochemistry, Statens Serum Institut, Copenhagen, Denmark, 2Department of Biomedical Sciences, Stellenbosch University, Cape Town, South Africa, 3Department of Fetal Medicine, Rigshospitalet, Copenhagen, Denmark, 4Department of Obstetrics and Gynecology, Roskilde Hospital, Roskilde, Denmark, 5Department of Obstetrics and Gynecology, Hillerød Hospital, Hillerød, Denmark Objective: To examine whether resistin levels in first trimester maternal serum are associated with insulin resistance or preeclampsia (PE). Methods: A case-control study of maternal serum resistin concentration conducted using 285 normal pregnancies and 123 PE pregnancies matched for gestational age, parity and maternal age. Samples were taken in gestational weeks 10+0–13+6. Results: There was a negative correlation between resistin and clinical severity of PE, but no correlation with IS, TNF-a, body mass index, birth weight and pregnancy length. Conclusions: Resistin is reduced in first trimester of PE pregnancies, particularly in severe PE. Inflammation and IS cannot explain this phenomenon. Keywords placenta, pre-eclampsia, prenatal screening
INTRODUCTION Resistin is a cysteine-rich 12.5 kD adipokine (1). It circulates in the serum in trimeric and higher oligomeric forms (1,2). In rodents, resistin is predominantly synthesised by adipocytes, it is elevated in the serum in obesity and increases insulin resistance (IR) through interference with liver glucose uptake; it is believed to be the link between obesity and diabetes (3). Human resistin is 56% and 54% identical, at an amino acid level, with mouse and rat resistin, respectively. Human resistin is synthesised by mononuclear cells and
Correspondence: Michael Christiansen, MD, Research Director, Chief Physician, Department of Clinical Biochemistry and Immunology, Statens Serum Institut, 5 Artillerivej DK2300S, Copenhagen, Denmark. E-mail: [email protected]
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macrophages (4), the bone marrow (5), adipocytes and muscle (6), islets cells in the pancreas (7) and placenta (8–10). The function of resistin in humans is not precisely known, but elevated levels have been found in inflammatory disorders (11,12), whereas the role of resistin in IR is more controversial with some studies showing a positive correlation between resistin and markers of IR and metabolic syndrome (13–16), and others showing a negative correlation (17). Pregnancy is characterised by major changes in carbohydrate and energy metabolism (18). Maternal IR is believed to ensure availability of nutrients to the foetus and is a characteristic of second and third trimester (18), which normalises following parturition (19). However, in first trimester, relative insulin sensitivity occurs, most likely to ensure maternal energy stores for later in pregnancy (18,20). How these changes in maternal metabolism are effectuated is not precisely known, but a combined effect of anti-insulinergic hormones, e.g. progesterone, estrogens, cortisol, human placental lactogen and human placental growth hormone, and maternal and placental adipokines are believed to be the major determinants of IR in pregnancy (19,21). Increased IR in pregnancy has not only been associated with various pathological conditions, most notably gestational diabetes mellitus (22) but also preeclampsia (PE) (23). PE is a pregnancy complication presenting – after gestational week 20 – with elevated blood pressure, proteinuria and in severe cases widespread organ failure (24). The worldwide prevalence of PE is estimated to be 2–10% (25), the incidence of PE differs considerably between developed countries (0.4% live births) and developing countries (2.8% live births) (26) and the condition is believed to cause 100 000 annual maternal deaths worldwide (24). Up to 25% of babies born with a very low (51500 grams) birth weight are a consequence of PE. Thus, PE is a major contributor to maternal as well as foetal morbidity and mortality. The aetiology of PE is probably heterogeneous and not completely clarified, but it is believed that the disease starts as a placental condition resulting in insufficient placental perfusion followed by a maternal syndrome with endothelial dysfunction and hypertension (27). Interestingly, PE is associated with an elevated risk of metabolic syndrome later in life (24), just as pre-pregnancy metabolic syndrome increases the risk of PE (24). Recently, it has been established that prophylactic anti-inflammatory and anti-thrombotic treatment with low dose aspirin, instituted prior to week 16, may reduce the risk of severe PE (28). This finding makes it highly relevant to identify early first trimester markers of PE. Resistin has been found to be elevated in second and third trimester of pregnancies developing PE (27), but very little is known about the maternal serum concentration of resistin in normal pregnancies and nothing has been reported on the first trimester level in pregnancies later developing PE. With the recent finding that perturbations in other adipokines, i.e. leptin (Lp) and free Lp index (29), are seen in first trimester of PE pregnancies, the role of other adipokines and potential regulators of IR and inflammation becomes relevant to study. The purpose of this study was threefold. The first aim was to establish the concentration of resistin in maternal serum in late first trimester normal pregnancy. The second aim was to assess resistin’s relation to maternal
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pregnancy parameters, e.g. body mass index (BMI), parity, age and markers of inflammation and IS, duration of pregnancy, gestational age and occurrence of PE. The third and final aim was to examine whether resistin could be used as a first trimester marker of PE.
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MATERIALS AND METHODS Pregnant Women Serum samples from 408 pregnant women (123 of whom later developed PE) were collected in gestational weeks 10+3–13+6. These women were enrolled from 1997 to 2001 in the Copenhagen First Trimester Screening Study (30). Gestational age was determined by crown-rump length. All samples were collected in dry containers and kept at 4 C for a maximum of 48 h until delivery to Statens Serum Institut. Samples were subsequently stored at 20 C. The samples had been subjected to four freeze–thaw cycles prior to the measurement of resistin. The criteria of The International Society for the Study of Hypertension in Pregnancy were used to define PE (31). Thus, the diagnosis of PE required hypertension, defined as either a systolic blood pressure 4140 mm Hg or a diastolic blood pressure 490 mm Hg occurring in a previously normotensive woman after 20 weeks of gestation in combination with proteinuria (40.3 g per 24 h or dipstick urine analysis 41+). Severe PE was defined by a diastolic blood pressure 4110 mm Hg in combination with subjective symptoms and/or abnormal laboratory findings, i.e. oliguria (5400 mL per 24 h), severe proteinuria (43 g per 24 h), elevated liver enzymes, raised s-bilirubin, s-urate or s-creatinine, thrombocytopenia, disseminated intravascular coagulation, haemolysis and hypercoagulability. The presence of haemolysis, elevated liver enzymes and low platelets defined the HELLP syndrome. Early PE was defined as a PE pregnancy with delivery prior to week 34. Controls were 285 women with an uncomplicated pregnancy. Demographic and clinical parameters of the women included in the PE group and controls are listed in Table 1; the PE group parameters are broken down with respect to clinical severity in Table 2. Table 1. Demographic and Clinical Characteristics of Preeclamptic and Control Pregnancies.
Parameter Age (years) median (range) Weight (kg) median (range) BMI (kg/m2) median (range) Gravida (=1(%), 2(%), 3(%), 43(%)) Birth weight (g) median (range) GA at birth (d) median (range) GA sampling (d) median (range) Resistin (ug/l) median (range)
Controls (n = 285)
Pre-eclampsia (n = 123)
30.1 (18.9–45.1) 62 (41–137) 21.7 (14.5–37.2) 156(58), 78(28), 34(12), 17(5) 3600 (2650–5010) 284 (260–300) 90 (73–97) 25.4 (4.9–83.0)
30.5 (18.0–45.9) 68 (47–112) 24.0 (19.1–40.4) 73(59), 27(22), 15(13), 8(7) 3200 (465–5000) 276 (167–304) 91 (75–97) 22.2 (7.5–58.0)
p* Ns 50.001 50.001 Ns 50.001 50.001 Ns 0.002
*Mann–Whitney U test, except for gravida where 2 was used and Resistin, where one-way ANOVA was used.
Resistin in First Trimester Pregnancy Table 2. Demographic and Clinical Characteristics of Severe Pre-eclamptic, Non-severe Pre-eclamptic and HELLP Pregnancies. Parameter
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Age (years) median (range) Weight (kg) median (range) BMI (kg/m2) median (range) Gravida (=1(%), 2(%), 3(%)) Birth weight (g) median (range)* GA at birth (d) median (range)* GA sampling (d) median (range) Resistin (ug/l) median (range)y
Mild (n = 96) 31 67 24.0 56
(18–42) (47–111) (19.1–40.4) (58), 20 (21), 20 (20) 3274 (753–5000)
Severe (n = 21) (21–37) (50–95) (20.0–32.8) (52), 7 (33), 3 (14) 2682 (465–4500)
(23–46) (50–112) (19.6–34.6) (100), 0(0), 0 (0) 2259 (990–3700)
278 (195–304)
264 (167–286)
254 (203–290)
90 (75–97)
91 (80–97)
91 (90–93)
21. 9 (7.5–58.0)
29 72 24.5 11
HELLP (n = 6)
22.4 (11.1–46.4)
31 67 23.1 6
24.3 (11.6–33.3)
*ANOVA, F-test, p50.001. yANOVA, F-test, p = 0.02.
Resistin Measurement The resistin concentration was determined in singlo using the Human Resistin ELISA development kit, Duo Set (DY1359, R&D Systems, Minneapolis, MN) as described by the manufacturer following appropriate sample dilution. The functional detection limit of the assay was 31.25 pg/mL resistin. The intraassay coefficient of variation (CV) was 55% and the inter-assay CV 510%. The calibration was done using a highly purified Escherichia coli-expressed recombinant human resistin produced by the kit manufacturer. The resistin concentration in serum samples was stable for at least 56 h at room temperature and 10 freeze–thaw cycles. TNF-a Measurement The concentration of TNF-a was determined in singlo using the human TNF-a/TNFS1A Duoset ELISA kit (DY210, R&D Systems), as described by the manufacturer using appropriately diluted samples. The functional detection limit was 15.6 pg/mL. The assay was calibrated using a recombinant TNF-a preparation provided with the kit. The intra- and inter-assay CVs were 510% and 520%, respectively. Serum samples were stable for 48 h at 23 C and 10 freeze–thaw cycles. Measurement of the Adiponectin and Lp The concentrations of adiponectin (ApN) and Lp were established using the commercially available ELISA kits DY1065 ApN and DY398 Lp (R&D Systems), respectively. Samples were analysed in singlo following appropriate dilutions. The functional detection limit of ApN was 50 pg/mL and 31.25 pg/mL for Lp. The intra- and inter-assay CV’s were 55% and 510%, respectively for both assays. Calibration was performed using recombinant ApN expressed in NS0 cells and Lp expressed in E. coli. Following analysis of each of the two analytes, the ApN/Lp ratio was calculated
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Data Analysis The relation between the maternal serum concentration of resistin and other parameters, e.g. gestational age and maternal weight was examined by Loess non-parametric regression or non-parametric correlation analysis a.m. Spearman. Log transformations were used to achieve compatibility with the normal distribution, which was in turn assessed by normal probability plots and Kolmogorov–Smirnov’s test. Medians were compared using the Mann–Whitney U-test. Comparison of multiple groups was performed using Kruskal–Wallis test. Assessment of discriminatory ability was performed using empirical receiver operator characteristics (ROC) curves. Log-linear multiple regression was used to construct discriminatory indices. p = 0.05 was selected as the level of significance. Statistical analyses were performed using SPSS version 19.0 (SPSS Inc., Chicago, IL). Ethics Blood sampling was approved as part of the Copenhagen First trimester Study by the Scientific Ethics committee for Copenhagen and Frederiksberg Counties (No. (KF) 01-288/97).
RESULTS The maternal serum resistin concentration was significantly (p50.002) lower in PE pregnancies than in controls (Table 1). The relation between maternal serum resistin and gestational age is shown in Figure 1 for controls and PE pregnancies. The Loess regression curves show that serum resistin was
Figure 1. Scatter plots and Loess regression lines depicting the relation between maternal serum resistin concentrations and gestational age in controls (n = 285) and PE cases (123). The resistin concentration in controls decreases throughout the examined period and after day 95 becomes lower than in PE pregnancies.
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independent of gestational age in PE pregnancies (Spearman’s rho = 0.008, p = 0.32, n = 123) in the whole examined period, whereas the resistin concentration decreased significantly (Spearman’s rho = 0.225, p50.001, n = 285) in control pregnancies. However, the resistin concentration in controls only dropped markedly after day 90; prior to that time the decrease with gestation was not significant (Spearman’s rho = –0.112, p = 0.15, n = 145); and from day 95, the values in PE pregnancies were higher than the values in controls with a median resistin concentration of 18.3 ng/mL (range: 7.8–53.6 ng/mL, n = 65) in controls and a median of 23.4 ng/mL (range: 13.3–44.0 ng/mL, n = 21) in PE pregnancies, but this difference was not significant (p = 0.8), so the low number of samples preclude any firm conclusions on this. In the gestational age window 10+0–12+6 weeks, where the resistin level was fairly constant in controls, the median resistin concentration in controls was 27.7 ng/mL (range: 4.9–89.0 ng/mL) and significantly lower, median 22.3 ng/mL (range: 7.5–58.0 ng/mL), in PE pregnancies (p = 0.002). This means that maternal serum resistin is 15–20% reduced in PE pregnancies in week 10+0–12+6. In neither the control nor PE pregnancies, were there any significant correlation between serum resistin and maternal weight, maternal BMI, maternal age or birth weight (p40.6, in all examinations). There was no correlation between the resistin concentration and the concentration of TNF-a in the pregnancies where TNF-a was measurable (415.6 pg/mL), irrespective of whether the pregnancy developed PE or not. Likewise, there was no correlation between IS, as determined by the ApN–Lp ratio, and resistin in either controls or PE pregnancies (Figure 2).
Figure 2. Scatter plots and Loess regression lines depicting the relation between IS (expressed by the ApN/Lp-ratio) and maternal serum resistin concentrations. There was no significant correlation between IS and resistin levels in either controls or PE pregnancies.
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Figure 3. Boxplot of the maternal serum resistin concentration levels in controls and PE cases in gestational week 10+3–13+0, grouped according to clinical severity. The difference between groups was significant, p = 0.018, ANOVA and there was a significant linear negative trend, p = 0.003.
The relation between maternal serum resistin in gestational week 10+3–13+5 and clinical severity of PE is shown to be significant in Table 2. In week 10+3– 12+6, where the resistin concentration in both controls and PE pregnancies was constant, the relation was as shown in Figure 3. The median (range) resistin concentration decreases significantly (p = 0.005, Kruskal–Wallis test), from 27.7 ng/mL (4.9–83.0) in controls (n = 163) to 22.3 ng/mL (6.2–58.0 ng/mL) in mild pre-eclampsia (n = 63) and 21.3 ng/mL (11.1–36.5 ng/mL) in severe preeclampsia (n = 11) to reach 17.6 ng/mL (11.6–33.3 ng/mL) in HELLP pregnancies (n = 4). Ten PE pregnancies with delivery prior to week 34, where only five of them had blood sampled prior to week 13, exhibit a median resistin concentration of 17.3 ng/mL (range: 11.6–36.1 ng/mL), and this level is lower than that of HELLP pregnancies, but the small number precluded that the resistin difference between early and late PE pregnancies could reach significance (p = 0.46). The ability of maternal serum resistin to discriminate between pregnancies that developed PE and normal pregnancies in week 10+0–12+6 was analysed by empirical ROC curve analysis (Figure 4). The area under the curve (AUC) was 0.617 (95% confidence interval: 0.539–0.695, p = 0.008). The empirical detection rate for false-positive rates of 20% and 10% was around 23% and 20%, respectively. In order to assess the potential of combining resistin with other first trimester PE markers, we performed a linear regression of the parameters, log10 resistin, log10 PAPP-A, BMI and gestational age using the dummy variables 1 and 2 for controls and PE pregnancies, respectively. We used the 206 pregnancies (61 PE pregnancies and 145 controls) that had blood sampled prior to week 13. It was found that only log10 resistin and BMI contributed independently and significantly to the discrimination between PE and control pregnancies. The discrimination index = 0.311 log10 resistin + 0.036 BMI + 1.84 had an R2 of 0.107. Figure 4 depicts the ROC curves of BMI, log10 resistin and the discriminatory index. BMI had an
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Figure 4. Receiver operator characteristics (ROC) curve of maternal serum resistin as a marker for pre-eclampsia in gestational week 10+3–13+0. The area under the curve was 0.616 with a 95% confidence interval: 0.545–0.695, p = 0.003. The ROC curves for BMI and the combination of resistin and BMI (discriminatory index) are also depicted.
AUC of 0.679 (range: 0.600–0.757) and the index an AUC of 0.706 (range: 0.629–0.783). BMI was thus a better discriminator than log10 resistin, and the addition of log10 resistin to BMI did increase the AUC, but not significantly so. This is probably a result of the relatively small number of pregnancies.
DISCUSSION This is – to our knowledge - the first report of reduced first trimester maternal serum resistin concentrations in pregnancies that later develop PE. The reduction is in the order of 15–20% of the resistin concentration seen in control pregnancies; in HELLP pregnancies, the reduction is 430%, in both cases, prior to week 13. Previous studies have reported elevated or un-perturbed resistin concentrations in second and third trimester PE pregnancies (8,32,33). The period from day 90 and onwards, where the maternal serum concentration of resistin decreases in controls and remains constant in PE pregnancies, suggests that resistin after day 95 will be elevated in PE pregnancies. This is in agreement with the cited studies. This is also – to our knowledge – the first report of a decreasing maternal serum resistin through first trimester in normal pregnancies. Other studies have reported on resistin concentrations in pregnancy (8,10,13,33–38), but the first measurements in these studies are from late first or early second trimester. One study found no gestational age dependence (13), but another did (36), whereas a third only found the levels increased in third trimester (30). As the resistin concentrations measured in this study in first trimester are
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substantially higher than those reported in other studies from late first and early second trimester, i.e. 20–25 ng/mL compared to 10–18 ng/mL (8,10,13,33–38), it does seem that first trimester is characterised by high resistin levels. PE pregnancies are then characterised by not reaching this level. The lack of an association between resistin and maternal BMI or IS (Figure 2), also reported by others in later parts of pregnancy (33,36), suggests that the maternal serum concentration of resistin is not defined by the metabolic status of the mother. An alternative reason for the variations in resistin could, however, be that it is a result of changes in the distribution of body fat in the mother – as the intra-abdominal fat increases during pregnancy (39) and resistin is preferentially synthesised in abdominal fat (40,41). The low resistin levels in the mother could be a result of a complicated endocrine dysfunction in the mother. Furthermore, resistin levels have been reported to be significantly heritable. Pantsulaia et al., 2007 reported that 66% of resistin variation could be attributed to genetic factors (40). We did not find a significant association between resistin and the concentration of the inflammatory marker TNF-a in maternal serum, suggesting that resistin is not increased due to low-level inflammation. As resistin is synthesised by the foetus and present in high concentrations in umbilical cord blood (35) at term, and in even higher concentrations in PE pregnancies (32), it would seem reasonable that the physiological significance of resistin is related to foetal development. This could also explain that the level is very high in first trimester where the foetus does not have a circulation that can ensure the presence of resistin in the foetal adipose tissue. However, this line of reasoning is hypothetical and will need genuine experimental support. Furthermore, if PE pregnancies are characterised by a relative resistin deficiency, this could explain why PE is associated with reprogramming of the foetus leading to an increased risk of metabolic syndrome. The ROC curve analysis (Figure 4) shows that resistin is a very poor marker of PE. Thus, it must be considered clinically irrelevant, except for the suggested ability of resistin to selectively identify HELLP and early PE. If PE screening is introduced, it is very important that the screening identifies pregnancies with a risk of morbidity and not the many mild cases that have no clinical significance. If more efficient screening algorithms could be combined with markers, like resistin, that more selectively identifies severe, clinically relevant, cases, it would greatly improve the ethical argument for introducing PE screening despite the dangers of increasing parental anxiety and interference with the incentive to seek timely medical care in false-negative cases. In conclusion, we have shown that maternal serum resistin is significantly reduced in PE pregnancies in week 10+3–12+6 and that this reduction is more pronounced with increasing severity of disease. Furthermore, resistin is not suitable as a marker of PE, but may have a role in defining the risk for clinically severe PE.
ACKNOWLEDGMENTS We gratefully acknowledge the expert technical assistance of Pia Lind and Pernilla Rasmussen.
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FUNDING The authors also gratefully acknowledge the financial support of the Danish Medical Research Council, Copenhagen University, The John and Birthe Meyer Foundation, The Ivan Nielsen Foundation, The Else and Mogens Wedell-Wedellsborg Foundation, The Dagmar Marshall Foundation, The Egmont Foundation, The Fetal Medicine Foundation, The Augustinus Foundation, The Gangsted Foundation, The A.P. Møllerller Foundation, The Mads Clausens Foundation, The Copenhagen Hospital Corporation, SAFE Network of Excellence and Statens Serum Institut.
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DECLARATION OF INTEREST The authors report no conflict of interest. The authors alone are responsible for the content and writing of the paper.
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34. Chen D, Dong M, Fang Q, et al. Alterations of serum resistin in normal pregnancy and pre-eclampsia. Clin Sci (Lond). 2005;108:81–4. Epub 2004/09/21. 35. Cho GJ, Yoo SW, Hong SC, et al. Correlations between umbilical and maternal serum resistin levels and neonatal birth weight. Acta Obstet Gynecol Scand 2006; 85:1051–6. Epub 2006/08/25. 36. Nien JK, Mazaki-Tovi S, Romero R, et al. Resistin: a hormone which induces insulin resistance is increased in normal pregnancy. J Perinat Med 2007;35:513–21. Epub 2007/10/09. 37. Palik E, Baranyi E, Melczer Z, et al. Elevated serum acylated (biologically active) ghrelin and resistin levels associate with pregnancy-induced weight gain and insulin resistance. Diabetes Res Clin Pract 2007;76:351–7. Epub 2006/10/03. 38. Verhaeghe J, van Bree R, Lambin S, Caluwaerts S. Adipokine profile and C-reactive protein in pregnancy: effects of glucose challenge response versus body mass index. J Soc Gynecol Investig 2005;12:330–4. Epub 2005/06/28. 39. Kinoshita T, Itoh M. Longitudinal variance of fat mass deposition during pregnancy evaluated by ultrasonography: the ratio of visceral fat to subcutaneous fat in the abdomen. Gynecol Obstet Invest 2006;61:115–18. Epub 2005/11/08. 40. McTernan CL, McTernan PG, Harte AL, et al. Resistin, central obesity, and type 2 diabetes. Lancet 2002;359:46–7. Epub 2002/01/26. 41. McTernan PG, McTernan CL, Chetty R, et al. Increased resistin gene and protein expression in human abdominal adipose tissue. J Clin Endocrinol Metab 2002;87: 2407. Epub 2002/05/08.
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ISSN: 1064-1955 (Print) 1525-6065 (Online) Journal homepage: http://www.tandfonline.com/loi/ihip20
Maternal Serum Resistin Is Reduced in First Trimester Preeclampsia Pregnancies and Is a Marker of Clinical Severity Michael Christiansen, Paula L. Hedley, Sophie Placing, Karen R. Wøjdemann, Anting L. Carlsen, Jennifer M. Jørgensen, Anne-Cathrine Gjerris, AnneCathrine Shalmi, Line Rode, Karin Sundberg & Ann Tabor To cite this article: Michael Christiansen, Paula L. Hedley, Sophie Placing, Karen R. Wøjdemann, Anting L. Carlsen, Jennifer M. Jørgensen, Anne-Cathrine Gjerris, Anne-Cathrine Shalmi, Line Rode, Karin Sundberg & Ann Tabor (2015) Maternal Serum Resistin Is Reduced in First Trimester Preeclampsia Pregnancies and Is a Marker of Clinical Severity, Hypertension in Pregnancy, 34:4, 422-433, DOI: 10.3109/10641955.2014.913615 To link to this article: http://dx.doi.org/10.3109/10641955.2014.913615
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Date: 10 December 2015, At: 00:06
Hypertens Pregnancy, 2015; 34(4): 422–433 ! Informa Healthcare USA, Inc. ISSN: 1064-1955 print / 1525-6065 online DOI: 10.3109/10641955.2014.913615
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Maternal Serum Resistin Is Reduced in First Trimester Preeclampsia Pregnancies and Is a Marker of Clinical Severity Michael Christiansen,1 Paula L. Hedley,1,2 Sophie Placing,1 Karen R. Wøjdemann,3,4 Anting L. Carlsen,1 Jennifer M. Jørgensen,1 Anne-Cathrine Gjerris,3,5 Anne-Cathrine Shalmi,3,5 Line Rode,3 Karin Sundberg,3 and Ann Tabor3 1
Department of Clinical Biochemistry, Statens Serum Institut, Copenhagen, Denmark, 2Department of Biomedical Sciences, Stellenbosch University, Cape Town, South Africa, 3Department of Fetal Medicine, Rigshospitalet, Copenhagen, Denmark, 4Department of Obstetrics and Gynecology, Roskilde Hospital, Roskilde, Denmark, 5Department of Obstetrics and Gynecology, Hillerød Hospital, Hillerød, Denmark Objective: To examine whether resistin levels in first trimester maternal serum are associated with insulin resistance or preeclampsia (PE). Methods: A case-control study of maternal serum resistin concentration conducted using 285 normal pregnancies and 123 PE pregnancies matched for gestational age, parity and maternal age. Samples were taken in gestational weeks 10+0–13+6. Results: There was a negative correlation between resistin and clinical severity of PE, but no correlation with IS, TNF-a, body mass index, birth weight and pregnancy length. Conclusions: Resistin is reduced in first trimester of PE pregnancies, particularly in severe PE. Inflammation and IS cannot explain this phenomenon. Keywords placenta, pre-eclampsia, prenatal screening
INTRODUCTION Resistin is a cysteine-rich 12.5 kD adipokine (1). It circulates in the serum in trimeric and higher oligomeric forms (1,2). In rodents, resistin is predominantly synthesised by adipocytes, it is elevated in the serum in obesity and increases insulin resistance (IR) through interference with liver glucose uptake; it is believed to be the link between obesity and diabetes (3). Human resistin is 56% and 54% identical, at an amino acid level, with mouse and rat resistin, respectively. Human resistin is synthesised by mononuclear cells and
Correspondence: Michael Christiansen, MD, Research Director, Chief Physician, Department of Clinical Biochemistry and Immunology, Statens Serum Institut, 5 Artillerivej DK2300S, Copenhagen, Denmark. E-mail: [email protected]
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macrophages (4), the bone marrow (5), adipocytes and muscle (6), islets cells in the pancreas (7) and placenta (8–10). The function of resistin in humans is not precisely known, but elevated levels have been found in inflammatory disorders (11,12), whereas the role of resistin in IR is more controversial with some studies showing a positive correlation between resistin and markers of IR and metabolic syndrome (13–16), and others showing a negative correlation (17). Pregnancy is characterised by major changes in carbohydrate and energy metabolism (18). Maternal IR is believed to ensure availability of nutrients to the foetus and is a characteristic of second and third trimester (18), which normalises following parturition (19). However, in first trimester, relative insulin sensitivity occurs, most likely to ensure maternal energy stores for later in pregnancy (18,20). How these changes in maternal metabolism are effectuated is not precisely known, but a combined effect of anti-insulinergic hormones, e.g. progesterone, estrogens, cortisol, human placental lactogen and human placental growth hormone, and maternal and placental adipokines are believed to be the major determinants of IR in pregnancy (19,21). Increased IR in pregnancy has not only been associated with various pathological conditions, most notably gestational diabetes mellitus (22) but also preeclampsia (PE) (23). PE is a pregnancy complication presenting – after gestational week 20 – with elevated blood pressure, proteinuria and in severe cases widespread organ failure (24). The worldwide prevalence of PE is estimated to be 2–10% (25), the incidence of PE differs considerably between developed countries (0.4% live births) and developing countries (2.8% live births) (26) and the condition is believed to cause 100 000 annual maternal deaths worldwide (24). Up to 25% of babies born with a very low (51500 grams) birth weight are a consequence of PE. Thus, PE is a major contributor to maternal as well as foetal morbidity and mortality. The aetiology of PE is probably heterogeneous and not completely clarified, but it is believed that the disease starts as a placental condition resulting in insufficient placental perfusion followed by a maternal syndrome with endothelial dysfunction and hypertension (27). Interestingly, PE is associated with an elevated risk of metabolic syndrome later in life (24), just as pre-pregnancy metabolic syndrome increases the risk of PE (24). Recently, it has been established that prophylactic anti-inflammatory and anti-thrombotic treatment with low dose aspirin, instituted prior to week 16, may reduce the risk of severe PE (28). This finding makes it highly relevant to identify early first trimester markers of PE. Resistin has been found to be elevated in second and third trimester of pregnancies developing PE (27), but very little is known about the maternal serum concentration of resistin in normal pregnancies and nothing has been reported on the first trimester level in pregnancies later developing PE. With the recent finding that perturbations in other adipokines, i.e. leptin (Lp) and free Lp index (29), are seen in first trimester of PE pregnancies, the role of other adipokines and potential regulators of IR and inflammation becomes relevant to study. The purpose of this study was threefold. The first aim was to establish the concentration of resistin in maternal serum in late first trimester normal pregnancy. The second aim was to assess resistin’s relation to maternal
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pregnancy parameters, e.g. body mass index (BMI), parity, age and markers of inflammation and IS, duration of pregnancy, gestational age and occurrence of PE. The third and final aim was to examine whether resistin could be used as a first trimester marker of PE.
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MATERIALS AND METHODS Pregnant Women Serum samples from 408 pregnant women (123 of whom later developed PE) were collected in gestational weeks 10+3–13+6. These women were enrolled from 1997 to 2001 in the Copenhagen First Trimester Screening Study (30). Gestational age was determined by crown-rump length. All samples were collected in dry containers and kept at 4 C for a maximum of 48 h until delivery to Statens Serum Institut. Samples were subsequently stored at 20 C. The samples had been subjected to four freeze–thaw cycles prior to the measurement of resistin. The criteria of The International Society for the Study of Hypertension in Pregnancy were used to define PE (31). Thus, the diagnosis of PE required hypertension, defined as either a systolic blood pressure 4140 mm Hg or a diastolic blood pressure 490 mm Hg occurring in a previously normotensive woman after 20 weeks of gestation in combination with proteinuria (40.3 g per 24 h or dipstick urine analysis 41+). Severe PE was defined by a diastolic blood pressure 4110 mm Hg in combination with subjective symptoms and/or abnormal laboratory findings, i.e. oliguria (5400 mL per 24 h), severe proteinuria (43 g per 24 h), elevated liver enzymes, raised s-bilirubin, s-urate or s-creatinine, thrombocytopenia, disseminated intravascular coagulation, haemolysis and hypercoagulability. The presence of haemolysis, elevated liver enzymes and low platelets defined the HELLP syndrome. Early PE was defined as a PE pregnancy with delivery prior to week 34. Controls were 285 women with an uncomplicated pregnancy. Demographic and clinical parameters of the women included in the PE group and controls are listed in Table 1; the PE group parameters are broken down with respect to clinical severity in Table 2. Table 1. Demographic and Clinical Characteristics of Preeclamptic and Control Pregnancies.
Parameter Age (years) median (range) Weight (kg) median (range) BMI (kg/m2) median (range) Gravida (=1(%), 2(%), 3(%), 43(%)) Birth weight (g) median (range) GA at birth (d) median (range) GA sampling (d) median (range) Resistin (ug/l) median (range)
Controls (n = 285)
Pre-eclampsia (n = 123)
30.1 (18.9–45.1) 62 (41–137) 21.7 (14.5–37.2) 156(58), 78(28), 34(12), 17(5) 3600 (2650–5010) 284 (260–300) 90 (73–97) 25.4 (4.9–83.0)
30.5 (18.0–45.9) 68 (47–112) 24.0 (19.1–40.4) 73(59), 27(22), 15(13), 8(7) 3200 (465–5000) 276 (167–304) 91 (75–97) 22.2 (7.5–58.0)
p* Ns 50.001 50.001 Ns 50.001 50.001 Ns 0.002
*Mann–Whitney U test, except for gravida where 2 was used and Resistin, where one-way ANOVA was used.
Resistin in First Trimester Pregnancy Table 2. Demographic and Clinical Characteristics of Severe Pre-eclamptic, Non-severe Pre-eclamptic and HELLP Pregnancies. Parameter
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Age (years) median (range) Weight (kg) median (range) BMI (kg/m2) median (range) Gravida (=1(%), 2(%), 3(%)) Birth weight (g) median (range)* GA at birth (d) median (range)* GA sampling (d) median (range) Resistin (ug/l) median (range)y
Mild (n = 96) 31 67 24.0 56
(18–42) (47–111) (19.1–40.4) (58), 20 (21), 20 (20) 3274 (753–5000)
Severe (n = 21) (21–37) (50–95) (20.0–32.8) (52), 7 (33), 3 (14) 2682 (465–4500)
(23–46) (50–112) (19.6–34.6) (100), 0(0), 0 (0) 2259 (990–3700)
278 (195–304)
264 (167–286)
254 (203–290)
90 (75–97)
91 (80–97)
91 (90–93)
21. 9 (7.5–58.0)
29 72 24.5 11
HELLP (n = 6)
22.4 (11.1–46.4)
31 67 23.1 6
24.3 (11.6–33.3)
*ANOVA, F-test, p50.001. yANOVA, F-test, p = 0.02.
Resistin Measurement The resistin concentration was determined in singlo using the Human Resistin ELISA development kit, Duo Set (DY1359, R&D Systems, Minneapolis, MN) as described by the manufacturer following appropriate sample dilution. The functional detection limit of the assay was 31.25 pg/mL resistin. The intraassay coefficient of variation (CV) was 55% and the inter-assay CV 510%. The calibration was done using a highly purified Escherichia coli-expressed recombinant human resistin produced by the kit manufacturer. The resistin concentration in serum samples was stable for at least 56 h at room temperature and 10 freeze–thaw cycles. TNF-a Measurement The concentration of TNF-a was determined in singlo using the human TNF-a/TNFS1A Duoset ELISA kit (DY210, R&D Systems), as described by the manufacturer using appropriately diluted samples. The functional detection limit was 15.6 pg/mL. The assay was calibrated using a recombinant TNF-a preparation provided with the kit. The intra- and inter-assay CVs were 510% and 520%, respectively. Serum samples were stable for 48 h at 23 C and 10 freeze–thaw cycles. Measurement of the Adiponectin and Lp The concentrations of adiponectin (ApN) and Lp were established using the commercially available ELISA kits DY1065 ApN and DY398 Lp (R&D Systems), respectively. Samples were analysed in singlo following appropriate dilutions. The functional detection limit of ApN was 50 pg/mL and 31.25 pg/mL for Lp. The intra- and inter-assay CV’s were 55% and 510%, respectively for both assays. Calibration was performed using recombinant ApN expressed in NS0 cells and Lp expressed in E. coli. Following analysis of each of the two analytes, the ApN/Lp ratio was calculated
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Data Analysis The relation between the maternal serum concentration of resistin and other parameters, e.g. gestational age and maternal weight was examined by Loess non-parametric regression or non-parametric correlation analysis a.m. Spearman. Log transformations were used to achieve compatibility with the normal distribution, which was in turn assessed by normal probability plots and Kolmogorov–Smirnov’s test. Medians were compared using the Mann–Whitney U-test. Comparison of multiple groups was performed using Kruskal–Wallis test. Assessment of discriminatory ability was performed using empirical receiver operator characteristics (ROC) curves. Log-linear multiple regression was used to construct discriminatory indices. p = 0.05 was selected as the level of significance. Statistical analyses were performed using SPSS version 19.0 (SPSS Inc., Chicago, IL). Ethics Blood sampling was approved as part of the Copenhagen First trimester Study by the Scientific Ethics committee for Copenhagen and Frederiksberg Counties (No. (KF) 01-288/97).
RESULTS The maternal serum resistin concentration was significantly (p50.002) lower in PE pregnancies than in controls (Table 1). The relation between maternal serum resistin and gestational age is shown in Figure 1 for controls and PE pregnancies. The Loess regression curves show that serum resistin was
Figure 1. Scatter plots and Loess regression lines depicting the relation between maternal serum resistin concentrations and gestational age in controls (n = 285) and PE cases (123). The resistin concentration in controls decreases throughout the examined period and after day 95 becomes lower than in PE pregnancies.
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independent of gestational age in PE pregnancies (Spearman’s rho = 0.008, p = 0.32, n = 123) in the whole examined period, whereas the resistin concentration decreased significantly (Spearman’s rho = 0.225, p50.001, n = 285) in control pregnancies. However, the resistin concentration in controls only dropped markedly after day 90; prior to that time the decrease with gestation was not significant (Spearman’s rho = –0.112, p = 0.15, n = 145); and from day 95, the values in PE pregnancies were higher than the values in controls with a median resistin concentration of 18.3 ng/mL (range: 7.8–53.6 ng/mL, n = 65) in controls and a median of 23.4 ng/mL (range: 13.3–44.0 ng/mL, n = 21) in PE pregnancies, but this difference was not significant (p = 0.8), so the low number of samples preclude any firm conclusions on this. In the gestational age window 10+0–12+6 weeks, where the resistin level was fairly constant in controls, the median resistin concentration in controls was 27.7 ng/mL (range: 4.9–89.0 ng/mL) and significantly lower, median 22.3 ng/mL (range: 7.5–58.0 ng/mL), in PE pregnancies (p = 0.002). This means that maternal serum resistin is 15–20% reduced in PE pregnancies in week 10+0–12+6. In neither the control nor PE pregnancies, were there any significant correlation between serum resistin and maternal weight, maternal BMI, maternal age or birth weight (p40.6, in all examinations). There was no correlation between the resistin concentration and the concentration of TNF-a in the pregnancies where TNF-a was measurable (415.6 pg/mL), irrespective of whether the pregnancy developed PE or not. Likewise, there was no correlation between IS, as determined by the ApN–Lp ratio, and resistin in either controls or PE pregnancies (Figure 2).
Figure 2. Scatter plots and Loess regression lines depicting the relation between IS (expressed by the ApN/Lp-ratio) and maternal serum resistin concentrations. There was no significant correlation between IS and resistin levels in either controls or PE pregnancies.
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Figure 3. Boxplot of the maternal serum resistin concentration levels in controls and PE cases in gestational week 10+3–13+0, grouped according to clinical severity. The difference between groups was significant, p = 0.018, ANOVA and there was a significant linear negative trend, p = 0.003.
The relation between maternal serum resistin in gestational week 10+3–13+5 and clinical severity of PE is shown to be significant in Table 2. In week 10+3– 12+6, where the resistin concentration in both controls and PE pregnancies was constant, the relation was as shown in Figure 3. The median (range) resistin concentration decreases significantly (p = 0.005, Kruskal–Wallis test), from 27.7 ng/mL (4.9–83.0) in controls (n = 163) to 22.3 ng/mL (6.2–58.0 ng/mL) in mild pre-eclampsia (n = 63) and 21.3 ng/mL (11.1–36.5 ng/mL) in severe preeclampsia (n = 11) to reach 17.6 ng/mL (11.6–33.3 ng/mL) in HELLP pregnancies (n = 4). Ten PE pregnancies with delivery prior to week 34, where only five of them had blood sampled prior to week 13, exhibit a median resistin concentration of 17.3 ng/mL (range: 11.6–36.1 ng/mL), and this level is lower than that of HELLP pregnancies, but the small number precluded that the resistin difference between early and late PE pregnancies could reach significance (p = 0.46). The ability of maternal serum resistin to discriminate between pregnancies that developed PE and normal pregnancies in week 10+0–12+6 was analysed by empirical ROC curve analysis (Figure 4). The area under the curve (AUC) was 0.617 (95% confidence interval: 0.539–0.695, p = 0.008). The empirical detection rate for false-positive rates of 20% and 10% was around 23% and 20%, respectively. In order to assess the potential of combining resistin with other first trimester PE markers, we performed a linear regression of the parameters, log10 resistin, log10 PAPP-A, BMI and gestational age using the dummy variables 1 and 2 for controls and PE pregnancies, respectively. We used the 206 pregnancies (61 PE pregnancies and 145 controls) that had blood sampled prior to week 13. It was found that only log10 resistin and BMI contributed independently and significantly to the discrimination between PE and control pregnancies. The discrimination index = 0.311 log10 resistin + 0.036 BMI + 1.84 had an R2 of 0.107. Figure 4 depicts the ROC curves of BMI, log10 resistin and the discriminatory index. BMI had an
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Figure 4. Receiver operator characteristics (ROC) curve of maternal serum resistin as a marker for pre-eclampsia in gestational week 10+3–13+0. The area under the curve was 0.616 with a 95% confidence interval: 0.545–0.695, p = 0.003. The ROC curves for BMI and the combination of resistin and BMI (discriminatory index) are also depicted.
AUC of 0.679 (range: 0.600–0.757) and the index an AUC of 0.706 (range: 0.629–0.783). BMI was thus a better discriminator than log10 resistin, and the addition of log10 resistin to BMI did increase the AUC, but not significantly so. This is probably a result of the relatively small number of pregnancies.
DISCUSSION This is – to our knowledge - the first report of reduced first trimester maternal serum resistin concentrations in pregnancies that later develop PE. The reduction is in the order of 15–20% of the resistin concentration seen in control pregnancies; in HELLP pregnancies, the reduction is 430%, in both cases, prior to week 13. Previous studies have reported elevated or un-perturbed resistin concentrations in second and third trimester PE pregnancies (8,32,33). The period from day 90 and onwards, where the maternal serum concentration of resistin decreases in controls and remains constant in PE pregnancies, suggests that resistin after day 95 will be elevated in PE pregnancies. This is in agreement with the cited studies. This is also – to our knowledge – the first report of a decreasing maternal serum resistin through first trimester in normal pregnancies. Other studies have reported on resistin concentrations in pregnancy (8,10,13,33–38), but the first measurements in these studies are from late first or early second trimester. One study found no gestational age dependence (13), but another did (36), whereas a third only found the levels increased in third trimester (30). As the resistin concentrations measured in this study in first trimester are
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substantially higher than those reported in other studies from late first and early second trimester, i.e. 20–25 ng/mL compared to 10–18 ng/mL (8,10,13,33–38), it does seem that first trimester is characterised by high resistin levels. PE pregnancies are then characterised by not reaching this level. The lack of an association between resistin and maternal BMI or IS (Figure 2), also reported by others in later parts of pregnancy (33,36), suggests that the maternal serum concentration of resistin is not defined by the metabolic status of the mother. An alternative reason for the variations in resistin could, however, be that it is a result of changes in the distribution of body fat in the mother – as the intra-abdominal fat increases during pregnancy (39) and resistin is preferentially synthesised in abdominal fat (40,41). The low resistin levels in the mother could be a result of a complicated endocrine dysfunction in the mother. Furthermore, resistin levels have been reported to be significantly heritable. Pantsulaia et al., 2007 reported that 66% of resistin variation could be attributed to genetic factors (40). We did not find a significant association between resistin and the concentration of the inflammatory marker TNF-a in maternal serum, suggesting that resistin is not increased due to low-level inflammation. As resistin is synthesised by the foetus and present in high concentrations in umbilical cord blood (35) at term, and in even higher concentrations in PE pregnancies (32), it would seem reasonable that the physiological significance of resistin is related to foetal development. This could also explain that the level is very high in first trimester where the foetus does not have a circulation that can ensure the presence of resistin in the foetal adipose tissue. However, this line of reasoning is hypothetical and will need genuine experimental support. Furthermore, if PE pregnancies are characterised by a relative resistin deficiency, this could explain why PE is associated with reprogramming of the foetus leading to an increased risk of metabolic syndrome. The ROC curve analysis (Figure 4) shows that resistin is a very poor marker of PE. Thus, it must be considered clinically irrelevant, except for the suggested ability of resistin to selectively identify HELLP and early PE. If PE screening is introduced, it is very important that the screening identifies pregnancies with a risk of morbidity and not the many mild cases that have no clinical significance. If more efficient screening algorithms could be combined with markers, like resistin, that more selectively identifies severe, clinically relevant, cases, it would greatly improve the ethical argument for introducing PE screening despite the dangers of increasing parental anxiety and interference with the incentive to seek timely medical care in false-negative cases. In conclusion, we have shown that maternal serum resistin is significantly reduced in PE pregnancies in week 10+3–12+6 and that this reduction is more pronounced with increasing severity of disease. Furthermore, resistin is not suitable as a marker of PE, but may have a role in defining the risk for clinically severe PE.
ACKNOWLEDGMENTS We gratefully acknowledge the expert technical assistance of Pia Lind and Pernilla Rasmussen.
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FUNDING The authors also gratefully acknowledge the financial support of the Danish Medical Research Council, Copenhagen University, The John and Birthe Meyer Foundation, The Ivan Nielsen Foundation, The Else and Mogens Wedell-Wedellsborg Foundation, The Dagmar Marshall Foundation, The Egmont Foundation, The Fetal Medicine Foundation, The Augustinus Foundation, The Gangsted Foundation, The A.P. Møllerller Foundation, The Mads Clausens Foundation, The Copenhagen Hospital Corporation, SAFE Network of Excellence and Statens Serum Institut.
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DECLARATION OF INTEREST The authors report no conflict of interest. The authors alone are responsible for the content and writing of the paper.
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