Meconium Aspiration Syndrome: A Role for Fetal Systemic Inflammation

Abstract

Background

Meconium aspiration syndrome (MAS) is a leading cause of morbidity and mortality in term infants. Meconium-stained amniotic fluid (MSAF) occurs in approximately one of every seven pregnancies, but only 5% of neonates exposed to MSAF develop MAS. Why some infants exposed to meconium develop MAS while others do not is a fundamental question. Patients with MSAF have a higher frequency of intra-amniotic infection/inflammation than those with clear fluid. We propose that fetal systemic inflammation is a risk factor for the development of MAS in patients with MSAF.

Objective

To investigate whether intra-amniotic inflammation and funisitis, the histopathologic landmark of a fetal inflammatory response, predispose to MAS.

Study Design

A prospective cohort study was conducted from 1995 through 2009. Amniotic fluid (AF) samples (n=1,281) were collected at the time of cesarean delivery from women who delivered singleton newborns at term (gestational age ≥38 weeks). Intra-amniotic inflammation was diagnosed if the AF concentration of matrix metalloproteinase-8 (MMP-8) was >23 ng/ml. Funisitis was diagnosed by histologic examination if inflammation was present in the umbilical cord.

Results

The prevalence of MSAF was 9.2% (118/1,281), and 10.2% (12/118) of neonates exposed to MSAF developed MAS. There were no significant differences in the median gestational age or umbilical cord arterial pH at birth between neonates who developed MAS and those who did not (each p > 0.1). Mothers whose newborns developed MAS had a higher median AF MMP-8 (456.8 ng/ml vs.157.2 ng/ml; p < 0.05). Newborns exposed to intra-amniotic inflammation had a higher rate of MAS than those who were not exposed to intra-amniotic inflammation [13.0% (10/77) vs. 0% (0/32), p = 0.03], as did those exposed to funisitis [31.3% (5/16) vs. 7.3% (6/82), relative risk, 4.3; 95% confidence interval, 1.5–12.3]. Among the 89 newborns for whom both AF and placental histology were available, MAS was more common in patients with both intra-amniotic inflammation and funisitis than in those without intra-amniotic inflammation and funisitis [28.6% (4/14) vs. 0% (0/28), p = 0.009], while the rate of MAS did not show a significant difference between patients with intra-amniotic inflammation alone (without funisitis) and those without intra-amniotic inflammation and funisitis [10.9% (5/46) vs. 0% (0/28)].

Conclusion

The combination of intra-amniotic inflammation with fetal systemic inflammation is an important antecedent of MAS. This concept has implications for the understanding of the mechanisms of disease responsible for MAS and for the development of prognostic models and therapeutic interventions for this disorder.

Introduction

Meconium aspiration syndrome (MAS) is a major cause of neonatal morbidity and mortality, and is a leading cause of neonatal respiratory distress.^1–12 An estimated 1,000 to 1,500 infants die in the U.S. each year as a result of meconium aspiration.^{1, 13, 14} Meconium-stained amniotic fluid (MSAF) is present in one of every seven pregnancies (range: 5%–20%; 400,000–600,000 deliveries in the U.S. per year),^{1, 15–20} but only approximately 5% (20,000–30,000) of neonates born to mothers with MSAF develop MAS.^{1, 15, 21–23} It is not known why only some, or which, neonates exposed to MSAF will develop MAS.^{1, 18, 24, 25} Attempts to prevent MAS have included oropharyngeal,^{26, 27} nasopharyngeal,²⁶ and tracheal^28–30 suctioning and amnioinfusion in women who have MSAF,^{31, 32} but none of these interventions has proven effective.^33–36

Typically, MAS affects term newborns with low Apgar scores (< 7 at 5 minutes).¹ Low Apgar scores are believed to be secondary to an intrauterine event that causes fetal hypoxia, which then causes meconium to be passed in utero,^{9, 11, 37–48} fetal gasping,^49–54 and aspiration of the meconium before birth. However, MAS occurs in the absence of umbilical artery acidemia; therefore, other mechanisms must be involved.^55–64

Clinical and experimental evidence suggests that lung inflammation induced by meconium plays a central role in the pathogenesis of MAS.^{4, 65–69} The pathophysiology has been attributed to: 1) the mechanical effect of meconium, which can obstruct the airways, and 2) the inflammatory effect of meconium.^{68, 70} We propose that intra-amniotic inflammation due to intra-amniotic infection or sterile intra-amniotic inflammation accompanied by a fetal inflammatory response predisposes fetuses exposed to MSAF to develop MAS. This concept is based on previous observations that MSAF is more likely to contain bacteria,^71–73 endotoxin,^73,74 and higher concentrations of inflammatory mediators, such as interleukin (IL)-1, tumor necrosis factor (TNF)-α,^73,75,76 IL-8,^67,76 and phospholipase-A2.⁷⁷ Thus, meconium (with its pro-inflammatory properties), when aspirated before birth and combined with a fetal systemic inflammatory response involving the fetal lungs, could predispose to MAS. If this is correct, fetuses with MSAF and fetal inflammatory response syndrome (FIRS) should have a higher rate of MAS than those without FIRS. The purpose of this study was to determine whether the combination of intra-amniotic inflammation and a fetal systemic inflammatory response is associated with MAS in neonates who have been exposed to MSAF.

Material and methods

Study design

A prospective cohort study was conducted to establish a perinatal biobank to facilitate investigation of contributors to obstetric diseases. One of the features recorded was whether the Amniotic fluid (AF) was clear or meconium stained. AF samples were collected from consecutively enrolled women at term, undergoing cesarean deliveries at the Seoul National University Hospital from July 1995 through June 2009, who met the following inclusion criteria: 1) singleton pregnancy; 2) term gestation (gestational age ≥38 weeks); 3) AF obtained at the time of cesarean delivery; and 4) MSAF identified at delivery. Exclusion criteria were: 1) multiple gestation; 2) stillbirth or fetal death; and 3) presence of major congenital malformations. Written informed consent was obtained from all patients prior to the retrieval of AF.

The Institutional Review Board of Seoul National University Hospital approved the collection and use of these samples and information for research purposes. Seoul National University has a Federalwide Assurance with the Office for Human Research Protections of the U.S. Department of Health and Human Services.

Laboratory studies

AF was collected under direct ultrasound visualization at the time of hysterotomy during the course of a cesarean delivery, and an aliquot was cultured for aerobic and anaerobic bacteria and for genital mycoplasmas (Mycoplasma hominis and Ureaplasma species). The remaining fluid was centrifuged and stored in polypropylene tubes at −70°C. A concentration of matrix metalloproteinase-8 (MMP-8) was measured using a commercially available enzyme-linked immunosorbent assay (Amersham Pharmacia Biotech, Inc, Bucks, UK), following the instructions of the manufacturer.⁷⁸ MMP-8 was assayed in duplicate per analytic run. The sensitivity of the test was 0.3 ng/mL, and the intra- and inter-assay coefficients of variation were <10%. Gram staining of amniotic fluid was not performed.

Histologic examination

Samples of the chorioamniotic membranes, chorionic plate, and umbilical cord were obtained from each placenta. Samples were fixed in 10% neutral-buffered formalin and embedded in paraffin. Sections of tissue blocks were stained with Hematoxylin and Eosin. Histopathologic examinations were performed by a pathologist blinded to the clinical information.

Clinical definitions

The presence or absence of meconium was evaluated at the time of cesarean delivery under visual observation, consistent with standard practice in labor and delivery units. Intra-amniotic inflammation was defined as an elevated AF MMP-8 concentration (>23 ng/mL).^78–84 A fetal systemic inflammatory response was defined by the presence of funisitis; i.e. the presence of neutrophil infiltration in the umbilical vessel walls or Wharton’s jelly, as previously described. ^85–93 MAS was defined as respiratory distress in an infant, born to a mother with MSAF, requiring assisted mechanical ventilation or oxygen at a concentration of ≥40% for at least 48 hours, and by radiographic findings consistent with MAS and symptoms that could not otherwise be explained.^1,31,94–96

Statistical analysis

Proportions were compared using the Fisher’s exact test. The Mann-Whitney U test was used for between-group comparisons of continuous variables. Poisson regression models with robust variance estimators were fitted to estimate relative risk (RR) and corresponding 95% confidence intervals (CI). These analyses were performed using SPSS, Version 19.0 (SPSS Inc., Chicago, IL, USA) and SAS, Version 9.3 (SAS Institute Inc., Cary, N.C., USA). A p-value of <0.05 was considered significant.

Results

Characteristics of the study population

AF was retrieved at the time of cesarean delivery from 1,281 patients with singleton pregnancies who delivered live-born term newborns (gestational age ≥38 weeks) without major congenital anomalies during the study. Altogether, 118 (9.2%) of the 1,281 women participating in this study had MSAF. Indications for cesarean delivery presented by the 118 women included the following: failure to progress during labor (n=76), non-reassuring fetal heart rate tracing (n=18), previous uterine surgery (n=15), fetal malpresentation (n=4), and other indicators (n=5); 12 (10.2%) of the 118 neonates developed MAS.

Table 1 describes the characteristics of the study population stratified according to the presence/absence of MAS; Table 2 shows the clinical characteristics of the 12 cases with MAS. The frequency of low Apgar scores (<7) at 5 minutes following birth was significantly higher in newborns who developed MAS than in those who did not [16.7% (2/12) vs. 0.9% (1/106), p<0.05]. There were no significant differences in the median of gestational age or umbilical cord arterial pH at birth between neonates who developed MAS and those who did not (40.4 vs. 40.4 weeks and 7.2 vs. 7.2, respectively, p >0.1 for each comparison). There was no difference in the rate of rupture of the membranes (ROM) at the time of cesarean delivery between mothers whose newborns developed MAS and those whose newborns did not [83.3% (10/12) vs. 65.7% (69/105), p>0.3]. However, patients whose newborns developed MAS had significantly higher rates of acute histologic chorioamnionitis [80% (8/10) vs. 32.6% (28/86)] and funisitis [45.5% (5/11) vs. 12.6% (11/87)] than those whose newborns did not develop MAS (p<0.05 for each).

Table 1.

Clinical characteristics of the study population according to the presence or absence of meconium aspiration syndrome in the context of meconium-stained amniotic fluid

	Meconium Aspiration Syndrome		P value
	Absence N=106	Presence N=12
Maternal age (years)*	31 (25–44)	32(27–38)	0.655
Nulliparity (%)	78.3 (83/106)	100.0 (12/12)	0.120
Indications for cesarean delivery (%)			0.056
Previous cesarean delivery	14.2 (15/106)	0.0 (0/12)
Failure to progress	66.0 (70/106)	50.0 (6/12)
Fetal malpresentation	3.8 (4/106)	0.0 (0/12)
Non-reassuring FHR pattern	12.3 (13/106)	41.7 (5/12)
Other	3.8 (4/106)	8.3 (1/12)
Presence of labor at amniocentesis (%)	76.4 (81/106)	91.7 (11/12)	0.461
Gestational age at delivery (weeks)*	40.4 (38.0–42.7)	40.4 (39.4–42.0)	0.742
Birth weight (g)*	3445 (2160–4520)	3660 (2800–4850)	0.134
Infant male gender (%)	56.6 (60/106)	91.7 (11/12)	0.026
Apgar score <7 (%)
1 minute	13.2 (14/106)	25.0 (3/12)	0.377
5 minute	0.9 (1/106)	16.7 (2/12)	0.027
Umbilical arterial pH*†	7.238(7.014–7.409)	7.219(6.950–7.295)	0.175
<7.00 (%)	0.0 (0/102)	8.3 (1/12)	0.105
<7.10 (%)	2.9 (3/102)	16.7 (2/12)	0.085
<7.20 (%)	24.5 (25/102)	41.7 (5/12)	0.296
NICU admission (%)	2.8 (3/106)	100.0 (12/12)	<0.001
Positive amniotic fluid culture (%)	20.4 (21/103)	40.0 (4/10)	0.224
MMP-8 > 23 ng/mL (%)	67.7 (67/99)	100.0 (10/10)	0.032
Acute histologic chorioamnionitis (%)	32.6 (28/86)	80.0 (8/10)	0.005
Funisitis (%)	12.6 (11/87)	45.5 (5/11)	0.016

FHR, fetal heart rate; MMP, matrix metalloproteinase; NICU, neonatal intensive care unit

^*Values are presented as the median (range).

^†Four cases without a result of an umbilical arterial blood gas analysis were excluded from analysis.

Table 2.

Characteristics of 12 cases with neonatal meconium aspiration syndrome

Case	GA (weeks)	Birth-weight (g)	Cord arterial pH	Apgar Score		Amniotic fluid			Placental pathology
				1 min	5min	MMP-8 (ng/mL)	Culture	Culture	Chorio-amnionitis	Funisitis
1	40.2	3660	6.950	5	6	2153.4	(−)		NA	NA
2	40.3	4450	7.190	6	8	61.7	(+)	Candida albicans, Streptococcus viridans	(+)	(+)
3	39.3	3660	7.082	8	7	2383.0	NA		(+)	(−)
4	41.2	3760	7.280	8	9	159.9	(−)		(−)	(+)
5	39.6	3100	7.210	7	9	11,754.7	NA		(+)	(+)
6	41.3	4850	7.238	9	9	212.8	(−)		(+)	(−)
7	40.1	3270	7.244	8	9	266.9	(+)	Streptococcus agalactiae	NA	(−)
8	40.6	3940	7.295	7	8	NA	(+)	Ureaplasma urealyticum	(+)	(+)
9	40.1	3550	7.178	7	7	646.7	(+)	Streptococcus mitis (Viridans Streptococcus)	(−)	(−)
10	40.4	3320	7.146	8	8	NA	(−)		(+)	(−)
11	41.0	2800	7.228	2	5	36.6	(−)		(+)	(−)
12	39.6	4360	7.294	8	9	3292.6	(−)		(+)	(+)

GA, gestational age; MMP, matrix metalloproteinase; NA, not available.

Microbial invasion of the amniotic cavity

The rate of positive AF cultures was two-fold higher in the MAS group than in the group without MAS. While this comparison did not reach statistical significance [40% (4/10) vs. 20.4% (21/103), p=0.2], the risk of a type II error is 67.5%. Microorganisms isolated from the AF using cultivation techniques included: Ureaplasma species (n=8), Escherichia coli (n=3), Enterococcus faecalis (n=3), Streptococcus anginosus (n=3), Staphylococcus epidermidis (n=3), and one isolate each of coagulase-negative staphylococci, Klebsiella pneumoniae, Streptococcus intermedius, Staphylococcus sciuri, Candida albicans, Streptococcus agalactiae, Lactobacillus jensenii, Lactobacillus species, and Pseudomonas. There were two cases with cultures positive for Gram-positive cocci and one case with Gram-negative rods; however, the precise organisms could not be identified at the genus level by the clinical laboratory.

Intra-amniotic inflammation

The median of AF MMP-8 concentrations was significantly higher in mothers whose newborns developed MAS than in those whose newborns did not [median 456.8 ng/mL (range 36.6–11,754.7 ng/mL) vs. median 157.2 ng/mL (range 0.3 –7163.4 ng/mL), p<0.05] (Figure 1). Similarly, intra-amniotic inflammation (defined as an MMP-8 concentration >23ng/mL) was significantly more frequent in patients whose newborns developed MAS than in those who did not [100% (10/10) vs. 67.7% (67/99), p<0.05]. MAS did not occur in the absence of intra-amniotic inflammation. There was no difference in the rate of intra-amniotic inflammation (AF MMP-8 concentration >23 ng/mL) according to the presence or absence of oligohydramnios [75.0% (6/8) vs. 68.8% (55/80), p>0.99]. In addition, the frequency of oligohydramnios before ROM did not differ between mothers whose newborns developed MAS and those whose newborns did not [9.1% (1/11) vs. 9.5% (8/84), p>0.99].

Figure 1.

Amniotic fluid (AF) matrix metalloproteinase-8 (MMP-8) in the context of neonatal development of meconium aspiration syndrome (MAS). The median AF MMP-8 was higher in newborns with MAS than in those without MAS (median 456.8 ng/mL; range, 36.6 to 11,754.7 ng/mL vs. median 157.2 ng/mL; range, 0.3 to 7163.4 ng/mL; p < 0.05).

Meconium aspiration syndrome in patients with and without funisitis

Newborns with funisitis were at a greater than four-fold greater risk of developing MAS than those without funisitis [31.3% (5/16) vs. 7.3% (6/82), RR, 4.3; 95% CI: 1.5–12.3]. Among the 89 newborns for whom both AF and placental histology were available, the rate of MAS in cases without intra-amniotic inflammation and funisitis (n=28), with intra-amniotic inflammation alone (n=46), and with both intra-amniotic inflammation and funisitis (n=14) was 0%, 10.9%, and 28.6%, respectively. Only 1 case had isolated funisitis without intraamniotic inflammation. MAS was more common in patients with both intraamniotic inflammation and funisitis than in those without intraamniotic inflammation and funisitis [28.6% (4/14) vs. 0% (0/28), P = .009], while the rate of MAS did not show a significant difference between patients with intraamniotic inflammation alone (without funisitis) and those without intraamniotic inflammation and funisitis [10.9% (5/46) vs. 0% (0/28), P = .15]. A Chi-squared test for trend showed that the frequency of MAS increased as the breadth of inflammation increased (from no inflammation to inflammation restricted to the amniotic cavity, or the combination of inflammation of the amniotic cavity and fetal inflammation; P = .004, from the analysis of linear-by-linear association, SPSS) (Figure 2).

Figure 2.

The frequency of neonatal development of meconium aspiration syndrome (MAS) in the context of intra-amniotic inflammation and funisitis. Neonates exposed to both intra-amniotic inflammation and funisitis were at significantly greater risk of MAS than newborns exposed to neither of these two conditions [28.6% (4/14) vs. 0% (0/28); p = 0.009]. In contrast, newborns exposed to only intra-amniotic inflammation, without funisitis, were not at greater risk of MAS than newborns exposed to neither of these two conditions [10.9% (5/46) vs. 0% (0/28)]. MAS did not occur in the absence of the intra-amniotic inflammation.

Comment

Principal finding of the study

The combination of intra-amniotic inflammation and fetal systemic inflammation (assessed by the presence of funisitis) predisposes to MAS.

Meconium-stained amniotic fluid is necessary but not sufficient to cause meconium aspiration syndrome

MSAF occurs in about one of every seven pregnancies^1,15–20; Yet, only 5% of exposed infants develop MAS.^1,15,21–23 The causes of MAS in newborns exposed to MSAF are unknown. Asphyxia (ante-partum and/or intra-partum) has been implicated in the pathogenesis of MAS,^{9,11,37–46,97} since fetal gasping can lead to aspiration of MSAF.^49–54 Yet, a large fraction of neonates with MAS have no evidence of asphyxia at birth, which suggests the existence of alternative etiologies.^55–64 Indeed, in the present study, we found that only two of 12 neonates with MAS had an Apgar score <7 at 5 minutes following birth, and only two of the 12 cases had an umbilical artery pH <7.1. Therefore, we focused on an alternative mechanism of disease: inflammation.

Inflammation predisposes to meconium aspiration syndrome

This focus is based on a considerable body of clinical and experimental evidence suggesting that inflammation plays a role in the pathogenesis of MAS,^{4,65–68,98–105} and that intra-amniotic inflammation occurs more commonly in MSAF than in clear AF.^{71,72,106–109} The key finding of our study is that newborns diagnosed with funisitis, a histopathologic hallmark of FIRS, were at more than four times the risk of MAS than those without funisitis. In contrast, intra-amniotic inflammation without funisitis was not significantly associated with MAS in this study. Together, these findings support the view that, in term newborns exposed to MSAF, a fetal inflammatory response predisposes to MAS.

A proposed pathophysiology for meconium aspiration syndrome

How could fetal systemic inflammation play a role in MAS? We propose that fetal swallowing of AF containing bacteria,^110–112 endotoxin (or other microbial products),^71,73,77 danger signals or alarmins,^{110,111,113–120} and other pro-inflammatory mediators^{67,73,75–77} can lead to increased bowel peristalsis and passage of meconium that can then be aspirated by the fetus. Meconium per se can block the airways and elicit a local inflammatory response in the lung (pneumonitis); yet, another component of the pathogenesis of MAS may be a systemic fetal inflammatory response. This may enhance the local effects of meconium, bacteria, and inflammatory mediators in the lungs, which may extend to the pulmonary circulation. Experimental evidence indicates that exposure to inflammatory mediators, such as endotoxin, induces vascular changes that predispose to persistent pulmonary hypertension.^{112,121–126} The combination of pneumonitis (caused by AF containing meconium and inflammatory mediators) and capillary damage/leakage developed during the course of fetal systemic inflammation could explain the association among MSAF, intra-amniotic inflammation, funisitis, and MAS. It has also been proposed that inflamed fetal vessels are more vulnerable to compression,¹²⁷ and this may play a role in predisposing newborns with fetal systemic inflammation to MAS. Additionally, AF has physiologic antimicrobial properties impaired by the addition of meconium.⁷¹ Hence, it is also possible that the increased prevalence of microbial invasion of the amniotic cavity in women with MSAF is related to an alteration of the host-defense mechanisms due to the presence of meconium. Further studies are needed to establish time-order.

Clinical implications

The clinical course of MAS is unpredictable, and knowledge of the presence of systemic inflammation may be useful to identify newborns at the greatest risk. This is important because, among neonates born with MSAF, it is not easy to differentiate between those who will have transient respiratory distress from those who will develop full-blown MAS.

Therefore, the findings reported herein may have practical implications for the clinical management of newborns with meconium as it is now possible to assess the presence or absence of fetal systemic inflammation by examining the concentration of IL-6 and C-reactive protein in umbilical cord blood. Some investigators have proposed examination of frozen sections of the umbilical cord to assess the likelihood of systemic fetal inflammation.^128–130 Determination of umbilical cord cytokines and acute phase reactants may be easier than examining frozen sections. The assessment of fetal systemic inflammation can be targeted to neonates born to mothers with MASF who have a clinical course suspicious for MAS. The rationale for this approach is that, among neonates who did not have funisitis (a hallmark of FIRS) only 7.3% (6/82) developed MAS, but the risk quadrupled to 31.3% (5/16) in neonates who had funisitis. Further studies are needed to determine whether the assessment of fetal systemic inflammation with biomarkers (cytokines, chemokines, acute phase reactants, or pathologic findings) can be used to predict MAS in neonates exposed to MSAF.

Strengths and limitations

This is the first study to report the relationship between MAS and the fetal, intra-amniotic, and placental inflammatory responses. Several markers of infection and/or inflammation were assessed, including AF culture, AF MMP-8 concentration, and placental pathology (funisitis). The observed prevalence of MSAF (9.2%, 118/1281) is consistent with that reported in the literature (5–20%).^1,7,24 Yet, the prevalence of MAS (10.2%, 12/118) was somewhat higher than that previously reported.^1,5,18,131 This is most likely attributable to our exclusive focus on term newborns with MSAF, whereas most prior studies included term and preterm neonates. In addition, all cases included in the current cohort study were delivered by cesarean section, a risk factor for the development of MAS among neonates with MSAF.^13,132

During the study period from 1995 through 2009, the clinical management of neonates with MSAF changed at our hospital to follow the American Academy of Pediatrics-Neonatal Resuscitation Program (AAP-NRP) recommendations,⁵ and this might have influenced our findings.

In the present study, there was no difference in the rate of MAS according to the presence or absence of a positive AF culture, nor did we find a statistically significant association between intra-amniotic inflammation alone (without funisitis) and MAS; both findings might be attributed to type II error. A post-hoc power calculation indicated that there was 32% power (type II error: 68%) for detecting the association between a positive AF culture and MAS, and 42% power (type II error: 58%) for detecting the association between intra-amniotic inflammation alone (without funisitis) and MAS.

In our data, male neonates were at a higher risk of MAS than female neonates; we believe that this is an incidental finding. Moreover, male neonates did not show a higher rate of intraamniotic inflammation and funisitis than female neonates (for intraamniotic inflammation: 73.1% vs. 66.7%; for funisitis: 16.9% vs. 15.4%). Whether the male sex is a risk factor for MAS is controversial. Two previous studies have reported that male neonates were at higher risk of MAS than female neonates,^133,134 while another study found no association between the male sex and MAS (OR 1.0; 95% CI, 0.92–1.2).¹

Like all observational studies, causation cannot be inferred from the associations reported herein, and experimental studies would be required to explore this hypothesis.

Conclusion

We propose that the combination of intra-amniotic inflammation with fetal systemic inflammation is an important antecedent of MAS. This concept has implications for the understanding of the mechanisms of disease and development of therapeutic interventions.