Bugatto, Fernando MD, PhD; Fernández-Deudero, Álvaro; Bailén, Ángeles MD; Fernández-Macías, Rosa MD; Hervías-Vivancos, Blas MD, PhD; Bartha, José L. MD, PhD
The incidence of obesity is increasing worldwide and is considered a chronic inflammatory condition with serious health consequences. The adipose tissue is considered an endocrine and paracrine organ, producing a number of molecules called adipokines, such as leptin, resistin, tumor necrosis factor (TNF)-α, and interleukins. Many of the metabolic disturbances associated with obesity and the metabolic syndrome, such as insulin resistance, dyslipidemia, hypertension, and premature heart disease, may be attributable to cytokine production by adipocytes.1 In addition, pregnancy is characterized by the physiologic activation of maternal leukocytes and increased systemic concentration of acute phase reactants and cytokines.2,3
Combination of obesity and pregnancy results in an exaggerated inflammatory response in the placenta, with accumulation of macrophages and production of proinflammatory mediators.4 Comprehensive data have demonstrated that obesity in pregnancy is associated not only with marked hyperinsulinemia and dyslipidemia but also with impaired endothelial function, higher blood pressure, and inflammatory up-regulation.5,6 The resulting inflammatory milieu in which the fetus develops may be responsible for many adverse perinatal outcomes associated with obese women.4,7
Obesity may negatively affect women’s reproductive health, from before conception until the postpartum period, and may include frequent menstrual cycle disturbances; infertility problems; and increased maternal, fetal, and neonatal complications.8 Obese women appear to be at increased risk of miscarriage, hypertensive disorders, gestational diabetes mellitus, venous thromboembolism, delivery by cesarean birth, neural tube defects and other congenital anomalies, antepartum stillbirth, macrosomia, and admission to the neonatal intensive care unit.8–10 It has also been reported that children exposed to maternal obesity are at increased risk of developing insulin resistance and metabolic syndrome.11,12 Thus, maternal obesity may be a serious condition that significantly impacts not only the mother’s health but also the health and future of her child. However, these adverse perinatal outcomes are directly related to the level of obesity, with class II and class III representing significantly higher risk.13,14
Recently, an increased expression of proinflammatory cytokines has been found in the placenta and in the newborn cord blood of obese mothers compared with that of lean women.4,12 The aim of the present study is to determine some common proinflammatory interleukins and adipokines in the amniotic fluid of normal, overweight, and obese pregnant women, to test the hypothesis of the existence of a relationship between maternal body mass index (BMI), which is calculated as weight (kg)/height (m)2, and a state of inflammatory exposure of the fetus.
MATERIALS AND METHODS
Amniotic fluid (AF) was obtained from 70 singleton pregnant women undergoing elective amniocentesis at 15–20 weeks of gestation between January and July 2008 for karyotype analysis (range 19–45 years, median 37 years). Women were asked to give an extra amount of 3 mL of AF for the study. Informed consent was obtained from all patients. Likewise, all the proposals of the ethical commission of our center were respected, and the personal data of the women included in the assay were protected. The study was approved by the local ethical and research committees.
Indications for genetic amniocentesis included advanced maternal age (n=41), maternal request (n=11), abnormal first- or second-trimester serum screening (n=8), markers seen on ultrasound (n=6), or family history of chromosome abnormalities (n=4). Women were divided into two groups according to their prepregnancy BMI: the control group, composed of 35 normal weight women; and the case group, composed of 35 overweight (n=22) or obese (n=13) pregnant women. The classifications for each group were determined considering the following conditions for each group, as stated by the World Health Organization15: normal (BMI 20–24.9; n=35), overweight (BMI 25–29.9; n=22), and obese (BMI 30 or more; n=13). Exclusion criteria were abnormal karyotype, fetal malformations, multiple pregnancy, or maternal diseases, including pregestational diabetes or hypertension.
The AF samples were centrifuged to remove cellular debris and stored at –20°C in 1.5-mL sterilized Eppendorf tubes immediately after collection. No freeze–thawing cycles were allowed. The following proinflammatory adipokines/parameters were selected: TNF-α, interleukin (IL)-8, IL-10, monocyte chemoattractant protein-1, leptin, resistin, and C-reactive protein (CRP). The measurements of the proinflammatory parameters were obtained simultaneously by using Multi Analyte immunoassay kits (Linco Research, Inc., St. Charles, MO) following the instructions of the manufacturer. This technique allows the simultaneous quantification of up to 100 proteins in the same well of reaction using 25 microliters of sample. The first five parameters were analyzed simultaneously, whereas resistin was studied separately because it is incompatible with the others in the Multiplex panel process. Multi Analyte profiling was performed on the Luminex-100 system (Luminex Corp., Austin, TX), and analysis of the data was performed with Luminex 100 IS v1.0 computer software. The sensitivities of the tests were 0.05 pg/mL for TNF-α, 0.11 pg/mL for IL-8, 0.15 pg/mL for IL-10, 0.14 pg/mL for monocyte chemoattractant protein-1, 0.085 ng/mL for leptin, and 0.006 ng/mL for resistin. The intraassay and interassay coefficients of variation were 3.49% and 3.78% for TNF-α, 3.26% and 6.48% for IL-8, 3.31% and 11.84% for IL-10, 4.65% and 11.75% for monocyte chemoattractant protein-1, 5.11% and 8.7% for leptin, and 4.25% and 8.2% for resistin, respectively.
A high-sensitivity CRP assay (Roche Diagnostics, Mannheim, Germany) was used for the quantification of AF CRP. The principle of the test is that CRP agglutinates with particles of latex covered with anti-CRP monoclonal antibodies. The formed precipitate is read by means of turbidimetry at 546 nm using the Roche Cobas C 501 module analyzer. The sensitivity was 0.015 mg/dL. The intraassay and interassay coefficients of variation were 1.4% and 4.85%, respectively.
Sample size was calculated using data from a pilot study, with the level of AF TNF-α being the main outcome measure. There was a difference of 1.2 pg/mL between the control group and the overweight group, with standard deviations 1.8 and 1.5 in each group. Using these data for an α value of 0.05 and a power (1–β) of 0.80, we calculated that a sample of 30 women in each group was needed. To test the influence of the degree of overweight on the maternal levels of this cytokine, we calculated the sample size using the relationship between BMI and TNF-α as a continuous variable, also found in the pilot study. Using a correlation coefficient of 0.35, a power (1–β) of 0.80, and an α value of 0.05, we calculated that the minimal sample size required for this analysis was 61 women. We included 70 women, 35 in each group, to account for withdrawals and for secondary analysis, including subgroups analysis by means of analysis of variance.
Statistical analysis was performed using the SPSS 15.0 for Windows (SPSS, Inc., Chicago, IL) computer statistics program. Distributions were checked with a histogram and the Kolmogorov-Smirnov test. When a variable was distributed normally, data were presented as mean and standard deviation. In cases of nonnormal distribution, data were shown as median and interquartile range. Qualitative variables were expressed as numbers and percentages.
Comparisons between two groups were performed by using either the Student t test or the Mann-Whitney U test (two-tailed) according to the normal or nonnormal distributions of the variables. Comparisons among groups were performed using either one-way analysis of variance or the Kruskal-Wallis test (H-test) when comparing normal or nonnormal data, respectively. The Bonferroni test was used for the post hoc analysis for comparison between the groups. Proportions were compared by using the χ2 and the Fisher’s exact tests.
The relationships between variables were analyzed using the Pearson’s or Spearman’s correlation coefficient when using parametric or nonparametric data, respectively. Statistical significance was set at the 95% level (P<.05).
Demographic and clinical characteristics of the 70 pregnant women who took part in the study are listed in Table 1. There were statistically significant differences among groups only in terms of body mass index and maternal weight, as expected.
There were no statistically significant differences in any of the studied variables between the control and the case groups, except for the AF resistin levels (P=.01). However, when analyzing the subgroups of overweight and obese pregnant women separately, significant differences in AF CRP and TNF-α levels among these groups (P=.007 and P=.003, respectively) (Table 2 and Fig. 1) and a borderline significant difference in AF resistin levels (P=.05) were found. Post hoc analysis showed that there were significant differences in AF CRP and TNF-α levels between normal and obese groups (0.018 [±0.010] compared with 0.035 [±0.028] mg/dL, P=.008; and 3.98 [±1.63] compared with 5.46 [±1.69] pg/mL, P=.01, respectively) and between overweight and obese groups (mean 0.019 [±0.013] compared with 0.035 [±0.028] mg/dL, P=.01; and mean 3.53 [±1.38] compared with 5.46 [±1.69] pg/mL, P=.003, respectively). There were no differences in AF IL-8, IL-10, monocyte chemoattractant protein-1, or leptin levels among these three groups (Table 2).
In addition, there were significant correlations between maternal body mass index and AF CRP (r=0.396; P=.001), TNF-α (r=0.357; P=.003), and resistin (r=0.353; P=.003) (Figs. 2 and 3). There were statistically significant correlations between TNF-α and CRP (r=0.249; P=.03), IL-8 (r=0.589; P<.001), and IL-10 levels (r=0.695; P<.001). Similarly, CRP levels were correlated with those of monocyte chemoattractant protein-1 (r=0.257; P=.03) and leptin (r=0.318; P=.008). We found significant positive correlations between IL-8 and IL-10 (r=0.556; P<.001), monocyte chemoattractant protein-1 (r=0.424; P<.001), and resistin levels (r=0.439; P<.001). Finally, leptin and monocyte chemoattractant protein-1 levels were also correlated (r=0.290; P=.01).
Amniotic fluid cytokine levels were compared between male and female fetuses. In total, there were 38 male and 32 female fetuses. We did not find significant differences for CRP, TNF-α, IL-10, IL-8, resistin, monocyte chemoattractant protein-1, or leptin (P=.86, P=.69, P=.95, P=.29, P=.11, P=.09 and P=.06, respectively). Transplacental amniocentesis was performed in no patients in the overweight group compared with eight patients in the control group. We did not find significant differences in any of the studied cytokines according to the amniocentesis technique when comparing the transamniotic with the transplacental method for CRP, TNF-α, IL-10, IL-8, monocyte chemoattractant protein-1, resistin, and leptin (P=.76, P=.48, P=.86, P=.95, P=.27, P=.47, and P=.10, respectively).
Maternal obesity is associated with changes in maternal serum cytokines and acute phase proteins in the second trimester of pregnancy.16 However, the role of intrauterine inflammatory cytokines has not been elucidated. We have measured some common adipokines (produced mainly by adipose tissue) and proinflammatory mediators in AF samples of overweight and obese women.
C-reactive protein increases rapidly in the presence of inflammatory processes, and maternal serum levels have been associated with maternal BMI.16 Increased concentrations of AF CRP are associated with a higher risk of intraamniotic infection, chorioamnionitis, and funisitis.17 TNF-α is secreted by activated macrophages, and obese pregnant women present a higher accumulation of macrophages and production of inflammatory mediators in the placenta.4 We observed substantial increases of TNF-α in the amniotic fluid of women with intrauterine infection,18 but no differences in maternal serum TNF-α levels have been reported between normal and obese pregnant women.6 Resistin is expressed in the human placenta, and its proinflammatory properties may indicate a role in inflammatory processes. Some studies have found that umbilical cord resistin levels are significantly higher than those of the maternal circulation, and a correlation between maternal serum resistin and BMI has not been found.19,20 Recently, Kusanovic et al have measured resistin in AF and found an increase with advancing gestation and elevated levels in the presence of intraamniotic infection.21
We have found higher AF CRP and TNF-α levels in obese mothers than in overweight and lean ones and higher AF resistin levels in the case group compared with the control group. We also found a significant correlation between AF CRP, TNF-α, and resistin levels and the BMI of women in the study. These results would mean higher exposure of fetuses to these acute phase reactant and proinflammatory cytokines in direct relationship to maternal weight.
Monocyte chemoattractant protein-1 is produced predominantly by macrophages and increased macrophage accumulation, and heightened levels of proinflammatory cytokines have also been demonstrated in the placenta of pregravid obese women compared with normal controls.4 Increased maternal serum monocyte chemoattractant protein-1 has been found in the morbid obesity category, and maternal serum leptin levels have shown good correlation with maternal BMI.16 Intraamniotic infection has also been associated with increased amniotic fluid concentrations of IL-8.18 However, in our study, monocyte chemoattractant protein-1, leptin, and IL-8 levels did not differ among the three studied groups, and a correlation with BMI was not found. According to our findings, these cytokines do not seem to be related to maternal obesity. Interestingly, AF monocyte chemoattractant protein-1 levels were correlated with those of CRP, the well-known acute phase reactant.
Interleukin-10 is an antiinflammatory cytokine produced at the maternal–fetal interface, and its production is important for preventing excessive inflammation.22 Amniotic fluid IL-10 levels were correlated with those of the proinflammatory cytokines TNF-α, IL-8, and monocyte chemoattractant protein-1, suggesting a compensatory mechanism to dampen inflammation.
Inflammatory processes result in the increase of multiple cytokines independent of their role (ie, proinflammatory or modulatory) and, depending on the trigger mechanism of inflammation, particular cytokine activation patterns may appear. It is their overall balance that may determine clinical outcome. We do not know the reasons for this particular cytokine pattern found in obese women. Further immunologic studies are needed to understand the underlying molecular interactions. Proinflammatory cytokines are being emergently considered as markers of amniotic fluid infection and for use in making clinical decisions in women with preterm labor or preterm premature rupture of membranes.17,18,21 In view of our results, a patient’s BMI should be considered when interpreting cytokine levels. We suggest that other amniotic fluid parameters such as glucose or white blood cell count should be taken into account when considering the diagnosis of subclinical chorioamnionitis and not only cytokine levels, especially in obese women. Otherwise, amniotic fluid cytokine levels should be adjusted by maternal BMI to better understand the clinical significance of these levels.
There is an ongoing controversy about the influence of fetal sex on amniotic fluid levels of inflammatory cytokines.23,24 We compared amniotic fluid cytokine levels between male and female fetuses and found no significant differences, even though the levels of both monocyte chemoattractant protein-1 and leptin were at borderline significant difference. These results should not influence the main results, as neither monocyte chemoattractant protein-1 nor leptin was significantly different in our studied groups.
Sample size was calculated by using comparison between the two principal groups and the correlation coefficient between TNF-α and BMI. Therefore, nonsignificant results when analyzing the rest of the cytokines or when comparing the subgroups should be considered carefully. This is a limitation of the present study. By virtue of the fact that in obese women the needle passes through far more subcutaneous and adipose tissue than in control women, there is at least the possibility that maternal cellular contamination is a confounding factor for the results. A count of cells in the AF samples could not be performed. However, when performing an amniocentesis, the first 2–3 mL was always discarded. Nevertheless, this fact should be taken into account as one of the limitations of the study that should be addressed in further studies.
In conclusion, we have found increased AF levels of TNF-α and CRP in obese women compared with lean and overweight women. Our results suggest that early pregnancy in utero exposure to higher proinflammatory adipocytokines and mediators exists in fetuses of overweight and obese women. This proinflammatory milieu may contribute to fetal programming, may be responsible for many complications of pregnancy, and may impact health status during future adult life (eg, increased risk of developing insulin resistance and metabolic syndrome).12,25
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© 2010 by The American College of Obstetricians and Gynecologists.