Implication of Meconium-aspirated Lungs in Stillborns and Neonatal Deaths

Research Article

Austin J Forensic Sci Criminol. 2015; 2(4): 1034.

Implication of Meconium-aspirated Lungs in Stillborns and Neonatal Deaths

Satoh F, Kakimoto Y, Miyashita K, Tsuboi A, Seto Y and Osawa M*

Department of Forensic Medicine, Tokai University School of Medicine, Japan

*Corresponding author: Motoki Osawa, Department of Forensic Medicine, Tokai University School of Medicine, Shimokasuya 143, Isehara, Kanagawa 259- 1193, Japan

Received: July 16, 2015; Accepted: August 08, 2015; Published: August 11, 2015


Intrauterine passage of meconium into amniotic fluid is thought to occur in response to fetal distress, in which meconium aspiration syndrome is a serious complication in delivery. This retrospective study describes postmortem histological examination to detect amniotic elements in the lungs of stillborns and neonatal deaths. Alcian blue stain and immunohistochemistry using antibodies to sialyl Tn and cytokeratin were performed for formalin-fixed tissues obtained from nine subjects: four stillborns and five neonates. Meconium masses occluded the bronchiolar and alveolar spaces in the lungs of five infants to varying extents. One case of clinically diagnosed meconium aspiration syndrome and another of an abandoned neonate exhibited a high degree of meconium occlusion. In the latter case, fetal distress was presumed to have occurred, as inferred from the histopathological features. The other three stillborns demonstrated a mild degree of small meconium, distributed mainly to alveolar spaces, in which it remained unknown whether this amount of meconium affected stillbirth. As another perspective, amniotic squames were evaluated by immunohistochemistry. Even fewer squames were present in alveoli of the lungs of all live-born subjects, indicating that complete exhalation or degeneration of amniotic elements occurs after several days following birth. Forensic autopsy cases of deceased infants often lack clinical management and detailed information. Meconium contamination in the lung sections is a finding that can indicate antepartum or intrapartum compromised status of infants.

Keywords: Meconium aspiration syndrome; Fetal distress; Stillbirth; Forensic autopsy; Immunohistochemistry


It is meaningful to confirm the presence of amniotic fluid contents in forensic autopsy and subsequent histological examination. For instance, patients with amniotic fluid embolism exhibit dyspnea and cyanosis after broken waters, which is sometimes fatal. After invasion of amniotic fluid into the maternal circulation, it can occlude the pulmonary microcirculation. In postmortem diagnosis, various fetal elements, such as epithelial squames from the fatal skin, lanugo hairs, vernix caseosa, and mucin from respiratory and gastrointestinal tracts, are evident within the pulmonary vasculature. Histopathological procedures to assist in detection of the elements have been established [1-3], in which special stains, such as Alcian blue for acid muco-polysaccharide in mucin, Attwood for squamous cells and mucin, and Sudan Black or Oil Red O for lipid droplets, are effective. Immunohistochemistry using antibody to cytokeratin is also employed to demonstrate squames [4,5]. For another instance related to infants, microscopic examination often reveals amniotic fluid in the lung sections of stillborns and neonates [6]. Innocuous amniotic fluid is filled from the bronchi to alveoli because of spontaneous intrauterine respiratory movements [7,8]. Moreover, Meconium Aspiration Syndrome (MAS) is a severe complication in delivery [9]. Infants who have inhaled meconium develop respiratory distress, including airway obstruction, surfactant dysfunction, and pneumonitis. Meconium is normally stored in the intestines until after birth, but it is expelled into the amniotic fluid prior to birth in response to fetal compromise. Hypoxia in utero has been considered the cause of intestinal contraction and relaxation of the anal sphincter [10]. Amniotic fluid is normally clear, but becomes greenish because of the admixture of meconium. Therefore, the presence of meconiumstained amniotic fluid may be a serious sign of fetal distress. The passage of meconium occurs in a median of 14% (range, 6–25%) of all births [11,12]. In histology, Alcian blue staining of the lung sections of infants is effective to detect a plug of meconium, in contrast to less specific detection by staining with hematoxylin and eosin. Moreover, sialyl Tn of NeuAc α2-6GalNAc, which is produced by goblet cells of the intestine, is well known as an antigen that reacts with mucintype glycoprotein in meconium. The sensitive monoclonal antibody directed to sialyl Tn is TKH-2 [13], which specifically confirms meconium in immunohistochemistry. However, to our knowledge, the morphological appearance of amniotic fluid and meconium in the infant lung sections has not been fully evaluated from the perspective of forensic pathology [14-16]. In this retrospective study, to evaluate the amniotic fluid contents in the lung sections using histology and immunohistochemisry, we examine acute deaths of neonates and infants at or near full term from our forensic autopsy file. Furthermore, we compare observations related to stillbirths and live-births.

Materials and Methods

In all, nine cases were retrieved from the autopsy file of our department for a four-year period of 2008 to 2012, including four stillbirths and five live-births. Lung tissues were fixed in 10% buffered formalin for one to two weeks; then paraffin-embedded tissues were prepared. In each case, multiple sections along the parallel planes from four blocks including all lung lobes were examined. For histological evaluation using microscopy, 4-μm-thick sections were stained with Mayer’s hematoxylin and eosin, and Alcian blue. The immunohistochemical evaluation was performed as described previously [17]. Briefly, mouse monoclonal antibody to sialyl Tn (TKH-2; Novocastra Laboratories Ltd., Newcastle, UK), was incubated with autoclave-treated sections. Prior to application of anti-cytokeratin antibody AE1/AE3 (Dako Cytomation, Carpinteria, CA), sections were treated with 0.1% trypsin at 37oC for 30 min. No pretreatment was performed for anti-surfactant apoprotein antibody, SP-A (Dako). The antibodies were incubated at a dilution of 1:50 or 1:100 for 1 h at room temperature, followed by introduction of the biotinylated second antibody and streptavidin-conjugated peroxidase (LSAB System; Dako). After washing, enzyme activity was visualized with 0.02% 3, 3’-diaminobenzidine containing 0.002% hydrogen peroxide. Sections using TKH-2 and AE1/AE3 were counter-stained respectively with hematoxylin and methylgreen. The immunohistochemical profile of TKH-2 for meconium was evaluated semi quantitatively as eight fields per section with 200× magnification using the following scores: negative stained, –; positive in less than 50% fields, +; 50%–75%, 2+; and more than 75%, 3+. That of cytokeratin for squames was evaluated as the mean number of positive stains in four fields per section with 400× magnification using the following scores: negative stained, –; in less than 10 stains per field, +; 10–20 stains, 2+; and more than 20 stains, 3+.


Case summary

(Table 1) summarizes the causes of death, results of the hydrostatic tests, and findings at forensic autopsy and histological examinations, with numbering of the nine cases. The subjects’ age of gestation was presumed to be from 31-42 weeks. Four infant deaths were judged as stillbirths because no portion of the lungs, even the stomach, floated in water during hydrostatic testing, except for those of one subject #3, who had received clinical management. Perinatal deaths during delivery or fresh stillbirths attributable to intrapartum death were evident in all cases because of little or no maceration. Hypoxia and bleeding caused by antepartum placental abruption was suspected as the cause of death in two subjects, #1 and #4; the mother of subject #4 was also found death at the scene. Subject #3 died of umbilical cord looping around the neck during delivery. Among the other five neonates, subject #5 had been diagnosed with MAS at a hospital, in which greenish meconium debris was present in the external ear and nasal cavities. Suctioning of the trachea was done after birth. In subject #6, with congenital diaphragm herniation, cyanosis occurred after normal delivery. For #9, asphyxia by smothering after normal birth was suspected. In the other two cases, the cause of death was unknown, but neither malformation nor inflammation was evident. One subject #8 had been abandoned. For that reason, the precise alive time after birth was unknown, but it appeared to have been for a short period. Overall, of the nine cases, conditions of fetal distress were presumed to pertain in four cases, #2-#5, based on their clinical courses and death-scene investigations.