Objective: To show that meconium causes fetal morbidity and death at earlier gestations than reported previously.
Methods: We searched for specimens from 1997 and 1998 with pathologic diagnosis of meconium-induced umbilical vascular necrosis in placentas of nonmalformed fetuses and newborns. Because intra-amniotic infection is known to activate cytokines, and blood pigment is often microscopically indistinguishable from meconium, we removed those confounding considerations by excluding placentas with chorioamnionitis and signs of intra-amniotic bleeding. Light microscopic identification of vacuolar amniotic epithelial degeneration was used to select specimens with meconium because blood does not cause that histopathologic abnormality. We used histochemical procedures to show absence of hemosiderin and presence of bilirubin, and immunocytochemical labeling with interleukin-1β to show cytokine.
Results: Four cases had meconium-induced umbilical vascular necrosis. The gestational ages were 16, 19, 29, and 38 weeks. Two cases were abortuses, the third was stillborn, and the fourth was a small-for-gestational-age liveborn, delivered by cesarean because of repetitive variable decelerations. Luna-Ishak staining showed bilirubin in macrophages between umbilical vascular myocytes and in the Wharton's jelly. Immunocytochemical methods showed interleukin-1β in those same macrophages.
Conclusion: Cytokines and other meconium-associated factors may contribute to the pathogenesis of fetal death. Survivors may suffer intraventricular hemorrhage, periventricular leukomalacia, and other morbidity.
Exposure of umbilical and superficial placental vessels to intra-amniotic meconium is ultimately complicated by vascular necrosis.1,2 Clinicopathologic correlations found that those lesions are variably associated with persistent fetal circulation, anoxic-ischemic encephalopathy, renal hemorrhage, liver necrosis, myocardial ischemic lesions, and necrotizing enterocolitis.3 The mechanism of fetal damage probably includes vasoactivity of the vessels before development of vascular necrosis. Benirschke4 affirmed that histopathology when he wrote, “I have observed the lesion many times and it was always associated with prolonged meconium discharge before birth.”
Recently we observed severe meconium-induced umbilical vascular necrosis in all three vessels of the umbilical cord of a fetus whose gestational age was 16 weeks. The present study reports that meconium-induced umbilical vascular necrosis can occur in immature fetuses and associated cytokines might cause their deaths during or beyond the second trimester.
In the second trimester and later, fetal mortality or morbidity might result from meconium, umbilical vascular necrosis, and access of cytokines to fetal circulation.
Department of Pathology, the University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma.
Address reprint requests to: Geoffrey Altshuler, MD, Children's Hospital of Oklahoma, Department of Pathology, 940 NE 13th Street, Oklahoma City, OK 73104
Received November 12, 1998. Received in revised form February 16, 1999. Accepted February 24, 1999.
We reviewed records from 1997 and 1998, in which meconium-induced vascular necrosis had been documented by examination of slides stained with hematoxylin-eosin. Autolytic umbilical vascular tissue is characterized by elongated, thin cells (Figure 1). Diagnosis of vascular media necrosis requires presence of numerous ovoid or globoid cells with dark nuclei and hypereosinophilic cytoplasm or cytoid eosinophilic cells with ghostlike or without nuclei (Figures 2 and 3). In the context of the present cases, by light microscopy, macrophages are characterized by large cytoplasm inclusive of bilirubin (Figure 4) and by content of immunohistochemically evident cytokine.
There are many reports of cytokines that accompany intrauterine infection, but few reports of cytokine activity with intra-amniotic meconium and reduced uterine blood flow states. To focus on cytokines unrelated to infection, we excluded all specimens that had deciduitis, chorioamnionitis, or villitis. Meconium pigment is often indistinguishable from blood pigment so to avoid the confounding presence of altered blood pigment, we excluded cases with clinical or pathologic signs of abruptio placentae or intra-amniotic hemorrhage. In the three cases that had amniotic epithelium, enlarged and vacuolated amniotic epithelium typified meconium-induced degeneration. Placentas in which subamniotic connective tissue has blood pigment did not have those light microscopic features. For all four cases we used a standard prussian-blue stain to establish that no hemosiderin was present. To show bilirubin optimally, we used the Luna-Ishak procedure,5 a histochemical method originally developed to show bile canaliculi in liver biopsy tissue, but which effectively shows bilirubin in necrobiotic and necrotic umbilical and superficial placental vascular media.
For cytokine labeling we chose antihuman interleukin-1β rabbit polyclonal antibody (P-420B; Endogen Inc, Woburn, MA), a reagent that is recommended for use with paraffin-embedded tissue. Sections 5 μm each were cut from formalin-fixed (10% buffered formalin), paraffin-embedded blocks, placed on Superfrost/Plus glass slides (Fisher Scientific, Pittsburgh, PA), deparaffinized, and rehydrated with water. The slides were subjected to xylene (two changes every 5 minutes), 100% alcohol (two changes every 5 minutes), 95% alcohol (one change at 5 minutes), 70% alcohol (one change at 5 minutes), and finally rinsed in distilled water (two changes every 3 minutes). For microwave antigen recovery, the slides were put in a loosely covered thermoresistant plastic Coplin jar with 200 mL of 10 mM citrate buffer at pH 6. They were exposed to microwaves at high setting (800 W with turntable), for 5 minutes. For the immunostains, the manufacturer's instructions were followed (Santa Cruz ABC Immunostain Systems; Santa Cruz Biotechnology Inc, Santa Cruz, CA). The sections were incubated for 1 hour with normal rabbit serum diluted in phosphate-buffered normal saline then incubated (alternatively for 60 minutes or 16 hours) with primary antibody (1:50 or 1:100, respectively, in diluted blocking serum). Next, the slides were incubated for 3 minutes in stable diaminobenzidine (Research Genetics Inc, Huntsville, AL). The sections were counterstained with conventional hematoxylin solution, cleared, and mounted. Negative controls initially consisted of tissue processed without inclusion of the primary antibody. Light microscopic slides with chorioamnionitis, labeled with interleukin-1β by the same method, were used as positive controls.
For additional negative controls, we collected umbilical cord segments from two macerated spontaneously aborted fetuses whose gestational ages were 16 and 31 weeks. The first umbilical cord specimen was associated with a termination of pregnancy in which the fetus had an encephalocele. The other specimen originated from a mother who had preeclampsia; the placenta had ischemic change, multiple infarcts, and multifocal decidual necrosis.
Features of clinical and pathologic aspects of the four cases are tabulated in Tables 1 and 2. All four cases, including two immature fetuses (cases 1 and 2), had meconium-associated umbilical vascular necrosis and demonstrable bilirubin and interleukin-1β in morphologically apparent macrophages.
The obstetric and neonatal literature has obscured the historic importance of meconium. There have been contradictory opinions on the significance of fetal exposure to meconium and assertions that the immature fetus is incapable of passage of meconium. More than 50 years ago, Clifford6 observed that yellow staining of the vernix caseosa, skin, nails, and umbilical cords of newborns resulted from prolonged exposure to meconium. He hypothesized that “yellow vernix syndrome” resulted from episodes of fetal asphyxia days or weeks before labor. He suggested that “the principal cause of the prenatal asphyxia is to be found in the placenta.” In our opinion, Clifford's observation and hypothesis remain true.
We found that the degree of meconium-associated risks and the color of meconium correlate with the time the fetus has been exposed to potent soluble biologic components. The most clinically significant meconium does not have a greenish color. Long-term placental staining, ie, longer than 6 hours of fetal and placental exposure to meconium, progressively manifests as a greenish tan, muddy brown, and light tan color.
Newborns with meconium aspiration syndrome excrete dark urine that is spectrophotometrically similar to meconium.7 Two accompanying facts are that umbilical cords of affected newborns are grossly discolored by meconium and pigment is seen by light microscopy in multiple and seemingly migratory macrophages. Those pathologic appearances indicate that color intensity of excreted urine results from clearance of circulating soluble meconium components. We doubt that it results exclusively from lymphatic drainage of intrapulmonary meconium.
Reviews of articles show the substantial discordance on the incidence and significance of meconium.8,9 A review by Katz and Bowes8 found that 5% of neonates with meconium aspiration syndrome are born through thin-consistency meconium, whereas Wiswell9 found instances of 38% and 41%. The extent to which this is confusing is aptly expressed by the titles of the aforementioned contributions, respectively, “Reflections on a murky subject” and “Meconium aspiration syndrome made murkier.” Lack of placental studies and epidemiologic investigations has contributed to the confusion. In a recent article, Wiswell10 comprehensively reviewed those issues, including placental considerations.
Abramovich and Gray11 studied distribution of meconium throughout various parts of intestinal tract specimens from 31 midpregnancy fetuses. They concluded that before 16 weeks' gestation, fetuses physiologically pass meconium into the amniotic fluid. At midgestation, many fetuses are exposed to intra-amniotic microbes and cytokines, and for those and other fetuses, passage of meconium is not necessarily physiologic.
In 4709 consecutive amniocentesis specimens obtained for genetic testing in the second trimester, Allen12 found 79 cases (1.67%) with greenish meconium. The accompanying fetal mortality rate was 5.06%. Allen acknowledged that the incidence of mortality was substantially less than that reported by other investigators. We suggest that exclusion of cases in which amniotic fluid was brownish explains the low mortality rate.12 Brownish fluid in many of the excluded cases probably contained pigments of old meconium.
Because there are more stillbirths in women with cholestatic hepatopathy, Sepulveda and colleagues13 investigated how circulating maternal bile agents might be fatal to fetuses. With methods similar to ours, they found that cholic acid had a dose-dependent vasoconstrictive effect on isolated human placental chorionic veins, and they postulated that it could cause fetal asphyxia.13 Using significantly different methods, Montgomery and colleagues14 found contradictory results.1,13–15 The importance of dose-dependent considerations, however, is substantiated by Bomzon and Ljuburcic.16 They found that low concentrations of bile acids produce vasodilation, not vasoconstriction.16
Falciglia and colleagues17 reported, to their knowledge, the first case of fatal meconium aspiration syndrome in an infant born at 27 weeks' gestation. They cited other authors' findings that in utero meconium is passed in only 3% of infants of gestational ages younger than 36 weeks, an opinion that accords well with previous statement by Matthews and Warshaw,18 who stated that there was meconium staining of amniotic fluid in 251 (7.5%) of 3374 admissions and that no case was less than 34 weeks' gestation.
Combined clinical and placental findings of fetal meconium exposure provide more meaningful diagnoses than do clinical features alone. A relevant publication includes epidemiologic analysis of placental pathology and clinical features of 1252 placentally studied cases.19 That and other data provided at least three noteworthy items: meconium staining of the amniotic fluid was present in 27% of 1252 cases, as opposed to the 7.5% of the aforementioned investigators' 3374 cases;18 in our previous clinicopathologic study,19 although we had little more than a third of the cases of other investigators,18 we found in utero passage of meconium in 13 infants less than 34 weeks' gestation, whereas they reported no such cases; and we found that infants of gestational age younger than 36 weeks have an incidence of in utero meconium passage of 6.7%, compared with the incidence of 3% which Falciglia and colleagues17 cited.
The pathophysiologic mechanism by which fetuses defecate remains unknown. As stated by Resnik, in discussion of Allen's contribution,12 it used to be thought that immature fetuses do not defecate because they have insufficient motilin secreted from the intestinal tract. In samples collected from human fetuses at 19–21 weeks' gestation, motilin is present in the fetal circulation at 60% of maternal levels.20 Thus, relative to the respective amounts of intestinal tract content in mother and fetus, fetal motilin levels should be sufficient to enable discharge of meconium. As cited by Mahmoud et al,21 several hormones are known to initiate the migrating motor complex along the duodenum. Kowalewska-Kantecka22 reported that no statistically significant differences in motilin were found between preterm and term fetuses. However, term newborns who have fetal distress have a fourfold greater amount of plasma motilin in umbilical cord specimens than normal term neonates.23
Our present findings are similar to those of Baergen and colleagues.24 In meconium-induced necrotic umbilical vascular tissue from specimens of mature fetuses, they found immunocytochemically evident interleukin-1 and interleukin-1 receptor antagonist, which is important because it shows means by which cytokine and soluble meconium components achieve augmented access to fetal circulation.
Complications of fetal meconium discharge can be hazardous to immature and extremely preterm fetuses, not just to mature and postmature fetuses. In three of the four placentas available for light microscopic study, we found histopathologic signs of reduced uterine blood flow, which might have caused meconium discharge that led to umbilical vascular necrosis, substantial access of cytokine to vital organs, and, in three of the cases, fetal death. We acknowledge published hypotheses that cytokines might provide insight into mechanisms of sudden infant death syndrome.24
1. Altshuler G, Hyde S. Meconium-induced vasoconstriction: A potential cause of cerebral and other fetal hypoperfusion and of poor pregnancy outcome. J Child Neurol 1989;4:137–42.
2. Altshuler G, Arizawa M, Molnar-Nadasdy G. Meconium-induced umbilical cord vascular necrosis and ulceration: A potential link between the placenta and poor pregnancy outcome. Obstet Gynecol 1992;79:760–6.
3. Altshuler G, Hyde S. Clinicopathologic implications of placental pathology. Clin Obstet Gynecol 1996;39:549–70.
4. Benirschke K. Obstetrically important lesions of the umbilical cord. J Reprod Med 1994;39:262–72.
5. Luna LG, ed. Manual of histologic staining methods of the Armed Forces Institute of Pathology. 3rd ed. New York:McGraw-Hill, 1968:74.
6. Clifford SH. Clinical significance of yellow staining of the vernix caseosa, skin, nails and umbilical cord. Am J Dis Child 1945;69:327.
7. Dehan M, Francoual J, Londenbaum A. Diagnosis of meconium aspiration by spectrophotometric analysis of urine. Arch Dis Child 1978;53:74–6.
8. Katz VL, Bowes WA Jr. Meconium aspiration syndrome: Reflections on a murky subject. Am J Obstet Gynecol 1992;166:171–3.
9. Wiswell TE. Meconium aspiration syndrome made murkier. Am J Obstet Gynecol 1992;167:1914–6.
10. Wiswell TE. Meconium staining and the meconium aspiration syndrome. In: Stevenson DK, Sunshine P, eds. Fetal and neonatal brain injury. Mechanisms, management and the risks of practice. New York: Oxford University Press, 1997:539–63.
11. Abramovich DR, Gray ES. Physiologic fetal defecation in midpregnancy. Obstet Gynecol 1982;60:294–6.
12. Allen R. The significance of meconium in midtrimester genetic amniocentesis. Am J Obstet Gynecol 1985;152:413–7.
13. Sepúlveda WH, Gonzalez C, Cruz MA, Rudolph MI. Vasoconstrictive effect of bile acids on isolated human placental chorionic veins. Eur J Obstet Gynecol Reprod Biol 1991;42:211–5.
14. Montgomery LD, Belfort MA, Saade GR, Moise KJ Jr., Vedernikov YP. Meconium inhibits the contraction of umbilical vessels induced by the thromboxane A2 analog U46619. Am J Obstet Gynecol 1995;173:1075–8.
15. Hyde S, Altshuler G. Need for placental and experimental pathologic examination to determine pathogenetic influences of chronically present intraamniotic meconium. Am J Obstet Gynecol 1996;174:1669–70.
16. Bomzon A, Ljubucic P. Bile acids as endogenous vasodilators. Biochem Pharmacol 1995;49:581–9.
17. Falciglia HS, Kosmetatos D, Brady K, Wesseler TA. Intrauterine meconium aspiration in an extremely premature infant. Am J Dis Child 1993;147:1035–7.
18. Matthews TG, Warshaw JB. Relevance of the gestational age distribution of meconium passage in utero. Pediatrics 1979;64:30–1.
19. Beebe LA, Cowan LD, Altshuler G. The epidemiology of placental features: Associations with gestational age and neonatal outcome. Obstet Gynecol 1996;87:771–8.
20. Adrian TE, Soltesz G, MacKenzie IZ, Bloom SR. Gastrointestinal and pancreatic hormones in the human fetus and mother at 18–21 weeks gestation. Biology Neonate 1995;67:47–53.
21. Mahmoud EL, Benirschke K, Vaucher YE, Poitras P. Motilin levels in term neonates who have passed meconium prior to birth. J Pediatr Gastroenterol Nutr 1988;7:95–9.
22. Kowalewska-Kantecka B. Motilin in umbilical blood. Rocz Akad Med Bialymst 1995;40:662–6.
23. Lucas A, Christofides ND, Adrian TE, Bloom SR, Aynsley-Green A. Fetal distress, meconium, and motilin. Lancet 1979;2:718.
24. Baergen R, Benirschke K, Ulich TR. Cytokine expression in the placenta. The role of interleukin 1 and interleukin 1 receptor antagonist expression in chorioamnionitis and parturition. Arch Pathol Lab Med 1994;118:52–5.
25. Sayers NM, Drucker DB, Grencis RK. Cytokines may give insight into mechanisms of death in sudden infant death syndrome. Med Hypotheses 1995;45:369–74.