Secreted phospholipase A₂ is increased in meconium-stained amniotic fluid of term gestations: potential implications for the genesis of meconium aspiration syndrome

Abstract

Background: Meconium-stained amniotic fluid (MSAF) represents the passage of fetal colonic content into the amniotic cavity. Meconium aspiration syndrome (MAS) is a complication that occurs in a subset of infants with MSAF. Secreted phospholipase A₂ (sPLA₂) is detected in meconium and is implicated in the development of MAS. The purpose of this study was to determine if sPLA₂ concentrations are increased in the amniotic fluid of women in spontaneous labor at term with MSAF.

Materials and methods: This was a cross-sectional study of patients in spontaneous term labor who underwent amniocentesis (n = 101). The patients were divided into two study groups: (1) MSAF (n = 61) and (2) clear fluid (n = 40). The presence of bacteria and endotoxin as well as interleukin-6 (IL-6) and sPLA₂ concentrations in the amniotic fluid were determined. Statistical analyses were performed to test for normality and bivariate analysis. The Spearman correlation coefficient was used to study the relationship between sPLA₂ and IL-6 concentrations in the amniotic fluid.

Results: Patients with MSAF have a higher median sPLA₂ concentration (ng/mL) in amniotic fluid than those with clear fluid [1.7 (0.98–2.89) versus 0.3 (0–0.6), p < 0.001]. Among patients with MSAF, those with either microbial invasion of the amniotic cavity (MIAC, defined as presence of bacteria in the amniotic cavity), or bacterial endotoxin had a significantly higher median sPLA₂ concentration (ng/mL) in amniotic fluid than those without MIAC or endotoxin [2.4 (1.7–6.0) versus 1.7 (1.3–2.5), p < 0.05]. There was a positive correlation between sPLA₂ and IL-6 concentrations in the amniotic fluid (Spearman Rho = 0.3, p < 0.05).

Conclusion: MSAF that contains bacteria or endotoxin has a higher concentration of sPLA₂, and this may contribute to induce lung inflammation when meconium is aspirated before birth.

• Acute phase protein reactant
• interleukin-6
• intra-amniotic inflammation/infection
• prostaglandins
• sPLA₂

Introduction

Meconium-stained amniotic fluid (MSAF) represents the passage of fetal colonic content into the amniotic cavity [1–13]. MSAF is a risk factor for maternal infection-related complications (e.g. chorioamnionitis [8,14–20], puerperal endomyometritis [16,17,20,21]), neonatal sepsis [3,22–25], cerebral palsy [26–29], hypoxic-ischemic encephalopathy [3,30–33], meconium aspiration syndrome (MAS) [3,6,8,12,13,34–54], and fetal death [55–57].

MAS occurs in a subset of infants born to mothers with MSAF [3,6,8,12,13,34–54]. However, why some infants with MSAF develop MAS, and others do not, remains an open question [6,38,41–43,45,51]. Meconium-induced lung injury has been attributed to mechanical obstruction [51,52,58–60], chemical injury [58,61–66], pulmonary cell apoptosis [35,36,60,65,67–70] and an inflammatory response [35,59,67,71–87]. A series of experimental and clinical studies have made a strong case for a role of secreted phospholipase A2 (sPLA₂) in MAS [67,88–95]. This enzyme can exert deleterious effects by eliciting inflammation [92,93,96–112] and inactivating lung surfactant [89,90,113–115]. The purpose of this study was to determine if sPLA₂ concentration is increased in the amniotic fluid of women in spontaneous labor at term with MSAF.

Materials and methods

Study design and population

A cross-sectional study was conducted which included patients at term with MSAF (n = 61) and clear amniotic fluid (n = 40, controls). Inclusion and exclusion criteria for the study population were similar to a previous report [116]. All women provided written informed consent before collection of the amniotic fluid samples. The collection and utilization of the samples was approved by the Human Investigation Committee of the participating institutions and the IRB of the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD/NIH/DHHS). The clinical definitions, sample collection, microbiological studies, detection of endotoxin, and statistical analysis have been described in a previous report [116]. sPLA₂ immunoassay was performed according to the methods defined by Stoner et al. [117,118].

Results

Among women with spontaneous labor at term, 60.4% (61/101) had MSAF and 39.6% (40/101) had clear amniotic fluid. The median maternal age was significantly higher in patients with MSAF than in those with clear fluid (p = 0.03). Otherwise, the clinical characteristics of the two study groups were similar (p > 0.05).

Microorganisms in the AF were identified in 16.4% (10/61) of patients in the MSAF group and in 5% (2/40) of those with clear fluid (p < 0.05). The most common microorganisms were Gram-negative rods (n = 6), followed by Ureaplasma urealyticum (n = 2), Gram-positive rods (n = 2) and Mycoplasma hominis (n = 1). One patient’s amniotic fluid had both a Gram-positive rod and M. hominis. Two patients with clear amniotic fluid had positive cultures for bacteria (U. urealyticum).

The Limulus amebocyte lysate (LAL) assay for bacterial endotoxin in the amniotic fluid was positive in 32.8% (20/61) of patients with MSAF, but in only 2.5% (1/40) of those with clear amniotic fluid (p < 0.001). After heat treatment to eliminate the effect of trypsin [119], the frequency of a positive LAL assay was still significantly higher in the MSAF group compared to those with clear amniotic fluid, even after heat treatment [19.7% (12/61) versus 2.5% (1/40); p < 0.05].

Patients with MSAF had a significantly higher median amniotic fluid sPLA₂ concentration (ng/mL) than those with clear amniotic fluid [1.7 (0.98–2.89) versus 0.3 (0–0.6); p < 0.001] (Figure 1). Moreover, in the MSAF group, those with endotoxin or microorganisms (defined by LAL or amniotic fluid gram stain or positive amniotic fluid culture) had a significantly higher median amniotic fluid sPLA₂ concentration (ng/mL) than those with the absence of endotoxin or microorganisms [2.4 (1.7–6.9) versus 1.7 (1.3–2.5); p = 0.049] (Figure 2). Amniotic fluid sPLA₂ concentration had a significant positive correlation with amniotic fluid IL-6 concentration (Spearman Rho = 0.3, p = 0.045).

Figure 1. Amniotic fluid secreted phospholipase A₂ concentrations (sPLA₂) in women at term with clear amniotic fluid and MSAF. Patients with MSAF had a significantly higher median amniotic fluid secreted phospholipase A₂ concentration (ng/mL) than those with clear amniotic fluid [1.7 (1–2.9) versus 0.3 (0–0.6); p < 0.001].

Figure 2. Amniotic fluid secreted phospholipase A₂ concentration (sPLA₂) among women with MSAF at term with presence and absence of endotoxin or microorganisms. Patients with MSAF and intra-amniotic inflammation/infection at term had a significantly higher median secreted phospholipase A₂ concentration (ng/mL) than those without intra-amniotic inflammation/infection [2.4 (1.7–6.99) versus 1.7 (1.3–2.5); p = 0.049].

Discussion

Principal findings of the study

(1) Patients with MSAF in spontaneous labor at term had a higher median sPLA₂ concentration in amniotic fluid than those with clear amniotic fluid; (2) among patients with MSAF, women with either microbial invasion of the amniotic cavity (MIAC; defined as a positive amniotic fluid culture for microorganisms) or the presence of endotoxin in the amniotic cavity had a higher median sPLA₂ concentration in the amniotic fluid than those without MIAC or bacterial endotoxin; and (3) there was a positive correlation between amniotic fluid sPLA₂ and amniotic fluid IL-6 concentration. Since sPLA₂ is an acute-phase reactant protein induced by IL-6, this observation suggests that an inflammatory response is associated with an increase in sPLA₂.

What are phospholipases A₂?

Phospholipase A2 (PLA₂) is a family of enzymes that hydrolyze the ester bond at the sn-2 position of phospholipids to generate arachidonic acid and lysophospholipids, which are precursors of eicosanoids and other lipid mediators (leukotrienes and prostaglandins) [110,112,120–135]. These enzymes are broadly classified into two groups: (1) intracellular or cytosolic PLA₂ (cPLA₂) and (2) extracellular or secreted PLA₂ (sPLA₂) [112,128]. PLA₂ participates in the production of prostaglandins, which are major mediators of the onset of spontaneous labor at term [136–164], as well as preterm labor [151,159,161,163,165–168]. cPLA₂ is an intracellular enzyme, while sPLA₂ (in particular, group IIA isoform) is an acute phase reactant protein released in response to tissue damage and infection [169–171]. IL-6 can induce the expression of group II sPLA₂ from hepatic cells in culture [172]. The properties and functions of cPLA₂ and sPLA₂ have been reviewed [112,124,128,130,132–134]. Recently, sPLA₂ has been implicated in the pathophysiology of meconium-induced lung injury (see below).

More than 10 isoforms of sPLA₂ have been described (e.g. groups I, II, III, V, etc.) [110,112,130,132–134]. Individual sPLA₂ enzymes act on both cellular membrane phospholipids and non-cellular phospholipids (e.g. surfactant and lipoproteins) including foreign phospholipids (e.g. bacterial membranes and dietary phospholipids) [133]. The functions of sPLA₂ depend on: (1) specific sPLA₂ isoform; (2) specific target phospholipid or membrane; (3) lipid mediators produced by enzymatic activity; (4) the mechanisms responsible for the activation of sPLA₂; and (5) the specific circumstances and site at which a particular sPLA₂ isoform is present [133]. For example, the group I sPLA₂ isoform is produced in the pancreas, and its primary function is the catalytic cleavage of dietary lipids [173]. The group II sPLA₂ isoform is largely expressed and stored in inflammatory cells including neutrophils [174], eosinophils [175,176], T-lymphocytes [177,178], monocytes [179,180], macrophages [181], mast cells [182] and platelets [183]. This particular isoform (group II sPLA₂) is detected in high concentrations in biological fluids in the context of inflammation (e.g. synovial fluid in rheumatoid arthritis [105,184–188], bronchoalveolar lavage (BAL) in patients with acute respiratory distress syndrome (ARDS) [100,114], and serum/plasma of patients with septic shock [189], Crohn’s disease [190], ulcerative colitis [191], acute pancreatitis [192–194], and rheumatoid arthritis [195].

The group II sPLA₂ isoform has potent antimicrobial activity [112,171,196–207]. Elsbach et al. purified sPLA₂ from polymorphonuclear leukocytes of rabbits, and reported that sPLA₂ was bactericidal against Escherichia coli and Salmonella typhimurium, acting in concert with a “bactericidal/permeability increasing protein” [196]. Subsequently, Weinrauch et al. extracted group II sPLA₂ from sterile peritoneal fluid of rabbits, and demonstrated that it had potent antimicrobial activity against Staphylococcus aureus [198,208]. Similarly, group II sPLA₂ isolated from the plasma of baboons after a challenge with E. coli has potent bactericidal properties against S. aureus and Streptococcus pyogenes [198,203]. Such activity can be blocked by a monoclonal antibody against the enzyme [198]. Other investigators have shown antimicrobial activity against Listeria monocytogenes [197,209] and Bacillus anthracis [203,204]. sPLA₂ may also participate in host defense against viruses [210–212] and parasites [213]. The presence of high sPLA₂ concentrations in biological fluids (e.g. tears [214,215], semen [216], intestinal lumen [197,217,218], inflammatory exudates [105,184–188], bronchoalveolar lavage [100,114], and serum [189,219]) of both animals and humans with bacterial infections has been interpreted as indicating that sPLA₂ is part of the host defense against microbial invasion [112,171].

Group II sPLA₂ can induce activation of human neutrophils [101,104], exocytosis in human lung macrophages [102], neutrophils [104], eosinophils [176], and degranulation of mast cells [99]. Triggiani et al. reported that sPLA2 [group I sPLA₂ from cobra venom and group II (recombinant synovial fluid) sPLA₂] can increase the expression of IL-6 mRNA and the rate of secretion of IL-6 from human lung macrophages, as well as the release of β-glucuronidase (a cytosolic enzyme used as a surrogate marker for cellular exocytosis) [102]. Groups I and II sPLA₂ generate an intracellular response that activates both exocytosis and cytokine gene expression in macrophages [92,102,111]. Other investigators have reported that different isoforms of sPLA₂ (group IA, IB, IIA, V and X) induce the production of cytokines (e.g. IL-6, TNFα and IL-10) and chemokines [e.g. monocyte chemotactic protein-1(MCP-1)/chemokine (C-C motif) ligand 2 (CCL2), macrophage inflammatory protein-1 (MIP-1α)/CCL3 and MIP1-β/CCL4] from inflammatory cells such as monocytes [105], neutrophils [108] and eosinophils [176]. These observations collectively suggest that sPLA₂ has an important role in inflammation. The catalytic action of sPLA₂, cleaving membrane phospholipids to generate eicosanoid precursors (arachidonic acid, leukotrienes and prostaglandins), has been implicated in the generation of an inflammatory state [112,114,170,220].

PLA₂ have been localized in lysosomes of chorioamnion [221], decidua [222,223] and amniotic fluid [224,225]. Moreover, its activity in fetal membranes was increased before the onset of labor [221]. Group II sPLA₂ mRNA expression and immunoreactivity has been demonstrated in amnion, choriodecidua and placenta [226–228]. Rice et al. concluded that this isoenzyme is a major contributor of the net tissue sPLA₂ activity in the human placenta and may contribute to the production of prostaglandins during labor [227]. The expression of this enzyme is increased in placentas of women in labor [228].

Phospholipase A₂ in meconium and meconium-induced lung injury

sPLA₂ has been reported in meconium [67,88,90,93]. The administration of meconium into the trachea of neonatal pigs results in severe histologic lung inflammation, increased apoptosis and increased lung sPLA₂ activity (measured by the concentration of arachidonic acid following incubation of lung homogenates with 1,2-dipalmitoylphosphatidylcholine (DPPC), a substrate that is specific to sPLA₂) [67].

sPLA₂ activity has been detected in meconium (determined by measuring DPPC metabolites in suspensions of this material before and after mixing with the substrate) [90]. Enzymatic activity is attributed to sPLA₂ (rather than other phospholipases), and has been demonstrated by the formation of lysophosphatidylcholine after samples had been heat-treated (sPLA₂ is heat-stable – other lipolytic enzymes are heat-sensitive). sPLA₂ extracted from meconium inhibits surfactant activity in vitro [90].

sPLA₂ activity in lung tissues can be induced by meconium and bile acids [115]. sPLA₂ is locally produced in lung tissue and contributes to the total PLA₂ activity during MAS [53,93]. Collectively, the evidence suggests that: (1) meconium contains sPLA₂ activity; (2) the lungs of neonates affected with MAS contain higher amounts of sPLA₂; (3) cPLA₂ was not detected in meconium or alveolar fluid; and (4) there is a correlation between sPLA₂ activity and TNF-α concentrations in bronchoalveolar lavage [53,67,90,92,93,115].

Phospholipase A₂ in amniotic fluid with MSAF and microbial invasion of the amniotic cavity

The findings reported herein suggest that the concentration of sPLA₂ is higher in MSAF than in clear amniotic fluid among patients in labor at term. After exclusion of samples with MSAF with either bacteria or endotoxin, the difference between clear amniotic fluid and MSAF disappeared. Moreover, sPLA₂ concentrations in amniotic fluid correlated positively with IL-6 concentrations. These observations suggest that the elevation in sPLA₂ can be attributed to the consequences of MIAC or the resulting inflammatory process.

Our findings and interpretation are consistent with those reported by Koyama et al., indicating that sPLA₂ activity (measured by high-performance liquid chromatography) and group II sPLA₂ concentration in amniotic fluid were higher in patients with preterm labor (with or without chorioamnionitis) than in preterm controls (i.e. pregnant women without labor who underwent amniocentesis for chromosomal studies between 17–30 weeks of gestation) [229].

We recently reported that the frequency of MIAC and bacterial endotoxin in amniotic fluid is higher among women in spontaneous labor at term with MSAF than in those with clear amniotic fluid [116]. We proposed that microorganisms or microbial products, such as endotoxin, present in amniotic fluid can be swallowed by the fetus, resulting in increased fetal peristalsis and intrauterine passage of meconium. Aspiration of meconium with microorganisms and inflammatory mediators during fetal life could predispose to MAS. Since sPLA₂ has been proposed to be a major mediator of lung injury in MAS, our findings suggest that the meconium of patients with MIAC or endotoxin contains higher concentrations of sPLA₂. Although exposure to sPLA₂ may begin during fetal life, aspirated meconium and microbial products contained in such meconium, as well as inflammatory mediators, may induce further production of sPLA₂ and other inflammatory mediators by the lung that may eventually lead to lung injury and respiratory insufficiency observed in MAS.

Conclusion

Term meconium-stained amniotic fluid that contains bacteria or endotoxin has a higher concentration of secreted phospholipase A₂, and this may contribute to induce lung inflammation when meconium is aspirated before birth.

Declaration of interest

The authors report no conflicts of interest.

This research was supported, in part, by the Perinatology Research Branch, Division of Intramural Research, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Department of Health and Human Services (NICHD/NIH); and, in part, with Federal funds from NICHD, NIH under Contract No. HHSN275201300006C. The research was also supported by a grant from the Walter Scott Foundation for Medical Research.