ADIPONECTIN IN AMNIOTIC FLUID IN NORMAL PREGNANCY, SPONTANEOUS LABOR AT TERM, AND PRETERM LABOR: A NOVEL ASSOCIATION WITH SUBCLINICAL INTRAUTERINE INFECTION/INFLAMMATION

Abstract

Objective

Adiponectin, an anti-inflammatory and anti-diabetogenic adipokine, has an important regulatory effect on both the innate and adaptive limbs of the immune response. The objective of this study was to determine whether adiponectin is present in amniotic fluid (AF) and if its concentration changes with gestational age, in the presence of labor and in the presence of intra-amniotic infection (IAI) in patients with spontaneous preterm labor (PTL) and intact membranes.

Study design

This cross-sectional study included 468 patients in the following groups: 1) women in the mid-trimester of pregnancy (14–18 weeks) who underwent amniocentesis for genetic indications and delivered a normal neonate at term (n=52); 2) normal pregnant women at term with (n=49) and without (n=41) spontaneous labor; 3) patients with an episode of PTL and intact membranes who were classified into: a) PTL who delivered at term (n=149); b) PTL who delivered preterm (<37 weeks gestation) without IAI (n=108); and c) PTL with IAI (n=69) Adiponectin concentration in AF was determined by ELISA.

Results

1) The median AF adiponectin concentration at term was significantly higher than in the mid-trimester (35.6 ng/mL, interquartile range [IQR] 26.4–52.7 vs. 29.9 ng/mL, IQR 19.9–35.2; p=0.01); 2) among women with PTL and intact membranes, the median amniotic fluid adiponectin concentration was significantly higher in patients with IAI than in those without IAI who delivered either at term (54.3 ng/mL, 39.0–91.8 vs. 50.1 ng/mL, 33.2–72.8; p = 0.02) or preterm (47.6 ng/mL, 32.6–74.6; p = 0.01); and 3) among women at term, there was no significant difference in the median amniotic fluid adiponectin concentration between those with and without labor (33.7 ng/mL, IQR 21.7–53.9 vs. 35.6 ng/mL IQR 26.4–52.7; respectively p=0.5).

Conclusions

1) Adiponectin is a physiologic constituent of AF; and 2) adiponectin concentrations in AF are increased significantly with advancing gestation and in the presence of IAI. Collectively, these findings suggest that adiponectin plays a dynamic role in normal gestation and in the presence of IAI.

Introduction

Spontaneous preterm parturition is syndromic in nature.^1;2 Consistent with this view, several mechanisms of disease have been implicated in the pathogenesis of this condition, including intra-amniotic infection/inflammation (IAI),^3–9 uteroplacental ischemia,^10–12 uterine overdistention,^13;14 allergic reactions,^15;16 cervical insufficiency,^2;17–19 hormonal disorders²⁰ and others. Despite the strong experimental and epidemiologic evidence suggesting an association between the aforementioned etiological factors and spontaneous preterm parturition, intra-amniotic infection/inflammation is the only pathological process for which a solid body of evidence supports a cause and effect relationship with preterm parturition.^9;21

The mechanism by which microbial invasion of the amniotic cavity (MIAC) and intra-amniotic inflammation induce preterm parturition involves the production and secretion of a wide range of pro-inflammatory cytokines^2;22–29 These cytokines and chemokines include: interleukin (IL)-1,^30–36 IL-6,^35;37–41 tumor necrosis factor (TNF)-α,^{34;35;42–47} IL-18,⁴⁸ 1L-16,⁴⁹ IL-8,^50;51 colony-stimulating factors,⁵² macrophage migration inhibitory factor,⁵³ monocyte chemotactic protein-1 (MCP-1),⁵⁴ macrophage inflammatory protein-1α (MIP-1 α),⁵⁵ RANTES,⁵⁶ epithelial cell-derived neutrophil-activating peptide-78,⁵⁷ CXCL6,⁵⁸ CXCL13⁵⁹ and CCL20.⁶⁰ Several lines of evidence support the causal link between cytokines and prematurity: 1) exposure of human decidua to bacterial products results in increased production of IL-1, IL-6 and TNF-α;^47;61;62 2) TNF-α,^63;64 IL-1^63;65 and IL-1β^66;67 can stimulate uterine contractility by induction of prostaglandin production;^65;68 3) matrix metalloproteinases (MMPs) have been implicated in membrane rupture^69–71 and cervical ripening.⁷² TNF-α can stimulate MMPs production;⁷² 4) administration of IL-1 can induce preterm labor and preterm birth in pregnant mice.³⁶ Moreover, treatment of these mice with the natural antagonist of IL-1 (IL-1 receptor antagonist) abrogates preterm parturition;³² 5) amniotic fluid concentrations of immunoreactive IL-1,^30;33 TNF-α,^42;43;45;61 IL-6,^{37;38;41;73;74} IL-18,⁴⁸ IL-16,⁴⁹ IL-8,^50;51 MCP-1,⁵⁴ MIP-1α,⁵⁵ RANTES,⁵⁶ are elevated in women with preterm labor in the presence of infection and/or inflammation.

Adiponectin is a member of a growing group of peptides and proteins secreted by adipose tissue, termed adipocytokine.⁷⁵ Some of these active molecules are produced mainly by adipose tissue (e.g. leptin,⁷⁶ adiponectin,⁷⁷), while others are shared with other systems (e.g. TNF-α,⁷⁸ IL-6⁷⁹). Adiponectin, identified independently by four groups,^80–83 took position as an important adipocytokines with a wide range of biological actions including insulin-sensitizing,⁸⁴ anti-atherogenic⁸⁵ and angiogenic⁸⁶ properties. In addition to its well-established role in glucose metabolism and regulation of vasculature, adiponectin also has a potent anti-inflammatory effect.⁸⁷

Despite its pleiotropic effects on metabolism, inflammatory and immune responses, only one report concerning amniotic fluid concentrations of this adipocytokine has been published.⁸⁸ Moreover, no data exist regarding amniotic fluid concentrations of adiponectin in patients at term, or in labor (either term or preterm). Thus, the objective of this study was to determine whether adiponectin is present in amniotic fluid (AF) and if its concentration changes with gestational age, in the presence of labor and in the presence of intra-amniotic infection (IAI) in patients with spontaneous preterm labor (PTL) and intact membranes.

Materials and Methods

Study design and population

A cross-sectional study was conducted by searching our clinical database and bank of biological samples and included 468 patients in the following groups: 1) women in the mid-trimester of pregnancy (14–18 weeks) who underwent amniocentesis for genetic indications and delivered a normal neonate at term (n=52); 2) normal pregnant women at term with (n=49) and without (n=41) spontaneous labor; 3) patients with an episode of PTL and intact membranes who were classified into: a) PTL who delivered at term (n=149); b) PTL who delivered preterm (<37 weeks gestation) without IAI (n=108); and c) PTL with IAI (n=69).

All participating women provided written informed consent prior to the collection of amniotic fluid. The collection and utilization of amniotic fluid for research purposes was approved by the Institutional Review Boards of the participant institutions and the Eunice Kennedy Shriver National Institute of Child Health and Human Development, NIH, DHHS. Many of these samples have been previously used to study the biology of inflammation, hemostasis, and growth factor concentrations in normal pregnant women and those with pregnancy complications.

Definitions

Patients were considered to have a normal pregnancy outcome if they did not have any medical, obstetrical, or surgical complication, and delivered a term neonate (≥37 weeks) of appropriate birth weight for gestational age^89;90 without complications. Spontaneous preterm labor was defined by the presence of regular uterine contractions occurring at a frequency of at least two every 10 minutes associated with cervical change before 37 completed weeks of gestation that required hospitalization. Intra-amniotic infection was defined as a positive amniotic fluid culture for micro-organisms. Intra-amniotic inflammation was diagnosed by an amniotic fluid IL-6 concentration ≥2.6 ng/mL.⁹¹

Sample collection

Amniotic fluid samples were obtained by transabdominal amniocentesis performed for genetic indication, evaluation of microbial status of the amniotic cavity and/or assessment of fetal lung maturity in patients approaching term. Women at term in labor consisted of women who were admitted for suspected preterm labor because of uncertain dates and had an amniocentesis for the assessment of fetal lung maturity. The criteria for considering that these patients were at term in labor was derived retrospectively if the following criteria were met: 1) spontaneous labor; 2) delivery within 24 hours from amniocentesis; 3) analysis of amniotic fluid consistent with maturity; 4) birthweight >2500 grams; 5) absence of respiratory distress syndrome or other complications of prematurity; and 6) physical examination of the newborn by pediatricians consistent with a term neonate. Samples of amniotic fluid were transported to the laboratory in a sterile capped syringe and cultured for aerobic/anaerobic bacteria and genital mycoplasmas. White blood cell (WBC) count, glucose concentration and Gram-stain were also performed shortly after collection as previously described^92–94. The results of these tests were used for clinical management. Amniotic fluid IL-6 concentrations were used only for research purposes. Amniotic fluid not required for clinical assessment was centrifuged for 10 minutes at 4°C and the supernatant was aliquoted and stored at −70°C until analysis.

The correlation between amniotic fluid adiponectin concentrations and maternal age, BMI, amniotic fluid WBC count, amniotic fluid concentrations of glucose and IL-6 as well as with birthweight was determined among patients with spontaneous preterm labor with intact membranes who delivered within 48 hours. The 48 hours interval was chosen to preserve a meaningful temporal relationship between amniotic fluid adiponectin concentration and amniotic fluid concentrations of glucose, IL-6 as well as with birthweight

Determination of human adiponectin concentration in amniotic fluid

Specific and sensitive enzyme-linked immunoassays were used to determine concentrations of adiponectin in human amniotic fluid. Immunoassays for human adiponectin were purchased from Linco Research (Human Adiponectin ELISA, LINCO Research Inc, St Charles, MO, USA). Adiponectin assays were validated for use in human amniotic fluid in our laboratory prior to their use in this study. The calculated inter-assay and intra-assay coefficients of variation for resistin in our laboratory were 1.6% and 3.4% respectively. The sensitivity was 0.47 ng/ml.

Statistical analysis

The normality of the data was tested using the Shapiro-Wilk and Kolmogorov-Smirnov tests. Because amniotic fluid adiponectin concentrations were not normally distributed, non-parametric tests were used for analyses. Comparisons between proportions were performed with the Chi-square test. Kruskal-Wallis with post-hoc analysis and Mann-Whitney U tests were used for continuous variables. Adjustment for multiple comparisons was performed using the Bonferroni method. Spearman rank correlation was utilized to assess correlations between amniotic fluid concentration of adiponectin, WBC count and IL-6. A p-value of 0.05 was considered statistically significant. The statistical package used was SPSS v.14.0 (SPSS Inc., Chicago, IL, USA).

Results

Demographic and clinical characteristics of the study population

Table I presents the demographic and clinical characteristics of patients in the mid-trimester, term not in labor and term in labor groups. Table II displays the demographic and clinical characteristics of patients with spontaneous preterm labor and intact membranes. Among patients with PTL, those with IAI had a significantly lower median gestational age at amniocentesis than those without IAI who delivered preterm and those who delivered at term (Table II).

Table I.

Demographic and clinical characteristics of patients in the midtrimester and those at term with and without spontaneous labor

	Mid-trimester (n=52)	pa	Term No labor (n=41)	Term In labor (n=49)	pb
Maternal age (years)	36 (35 – 38)	<0.001	27 (21 – 32)	22 (19 – 26)	0.007
GA at amniocentesis (weeks)	16 (16 – 17)	<0.001	38 (38 – 39)	38 (37.7 – 39.3)	NS
GA at delivery (weeks)	39 (38 – 40)	NS	38 (38 – 39)	38.5 (38 – 39)	NS
Birth weight (grams)	3344 (3144–3589)	NS	3260 (3070–3668)	3360 (3085–3550)	NS

Values are expressed as median (interquartile range).

NS: not significant.

p^a: comparison between patients in the mid-trimester and those at term not in labor

p^b: comparison between patients at term not in labor and those at term in labor

Table II.

Demographic and clinical characteristics of patients presenting with spontaneous preterm labor with intact membranes

	PTL without IAI Term delivery (n=149)	p	PTL without IAI Preterm delivery (n=108)	pa	PTL with IAI Preterm delivery (n=69)	pb
Maternal age (years)	22 (19 – 30)	NS	22 (19 – 30)	NS	23 (20 – 27)	NS
GA at amniocentesis (weeks)	31.8 (29.4 – 33.3)	NS	31.9 (29.8 – 33.0)	<0.001	28.5 (24.9 – 32.7)	<0.001
GA at delivery (weeks)	38.7 (37.9 – 39.7)	<0.001	34.6.5 (33.1 – 35.5)	<0.001	29.4 (25.2 – 32.9)	<0.001
Birth weight (grams)	3165 (2899–3532)	<0.001	2335 (1940–2677)	<0.001	1140 (699–1995)	<0.001

Values expressed as median (interquartile range)

p: comparison between PTL who delivered at term and PTL without IAI

p^a: comparison between PTL who delivered preterm without IAI and PTL with IAI

p^b: comparison between PTL who delivered at term and PTL with IAI

PTL: preterm labor; GA: gestational age; IAI: intra-amniotic infection/inflammation

NS: not significant

Amniotic fluid adiponectin concentrations in the mid-trimester and at term

The median amniotic fluid concentration of adiponectin was significantly higher in patients at term not in labor than in those in the mid-trimester of pregnancy (35.6 ng/mL, interquartile range [IQR] 26.4–52.7 vs. 29.9 ng/mL, IQR 19.9–35.2; p=0.01, Figure 1). Among women at term, there was no significant difference in the median amniotic fluid adiponectin concentration between those with and without labor (33.7 ng/mL, IQR 21.7–53.9 vs. 35.6 ng/mL IQR 26.4–52.7 respectively; p=0.5, Figure 1).

Figure 1. Amniotic fluid adiponectin concentrations in women with a normal pregnancy in the mid-trimester and in those at term not in labor.

The median amniotic fluid concentration of adiponectin was significantly lower in the mid-trimester than at term. Among women at term, there was no significant difference in the median amniotic fluid concentration of adiponectin between patient in labor and those not in labor.

Amniotic fluid adiponectin concentrations in spontaneous preterm labor and intact membranes

Among women with PTL and intact membranes, the median amniotic fluid adiponectin concentration was significantly higher in patients with IAI than in those without IAI who delivered either at term (54.3 ng/mL, 39.0–91.8 vs. 50.1 ng/mL, 33.2–72.8; p = 0.02, Figure 2) or preterm (54.3 ng/mL, 39.0–91.8 vs. 47.6 ng/mL, 32.6–74.6; p = 0.01, Figure 2). Among women with preterm labor without IAI, no significant difference was found in the median amniotic fluid concentration of adiponectin between women who delivered at term and those who delivered preterm (p=0.7, Figure 2).

Figure 2. Amniotic fluid concentration of adiponectin in women with spontaneous preterm labor and intact membranes.

Among women with preterm labor and intact membranes, the median amniotic fluid adiponectin concentration was significantly higher in patients with IAI than in those without IAI who delivered either at term or preterm.

Amniotic fluid adiponectin concentrations and intra-amniotic infection/inflammation

Amniotic fluid adiponectin concentrations correlated with amniotic fluid WBC count (Spearman rho coefficient: 0.26, p=0.01), amniotic fluid concentrations of glucose concentration (r=−0.37, p=0.002), and IL-6 (r=0.34, p<0.001).

Discussion

Principal findings of the study

1) adiponectin is a constituent of the amniotic fluid; 2) the median amniotic fluid concentration of adiponectin is higher at term that in the mid-trimester; 3) among women with spontaneous PTL and intact membranes, those with IAI had a significantly higher median amniotic fluid adiponectin concentration than those without IAI who delivered either at term or preterm; 4) amniotic fluid adiponectin concentrations were positively correlated with amniotic fluid WBC count and IL-6 concentrations; and 5) amniotic fluid adiponectin concentrations were negatively correlated with amniotic fluid glucose concentrations.

Adiponectin is a potent anti-inflammatory adipocytokine

In addition to its well-established role in metabolic regulation, adiponectin has emerged as a potent anti-inflammatory adipocytokine. The evidence in support of this view includes: 1) adiponectin induces production of anti-inflammatory cytokines (IL-10, IL-1 receptor antagonist⁹⁵) by human monocytes, macrophages and dendritic cells; 2) adiponectin suppresses macrophage production of pro-inflammatory cytokines (TNF-α,⁹⁶ interferon-gamma,⁹⁵ IL-6) and mitigates their phagocytic activity in response to stimulation with lipopolysaccharide (LPS);⁹⁷ 3) adiponectin abates T-cell ability to evoke an allogenic T-cell response;⁹⁵ 4) adiponectin inhibits activation of the nuclear transcription factor NF-κB in endothelial cells;⁹⁸ and 5) alterations in adiponectin concentrations characterize systemic inflammatory conditions such as overweight/obesity ^99;100 insulin resistance,¹⁰¹ and systemic lupus erythematosus.¹⁰² The importance of adiponectin in major metabolic pathways, as well as in the innate and adaptive limbs of the immune response suggests that this adipocytokine plays a role in the regulation of the intricate interface between inflammation and metabolism.

Adiponectin and human pregnancy

Congruent with studies in non-pregnant individuals, adiponectin has been implicated in the metabolic adaptations to gestation ^103–106, as well as in complications of pregnancy such as GDM^107;108 and preeclampsia.^109–114 Indeed, the importance of adiponectin in human pregnancy has been corroborated in the following reports: 1) normal pregnancy is associated with alterations in circulating adiponectin;^{104;115–119} 2) circulating maternal adiponectin correlates with insulin resistance indices during pregnancy;^103;120;121 3) gestational diabetes mellitus (GDM) is associated with decreased maternal concentrations of adiponectin as compared to normal pregnant women;^121–124 4) overweight pregnant women have a lower plasma concentration of adiponectin^125;126 than non-obese pregnant women; and 5) preeclampsia is associated with altered maternal circulating adiponectin.^127–131

Adiponectin is a physiological component of the amniotic fluid

Adiponectin was detected in the amniotic fluid of all patients included in this study, from the early second trimester until 42 weeks of gestation, suggesting that adiponectin is a physiologic component of human amniotic fluid. This is a novel finding. Amniotic fluid adiponectin concentrations were determined in only one study.⁸⁸ Baviera et al.⁸⁸ reported the presence of adiponectin in 50 normal pregnant patients between 15–18 weeks of gestation. The results of the present study are in agreement with the latter study. In both studies adiponectin was detected in all samples. Moreover the mean and IQR of patients in mid-trimester are remarkably similar: 29.0 ng/mL, 19.9–35.2 in the present study and 26.8 13.9–37.3 ng/mL in the study conducted by Baviera et al.⁸⁸ The results reported herein extend our knowledge by demonstrating that adiponectin is present in amniotic fluid in a wide range of gestational ages, at term and during preterm and term labor. Moreover, we were able to report higher amniotic fluid adiponectin concentrations with advancing gestation and in the presence of IAI.

Our findings characterize adiponectin as a novel physiologic constituent of amniotic fluid. Adiponectin is argued to be produced exclusively by adipocytes. Thus its presence in amniotic fluid deserves comment. A possible explanation could be a contamination from the maternal and fetal circulation. However, this possibility is highly unlikely from the following reasons: 1) contamination at the time of amniocentesis is rare, with maternal origin ranging from 0.3–10.8%, whereas fetal injury rates range between 0.6–2%.¹³² Of note, these data were obtained from a review that was published before the widespread use of ultrasound. Adiponectin was detected in all samples in the present report as well as in the study by Baviera et al;⁸⁸ 2) adiponectin concentrations are higher in the presence of IAI and correlate with amniotic fluid WBC count IL-6 and glucose concentrations; and 3) the mean and IQR of amniotic fluid adiponectin concentration of patients in mid-trimester are remarkably similar in the present study and the one conducted by Baviera et al.⁸⁸ Collectively these data strongly suggest that adiponectin is a genuine component of amniotic fluid and its presence in that compartment cannot be attributed to contamination by maternal or fetal blood.

High amniotic fluid concentrations of adiponectin at term and in the presence of intra-amniotic infection: possible ontology and etiology

The presence of adiponectin in amniotic fluid is an intriguing finding since it has been argued that this adipokine is produced exclusively by adipocytes. Nevertheless, adiponectin have been detected in other body fluids including synovial,¹³³ cerebrospinal,^134;135 peritoneal,¹³⁶ saliva¹³⁷ and urine.^102;138 Thus, although, the source for amniotic fluid adiponectin remains unknown, putative origins include fetal urine and fetal membranes.

Several lines of evidence support fetal urine as a source for amniotic fluid adiponectin. Circulating fetal adiponectin concentrations are very high.^{115;116;119;139;140} Indeed, they are two-to-four fold higher than the adults population.^104;116;119 Since adiponectin is secreted in the urine^102;138 (in its native conformation¹⁴¹) it is conceivable that amniotic fluid adiponectin originate from fetal urine. Moreover, term newborns have higher circulating adiponectin than preterm neonates.^142;143 Thus, the higher median amniotic fluid concentrations of adiponectin at term than in mid trimester reported herein, can be explained by higher urine adiponectin in mature fetuses. Finally, fetal urine origin of adiponectin can also account for the higher amniotic fluid adiponectin in the presence of IAI. Renal dysfunction is associated with increased urinary adiponectin secretion.^102;138 Fetal renal insult has been associated with intra-amniotic infection inflammation.^144;145 Taken together, it is possible that renal dysfunction in fetuses affected by the inflammatory process results in increased fetal urine adiponectin concentration which in turn leads to high amniotic fluid adiponectin.

An additional explanation for the presence of adiponectin in the amniotic fluid could be the production and secretion by the fetal membranes. Lappas et al.¹⁴⁶ have demonstrated an ex-vivo secretion of adiponectin by human amnion. Consistent with the findings of the latter report, we have recently found mRNA expression of adiponectin in human amnion.¹⁴⁷ In additional, adiponectin was detected in the amniotic fluid as early as the 15^th week of gestation, at which period the major contribution for amniotic fluid is not the fetal urine. Collectively, these data strongly suggest that the amnion is a source for amniotic fluid adiponectin. Secretion of adiponectin by the amnion can account for the higher concentrations at term since the surface area of the amnion is significantly larger at term than in the mid trimester. Finally, pathway analysis of human amnion gene expression identified adipocytokine signaling pathway as one of the signaling pathways associated with labor, which is an inflammatory process.¹⁴⁸ Thus, it is tempting to suggest that the increased of amniotic fluid adiponectin in the presence of IAI is regulated by the amnion. Further studies will be needed in order to establish the intriguing relationship between the amnion and adiponectin in the context of IAI. These putative sources of adiponectin, i.e. fetal urine and amnion and the suggested explanations for the increased adiponectin at term and in the presence of IAI are not necessarily mutually exclusive and it is possible that both are involved in the regulation of adiponectin.

In conclusion, this is the first study describing the presence of adiponectin in amniotic fluid of term and preterm women and its association with IAI. These observations are in line with, and lend credence to, our previous studies that suggest an association between amniotic fluid adipokines and IAI. ^149–151 Collectively, these novel findings suggest that adipokines play a dynamic, and hitherto unrecognized role in both normal gestation and in intra-amniotic infection. Concomitantly, the source of amniotic fluid adiponectin is unknown. Identification of amniotic fluid origin is of major importance in order to further elucidate the interaction between the IAI, adiponectin and fetal-placental compartments.