Among singleton term neonates with meconium staining at birth (n=835) from all live deliveries (n=16,264) in our institution from January 2006 to June 2014, a total of 160 cases whose placentas were available for histological examination were included in this retrospective study. The study was approved as a retrospective study without informed consent by the institutional review board of the hospital (2011-07-063). Multiple gestation, fetal deaths in utero, or other major anomalies were excluded. Clinical characteristics of the study population were reviewed in three categories: (1) maternal factors (age, parity, gestational age at delivery, mode of delivery); (2) neonatal factors (weight, sex, presence of fetal growth restriction, Apgar score, grade of meconium staining, neonatal intensive care unit admission and duration, arterial blood gas analysis of cord blood); and (3) intrapartum factors (presence of fever [≥38°C], rupture of membrane to delivery interval, oligohydramnios, use of labor epidural anesthesia, use of oxytocin, and fetal heart rate patterns). MAS was classified as follows: mild, requiring <40% oxygen for <48 hours; moderate, requiring ≥ 40% oxygen for at least 48 hours; or severe, requiring assisted mechanical ventilation for more than 48 hours [5,6].

The tissue samples (the placental disc in full thickness, a cross section of the umbilical cord, and a roll of the chorioamniotic membranes) were fixed in 10% formalin and embedded in paraffin. The tissue sections stained with hematoxylin and eosin were reviewed by a pathologist who was blind to the clinical information. Placental histologic findings were categorized as those associated with amniotic fluid infection, maternal underperfusion, and fetal vascular obstructive lesions, according to the criteria proposed by the perinatal section of the Society for Pediatric Pathology [12-14]. Chronic placental inflammation such as chronic villitis, chronic chorioamnionitis, and chronic deciduitis were diagnosed according to the criteria reported previously [14,20,21]. Meconium-laden macrophages, amnion degeneration, chorionic vascular muscle necrosis were included as histological findings related to meconium staining (Fig. 1) [15-17]. Tables 1 and 2 summarize the histological findings and their grading systems (if included).

For statistical analysis, SPSS Statistics ver. 23 (IBM Corp., Armonk, NY, USA) was used. For evaluation of the significance of clinical variables in MAS, we used chi-square test and Fisher exact test for the proportions and Kruskal-Wallis test and Mann-Whitney U test for the continuous variables. The relationship between histologic findings and MAS was verified by chi-square test and Fisher exact test. For evaluation of the correlation between the grades of histopathologic findings and the severity of MAS, linear-by-linear-association was used. The p-values less than .05 were considered to be statistically significant.

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This study demonstrated that acute funisitis, histological findings associated with maternal underperfusion, and chronic deciduitis were frequently found in MAS. The grade of funisitis and chorionic vascular muscle necrosis was correlated with the severity of MAS.

Funisitis, which is characterized by the migration of polymorphonuclear leukocytes from the lumen to the wall of the umbilical vessels, was the most significant histological factor associated with MAS [12]. Funisitis is most commonly associated with intraamniotic infection as is acute chorioamnionitis. Acute chorioamnionitis represents a maternal inflammatory response to intraamniotic infection, whereas funisitis is an evidence of a fetal inflammatory response and reflects a systemic fetal inflammatory response syndrome [22-28]. Meconium may induce local inflammation following tissue degeneration in the fetal membranes (chorioamnionitis) and umbilical cord (funitis) by its leukotactic activity, but it is less severe than that by intraamniotic infection is [17]. Intraamniotic inflammation occurs more frequently in meconium-stained amniotic fluid than in clear amniotic fluid [29-34]. The significant association of funisitis with MAS but not acute chorioamnionitis, in spite of the presence of both acute chorioamnionitis and funisitis in intraamniotic infection/inflammation, suggests that a fetal inflammatory response is crucial to MAS but not a general intraamniotic inflammatory response. It has been proposed that the combination of a local inflammatory response in the lung by meconium and capillary damage/leakage as a manifestation of systemic fetal inflammatory response explains the pathogenesis of MAS [19]. Upregulation of adhesion molecules in the umbilical cord was observed in the presence of funisitis, which was thought to result in increased concentrations of cytokines and soluble adhesion molecules in the fetal circulation. Activation of endothelium may affect not only the umbilical cord but also other fetal organs [25]. Funisitis is associated with adverse neonatal outcome including an Apgar score <7 at 1 minute, small for gestational age and the rate of admission to the neonatal intensive care unit. It is also related to various clinical diseases such as chronic lung disease, intracranial hemorrhage, cerebral palsy, and negative long-term neurologic outcome [25,35-45]. The significant relationship of funisitis with MAS was supported by previous studies, but our study is the first to demonstrate the correlation of the severity between funisitis and MAS [19].

The histological findings associated with maternal underperfusion were identified more frequently in MAS than in non-MAS placentas. Maternal blood from the spiral arteries of the basal plate flows into the intervillous spaces, and the exchange of oxygen and nutrients occurs with fetal blood in the villi. With maternal underperfusion, villi are less perfused, resulting in hypoxic damage and hence increased syncytial knots and villous agglutination [13,46-48]. Circulatory stasis due to underperfusion also induces intervillous fibrin [13,46,47]. Long-standing severe hypoxia may result in distal villous hypoplasia, manifested as decreased number, size, and branching [13,49-53]. Inadequate vascular remodeling and/or structural abnormality of maternal arteries, including mural hypertrophy of arterioles and persistent muscularization of arteries in the basal plate is a known cause of maternal underperfusion such as preeclampsia [13,54-58]. Villous ischemic changes were previously mentioned as a placental finding of MAS, but they were not studied thoroughly.59 Maternal underperfusion is a major risk factor of fetal growth restriction, preterm rupture of membrane, and preterm labor [13,60-63]. Especially, it is frequently related to preeclampsia [13,64]. Chronic asphyxia is one of the possible causes of MAS [5]. Preeclampsia and maternal hypertension which bring about maternal underperfusion are known as risk factors of MAS [65]. Because maternal underperfusion can lead to pulmonary hypoplasia and immaturity, this situation is vulnerable to meconium-induced inflammation. Thus, conditions that can cause maternal underperfusion might increase the risk of MAS.

Chronic deciduitis is defined as the presence of lymphocytes and plasma cells in the basal plate of the placenta [21,66]. Chronic deciduitis has been associated with preterm labor and is also related to adverse outcomes such as intrauterine growth restriction and fetal death [67,68]. The etiology of chronic deciduitis is still not clear; however, chronic microbial infection and immune response to fetus antigen are thought to be involved [66]. The latter is supported by the association between chronic deciduitis and basal villitis, its presence in the placenta of pregnancies by egg donation, and the proximity of the plasma cells to trophoblast cells [66]. The relationship between chronic deciduitis and MAS has not been described before. An immunological predisposing factor to MAS may be suggested; however, the absence of significant association of MAS with chronic villitis and chronic chorioamnionitis, which are also representative of immune response to fetal antigens, does not support the hypothesis. It requires further studies to clarify.

Chorionic vascular muscle necrosis was frequent in MAS, and the severity was correlated with that of MAS. Chorionic vascular muscle necrosis can be diagnosed by observing eosinophilic cytoplasmic degeneration, nuclear pyknosis, discohesion, and rounding of peripheral vascular smooth muscle cells in vessels, resulting from apoptosis in chorionic vessels by prolonged meconium exposure [69]. It is associated with placental lesions resulting from hypoxia and poor neonatal outcome, including intrauterine growth restriction, intrauterine fetal demise, and fetal distress [70].

Collectively, our data support that fetal inflammatory response evidenced by funisitis and chorionic vascular muscle necrosis occurs prenatally in MAS. It is unlikely that MAS is simply caused by aspiration of meconium in utero or during the intrapartum period, which is relatively short, because these placental pathologic findings require a certain time period to be raised. Thureen et al. [59] reported that MAS is a kind of prenatal disease based on histopathologic pulmonary and placental evidences. Meconium passage normally occurs within the first 24 to 48 hours after birth, especially when the babies are 37 weeks or older. But, it is known to be associated with fetal distress such as hypoxia and infection in near-term or term babies [2]. There is also a hypothesis that the fetal swallowing of amniotic fluids containing various factors which evoke inflammation can lead to bowel peristalsis and meconium passage [19]. There are a variety of hypotheses on how meconium raises the respiratory distress in neonates. Nitric oxide (NO) has been known to damage lung epithelial cells responding to meconium. As such, Muller et al. [71] found that lipopolysaccharide and meconium-induced NO production is positively regulated by DMBT1. Ghidini and Spong [5] suggested that alternative mechanisms, such as chronic asphyxia, infection, and acute asphyxia, cause severe MAS that can result in meconium passage in utero and lung damage. There is a possibility that the above mechanisms are causative of both meconium passage and severe MAS, rather than that the meconium passage would be a direct cause of severe MAS [5].

The limitations of this study include the followings: first, placentas were not collected consecutively from all births, and the placentas from neonates with MAS were enrolled for pathological examination at a higher rate than those without MAS. This selection bias explains the high frequency of MAS and inconsistent statistics of clinical characteristics in this study compared with those reported previously [1-4,7]. Second, the relatively small number of moderate and severe MAS cases included may have limited the statistical significance of the results. This is partly supported by that some clinical factors found to be significantly different between mild MAS and non-MAS did not show statistically significant difference between moderate/severe MAS and non-MAS. Further studies with consecutive collection of a larger number of cases could confirm our results and also may find additional placental findings as risk factors.

Our results suggest that fetal inflammatory response evidenced by funisitis occurs prenatally in MAS, and the stage of funisitis and the severity of chorionic vascular muscle necrosis may be predictive markers of the severity of MAS. If meconium staining is detected during delivery and MAS is clinically suspected, the placenta should be fully examined to determine whether these abnormal findings exist and at what stages they are. If the placenta shows high-grade funisitis or extensive chorionic vascular muscle necrosis, the neonate should be treated aggressively from the very beginning to prevent severe MAS.

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