Editor-in-Chief
EDWARD E LAWSON, MD
Johns Hopkins Hospital
Baltimore, Maryland
Book Review Editor
JAYANT P SHENAI, MD
Nashville, Tennessee
Editorial Board
DAVID H ADAMKIN, MD
Louisville, Kentucky
H FRANK ANDERSEN, MD
Dallas, Texas
RONALD ARIAGNO, MD
Stanford, California
KEITH BARRINGTON, MD
Montreal, QC, Canada
MORAYE BEAR, MA, MS
Woodland Hills, California
JATINDER BHATIA, MD
Augusta, Georgia
LUC BRION, MD
Bronx, New York
ANITA J CATLIN, DNSc, FNP, FAAN
Rohnert Park, California
IRA CHASNOFF, MD
Chicago, Illinois
DOUGLAS CUNNINGHAM, MD
Orange, California
WILLIAM A ENGLE, MD
Indianapolis, Indiana
GABRIEL ESCOBAR, MD
Oakland, California
AVROY A FANAROFF, MD
Cleveland, Ohio
GILBERT I FURMAN, MD
West Covina, California
JANUSZ GADZINOWSKI, MD, PhD
Poznan, Poland
Editor, Emeritus
GILBERT I MARTIN, MD
West Covina, California
Imaging Section Editor
MARILYN SIEGEL, MD
St. Louis, Missouri
JEFFREY S GERDES, MD
Philadelphia, Pennsylvania
JAY P GOLDSMITH, MD
New Orleans, Louisiana
JOSEPH HAGEMAN, MD
Evanston, Illinois
JOHN HARTLINE, MD
Kalamazoo, Michigan
DIANE HOLDITCH-DAVIS, PhD
Chapel Hill, North Carolina
THOMAS C HULSEY, MSPH, ScD
Charleston, South Carolina
MICHAEL T HYNAN, PhD
Milwaukee, Wisconsin
ROBERT KOPOTIC, MSN, RN, RRT
Jamul, California
HUGO LAGERCRANTZ, MD
Stockholm, Sweden
MALCOLM LEVENE, MD
Leeds, United Kingdom
KAREN H MORIN, DSN, RN
Newark, Delaware
HIROSHI NISHIDA, MD
Tokyo, Japan
JOHN PARIS, SJ
Chestnut Hill, Massachusetts
VINOD K PAUL, MD, PhD
New Delhi, India
JEFFREY J POMERANCE, MD, MPH
Pasadena, California
International Editors
GARY DARMSTADT, MD, MS
Baltimore, Maryland
ARTHUR I EIDELMAN, MD
Jerusalem, Israel
Maternal-Fetal Medicine Editor
AARON B CAUGHEY, MD, MPP, MPH, PhD
San Francisco, California
ARUN PRAMANIK, MD
Shreveport, Louisiana
JOANN PRAUSE, PhD
Laguna Hills, California
KARI RAIVIO, MD, PhD
Helsinki, Finland
WILLIAM E ROBERTS, MD
Jackson, Mississippi
WARREN ROSENFELD, MD
Mineola, New York
OLA DIDRIK SAUGSTAD, MD, PhD
Oslo, Norway
MICHAEL E SPEER, MD
Houston, Texas
JOSEPH SPINNATO, MD
Cincinnati, Ohio
ANN R STARK, MD
Houston, Texas
DAVID K STEVENSON, MD
Palo Alto, California
DHARMAPURI VIDYASAGAR, MD
Chicago, Illinois
MICHELE C. WALSH, MD
Cleveland, Ohio
DAVID D WIRTSCHAFTER, MD
Los Angeles, California
THOMAS E WISWELL, MD
Orlando, Florida
THE OFFICIAL JOURNAL OF THE SECTION ON PERINATAL PEDIATRICS
AMERICAN ACADEMY OF PEDIATRICS

Volume 28 Supplement 3 December 2008

Proceedings of the First International

Conference for Meconium Aspiration Syndrome
and Meconium-induced Lung Injury

GUEST EDITORS

Gilbert I Martin, MD
Dharmapuri Vidyasagar, MD

This supplement is sponsored by the Division of Neonatology, Department of Pediatrics

at the University of Illinois, NIH, INO Therapeutics, Thrasher Research Fund,
and Section on Perinatal Pediatrics, American Academy of Pediatrics

Journal of Perinatology is published by Nature Publishing Group, a division of Macmillan
Publishers Ltd. The Journal is the official journal of the Section on Perinatal Pediatrics,
American Academy of Pediatrics. It is also the official publication of the National Perinatal
Association.
Scope. Journal of Perinatology provides all members of the perinatal/neonatal healthcare team
with original information pertinent to improving maternal, fetal and neonatal care. The scope of
the Journal reflects the multidisplinary nature of the subject; its coverage includes maternal and
fetal medicine, neonatal medicine, and follow-up of the high-risk infant. Journal of Perinatology
publishes peer-reviewed clinical research articles, state-of-the-art reviews relevant to clinical
management, commentaries, letters to the editor, and brief communications including case
reports and educational vignettes. These articles embrace the full scope of the specialty, including
clinical, professional, political, administrative and educational aspects. The Journal also explores
legal and ethical issues, neonatal technology and product development. All manuscripts are
original and initially published exclusively in the Journal. The audience includes all those who
participate in perinatal/neonatal care, including: neonatologists, perinatologists, pediatricians,
obstetricians/gynecologists, neonatal and perinatal nurses, respiratory therapists, pharmacists,
social workers and nutritionists.
This journal is covered by Index Medicus, MEDLARS, Cambridge Scientific Abstracts, Elsevier
BIOBASE/Current Awareness in Biological Sciences, Combined Cumulative Index to
Pediatrics, and CINAHL Information System.
Editorial. Manuscripts should be submitted online at http://www.mts-jper.nature.com. Detailed
‘Instructions to Authors’ are available at this website, or in print in the first issue of the volume.
Publisher. All business correspondence including enquiries about supplement publication and
sponsorship opportunities should be sent to Journal of Perinatology, Nature Publishing Group,
75 Varick Street, 9th Floor, New York, NY 10013-1917, US. Tel: +1 212 726 9296.
Fax: +1 646 563 7057.
Executive Editor: Joyce-Rachel John
Production Editor: Gene Wu
Visit the Journal’s home pages for details of the aims and scope, readership, instructions to
authors and how to contact the Editor and publishing staff. Use the website to order a
subscription, reprints, a sample copy or individual articles.
Free to all readers. The complete text of all articles in the back archive 4 years after
publication; table of contents and abstracts for all articles published within the last 4 years
and the complete text of the January 2008 issue. Register to receive the table of contents by e-mail
as each issue is published.
2008 Subscriptions
Institutional Subscriptions
New institutional policy
NPG has moved to a site license policy for institutional online access. Institutions may also
purchase a separate print subscription.
Subscribing to a site license
Contact your local sales representative for a tailored price quote for your institution. You will be
required to complete a NPG site license agreement. More information and contact details are
available at the http://www.nature.com/libraries.
Institutional print subscriptions
Orders can be placed with your regular subscription agent or through NPG – either online at
www.nature.com/jp or by contacting our customer services department. Prices are as follows:
The Americas $423.00 Europe h336.00 UK/Rest of World d217.00
Personal Subscriptions
Personal customers who pay by personal check or credit card can either purchase a combined
print plus online subscription or an online-only subscription. Prices are as follows:
Combined (print plus online)
The Americas $177.00 Europe h141.00 UK/Rest of World d91.00
Online-only
The Americas $160.00 Europe h127.00 UK/Rest of World d82.00
Whilst every effort is made by the publishers and editorial board to see that no inaccurate or misleading data, opinion or statement appears in this publication, they wish to make it clear that the data and
opinions appearing in the articles and advertisements herein are the responsibility of the contributor or advertiser concerned. Accordingly, the Publisher, the editorial committee and their respective
employees, officers and agents accept no liability whatsoever for the consequences of any such inaccurate or misleading data, opinion or statement. Whilst every effort is made to ensure that drug doses
and other quantities are presented accurately, readers are advised that new methods and techniques involving drug usage, and described within this publication, should only be followed in conjunction
with the drug manufacturer’s own published literature, and a medically qualified and licensed physician.
Contact information
SITE LICENCES
The Americas:
Tel: +1 800 221 2123. Fax: +1 212 689 9711. E-mail: institutions@natureny.com
Asia Pacific:
Tel: +81 3 3267 8769. Fax: +81 3 3267 8746. E-mail: institutions@naturejpn.com
Australia and New Zealand:
Tel: +61 3 9825 1160. Fax: +61 3 9825 1010. E-mail: nature@macmillan.com.au
India:
Tel: +91 124 288 1054. Fax: +91 124 288 1053. E-mail: harpal@nature.com
Rest of the World:
Nature Publishing Group, The Macmillan Building, 4 Crinan Street, London, N1 9XW, UK
Tel: +44 (0) 20 7843 4759. Fax: +44 (0) 20 7843 4998. E-mail: institutions@nature.com
PRINT SUBSCRIPTIONS
All customers (excluding Japan, Korea and China):
Customer Service Department, Nature Publishing Group, Houndmills, Basingstoke, Hants, RG21 6XS,
UK. Tel: +44 (0) 1256 329242. Fax: +44 (0) 1256 812358. E-mail: subscriptions@nature.com
Japan, Korea and China:
Nature Publishing Group, Nature Japan, Chiyoda Building, 2-37 Ichigayatamachi, Shinjuku-ku,
Tokyo 162-0843, Japan. Tel: +81 3 3267 8751. Fax: +81 3 3267 8746.
E-mail: institutions@naturejpn.com
Prices are applicable in the following regions: US dollars ($) for North, Central, South America
and Canada; Euros (h) for all European countries (excluding the UK); Sterling (d) for UK and
rest of world. Please ensure you use the appropriate currency. All prices, specifications and details
are subject to change without prior notification. Single issues are available. For information,
please contact: Subscriptions Dept, Nature Publishing Group, Brunel Road, Houndmills,
Basingstoke, Hampshire RG21 9XS, UK. Tel: +44 (0) 1256 329242.
Fax: +44 (0) 1256 812358. E-mail: subscriptions@nature.com
Journal of Perinatology. (ISSN 0743-8346) is published monthly by Nature Publishing Group,
75 Varick Street, 9th Floor, New York, NY 10013-1917. Periodicals postage is paid at New York, NY,
US and additional offices. POSTMASTER: please send address changes to Journal of
Perinatology, Nature Publishing Group, 342 Broadway PMB 301, New York, NY
10013-3910.
Reprints and Permissions. For detailed information on reprints and permission requests,
including instructions for obtaining these online, please visit http://www.nature.com/reprints/
index.html. Alternatively, for reprints, please e-mail: reprints@nature.com. For North America:
Tel: +1 212 726 9631. Fax: +1 212 679 0843. Outside North America: Tel: +44 (0) 20 7843 4967.
Fax: +44 (0) 20 7843 4791. For permissions, please e-mail: ajpermissions@nature.com: Tel: +44
(0) 20 7843 4613.
Advertisements. Enquiries concerning advertisements and supplements should be addressed
to: Alf Anderson, Advertising Manager, Nature Publishing Group, 75 Varick Street, 9th Floor,
New York, NY 10013-1917, US. Tel: +1 617 475 9231, Fax: +1 617 494 4960.
E-mail: aanderson@boston.nature.com
Supplements. Enquiries concerning supplements should be addressed to: Michelle Libby, Commercial
Projects Executive. Tel: +1 617 475 9230. Fax: +1 617 494 4960. E-mail: m.libby@boston.nature.com
Copyright r 2008 Nature Publishing Group
ISSN 0743-8346
EISSN 1476-5543
All rights of reproduction are reserved in respect of all papers, articles, illustrations, etc, published
in this journal in all countries of the world. All material published in this journal is protected by
copyright, which covers exclusive rights to reproduce and distribute the material. No material
published in this journal may be reproduced or stored on microfilm or in electronic, optical or
magnetic form without the written authorization of the publisher.
Authorization to photocopy material for internal or personal use, or internal or personal use of
specific clients, is granted by Nature Publishing Group to libraries and others registered with the
Copyright Clearance Center (CCC) Transactional Reporting Service, provided the relevant
copyright fee is paid direct to CCC, 222 Rosewood Drive, Danvers, MA 01923, US. Identification
code for Journal of Perinatology: 0743-8346/08.
Apart from any fair dealing for the purposes of research or private study, or criticism or review, as
permitted by Sections 107 or 108 of the US Copyright Law, this publication may be reproduced, stored or
transmitted in any form or by any means, only with the prior permission in writing of the publishers.
Typeset by Macmillan India Ltd, Bangalore, India
THE OFFICIAL JOURNAL OF THE SECTION ON PERINATAL PEDIATRICS
AMERICAN ACADEMY OF PEDIATRICS

Volume 28 Supplement 3 December 2008

Contents

REVIEWS

S1 Introduction: Proceedings of the First International Conference for Meconium Aspiration Syndrome
and Meconium-induced Lung Injury
GI Martin and D Vidyasagar

S3 Meconium aspiration syndrome: historical aspects

AA Fanaroff

S8 Mechanism(s) of in utero meconium passage

J Lakshmanan and MG Ross

S14 Obstetric approaches to the prevention of meconium aspiration syndrome

H Xu, S Wei and WD Fraser

S19 Delivery room management of the meconium-stained newborn

TE Wiswell

S27 Neonatal resuscitation guidelines for ILCOR and NRP: evaluating the evidence and developing a
consensus
J Kattwinkel

S30 Developing a systems approach to prevent meconium aspiration syndrome: lessons learned
from multinational studies
VK Bhutani

S36 Meconium aspiration syndrome requiring assisted ventilation: perspective in a setting with limited resources
S Velaphi and A Van Kwawegen

S43 Meconium aspiration syndrome: experiences in Taiwan

HC Lin, SY Wu, JM Wu and TF Yeh

S49 Continuous positive airway pressure and conventional mechanical ventilation in the treatment of meconium
aspiration syndrome
JP Goldsmith

Contents continuedy
S56 Treatment of MAS with PPHN using combined therapy: SLL, bolus surfactant and iNO
J Gadzinowski, K Kowalska and D Vidyasagar

S67 Nitric oxide and beyond: new insights and therapies for pulmonary hypertension
RH Steinhorn

S72 Pharmacotherapy for meconium aspiration

A Asad and R Bhat

S79 Extracorporeal membrane oxygenation: use in meconium aspiration syndrome

BL Short

S84 Role of iNO in the modulation of pulmonary vascular resistance

A Bin-Nun and MD Schreiber

S93 Neurodevelopmental outcome of infants with meconium aspiration syndrome: report of a study
and literature review
N Beligere and R Rao

S102 Studies of meconium-induced lung injury: inflammatory cytokine expression and apoptosis
D Vidyasagar and A Zagariya

S108 Angiotensin II in apoptotic lung injury: potential role in meconium aspiration syndrome
BD Uhal and A Abdul-Hafez

S113 Toxic effects of different meconium fractions on lung function: new therapeutic strategies for
meconium aspiration syndrome?
OD Saugstad, PA Tølløfsrud, P Lindenskov and CA Drevon

S116 The role of complement in meconium aspiration syndrome

TE Mollnes, A Castellheim, PHH Lindenskov, B Salvesen and OD Saugstad

S120 Phospholipase A2 in meconium-induced lung injury

P Kääpä and H Soukka

S123 Meconium-induced release of nitric oxide in rabbit alveolar cells

R Fontanilla, A Zagariya and D Vidyasagar

S127 Intracellular and extracellular serpins modulate lung disease

DJ Askew and GA Silverman

Copyright r 2008 Nature Publishing Group

Subscribing organisations are encouraged to copy and distribute
this table of contents for internal non-commercial purposes
This issue is now available at:
www.nature.com/jp
 Journal of Perinatology (2008) 28, S1–S2
r 2008 Nature Publishing Group All rights reserved. 0743-8346/08 $30
www.nature.com/jp
REVIEW
Introduction: Proceedings of the First International Conference
for Meconium Aspiration Syndrome and Meconium-induced
Lung Injury
GI Martin and D Vidyasagar
Journal of Perinatology (2008) 28, S1–S2; doi:10.1038/jp.2008.176
The history of the word ‘meconium’ holds interest for all of us
involved in perinatal care. It is believed that Aristotle coined this
term, which has been anglicized to mean ‘opium like.’ As opium is
derived from poppy seeds and when processed the resulting poppy
seed sludge is black and tarry the word was an appropriate
description.
Meconium-stained amniotic fluid occurs in approximately 13%
of live births. Meconium aspiration syndrome (MAS) occurs in
5 to 10% of infants born through meconium-stained amniotic
fluid. When MAS occurs, there is an increase in neonatal mortality
and morbidity. Great progress has been made in the improvement
of survival of infants with MAS. Great progress in management has
been made since first description of the pathophysiology and poor
outcome of infants with MAS in 1975.1 These include improved
intrapartum and post-delivery management of MAS. Although there
is a significant decrease in the occurrence of MAS and associated
mortality in developed countries MAS remains a major problem in
developing countries.
The physiological effects of MAS owing to airway obstruction
and associated increased pulmonary vascular resistance are well
studied. However, the mechanism of meconium-induced
inflammation and subsequent meconium-induced lung injury
(MILI) are just being investigated. In recent years, there is an
accumulation of evidence from investigators from different
institutions that MAS also causes inflammatory reaction and
cellular injury. These are new findings; the impact of these
findings on management is yet to be determined.
‘The First International Meeting for MILI’ was held on 23 and
24 March 2007 in Chicago. The organizing committee included:
Vidyasagar, Bhat, Bhutani, Wiswell, Gadzinowski, Saugsted and
Zagariya. The conference was partially funded by a grant (to DV)
from NHLBI (Grant no. HL090484-01) to promote the international
collaboration of researchers in the field. The symposium provided a
Correspondence: Dr D Vidyasagar, Division of Neonatology, Department of Pediatrics, The
University of Illinois at Chicago, 840 South Wood Street, Chicago, IL 60517, USA.
E-mail: dsagar@uic.edu
forum for discussion of new findings and also identified knowledge
gaps in management and pathophysiology of MILI. Clinical and
laboratory investigators around the world were invited to discuss
their experiences and research in this field. The Editorial Board of
the Journal of Perinatology kindly agreed to publish the articles
from the speakers at the symposium in this special supplement.
A summary of conclusions from the presentations at the
conference is presented below.
The conference was divided into four sessions. The first session
was devoted to discussions regarding the history of MAS and the
global burden of MAS. Developed countries have noticed a steady
decrease in mortality and morbidity from MAS, but developing
countries such as India and Africa share a major burden of birth
asphyxia and MAS. To reduce unacceptably high infant mortality rate
in developing countries, it was concluded that more research is
needed both in clinical management and in understanding of
MAS and MILI.
The second session was devoted to understand the mechanisms
of in utero passage of meconium in the fetus and potential
pathways that can be blocked to reduce MAS. Mechanisms of
vascular remodeling in the fetus that may contribute to severe
persistent pulmonary hypertension of newborn was described in
infants with severe MAS. Speakers identified several potential areas
of research in the future.
The third session was devoted to review the current practices of
perinatal, intrapartum and post-neonatal management of MAS.
It was concluded that the existing evidence from the literature
indicates that amnioinfusion in the presence of meconium-stained
amniotic fluid or oropharyngeal suction of fetus being delivered
and routine post-delivery endotracheal suctioning have no
beneficial effects in preventing or reducing MAS. There were
questions in developing countries, where resources are limited for
monitoring the fetus to the above therapies. Therefore, there is a
need for studies in developing countries to evaluate the effectiveness
of these practices.
The investigators from India and South Africa stated that
developing countries with limited resources of fetal monitoring and
delivery room care should develop strategies appropriate for their
 Introduction
S2
circumstance. They expressed that there is a need for collaborative
studies to improve the efficacy of clinical management of MAS
babies.
In the fourth session, research data regarding MILI and
apoptosis was presented. It was clear that meconium is a strong
inducer of inflammation and cellular apoptosis. Several pathways
were identified for the induction of apoptosis. This session laid
a firm foundation for future studies and potential collaboration
among interested investigators. The last session was devoted to
reviewing the newer treatment modalities (iNO therapy, ECMO)
and long-term outcomes. It was stressed that iNO therapy
successfully decreases PPHV.
ECMO was ultimate therapy for non-responding patients. It was
noted that in developed countries the need for ECMO in MAS has
decreased. Other discussions included the use of room air
resuscitation of MAS (particularly in developing countries), use of
antibiotics, steroids and other supportive pharmacologic
treatments. These modalities are of significance in developing
countries and need to be studied.
The long-term outcome of infants with MAS was reviewed.
Existing reports consist of diverse population and mixed therapies.
It was noted that there was an increased incidence of
neurodevelopmental delay in babies with MSAF and MAS. These
observations indicate the need for larger follow-up studies of
infants with MSAF and MAS.
In summary, the conference, which was the first of its kind on
MAS, brought together established investigators in the field around
Journal of Perinatology
the globe. The conference allowed free exchange of ideas among
the researchers. In this supplement of the Journal of Perinatology
we are publishing the articles submitted by the speakers. The
articles are arranged into two groups: the first 15 articles deal with
history, epidemiology and clinical aspects of MAS. The next seven
articles deal with the cellular mechanisms of MILI. We hope the
readers of the Journal of Perinatology will find the information
timely and useful.
Acknowledgments
We thank all the authors for their patience and for their cooperation.
We also thank the reviewers for their diligent efforts. And special thanks to the
editorial staff Ms Sandy Skelley for her valuable efforts in making this project
a success. We gratefully acknowledge the grant support from, iNO therapeutics,
and the NIH for enabling us to conduct the symposium and The Thrasher
Foundation and the Perinatal Section of AAP for their support to publish this
Supplement.
Disclosure
D Vidyasagar has received grant support from INO Therapeutics and NIH.
GI Martin has declared no financial interests.
Reference
1 Vidyasagar D, Harris V, Pildes RS. Assisted ventilation in infants with meconium
aspiration syndrome Pediatrics 1975; 56(2): 208–213.
 Journal of Perinatology (2008) 28, S3–S7
r 2008 Nature Publishing Group All rights reserved. 0743-8346/08 $30
www.nature.com/jp
REVIEW
Meconium aspiration syndrome: historical aspects
AA Fanaroff
Department of Pediatrics, Case Western Reserve University, Rainbow Babies & Children’s Hospital/University Hospitals
Case Medical Center, Cleveland, OH, USA
The meconium aspiration syndrome (MAS) is a common problem that
continues to concern perinatologists and neonatologists. MAS is defined as
respiratory distress in an infant born through meconium-stained amniotic
fluid (MSAF) whose symptoms cannot be otherwise explained. This disorder
may be life threatening, complicated by respiratory failure, pulmonary air
leaks and persistent pulmonary hypertension. Approaches to the prevention
of MAS have changed over time with collaboration between obstetricians
and pediatricians forming the foundations for care. This report details the
management of babies delivered with associated MSAF before the
accumulation of evidence for best practice through appropriately powered,
prospective randomized controlled trials.
Journal of Perinatology (2008) 28, S3–S7; doi:10.1038/jp.2008.162
Background
Meconium, the fecal material that accumulates in the fetal
colon throughout gestation, is a term derived from the Greek
mekoni, meaning poppy juice or opium. Commencing with
Aristotle’s observation of the association between meconium
staining of the amniotic fluid and a sleepy state or neonatal
depression, obstetricians have been concerned about fetal well
being in the presence of meconium-stained amniotic fluid (MSAF).
Fetal hypoxic stress may stimulate colonic activity, resulting in the
passage of meconium, and also may stimulate fetal gasping
movements that result in meconium aspiration.
Meconium is a sterile, thick, black-green, odorless material first
observed in the fetal intestine during the third month of gestation.
Meconium results from the accumulation of debris, including
desquamated cells from the intestine and skin, gastrointestinal
mucin, lanugo hair, fatty material from the vernix caseosa,
amniotic fluid and intestinal secretions. It comprises 72 to 80%
water and contains cholesterol and its precursors, lipids, enzymes
including pancreatic phospholipase A2, mucopolysaccharides,
Correspondence: Dr AA Fanaroff, Department of Pediatrics, MS RBC 6040, Rainbow Babies &
Children’s Hospital, 11100 Euclid Avenue, Cleveland, OH 44106, USA.
E-mail: aaf2@case.edu
protein, bile acids and salts as well as drug metabolites.
The black-green color results from bile pigments. When
aspirated into the lung, either in the fetus or newly born infant,
meconium may stimulate the release of cytokines and other
vasoactive substances that lead to cardiovascular and inflammatory
responses.
In the fetus, passage of meconium occurs physiologically early
in gestation, when it contributes to alkaline phosphatase in
amniotic fluid. Abramovich1 noted that fetal defecation diminishes
after 16 weeks and ceases by 20 weeks, concurrent with innervation
of the anal sphincter. At that time, the rectum appears to be filled
with meconium. From approximately 20 to 34 weeks, fetal passage
of meconium was infrequent.2 Most newborn infants who pass
meconium are mature (term). Many are postmature and the
babies may exhibit peeling skin, long fingernails and decreased
vernix. The vernix, umbilical cord and nails may be meconium
stained, depending upon how long the infant has been exposed
in utero. In general, nails will become stained after 6 h and vernix
after 12 to 14 h of exposure.
The passage of meconium normally occurs within the first 24 to
48 h after birth. However, the passage of fetal meconium, resulting
in MSAF, occurs in approximately 8 to 25% of all deliveries,
primarily in situations of advanced fetal maturity or fetal stress.
Most infants who are delivered with MSAF are beyond the 37 weeks
of gestation and meconium rarely appears in amniotic fluid before
32 weeks of gestation. The premature infant lacks ‘motilin,’ while
assists in moving meconium through the lower gastrointestinal
track. The meconium aspiration syndrome (MAS), associated with
aspiration or perhaps diffusion of meconium into the fetal airways,
occurs in about 5% of these infants.3,4
Meconium aspiration syndrome is defined as respiratory distress
in an infant born through MSAF whose symptoms cannot be
otherwise explained. Aspirated meconium can interfere with
normal breathing by several mechanisms. They include airway
obstruction, chemical irritation, infection and surfactant
inactivation (see Figure 1). When aspirated into fetal lungs,
meconium particles mechanically obstruct the small airways.
Meconium or the chemical pneumonitis it causes inhibits
surfactant function, and inflammation of lung tissue
contributes further to small airway obstruction. The chest
 Meconium aspiration syndrome
AA Fanaroff
S4
Figure 1 Pathophysiology of meconium aspiration syndrome. From
Fanaroff and Martin’s Neonatal Perinatal Medicine, Diseases of the Fetus
and Infant 8th edition, edited by R Martin, A Fanaroff and M Walsh, Mosby
Elsevier, 2006.
typically appears barrel-shaped, with an increased anterior–
posterior diameter caused by overinflation. Auscultation reveals
rales and rhonchi. These signs usually are seen immediately
after birth.
The respiratory manifestations include respiratory distress,
tachypnea, cyanosis, and air trapping together with reduced
pulmonary compliance. Cleary and Wiswell3 have proposed severity
criteria to define MAS: (1) mild MAS is disease that requires less
than 40% oxygen for less than 48 h, (2) moderate MAS is disease
that requires more than 40% oxygen for more than 48 h with no
air leak and (3) severe MAS is disease that requires assisted
ventilation for more than 48 h and is often associated with
persistent pulmonary hypertension. Of infants in whom the MAS
develops, more than 4% die, accounting for 2% of all perinatal
deaths.
An estimated 25 000 to 30 000 cases and 1000 deaths related to
MAS occur annually in the United States with many more
cases in developing countries. However Yoder et al.5 documented
a decline in the incidence of MAS in the United States
(from 5.8 to 1.5%Falmost a fourfold reduction) during the
period 1990 to 1997, which they attributed to a 33% reduction in
the incidence of births at more than 41 weeks’ gestation. This
probably can be explained by reductions in the number of infants
passing meconium (in association with advanced maturity) and in
the rate of intrauterine hypoxia (for which the risk is increased
in post-term pregnancies).
Acute intrapulmonary meconium contamination induces a
concentration-dependent pulmonary hypertensive response, with
15 to 20% of infants with the MAS showing persistent pulmonary
hypertension. However, evidence of a long-term process of
muscularization of distal pulmonary arterioles in infants with the
MAS who died suggests that factors other than meconium
aspiration (for example, chronic intrauterine hypoxemia) may
contribute to the pulmonary symptoms.6
Journal of Perinatology
Rationale for routine endotracheal intubation to
prevent MAS
The foundations for routine endotracheal intubation of infants for
MSAF was based on what today would be considered entirely
anecdotal evidence. Gregory et al.7 wished to document the
prevalence of MSAF, the nature of the MSAF (thick or watery, thick
but still liquid, particulate) and the incidence of respiratory distress
when there was meconium present. In a prospective collection of
data they reported MSAF in 8.8% of 1000 consecutive newborn
infants over a 6 month time period. They aspirated the upper and
lower airways as soon as possible ‘sometimes before the first breath’
in 80 of the 88 infants with MSAF.7 If the trachea contained
meconium, intubation and suctioning were repeated until the
airway had been cleared. Fifty-seven infants had meconium in
their mouth, 62 had meconium in the stomach and 8 had
meconium in the trachea but none in the mouth or larynx. Half of
the infants with meconium in the airway (23 of 46) had abnormal
chest X-rays and 16 (70%) of these were symptomatic. They
subsequently compared incidence of air leaks in 35 hospitalized
infants with MAS from their institution (2 of 35; 6%) with
15 outborn infants with MAS including 6 of the 15 (40%) with air
leaks. They concluded that ‘all infants born through thick,
particulate or ‘pea soup’ meconium should have their trachea
aspirated immediately after birth by the most experienced person in
the delivery room and receive thoracic physical therapy and
postural drainage for the first 8 h of life, if the meconium is
recovered from the trachea and/or the chest X-ray is consistent with
aspiration.’ Ting and Brady8 a year later following a retrospective
chart review noted that of 97 infants with thick meconium who
received endotracheal suctioning at birth, 27 became symptomatic
and only one died. On the other hand, 28 infants with thick
meconium who were not suctioned, 16 became symptomatic and
7 died. Soon thereafter Carson et al.9 published their combined
obstetric and pediatric approach to prevent MAS. In a study
originally intended to examine the effectiveness of pulmonary
lavage for MAS, they performed routine intrapartum pharyngeal
suctioning with a DeLee catheter with the infants head on
the perineum, before the onset of respirations, thereafter
inspecting the larynx when the baby delivered and intubating
and suctioning if meconium was present. Their treatment group
was compared to historical controls, MAS occurred in 1 of 273
(0.4%) with routine suctioning and 18 of 947 (1.9%) (P ¼ 0.07) in
the non-suctioned group. They noted that routine suctioning
of the trachea under direct vision after delivery is rarely necessary
but should be performed if meconium is visualized at the vocal
cords, and tracheobronchial lavage with saline may add to the
respiratory morbidity. They indicated that ‘No deaths or severe
cases of MAS have occurred since institution of the obstetric
suctioning procedure’ and concluded that ‘routine intrapartum
pharyngeal suctioning with a DeLee catheter of infants with
meconium staining has significantly reduced the incidence and

severity of MAS’ despite a P-value not supporting such a
conclusion.
On the basis of these studies by Gregory,7 Ting8 and Carson,9
the commonest approach to MSAF, whether thick, watery or
particulate, was for the obstetrician to immediately suction the
nasopharynx as soon as the head appeared on the perineum.
Thereafter, visualization of the cords by direct laryngoscopy and
suctioning of the trachea through the endotracheal tube if
meconium was detected was the routine adopted at most centers.
This was performed before stimulation of the infant or
administration of positive pressure ventilation. This approach
remained the standard of care until challenged by a series of
prospective randomized trials.
Corticosteroids for MAS
Yeh et al.10 evaluated the efficacy of glucocorticoids in the
treatment of infants with MAS. Using a ‘double blind’ technique
they randomized 35 infants with MAS to hydrocortisone or a lactose
placebo. No significant differences in arterial PO2, PCO2, pH,
A-aDO2 gradients, requirement for assisted ventilation, or in
survival were observed between the groups. In the placebo group, a
significant decrease in respiratory distress score occurred at 48 to
72 h of age (P<0.01); in hydrocortisone-treated infants, it was seen
only after 72 h. The infants in the steroid group took a significantly
longer period of time to wean to room air than those in the control
group (68.9±9.6 vs 36.6±6.9 h) (P<0.01). They concluded that
hydrocortisone is not recommended for the treatment of MAS.
MAS and pulmonary hypertension
Pulmonary hypertension was a significant comorbidity in infants
with MAS and frequently the cause of death. The management of
pulmonary hypertension was haphazard and the outcomes
unpredictable. Fox et al.11 reported on 10 patients clinically
diagnosed as having perinatal aspiration syndromes together with
pulmonary hypertension. These infants were either term or
postmature babies and at catheterization had the following
characteristics: (1) systemic or supra-systemic levels of pulmonary
artery pressure (PAP) (range, 50 to 117 mm Hg); (2) a degree of
pulmonary hypertension not related to the degree of aspiration
evident on chest roentgenograms; (3) evidence of right-to-left
shunting at the ductal or foramen ovale level and (4) sustained
severe hypoxemia despite 100% inspired oxygen concentration.
Indwelling pulmonary artery catheters were used for continuous
monitoring of PAP in these 10 infants with severe persistent
pulmonary hypertension of the newborn. The labile nature of PAP,
with changes up to 50 mm Hg, was documented. PAP in the eight
infants with supra-systemic pulmonary hypertension was analyzed
at the time of maximum decrease in pressure (mean 36.1 mm Hg)
and physiologic measurements were compared over an 8-h period.
Meconium aspiration syndrome
AA Fanaroff
S5
During the study period when the infants were hyperventilated, as
the PaCO2 decreased from 48.9 to 28.3 mm Hg (P<0.02), the mean
PAP decreased by 36 mm Hg (P<0.001) to sub-systemic pressure
levels, and the mean alveolar/arterial oxygen difference decreased
by 146 mm Hg (P<0.001). Following the decrease in PAP, patients
were mechanically ventilated to maintain PaCO2 in the range of
25 to 30 mm Hg until pulmonary hypertension gradually resolved
in the six survivors. On the basis of this small uncontrolled study,
hyperventilation to lower pCO2 levels became the standard of care,
and in light of present knowledge probably contributed to a
number of deaf survivors.
Extracorporeal membrane oxygenation and MAS
The history of the introduction of extracorporeal membrane
oxygenation (ECMO) into neonatal perinatal medicine is closely
aligned with the management of MAS. Bartlett et al.12 documented
16 moribund newborn infants with respiratory failure who were
treated with ECMO for 1 to 8 days. Their diagnoses and outcomes
included respiratory distress syndrome, four patients (two improved,
one survived); MAS, eight patients (four improved, three survived);
persistent fetal circulation (some with diaphragmatic hernia), four
patients (three improved, two survived). Intracranial bleeding
occurred in 43% accounting for most of the deaths. In a parallel
series of 21 infants treated with conventional ventilator therapy, the
mortality rate was 90% and intracranial bleeding occurred in 57%.
They modestly concluded that ECMO provided life support and
gains time in newborn respiratory failure. In high mortality risk
infants, the rate of survival is higher and that of intracranial
bleeding is lower with ECMO than with optimal ventilator
management. So dawned the ECMO era, and ultimately the
survival of many moribund infants with pulmonary hypertension
and respiratory failure, including those with MAS. Indeed, in the
ECMO registry, the highest survival rates (>90%) were seen
in the patients with MAS who qualified for ECMO. With the
evidence-based approach to prevention of MAS and the initiation of
better less-invasive therapies, including inhaled nitric oxide,
surfactant therapy, surfactant lavage and various modes of
mechanical ventilation, including high-frequency ventilation, it is
rare for an infant to require ECMO for MAS.13–16
Evidence-based practice to prevent MAS
On the basis of the evidence from non-randomized studies,6–8 to
reduce the incidence and severity of MAS the recommendation was
to intubate and suction all babies born through thick meconium.
However, for term babies who are vigorous at birth endotracheal
intubation may be both difficult and unnecessary. This concept of
routine intubation was challenged first by Faciglia using a
concurrent observational study.17,18 He showed no differences in
outcome with and without routine intubation. Linder et al.19 in a
Journal of Perinatology
 Meconium aspiration syndrome
AA Fanaroff
S6
prospective controlled trial, perhaps inadequately randomized,
assessed the need for intubation of vigorous term meconium-
stained infants. Of 572 infants, meconium-stained infants,
delivered vaginally, with 1-min Apgar score of 9 or 10, none of 264
expectantly managed infants developed MAS and 2 of 308 intubated
infants developed MAS. They concluded that intubation may be
superfluous in a vigorous infant born through MSAF and practice
started to change.
A decade later in international prospective, randomized,
controlled trial assessing need for intubation of apparently vigorous
meconium-stained infants, Wiswell et al.20 enrolled 2094 babies
born with MSAF to either intubation and suctioning or to expectant
management. They found no significant differences in either MAS
or any respiratory disorders supporting the concept that it was not
necessary to intubate vigorous term infants irrespective of the
nature of the MSAF (thick or thin). Halliday21 accumulated four
randomized controlled trials of endotracheal intubation at birth in
vigorous term meconium-stained babies, which were identified.
Meta-analysis of these trials does not support routine use of
endotracheal intubation at birth in vigorous meconium-stained
babies to reduce mortality, MAS, other respiratory symptoms or
disorders, pneumothorax, oxygen need, stridor, HIE and
convulsions. However, the event rates of many of these outcomes is
low in the reported trials making reliable estimates of treatment
effect impossible.
Vain et al.22 finally shattered all the anecdotal data when they
reported on their prospective randomized trial that routine
intrapartum oropharyngeal and nasopharyngeal suctioning of
term-gestation infants born through MSAF does not prevent MAS.
Amnioninfusion
Amnioinfusion has been advocated as a technique to reduce the
incidence of meconium aspiration and to improve neonatal
outcome. However, meconium passage may predate labor so that a
large proportion of women with MSAF have infants with meconium
in the trachea or bronchioles before meconium passage has been
noted clinically and before amnioinfusion can be performed.23
Fraser et al.24 reported that for women in labor who have thick
meconium staining of the amniotic fluid, amnioinfusion did not
reduce the risk of moderate or severe MAS, perinatal death, or other
major maternal or neonatal disorders.25 Xu et al.26 summarized
the evidence for the use of amnioinfusion to prevent MAS. They
concluded that in clinical settings with standard peripartum
surveillance, the evidence does not support the use of
amnioinfusion for MSAF. In settings with limited peripartum
surveillance, where complications of MSAF are common,
amnioinfusion appears to reduce the risk of MAS. As always they
noted that further studies were needed.
Journal of Perinatology
Long-term consequences
The initial follow-up report on follow-up of MAS by Marshall
et al.27 in 1978 included a 1-year cohort of 17 patients,
representing 3.7% of all admissions to their unit. Four patients
(23.5%) died of acute respiratory failure and two patients with MAS,
and persistence of the fetal circulation required cardiac
catheterization to exclude cyanotic congenital heart disease. None
of the survivors had persistent chronic lung disease but two of three
patients with MAS and seizures had significant psychomotor
retardation at follow-up examination. The current data indicate
that there is an increased prevalence of asthmatic symptoms and
abnormal bronchial reactivity among survivors of the MAS.
Conclusion
Meconium aspiration syndrome remains a challenging condition
most notably in developing countries. In the USA avoidance of
post-term pregnancies, improved intrapartum monitoring and
amnioinfusion have dramatically reduced the incidence of MAS.
Whereas there is inconclusive evidence as to the optimal ventilator
strategy for MAS, surfactant replacement therapy has been
beneficial and surfactant lavage is still being explored. Inhaled
nitric oxide has significantly enhanced the treatment of pulmonary
hypertension and substantially reduced the need for ECMO. Despite
optimal perinatal management, infants born through MSAF may
still develop MAS and have morbid or even fatal outcomes.
Disclosure
Avroy Fanaroff has received consulting fees from NATUS.
References
1 Abramovich DR, Gray ES. Physiologic fetal defecation in midpregnancy. Obstet
Gynecol 1982; 60: 294.
2 Ramon Y, Cajal CL, Martinez RO. Defecation in utero: a physiologic fetal function. Am
J Obstet Gynecol 2003; 188: 153.
3 Cleary GM, Wiswell TE. Meconium-stained amniotic fluid and the meconium
aspiration syndrome: an update. Pediatr Clin North Am 1998; 45: 511–529.
4 Dargaville PA, Copnell B. The epidemiology of meconium aspiration syndrome:
incidence, risk factors, therapies, and outcome. Pediatrics 2006; 117: 1712.
5 Yoder BA, Kirsch EA, Barth WH, Gordon MC. Changing obstetric practice associated
with decreasing incidence of meconium aspiration syndrome. Obstet Gynecol 2002;
99: 731–739.
6 Murphy JD, Vawter GF, Reid LM. Pulmonary vascular disease in fatal meconium
aspiration. J Pediatr 1984; 104: 758–762.
7 Gregory GA, Gooding CA, Phibbs RH, Tooley WH. Meconium aspiration in infantsFa
prospective study. J Pediatr 1974; 85: 848–852.
8 Ting P, Brady JP. Tracheal suction in meconium aspiration. Am J Obstet Gynecol
1975; 122: 767–771.
9 Carson B, Losey R, Bowes Jr WA, Simmons MA. Combined obstetric and pediatric
approach to prevent meconium aspiration syndrome. Am J Obstet Gynecol 1976;
126: 712.

10 Yeh TF, Srinivasan G, Harris V, Pildes RS. Hydrocortisone therapy in meconium
aspiration syndrome: a controlled study. J Pediatr 1977; 90: 140–143.
11 Fox WW, Gewitz MH, Dinwiddie R, Drummond WH, Peckham GJ. Pulmonary
hypertension in the perinatal aspiration syndromes. Pediatrics 1977; 59: 205–211.
12 Bartlett RH, Gazzaniga AB, Huxtable RF, Schippers HC, O’Connor MJ, Jefferies MR.
Extracorporeal circulation (ECMO) in neonatal respiratory failure. J Thorac
Cardiovasc Surg 1977; 74: 826–833.
13 El Shahed AI, Dargaville P, Ohlsson A, Soll RF. Surfactant for meconium aspiration
syndrome in full term/near term infants. Cochrane Database Syst Rev 2007:
CD002054.
14 Greenough A, Pulikot A, Dimitriou G. Prevention and management of meconium
aspiration syndromeFassessment of evidence based practice. Eur J Pediatr 2005;
164: 329–330.
15 Wiswell TE, Knight GR, Finer NN, Donn SM, Desai H, Walsh WF et al. A multicenter,
randomized, controlled trial comparing surfaxin (lucinactant) lavage with standard
care for treatment of meconium aspiration syndrome. Pediatrics 2002; 109:
1081–1087.
16 Nakamura T, Matsuzawa S, Sugiura M, Tamura M. A randomised control study of
partial liquid ventilation after airway lavage with exogenous surfactant in a meconium
aspiration syndrome animal model. Arch Dis Child Fetal Neonatal Ed 2000; 82:
F160–F162.
17 Falciglia H. Failure to prevent meconium aspiration syndrome. Obstet Gynecol 1988;
71: 349–353.
18 Falciglia HS, Henderschott C, Potter P, Helmchen R. Does DeLee suction at the
perineum prevent meconium aspiration syndrome? Am J Obstet Gynecol 1992; 167:
1243–1249.
Meconium aspiration syndrome
AA Fanaroff
S7
19 Linder N, Aranda JV, Tsur M, Matoth I, Yatsiv I, Mandelberg H et al. Need for
endotracheal intubation and suction in meconium-stained neonates. J Pediatr 1988;
112: 613–615.
20 Wiswell TE, Gannon CM, Jacob J, Goldsmith L, Szyld E, Weiss K et al.
Delivery room management of the apparently vigorous meconium-stained neonate:
results of the multicenter, international collaborative trial. Pediatrics 2000; 105
(1 Part 1): 1–7.
21 Halliday HL. Endotracheal intubation at birth for preventing morbidity and mortality in
vigorous, meconium-stained infants born at term. Cochrane Database Syst Rev 2001:
CD000500.
22 Vain NE, Szyld EG, Prudent LM, Wiswell TE, Aguilar AM, Vivas NI. Oropharyngeal and
nasopharyngeal suctioning of meconium-stained neonates before delivery of their
shoulders: multicentre, randomised controlled trial. Lancet 2004; 364: 597–602.
23 ACOG Committee Obstetric Practice. ACOG Committee Opinion Number 346, October
2006: amnioninfusion does not prevent meconium aspiration syndrome. Obstet
Gynecol 2006; 108: 1053.
24 Fraser WD, Hofmeyr J, Lede R, Faron G, Alexander S, Goffinet F et al., Amnioinfusion
Trial Group. Amnioinfusion for the prevention of the meconium aspiration syndrome.
N Engl J Med 2005; 353: 909–917.
25 Ross MG. Meconium aspiration syndromeFmore than intrapartum meconium.
N Engl J Med 2005; 353: 946–948.
26 Xu H, Hofmeyr J, Roy C, Fraser WD. Intrapartum amnioinfusion for meconium-stained
amniotic fluid: a systematicreview of randomised controlled trials. British J Obstet
Gynecol 2007; 114: 383–390.
27 Marshall R, Tyrala E, McAlister W, Sheehan M. Meconium aspiration syndrome.
Neonatal and follow up study. Am J Obstet Gynecol 1978; 131: 672–676.
Journal of Perinatology
 Journal of Perinatology (2008) 28, S8–S13
r 2008 Nature Publishing Group All rights reserved. 0743-8346/08 $30
www.nature.com/jp
REVIEW
Mechanism(s) of in utero meconium passage
J Lakshmanan and MG Ross
Department of Obstetrics and Gynecology, Harbor-UCLA Medical Center, Torrance, CA, USA
To use sheep and rat models and demonstrate that stressors activate fetal
glucocorticoid (GC) system, corticotrophin-releasing factor (CRF) system
and cholinergic neurotransmitter system (ChNS) leading to propulsive
colonic motility and in utero meconium passage. Immunohistochemical
studies (IHS) were performed to localize GC-Receptors, CRF-receptors and
key molecules of ChNS in sheep fetal distal colon. CRF expression in
placenta and enteric endocrine cells in fetal rat system were examined and
the effects of acute hypoxia on in utero meconium passage was tested. IHS
confirmed localization and gestation dependent changes in GC-Rs, CRF-Rs
and cholinergic markers in sheep fetal colon. Rat placenta and enteric
endocrine cells express CRF and gastrointestinal tract express CRF-Rs.
Hypoxia is a potent inducer of meconium passage in term fetal rats. Stress is
a risk factor for in utero meconium passage and laboratory animal models
can be used to develop pharmacotherapy to prevent stress-induced in utero
meconium passage.
Journal of Perinatology (2008) 28, S8–S13; doi:10.1038/jp.2008.144
Introduction
Newborn meconium passage, a developmentally programmed
event, normally occurs within the first 24–48 h after birth,
although little is known of the regulatory process. Clinically, a
delay in newborn meconium passage has been observed in infants
born with Hirschprung disease, a defect involving the absence of
intrinsic ganglia in colon.1 In contrast, we have no similar
anatomical or neurochemical explanations for the meconium
staining observed in 7–22% of all term deliveries.2 Greater than
90% of cases of clinically observed meconium-stained amniotic
fluid are noted in fetuses at or following 37 weeks gestation, being
uncommon in preterm deliveries.3 Passage of meconium occurs
most often in deliveries beyond 40 weeks of gestation.4 An increased
incidence of meconium passage in amniotic fluid is noted in the
presence of fetomaternal stress factors such as hypoxia and
infection, independent of fetal maturation.5,6 On the basis of the
clinical observations, fetal stress and gastrointestinal maturation
Correspondence: Dr MG Ross, Department of Obstetrics and Gynecology, Harbor-UCLA
Medical Center, 1000 W. Carson Street, Box 3, Torrance, CA 90502, USA.
E-mail: mikeross@ucla.edu
have been attributed as risk factors responsible for meconium
passage as well as the potential meconium aspiration in utero.
Despite these risk factors, presently very little is known of the
cascade of events that leads to meconium passage in the
newborn immediately after birth or to the mechanisms
contributing ‘premature’ meconium passage in utero. There
are no small or large animal models that could be extrapolated
to humans. We review here recent studies gathered in our
laboratory in rat and sheep models directed toward understanding
the cellular and molecular basis of stress-induced in utero
meconium passage.
Stress and gut motility: lessons learned from
laboratory animals
Although impact of emotional stress on gastric and colonic motor
activity has long been known in humans only during the past two
decades, much attention has been paid to understand the neural
circuitry and hormonal effectors that mediate effects of stress on
gut functions in animal models (see review7,8). The rat is a widely
used animal model for stress-related alteration on gut motor
functions, although limited investigation has also been performed
in mouse models.9 Inhibition of gastric emptying, gastric secretion,
gastric contraction and stimulation of colonic motility and
defecation have now been well established in adult rats as the
hallmarks of neurovisceral motor responses to stressors of
psychological, physical, chemical and immunological origin.10–13
Members of stress hormone family, more specifically
corticotrophin-releasing factor (CRF) and urocortin, have been
shown to mimic multifaceted acute responses to stress.8 Both
hormones mediate their actions through CRF-receptor subtype 1
(CRF-R1) and CRF-receptor subtype 2 (CRF-R2). Both receptors
are encoded by distinct genes and both are G protein-coupled
receptors linked to multiple intracellular signaling pathways.14
Of the two CRF receptor subtypes, CRF-R1 stimulates colonic
motility and defecation, whereas CRF-R2 inhibits gastric emptying
and gastric contraction at times of stress.15 Anatomical,
physiological, pharmacological and biochemical studies have
provided unambiguous evidence that the CRF system

(CRF, urocortins, CRF-R1 and CRF-R2) is the key player of
gastrointestinal motility disturbance during times of stress.7,16
Hypothesis: in utero meconium passage is a fetal
neurovisceral motor response to stress
We hypothesize that in utero meconium passage is a neurovisceral
motor response to fetal stress and that the CRF system plays a
critical role in the stimulation of cholinergically mediated fetal
colonic propulsive motor functions that trigger meconium passage.
An integrated model proposed for fetal in utero meconium passage
is shown in Figure 1. The salient points of our hypothesis are as
follows: fetal–maternal stress leads to increased fetal plasma
glucocorticoids, which stimulate the synthesis of placental CRF.17
Stress-induced increase in glucocorticoids are also speculated to
upregulate the CRF-R1 density18 on the myenteric neurons as well
as the expression of peripheral choline acetyltransferase,19 the
enzyme that catalyzes the synthesis of acetylcholine, the principle
neurotransmitter that regulates the motor activity of
gastrointestinal system. In conditions of stress, increased maternal
and/or fetal sympathetic tone will lead to increased circulating
levels of catecholamines, which are known stimulators of placental
CRF release.20 Thus, stress-induced activation of the CRF–CRF-R1
system at the level of myenteric neurons will evoke release of
acetylcholine, increased colonic smooth muscle motility and
in utero meconium passage.
Stress is a potent stimulator of in utero meconium
passage in term fetal rats
Although clinical signs of stress due to hypoxia are frequently
observed in human neonates born with meconium staining, there
is no direct evidence to support the fact that hypoxic stress is a risk
factor for in utero meconium passage. We recently developed a
Figure 1 An integrated model for stress-induced in utero meconium passage.
An interconnected pathway involving glucocorticoids, CRF and cholinergic
neurocircuitry systems stimulate colonic motility leading to in utero meconium
passage (detailed in the text).
Mechanism of in utero meconium passage
J Lakshmanan and MG Ross
S9
novel hypoxic stress paradigm to demonstrate that hypoxic stress at
term will provoke in utero meconium passage.21 The experimental
design includes exposure of term pregnant rats (term ¼ 22 days)
at 22 days of gestation to decreasing concentration of oxygen in a
stepwise manner as illustrated in Figure 2a. Fetuses removed from
uterine horn at the end of hypoxic exposure were alive and
exhibited thick meconium staining as shown in Figure 2b, whereas
control fetuses did not exhibit meconium.
Examination of fetal and maternal plasma revealed a marked
elevation in CRF levels,21 suggesting that the peripheral CRF
pathway may mediate the stimulation of fetal colonic motility and
meconium passage. As acute hypoxia-induced meconium passage
has a rapid onset, the mechanisms underlying the peripheral
pathway most likely involve the activation (directly and neurally)
of fetal adrenal epinephrine and norepinephrine secretion as well
as sympathetic nervous system norepinephrine release, which in
turn stimulate placental CRF exoctyosis. Recent studies in our
laboratory indicate that rat placenta, similar to human placenta, is
a site of CRF synthesis.22,23 In addition, enteric endocrine cells
expressing CRF in term rat fetal gut could also function as a
potential local source, as speculated in humans.24,25 Increased
peripheral CRF in the fetus stimulates colonic motility through
myenteric CRF-R1 and rapid meconium passage (Figure 3a).
The central CRF pathway involves activation of CRF neurons
in paraventricular brain nuclei directly innervating colonic
segments, as has been demonstrated in adult rats (Figure 3b).
Thus, acute hypoxic stress-induced in utero meconium passage
involves release of stored CRF protein pool from peripheral and/or
central tissues. Ongoing studies in our laboratory also support the
expression of CRF-R1 in smooth muscle and enteric neuronal
circuitry in term rat fetal gastrointestinal tract,26 lending strong
anatomical support for our hypothesis that intrauterine stress may
impact gut motility through CRF-R1 in a manner analogous to
stress-induced stimulation of colonic motility and defecation in
adult rats.15
Lessons learned from sheep model in support of
stress-induced in utero meconium passage
Sheep are used in our laboratory as a large animal model for
understanding the molecular mechanism of in utero meconium
passage.27–29 The larger size of the gut and longer
gestational age permit in vivo studies exploring gestation- and
hormone-dependent changes in gastrointestinal motility, and
in vitro organ bath studies examining smooth muscle contractility.
The large fetal size permits withdrawal of blood in sufficient
quantities for evaluating circulating hormone and cytokine levels.
In the following section, we summarize some of our investigations
relevant to hormonal regulation of sheep fetal distal colonic
motility as well as recent progress made on the ontogeny of
glucocorticoid receptor system, CRF system and cholinergic
Journal of Perinatology
 Mechanism of in utero meconium passage
J Lakshmanan and MG Ross
S10

Oxygen content in chamber

25

20
% Oxygen
15

10

21
24
27

33
36
39
30
12
15
18

42
6
8
3
0

Time (min)

Figure 2 Hypoxic stress paradigm in term fetal rat with meconium passage. Oxygen content in hypoxic chamber is reduced in a stepwise manner from 21 to 8%
as indicated in the figure (left panel). A photograph of a fetal rat with meconium passage (arrows; right panel).

a b

Figure 3 Hypothesized mechanisms for hypoxia-induced in utero meconium passage. Acute hypoxia-induced in utero meconium passage could be mediated either by
(a) placental CRF release or (b) central hypothalamic paraventricular nucleus CRF. In both circumstances, CRF directly stimulates CRF-R1 localized on
cholinergic myenteric neurons.

synaptic circuitry system in fetal distal colonic segments. In
addition, we discuss our recent advances in support of knowledge of
CRF signaling system in the prevention of in utero meconium
passage before term.
Sheep fetal colon is a glucocorticoid target organ
As the incidence of meconium-stained amniotic fluid increases
with increase in gestational ages and fetal stress, we hypothesized
that fetal colon could be a glucocorticoid target organ with fetal
colonic motility function regulated by glucocorticoids.27 We
examined in vivo effects of betamethasone (a synthetic
glucocorticoid) in the presence and absence of thyroxine on
in vitro colonic smooth muscle cholinergic contractility.27 Muscle
strips prepared from sheep fetuses that received intramuscular
injection of betamethasone alone or in combination with thyroxine
exhibited greater contractile responsiveness to bethanechol
(a cholinergic agonist) than muscle strips prepared from
vehicle-treated fetuses. These glucocorticoid-specific changes were
noticed 48 h after intramuscular injections of betamethasone,
suggesting that glucocorticoids regulate cholinergic contractile
maturation through ‘genomic’ actions. The findings obtained
following glucocorticoid administration has provided partial
support for our hypothesis that stress-mediated glucocorticoid
release may stimulate colonic motility and meconium passage.
We also examined the in vivo effect of bethanechol on colonic
muscular contractile and electromyogram activity in the sheep
fetus.28 We noticed increased strain gauze activity in response to
bethanechol infusion in transverse but not in distal colon at 0.9
gestation (135 days gestation, term ¼ 150 days). This finding
suggested the presence of a local inhibitor system for
cholinergic-dependent contractility in the distal colon before term,
perhaps serving as an intrinsic mechanism to prevent premature
meconium passage.
Localization and gestation-dependent changes in glucocorticoid
receptor expression in sheep fetal distal colon
To validate our hypothesis that sheep fetal colon is glucocorticoid
target organ, we undertook studies to evaluate the cellular

Journal of Perinatology

localization and gestation-dependent changes in glucocorticoid
receptors in sheep fetuses at very preterm (118–120 days), preterm
(130–132 days), near term (140–142 days) and term (146–147
days) gestation.30 Glucocorticoid receptor antibody elicited positive
staining in smooth muscle layers as well as in myenteric and
submucosal neurons at all stages of development with maximal
expression noticed at near term, suggesting the profound changes
in nuclear transcription machineries begin to occur at near term.
We also noticed a robust change in fetal plasma cortisol levels at
term, suggesting that the expression of glucocorticoid receptors
could be turned ‘off’ by glucocorticoids at term, or immediately
after birth, as circulating levels of glucocorticoids reach basal levels
within 24 h after birth in many species including humans.
Evidence for prereceptor metabolism of glucocorticoids
in fetal sheep distal colon
Glucocorticoid actions at the cellular level are controlled by 11-beta
hydroxysteroid hydrogenase (11bHSD) type 1 and type 2, enzymes
that regulate the interconversion of active glucocorticoid and their
inactive 11-keto metabolites. We recently examined the presence of
11 bHSD-1 and 11 bHSD-2 in sheep fetal colonic enteric nervous
system as evidence that these enzymes provide a novel means for
regulation of glucocorticoid-mediated ‘genomic’ activities.31 The
antibodies to 11bHSD-1 and 11 bHSD-2 elicited positive
immunostaining of both myenteric and submucosal ganglia at all
gestation ages. The percentage of myenteric ganglia expressing
11bHSD-1 and 11bHSD-2 were maximal at term. The percentage of
submucosal ganglia expressing 11bHSD-1 was maximal at term,
whereas the expression of 11bHSD-2 remained constant throughout
gestation. We speculate that increasing levels of 11bHSD-1, and thus
increased active glucocorticoid levels, at term could contribute to
glucocorticoid-dependent colonic contractile maturation.
CRF system
Identification and gestation-dependent changes in CRF
system in sheep fetal distal colon
We recently undertook a systematic investigation to obtain
anatomical support that CRF system is an integral component of
distal colonic contractility apparatus.32 Our findings can be
summarized as follows: CRF-R1- and CRF-R2-specific antibodies
strongly immunostained longitudinal and circular smooth muscles
in addition to muscularis mucosa in colonic segments at all
gestational ages. A marked decrease in CRF-R2 immunostaining
occurred in smooth muscle layers close to term. CRF-R1 antibody
immunostained neuronal somas both in myenteric and
submucosal ganglia, and the percentage of ganglia expressing
CRF-R1 were maximal at term gestation. On the basis of the
reciprocal changes in CRF-R1 and CRF-R2 expression in the
smooth muscle-enteric unit, we speculate that CRF-R1 could be
hyper-responsive to stressors that stimulate the release of
Mechanism of in utero meconium passage
J Lakshmanan and MG Ross
S11
endogenous CRF leading to increased motor activity and
meconium passage.
Cellular localization of CRF-binding protein in sheep
distal colon
Corticotrophin-releasing factor-binding protein is a protein known
to compete with CRF-R1 and CRF-R2 for CRF and urocortin.33 Our
immunohistochemical investigation revealed expression of
CRF-binding protein in the smooth muscle-enteric unit.34 We
observed a marked decrease in smooth muscle expression of
CRF-binding protein and a simultaneous increase in myenteric
neuronal somas at term, indicating that this protein is a critical
regulator of CRF and urocortin functions at the level of smooth
muscle-enteric unit in sheep fetal distal colon.
CRF signaling system may play a role in preventing
in utero meconium passage before term
We performed in vitro study of sheep fetal colonic contractility to
assess the significance of CRF receptor system.29 In these studies,
colonic smooth muscle strips prepared from preterm sheep fetal
distal colonic segments were subjected to cholinergically dependent
contractility in an organ bath system following pre-incubation with
CRF or urocortin. Both neuropeptides significantly decreased the
cholinergically stimulated contractility. Immunohistochemical
studies confirmed the expression of muscarinic receptor subtype
3 and CRF-R2 in preterm sheep fetal colon. Additional studies
revealed positive immunostaining for CRF and urocortin in smooth
muscle and enteric neurons, indicating the local availability of
neuropeptides to inhibit smooth muscle motility during the time of
stress.29 All in all, stress hormones (CRF and urocortin) and stress
hormone receptors (CRF-R1 and CRF-R2) appear to function in
parallel or series pathways, and CRF-R2 plays a central role in
preventing stress-induced in utero meconium passage before term.
Cholinergic circuitry system
Maturational changes in sheep fetal colonic cholinergic circuitry
system parallels plasma glucocorticoid surge
We utilized antibodies specific to vesicular acetylcholine
transporter, a high-affinity choline transporter, and peripheral
choline acetyl transferase as markers for cholinergic synaptic
circuitry system to examine the maturation of cholinergic system
in distal colon of sheep fetuses from very preterm to term
gestation.35 The percentage of myenteric ganglia expressing
cholinergic markers in term fetuses were significantly higher than
those observed in preterm. The marked increase observed in
cholinergic markers in myenteric ganglia in fetal distal colon at
term is possibly mediated by effects of endogenous glucocorticoid
levels, which peak at term. We speculate that glucocorticoid is the
physiological hormone that regulates enteric cholinergic neural
synaptic circuitry system similar to their actions in central nervous
system regions.
Journal of Perinatology
 Mechanism of in utero meconium passage
J Lakshmanan and MG Ross
S12
Localization of inhibitory and stimulatory muscarinic
receptor subtypes in sheep fetal distal colon: implication
for meconium passage
Acetylcholine mediates gastrointestinal motility through muscarinic
receptors. To date, five muscarinic receptors (M1–M5) have been
cloned, with M3 confirmed as the prominent mediator of smooth
muscle contraction. Of the other subtypes, M1, M2 and M4 have
been shown to function as potent autoinhibitor (that is, inhibitory
presynaptic muscarinic receptor) of Ach release from myenteric
plexus. We examined the maturation and topographical
distribution of muscarinic receptor subtypes in distal colon from
very preterm to term.36 M1–M5 antibodies elicited positive staining
in colonic sections at all gestational ages. M4 is expressed most
abundantly in circular and longitudinal smooth muscle layers,
whereas M3 is expressed in rather low levels. Positive
immunostaining for both M5 and M1 in smooth muscle layers are
similar, but their immunostaining intensity was significantly lower
compared with M3, M4 and M2. The percentage of neurons
expressing inhibitory autoreceptors (M1, M2 and M4) in the enteric
ganglia was markedly higher in distal colonic sections of preterm
and near-term fetuses, with expression declining rapidly and
markedly at term. Our results indicate that before term, muscarinic
autoreceptors may inhibit presynaptic release of Ach and
consequently limit the cholinergically mediated smooth muscle
contractility. This could represent a physiological mechanism
analogous to CRF-R2 functions in preventing stress-induced
in utero meconium passage before term. The rapid decline of
muscarinic inhibitory autoreceptors noticed at term most
likely relieves the colonic contractile machinery from
auto-inhibition and will increase efficacy of cholinergic synaptic
transmission, facilitating ready responsiveness of colonic contractile
machinery to Ach.
In summary, we have established for the first time a rat model
for stress-induced in utero meconium passage. We have further
obtained anatomical support for the expression of key molecules of
glucocorticoid system, CRF system and cholinergic circuitry system
in sheep fetal distal colon. All three systems seem positioned
in distal colon readily to respond to stress. Our studies unexpectedly
revealed the presence of potential mechanisms to prevent the
in utero meconium passage before term, including the
expression of CRF-R2. Future studies should improve our
understanding of the mechanisms of in utero meconium passage
and possibly to develop pharmacotherapy to prevent stress-induced
in utero meconium passage and consequences of meconium
aspiration.
Acknowledgments
This research was supported by grants from the March of Dimes Foundation
(MGR) and the National Institutes of Health (JL).
Journal of Perinatology
Disclosure
Jayaraman Lakshmanan has received grant funding from NIH R03. Michael Ross
has received grant funding from the March of Dimes.
References
1 Momoh JT. Hirschsprung’s disease: problems of diagnosis and treatment. Ann Trop
Paediatr 1982; 2: 31–35.
2 Wiswell TE, Tuggle JM, Turner BS. Meconium aspiration syndrome: have we made a
difference? [see comments]. Pediatrics 1990; 85: 715–721.
3 Ahanya SN, Lakshmanan J, Morgan BL, Ross MG. Meconium passage in utero:
mechanisms, consequences, and management. Obstet Gynecol Surv 2005; 60:
45–56.
4 Usher RH, Boyd ME, McLean FH, Kramer MS. Assessment of fetal risk in postdate
pregnancies. Am J Obstet Gynecol 1988; 158: 259–264.
5 Miller FC, Read JA. Intrapartum assessment of the postdate fetus. Am J Obstet Gynecol
1981; 141: 516–520.
6 Richey SD, Ramin SM, Bawdon RE, Roberts SW, Dax J, Roberts J et al. Markers of
acute and chronic asphyxia in infants with meconium-stained amniotic fluid.
Am J Obstet Gynecol 1995; 172: 1212–1215.
7 Tache Y, Bonaz B. Corticotropin-releasing factor receptors and stress-related alterations
of gut motor function. J Clin Invest 2007; 117: 33–40.
8 Tache Y, Martinez V, Million M, Rivier J. Corticotropin-releasing factor and the brain-
gut motor response to stress. Can J Gastroenterol 1999; 13(Suppl A): 18A–25A.
9 Martinez V, Wang L, Rivier J, Grigoriadis D, Tache Y. Central CRF, urocortins and
stress increase colonic transit via CRF1 receptors while activation of CRF2 receptors
delays gastric transit in mice. J Physiol 2004; 556: 221–234.
10 Million M, Wang L, Martinez V, Tache Y. Differential Fos expression in the
paraventricular nucleus of the hypothalamus, sacral parasympathetic nucleus and
colonic motor response to water avoidance stress in Fischer and Lewis rats. Brain Res
2000; 877: 345–353.
11 Williams CL, Villar RG, Peterson JM, Burks TF. Stress-induced changes in intestinal
transit in the rat: a model for irritable bowel syndrome. Gastroenterology 1988; 94:
611–621.
12 Miampamba M, Sharkey KA. c-Fos expression in the myenteric plexus, spinal cord and
brainstem following injection of formalin in the rat colonic wall. J Auton Nerv Syst
1999; 77: 140–151.
13 la Fleur SE, Wick EC, Idumalla PS, Grady EF, Bhargava A. Role of peripheral
corticotropin-releasing factor and urocortin II in intestinal inflammation and motility
in terminal ileum. Proc Natl Acad Sci USA 2005; 102: 7647–7652.
14 Hillhouse EW, Grammatopoulos DK. The molecular mechanisms underlying the
regulation of the biological activity of corticotropin-releasing hormone receptors:
implications for physiology and pathophysiology. Endocr Rev 2006; 27: 260–286.
15 Maillot C, Million M, Wei JY, Gauthier A, Tache Y. Peripheral corticotropin-releasing
factor and stress-stimulated colonic motor activity involve type 1 receptor in rats.
Gastroenterology 2000; 119: 1569–1579.
16 Grundy D, Al-Chaer ED, Aziz Q, Collins SM, Ke M, Tache Y et al. Fundamentals of
neurogastroenterology: basic science. Gastroenterology 2006; 130: 1391–1411.
17 Jones SA, Brooks AN, Challis JR. Steroids modulate corticotropin-releasing hormone
production in human fetal membranes and placenta. J Clin Endocrinol Metab 1989;
68: 825–830.
18 Bruder ED, Jacobson L, Raff H. Plasma leptin and ghrelin in the neonatal rat:
interaction of dexamethasone and hypoxia. J Endocrinol 2005; 185: 477–484.
19 Takahashi LK, Goh CS. Glucocorticoid facilitation of cholinergic development in the rat
hippocampus. Neurosci 1998; 83: 1145–1153.
20 Coukos G, Monzani A, Saletti C, Petraglia F. [Noradrenaline and interleukin-1
stimulate CRF secretion from human placental cells in culture]. Medicina (Firenze)
1988; 8: 441–442.

21 Lakshmanan J, Ahanya SN, Rehan V, Oyachi N, Ross MG. Elevated plasma
corticotrophin release factor levels and in utero meconium passage. Pediatr Res 2007;
61: 176–179.
22 Lakshmanan J, Salido E, Amidi F, Amidi E, Raj R, Ross MG. Rat placenta expresses
corticotrophin releasing factor protein and mRNA. Reproductive Sciences 2007;
14(1 Suppl): 175A.
23 Frim DM, Emanuel RL, Robinson BG, Smas CM, Adler GK, Majzoub JA.
Characterization and gestational regulation of corticotropin-releasing hormone
messenger RNA in human placenta. J Clin Invest 1988; 82: 287–292.
24 Lakshmanan J, Richard JD, Liu GL, Ross MG. Corticotrophin releasing factor is a fetal
gut hormone. Reprod Sci 2007; 14(1 Suppl): 251A.
25 Kawahito Y, Sano H, Kawata M, Yuri K, Mukai S, Yamamura Y et al. Local secretion of
corticotropin-releasing hormone by enterochromaffin cells in human colon.
Gastroenterology 1994; 106: 859–865.
26 Richard JD, Lakshmanan J, John TA, Ross MG. Rat fetal gastrointestinal tract is a target
organ for corticotrophin-releasing factor family neuropeptides. Am J Obstet Gyn 2006;
195(6 Suppl): S214.
27 Ross B, Bradley K, Nijland MJ, Polk DH, Ross MG. Increased fetal colonic muscle
contractility following glucocorticoid and thyroxine therapy: implications for
meconium passage. J Matern Fetal Med 1997; 6: 129–133.
28 Acosta R, Oyachi N, Lee JJ, Lakshmanan J, Atkinson JB, Ross MG.
Mechanisms of meconium passage: cholinergic stimulation of
electromechanical coordination in the fetal colon. J Soc Gynecol Investig
2005; 12: 169–173.
Mechanism of in utero meconium passage
J Lakshmanan and MG Ross
S13
29 Lakshmanan J, Oyachi N, Ahanya SA, Liu G, Mazdak M, Ross MG.
Corticotropin-releasing factor inhibition of sheep fetal colonic contractility: mechanisms
to prevent meconium passage in utero. Am J Obstet Gynecol 2007; 196: 357.
30 Lakshmanan J, Oyachi N, Liu GL, Choi GY, Ross MG. Fetal colonic enteric nervous
system is a site of glucocorticoid-induced gastrointestinal maturation. Reprod Sci 2007;
14(1 Suppl): 287A.
31 Lakshmanan J, Liu GL, Oyachi N, Ross MG. Evidence for pre-receptor metabolism of
glucocorticoids in ovinee fetal distal colonic enteric nervous system. Reprod Sci 2007;
14(1 Suppl): 288A.
32 Lakshmanan J, Liu GL, Oyachi N, Ross MG. Localization and gestation-dependent
pattern of CRF-receptos (R1, R2) expression in ovine fetal distal colon. Reprod Sci
2007; 14(1 Suppl): 258A.
33 Seasholtz AF, Valverde RA, Denver RJ. Corticotropin-releasing hormone-binding
protein: biochemistry and function from fishes to mammals. J Endocrinol 2002; 175:
89–97.
34 Lakshmanan J, Liu GL, Oyachi N, Ross MG. Cellular localization of corticotrophin
releasing factor binding protein (CRF-BP) in fetal ovine distal colon: a possible local
inhibitor of stress-induced colonic motility. Reprod Sci 2007; 14(1 Suppl): 288A.
35 Lakshmanan J, Lips KS, Liu GL, Ross MG. Maturational changes in ovine fetal colonic
cholinergic circuitry parallels plasma glucocorticoid surge. Reprod Sci 2007;
14(1 Suppl): 249A.
36 Lakshmanan J, Liu GL, Ross MG. Localization of inhibitory and stimulatory muscarinic
receptor subtypes in ovine fetal distal colon: implications for meconium passage.
Reprod Sci 2007; 14(1 Suppl): 168A.
Journal of Perinatology
 Journal of Perinatology (2008) 28, S14–S18
r 2008 Nature Publishing Group All rights reserved. 0743-8346/08 $30
www.nature.com/jp

REVIEW
Obstetric approaches to the prevention of meconium aspiration
syndrome
H Xu, S Wei and WD Fraser
Department of Obstetrics and Gynecology, Hoˆpital Sainte-Justine, Universite´ de Montre´al, Montreal, QC, Canada

Definition of meconium aspiration syndrome

Meconium aspiration syndrome (MAS) is associated with increased
risk for perinatal mortality and morbidities. To provide an overview of the
advances in our knowledge concerning the obstetric approaches to the
prevention of MAS. The evidence of the effectiveness of intrapartum
surveillance, amnioinfusion, and delivery room management in the
prevention of MAS are reviewed in the present paper. Meconium aspiration
syndrome remains one of the most common but challenging conditions
for obstetricians and pediatricians. The available evidence did not
demonstrate a beneficial effect of either of obstetric strategies in the
prevention of MAS.
Journal of Perinatology (2008) 28, S14–S18; doi:10.1038/jp.2008.145
Introduction
Meconium staining of the amniotic fluid (MSAF) occurs in <5% of
preterm, 7 to 22% of term deliveries, increasing to between 23 and
52% of births at >42 weeks.1–7 The mechanisms influencing
meconium passage are complex, involving hormonal and
neuroregulatory functions, chronic hypoxia or reflecting
maturation of the fetal gastrointestinal system.8–11 MSAF is
associated with an increased risk of neonatal morbidity and
mortality. Meconium aspiration syndrome (MAS) is the most
serious neonatal pathology associated with MSAF. It has been
reported to occur in 1.7 to 35.8% of cases complicated with MSAF,
and 1 to 3% of liveborn infants.1,12–14 The case fatality rate of MAS
has been reported to be high, ranging from 5 to 40%.1,13–17
Approximately one-third of babies with MAS require intubation and
mechanical ventilation, and other new therapies such as high
frequency ventilation, inhaled nitric oxide and surfactant
administration, although the effectiveness of certain of these
technologies remain controversial.2,17–19 Serious complications
resulting from MAS include pneumothorax, convulsion
and death.
Correspondence: Dr WD Fraser, Department of Obstetrics and Gynecology, Hôpital Sainte-
Justine, Université de Montréal, 3175 Chemin de la Côte Sainte-Catherine, Montreal, QC,
Canada H3T 1C5.
E-mail: william.fraser@umontreal.ca
Rossi et al.20 has defined MAS as respiratory distress in the first 4 h
after birth with oxygen requirement and chest roentgenogram
showing characteristic features of MAS. Recently, Cleary and
Wiswell have defined MAS as respiratory distress in an infant born
through MSAF, which cannot be otherwise explained. Mild MAS has
been defined as requiring <40% oxygen for <48 h, moderate MAS
as requiring >40% concentration of oxygen therapy for at least
48 h, and severe MAS if requiring assisted mechanical ventilation,
that is, often associated with persistent pulmonary hypertension.1,20
They further pointed out that the severity of MAS does not
necessarily correspond to the degree of chest radiographic
abnormality. MAS is associated with a range of radiographic
features including coarse, patchy infiltrates, consolidation,
atelectasis, pleural effusions, air leaks, hyperinflation, a wet-lung
picture and hypovascularity.1 In some cases, the chest film may be
interpreted as normal.1
Pathophysiology and risk factors
The pathophysiology of MAS is complex and remains controversial.
Many factors, such as airway obstruction, alveolar or parenchymal
inflammation, impaired surfactant production and function and
direct toxicity of meconium constituents could be involved in the
pathophysiology of the MAS.1,21,22 It has been suggested that the
extent of lung destruction is not closely correlated to the quantity of
meconium in lung tissue but rather to the degree of hypoxia and
acidosis present at delivery.23 Ghidini and Spong22 postulated that
the pathologic events leading to mild, moderate or severe cases of
MAS may be different. Severe MAS may not be in fact causally
related to the aspiration of meconium but rather may be caused by
other pathologic processes occurring in utero, such as chronic
asphyxia, infection or persistent pulmonary hypertension.22 The
hypothesis is in fact supported by the lack of evidence that the
severity of MAS directly correlates with the amount of meconium
aspirated, the consistency of meconium and the duration of
exposure to meconium.1,15,20,22,23 It is still unclear whether
obstruction of airways because of aspiration of meconium has a
pivotal role in the progress of MAS. MAS can occur before delivery,

even in the absence of labor, being reported in infants delivered by
elective cesarean section.24
Although the presence of meconium during labor is known to
be associated with an increased risk of perinatal morbidity and
mortality, most babies have favorable outcomes. Early recognition
of infants at the highest risk for the development of MAS could be
essential for optimizing the clinical preventive strategies. A vast
array of risk factors for the occurrence of MAS have been identified
either using unselected obstetrical populations or infants born
through MSAF. Those factors are heavy MSAF, nulliparity, postterm
delivery, fetal heart rate (FHR) abnormalities during labor,
presence of meconium below the vocal cords, cesarean delivery and
the low Apgar scores.13,14,20,25–30 It has been reported that there is
an apparent relationship between maternal ethnicity and risk of
MSAF, and the risk of MAS having been observed to be increased in
black Americans, Africans and Pacific Islanders.30–32
Intrapartum fetal monitoring
The goal of continuous electronic fetal heart rate monitoring
(EFM) is to detect fetal hypoxemia and therefore reduce the risk of
adverse neonatal outcomes. However, the effectiveness of this
approach to care has been questioned. Randomized trials of EFM,
with or without fetal blood gas and acid–base assessment, which
were conducted in the unselected obstetrical population, have
found no evidence that this approach to care reduces the risk of
fetal or neonatal mortality or morbidity.33–39
Intrapartum monitoring has been recommended to screen for
early signs of fetal hypoxia, a risk factor for MAS. Several authors
have noted an increase in the frequency of FHR abnormalities in
association with MSAF.15,20,40,41 It has been reported that, in the
presence of MSAF, fetal tachycardia, variable and late decelerations
and decreased long-term variability are risk factors for MAS.
Certain authors investigated the relationships among abnormal
cardiotocograms in labor, MSAF and adverse neonatal outcomes
such as low arterial cord blood pH, and low Apgar scores. The
authors did not find that the presence of abnormal FHR patterns
increased the overall correlation between MSAF and adverse
outcome.7,42 In contrast, Umstad et al.43 investigated the predictive
value of abnormal FHR patterns in early labor and found that the
presence of meconium in the amniotic fluid improved the
predictive properties of the test.
Amnioinfusion
Amnioinfusion (AI), or transcervical infusion of saline into the
amniotic cavity, was used first to relieve persistent variable FHR
decelerations during labor or to prevent the occurrence of
decelerations in presence of oligohydramnios.44 Results of
randomized controlled trials, including a meta-analysis indicate
that, in the presence of oligohydramnios, prophylactic intrapartum
Obstetric management of MAS
H Xu et al
S15
AI significantly reduces the risk of FHR decelerations and cesarean
section.45–52
AI has been also proposed as a method to reduce MAS. Potential
mechanisms through which AI could act include mechanical
cushioning of the umbilical cord, which could correct or prevent
recurrent umbilical compressions that lead to fetal acidemia, a
condition predisposing to MAS; and dilution of meconium that
could reduce its mechanical and inflammatory effects in the
pathogenesis of MAS.
To date, more than 15 randomized or quasi-randomized trials
of AI for MSAF have been reported, with conflicting results.53,54 The
methodological quality varied across studies. The largest trial (over
1998 participants) was an international trial performed in 56
centers where EFM and neonatal intubation and suctioning for
babies with respiratory difficulty were routinely available.55 Analysis
was by intention to treat. The primary outcome was a composite
indicator that included the occurrence of perinatal death and/or
moderate or severe MAS. The results indicated that AI showed no
effect on the primary outcome (relative risk; RR 1.26, 95%
confidence interval; CI 0.82 to 1.95). Furthermore, the frequencies
of oropharyngeal suctioning, laryngoscopy or intubation in the
delivery room were similar between groups, as were the proportion
of babies with meconium visualized below the vocal cords. There
were no differences between groups in the occurrence of the
combined outcome of perinatal mortality and/or serious morbidity
(RR 1.13, 95% CI 0.88 to 1.47). In addition, an analysis that
stratified for the presence or absence of variable FHR decelerations
before randomization found no effect on the primary outcome
either, although the study was underpowered to detect such effects
within strata.
We recently conducted a systematic review, integrating the
results of the largest trial.54 Studies were included if they were
randomized controlled trials that evaluated the effect of
prophylactic AI during labor with MSAF; treatment was randomly
allocated (AI versus controls). All included studies were further
subjected to a score-based quality assessment for randomized
studies that was adapted from the Jadad score. The main analysis
was based on the studies that were considered to be of high quality.
The meta-analysis included a total of 4030 women, 1999 allocated
to AI and 2031 allocated to control. Of these, 3178 women were
recruited in clinical settings with standard peripartum surveillance
and 852 women were randomized in centers with limited
peripartum surveillance, defined as the nonavailability of EFM
during labor. The methodological quality varied across studies.
Heterogeneity was noted across studies with respect to the AI
protocols and the end points evaluated. In the setting of standard
peripartum surveillance, the results failed to demonstrate a
reduction in the risk of MAS (RR 0.59, 95% CI 0.28 to 1.25),
Apgar-5 <7 (RR 0.90, 95% CI 0.58 to 1.41) or caesarean delivery
(RR 0.89, 95% CI 0.73 to 1.10). However, in clinical settings with
limited peripartum surveillance, AI appeared to reduce the risk of
Journal of Perinatology
 Obstetric management of MAS
H Xu et al
S16
MAS (RR 0.25, 95% CI 0.13 to 0.47). Several findings from this
meta-analysis are worthy of comment. Firstly, the observed
heterogeneity in the stratum of studies conducted in centers with
standard surveillance is largely attributable to our recent large
trial. The source of this heterogeneity remains largely unexplained.
The most significant finding of the meta-analysis is the apparent
discordance in the observed effect of AI on MAS between centers
with standard and limited peripartum surveillance. Continuous
EFM is a key component in the prevention of asphyxia in patients
with MSAF. Therefore, the application of this technology may
reduce the contribution of severe asphyxia to the risk of occurrence
of MAS. It would appear that in settings where this technology is
routinely used, AI confers no additional benefit over EFM in terms
of prevention of MAS. However, in settings where continuous EFM
is not routinely available, AI may be beneficial for the reduction of
MAS. Further studies in such settings are warranted to confirm this
hypothesis.
AI may not be without risk. The use of AI has been reported to
be associated with adverse events. Complications including uterine
overdistension and hypertonia, uterine rupture in association with
previous uterine scar, FHR abnormality, umbilical cord prolapse
and chorioamnionitis have been reported.56–59 Four cases of
maternal deaths have been reported associated with the AI.58,59
Several authors have reported the occurrence of excessive uterine
contractions or unusually rapid labor progress related to AI. In the
AI group of Fraser et al.’s55 trial, 10 women (1.1%) experienced
bleeding, and in 63 (6.9%) women, hypertonicity, hydramnios or
uterine overdistension was diagnosed during the procedure whereas
the incidence of other maternal complications were comparable
between AI and control groups.
American College of Obstetricians and Gynecologists has
recently published a Committee Opinion that concludes that
routine prophylactic AI for the dilution of MSAF should be carried
out only in the setting of additional clinical trials. However, they
state that AI remains a reasonable approach to the treatment of
repetitive variable decelerations, regardless of amniotic fluid
meconium status.60
Delivery room management
Routine oropharyngeal suctioning before delivery of the infants’
shoulders has long been involved in preventing MAS. The findings
of observational studies remain conflicting.20,26,61 –63 Falciglia
et al.26 compared infants with meconium-stained fluid who
underwent ‘early’ oronasopharyngeal DeLee suctioning with a
similar group of infants whose airways were suctioned ‘late’ (after
chest delivery). They found no evidence of benefit of oropharyngeal
suctioning in the prevention of MAS. Rossi et al.20 also reported the
similar rates of meconium visualized in the vocal cords despite
early oropharyngeal suctioning. In contrast, several authors
reported that intrapartum pharyngeal suctioning reduced the
Journal of Perinatology
severity of MAS and the risk of respiratory distress.61–63 They
suggested that combined approach of intrapartum oropharyngeal
suctioning and endotracheal suctioning was effective in the
reduction of MAS.
Vain et al. conducted a multicenter international trial to assess
the effectiveness of oropharyngeal and nasopharyngeal suctioning
before delivery of the shoulders for the prevention of MAS. They
found that the incidence of MAS, need for mechanical ventilation,
and neonatal mortality was similar between groups (suction versus
no suction). In addition, they found no evidence of a benefit of
intrapartum suctioning on the occurrence of MAS, MAS requiring
mechanical ventilation, or mortality.64
Regarding the postdelivery management such as routine
endotracheal suctioning and intubation, reports from observational
studies suggested that intratracheal suctioning could prevent the
occurrence of MAS for meconium-stained neonates and significantly
decreased the mortality subsequent to that disorder.65–67 Intubation
is not without risk and has been associated with hypoxia,
bradycardia and laryngeal stridor.67–70 Should endotracheal
suctioning and intubation be applied universally in infants born
through MSAF or selectively reserved for those who are depressed
after birth is another topic of controversy. Some investigators
suggested that a selective approach may be useful and
justified,6,8,71–73 whereas other suggested that universal intubation
and suctioning was the best strategy to prevent potential morbidity
and mortality related to meconium staining.67
Linder et al.68 suggested that nondepressed meconium-stained
infants did not benefit from immediate intratracheal suctioning
and such intervention could be harmful. Liu and Harrington
conducted a randomized trial to assess if intubation of the low-risk
newborn with thin meconium affects the incidence of respiratory
symptoms. They were unable to demonstrate the beneficial effect of
intubation and intratracheal suctioning in the infant with thin
meconium and an otherwise low-risk pregnancy.74 To address this
question, Wiswell et al.69 conducted a multicenter randomized
trial, involving a total of 2094 neonates, to investigate
whether intubation and suctioning of apparently vigorous,
meconium-stained neonates would reduce the risk of MAS. There
were no significant differences between intubation and expectant
management groups in the rates of MAS or other respiratory
disorders. Moreover, intratracheal suctioning showed no benefit
over expectant management even for infants born through the
thickest consistency MSAF. They further identified the independent
risk factors for the development of MAS using stepwise logistic
regression. The results indicated that the use of oropharyngeal
suctioning lead to a decreased risk of MAS (8.5% in infants who did
not have intrapartum suction versus 2.7% in infants with
intrapartum suctioning). The authors concluded that endotracheal
intubation and suctioning still be performed in infants born
through MSAF, if they are not vigorous, if they need positive
pressure ventilation or they develop symptoms of respiratory

distress. Furthermore, a recently published meta-analysis of four
randomized trials demonstrated no significant benefit of routine
endotracheal intubation and suctioning at birth over routine
resuscitation including oropharyngeal suction of vigorous,
meconium-stained infants born at term.75 The authors
recommended that intubation and suctioning be restricted to
depressed newborns, that is, those with a heart rate of <100 beats
per min, poor respiratory effort and poor tone.
Conclusion
MAS remains a challenging condition for obstetricians and
neonatologists. Despite the decreased risk of MAS and related
mortality and morbidity, the available evidence did not
demonstrate a beneficial effect of either of obstetric strategies in the
prevention of MAS. The suggested apparent disparity in the effect of
AI on MAS between centers with standard and limited peripartum
surveillance is worthy of attentions for clinicians. Additional
well-designed randomized controlled trials in settings of limited
peripartum surveillance are required to elucidate the optimal
management of MAS in this context.
Disclosure
WD Fraser has received grant support from Utah Medical Corporation. The
remaining authors have declared no financial interests.
References
1 Cleary GM, Wiswell TE. Meconium-stained amniotic fluid and the meconium
aspiration syndrome: an update. Pediatr Clin North Am 1998; 45: 511–529.
2 Wiswell TE, Tuggle JM, Turner BS. Meconium aspiration syndrome: have we made a
difference? Pediatrics 1990; 85: 715–721.
3 Scott H, Walker M, Gruslin A. Significance of meconium-stained amniotic fluid in the
preterm population. J Perinatol 2001; 21: 174–177.
4 Usher RH, Boyd ME, McLean FH, Kramer MS. Assessment of fetal risk in postdate
pregnancies. Am J Obstet Gynecol 1988; 158: 259–264.
5 Ostrea EM, Naqvi M. The influence of gestational age on the ability of the fetus to pass
meconium in utero. Acta Obstet Gynecol Scand 1982; 61: 275–277.
6 Eden RD, Seifert LS, Winegar A, Spellacy W. Perinatal characteristics of uncomplicated
postdate pregnancies. Obstet Gynecol 1987; 69: 296–299.
7 Steer PJ, Eigbe F, Lissauer TJ, Beard RW. Interrelationships among abnormal
cardiotocograms in labor, meconium staining of the amniotic fluid, arterial cord blood
pH, and Apgar scores. Obstet Gynecol 1989; 74: 715–721.
8 Lucas A, Adrian TE, Christofides N, Bloom SR, Aynsley-Green A. Plasma motilin,
gastrin and enteroglucagon and feeding in the human newborn. Arch Dis Child 1980;
55: 673–677.
9 Miller FC, Sacks DA, Yeh SY, Paul RH, Schifrin BS, Martin Jr CB et al. Significance of
meconium during labor. Am J Obstet Gynecol 1975; 122: 573–580.
10 Bochner CJ, Medearis AL, Ross MG, Oakes GK, Jones P, Hobel CJ et al. The role of
antepartum testing in the management of postterm pregnancies with heavy meconium
in early labor. Obstet Gynecol 1987; 69: 903–907.
11 Ahanya SN, Lakshmanan J, Morgan BL, Ross MG. Meconium passage in utero:
mechanisms, consequences, and management. Obstet Gynecol Surv 2005; 60: 45–56.
12 Brown BL, Gleicher N. Intrauterine meconium aspiration. Obstet Gynecol 1981; 57:
26–29.
Obstetric management of MAS
H Xu et al
S17
13 Davis RO, Phillips III JB, Harris Jr BA, Wilson ER, Huddleston JF. Fatal meconium
aspiration syndrome occurring despite airway management considered appropriate.
Am J Obstet Gynecol 1985; 151: 731–736.
14 Urbaniak KJ, McCowan LM, Townend KM. Risk factors for meconium aspiration
syndrome. Aust N Z J Obstet Gynaecol 1996; 36: 401–406.
15 Hernandez C, Little BB, Dax JS, Gilstrap III LC, Rosenfeld CR. Prediction of the severity
of meconium aspiration syndrome. Am J Obstet Gynecol 1993; 169: 61–70.
16 Falciglia HS. Failure to prevent meconium aspiration syndrome. Obstet Gynecol 1988;
71: 349–353.
17 Coltart TM, Byrne DL, Bates SA. Meconium aspiration syndrome: a 6-year retrospective
study. Br J Obstet Gynaecol 1989; 96: 411–414.
18 Bhutani VK, Chima R, Sivieri EM. Innovative neonatal ventilation and meconium
aspiration syndrome. Indian J Pediatr 2003; 70: 421–427.
19 Wiswell TE. Advances in the treatment of the meconium aspiration syndrome. Acta
Paediatr Suppl 2001; 90: 28–30.
20 Rossi EM, Philipson EH, Williams TG, Kalhan SC. Meconium aspiration
syndrome: intrapartum and neonatal attributes. Am J Obstet Gynecol 1989; 161:
1106–1110.
21 Katz VL, Bowes WA. Meconium aspiration syndrome: reflections on a murky subject.
Am J Obstet Gynecol 1992; 166: 171–183.
22 Ghidini A, Spong CY. Severe meconium aspiration syndrome is not caused by
aspiration of meconium. Am J Obsted Gynecol 2001; 185: 931–938.
23 Jovanovic R, Nguyen HT. Experimental meconium aspiration in guinea pigs. Obstet
Gynecol 1989; 73: 652–656.
24 Greenough A. Meconium aspiration syndromeFprevention and treatment. Early
Hum Dev 1995; 41: 183–192.
25 Meis PJ, Hall III M, Marshall JR, Hobel CJ. Meconium passage: a new classification for
risk assessment during labor. Am J Obstet Gynecol 1987; 131: 509–513.
26 Falciglia HS, Henderschott C, Potter P, Helmchen R. Does DeLee suction at the
perineum prevent meconium aspiration syndrome? Am J Obstet Gynecol 1992; 167:
1243–1249.
27 Alexander GR, Hulsey TC, Robillard PY, De Caunes F, Papiernik E. Determinants of
meconium stained amniotic fluid in term pregnancies. J Perinatol 1994; 14: 259–263.
28 Meydanli MM, Dilbaz B, Caliskan E, Dilbaz S, Haberal A. Risk factors for meconium
aspiration syndrome in infants born through thick meconium. Int J Gynaecol Obstet
2001; 72: 9–15.
29 Paz Y, Solt I, Zimmer EZ. Variables associated with meconium aspiration syndrome in
labours with thick meconium. Eur J Obstet Gynecol Reprod Biol 2001; 94:
27–30.
30 Dargaville PA, Copnell B, Australian and New Zealand Neonatal Network. The
epidemiology of meconium aspiration syndrome: incidence, risk factors, therapies, and
outcome. Pediatrics 2006; 117: 1712–1721.
31 Sriram S, Wall SN, Khoshnood B, Singh JK, Hsieh HL, Lee KS. Racial disparity in
meconium stained amniotic fluid and meconium aspiration syndrome in the United
States, 1989 to 2000. Obstet Gynecol 2003; 102: 1262–1268.
32 Sedaghatian MR, Othman L, Hossain MM, Vidyasagar D. Risk of meconium stained
amniotic fluid in different ethnic groups. J Perinatol 2000; 20: 257–261.
33 Shy KK, Larson EB, Luthy DA. Evaluating a new technology: the effectiveness of
electronic fetal heart rate monitoring. Annu Rev Public Health 1987; 8: 165–190.
34 Lumley J. Does continuous intrapartum fetal monitoring predict long-term
neurological disorders? Paediatr Perinat Epidemiol 1988; 2: 299–307.
35 Paneth N, Bommarito M, Stricker J. Electronic fetal monitoring and later outcome.
Clin Invest Med 1993; 16: 159–165.
36 Neilson JP. Electronic fetal heart rate monitoring during labor: information from
randomized trials. Birth 1994; 21: 101–104.
37 Graham EM, Petersen SM, Christo DK, Fox HE. Intrapartum electronic fetal heart rate
monitoring and the prevention of perinatal brain injury. Obstet Gynecol 2006; 108:
656–666.
38 Thacker SB, Stroup DF, Peterson HB. Efficacy and safety of intrapartum electronic fetal
monitoring: an update. Obstet Gynecol 1995; 86: 613–620.
Journal of Perinatology
 Obstetric management of MAS
H Xu et al
S18
39 Alfirevic Z, Devane D, Gyte GM. Continuous cardiotocography (CTG) as a form of
electronic fetal monitoring (EFM) for fetal assessment during labour. Cochrane
Database Syst Rev 2006; 3: CD006066.
40 Starks GC. Correlation of meconium-stained amniotic fluid, early intrapartum pH,
and Apgar scores as predictors of perinatal outcome. Obstet Gynecol 1980; 56:
604–609.
41 Hageman JR. Meconium staining of the amniotic fluid: the need for reassessment of
management by obstetricians and pediatricians. Curr Probl Pediatr 1993; 23:
396–401.
42 Krebs HB, Petres RE, Dunn LT, Jordaan HVF, Segreti A. Intrapartum Fetal Heart Rate
Monitoring. III. Association of meconium with abnormal fetal heart rate patterns. Am J
Obstet Gynecol 1980; 137: 936–943.
43 Umstad MP. The predictive value of abnormal fetal heart rate patterns in early labour.
Aust NZJ Obstet Gynaecol 1993; 33: 145–149.
44 Miyazaki FS, Nevarez F. Saline amnioinfusion for relief of repetitive variable
decelerations. A prospective randomized study. Am J Obstet Gynecol 1985; 153:
301–306.
45 Wang CC, Rogers MS. Lipid peroxidation in cord blood: a randomised sequential airs
study of prophylactic saline amnioinfusion for intrapartum oligohydramnios. Br J
Obstet Gynaecol 1997; 104: 1145–1151.
46 Nageotte MP, Freeman RK, Garite TJ, Dorchester W. Prophylactic intrapartum
amnioinfusion in patients with premature rupture of membranes. Am J Obstet Gynecol
1985; 153: 557–562.
47 Nageotte MP, Bertucci L, Towers CV, Lagrew DL, Modaniou H. Prophylactic
amnioinfusion in pregnancies complicated by oligohydramnios: a prospective study.
Obstet Gynecol 1991; 77: 677–680.
48 Owen J, Henson BV, Hauth JC. A prospective randomized study of saline solution
amnioinfusion. Am J Obstet Gynecol 1990; 162: 1146–1149.
49 MacGregor SM, Banzhaf WC, Silver RK, Depp R. A prospective randomized evaluation
of intrapartum amnioinfusion. Fetal acid–base status and cesarean delivery. J Reprod
Med 1991; 36: 69–73.
50 Schrimmer DB, Macri CJ, Paul RH. Prophylactic amnioinfusion as a treatment for
oligohydramnios in laboring patients: a prospective, randomized trial. Am J Obstet
Gynecol 1991; 165: 972–975.
51 Chauhan SP, Rutherford SE, Hess LW, Morrison JC. Prophylactic intrapartum
amnioinfusion for patients with oligohydramnios. J Reprod Med 1992; 37:
817–820.
52 Pitt C, Sanchez-Ramos L, Kaunitz AM, Gaudier F. Prophylactic amnioinfusion for
intrapartum oligohydramnios: a meta-analysis of randomized controlled trials. Obstet
Gynecol 2000; 96: 861–866.
53 Hofmeyr GJ. Amnioinfusion for meconium-stained liquor in labor. Cochrane Database
Syst Rev 2002; 1: CD000014.
54 Xu H, Hofmeyr J, Roy C, Fraser W. Intrapartum amnioinfusion for meconium stained
amniotic fluid: a systematic review of randomised controlled trials. BJOG 2007; 114:
383–390.
55 Fraser WD, Hofmeyr GJ, Lede R, Faron G, Alexander S, Goffinet F et al. Amnioinfusion
for the prevention of the meconium aspiration syndrome. N Engl J Med 2005; 353:
909–917.
56 Wenstrom K, Andrews WW, Maher JE. Amnioinfusion survey: prevalence, protocols, and
complications. Obstet Gynecol 1995; 86: 572–576.
Journal of Perinatology
57 Dragich DA, Ross AF, Chestnut DH, Wenstrom K. Respiratory failure associated with
amnioinfusion during labor. Anesth Analg 1991; 72: 549–551.
58 Maher JE, Wenstrom KD, Hauth JC, Meis PJ. Amniotic fluid embolism after saline
amnioinfusion: two cases and review of the literature. Obstet Gynecol 1994; 83:
851–854.
59 Dorairajan G, Soundararaghavan S. Maternal death after intrapartum
saline amnioinfusion-report of two cases. Br J Obstet Gynaecol 2005; 112:
1331–1333.
60 ACOG Committee Obstetric Practice. ACOG Committee Opinion Number 346, October
2006: amnioninfusion does not prevent meconium aspiration syndrome. Obstet
Gynecol 2006; 108: 1053.
61 Carson BS, Losey RW, Bowes Jr WA, Simmons MA. Combined obstetric and pediatric
approach to prevent meconium aspiration syndrome. Am J Obstet Gynecol 1976; 126:
712–715.
62 Yoder BA, Kirsch EA, Barth WH, Gordon MC. Changing obstetric practices associated
with decreasing incidence of meconium aspiration syndrome. Obstet Gynecol 2002;
99: 731–739.
63 Chishty AL, Alvi Y, Iftikhar M, Bhutta TI. Meconium aspiration in neonates: combined
obstetric and paediatric intervention improves outcome. J Pak Med Assoc 1996; 46:
104–108.
64 Vain NE, Szyld EG, Prudent LM, Wiswell TE, Aguilar AM, Vivas NI. Oropharyngeal
and nasopharyngeal suctioning of meconium-stained neonates before delivery
of their shoulders: multicentre, randomised controlled trial. Lancet 2004; 364:
597–602.
65 Gregory GA, Gooding CA, Phibbs RH, Tooley WH. Meconium aspiration in
infantsFa prospective study. J Pediatr 1974; 85: 848–852.
66 Ting P, Brady JP. Tracheal suction in meconium aspiration. Am J Obstet Gynecol
1975; 122: 767–771.
67 Wiswell T, Henley MA. Intratracheal suctioning, systematic infection, and the
meconium aspiration syndrome. Pediatrics 1992; 89: 203–206.
68 Linder N, Aranda JV, Tsur M, Matoth I, Yatsiv I, Mandelberg H et al. Need for
endotracheal intubation and suction in meconium-stained neonates. J Pediatr 1988;
112: 613–615.
69 Wiswell TE, Gannon CM, Jacob J, Goldsmith L, Szyld E, Weiss K et al. Delivery room
management of the apparently vigorous meconium-stained neonate: results of the
multicenter, international collaborative trial. Pediatrics 2000; 105: 1–7.
70 Kresh MJ, Brion LP, Fleishman AR. Delivery room management of meconium stained
neonates. J Perinatol 1991; 11: 46–48.
71 Bent RC, Wiswell TE, Chang A. Removing meconium from infant tracheae. Am J Dis
Child 1992; 146: 1085–1089.
72 Peng TCC, Gutcher GR, Van Dorsten JP. A selective aggressive approach to the neonate
exposed to meconium-stained amniotic fluid. Am J Obstet Gynecol 1996; 175:
296–303.
73 Yoder BA. Meconium-stained amniotic fluid and respiratory complications: impact of
selective tracheal suction. Obstet Gynecol 1994; 83: 77–84.
74 Liu WF, Harrington T. The need for delivery room intubation of thin meconium in the
low-risk newborn: a clinical trial. Am J Perinatol 1998; 15: 675–682.
75 Halliday HL, Sweet D. Endotracheal intubation at birth for preventing morbidity and
mortality in vigorous, meconium-stained infants born at term. Cochrane Database
Syst Rev 2001; 1: CD000500.
 Journal of Perinatology (2008) 28, S19–S26
r 2008 Nature Publishing Group All rights reserved. 0743-8346/08 $30
www.nature.com/jp
REVIEW
Delivery room management of the meconium-stained newborn
TE Wiswell
Center for Neonatal Care, Orlando, FL, USA
Review of all medical literature dealing with delivery room management of
meconium-stained infants. Additionally, the author contacted multiple
individuals involved historically or clinically with the published studies or
the persons who developed treatment guidelines. Although many therapies
have been suggested as being effective, none have been definitively proven
efficacious by the gold standard: a large, randomized, controlled trial
(RCT). Further adequate investigations (RCTs) need to be performed to
assess whether proposed management schemes are of benefit in the care of
meconium-stained newborn infants.
Journal of Perinatology (2008) 28, S19–S26; doi:10.1038/jp.2008.143
Introduction
Meconium-stained amniotic fluid (MSAF) is noted in 10 to 15% of
all deliveries (B500 000 annually in the United States).1,2
Approximately 3 to 4% of meconium-stained infants will develop
the meconium aspiration syndrome (MAS). Neonates that are
depressed and are born through thick-consistency MSAF are at the
highest risk for developing MAS. A considerable proportion of
infants with MAS require mechanical ventilation, whereas many
develop pulmonary air leaks. In addition, MAS is frequently
associated with persistent pulmonary hypertension of the newborn.
The mortality rate among infants with the disorder may be as high
as 5 to 10%. Humans are not the only species affected by MAS; it is
seen frequently and is a leading cause of death in domestic farm
animals (pigs, cattle, and so on).
In an effort to prevent or mitigate the course of MAS, many
methods have been suggested to remove aspirated meconium from
the airways. Historically, farmers would grasp meconium-stained
animals by their hindquarters and then swing them around their
heads. Centrifugal forces would move the meconium-stained fluid
toward the head of the rotating animal and the material would
then be manually extracted. In 1871, Dr Bernard Schultze
described a resuscitation maneuver, one of the benefits of which
purportedly included the removal of meconium from the airways3.
Caregivers would grasp affected infants by the shoulders vertically
at the level of the adult’s knees. The caregiver would then sweep
Correspondence: Dr TE Wiswell, Center for Neonatal Care, 2718 North Orange Avenue,
Suite B, Orlando, FL 32804, USA.
E-mail: Thomas_Wiswell@yahoo.com
the baby upward inverting them by 1801 and then swinging them
back down to their original position. This maneuver would be
repeated as many as 10 to 12 times. This untested therapy was
widely practiced through the 1920s.
Intratracheal suctioning
This author has not found any written descriptions concerning
intubation and suctioning of meconium-stained infants before the
year 1960. In a textbook of neonatal resuscitation published that
year,4 Dr L Stanley James (Figure 1) stated that if meconium had
been aspirated into the trachea, it should be suctioned out. He then
suggested using an endotracheal tube as a suction device. Dr Jack
Sinclair (personal communication) states that when he arrived as
a resident in Pediatrics at Columbia Hospital for Children in New
York City in 1961, both Dr James and Dr Virginia Apgar were
teaching and advocating intubation and suctioning of
meconium-stained babies. The latter individuals even made a
teaching movie of this procedure. Dr Lillian Blackmon (personal
communication), while training at Columbia Hospital for
Children, did an elective with Dr William Tooley at the University
of California at San Francisco (UCSF) in 1966. At that time,
physicians at UCSF were not suctioning the airways of meconium-
stained neonates. Dr Blackmon subsequently returned to UCSF in
1968. At virtually the same time, Dr William Silverman (Figure 2),
the long-time director of Neonatology at Columbia Hospital for
Children, also moved west and became chief of neonatology of San
Francisco Children’s Hospital. Dr Blackmon states that the two of
them began training residents and clinicians in San Francisco to
intubate and suction meconium-stained neonates.
Dr George Gregory (Figure 3) and colleagues5 are given credit
for the wide dissemination of the intubation and suctioning
technique in their seminal 1974 publication. These individuals
described their prospective management of meconium-stained
infants at UCSF over a 6-month period. Eighty such infants were
intubated and had their tracheas suctioned. In addition, the infants
subsequently were treated with aerosolized distilled water, chest
physiotherapy (CPT) and postural drainage (positional therapy
alternating between head up and head down, as well as side to
side). Sixteen (20%) of the infants were deemed to be ‘sick’, six
(38%) of whom had thin-consistency meconium suctioned from
 DR management of meconium-stained newborns
TE Wiswell
S20
their airways. Of the 80 infants, 2 required mechanical ventilation,
but none were treated with continous positive airway pressure.
Seven neonates developed pneumothorax and/or
pneumomediastinum. The authors stated that none of the 35
meconium-stained babies managed in this manner subsequent to
the study period and admitted to their Newborn Intensive Care Unit
required ventilation. The authors also presented data from 15
infants with MAS transferred to their unit subsequent to the study
period. Of the 15 infants, 5 (33%) required either mechanical
ventilation or continous positive airway pressure, whereas 6 (40%)
developed a pneumothorax. On the basis of their experience, the
authors recommended intratracheal suctioning of all infants born
Figure 1 Dr L Stanley James (photograph courtesy of Dr Richard Polin).
Figure 2 Drs Lillian Blackmon and William Silverman (photograph courtesy of Dr Blackmon).
Journal of Perinatology
through thick, particulate meconium (despite almost 40% of their
‘sick’ population having only thin-consistency meconium
retrieved). They also recommended subsequent CPT and postural
drainage for those infants from whom meconium-stained material
is retrieved from the trachea, as well as for those with abnormal
chest roentgenograms.
Although Gregory and colleagues are frequently cited as the
original investigators who showed benefits of intubation and
suctioning, Burke-Strickland and Edwards6 (Figure 4) published
the results of their prospective trial of the technique a full year
earlier. The latter authors intubated and suctioned 84 meconium-
stained infants over a 2-year period and compared their outcomes
with 17 such infants who did not receive tracheal cleansing.
Overall 70% of intubated infants also had saline lavage. The non-
intubated infants had more prolonged respiratory distress and
longer hospital stays compared with their intubated counterparts.
Burke-Strickland and Edwards recommended that all infants born
through MSAF of any consistency should be intubated and
suctioned.
Interestingly, 1 year after the Gregory publication, the results of
another study also based out of San Francisco were published.
Ting (Figure 5) and Brady7 did a retrospective review of outcomes
of 125 meconium-stained infants born over a 3-year period; of
these 28 babies were not intubated. Of these, 16 (57%)
developed MAS and 7 (25%) died. The remaining 97 infants were
intubated and suctioned; of these 27 (28%) developed MAS and
1 (1%) died. These investigators concluded that all infants born
through MSAF of any consistency should be intubated and
suctioned.
On the basis of their anecdotal experience, Carson et al.8
(Figure 6) suggested a different approach. They proposed that
 DR management of meconium-stained newborns
TE Wiswell
S21

Figure 5 Dr Pauline Ting (photograph courtesy of Dr Ting).

Figure 3 Dr George A Gregory (photograph courtesy of Dr George Gregory).

Figure 4 Dr Nancy Edwards Dow (photograph courtesy of Dr Dow). Figure 6 Dr Bonita Carson (photograph courtesy of Dr Carson).

Journal of Perinatology
 DR management of meconium-stained newborns
TE Wiswell
S22

Figure 7 Clinician suctioning endotracheal tube directly with his mouth.

Figure 8 Dr Rebecca Bent (photograph courtesy of Dr Bent).

clinicians should visualize the hypopharynx and vocal cords of
meconium-stained infants after oropharyngeal suctioning. These
authors recommended that tracheal suctioning should be
performed only if meconium could be visualized at the vocal cords.
Subsequent to these publications, there was widespread
acceptance of the practice of intubation and suctioning of the
trachea of all meconium-stained infants. This approach was
recommended in numerous textbooks and journals. The most
common technique (‘straw method’) consisted of providing
negative pressure at the hub of the endotracheal tube using one’s
mouth (Figure 7). Essentially, this practice was how residents in
pediatrics gained and improved their intubation skills. Tasting
meconium became a rite of passage for pediatric trainees.
Fortunately, most clinicians ultimately realized that a face mask

Journal of Perinatology
 DR management of meconium-stained newborns
TE Wiswell
S23

could be interposed between one’s mouth and the endotracheal

tube and obviated this culinary delight. In the decade following the
aforementioned publications, the incidence of MAS and deaths
attributable to the disorder appeared to decline significantly.9
It was recognized that there was a wide variation in the amount
of negative pressure produced by different individuals when directly
suctioning the endotracheal tube. Moreover, the infectious risks of
this practice were obvious. Some chose to directly place a standard
suction catheter or a de Lee suction catheter into the larynx. Many
devices (at least 20) were commercially developed as alternatives to
the straw method. Bent (Figure 8) et al.10 assessed the various
methods and found one to be consistently the best.
In 1988, Linder (Figure 9) and colleagues11 reported that a
selective approach might be more appropriate than a universal
one. They performed a trial in which apparently vigorous
meconium-stained infants were quasi-randomized to either
intratracheal suctioning or expectant management. Their results
were questioned because of major design flaws in the trial.
Although some suggested that a selective approach could be
deleterious, others found it to result in good outcomes. In the late
1990s, Wiswell et al.12 (Figure 10) spearheaded a large
Figure 10 Dr Thomas Wiswell (photography courtesy of Dr Wiswell).
international prospective, randomized controlled trial to assess a
selective approach. Almost 2100 meconium-stained neonates were
enrolled. During the 10 to 15 s after delivery, these babies had to be recommendations and advised that apparently vigorous
apparently vigorous (defined as having a heart rate >100 beats per meconium-stained infants do not need intubation and
min, having spontaneous respirations and having reasonable intratracheal suctioning.
tone). They were randomized either to be intubated and suctioned A future question that needs to be answered is whether or not
or to expectant management. The results of this approach are depressed meconium-stained infants actually benefit from having
given in the Table 1. There appeared to be no benefit to tracheal their airways intubated and suctioned. As many clinicians believe
cleansing of this population. Subsequent to this publication, the that most cases of MAS are due to in utero aspiration of MSAF, the
Neonatal Resuscitation Program (NRP) changed its potential efficacy of tracheal cleansing needs to be assessed in a
well-conducted randomized, controlled trial. Another issue that
needs to be addressed is the training of pediatric residents in
intubation skills. Historically, most residents honed their skills
during training by intubating virtually all meconium-stained
infants. However, by eliminating the need to intubate the majority
of the latter population, many believe that residents are no longer
gaining or maintaining these skills.

Intrapartum suctioning of the nasopharynx and oropharynx

Figure 9 Dr Nehama Linder (photograph available from the following website:
http://www.nacion.com/ln_ee/2007/octubre/16/_Img/1762242_0.jpg).
Intrapartum suctioning consists of using either a suction catheter
or a bulb syringe to remove material from a meconium-stained
infant’s oropharynx and nasopharynx after the delivery of baby’s
head, but prior to delivery of the thorax. In the mid-1970s, Carson
et al.8 performed the previously mentioned study, the original goal
of which was to assess the value of tracheobronchial saline lavage
(Dr Carson, personal communication). They compared
meconium-stained infants managed differently during three
separate time periods. Group 1 infants (n ¼ 947) had no
intrapartum suctioning, no saline lavage and inconsistent
intratracheal suctioning. Group 2 infants (n ¼ 381) had

Journal of Perinatology
 DR management of meconium-stained newborns
TE Wiswell
S24

Table 1 Incidence of MAS among 2094 apparently vigorous meconium-stained infants randomized to either intubation and suctioning or expectant management

Consistency of MSAF Intubation and suction group Expectant management group Significance
(n ¼ 1051) (%) (n ¼ 1043) (%)

Thin 5/447 (1.1) 2/453 (0.4) Not significant

Moderately thick 7/301 (2.3) 6/307 (2.0) Not significant
Thick 22/303 (7.3) 20/283 (7.1) Not significant
Overall 34/1051 (3.2) 28/1043 (2.7) Not significant
Abbreviations: MAS, meconium aspiration syndrome; MSAF, meconium-stained amniotic fluid.

were not statistically significant! Nonetheless, there was a

Figure 11 Dr Horatio Falciglia (photograph available from the following
website: http://www.cincinnatichildrens.org/research/div/neonatology/fs/fac/
horacio-falciglia.htm?pres ¼ Hrs).
intrapartum suctioning, intubation and tracheal suctioning, and
saline lavage. Group 3 infants (n ¼ 273) had intrapartum
suctioning and selective tracheal intubation/suctioning (if
meconium was present at the vocal cords). The authors intended to
randomize those babies in group 3 who required intubation to
saline lavage or no lavage. However, only 2 of 273 group 3 infants
had meconium noted at the cords and were eligible to be lavaged.
Hence, the authors were unable to assess whether saline lavage was
of benefit. The incidence of MAS in these populations was 1.9%
(group 1), 1.8% (group 2) and 0.4% (group 3). These differences
widespread assumption that the lower incidence of MAS in group 3
babies was due to the intrapartum suctioning. There was worldwide
acceptance of this hypothesis and it became the standard of care
for almost three decades.
Subsequent studies were unable to replicate Carson group’s
remarkably low incidence of MAS after intrapartum suctioning.
Most notable were the reports of Falciglia (Figure 11) and
colleagues. Initially, he performed a historical comparison
evaluation.13 He assessed the outcomes of meconium-stained
infants born in two different years (1975 and 1983, respectively).
During the earlier year, no intrapartum suctioning was customarily
performed, in contrast with the latter year when it was performed
routinely. There was no difference in the incidence of MAS in 1975
(2.0% of 742) compared with 1983 (2.1% of 755). Subsequently,
Falciglia et al.14 performed a concurrent observational study
during which an independent observer (unknown to the
obstetrician) documented whether or not obstetricians performed
‘early’ oronasopharyngeal suctioning while the baby’s head was at
the perineum (prior to delivery of the child’s thorax) compared
with those that received ‘later’ suctioning subsequent to delivery.
MAS was actually more common in the group that underwent
‘early’ suctioning (23/221, 10.4%) compared with those
undergoing ‘later’ suctioning (15/227, 6.9%).
To definitively assess whether or not this procedure was of
benefit to neonates, Vain (Figure 12) and colleagues15 performed
an international prospective, randomized controlled trial. The
population consisted of 2514 infants born through MSAF of any
consistency, gestational age X37 weeks and vertex (cephalic)
presentation. Subjects were randomized either to (1) suctioning of
the oropharynx and nasopharynx prior to delivery of the thorax or
to (2) no suctioning prior to delivery. No differences were found in
the incidence of MAS between suctioned (4.1%) and non-suctioned
(3.8%) infants. Moreover, there were no differences in other key
outcome variables including mortality, the need for mechanical
ventilation, duration of mechanical ventilation and duration of
supplemental oxygen use. Subsequent to this publication, both the
NRP and the American College of Obstetricians and Gynecologists
(ACOG) changed their stances and no longer recommend
intrapartum oropharyngeal and nasopharyngeal suctioning prior
to delivery when MSAF is present.

Journal of Perinatology

Figure 12 Drs Nestor Vain and Luis Prudent (photograph courtesy of Dr Vain).
Other proposed delivery room therapies
CPT
Chest physiotherapy is performed in an effort to prevent
accumulation of debris and to improve mobilization of airway
secretions. CPT consists of techniques such as percussion,
vibration, postural drainage, saline administration and suctioning.
Theoretically, CPT should help remove meconium from the
airways, prevent its consequences and improve gas exchange. It is
widely performed on infants born through MSAF, as well as those
with MAS. Nevertheless, there are virtually no data to support this
therapy in meconium-stained neonates, either in the delivery room
or thereafter. One must remember that there are potential
complications of CPT (pneumothorax, hypoxemia, arrhythmia,
airway perforation and tissue damage).
Cricoid pressure, epiglottal blockage and thorax compression
Several other maneuvers have been suggested as being of benefit in
preventing MAS.16 Cricoid pressure involves the application of
pressure to the neonate’s airway in an effort to compress it and
prevent intratracheal meconium from migrating distally. Epiglottal
blockage entails the placement of one or more fingers into the
infant’s hypopharynx in an attempt to apply pressure on the
epiglottis and block the meconium passage downward. The latter two
therapies have the potential to traumatize the airway. Both are likely
to produce a vagal response in a potentially compromised infant.
Thorax compression consists of manually encircling a
meconium-stained infant’s chest and applying pressure in an
DR management of meconium-stained newborns
TE Wiswell
S25
effort to prevent the child from inhaling deeply and aspirating fluid.
None of the aforementioned maneuvers have ever been assessed
in clinical trials. All are potentially dangerous and none are
recommended.
Gastric suctioning
Karlowicz17 suggested suctioning of the stomachs of all
meconium-stained neonates soon after delivery. He hypothesized
that some cases of MAS could potentially be caused by the postnatal
reflux of gastric contents into the oropharynx. The material could
then potentially be aspirated into the airways. Suctioning of the
stomach within minutes of delivery would obviate this from
occurring, although an intriguing hypothesis, gastric suctioning,
has never been evaluated for efficacy in preventing MAS.
Summary
Multiple delivery room therapies have been proposed as being
effective in preventing or ameliorating MAS. However, to date, none
of the therapies assessed by randomized, controlled trials have been
definitively proven to be effective. An important question remains:
do depressed, meconium-stained neonates benefit by being
intubated and suctioned?
Disclosure
TE Wiswell has declared no financial interests.
Journal of Perinatology
 DR management of meconium-stained newborns
TE Wiswell
S26
References
1 Wiswell TE, Bent RC. Meconium staining and the meconium aspiration syndrome.
Pediatr Clin North Am 1993; 40: 955–981.
2 Wiswell TE, Fuloria M. Management of meconium stained amniotic fluid. Clin
Perinatol 1999; 26: 659–668.
3 O’Donnell CPF, Gibson AT, Davis PG. Pinching, electrocution, ravens’ beaks, and
positive pressure ventilation: a brief history of neonatal resuscitation. Arch Dis Child
2006; 91: F369–F373.
4 James LS. Resuscitation procedures in the delivery room. In: Abramson H (ed).
Resuscitation of the Newborn Infant. CV Mosby Co: St Louis, 1960, pp 141–161.
5 Gregory GA, Gooding CA, Phibbs RH, Tooley WH. Meconium aspiration in infants: a
prospective study. J Pediatr 1974; 85: 848–852.
6 Burke-Strickland M, Edwards NB. Meconium aspiration in the newborn. Minn Med
1973; 57: 1031–1035.
7 Ting P, Brady JP. Tracheal suction in meconium aspiration. Am J Obstet Gynecol
1975; 122: 767–771.
8 Carson B, Losey RW, Bowes Jr WA, Simmons MA. Combined obstetric and pediatric approach
to prevent meconium aspiration syndrome. Am J Obstet Gynecol 1976; 126: 712–715.
9 Wiswell TE, Tuggle JM, Turner BS. Meconium aspiration syndrome: have we made a
difference? Pediatrics 1990; 85: 715–721.
Journal of Perinatology
10 Bent RC, Wiswell TE, Chang A. Removing meconium from infant trachea. What works
best? Am J Dis Child 1992; 146: 1085–1089.
11 Linder NJ, Aranda V, Tsur M, Matoth I, Yatsiv I, Mandelberg H et al. Need for
endotracheal intubation and suction in meconium stained neonates. J Pediatr 1998; 112:
613–615.
12 Wiswell TE, Gannon CM, Jacob J, Goldsmith L, Szyld E, Weiss K et al. Delivery room
management of the apparently vigorous meconium-stained neonate: results of the
multicenter, international collaborative trial. Pediatrics 2000; 105: 1–7.
13 Falciglia HS. Failure to prevent meconium aspiration syndrome. Obstet Gynecol 1988;
71: 349–353.
14 Falciglia HS, Henderschott C, Potter P, Helmchen R. Does DeLee suction at the
perineum prevent meconium aspiration syndrome? Am J Obstet Gynecol 1992; 167:
1243–1249.
15 Vain N, Szyld E, Prudent L, Wiswell TE, Aguilar AM, Vivas NI. Oro- and nasopharyngeal
suctioning of meconium-stained neonates before delivery of their shoulders: results
of the international, multicenter, randomized, controlled trial. Lancet 2004; 364:
597–602.
16 Cleary GM, Wiswell TE. Meconium-stained amniotic fluid and the meconium
aspiration syndrome. An update. Pediatr Clin North Am 1998; 45: 511–529.
17 Karlowicz MG. More on meconium aspiration. Pediatrics 1990; 86: 1007–1008.
 Journal of Perinatology (2008) 28, S27–S29
r 2008 Nature Publishing Group All rights reserved. 0743-8346/08 $30
www.nature.com/jp
REVIEW
Neonatal resuscitation guidelines for ILCOR and NRP: evaluating
the evidence and developing a consensus
J Kattwinkel
Division of Neonatology, Department of Pediatrics, University of Virginia Health System, Charlottesville, VA, USA
Journal of Perinatology (2008) 28, S27–S29; doi:10.1038/jp.2008.146
I have been asked to describe the process used by the American
Academy of Pediatrics (AAP) and American Heart Association (AHA)
to evaluate the evidence for the purpose of developing
recommendations for the Neonatal Resuscitation Program. This
symposium is primarily interested in the recommendations
regarding management of meconium during neonatal
resuscitation, but I will first describe the neonatal program and the
process used in development of all AHA and AAP resuscitation
recommendations.
The Neonatal Resuscitation Program (NRP) is a standardized
instruction in neonatal resuscitation. It includes a
self-instructional textbook and a set of skills taught by instructors.
The instructors first involved national faculty who were given the
responsibility of training regional instructors in their respective
states, who then trained hospital-based instructors, who in turn
trained participants. The program was developed by collaboration
of the AHA and AAP and is managed and revised by the NRP
Steering Committee administered by the AAP. There have been over
2.2 million participants in the United States since inception of the
NRP nearly 20 years ago, and the program has also been
implemented in 106 other countries. Revisions are made
approximately every 5 years.
The revision process involves six steps: define the issues, review
the literature, debate the evidence, publish guidelines, produce
educational materials, and deliver the program.1 The first three
steps are conducted in collaboration with the International Liaison
Committee on Resuscitation (ILCOR), which then publishes a
document entitled Consensus on Science and Treatment
Recommendations (COSTR). Each of the resuscitation councils
represented on ILCOR then develops separate resuscitation
guidelines appropriate for their respective countries.
The neonatal resuscitation issues considered during the most
recent review included topics such as the use of 100% oxygen
Correspondence: Dr J Kattwinkel, Division of Neonatology, Department of Pediatrics,
University of Virginia Health System, PO Box 800386, Charlottesville, VA 22908, USA.
E-mail: jk3f@virginia.edu
versus room air, various issues related to temperature
management, ventilation strategies during resuscitation,
components and timing associated with volume expansion, glucose
management following intensive resuscitation, use of CO2 detectors
for confirmation of tube placement, epinephrine dosing and routes
of administration, ethical considerations regarding initiating and
discontinuing resuscitation, as well as various aspects of managing
meconium prior to, during and following delivery.
After the issues are defined, ILCOR representatives and
consultants are asked to begin the evidence evaluation process by
preparing worksheets on each topic. Worksheet development begins
with an intensive review of the literature. Over 30 000 abstracted
and critiqued references are cataloged in an Endnotes database
maintained at the AHA. Pertinent studies are then classified
according to their level of evidence, ranging from level 1
(randomized controlled trial), through levels 4 (historic,
nonrandomized cohort study) and 6 (animal or mechanical model
study), to level 8 (rational conjecture or common practice). Each
study is also ranked as to the quality of evidence (fair, good or
excellent) and whether the study is supportive, neutral or opposes
the evidence. The neonatal resuscitation worksheets are available
for review by accessing the NRP website (http://www.aap.org/nrp/
nrpmain), clicking on ‘Science’ and ‘Evidence-Based Guidelines’,
and then accessing the link to the worksheets maintained on the
AHA website. There are three worksheets addressing issues about
meconium.
The worksheets serve as the focus for a series of ILCOR debates
that take place over approximately 3 years, which culminates in
the Evidence Evaluation (E-2) conference during which consensus
is reached on the science. This conference forms the basis for the
COSTR document, the most recent version of which was jointly
published in Circulation2 and Resuscitation3 and the pediatric
and neonatal portions republished in Pediatrics.4 The NRP
Steering Committee then considered the practical implications of
COSTR in drafting the NRP Resuscitation Guidelines that were also
published in Circulation5 and Pediatrics.6
Three resuscitation-associated meconium issues were examined
during the most recent evidence evaluation process.
 Neonatal resuscitation guidelines for ILCOR and NRP
J Kattwinkel
S28
Does amnioinfusion reduce the incidence or severity
of meconium aspiration syndrome?
It has been hypothesized that infusion of saline into
meconium-contaminated amniotic fluid will dilute the meconium
and decrease the potential for developing obstruction of airways
and development of meconium aspiration syndrome. Review of the
literature revealed 23 reports supporting the hypothesis:
amnioinfusion significantly reduced the frequency of meconium
aspiration syndrome, of meconium below the cords and neonatal
acidemia, and was associated with a lower overall cesarean section
rate. Nine studies showed no significant benefit of amnioinfusion.
Three reports suggested that any demonstrated benefit of
amnioinfusion may have been a reflection of reversing fetal heart
rate decelerations rather than due to dilution of meconium. There
were also case reports of uterine rupture when amnioinfusion was
administered during a trial of labor. In view of the conflicting data,
insufficient input from the obstetrics profession, and knowledge of
an ongoing randomized control trial, the Committee decided to
take no position on amnioinfusion during this evidence evaluation
cycle. Although the Guidelines were in press, the Fraser et al.7 trial
was published (as also reported at this conference), and the
American College of Obstetrics and Gynecology released a
Committee Opinion that ‘routine prophylactic amnioinfusion for
the dilution of meconium-stained amniotic fluid is not
recommended.’8
Should the current recommendation, to always
perform intrapartum suctioning, be continued?
Since the Carson et al.9 study in 1976, there has been a generally
accepted recommendation that babies born with meconium-stained
amniotic fluid should have their nose, mouth and pharynx
suctioned after delivery of the head, but before delivery of the
shoulders (intrapartum suctioning). The recommendation was
based on a probably erroneous assumption that most meconium
aspiration syndrome (MAS) aspiration occurred during the
establishment of air breathing and that removal of the meconium
while the chest was still compressed in the birth canal could
prevent MAS. The Carson et al. study reviewed retrospectively all
births that occurred at the University of Colorado during three time
periods (Table 1).
In total, 12 to 16% of the babies born in 1970 to 1975 had
meconium in their amniotic fluids. For the first 46 months
(period 1), the standard had been not to perform intrapartum
suctioning, but to perform direct endotracheal suctioning of all
meconium-stained babies after delivery. This post-delivery practice
had been initiated following the observation of Gregory et al.10 in
1974 that 56% of meconium-stained neonates had meconium
recovered from below the cords. For the next 10 months (period 2),
Carson et al. performed intrapartum suctioning, continued to
Journal of Perinatology
Table 1 Experience with meconium births, as reported by Carson et al.9
7/1970–4/1974 5/1974–3/1975 4/1975–11/1975
Births 7585 2320 1681
Meconium in 12.5% 16.4% 16.2%
amniotic fluid
MAS 18* 7** 1*,**
MAS deaths 5 0 0
Abbreviation: MAS, meconium aspiration syndrome.
Period 1: endotracheal suctioning under direct vision. Period 2: intrapartum
suction+tracheobronchial lavage. Period 3: intrapartum suction+endotracheal
suction (2)±lavage.
*P ¼ 0.18; **P ¼ 0.25.
suction the trachea immediately following birth, and then
performed a tracheobronchial lavage with normal saline. During
the third epoch of 8 months (period 3), the intrapartum suctioning
continued to be performed, but neonatal suction and lavage were
less common. Even though there were no significant differences in
the incidence of meconium aspiration syndrome among any of the
epochs, the practice of intrapartum suctioning was recommended
and adopted by the vast majority of clinicians for the next 30 years,
probably because the practice appeared to be noninvasive and
seemed logical.
However, the practice of intrapartum suctioning continued
to be questioned by various obstetricians and neonatologists, and in
2000 to 2001 Vain et al.11 conducted a randomized, multicenter,
control trial of 2514 meconium-stained deliveries occurring in
10 centers in Argentina and 1 in the United States.
Meconium-stained births were randomized to receive either
pharyngeal suctioning of the fetus before delivery of the shoulders
or no intrapartum suctioning. Although there were 5% protocol
deviations, an intent-to-treat analysis revealed no significant
difference in the incidence of meconium aspiration syndrome
between the two groups. The relative risk was 0.9, with 95%
confidence limits of 0.6 to 1.3. Primarily as a result of the Vain
study, ILCOR and the latest edition of NRP have stated the
following: ‘Current recommendations no longer advise routine
intrapartum oropharyngeal and nasopharyngeal suctioning for
infants born to mothers with meconium staining of amniotic fluid
(class I).’ It should be noted that, although there was some
disagreement during development of the recommendation, the
NRP Steering Committee is on record as emphasizing that the new
recommendation should not be interpreted to mean that
intrapartum suctioning of meconium-stained babies is
contraindicated, but merely that the previously recommended
practice of routinely performing suctioning in such babies is no
longer recommended. Several members felt that clearing of the
oropharynx to facilitate the possible need for subsequent
visualization of the trachea for direct suctioning, still seems
reasonable.

Is the current recommendation, to limit endotracheal
suctioning only to the nonvigorous newborn, still valid?
Traditional teaching has recommended that meconium-stained
infants have endotracheal intubation immediately following birth
and that suction be applied to the endotracheal tube as it is
withdrawn. As noted above, this recommendation stemmed from
the observation of Gregory et al.10 who reported on experience with
1000 births, of whom 8.8% were meconium stained and 91% of
those 88 had received direct endotracheal suction. Meconium
was recovered from the trachea in 56% (46 babies) and 17%
(8 patients) of those had had none in ‘mouth or larynx’ during
laryngoscopy. Although this was not a control trial, from these
observations, these clinician investigators recommended that, ‘all
infants born through thick or ‘pea soup’ meconium should have
their trachea aspirated immediately after birthy’. Again, the
recommendation was universally accepted and became the
standard until Wiswell et al.12 conducted a randomized multicenter
control trial comparing the practice of performing endotracheal
suctioning on all babies, versus only suctioning those babies who
were nonvigorous. Vigorous was defined as strong respiratory effort,
good muscle tone, and a heartrate greater than 100 beats per
minute. Over 26 months, at 12 centers, 2094 meconium-stained
neonates who were classified as ‘vigorous’ at birth, were
randomized to receive endotracheal suctioning versus conservative
therapy. There was no significant difference in the incidence of
meconium aspiration syndrome or other lung disease between
those suctioned versus those not suctioned. As a result, ILCOR and
the NRP revised their recommendation in 2000 to limit
endotracheal suctioning only to those babies who were meconium
stained and not vigorous at birth. We finally stopped the practice of
chasing the healthy baby around the delivery room with a
laryngoscope.
There are still several unanswered questions regarding the
appropriate management of the meconium-stained baby at birth
that have not received definitive answers in the most recent COSTR
document and the 2005 NRP Guidelines. First, is amnioinfusion an
effective procedure for diluting meconium and reducing meconium
aspiration syndrome? The above-mentioned Fraser et al.7 trial and
American College of Obstetrics and Gynecology statement8 appear
to have answered that question and will likely influence the next
evidence evaluation documents. Second, is intrapartum suctioning
contraindicated, rather than simply not recommended as a routine
procedure? It seems unlikely that another larger control trial will
be conducted to answer this question and there appears to be little
evidence suggesting that intrapartum oropharyngeal suctioning is
particularly hazardous. And finally, is direct endotracheal
suctioning of the newborn of any description indicated following
birth? As the original observation that formed the basis for the
Neonatal resuscitation guidelines for ILCOR and NRP
J Kattwinkel
S29
procedure was uncontrolled and was published over 30 years ago,
and as there is increasing evidence that much of meconium
aspiration occurs well before birth and as essentially every report of
endotracheal suctioning since the report of Gregory et al. reports
some morbidity associated with the procedure, it would seem well
justified for there to be a modern-day large, randomized,
multicenter trial comparing endotracheal suctioning versus no
endotracheal suctioning of the nonvigorous meconium-stained
newborn immediately following birth. Until such a study is
performed, it is likely that subsequent NRP guidelines will continue
to recommend that the procedure be performed.
Disclosure
J Kattwinkel has received grant support from the American Academy of Pediatrics
and the National Institutes of Health.
References
1 Perlman JM, Kattwinkel J. Delivery room resuscitation: past, present, and future. Clin
Perinatol 2006; 33: 1–9.
2 International Liaison Committee on Resuscitation. 2005 International Consensus on
Cardiopulmonary resuscitation and emergency cardiovascular care science with
treatment recommendations. Circulation 2005; 112: III-1–III-136.
3 International Liaison Committee on Resuscitation. 2005 International Consensus on
Cardiopulmonary resuscitation and emergency cardiovascular care science with
treatment recommendations. Part 7. Neonatal resuscitation. Resuscitation 2005; 67:
293–303.
4 The International Liaison Committee on Resuscitation (ILCOR) consensus on science
with treatment recommendations for pediatric and neonatal patients: neonatal
resuscitation. Pediatrics 2006; 117: e978–e988.
5 American Heart Association (AHA) Guidelines for Cardiopulmonary Resuscitation
(CPR) and Emergency Cardiovascular Care (ECC) of pediatric and neonatal patients:
neonatal resuscitation guidelines. Circulation 2005; 112: IV-188–IV-195.
6 American Heart Association (AHA) Guidelines for Cardiopulmonary Resuscitation
(CPR) and Emergency Cardiovascular Care (ECC) of pediatric and neonatal patients:
neonatal resuscitation guidelines. Pediatrics 2006; 117: e1029–e1038.
7 Fraser WD, Hofmeyr J, Lede R, Faron G, Alexander S, Goffinet F et al. Amnioinfusion
for the prevention of the meconium aspiration syndrome. N Engl J Med 2005; 353:
909–917.
8 American College of Obstetrics and Gynecology (ACOG). Committee Opinion no. 346;
October, 2006.
9 Carson BS, Losey RW, Bowes WA, Simmons MA. Combined obstetric and pediatric
approach to prevent meconium aspiration syndrome. Am J Obstet Gynecol 1976; 126:
712–715.
10 Gregory GA, Gooding CA, Phibbs RH, Tooley WH. Meconium aspiration in
infantsFa prospective study. J Pediatr 1974; 85: 848–852.
11 Vain NE, Szyld EG, Prudent LM, Wiswell TE, Aguilar AM, Vivas NI. Oropharyngeal and
nasopharyngeal suctioning of meconium-stained neonates before delivery of their
shoulders: multicentre, randomised controlled trial. Lancet 2004; 364: 597–602.
12 Wiswell TE, Gannon CM, Jacob J, Goldsmith L, Szyld E, Weiss K et al. Delivery room
management of the apparently vigorous meconium-stained neonate: results of the
multicenter, international collaborative trial. Pediatrics 2000; 105: 1–7.
Journal of Perinatology
 Journal of Perinatology (2008) 28, S30–S35
r 2008 Nature Publishing Group All rights reserved. 0743-8346/08 $30
www.nature.com/jp
REVIEW
Developing a systems approach to prevent meconium aspiration
syndrome: lessons learned from multinational studies
VK Bhutani
Division of Neonatal and Developmental Medicine, Department of Pediatrics, Lucile Salter Packard Children’s Hospital, Stanford
University School of Medicine, Stanford, CA, USA
Passage of fetal bowel movement (meconium) is common (in about one
out of six births), and in some the staining of the amniotic fluid is a sign of
fetal distress. Inhalation of meconium (aspiration syndrome, in upto one
out of five to eight such births) just before or at birth may be preventable by
a coordinated approach by well-trained and informed birth attendants.
Respiratory failure secondary to meconium aspiration syndrome (MAS)
remains a major cause of morbidity and mortality in the neonatal
population. Infants with hypoxemic respiratory failure because of MAS,
persistent pulmonary hypertension of the newborn and pneumonia/sepsis
have an increased survival with extracorporeal membrane oxygenation
(ECMO). Other treatment options earlier limited to inotropic support,
continuous airway pressure (CPAP), conventional ventilatory management,
respiratory alkalosis, paralysis and intravenous vasodilators have been
replaced by synchronized intermittent mandatory ventilation (SIMV),
high-frequency oscillatory ventilation (HFOV), surfactant and inhaled nitric
oxide (iNO). HFOV has been advocated for use to improve lung inflation
while potentially decreasing lung injury through volutrauma. Other reports
describe the enhanced efficacy of HFOV when combined with iNO.
Subsequent to studies reporting that surfactant deficiency or inactivation
may contribute to neonatal respiratory failure, exogenous surfactant therapy
has been implemented with apparent success. Recent studies have shown
that iNO therapy in the neonate with hypoxemic respiratory failure can
result in improved oxygenation and decreased need for ECMO. However,
these innovative interventions are costly, require a sophisticated
infrastructure and are not universally accessible. In this paper, a context of
systems-approach for prenatal, natal and postnatal management of babies
delivered through meconium stained amniotic fluid (MSAF) so that adverse
outcomes are minimized and the least number of babies require innovative
ventilatory support is described. Previously reported data from a single
urban perinatal center (Philadelphia, PA, USA), over a 6-year period
(1995–2000), demonstrated that 14.5% (3370/23 175 of live births babies
were delivered with MSAF. These data also showed that 4.6% of babies (155/
3370) with MSAF sustained MAS. Overall, 26% of babies (40/155) with MAS
needed ventilatory support (or 0.17% of all live births); of these, only 20%
Correspondence: Dr VK Bhutani, Division of Neonatal and Developmental Medicine, Lucile
Salter Packard Children’s Hospital at Stanford University School of Medicine, 750 Welch Ave,
#315, Palo Alto, CA 94305, USA.
E-mail: bhutani@stanford.edu
(8/40 or 0.035% of live births) needed innovative ventilatory support. None
died or needed ECMO. These data describe the components for a systems
approach to prevent and manage adverse outcomes related to MSAF at the
regional level II or III perinatal center. Replication of a similar
strategy may be more relevant to cost containment and be a safer
approach for neonates at risk for MAS-related respiratory failure. This paper
assess the evidence for pivotal steps needed to prevent MAS and
ensuing neonatal death and disease in the context of diverse perinatal
health services.
Journal of Perinatology (2008) 28, S30–S35; doi:10.1038/jp.2008.159
Introduction
Optimizing the management of infants born with meconium
staining, relevant to the Indian macro- and micro-health
environment, may prevent adverse outcomes owing to perinatal
asphyxia, meconium aspiration syndrome (MAS), and ensuing
respiratory failure. The predictive risk factors for MAS in infants
delivered through meconium-stained amniotic fluid (MSAF) have
not been ascertained prospectively. On the basis of a retrospective
study reported by Usta et al.,1 odds ratio >3 was identified when
an infant’s delivery was associated with the (a) induction for
non-reassuring fetal heart rate (FHR) pattern, (b) need for
endotracheal intubation (ET), (c) Apgar p3 at 1 min or (d) need
for a Cesarean section (Table 1). This study informed and
influenced clinical delivery room practice for several years. In
addition, the management of meconium at the time of birthing
has been examined from two perspectives: (i) suctioning of the
meconium from an infant’s upper airway after delivery of the head
but before delivery of the shoulders (intrapartum suctioning,
oronasopharyngeal suctioning) and (ii) suctioning of an infant’s
trachea immediately after birth with an ET. Recommendations for
intrapartum suctioning for meconium had been based on
consensus of studies that have yielded conflicting results about the
value of intrapartum oronasopharyngeal suctioning of infants born
with MSAF. A more recent large multicenter randomized trial found
that intrapartum suctioning of meconium does not reduce the
incidence of MAS. The latter study has led the International Liaison

Table 1 Clinical risk factors for MAS in infants born through MSAF
Clinical risk factors
1 Induction for non-reassuring fetal heart rate
2 Need for intubation
3 Apgar p3 at age 1 min
4 Cesarean section
5 Previous cesarean section
Abbreviations: MAS, meconium aspiration syndrome; MSAF, meconium-stained amniotic
fluid.
Based on live births from 1990 to 1993 (Memphis, TN, USA) with MASF n ¼ 937 and
MAS in n ¼ 39 (4.2%).
Adapted from Usta et al.1
Committee on Resuscitation2 or ILCOR to recommend against
routine intrapartum oronasopharyngeal suctioning for infants born
with MASF. On the other hand, the current recommendation for ET
suctioning is based on a pivotal randomized, controlled trial that
showed that ET intubation and suctioning for vigorous infants at
birth offers no benefit. It is also known that the benefit of ET
suctioning in meconium-stained, depressed infants has not been
systematically studied. However, the current ILCOR treatment
recommendation is that meconium-stained, depressed infants
should receive ET suctioning immediately after birth and before
stimulation, presuming that the equipment and expertise is
available and that ET suctioning is not necessary for infants with
MASF who are vigorous. The relevance for application of ILCOR
recommendations to a population with diverse access to perinatal
health care has been questioned. The studies and
recommendations cited by ILCOR are reported from those
conducted in developed nations and nations with sophisticated
medicalized health-care systems. These recommendations may not
be applicable in areas with low income resources, births conducted
at home or limited perinatal access to evidence-based medical
care.3 Dramatic improvements in the overall birthing conditions
and the continuing advances in perinatal-neonatal practices
during the last decade have yet to universally affect the
unacceptably high risk of morbidity and mortality in the neonatal
population. Earlier experiences have led us to believe that building
an interdisciplinary leadership approach that lends itself to a
systems approach would provide innovative strategies to bridge the
existing access barriers in micro- and macro-health environments
of diverse social and cultural backgrounds.
Global perspective of births with MASF
Meconium-stained amniotic fluid is found in 7 to 20% of
pregnancies at the time of delivery.3–7 MSAF is associated with fetal
acidosis, abnormalities in FHRs and low Apgar scores, suggesting
hypoxia as the stimulant of passage of meconium in utero.8,9 The
most severe condition associated with meconium passage in utero,
Developing a systems approach to prevent MAS
VK Bhutani
S31
MAS, occurs in 2 to 9% of neonates born through MASF and has a
Odds ratio
high mortality rate of 40%.5,7,10–13 At delivery, meconium is found
below the vocal cords in 20 to 45% of neonates born through
6.9 MSAF.4,10,13,14 If it has not been aspirated into the lungs, the
4.9 removal of meconium from the airways before the first breath can
3.1 reduce the incidence of MAS. This preemptive removal of
3.0 meconium from the airways can be performed at the time of
2.5 delivery (intrapartum suctioning) or immediately after delivery
(postpartum suctioning). Intrapartum suctioning consists of
clearing the mouth, pharynx and nose with either a large-bore
suction catheter (12 to 14 mm caliber) or a bulb syringe as soon as
the head is delivered but before delivery of the shoulders.
Postpartum suctioning consists of intubating and suctioning the
trachea before performing the other steps of resuscitation.
Respiratory failure is a major cause of morbidity and mortality in
the neonatal population. Infants with hypoxemia develop
respiratory failure because of MAS, persistent pulmonary
hypertension of the newborn and pneumonia/sepsis. Recently,
Bhutani et al.15 suggested a system-based strategy comprising the
prenatal, natal and postnatal management of babies delivered
through MASF, so that the adverse outcomes are minimized and
the least number of babies require innovative ventilatory support.
At an urban perinatal center in Pennsylvania, over a 6-year period
(1995 to 2000), 14.5% (3370/23 175 of live-birth babies) were
delivered with MSAF (Figure 1). These data show that 4.6% of
babies (155/3370) with MSAF sustained MAS (Figure 2). Overall,
26% (40/155) of babies with MAS needed ventilatory support
(0.17% of all live births) (Figure 3); of these, only 20% (8/40 or
0.035% of live births) needed innovative ventilatory support. Infants
with low Apgar scores were more likely to need ventilatory support
(Figure 4). None died or needed extracorporeal life support.
Components of a systems approach to prevent MAS
Interdisciplinary health-care perspectives
Depending on the likelihood of MAS as a potential but significant
neonatal morbidity, the development of a ‘rapid response team’ was
envisioned as an institutional policy (prevalent in most US birthing
facilities) with the goal of modulating an intensive care nursery
census and also the risk of adverse neonatal outcomes. The
potential reduction in neonatal mortality and cost containment
would be the anticipated advantages of team-based interventions.
The team would constitute the obstetrician, pediatrician (and/or
neonatologist), the perinatal nurses (obstetrical and neonatal) and
the family, supported by the maternal child health administrators.
Although the obstetrician has a task-oriented responsibility to
take care of the mother and the family through the performance
and delivery of quality of care services in the delivery room, a
personal agenda is to strive for a ‘HAPPY’ family experience. This is
achieved through minimally atraumatic birthing with the
prevention of fetal hypoxemia and acidosis, and an effective
Journal of Perinatology
 Developing a systems approach to prevent MAS
VK Bhutani
S32

4500 90
83
4000 80
3500
70
Live births

3000

Number of babies
60
2500
2000 50
1500 40
1000 30 26
24
500
20
0 7 8 8
1995 1996 1997 1998 1999 2000 10
0
Figure 1 Annual incidence of meconium staining of the amniotic fluid (MSAF) Room air Oxygen CPAP SIMV SIMV+Surf HFV+iNO
(1995 to 2000) at a single US urban perinatal center: n ¼ 3370 infants with
MSAF/23 175 live births (15.5%). Of these, 945 infants (4.4%) were intubated Figure 3 Modes of respiratory support in newborns with meconium aspiration
(based on prevailing Neonatal Resuscitation Program recommendations). Open syndrome (MAS) (n ¼ 40 needed support/155 live births with MAS, 25.8%).
bars are annual live births; speckled bars are live births with MSAF.
120

100

Number of babies
Number of babies with MSAF

700
80
600
500 60
Ventilatory
400 Ventilatory
40 support (n =24/132)
support (n =16/23)
300
20
200 None
None
0
100 Low apgar score Normal apgar score

0
1995 1996 1997 1998 1999 2000 Figure 4 Effect of low Apgar score (<3 at age 1 min or <6 at age 5 min) on
need for ventilatory support in infants with meconium aspiration syndrome
Figure 2 Annual occurrence of meconium aspiration syndrome (MAS) (1995 to (MAS). Over a 6-year period, 1995 to 2000, n ¼ 24/132 (18.2%) infants with
2000) in infants delivered through meconium staining of the amniotic fluid normal scores need ventilatory support as compared with n ¼ 16/23 (69.5%) with
(MSAF) (n ¼ 155 infants with MAS/3370 infants with MSAF ((4.6%). Speckled low scores (P<0.001). Solid bars are infants with MAS who needed ventilatory
bars are infants with MSAF and solid bars are infants with MAS. support, and the open bars are infants who were observed for need of oxygen/
ventilatory support.

management of maternal and personnel stress. The perinatal
birthing nurse provides support, educates and cares for the family
during the changing birthing environment. A seemingly benign
process has the potential of disintegration as erstwhile birthing
plans are shelved. Participation in clinical management includes
fetal monitoring and appropriate availability and documentation of
instrumentation. A pediatrician needs to be accessible in a timely
manner for unpredictable and ‘stat’ calls and have the skills and
ability to perform (without anxiety) in an acute stressful response.
In addition, the pediatrician needs to clarify any confusion on the
amount of meconium staining and the status of fetal well-being,
and to make a decision of the likelihood of an elective neonatal
intubation. The family perspectives are overwhelmed by the sudden
medicalization of the birthing process and are aggravated by
anxiety, stress, interruption of bonding and loss of privacy with
unanticipated instrumentations and participation of often
unselected and unsolicited providers.
Performance and judgment of the ‘rapid response team’
Besides being awake and alert, the team needs to have a
physiologic understanding and risk assessment ability of the
clinical progression of MAS. Credentialing for neonatal
resuscitation, performance standards and intubation skills of the
team are now routine at all birthing facilities. The team needs to
acquire the prenatal history and the evolving perinatal events,
directly observe the natal events and render as well as execute
postnatal management decisions. The relevant prenatal history
acquired through the attending obstetrician and nurse would
include the intensity of meconium staining, status of fetal
well-being and FHR patterns and fetal pH (if measured) data on
the use of amnioinfusion, and risk factors for perinatal infections
and for any maternal-placental complications. The latter include a
prolonged second stage of labor, abruption of the placenta,
placenta previa or cord accidents (nuchal cords, true knots, cord
compression and cord prolapse), abnormal fetal presentations,
maternal hypertension, pre-eclampsia, intrauterine growth
retardation, post-maturity or placental calcifications. FHR
monitoring is a predominant method to assess fetal oxygenation in
labor; however, it is a nonspecific, indirect and inaccurate measure
of fetal hypoxia and acidosis. Electronic fetal monitoring is
associated with increased Cesarean section because of
‘non-reassuring’ FHR patterns. These include FHR between 100
and 120 with no accelerations, FHR <100 beats/min with
accelerations, increased heart rate variability of >25 beats/min for

Journal of Perinatology

>30 min, late decelerations (>1 per 30 min), persistent late
decelerations (>50% of contraction), fetal tachycardia
(>160 beats/min) or a sinusoidal pattern. Natal observations
provide an assessment of fetal handling during instrumentation
(such as vacuum extractions, forceps delivery and neck
manipulation as well as the handling of the baby’s head by the
obstetrician, and the presence of nuchal cords. Once the infant’s
head is delivered, the activity and vigor status and the resuscitation
process are initiated, as indicated. Once the infant is stabilized, the
umbilical cord status is ascertained meconium staining, presence
of venous clots and insertion at the placenta.
Postnatal management decisions
When meconium is present in the amniotic fluid, the current
ILCOR recommendations are to defer suctioning of the
hypopharynx on delivery of the head. If the meconium-stained
newly born infant has absent or depressed respirations, heart rate
or muscle tone, residual meconium should be suctioned from the
trachea. Ventilation is of primary concern. The role of intrapartum
suctioning has been a subject of controversy and recent research.
Studies in favor of intrapartum suctioning. Intrapartum
suctioning has been considered standard practice for more than
25 years on the basis of a study by Carson et al.16 In this
before-and-after trial, they reported an incidence of 1.9%
(n ¼ 947) for MAS with a mortality rate of 28% during a period
when only postpartum suctioning was performed, as compared
with MAS incidence of 0.4% (n ¼ 273, P ¼ 0.07) and no deaths
when intrapartum suctioning was also performed in addition
to postpartum suctioning. The effectiveness of intrapartum
suctioning was confirmed in a subset of patients enrolled in a
multicenter randomized controlled trial looking into the efficacy
of postpartum suctioning in vigorous newborns.17 In this study by
Wiswell et al., the incidence of MAS was 8.5% in infants who
did not have intrapartum suctioning (n ¼ 94), as compared
with 2.7% in infants who had intrapartum suctioning (n ¼ 54;
P ¼ 0.013).
Equivocal studies. Two prospective and non-randomized clinical
trials by Falciglia et al.10,14 compared early suctioning (suctioning
by the obstetrician before delivery of the thorax) and late
suctioning (suctioning by the obstetrician after delivery of the
thorax) and showed no difference in the incidence of MAS. In the
first study, no differences in the rate of meconium below the cords
(36 vs 37%) or the incidence of MAS (20% in each group) between
early and late suctioning were noted.8 The second study reported a
higher rate of meconium below the cords (53%) among the early
suctioning group compared with the late suctioning group, which
had a rate of 36% (P<0.001), but there was no difference in the
incidence of MAS between the two groups (P>0.05).13 Rossi et al.13
reported that despite intrapartum suctioning, meconium was
Developing a systems approach to prevent MAS
VK Bhutani
S33
present in the trachea in 37% (n ¼ 238) of neonates born through
MSAF, and MAS developed in 9.2% of cases.
Studies that do not recommend intrapartum suctioning.
A large multicenter, randomized controlled clinical trial enrolled
2514 patients with MSAF with the desired goal of assessing the
effectiveness of intrapartum suctioning for the prevention of MAS.18
This study also examined the effect of intrapartum suctioning in
high-risk subgroup infants (those with thick MSAF) with abnormal
FHR patterns, infants with delivery by Caesarian section and infants
needing resuscitation in the delivery room. This study showed that
intrapartum suctioning does not reduce the incidence of MAS
(relative risk (RR): 0.9, 95% confidence interval (CI): 0.6 to 1.3),
need for mechanical ventilation (RR: 0.8, 95% CI: 0.4 to 1.4) and
mortality (RR: 0.4, 95% CI: 0.1 to 1.5).
Current ILCOR recommendations. On the basis of these large
studies, the current guidelines of the American Academy of
Pediatrics and the American Heart Association through the
Neonatal Resuscitation Program and Pediatric Working Group of
the ILCOR1 no longer recommend routine intrapartum
oronasopharyngeal suctioning by an obstetrician before delivery of
the shoulder. Discretionary intrapartum suction also remains a
standard of care. This decision is not based on a meta-analysis,
but, on the basis of consensus of opinions that are most relevant to
the care of infants already affected, improved the dating of
pregnancy by a lowering of the gestational age at birth (fewer
deliveries for pregnancies >41 weeks), earlier recognition of fetal
distress and improved access to sophisticated birthing facilities in
developed nations. The other currently recommended approach to
prevent MAS is immediate postnatal ET suctioning in non-vigorous
(depressed) babies born through MSAF. In nations and
communities with improved perinatal health-care access, an
observational and non-intervention strategy is indicative of
eminent and cautious practice. Mandatory intrapartum suctioning
in babies born through MSAF is being discontinued, largely on the
basis of a single large randomized controlled study conducted in a
setting with facilities for intensive fetal monitoring, prompt
response to fetal distress and 24 h availability of personnel trained
in neonatal resuscitation, including postnatal intubation and ET
meconium suctioning.
Impact of different clinical settings. Most of the deliveries in
these countries take place either at home or at ill-equipped and
understaffed hospitals. The profile of women and their neonates
born in a developing country setting contrasts sharply with that of
developed countries. Even in the study by Vain et al.,18 which
included 11 sites in Argentina, a middle-income country, the
clinico-epidemiologic profile was not comparable with the
diverse environment and communities encountered in India
(see Table 2). The Indian experiences document high rates of
Journal of Perinatology
 Developing a systems approach to prevent MAS
VK Bhutani
S34
Table 2 Comparison of data from an Indian Database3 reported by Vain et al.18
Variable NNPD3
18 sites in India
Babies with MSAF (n ¼ 12 156)
Birth weight (g, mean±s.d.) 2646±552
Small for gestation 1566 (12.9%)
Cesarean section 5208 (42.8%)
Pregnancy-induced hypertension 961 (7.9%)
Eclampsia 177 (1.5%)
Oligohydramnios 360 (3.0%)
Fetal bradycardia (<120 b.p.m.) 1523 (12.5%)
Fetal tachycardia (>160 b.p.m.) 369 (3.0%)
Meconium aspiration syndrome 1896 (15.6%)
Abbreviations: MSAF, meconium-stained amniotic fluid; NNPD, National Neonatal Perinatal Database.
pregnancy-related complications such as pregnancy-induced
hypertension, oligohydramnios, intrauterine growth retardation
and post-maturity. In addition, in the Indian settings, postnatal
intubation and ET suctioning of non-vigorous infants could be
jeopardized if discretionary recommendations are made without
specific, practical and easily implemented algorithms.
Low- and middle-income nations account for the majority of
the 130 million live births and 4 million neonatal deaths occurring
each year.19 Therefore, we speculate that the relatively simpler and
easier techniques to perform intrapartum suctioning and the
availability of appropriate biotechnologies, such as large-bore,
portable, disposable and easily manipulated suction devices for the
mouth, nose and/or trachea that are non-traumatic, sterile and
independent of wall suction, may still have a role in the prevention
of MAS, whereas improvements of perinatal health-care access are
initiated and implemented.
Understanding immediate postnatal tachypnea and clinical
signs
The most frequent benign etiology for tachypnea is respiratory
compensation for metabolic acidosis as evidenced by hypocapnia
and normoxemia. On the other hand, onset of respiratory disease
would be evident by hypercapnia and hypoxemia. Worrisome
tachypnea could be due to air leaks (pneumothoraces and
pneumomediastinum), atelectasis, aspiration, pneumonia and,
most of all, concern for incipient persistent pulmonary
hypertension. Peripheral cyanosis (manifested by blue toes) would
require continuous pulse oximetry to document normoxemia and
observation for expectant care. Central cyanosis (as manifested by
blue lips) would require an evaluation for hemodynamic changes
with handling, evaluation of response to inspired oxygen and
continued observation in a nursery environment for continued
cardiorespiratory environment. Crystalloid infusion may be helpful
for infants with ‘benign tachypnea’ if the capillary perfusion is
Journal of Perinatology
Vain et al.18
11 sites in Argentina and 1 in the United States
Intrapartum suction+ (n ¼ 1263) Intrapartum suction (n ¼ 1251)
3413±483 3400±496
39 (3%) 48 (4%)
401 (32%) 398 (32%)
65 (5%) 65 (5%)
17 (1%) 20 (2%)
145 (11%) 130 (10%)
52 (4%) 47 (4%)
compromised. In such conditions, the measurements of arterial
blood gases or chest radiographs may not be necessary. If
metabolic acidosis is documented for persistent tachypnea with
hypoxemia, base correction with bicarbonate is not essential and
crystalloid infusion is likely to be sufficient. Persistent tachypnea-
associated hypoxemia and hypercapnia would be indications for a
chest radiograph evaluation. When an infant is stabilized, postnatal
gastric suction, presumably to prevent postnatal aspiration, is not
evidence-based and, similarly, gastric lavage is not necessary.
Infants do need to be monitored for airway stability and subsequent
choking/bradycardia.
Subsequent postnatal management
Infants are likely to need close glucose monitoring for hypoglycemia
because of concomitant risk factors. Intravenous dextrose infusions
and delayed enteral nutrition need to be considered for persistent
tachypnea (owing to hypoxemia and/or hypercapnia), low Apgar
scores and hypoglycemia. Screening for sepsis would be based on the
presence of known maternal risk factors or an abnormal chest
radiograph. The need for continued cardiorespiratory monitoring,
parenteral nutrition and concerns for sepsis are the most common
reasons for continued neonatal observation and management. Within
about 30 min, age, and color changes should have stabilized.
Similarly, within about 6 h, age, any transient grunting, flaring and
retractions should stabilize. Deviations from these time frames should
initiate a process of a more intense evaluation. The absence of any
signs of sepsis or cardiorespiratory distress beyond 24 h of age is likely
to exclude the MAS.
Conclusions
A systems approach that provides for a well-coordinated and trained
‘rapid response team’ for meconium-stained births is likely to
implement an evidence-based intervention and effectively manage

a potential catastrophic outcome for the infant, the family and the
staff. Classic practices of maintaining equipoise in the delivery
room require working (and communicating) with the attending
obstetrician and nursing staff and supporting the education of the
family. The team’s approach is to be expectant but prepared to
intubate the infant. The team’s role is to monitor the progress of
labor, fetal status, fetal data and family status and to be prepared to
interact, interpret, intervene and initiate support as needed. These
data from a single US center also illustrate the complex
interactions of technologies used during a birthing practice that
could have both potentially useful (airway clearance) and/or
deleterious effects because of the combinations of trauma,
introduction or aggravation of infection and/or interference of gas
exchange. Several of the components of clinical practice are based
on evidence, and their implementation in practice may need
further validation or resolution by consensus. There is a health and
societal need to establish guidelines for practice at birthing
facilities in the United States. Consistency in practice may lead to
cost containment, even though the cost-effectiveness of such an
approach has not been validated. Methodological inquiry,
evidentiary analysis and reassessment of both clinical and
biotechnological options would better inform the development of a
practical stepwise algorithm to optimize bedside care.
Disclosure
The author has declared no financial interests.
References
1 Usta IM, Mercer BM, Sibai BM. Predictive risk factors for meconium aspiration
syndrome. Obstet Gynecol 1996; 86: 230–234.
2 International Liaison Committee on Resuscitation (ILCOR). Consensus on science with
treatment recommendations for pediatric and neonatal patients: part 7Fneonatal
resuscitation. Pediatrics 2006; 117: e978–e988.
Developing a systems approach to prevent MAS
VK Bhutani
S35
3 National Neonatal-Perinatal Database (NNPD). Morbidity and mortality among
outborn neonates at 10 tertiary care institutions in India during the year 2000. J Trop
Pediatr 2004; 50: 170–174.
4 Dooley SL, Pesavento DJ, Depp R, Socol ML, Tamura RK, Wiringa KS. Meconium below
the vocal cords at delivery: correlation with intrapartum events. Am J Obstet Gynecol
1985; 153: 767–770.
5 Davis RO, Philips III JB, Harris Jr BA, Wilson ER, Huddleston JF. Fatal meconium
aspiration syndrome occurring despite airway management considered appropriate. Am
J Obstet Gynecol 1985; 151: 731–736.
6 Wiswell TE, Henley MA. Intratracheal suctioning, systemic infection, and the
meconium aspiration syndrome. Pediatrics 1992; 89: 203–206.
7 Chaturvedi P, Yadav B, Bharambe MS. Delivery room management of neonates born
through meconium stained amniotic fluid. Indian Pediatr 2000; 37: 1251–1255.
8 Mahomed K, Nyoni R, Masona D. Meconium staining of the liquor in a low-risk
population. Paediatr Perinat Epidemiol 1994; 8: 292–300.
9 Ziadeh SM, Sunna E. Obstetric and perinatal outcome of pregnancies with term labour
and meconium-stained amniotic fluid. Arch Gynecol Obstet 2000; 264: 84–87.
10 Falciglia HS. Failure to prevent meconium aspiration syndrome. Obstet Gynecol 1988;
71: 349–353.
11 Yoder BA. Meconium-stained amniotic fluid and respiratory complications: impact of
selective tracheal suction. Obstet Gynecol 1994; 83: 77–84.
12 Vidyasagar D, Harris V, Pildes RS. Assisted ventilation in infants with meconium
aspiration syndrome. Pediatrics 1975; 56: 208–213.
13 Rossi EM, Philipson EH, Williams TG, Kalhan SC. Meconium aspiration syndrome:
intrapartum and neonatal attributes. Am J Obstet Gynecol 1989; 161: 1106–1110.
14 Falciglia HS, Henderschott C, Potter P, Helmchen R. Does DeLee suction at the
perineum prevent meconium aspiration syndrome? Am J Obstet Gynecol 1992; 167:
1243–1249.
15 Bhutani VK, Chima R, Sivieri EM. Innovative neonatal ventilation and meconium
aspiration syndrome. Indian J Pediatr 2003; 70: 421–427.
16 Carson BS, Losey RW, Bowes Jr WA, Simmons MA. Combined obstetric and pediatric
approach to prevent meconium aspiration syndrome. Am J Obstet Gynecol 1976; 126:
712–715.
17 Wiswell TE, Gannon CM, Jacob J, Goldsmith L, Szyld E, Weiss K et al. Delivery room
management of the apparently vigorous meconium-stained neonate: results of the
multicenter, international collaborative trial. Pediatrics 2000; 105: 1–7.
18 Vain NE, Szyld EG, Prudent LM, Wiswell TE, Aguilar AM, Vivas NI. Oropharyngeal and
nasopharyngeal suctioning of meconium-stained neonates before delivery of their
shoulders: multicentre, randomised controlled trial. Lancet 2004; 364: 597–602.
19 Lawn JE, Cousens S, Zupan J. 4 million neonatal deaths: when? Where? Why? Lancet
2005; 365: 891–900.
Journal of Perinatology
 Journal of Perinatology (2008) 28, S36–S42
r 2008 Nature Publishing Group All rights reserved. 0743-8346/08 $30
www.nature.com/jp
REVIEW
Meconium aspiration syndrome requiring assisted ventilation:
perspective in a setting with limited resources
S Velaphi and A Van Kwawegen
Department of Paediatrics, Chris Hani Baragwanath Hospital, University of the Witwatersrand, Johannesburg, South Africa
To determine characteristics, management, complications and outcome of
neonates with meconium aspiration syndrome (MAS) requiring mechanical
ventilation (MV). A retrospective review of clinical data of neonates with
MAS who were admitted to a public hospital for MV between January 2004
and December 2006. Eighty-eight neonates were ventilated for MAS. Thirty-
one percent were postdates and 51% had no electronic fetal monitoring.
Postnatal suctioning of meconium was not performed according to protocol
in 47% of nonvigorous infants. High-frequency ventilation and surfactant
were used in 32 and 14% of cases, respectively. Persistent pulmonary
hypertension of the newborn (PPHN) and pneumothorax occurred in 57
and 24% of cases, respectively. Overall mortality rate was 33%. Neonates
suffering from MAS with PPHN had higher mortality rate of 48% compared
with 13% in those suffering from MAS without PPHN. Factors associated
with mortality were peak inspiratory pressure (P<0.001), pneumothorax
(P<0.001) and PPHN (P ¼ 0.001). Postdates, inadequate intrapartum
monitoring and limited use of adjunct respiratory therapies were common.
Severe MAS is associated with adverse outcome.
Journal of Perinatology (2008) 28, S36–S42; doi:10.1038/jp.2008.155
Introduction
Meconium aspiration syndrome (MAS) is associated with significant
morbidity and mortality. Common complications associated with
MAS include interstitial emphysema, pneumothorax, pneumonia
and persistent pulmonary hypertension of the newborn (PPHN).
Pulmonary air leaks have been observed to occur in 15 to 33% of
cases of MAS.1,2 Although MAS is still associated with significant
mortality, mortality rates seem to have decreased over the years.
Studies from 1970s and 1980s reported high mortality rates ranging
from 28 to 40%.3,4 Recent literature has reported mortality rates less
than 15%.1,2,5 This reduction in mortality rates appears to be related
(1) to reduction in incidence of MAS, which is related to changes in
obstetric practices6,7 and (2) to use of adjunct respiratory therapies,
such as exogenous surfactant, nitric oxide and high-frequency
ventilation in the management of neonates with MAS.8–10 Yoder
Correspondence: Dr S Velaphi, Department of Paediatrics, Chris Hani Baragwanath Hospital,
University of the Witwatersrand, PO Bertsham, Johannesburg 2013, South Africa.
E-mail: velaphisc@medicine.wits.ac.za
et al.6 reported that MAS decreased nearly fourfold between 1990
to1992 and 1997 to 1998, and this was associated with 33%
reduction in births more than 41 weeks gestation, more frequent
diagnosis of nonreassuring fetal heart rate patterns and greater use
of amnioinfusion. Approximately 50% of patients with MAS will have
severe MAS defined as the need for mechanical ventilation (MV).11
The reports on the outcome of patients with MAS, especially those
who require MV, originate mainly from developed countries. Factors
such as poor monitoring during labor and low Apgar scores that are
associated with the need for MV among infants with MAS11 occur
commonly in developing countries; therefore, patients with MAS in
developing countries might have worse outcomes compared with
those from developed countries. Secondly, the adjunct respiratory
therapies that have been associated with reduction in mortality are
not easily available in developing countries. The limitations of
assessing the outcomes of MAS in developing countries are related to
limited resources resulting in difficulties in making a diagnosis of
MAS, which requires the presence of X-ray changes. In developing
countries, patients with possible MAS might die before a chest X-ray
(CXR) can be taken or might improve and be discharged without
having had a CXR unless they are admitted to the neonatal
intensive care unit (NICU) for MV. Therefore, patients who are
admitted to NICU are more likely to have CXR. The aim of this
study was to describe characteristics, management and outcome of
neonates with MAS in a setting where resources are limited. Owing
to difficulties in reliably diagnosing MAS in patients who do not
require MV, this study was limited to those who required MV.
Methods
Infants with severe MAS were identified by review of discharge
diagnoses and hospital records of all patients who weighed
X2000 g and were admitted to the NICU of Chris Hani
Baragwanath Hospital, Johannesburg, South Africa, between
January 2004 and December 2006. Maternal and neonatal hospital
charts were reviewed for confirmation of the diagnosis and
pertinent clinical data.
Severe MAS was diagnosed if infants met the three following
criteria: meconium-stained amniotic fluid (MSAF), need for MV
and radiographic abnormalities consistent with MAS (Figure 1).

Figure 1 Chest X-ray representative of X-rays that were selected toward making a diagnosis of meconium aspiration syndrome.
Patients were excluded from the study if there was no
documentation of the presence of MSAF, if records were incomplete,
if CXR was not available from records and no comment was
present on CXR findings in doctor’s notes or if the baby had
congenital abnormalities.
During the study period, the protocol on postnatal management
of infants born through MSAF was according to the guidelines of
the International Liaison Committee on Resuscitation.12 The
guidelines stated that for all infants born through MSAF with
absent or depressed respiration, direct laryngoscopy should be
conducted immediately after birth for suctioning of residual
meconium from the hypopharynx and for intubation and
suctioning of the trachea. All patients who were admitted with
respiratory distress were started on antibiotics; patients who
required MV were started on synchronized intermittent mandatory
ventilation using a peak inspiratory pressure of 20 to 25 cm H2O,
positive end expiratory pressure of 4 to 5 cm H2O, inspiratory time
of 0.4 s and flow of 5 to 10 l min1. There were two models of
conventional ventilators used in the unit: the Bear Cub 750PSV
infant ventilator and Newport E100M ventilator. The mode of
control for the conventional ventilators was pressure limited and
time cycled. There were two ventilators available for high-frequency
ventilation, both of them were Sensor Medics 3100A oscillatory
ventilators. All patients had CXRs ordered immediately
postintubation or after insertion of umbilical arterial/venous
catheters or when patient deteriorated. Sometimes, there were
delays in having X-rays taken especially at night or over the
weekends because of staff (radiographers) shortages. Availability of
surfactant was limited to 5 vials per month in 2004, and this was
increased to 10 vials per month in 2005. Nitric oxide was made
available to the unit at the end of 2004. We are limited to use two
tanks of nitric oxide per year, with each tank of 10 l containing 900
parts per million of nitric oxide. Both the gas and the delivery and
monitoring system were bought from Sidewinder Medical, Cape
Town, South Africa. It was only used in patients who had hypoxia
thought to be secondary to persistent pulmonary hypertension of
the newborn, and it was stopped if there was no response in
Meconium aspiration syndrome and ventilation
S Velaphi and A Van Kwawegen
S37
60 min. PPHN was diagnosed either with a preductal versus
postductal pulse-oximeter oxygen saturation gradient of >10% or
on echocardiography.
Demographic characteristics, clinical data and neonatal
outcomes to NICU discharge were recorded. Comparison between
survivors and nonsurvivors was performed using w2 or Fisher exact
test for categorical variables and Student’s t-test for continuous
variables. Permission to conduct the study was received from the
University of the Witwatersrand Human Research Ethics
Committee.
Results
Among the 2157 infants who required MV from January 2004 to
December 2006, 800 weighed X2000 g. Of these 800 infants, 143
had a diagnosis of MAS or had an abnormal CXR with changes
suggestive of MAS and were admitted within 48 h of life. Among the
143 patients, 55 were excluded. The reasons for exclusion were
absence of a history of MSAF, incomplete hospital records, and
therefore inability to confirm or exclude the presence of MSAF, and
CXR not found or no comment in patient’s file regarding CXR
findings (Figure 2). There were 88 patients who had complete
records, with history of MSAF in mother’s or infant’s hospital file,
changes on CXR and were ventilated; these were included in the
study. These 88 patients accounted for 4% of all admissions
requiring MV and 11% of NICU admissions weighing X2000 g.
There were 61 399 live births over this 3-year period. The incidence
of severe MAS or of infants with MAS requiring MV was 1.43 per
1000 live births.
Characteristics of mothers to infants with MAS are shown in
Table 1. Ninety-eight percent of patients were born inside a
healthcare facility and only 2% were born at home. The maternal
HIV status was positive in 31% of cases, which was similar to the
institutional statistics from antenatal clinic. Thirty-one percent
were born at X41 weeks gestation. Electronic monitoring was
documented in 49% of cases, of which 78% had abnormal tracing
and the common abnormality was late decelerations. Forty-four
Journal of Perinatology
 Meconium aspiration syndrome and ventilation
S Velaphi and A Van Kwawegen
S38

Total number of live births from January 2004 to December 2006 Table 1 Maternal characteristics of infants with MAS requiring ventilation
(n= 61399)
Number (%)

Maternal age
<18 years 4 (5)
Total neonatal intensive care unit (NICU) admissions
(n = 2157) 18–35 years 68 (77)
>35 years 13 (15)
Not recorded 3 (3)

NICU admissions with birth weight ≥2kg Parity

(n = 800) 0 40 (45)
1–3 37 (42)
>3 6 (7)
Not recorded 5 (6)
Birth weight .2kg + Diagnosis of MAS and/ or chest X-ray (CXR)
suggestive of meconium aspiration syndrome (MAS) Human immunodeficiency virus
(n = 143)
Positive 27 (31)
Negative 50 (57)
Unknown 11 (12)
MSAF could not be MSAF MSAF
excluded or confirmed present absent
(n=12) (n=101) (n =30) Antenatal care
Yes 74 (84)
No 14 (16)

Monitoring with CTG

CXR available and No CXR or no record of
Yes, with abnormalities 33 (38)
abnormal X-ray findings
(n=88) (n =13) Yes, with no abnormalities 10 (11)
No 45 (51)
Figure 2 Number of live births, patients requiring mechanical ventilation and
those excluded or included in the study. Gestational age by dates
<35 weeks 2 (2)
percent were born by cesarian section, and the indication for the 35–37 weeks 6 (7)
cesarian section was fetal distress in 90% of cases. Although 38–40 weeks 39 (44)
consistency of MSAF was not recorded in 44% of patients, the X41 weeks 27 (31)
majority of them were born through thick meconium. Not recorded 14 (16)
Infant details are shown in Table 2. There were 38 (43%)
Place of birth
patients who required resuscitation, with almost all of them
Inborn 75 (85)
responding to bag mask ventilation. The number of patients who
Referrals from other hospitals 5 (6)
required resuscitation was greater than those with low Apgar score Clinic 6 (7)
(score p5) at 1 min, suggesting over-reading of Apgar scores or Home 2 (2)
reflecting our practice of withholding assisted ventilation from
infants with the lowest Apgar scores because of concerns about poor Mode of delivery
neurodevelopmental outcome. Among those who required Normal vaginal delivery 43 (49)
resuscitation, only 53% were recorded as having been intubated for Cesarian section 39 (44)
suctioning of meconium. Severity of the lung disease was moderate Vacuum 5 (6)
to severe in 64% of patients as defined by an oxygen index of more Breech 1 (1)
than 10. Twenty-three patients out of the 28 who did not have
Consistency of MSAF
C-reactive protein (CRP) results are those who died on day 1 of life
Thick 47 (53)
before CRP level could be done. Among those who had CRP results,
Thin 6 (7)
92% had CRP levels more that 10 mg l1.
Unrecorded 35 (40)
Morbidity and mortality of infants with MAS are shown in
Table 3. Common complications among patients with MAS were
PPHN (57%) and pneumothorax (24%). Of the 50 patients who

Journal of Perinatology
 Meconium aspiration syndrome and ventilation
S Velaphi and A Van Kwawegen
S39

Table 2 Characteristics of infants with MAS requiring mechanical infants Table 3 Complications, duration of stay in NICU and outcome of patients with
MAS requiring mechanical ventilation (n ¼ 88)
Number (%)
Number (%)
Birth weight (g) 3080 (2100–4330)a
Complications
Gender Pneumothorax 21 (24)
Female 45 (51) PPHN 50 (57)
Male 43 (49)
Median duration of stay in NICU (days) 4 (1–31)a
1 min Apgar score 6 (1–9) a p3 days 33 (38)
<5 19 (21) 4–7 days 35 (40)
X5 64 (73) 8–14 days 10 (11)
Not recorded 5 (6) >14 days 10 (11)

5 min Apgar score 8 (3–10)a Outcome among all patients (n ¼ 88)

<7 21 (24) Died 29 (33)
X7 54 (61) Survived 59 (67)
Not recorded 13 (15)
Outcome among those without PPHN (n ¼ 38)
Required resuscitation Died 5 (13)
No 50 (57) Survived 33 (87)
Yes 38 (43)
Bag mask ventilation 38 (43) Outcome among those with PPHN (n ¼ 50)
Bag mask ventilation+chest compression 1 (1) Died 24 (48)
Above+adrenalin 0 Survived 26 (52)

Intubated and suctioned for meconium 20/38 (53) Abbreviations: MAS, meconium aspiration syndrome; NICU, neonatal intensive care unit;
PPHN, persistent pulmonary hypertension of the newborn.
a
Median (range).
Median oxygen index (OI) (range) 15.17 (0.97–71.16)a
Number of patients with OI <10 32 (36)
Number of patients with OI between 10 and 20 42 (48)
Number of patients with OI >20 14 (16)
The use of exogenous surfactant, number of patients who were
C-reactive protein (CRP) (range) 52.9 (1–567) a put on high-frequency oscillatory ventilation and nitric oxide were
Number of patients with CRP<10 mg l1 5 (6) low during the study period, and it did not change significantly
Number of patients with CRP between 10 and 20 mg l1 8 (9) over the years (P-values all more than 0.100) (Table 5). Incidence
Number of patients with CRP >20 mg l1 47 (53) of pneumothorax, PPHN and mortality rate did not change
Number of patients with unrecorded CRP 28 (32) significantly over the years.
Abbreviations: CRP, C-reactive protein; MAS, meconium aspiration syndrome; OI, oxygen index.
a
Median (range).
Discussion
Severe MAS is defined as those cases that require assisted MV.13 It
has been reported that between 40 and 50% of infants with MAS
had PPHN, 47 were diagnosed by differential pulse oximetry will need MV.11,14,15 In public hospitals of developing countries,
saturations and only 17 (36%) had echocardiography, which offering MV is often restricted to those who are expected to have
confirmed the diagnosis of PPHN, and the other 30 did not have favorable outcomes, and among those who qualify, it is often
echocardiography because of limited human resources. Three were delayed due to limited bed availability in NICUs. Thus, patients
diagnosed only on echocardiography. Median duration of stay in who are ventilated in countries with limited resources are more
NICU was 4 days, with 22% of them staying for more than 7 days. likely to have poor outcome because of delays in instituting
One-third of patients with MAS did not survive. Factors associated treatment, limited adjunct respiratory therapies and high incidence
with mortality were female gender (P ¼ 0.046), maximum peak of having associated conditions such as infections. In this study, we
inspiratory pressure (P<0.001), development of pneumothorax sought to determine characteristics and outcome of infants who
(P<0.001) and PPHN (P ¼ 0.001) (Table 4). Most of the deaths were ventilated with MAS, in a setting where there are limited
occurred early during their NICU stay. resources.

Journal of Perinatology
 Meconium aspiration syndrome and ventilation
S Velaphi and A Van Kwawegen
S40

Table 4 Comparison between survivors and nonsurvivors

Nonsurvivors (n ¼ 29) Survivors (n ¼ 59) P-value

Characteristics
Mean birth weight 2976±546 3128±429 0.157
Gestational age 40 (32–43) 40 (35–43) 0.843
Apgar score at 1 min 6 (1–9) 7 (2–9) 0.582
Apgar score at 5 min 8 (3–10) 8 (4–10) 0.360
Male:female 1:2.1 1:0.74 0.046
Mother human immunodeficiency virus positive 21% 36% 0.238
Requiring resuscitation at birth 38% 46% 0.640
Intubated for suctioning at birth 10/11 (91%) 16/27 (59%) 0.121
Peak pressure required on admissiona,b 26 (25–29) 25 (25–29) 0.070
OI on admission 15.6 (8.1–21.7) 12.2 (7.6–18.5) 0.183

Patients at different levels of OIs at admission 0.413

Number of patients with OI <10 8 (28%) 24 (41%)
Number of patients with OI 10–20 11 (38%) 21 (35%)
Number of patients with OI >20 10 (34%) 14 (24%)

Median maximum peak pressurea,b 32 (30–35) 28 (25–32) <0.001

Complications
Pneumothorax 15 (52%) 6 (10%) <0.001
PPHN 24 (83%) 26 (44%) 0.001
Duration of stay in NICU 2 (1–12) 10 (2–25) <0.001
Abbreviations: NICU, neonatal intensive care unit; OI, oxygen index; PPHN, persistent pulmonary hypertension of the newborn.
a
Peak pressure is in cm H2O.
b
Median (25th to 75th percentile).

Table 5 Use of respiratory adjunct therapies, complications and mortality rate over the study period

Year 2004 (n ¼ 34) Year 2005 (n ¼ 22) Year 2006 (n ¼ 32) All

Number of patients who received surfactant 2 (6%) 4 (18%) 6 (19%) 12 (14%)

Number of patients who were put on high-frequency oscillation ventilator 7 (21%) 10 (45%) 11 (34%) 28 (32%)
Number of patients who were put on nitric oxide 2 (6%) 1 (3%) 2 (6%) 5 (6%)
Number of patients with PPHN 22 (64%) 15 (68%) 13 (41%) 50 (57%)
Number of patients with pneumothorax 10 (29%) 4 (18%) 7 (22%) 21 (24%)
Number of deaths 14 (41%) 5 (23%) 10 (31%) 29 (33%)
Abbreviation: PPHN, persistent pulmonary hypertension of the newborn.

About one-third of patients with severe MAS were born at 41
weeks gestation or more; and 51% of patients were born to mothers
who did not have electronic monitoring during labor despite
having a history of MSAF. Postmaturity may have contributed to
these patients developing MAS. The risk of MAS has been shown to
be higher in post-term than term infants with relative risk of 3.1
with 95% confidence interval of 2.6 to 3.7.16 Inadequate
monitoring might have led to delays in expediting delivery of
infants who were compromised, and this allowed for ongoing
hypoxia resulting in severe disease. The effect of postmaturity and
monitoring during pregnancy and labor on MAS is supported by
reduction of MAS in institutions where postmaturity has been
reduced and monitoring is intensive.6
The protocol of intubating and suctioning infants suffering
from absent or depressed respiration was not followed consistently,
as only 53% of infants who should have been intubated according
to the protocol were actually intubated. The possible reasons for
this low rate of suctioning could be poor documentation of what
happens during resuscitation, or that junior doctors who
commonly conduct neonatal resuscitation in our hospital were not

Journal of Perinatology

confident enough to perform intubations and therefore not
adhering to protocol. Inadequate suctioning of meconium could
have contributed to the development of severe MAS, as it has been
reported that meconium in the trachea is one of the major risk
factors associated with diagnosis of MAS.11,17 –20 It is also possible
that this did not affect the development of severe MAS, as some
studies have suggested that aspiration of meconium occurs
in utero and that damage in the lungs has already taken place by
the time the baby is born.1,19,21–23
The high mortality rate of 33% in this study could be related to
associated conditions, such as infections and PPHN, and low usage
of adjunct respiratory therapies. Among the patients who had their
CRP level assessed, 92% had CRP >10 mg l1 and 78% had CRP
>20 mg l1, suggesting the presence of infection or inflammation.
In vitro studies have suggested that meconium may activate
alveolar macrophages with subsequent release of inflammatory
cytokines resulting in an increase in CRP.24 Although only one
patient had positive culture, it is difficult to exclude infection when
there is abnormal CXR and high CRP. Some studies have reported
that clinical chorioamnionitis and neonatal sepsis are more
common in neonates with severe MAS.11,25,26 More than 50% of
patients in this study had PPHN. Should one exclude or include
infants with PPHN when one is looking at outcome of infants with
MAS? The literature suggests that these patients should be excluded.
The reason for this is that the cause of PPHN in patients with MAS
might have preceded the aspiration of meconium. This is supported
by findings in autopsies from cases dying from severe MAS within
48 h of birth, which revealed muscularization of distal pulmonary
arterioles, which requires 3 to 8 days to develop, making it unlikely
that PPHN was a complication of MAS.23,27,28 As PPHN on its own
is associated with high mortality rate, one would expect less
mortality rate from MAS if infants with PPHN are excluded. In this
study, the mortality rate was 73% lower in those with PPHN
compared to those without PPHN. The low mortality rate in
developed countries has been associated with an increase in the use
of exogenous surfactant, high-frequency ventilation and inhaled
nitric oxide, with >50% of infants receiving one or more of these
therapies.5 A postal survey of 227 neonatal units in the United
Kingdom revealed that 96, 42 and 29% of units were using
exogenous surfactant, high-frequency ventilation and inhaled
nitric oxide for MAS, respectively.8 In this study, use of exogenous
surfactant, high-frequency ventilation and inhaled nitric oxide
ranged from 6 to 19, 21 to 45 and 3 to 6%, respectively, over
the 3 years.
In conclusion, patients who were ventilated for MAS were more
likely to have been born postdates, to have not been monitored
electronically during labor, to have been born by cesarean section
and to have not been intubated for suctioning of meconium at
birth. MAS requiring MV is still associated with significant
morbidity and mortality in the setting where there are limited
resources. Reducing patients who deliver post-term, intensive
Meconium aspiration syndrome and ventilation
S Velaphi and A Van Kwawegen
S41
monitoring during labor and offering appropriate neonatal care
including exogenous surfactant will improve incidence of MAS and
those requiring ventilation, and therefore less strain on already
limited resources.
Disclosure
The authors have declared no financial interests.
References
1 Wiswell TE, Tuggle JM, Turner BS. Meconium aspiration syndrome: have we made a
difference? Pediatrics 1990; 85: 715–721.
2 Chinese collaborative study group for neonatal respiratory diseases. Treatment of severe
meconium aspiration syndrome with porcine surfactant: a multicentre, randomized,
controlled trial. Acta Paediatr 2005; 94: 896–902.
3 Carson BS, Losey RW, Bowes WA, Simmons MA. Combined obstetric and pediatric
approach to prevent meconium aspiration syndrome. Am J Obstet Gynecol 1976; 126:
712–715.
4 Davis RO, Philips JB, Harris BA, Wilson ER, Huddelston JF. Fetal meconium aspiration
syndrome occurring despite airway management considered appropriate. Am J Obstet
Gynecol 1985; 151: 731–736.
5 Dargaville PA, Copnell B, for the Australian and New Zealand Neonatal Network. The
epidemiology of meconium aspiration syndrome: incidence, risk factors, therapies and
outcome. Pediatrics 2006; 117: 1712–1721.
6 Yoder BA, Kirsch EA, Barth Jr WH, Gordon MC. Changing obstetric practices associated
with decreasing incidence of meconium aspiration syndrome. Obstet Gynecol 2002;
99: 731–739.
7 Mahomed K, Mulambo T, Woelk G, Hofmeyr J, Gulmezoglu AM. The collaborative
randomized amnioinfusion for meconium project (CRAMP): 2. Zimbabwe. Br J Obstet
Gynaecol 1998; 105: 309–313.
8 Greenough A, Pulikot A, Dimitriou G. Prevention and management of meconium
aspiration syndromeFassessment of evidence based practice. Eur J Pediatr 2005;
164: 329–330.
9 Finer NN, Barrington KJ. Nitric oxide for respiratory failure in infants born at or near
term. Cochrane Database Syst Rev 2006; 4: CD000399.
10 El Shahed AI, Dargaville P, Ohlsson A, Soll RF. Surfactant for meconium aspiration
syndrome in full term/near term infants. Cochrane Database Syst Rev 2007; 3:
CD002054.
11 Hernandez C, Little BB, Dax JS, Gilstrap LC, Rosenfeld CR. Prediction of the severity of
meconium aspiration syndrome. Am J Obstet Gynecol 1993; 169: 61–70.
12 Kattwinkel J, Niermeyer S, Nadkarni V, Tibballs J, Phillips B, Zideman D et al. An
Advisory Statement from the Pediatric Working Group of the International Liaison
Committee on Resuscitation. Pediatrics 1999; 103: e56.
13 Wiswell TE, Bent RC. Meconium staining and the meconium aspiration syndrome.
Pediatr Clin North Am 1993; 40: 955–981.
14 Liu WF, Harrington T. Delivery room risk factors for meconium aspiration syndrome.
Am J Perinatol 2002; 19: 367–378.
15 Adhikari M, Gouws E, Velaphi SC, Gwamanda P. Meconium aspiration syndrome:
importance of the monitoring of labor. J Perinatol 1998; 18: 55–60.
16 Clausson B, Cnattingius S, Axelsson O. outcomes of post-term births: the
role of fetal growth restriction and malformations. Obstet Gynecol 1999; 94:
758–762.
17 Yoder B. Meconium-stained amniotic fluid and respiratory complications: impact of
selective tracheal suction. Obstet Gynecol 1994; 83: 77–84.
18 Dooley S, Pesavanto D, Depp R, Socol M, Tamura R, Wiringa K. Meconium below the
vocal cords at delivery: correlation with intrapartum events. Am J Obstet Gynecol 1985;
153: 767–770.
Journal of Perinatology
 Meconium aspiration syndrome and ventilation
S Velaphi and A Van Kwawegen
S42
19 Rossi E, Philipson E, Williams T, Kalhan S. Meconium aspiration syndrome:
intrapartum neonatal attributes. Am J Obstet Gynecol 1989; 74: 715–721.
20 Usta I, Mercer B, Sibai B. Risk factors for meconium aspiration syndrome. Obstet
Gynecol 1995; 86: 230–240.
21 Flaciglia HS. Failure to prevent meconium aspiration syndrome. Obstet Gynecol 1988;
71: 349–353.
22 Cornish JD, Dreyer GL, Snyder GE, Kuehl TJ, Gerstmann DR, Null DM et al. Failure of
acute perinatal asphyxia or meconium aspiration to produce persistent pulmonary
hypertension in a neonatal model. Am J Obstet Gynecol 1994; 171: 43–49.
23 Thureen P, Hall DM, Hoffenberg A, Tyson RM. Fatal meconium aspiration in spite of
appropriate perinatal airway management: pulmonary and placental evidence of
prenatal disease. Am J Obstet Gynecol 1997; 176: 967–975.
Journal of Perinatology
24 Kojima T, Hattori K, Fujiwara T, Sasai-Takedatsu M, Kobayashi Y. Meconium-induced
lung injury mediated by activation of alveolar macrophages. Life Sci 1994; 54:
1559–1562.
25 Coughtrey H, Jeffery HE, Henderson Smart DJ, Storey B, Poulos V. Possible causes
linking asphyxia, thick meconium, and respiratory distress. Aust N Z J Obstet Gynaecol
1991; 31: 97–102.
26 Wiswell TE, Henley MA. Intratracheal suctioning, systemic infection, and the
meconium aspiration syndrome. Pediatrics 1992; 89: 203–206.
27 Perlman EJ, Moore GW, Hutchins GM. The pulmonary vasculature in meconium
vasculature in meconium aspiration. Hum Pathol 1989; 20: 701–706.
28 Murphy ID, Vawter GF, Reid LM. Pulmonary vascular disease in fatal meconium
aspiration. J Pediatr 1984; 104: 158–162.
 Journal of Perinatology (2008) 28, S43–S48
r 2008 Nature Publishing Group All rights reserved. 0743-8346/08 $30
www.nature.com/jp

REVIEW
Meconium aspiration syndrome: experiences in Taiwan
HC Lin1,2, SY Wu3, JM Wu4 and TF Yeh1,5
1
Department of Pediatrics, China Medical University Hospital, Taichung, Taiwan, ROC; 2School of Chinese Medicine, China Medical
University, Taichung, Taiwan; 3Department of Pediatrics, College of Medicine, National Cheng Kung University, Tainan, Taiwan;
4
Department of Pediatrics, John Stroger Hospital of Cook County, Chicago, IL, USA and 5Department of Pediatrics, College of Medicine,
China Medical University Hospital, Taichung, Taiwan, ROC

Incidence of MAS
Meconium aspiration syndrome (MAS) is still one of the most challenged
Table 2 compares the incidence and mortality of infants
diseases for the neonatologists. We reviewed our earlier studies of MAS in an
suffering from MAS between the United States and one of the
attempt to provide some idea for more understanding of MAS. This study is
tertiary centers in Taiwan (China medical university hospital,
a retrospective review and summarization of our earlier studies in MAS at
Taichung, Taiwan).1–3 It is interesting to note that data of the
two tertiary neonatal centers in Taiwan. Incidence of MAS was decreased
incidences of meconium staining of amniotic fluid, MAS
sharply in Taiwan. MAS infants who required resuscitation in the birth
and mortality are almost comparable with those from the
room being out-born, birth asphyxia and infants who developed persistent
United States. However, these data are obtained from one
pulmonary hypertension (PPHN) and pneumothrax were associated with
tertiary center with many high-risk maternal transfers; the
increasing mortality. In MAS infants who neither required mechanical
actual overall incidence in Taiwan could be much lower. The
ventilation nor had a history suggestive of perinatal infection, antibiotic
outborn infants have higher mortality rates (7.3%) than inborn
treatments would not affect the outcome of MAS. Dexamethasone did reduce
infants (0.81%) (Table 3).
inflammation response and improve cardiopulmonary perfusion. However,
steroids did not prevent the development of PPHN. Our review provided the
risk factors of mortality for MAS. Antibiotic treatments should not be a
Perinatal and postnatal factors and mortality in MAS
routine for every infant with MAS. Although steroids reduce pulmonary
inflammation, their role in the prevention of PPHN remains to be A review of 314 cases of MAS between 1995 and 20011,5 indicated
further studied. that infants who required resuscitation in the birth room, who
Journal of Perinatology (2008) 28, S43–S48. doi:10.1038/jp.2008.157 were first out-born or with birth asphyxia, and infants who
developed persistent pulmonary hypertension (PPHN) and
pneumothorax are important factors associated with increased
mortality in MAS (Table 4).

The incidence of meconium aspiration syndrome (MAS) varies

from population to population and from countries to countries.1–4
With the recent improvement in socioeconomic status and the
advances in prenatal care in Taiwan, the incidence of MAS in
Taiwan sharply decreased in the past decade.1 The adoption of
National Healthcare Insurance System is probably the most
important factor leading to better health access and care. Table 1
shows some of the factors that characterized health care in Taiwan.
At present, nearly 100% of the pregnant women have prenatal care
and majority of the deliveries are carried out in large hospitals.
However, the mean age of the mother at the time of first childbirth
is about 26 years, older than that in most of the developing and
developed countries.
Correspondence: Dr TF Yeh, Department of Pediatrics, College of Medicine, China Medical
University, 91 Hsueh Shih Rd., Taichung, Taiwan, ROC.
E-mail: tfyeh@mail.ncku.edu.tw
Management of meconium-stained amniotic fluid in
delivery room
Since 1995, the management of meconium-stained amniotic
fluid has followed a guideline as shown in Figure 1.6 This
guideline is not much different from the recent recommendations
of American Academy of Pediatrics.7,8 We do endotracheal
suction in infants who have thick meconium staining,
who have low Apgar score or who have respiratory distress shortly
after birth.9 We perform intrapartum oro- and nasopharyngeal
suction in all infants if there is meconium staining of amniotic
fluid.6,10 We feel that this procedure is simple and does not
carry significant adverse effects. Using this guideline, about
50% of meconium-stained infants required endotracheal suction.
We do amnioinfusion only if there is oligohydramnio and
thick meconium staining.11–15
 Meconium aspiration syndrome
HC Lin et al
S44

The role of antibiotics in MAS
Meconium has been shown to enhance bacterial growth
in vitro, particularly Gram-negative organisms. It is a
common practice to place these infants on antibiotics. We have
conducted a study on 259 MAS infants who did not require
mechanical ventilation and who did not have a history suggesting
perinatal infection.5 The infants were divided into two groups;
one received antibiotics and the other did not. Both groups were
comparable in their baseline data. There was no significant
difference between the groups in clinical course and mortality
(Table 5).
Thus, antibiotic treatments would not affect the clinical course
and outcome in MAS without perinatal risk factors for infection
and without ventilator use.

The role of pulmonary inflammation in the development

Table 1
of PPHN in MAS
Health care in Taiwan Pulmonary hypertension or PPHN of the newborn occurs
NHIS coverage in 10 to 15% of infants with MAS. This condition usually
Prenatal care 98–100% presents as persistent hypoxemia occurred at 6 to 24 h after
Average age of mother for first childF26 years birth. A spontaneous recovery usually occurs within 3 to
Uniform population and easy access for medical care 4 days if the patient survives, suggesting that a functional
Establishment of regionalization of perinatal care vascular constriction is probably involved in the
Minimal years of maternal education 9–12 years pathogenesis.
Average number of children per familyF1.2 Pulmonary hypertension or PPHN in infants with MAS
Abbreviation: NHIS, National Healthcare Insurance System. could be due to (1) hypertrophy or neo-muscularization of

Table 2 Incidences of MAS and mortality in the United States and Taiwan Table 4 Risk factors of mortality in MAS by logistic regression model
2 1
USA (Wiswell et al. ) Taiwan (Lin et al. ) Variable P-value Odds ratio CI lower CI upper
1997 1995–2001
Asphyxia 0.0032 0.026 0.002 0.293
Incidence of meconium staining 10–15% 13% Outborn 0.0869 0.124 0.011 1.353
MAS/meconium staining 5% 5.9% PPHN 0.0145 0.065 0.007 0.581
IMV/MAS 33% (inborn 0.81%) Shock 0.4246 2.50 0.263 23.771
Incidence of MAS/liveborn 0.5% 0.7% Pneumothorax 0.0143 0.158 0.036 0.692
Mortality/MAS 5% 4.8%
Abbreviations: MAS, meconium aspiration syndrome; PPHN, persistent pulmonary
Abbreviations: IMV, intermittent mandatory ventilation; MAS, meconium aspiration hypertension of the newborn.
syndrome. Adapted from Lin et al.1

Table 3 Incidence and mortality rate of MAS from 1995 to 2001

1995 1996 1997 1998 1999 2000 2001 Total

Inborn babies (n) 14 22 14 12 16 21 24 123

Outborn babies (n) 26 18 19 19 31 43 35 191
Survival (n) 36 39 32 29 43 63 57 299
Expired (n) 4 1 1 2 4 1 2 15
Mortality rate (%) 4/40 1/40 1/33 2/31 4/47 1/64 2/59 15/314
(10.0) (2.5) (3.0) (6.5) (8.5) (1.6) (3.4) (4.8)
Incidence of MAS in inborn (%) 14/154 22/24 14/23 12/216 16/24 21/26 24/23 123/15
9 59 72 3 06 58 60 967
(0.90) (0.89) (0.59) (0.55) (0.66) (0.79) (1.01) (0.77)
Incidence of mortality in inborn (%) 1/14 0/22 0/14 0/12 0/16 0/21 0/24 1/123
(7.14) (0) (0) (0) (0) (0) (0) (0.81)
Abbreviation: MAS, meconium aspiration syndrome.
Adapted from Lin et al.1

Journal of Perinatology
 Meconium aspiration syndrome
HC Lin et al
S45

postacinar pulmonary capillaries as a result of chronic intrauterine of this study, we made the following conclusions:
hypoxia, (2) functional pulmonary vasoconstriction as a result (1) Following meconium aspiration, elevation of pulmonary
of hypoxemia, hypercarbia or acidosis or (3) functional pulmonary arterial pressure appears to be biphasic, the early phase starting
vasoconstriction as a result of lung inflammation. We
hypothesized that lung inflammation may play an important
role in pulmonary vascular constriction and hypertension. We 35 Newborn piglets
therefore conducted a MAS study on newborn piglet models.16 (1–7 days)

The purposes of the study were to investigate the following:

(1) Does MAS in newborn piglets produce pulmonary hypertension? 7
15
13

(2) Does pulmonary inflammation play a role in the development Saline control MAS MAS +dexamethasone
of pulmonary hypertension. (3) Can anti-inflammatory agents, IMV, FIO2 0.3, RR 20/min, PIP/PEEP 15-20/4-5 cm H2O
Pulmonary artery catheter-PAP, PF
such as dexamethasone, prevent pulmonary hypertension. Figure 2 Femoral artery catheter–BP
3 ml kg -1 20% meconium mixed with saline
shows the method of the study on 35 newborn piglets Dexamethasone 0.5 mg kg -1 q 12h for 2 doses
(aged 1 to 7 days).16 We also administered dexamethasone early Tracheal aspirate daily for LTB4, LTC4, LTD4, thromboxane B2, 6-keto PGF-1α

in several infants.17 The results of piglet and newborn studies Figure 2 Method for Newborn Piglet MAS study. Adapted Wu et al.16
are shown in Tables 6 and 7. On the basis of the results

Table 6 Cardiac SV

MECONIUM STAINED A. F. Before 0–12 h 12–24 h d-2 d-3 d-4

SUCTION of Oro and NASOPHARYNX
S.V. (mg kg1)
PRIOR TO DELIVERY OF THORAX
C 1.5±0.4 1.4±0.5 1.5±0.4 1.6±0.5 1.6±0.4 1.9±0.8
MAS 1.4 0.4 1.2 0.3 1.1 0.2** 1.3±0.4* 1.6±0.3 1.8±0.4
± ± ±
MAS+D 1.4±0.5 1.3±0.4 1.3±0.3 1.4±0.3 1.5±0.5 1.9±0.3
THICK THIN
MECONIUM MECONIUM
mBP (mm Hg)
C 118±16 107±11 99±7 96±8 87±9 92±10
MAS 120±11 118±11 110±16 106 14** 103 8* 102±7
± ±
E. T. SUCTION GOOD APGAR LOW APGAR
MAS+D 119±17 120±12 124±15* 120±10** 118±8* 101±6
7-10

E. T. SUCTION
mPAP (cm H2O)
C 19±6 19±5 20±5 20±8 21±6 22±9
MAS 20±3 24±5* 23±5* 25±4* 24±7 22±6
NO RESP. DIST IN RESP. DIST MAS+D 20±6 27±10* 24±6* 24±8* 26±10 23±4
Abbreviations: MAS, meconium aspiration syndrome; MAS+D, meconium aspiration
OBSERVE E. T. syndrome+dexamethasone; mBP, mean blood pressure; mPAP, mean pulmonary arterial
SUCTION pressure; SV, stroke volume.
*P<0.05, **P<0.01 (as compared with saline control).
Figure 1 A guideline for the management of meconium staining of amniotic C: saline control, n ¼ 7; MAS, n ¼ 15; MAS+D, n ¼ 13.
fluid in the delivery room. Adapted from Wu et al.16

Table 5 MAS treated with or without antibiotics

No antibiotics (n ¼ 127) Antibiotics group (n ¼ 132) P-value

Tachypnea (days) 5.9±2.7 6.2±3.5 0.91

Duration of O2 (days) 4.6±1.7 3.6±1.3 0.46
Duration of CPAP (days) 2.2±1.8 3.0±2.2 0.65
Nasal CPAP (case) 29/127 33/132 0.68
Pulmonary air leaks (case) 4/127 7/132 0.39
Abbreviations: CPAP, continuous positive airway pressure; MAS, meconium aspiration syndrome.
Mean values±s.d.
Adapted from Lin et al.5

Journal of Perinatology
 Meconium aspiration syndrome
HC Lin et al
S46

Table 7 Respiratory status in MAS infants with and without dexamethasone therapy

Before d-1 d-2 d-3 d-4 d-5 d-7

IMV
C 9 6 5 5 4 3 2
D 12 6 4 1 0 0 0

FIO2
C 0.54±0.31 0.35±0.16 0.26±0.16 0.29±0.16 0.29±0.08 0.29±0.09 0.28±0.18
D 0.52±0.24 0.40±0.20 0.30±0.18 0.32±0.29 0.30±0.22 0.31±0.24 0.30±0.21

PO2 (mm Hg)

C 86±58 84±33 74±22 75±30 70±26 80±18 75±25
D 91±80 86±42 70±42 78±28 67±23 76±14 74±14

PCO2 (mm Hg)

C 43±12 36±9 36±8 39±5 43±8 37±8 41±2
D 42±12 34±7 32±5* 33±9* 30±7** 35±7 39±5

pH
C 7.28±0.12 7.41±0.07 7.40±0.05 7.40±0.07 7.39±0.09 7.39±0.06 7.45±0.17
D 7.30±0.70 7.39±0.05 7.40±0.05 7.45±0.03* 7.43±0.04* 7.40±0.06 7.44±0.05
Abbreviations: IMV, intermittent mandatory ventilation; MAS, meconium aspiration syndrome.
*P<0.05, **P<0.01.
C: control infants, n ¼ 23; D: dexamethasone-treated infants, n ¼ 27. Adapted from Yeh et al.17

Figure 3 Mean blood pressure during the study. MAS piglets that received dexamethasone had significantly higher mean blood pressure at 4, 16, 36, 72 and 82 h than
saline control piglets. *P<0.05. Adapted from Wu et al.16

from 2 to 6 h and the later phase starting from 48 h.
(2) A good correlation was seen between tracheal aspirate TXB2,
leukotriene D4 and mean pulmonary anterial pressure. (3) The
use of dexamethasone reduced tracheal aspirate TXB2 and
6-keto PGF1a. (4) Dexamethasone increased cardiac stroke
volume, increased pulmonary blood flow and improved
ventilation/perfusion match.
The role of pulmonary inflammation in the development of
pulmonary hypertension or PPHN in MAS and the possible role of steroids
in the prevention of PPHN remain to be further studied (Figures 3 to 7).

Journal of Perinatology

Figure 4 Mean pulmonary arterial pressure (PAP) changes during the study. Piglets with MAS and dexamethasone had significantly higher mean PAP than saline
control piglets at 4, 24, 36, 48 and 60 h after study. There were simultaneous increases in cardiac stroke volume, suggesting that the increases of mean PAP is
a result of increased pulmonary flow. *P<0.05. Adapted from Wu et al.16
Figure 5 Changes of 6-keto prostaglandin (PGF)1a in tracheal aspirate. MAS piglets
had significantly higher 6-keto PGF1a than saline controls. The use of
dexamethasone significantly decreased 6-keto PGF1a. Adapted from Wu et al.16
Figure 6 Changes of leukotriene (LTC4) in tracheal aspirate during the study. Piglets
with MAS had significantly higher LTC4 than saline controls at 24 h. The use of
dexamethasone significantly decreased LTC4 at 48 and 72 h. Adapted from Wu et al.16
Meconium aspiration syndrome
HC Lin et al
S47
Figure 7 Changes of leukotriene (LTE4) in tracheal aspirate during the study.
Piglets with MAS had significantly higher LTE4 than saline controls at 24 h. The
use of dexamethasone significantly decreased LTE4 at 48, 72 and 92 h. Adapted
from Wu et al.16
Disclosure
The authors have declared no financial interests.
References
1 Lin HC, Su BH, Lin TW, Peng CT, Tsai CH. Risk factors of mortality in meconium
aspiration syndrome. Acta Pediatr TW 2004; 45: 30–34.
2 Wiswell T, Henley MA. Intratracheal suctioning, systemic infection, and the meconium
aspiration syndrome. Pediatrics 1992; 89: 203–206.
3 Wiswell TE, Gannon CM, Jacob J, Goldsmith L, Szyld E, Weiss K et al. Delivery
room management of the apparently vigorous meconium-stained neonate:
results of the multicenter, international collaborative trial. Pediatrics 2000; 105:
1–7.
Journal of Perinatology
 Meconium aspiration syndrome
HC Lin et al
S48
4 Gregory GA, Gooding CA, Phibbs RH, Tooley WH. Meconium aspiration in infantsFa
prospective study. J Pediatr 1974; 85: 848–852.
5 Lin HC, Su BH, Tsai CH, Lin TW, Yeh TF. Role of antibiotics in management of non-
ventilated cases of meconium aspiration syndrome without risk factors for infection.
Biol Neonate 2005; 87: 51–55.
6 Yeh TF. Resuscitation in ‘Neonatal Therapeutics’, Yeh TF (ed), 2nd edn, Mosby-Year
Book: St Louis, 1992, pp 170–179.
7 Kattwinkel J, Niermeyer S, Nadkarni V, Tibballs J, Phillips B, Zideman D et al. An
advisory statement from the Pediatric Working Group of the International Liasion
Committee on Resuscitation. Pediatrics 1999; 103: e56–e69.
8 Kattwinkel J. Textbook of Neonatal Resuscitation, 4th edn. American Academy of
Pediatrics/American Heart Association: Elk Grove Village (IL)7, 2000.
9 Gooding CA, Gregory GA. Roentgenographic analysis of meconium aspiration of the
newborn. Radiology 1971; 100: 131–140.
10 Ting P, Brady JP. Tracheal suction in meconium aspiration. Am J Obstet Gynecol
1975; 122: 767–771.
11 Wenstrom KD, Parsons MT. The prevention of meconium aspiration in labor using
amnioinfusion. Obstet Gynecol 1989; 73: 647–651.
Journal of Perinatology
12 Spong CY, Ogundipe OA, Ross MG. Prophylactic amnioinfusion for meconium stained
amniotic fluid. Am J Obstet Gynecol 1994; 171: 931–935.
13 Sadovsky Y, Amon E, Bade ME, Petrie RH. Prophylactic amnioinfusion during labor
complicated by meconium: a preliminary report. Am J Obstet Gynecol 1989; 161:
613–617.
14 Hourdequin P, Kauffmann E, Gabriel R, Jotterand AD, Chatelet-Cheront C, Quéreux C
et al. Amnioinfusion during labour: experience and review of the literature. Contracept
Fertil Sex 1999; 27: 222–230.
15 Fraser WD, Hofmeyr J, Lede R, Faron G, Alexander S, Goffinet F, et al., Amnioinfusion
Trial Group. Amnioinfusion for the prevention of the meconium aspiration syndrome.
N Engl J Med 2005; 353: 909–917.
16 Wu JM, Yeh TF, Wang JY, Wang JN, Lin YJ, Hsieh WS et al. The role of pulmonary
inflammation in the development of pulmunary hypertension in newborn with
Meconium Aspiration Syndrome (MAS). Pediatr Pulmonol Suppl 1999; 18:
205–208.
17 Yeh TF, Lin YJ, Lin HC, Wu JM, Lin CH. Early postnatal dexamethasone therapy in
infants with Meconium Aspiration Syndrome (MAS)Fa randomized clinical trial.
Ped Res 1998; 43: 204A.
 Journal of Perinatology (2008) 28, S49–S55
r 2008 Nature Publishing Group All rights reserved. 0743-8346/08 $30
www.nature.com/jp
REVIEW
Continuous positive airway pressure and conventional
mechanical ventilation in the treatment of meconium aspiration
syndrome
JP Goldsmith
Department of Pediatrics, Tulane University, New Orleans, LA, USA
Meconium aspiration syndrome (MAS) is a complex syndrome that ranges
in severity from mild respiratory distress to severe respiratory failure,
persistent pulmonary hypertension of the newborn and sometimes death.
Understanding of the syndrome’s complicated pathophysiology will help
determine the appropriate treatment strategy, including the use of
continuous positive airway pressure (CPAP), conventional mechanical
ventilation (CMV) and other therapies. Approximately 30 to 50% of infants
diagnosed with MAS will require CPAP or mechanical ventilation. The
optimum modes of ventilation for MAS are not known. Very few studies have
been conducted to determine ‘best’ ventilatory strategies. Despite the
introduction, over the last two decades, of innovative ventilatory treatments
for this disease (for example, surfactant, high-frequency ventilation, inhaled
nitric oxide, extracorporeal membrane oxygenation), the majority of infants
can be successfully managed with CPAP or mechanical ventilation alone.
Journal of Perinatology (2008) 28, S49–S55; doi:10.1038/jp.2008.156
Introduction
Approximately 30 to 50% of infants diagnosed with meconium
aspiration syndrome (MAS) will require continuous positive airway
pressure (CPAP) or mechanical ventilation. The optimum modes of
ventilation for MAS are not known. Very few studies have been
conducted to determine ‘best’ ventilatory strategies. Retrospective
reviews of the application of conventional mechanical ventilation
(CMV) to infants with this malady span three decades, during
which time a host of adjunctive therapies have become available.1,2
Despite multiple therapies for this syndrome, there is a disturbingly
high-mortality rate (4 to 19%) historically. However, in the most
recent of these reviews, only 20% of 40 infants with MAS requiring
positive pressure assistance needed ‘innovative ventilatory support’
(defined as the use of high-frequency ventilation (HFV), inhaled
nitric oxide or surfactant) and no infants died or required
Correspondence: Dr JP Goldsmith, Department of Pediatrics, Tulane University, 1625 Joseph
Street, New Orleans, LA 70115-5034, USA.
E-mail: goldsmith.jay@gmail.com
extracorporeal membrane oxygenation.2 It appears that when used
optimally, conventional therapies are adequate to treat the majority
of infants with this disease. Neonatal MAS challenges the practicing
clinician to make the correct diagnosis, understand the changing
and complex pathophysiology of the disease, and tailor the
respiratory support to the specific alterations in pulmonary
mechanics.
Making the diagnosis of MAS
Approximately 8 to 19% of all term deliveries occur through
meconium-stained amniotic fluid (MSAF) and MAS develops in
5 to 33% of these infants.3 Respiratory complications of MAS are
often the consequence of hypoxia in utero with significant acidosis
and depressed respirations at birth. There also appears to be a
greater risk of developing MAS as well as all respiratory
complications (that is, tachypnea, pneumonia, pulmonary air
leaks) if the meconium is ‘thick’ as opposed to ‘thin’.4 However, a
recent meta-analysis of the use of amnioinfusion showed no
neonatal benefit when meconium was thinned through this process
except in settings with limited peripartal surveillance.5 Infants who
are vigorous at birth have a very low risk of developing MAS.6
Those who develop MAS are often greater than 41 weeks gestation
and have clinical signs of postmaturity (decreased subcutaneous
tissue, peeling skin, long nails and so on). The respiratory signs of
MAS are similar to other neonatal respiratory diseases. These
include tachypnea, retractions, grunting, nasal flaring and
cyanosis. If the meconium has been present in utero for greater
than 3 h, the infant may have meconium staining of the skin,
nails and umbilical cord. The anterior–posterior diameter of the
chest may be increased if there is significant air trapping.
The classic radiographic picture of MAS includes diffuse patchy
infiltrates with areas of atelectasis mixed with areas of
hyperinflation throughout the lung fields. However, initially the
chest X-ray may not be very diagnostic, as it may take many hours
for the chemical pneumonitis secondary to MAS to develop. Other
findings on X-ray include possible air leaks and cardiomegaly, if
significant perinatal asphyxia has resulted in cardiomyopathy.
 CPAP and CMV in the treatment of MAS
JP Goldsmith
S50

Later in the course of the disease, the MAS picture may be
complicated by the radiographic signs of surfactant deficiency
(atelectasis, air bronchograms, decreased lung volume) as the
surfactant system may be negatively impacted by the primary
disease.
Despite the classic clinical and radiological appearance of MAS,
it may be difficult in certain cases to distinguish MAS from other
neonatal respiratory diseases. Table 1 lists the differential
diagnosis.7 It is important diagnostically and physiologically to
determine if persistent pulmonary hypertension of the newborn
(PPHN) is present with significant right-to-left shunting of blood
through the foramen ovale and/or the ductus arteriosus resulting
in exaggerated hypoxemia. PPHN may result secondary to MAS or
may be present by itself (‘primary PPHN’) with concurrent MSAF
but no true aspiration. As noted by Goetzman8 in these cases of
primary PPHN, ‘when meconium is present, it is a fellow traveler
and not the cause of the PHN (PPHN) and hypoxemia, contrary to
Table 1 Differential diagnosis of MAS (when MSAF is present at delivery)
Transient tachypnea of the newborn
Aspiration of amniotic fluid or blood
Respiratory distress syndrome
Sepsis with pulmonary edema
Pneumonia
Congenital heart disease
Intrapartum asphyxia with cardiomyopathy
Persistent pulmonary hypertension of the newborn
Delayed transition
Abbreviation: MSAF, meconium-stained amniotic fluid.
Adapted from Fuloria M, Wiswell TE.7
what is often implied when MAS is used as the primary diagnosis.’
Importantly, the treatment of the baby’s respiratory disease will
depend on the presence and severity of pulmonary hypertension.
Thus the diagnosis of MAS may be assumed when three criteria
are met: (1) a history of meconium in the trachea; (2) clinical
evidence of significant respiratory distress; and (3) X-ray evidence
of aspiration pneumonia.1 The association of transient tachypnea
and MSAF without meconium in the airway or X-ray evidence of
pneumonitis is often misdiagnosed as MAS and may lead to
inappropriate treatment, including early positive pressure, which
then may result in inadvertent pulmonary air leaks.
Pulmonary pathophysiology of MAS
Multiple mechanisms are involved in the pulmonary
pathophysiology of MAS. Meconium has deleterious effects on the
airways, injures the pulmonary parenchyma and alveoli, inhibits
surfactant function and may cause hypoxic pulmonary
vasoconstriction resulting in PPHN. The complex pulmonary effects
of MAS are seen in Figure 1.9 Individual infants may manifest any
one or more of these effects and the degree of respiratory distress
may wax and wane according to the severity of the
pathophysiology. Often there is a vicious cycle of air trapping,
ventilation–perfusion mismatch, hypoxemia, right-to-left
shunting, acidosis and increased pulmonary vascular resistance,
which may be difficult to treat successfully with standard therapies.
Meconium may easily obstruct both the large and small airways
in the newborn lung. When the obstruction is partial, a ‘ball-valve’
mechanism may lead to air trapping and air leaks (Figure 2).10 On
inspiration the airway dilates allowing gas to enter, but during

Alveolar and Effects of

parenchymal mediators
Altered lung elastic
inflammation (cytokines,
forces (increased
and edema eicosanoids)
resistance, decreased Surfactant
compliance) dysfunction

Protein leak
Airway into the
obstruction airways
Meconium
aspiration
syndrome
Effects of in utero
Direct toxicity
hypoxemia (pulmonary
by meconium
vascular remodeling,
constituents
lung parenchymal
changes)
Pulmonary
vasoconstriction due
Altered to components
pulmonary of meconium
vasoreactivity

Figure 1 The complex pulmonary pathophysiology of meconium aspiration syndrome. From reference 9 with permission.

Journal of Perinatology

a b
Figure 2 Air trapping behind particulate matter (i.e. meconium) in an airway,
which leads to alveolar overexpansion and rupture. Tidal gas passes the
meconium on inspiration when the airway dilates (a), but does not exit on
expiration when the airways constrict (b). From reference 10 with permission.
expiration the airway constricts around the obstruction, thus
trapping air inside the lung. This may result in overexpansion of
the lung, ventilation–perfusion mismatch, decreased compliance
and possibly air leak. Complete obstruction of the airway by
meconium may result in atelectasis and resultant hypoxemia and
hypercapnia.
Meconium also causes direct pulmonary parenchymal and
alveolar injury resulting in a profound inflammatory response. The
release and migration of inflammatory cells and mediators, such
as cytokines and phospholipase A2, to the lung may cause direct
pulmonary injury such as epithelial cell necrosis, cell death and
apoptosis.11,12 These mediators may also result in constriction of
pulmonary vessels, alteration and impairment of neutrophil
function and cause capillary leakage and edema similar to
respiratory distress syndrome.9
Multiple investigations in both animals and humans have
shown the deleterious effects of meconium on endogenous
surfactant.12–14 Meconium may inhibit surfactant production,
displace surfactant from the alveolar surface and decrease its
surface tension lowering function. Surfactant inhibition is
concentration dependent and results from a variety of toxic
biochemical effects of the meconium constituents. The influx of
neutrophils into the airways subsequent to these toxic effects
produces proteases, which further degrade surfactant proteins and
frequently results in hemorrhagic edema of the lung.
Fox et al.15 initially reported the association of pulmonary
hypertension in association with perinatal aspiration syndromes.
The majority of cases of significant PPHN are associated with MAS.
The relationship of PPHN with MAS is complex and multifactorial.
Vasoconstriction of pulmonary vessels may be associated with
normal or increased muscularization of the vessel walls.16
Prolonged in utero hypoxia may result in vascular remodeling
secondary to hypertrophy of the medial musculature. Acute
maladaptation secondary to intrapartum asphyxia with normal
vessel anatomy is caused by the inhibition of endogenous
CPAP and CMV in the treatment of MAS
JP Goldsmith
S51
Table 2 Theoretical classification of neonatal pulmonary disorders
Atelectatic Obstructive
Example Respiratory distress syndrome Meconium aspiration syndrome
Physiology kLung volume mLung volume
kCompliance kCompliance
kFunctional residual capacity mFunctional residual capacity
Normal airway resistance mAirway resistance
Normal time constant mTime constant
Key: m, increased; k, decreased.
Adapted from Goldsmith JP, Karotkin EH.17
pulmonary vasodilators and an inflammatory response resulting
from MAS, which releases vasoconstrictive substances and causes
platelet aggregation thus increasing pulmonary vascular resistance.
Hypoxemia and acidosis also result in increased pulmonary artery
pressure. Thus in both the acute and chronic hypoxic states, with
normal or increased pulmonary vessel muscularization, the
inflammatory response to MAS may trigger the vicious cycle of
postnatal hypoxia, pulmonary vasoconstriction, right-to-left
shunting and further hypoxia. In the presence of chronic vascular
remodeling, the combination of MAS and PPHN is much more
difficult to treat.
Altered pulmonary mechanics of MAS
In the theoretical classification of neonatal pulmonary disorders,
MAS is considered an obstructive disease, although in reality the
condition is both obstructive and atelectatic, and changes with time
and treatment. Table 2 shows the physiologic differences between
‘pure’ atelectatic and obstructive lung disease.17 MAS is
characterized in this simplified chart as an obstructive disease with
increased lung volume, decreased compliance, increased functional
residual capacity and increased airway resistance and time
constants. The pathophysiology of MAS discussed in the previous
section alters the respiratory mechanics of the lung in four major
ways as seen in Table 3. Understanding the principles of normal
ventilation and the alteration in respiratory mechanics secondary
to MAS pathology is necessary to devise ventilation strategies to
treat affected infants with CPAP and/or CMV.
Meconium in the pulmonary airways leads to increased airway
resistance and prolonged time constants. The time constant is a
measure of how quickly the lung can inflate or deflate, or how
long it takes for alveolar and proximal airway pressures to
equilibrate. The expiratory time constant (Kt) is directly related to
both compliance (CL) and airway resistance (Raw) by the
equation: Kt ¼ CL  Raw. It takes three time constants to discharge
95% of the tidal volume of one breath. If the time constant is
prolonged secondary to increased airway resistance (without a
concomitant reduction in lung compliance), the patient is at risk
Journal of Perinatology
 CPAP and CMV in the treatment of MAS
JP Goldsmith
S52

Table 3 Ventilation strategies to match pathophysiology of MAS TLC

Pathophysiology Pulmonary Ventilation strategy

dynamics C

Airway obstruction; mLung volume Avoid ventilation on ‘C’ portion of high FRC
(overexpansion)
ball-valve mAirway resistance compliance curve; Adequate

Volume
phenomenon mTime constants expiratory time; Avoid inverse I:E
B
ratios
Parenchymal/ kCompliance Adequate CPAP or PEEP
alveolar injury Ventilation– Avoid high inflating pressures
perfusion mismatch normal FRC
Surfactant kCompliance; Adequate CPAP or PEEP; A
dysfunction Ventilation– Volume recruitment strategy; low FRC (atelectasis)
perfusion mismatch Avoid overdistension to k incidence
of PAL Pressure
FRC = functional residual capacity
Pulmonary Exaggerated Generous oxygenation
TLC = total lung capacity
hypertension hypoxia; (80 to 120 mm Hg);
Ventilation– Avoid significant acidosis Figure 3 Lung expansion or compliance curve with flattened areas (A and C) at
perfusion mismatch both ends. Area (C) represents the situation in an overexpanded lung as occurs
with air trapping. FRC, functional residual capacity; TLC, total lung capacity.
Abbreviations: m, increased; k, decreased; CPAP, continuous positive airway pressure; I:E, From reference 19 with permission.
inspiratory:expiratory; MAS, meconium aspiration syndrome; PAL, pulmonary air leak;
PEEP, Positive end expiratory pressure.

The deactivation of surfactant from MAS complicates this

for incomplete emptying of the previously inspired breath. This
condition may be exaggerated when the patient is on assisted
ventilation and there is insufficient ventilator time for exhalation
because of an abnormally long time constant. The result of this
‘inadvertent PEEP’will be gas trapping accompanied by an increase
in lung volume, a build up of pressure in the alveoli and distal
airways, and decreased compliance.18
Significant air trapping may result in hyperinflation and
flattening of the diaphragms on chest X-ray, increased anterior–
posterior diameter of the chest and even cardiovascular effects such
as decreased cardiac output, diminished blood pressure and signs
of poor perfusion. Babies with increased functional residual
capacity and high lung volumes have decreased compliance as
seen in the flattened portion or section ‘C’ of the standard
pressure–volume curve (Figure 3).19 Attempting to assist
ventilation at the upper end of the compliance curve (that is, at
high lung volumes) results in a further decrease in compliance
and may lead to pulmonary air leaks.
As noted above, meconium has a direct injurious effect on the
pulmonary airways, parenchyma and alveoli. This often results in
capillary leak and edema. The additional injury of baro-
volutrauma from assisted ventilation through an endotracheal tube
should be avoided if possible. Thus the use of oxygen by hood or
cannula and nasal CPAP is advocated to avoid intubation and the
bellows action of CMV. However, term and near-term infants may
not tolerate nasal CPAP despite adequate sedation. Moreover, the
use of a ‘permissive hypercapnia’ technique in these infants is
problematic because of the sensitivity of the pulmonary vasculature
to acidosis.
picture. It makes the use of PEEP mandatory during CMV despite
the high functional residual capacity, increased time constant and
air trapping that often occurs. Fox et al.20showed in a small
number of infants (n ¼ 14) that moderate levels of PEEP
(4 to 7 cm H2O) resulted in the greatest increase in oxygenation
in babies ventilated for MAS (Figure 4).
The addition of PPHN to the MAS pathophysiology further
complicates the pulmonary mechanics and makes ventilatory
strategies very difficult. Increased pulmonary vascular resistance
may result from hypoxia, acidosis and reduced cardiac output.
Ventilatory strategies must recognize these triggers. Allowing mild
hyperoxia in these infants may reduce the periods of hypoxemia
that occur with interventions or agitation. Pulmonary toilet must
be done judiciously with careful attention to the pulse oximeter.
Appropriate sedation and limiting interventions while nursing the
infant in a dimly lit and quiet area of the neonatal intensive care
unit are all helpful adjuncts to care. Although ‘gentle ventilation’
is recommended by some,21 permissive hypercapnia and the
resultant respiratory acidosis may worsen the pulmonary
hypertension. At the other extreme, very aggressive assisted
ventilation with hyperventilation in an attempt to improve
oxygenation may contribute to overdistension of the lung, air leaks
and decrease cardiac output, further aggravating the PPHN.
Conventional ventilation strategies
As there is little evidence from clinical trials on ventilator treatment
of MAS, conventional management is primarily based on
pulmonary pathophysiology. To avoid additional air trapping and

Journal of Perinatology

25
20
∆ PaO2 (torr/cm H2O EEP)
15
10
5 to decrease pulmonary vascular resistance.24 However, follow-up
0 barotrauma and adverse neurologic outcomes.25,26 Thus
Low Medium
(0-3) (4-7)
Intial EEP (cm H2O)
(mean ± SEM)
Figure 4 Incremental improvement in oxygenation with different levels of
positive end expiratory pressure in the treatment of meconium aspiration
syndrome. From reference 20 with permission.
possibly air leaks, many clinicians will treat initially with oxygen
alone without positive pressure of any type. An inspired oxygen
fraction (FiO2) of up to 1.0 has been advocated in the past to treat
hypoxemia. However, exposure to 100% oxygen for even brief
periods of time in the newborn lamb increased the contractile
response of small pulmonary arteries, presumably because of the
production of reactive oxygen species.22 Human and animal studies
have shown significant biochemical and clinical negative effects
after the use of 100% oxygen during resuscitation. Therefore, it
may be prudent to avoid oxygen concentrations greater than 80%
and switch to some form of positive pressure if concentrations
higher than this are necessary. An oxyhood is preferred to nasal
cannula as term and near-term infants may be less tolerant of a
cannula, and the percentage of inspired oxygen can be greater in a
hood. Sufficient oxygen should be given to maintain oxygen
saturations 90 to 95%. The partial pressure of oxygen (PaO2) target
may be as high as 90 mm Hg as the risk of eye and lung toxicity is
less in term infants, and the dangers of intermittent hypoxemia
may result in the development or worsening of PPHN.
Although CPAP or CMV may be necessary to treat hypoxemia,
hypercapnia and acidosis, there is no general agreement on blood
gas parameters to initiate these therapies. Ambalavanan et al.23
advise that infants with MAS who have PaO2 <50 mm Hg, partial
pressure of carbon dioxide (PaCO2) >60 mm Hg, or acidosis (pH
<7.25) in an oxygen-enriched environment with FiO2 >0.80 are
candidates for mechanical ventilation.
CPAP and CMV in the treatment of MAS
JP Goldsmith
S53
The complicated pulmonary pathophysiology resulting from
areas of atelectasis and areas of hyperinflation, in association with
ventilation–perfusion mismatch and airway compromise, makes
ventilator management of MAS one of neonatology’s greatest
challenges. The ventilator strategy used will depend on the stage
and severity of the disease, especially the presence or absence of
significant pulmonary hypertension. Clinicians vary in their desire
to keep blood gas values in various ranges. Prior to the use of
inhaled nitric oxide and HFV, some clinicians employed
hyperventilation to achieve alkalosis by hypocapnia in an attempt
studies on infants managed with this strategy have shown an
increased incidence of sensorineural hearing loss, pulmonary
hyperventilation has become a less-used strategy, as clinicians feel
that the newer modalities such as HFV and/or inhaled nitric oxide
High are less dangerous than sustained hypocapnia. Contemporary
(8-14)
management employs the acceptance of standard blood gas ranges
or ‘gentle ventilation,’ a strategy in which higher PaCO2 levels and
lower PaO2 levels are targeted in the belief that ventilator-induced
lung injury will be reduced.21 Other than the fear of intermittent
hypoxemia worsening PPHN, there is probably little reason to keep
babies hyperoxic. Moreover, catheterization data in older infants
with pulmonary hypertension secondary to bronchopulmonary
dysplasia suggest that oxygen tensions that exceed 70 to 80 mm Hg
do little to decrease pulmonary artery pressure.27 No trials have
compared any of these strategies in the treatment of infants with
MAS.
Infants with MAS without associated PPHN may be managed in
a relatively standard fashion with slightly higher oxygen targets
than used for premature infants with respiratory distress syndrome.
The pH should be maintained above 7.3, PaCO2 in the 40 to
50 mm Hg range and the PaO2 targeted at 70 to 80 mm Hg. This
may be achieved with a moderate peak inflating pressure preferably
not exceeding 25 cm H2O, a relatively rapid ventilator rate (40 to
60 breaths per minute), a moderate PEEP (4 to 6 cm H2O) and an
adequate expiratory time (0.5 to 0.7 s) to prevent air trapping.
This strategy requires a relatively short inspiratory time of 0.3 to
0.4 s. If the diaphragms on chest X-ray are flat and gas trapping is
suspected, the expiratory time should be increased to 0.7 to 0.9 s,
PEEP decreased to 3 to 4 cm H2O and the rate decreased to allow at
least a 0.25 inspiratory time. Borderline hypoxemia in this
situation may be acceptable as increased ventilator settings may
cause lung injury or the patient may be converted to HFV to
improve oxygenation.
In infants whose MAS is complicated by significant PPHN, the
ventilatory strategy is somewhat different. Intermittent hypoxemia
can be mitigated by allowing the PaO2 to be as high as 80 to
100 mm Hg without great concern for oxygen toxicity. As oxygen
saturations have never been studied in this context and are
surrogates for oxygen tensions, which do not correlate well when
Journal of Perinatology
 CPAP and CMV in the treatment of MAS
JP Goldsmith
S54
saturations are greater than 95%, monitoring by saturation alone is
not recommended. Some clinicians also prefer to target mild
hypocapnia (PaCO2 range of 30 to 35 mm Hg), which results in
mild alkalosis as a buffer to acidosis that may trigger a downward
PPHN spiral. In both clinical situations of MAS with and without
PPHN, a volume-recruitment strategy is preferred to recruit
atelectatic areas of the lung and treat ventilation–perfusion
mismatch. Such an approach resulted in improved oxygenation
and less ventilator-induced lung injury in an experimental piglet
model.28 This strategy has to be tempered with the knowledge that
such an approach may worsen air trapping in the face of
mechanical airway obstruction.
All types of conventional methods of ventilation have been used
in these cases. Most standard ventilators now allow the operator to
choose among intermittent mandatory ventilation, patient-
triggered ventilation, pressure support ventilation and/or volume
controlled ventilation modes. None of these modalities have been
compared with any other in a rigorous way, although each mode
of ventilation has its supporters. As non-asphyxiated term infants
may vigorously fight assisted ventilation, one approach has been to
use a patient-triggered mode (synchronized intermittent mandatory
ventilation or assist/control) in conjunction with adequate
sedation. Muscle relaxation is rarely necessary and has significant
drawbacks when this step is considered to achieve acceptable blood
gases and avoid high ventilatory pressures and possible
barotrauma, it may be time to move to alternative therapies (that
is, inhaled nitric oxide, HFV). Careful attention must be paid to the
environment in which these babies are nursed and ventilated.
Although it seems appropriate to provide chest physiotherapy and
pulmonary toilet, these interventions must be done with caution
(or not at all) as they may trigger hypoxemia, agitation and an
increase in pulmonary vascular resistance. Other nursing
interventions must be considered in the same context with
particular attention paid to minimize environmental stimuli such
as light and noise.
Conclusions
MAS is a complex syndrome that ranges in severity from mild
respiratory distress to severe respiratory failure, PPHN and
sometimes death. Understanding of the syndrome’s-complicated
pathophysiology will help determine the appropriate treatment
strategy, including the use of CPAP, CMV and other therapies. The
use of echocardiography, pulmonary function studies and graphics
will help guide the clinician in the choice of therapies. As shown by
Bhutani et al.2 at Pennsylvania Hospital over a period of 6 years,
the use of a systems-based strategy for babies delivered through
MSAF can minimize adverse outcomes. This review showed that
conventional respiratory therapies such as oxygen, CPAP and CMV
were adequate treatment for 80% of MAS-affected infants with no
infant requiring extracorporeal membrane oxygenation or dying
Journal of Perinatology
from the disease. In a time when the newest and most costly
therapy seems to find a use in every patient, we can only admire
and aspire to the benchmark that Dr Bhutani et al. have set.
Disclosure
JP Goldsmith has received consulting fees from Discovery Labs.
References
1 Vidyasagar D, Yeh TF, Harris V, Pildes RD. Assisted ventilation in infants with
meconium aspiration syndrome. Pediatrics 1975; 56: 208–213.
2 Bhutani VK, Chima R, Sivieri EM. Innovative neonatal ventilation and meconium
aspiration syndrome. Indian J Pediatr 2003; 70(5): 421–427.
3 Wiswell TE, Tuggle JM, Turner BS. Meconium aspiration syndrome: have we made a
difference? Pediatrics 1990; 85: 848–852.
4 Wiswell TE, Gannon CM, Jacob J, Goldsmith L, Szyld E, Weiss K et al. Delivery room
management of the apparently vigorous meconium-stained neonate: results of the
multicenter, international collaborative trial. Pediatrics 2000; 105(1 Part 1): 1–7.
5 Fraser WD, Hofmeyr GJ, Lede R, Faron G, Alexander S, Goffinet F et al. An
international trial for the prevention of meconium aspiration syndrome. New Engl J
Med 2005; 353: 909–917.
6 Halliday HL, Sweet D. Endotracheal intubation at birth for preventing morbidity and
mortality in vigorous, meconium-stained infants born at term (Cochrane Review). The
Cochrane Library, Issue 2, 2003. Oxford: Update Software.
7 Fuloria M, Wiswell TE. Managing meconium aspiration. Contemp Ob/Gyn 2000; July:
113–125.
8 Goetzman BW. Meconium aspiration. Am J Dis Child 1992; 146: 1282–1283.
9 Wiswell TE, Srinivasan P, Roberton NRC. Aspiration syndromes. In: Greenough A,
Milner AD (eds). Neonatal Respiratory Disorders, 2nd edn, Arnold: London, UK, 2003,
pp 334–354.
10 Harris TR, Herrick BR. Pneumothorax in the Newborn. Biomedical Communications,
Arizona Health Sciences Center: Tucson, AZ, 1978.
11 Davey AM, Becker JD, Davis JM. Meconium aspiration syndrome: physiologic and
inflammatory changes in a newborn piglet model. Pediatr Pulmonol 1993; 16:
101–108.
12 Zagariya A, Bhat R, Uhal B, Navale S, Freidine M, Vidyasagar D. Cell death and lung
cell histology in meconium aspirated newborn rabbit lung. Eur J Pediatr 2000; 159:
819–826.
13 Dargaville PA, South M, McDougall PN. Surfactant and surfactant inhibitors in
meconium aspiration syndrome. J Pediatr 2001; 138: 113–115.
14 Cleary GM, Wiswell TE. Meconium stained amniotic fluid and the meconium
aspiration syndrome: an update. Pediatr Clin North Am 1998; 45: 511–529.
15 Fox WW, Gewitz MH, Dinwiddie R, Drummond WH, Peckham GJ. Pulmonary
hypertension in the perinatal aspiration syndromes. Pediatrics 1977; 59: 205–211.
16 Walsh-Sukys M. Persistent pulmonary hypertension of the newborn: the black box
revisited. Clin Perinatol 1993; 20: 127–143.
17 Goldsmith JP, Karotkin EH. Introduction. In: Goldsmith JP, Karotkin EH (eds). Assisted
Ventilation of the Neonate, 4th edn, WB Saunders: Philadelphia, 2003, pp 1–14.
18 Simbruner G. Inadvertent positive end-expiratory pressure in mechanically ventilated
newborn infants: detection and effect on lung mechanics and gas exchange. J Pediatr
1986; 108: 589–595.
19 Wood BR. Physiologic principles. In: Goldsmith JP, Karotkin EH (eds). Assisted
Ventilation of the Neonate. 4th edn, WB Saunders: Philadelphia, 2003, pp 15–40.
20 Fox WW, Berman LS, Downes Jr JJ, Peckham GJ. The therapeutic application of
end-expiratory pressure in the meconium aspiration syndrome. Pediatrics 1975; 56:
214–217.
21 Gupta A, Rastogi S, Sahni R, Bhutada A, Bateman D, Rastogi D et al. Inhaled nitric
oxide and gentle ventilation in the treatment of pulmonary hypertension of the
newbornFa single-center, 5 year experience. J Perinatol 2002; 22: 435–441.

22 Lakshminrusimha S, Ruddell JA, Steinhorn RH, Ryan RM, Gugino SF, Morin FC et al.
Pulmonary arterial contractility in neonatal lambs increases with 100% oxygen
resuscitation. Pediatr Res 2006; 59(1): 137–141.
23 Ambalavanan N, Schelonka R, Carlo W. Ventilatory strategies. In: Goldsmith JP,
Karotkin EH (eds). Assisted Ventilation of the Neonate, 4th edn, WB Saunders:
Philadelphia, 2003, pp 249–259.
24 Drummond WH, Peckham GJ, Fox WW. The clinical spectrum of the newborn with
persistent pulmonary hypertension. Clin Pediatr 1977; 16: 335–341.
25 Bifano EM, Pfannenstiel A. Duration of hyperventilation and outcome in infants with
persistent pulmonary hypertension. Pediatrics 1988; 81: 657–666.
CPAP and CMV in the treatment of MAS
JP Goldsmith
S55
26 Walsh-Sukys MC, Tyson JE, Wright LL, Bauer CR, Korones SB, Stevenson DK et al.
PPHN in the era before NO: practice variation and outcomes. Pediatrics 2000;
105(1 Part 1): 14–20.
27 Mourani PM, Ivy DD, Gao D, Abman SH. Pulmonary vascular effects of inhaled nitric
oxide and oxygen tension in bronchopulmonary dysplasia. Am J Respir Crit Care Med
2004; 170(9): 1006–1013.
28 van Kaam AH, Haitsma JJ, De Jaegere A, van Aalderen WM, Kok JH, Lachmann B.
Open lung ventilation improves gas exchange and attenuates secondary lung
injury in a piglet model of meconium aspiration. Crit Care Med 2004; 32:
443–449.
Journal of Perinatology
 Journal of Perinatology (2008) 28, S56–S66
r 2008 Nature Publishing Group All rights reserved. 0743-8346/08 $30
www.nature.com/jp
REVIEW
Treatment of MAS with PPHN using combined therapy:
SLL, bolus surfactant and iNO
J Gadzinowski1, K Kowalska1 and D Vidyasagar2
1
Department of Neonatology, University of Medical Sciences, Poznan˜, Poland and 2Neonatal Division, Department of Pediatrics,
UIC, Chicago, USA
The objective of the study was to compare the effectiveness of surfactant
treatment either by bolus or surfactant lung lavage followed by inhaled
nitric oxide (iNO) therapy in infants with meconium aspiration syndrome
(MAS) complicated by persistent pulmonary hypertension (PPHN). In this
study, thirteen infants with diagnosis of MAS and PPHN were first treated
with conventional respiratory support. Then between 2 and 22 h of life they
were randomized either to bolus surfactant treatment (n=6) or surfactant
lung lavage (SLL, n=7) treatment. Then all infants were treated with iNO
therapy. The groups were compared with regard to their clinical course:
changes in PaO2, FiO2, MAP, OI, A-a oxygen gradient, duration of iNO
therapy, length of ventilation and hospitalization. Complications and
mortality were also compared. The results showed that infants treated with
SLL had significant improvements in oxygenation, decreases in MAP and
A-a gradients. But there were no significant differences in duration of
ventilation, iNO treatment, length of hospitalization or complications. In
conclusion these data show no advantage of SLL therapy over bolus
surfactant treatment in infants with MAS complicated by PPHN.
Journal of Perinatology (2008) 28, S56–S66; doi:10.1038/jp.2008.163
Introduction
Improved perinatal management of meconium aspiration has
contributed substantially to a decrease in incidence and mortality
because of meconium aspiration syndrome (MAS). In infants not
responding to conventional treatment of MAS, other methods such
as bolus surfactant, surfactant lung lavage (SLL) as well as
extracorporeal membrane oxygenation (ECMO) can be used.
Surfactant treatment by bolus may be associated with
inactivation by meconium in the airways, which may explain
non-responsiveness in earlier studies.1 It has been suggested that
the administration of surfactant followed by SLL may lead to a
washout of residual meconium both mechanically and because of
the improved mucocilliary transport of meconium. This approach
Correspondence: Professor J Gadzinowski, Department of Neonatology, Katedra Neonatologii
UM w Poznaniu, Ul. Polna 33, 60-535 Poznañ, Poland.
E-mail: jgadzin@gpsk.am.poznan.pl
of treatment may thus lead to a thorough removal of meconium
from the respiratory tracts and minimize the damaging effect of
meconium on the endogenous surfactant.1 Another major
complication of MAS is the development of persistent pulmonary
hypertension (PPHN). Inhaled nitric oxide (iNO) has been used
successfully to manage infants with PPHN. The effectiveness of iNO
would depend on unimpeded delivery of the gases to the
well-ventilated parts of the lungs. It is possible that iNO does not
reach the alveoli, which are covered with meconium. Therefore, we
hypothesize that SLL will clear the meconium, stabilize the
alveoli and increase the diffusion of iNO resulting in better
oxygenation.
The objective of the trial
The objective of our trial was to study the effectiveness of the
combined therapies of bolus surfactant treatment or SLL followed
by iNO in infants with meconium aspiration syndrome complicated
by pulmonary hypertension.
Materials
This study was conducted in the neonatal intensive care unit
(NICU) of the Neonatology Department of the Poznan University of
Medical Sciences, Poznan, Poland. We enrolled 13 neonates into
the study during the period between 1998 and 2004. The diagnosis
of MAS was based on clinical and radiological criteria. PPHN was
diagnosed on the basis of echocardiogram results.
Methodology of the trial
Randomization is shown in the diagram.
Group A1
Group A1 included seven newborns who were treated with a
combined therapy (SLL þ bolus surfactant þ iNO). Besides the
conventional treatment, SLL with a surfactant solution Survanta
was carried out between the 2nd and 22nd hour of life (average
time of treatment initiation 9.7±7.4 h). After the lavage

treatment, one dose of bolus Survanta at 100 mg of phospholipids
per kg of body weight was given.
Subsequently, echocardiogram assessment was carried out
for the diagnosis of PPHN; iNO was administered when
PPHN was diagnosed. The initial dose of nitric oxide was 20 p.p.m.
(Figure 1).
Group A2
Group A2 included six newborns treated conventionally. Between
the 2nd and 22nd hour of life (average time of treatment initiation
11±6.4 h), the newborns were given one dose of Survanta as a
bolus in the amount of 100 mg per kg of body weight. Infants did
not undergo SLL. After giving one dose of surfactant as well as
conducting an echocardiographic assessment and diagnosing with
PPHN, iNO was administered with an initial dose of NO at
20 p.p.m.
Lavage with a surfactant solution. Bronchoalveolar lavage with
saline natural surfactant solution was in newborns fulfilling the
inclusion criteria, which was carried out after sedation and initial
cardiopulmonary stabilization. The steps in surfactant lavage are
described below.
MAS +PPHN
A1- SLL+ BOLUS A2- BOLUS
iNO iNO
Figure 1 Scheme of the trial.
Surfactant
solution
Bodai valve
Figure 2 The surfactant lung lavage (SLL) technique.
Treatment of MAS with PPHN using combined therapy
J Gadzinowski et al
S57
(A) Preparation of surfactant solution for lavage2
1. A natural surfactant Survanta (Ross Abbott Laboratories,
Zwolle, The Netherlands) was used.
2. The dose of phospholipids for the lavage solution was 5 mg in
1 ml of physiological salt.
3. The volume of the solution was 15 ml per kg of the patient’s
body weight.
4. The calculated volume was divided into four parts.
5. A single lavage volume was 2 ml kg1.
Example of calculation: If the patient’s body weight was 3000 g,
45 ml of surfactant solution was used as a lavage: 9 ml of Survanta
supplemented with normal saline up to 45 ml.
(B) Lavage technique2
Installation of a closed circuit system was used for the
administration of lavage. This was possible using a Bodai valve in
the connector in the endotracheal tube. The surfactant solution
was instilled through the connector, whereas the lung fluid was
suctioned through the Bodai valve (Figure 2). This closed lavage
and suctioning system helped maintain both mechanical
ventilation and positive end-expiratory pressure (PEEP) at constant
levels. The prepared volume of the solution was divided into four
parts. Lavage and suctioning were conducted in four body
positions: on the right and left sides, and in the Trendelenburg and
anti-Trendelenburg positions, similar to the administration of the
Survanta dose given as treatment (in line with the producer’s
recommendations).
The surfactant solution was administered through the
endotracheal tube. After 2 ml of the solution was instilled, the
mechanical ventilation was continued. After 3 to 5 respiratory
cycles, the secretions were suctioned (Figure 2).
Treatment with bolus surfactant. Bolus surfactant was
administered through the endotracheal tube after sedation and
stabilization.
(A) Surfactant preparation
A natural surfactant Survanta (Ross Abbott Laboratories) was
used.
(B) The technique of surfactant administration
The surfactant was administered endotracheally with a catheter
through an intubation tube and through the Bodai valve placed in
the connector by the intubation tube. During the application, the
mechanical ventilation was continued. The dose of surfactant was
100 mg per kg of body weight. The surfactant was administered in
four positions: on the right and left sides, and in the Trendelenburg
and anti-Trendelenburg positions. Bolus surfactant was
administered once after SLL (group A1) and also once but without
SLL prior to it (group A2).
Journal of Perinatology
 Treatment of MAS with PPHN using combined therapy
J Gadzinowski et al
S58
Safety of the procedures. During the whole procedure, saturation
and heart rate were monitored. Saturation was considered safe if,
during lavage, it was within the range of 87 to X93%. If
saturation fell below 87%, the parameters of mechanical ventilation
were adjusted: peak pressure, fraction of inspired oxygen (FiO2)
and frequency of breaths. Lavage or surfactant was not given until
saturation reached a safe level. Once this was achieved, the
procedure was continued.
Parameters analyzed in the trial
The following parameters were analyzed in both groups:
(1) partial arterial oxygen pressure (PaO2);
(2) Fraction of inspired oxygen (FiO2);
(3) oxygenation index (OI);
(4) alveolar–arterial oxygen gradient (AaDO2).
The parameters listed above were analyzed at six time intervals:
 At 15 min before treatment initiation (0 h);
 after 1 h of combined therapy (1 h);
 after 2 h of combined therapy (2 h);
 after 4 h of combined therapy (4 h);
 after 24 h of combined therapy (24 h);
 after 48 h of combined therapy (48 h).
The following were also analyzed:
1. length of time on mechanical ventilation;
2. duration of iNO treatment;
3. length of hospital stay;
4. complications;
5. number of deaths.
Equipment. Intermittent mandatory ventilation (IMV) was
used as treatment for respiratory failure. Echocardiographic
examination was conducted by using HDI 3500 ATL and a
trans-receiving head with a frequency of 7 to 4 MHz. iNO was
administered by using SLE 3600 INOSYS. The initial and, at the
same time, maximum dose of NO was 20 p.p.m. Saturation was
measured by using pulse oximeters, and maintained in the range
between 87 and 93%. Arterial blood PO2 and PCO2 were measured.
Biochemical analysis was conducted by using Rapidlab.
Clinical classification and characteristics of recruited
newborns. After the diagnosis of MAS, the newborns were divided
into two groups: A1 and A2 (Figure 1) by randomization. In group
A1, infants received bolus surfactant treatment followed by iNO.
Group A2 received SLL followed by iNO therapy. There were no
statistically significant differences in the following parameters:
birth weight, gestational age as well as assessment of clinical
Journal of Perinatology
condition in the first minutes of life. No statistically significant
differences were observed in the frequency of Cesarean section
as well as the time for inclusion in the trial.
In group A1, the maturity of the newborns was 38.1 (±2.3)
weeks of gestation. The average birth weight was 3267 g (±822.4).
The overall condition of the newborns in the 1st and 5th minutes
of life was assessed according to the Apgar scale. The median in the
first minute of life was 5 points and it was 7 points in the fifth
minute of life.
In group A2, the maturity of the newborns was 37.8 (±2.1)
weeks of gestation. The average birth weight was 3128 g (±1155.4).
The overall condition of the newborns in the first and fifth minutes
of life was assessed on the basis of Apgar scale. The median in the
first minute of life was 4 points and it was 6 points in the fifth
minute of life.
Inclusion criteria. The trial was prospective and randomized
and included newborns who fulfilled the following criteria:
(1) Gestational ages X35 weeks of gestation;
(2) postnatal age p24 h;
(3) MAS was diagnosed on the following basis:
 presence of meconium-stained amniotic fluid below the
vocal cords;
 clinical symptoms: respiratory failure that required
treatment with mechanical ventilation (FiO2X40%) to
maintain PaO2 at 50 to 80 mm Hg and saturation of 87 to
93%;
 radiological findings consistent with MAS.
(4) Parental consent to enter the baby in the study.
Echocardiogram assessment was conducted for the diagnosis of
PPHN in all infants diagnosed as MAS. PPHN was diagnosed on the
basis of the following echocardiographic parameters:3–6
 mean pulmonary arterial pressure in the pulmonary trunk
X40 mm Hg;
 ductus arteriosus and foramen ovale;
 tricuspid valve insufficiency (TI);
 out of the wave A in M-mode pulmonary valve presentation.
The blood flow in the pulmonary artery was recorded by the
Doppler method using a pulse-wave technique and a trans-
receiving head with a high frequency of ultrasonic beams (7 to
4 MHz). The pressure in the pulmonary trunk was calculated on
the basis of the time from the initial blood flow wave until it
reached the maximum speed (acceleration time (AcT)). The
outflow wave was registered on the way from the right ventricle
next to the ring of the pulmonary valve or in the pulmonary trunk.
The flow acceleration time is shortened in the pulmonary artery.3
The mean pressure was calculated on the basis of a mathematical
 Treatment of MAS with PPHN using combined therapy
J Gadzinowski et al
S59

formula:4 80(0.5AcT). Mean pulmonary artery pressure (MPAP)
<40 mm Hg was defined as the norm.
Randomization. Infants were randomized into groups A1 and
A2. Each group of infants was managed by a different group of
neonatologists. Throughout the study, the infants stayed in the
hospital.
Statistical analysis. Parameters such as PaO2, FiO2, mean
airway pressure (MAP), OI, AaDO2 as well as duration of hospital
stay and the length of time on IMV and iNO were expressed in the
interval scale and described with the arithmetic mean value,
standard deviation as well as the minimum and maximum values
and confidential interval.
The normal distribution of these parameters was checked with
Shapiro–Wilk’s test. For the parameters that showed normal
distribution, the Student’s t-test for independent variables was used
for comparison of groups A1 and A2; otherwise, non-parametric
Mann–Whitney’s test was used.
For comparisons of values obtained at 0, 1, 2, 4, 24 and 48 h,
once the normal distribution was confirmed, the analysis of
variances for repeatable variables was carried out using the post
hoc Newman–Keuls test; if normal distribution was not confirmed,
the non-parametric Friedman test was used with Dunn’s test for
repeated comparisons. The Apgar scale as a parameter, expressed in
the ordinal number scale, was described with the median as well as
the minimum and maximum values. Apgar score between the groups
was compared using the non-parametric Mann–Whitney test.
The parameters expressed in the nominal scale, such as the
number of deaths and complications, were described with numbers
and corresponding percentages. Dependence was tested with
Fisher’s exact test. Statistical significance of a ¼ 0.05 was assumed
for verification of statistical tests.
Statistical calculations were performed using a statistical set
StatSoft Inc. (2005), STATISTICA, Tulusa, AZ, USA (data analysis
software system), version 7.1.
Results of the trial
PaO2
The mean initial (0 h) PaO2 before treatment initiation was
48.4 mm Hg (±15.0) in group A1. After 1 h of treatment, an
increase to 114.4 mm Hg (±49.8) was observed. After 2 h, the
value was 68.1 mm Hg (±15.3), after 4 h 69.6 mm Hg (±19.0),
after 24 h 78.7 mm Hg (±18.9) and after 48 h 67.9 mm Hg
(±12.3) (Table 1).
In group A2, the initial (0 h) oxygen pressure value before
treatment was 45.1 mm Hg (±14.3). No increase in this
parameter was observed, but after 1 h, its value was 46.7 mm Hg
(±14.9). An increase up to 51.1 mm Hg (±19.6) was observed
after 2 h, which further increased to 65.5 mm Hg (±38.6) after
4 h and a similar level of 61.9 mm Hg (±18.9) after 24 h and
64.0 mm Hg (±14.0) after 48 h (Table 2).

Table 1 Partial arterial oxygen pressure in group A1

PaO2 (mm Hg) N Mean value 95% confidence interval +95% confidence interval Standard deviation Minimum value Maximum value Median

0h 7 48.4 34.6 62.2 15.0 32.6 70.0 43.0

1h 7 114.4 68.3 160.4 49.8 36.4 203.0 116.0
2h 7 68.1 54.0 82.3 15.3 53.0 86.0 59.0
4h 7 69.6 52.0 87.2 19.0 52.0 108.0 67.0
24 h 7 78.7 61.2 96.2 18.9 58.0 112.0 74.0
48 h 7 67.9 56.5 79.3 12.3 51.0 83.0 62.5
A statistically significant increase of PaO2 in group A1 was observed between 0 and 4 h of treatment (P ¼ 0.0311).

Table 2 Partial arterial oxygen pressure in group A2

PaO2 (mm Hg) N Mean value 95% confidence interval +95% confidence interval Standard deviation Minimum value Maximum value Median

0h 6 45.1 30.0 60.1 14.3 36.6 74.0 40.2

1h 6 46.7 31.0 62.4 14.9 28.6 61.5 50.4
2h 6 51.1 30.7 71.6 19.6 31.0 80.0 46.7
4h 6 65.5 25.0 105.8 38.6 23.0 134.0 54.5
24 h 6 61.9 38.4 85.5 18.9 41.9 88.0 52.9
48 h 6 64.0 46.6 81.4 14.0 51.1 88.0 60.0
Increase of PaO2 in group A2, during the 48 h treatment was not statistically significant (P>0.05).

Journal of Perinatology
 Treatment of MAS with PPHN using combined therapy
J Gadzinowski et al
S60

140
P =0.0105
120
mmHg 100

80 PaO2 A1
60 PaO2 A2

40

20

0
0h 1h 2h 4h 24h 48h

Figure 3 Partial arterial oxygen pressure in groups A1 and A2.

Table 3 Fraction of inspired oxygen in group A1

FiO2 (%) N Mean value 95% confidence interval +95% confidence interval Standard deviation Minimum value Maximum value Median

0h 7 95.7 85.2 106.2 11.3 70.0 100.0 100.0

1h 7 79.4 57.9 100.7 23.2 50.0 100.0 90.0
2h 7 67.1 48.7 85.6 20.0 40.0 100.0 60.0
4h 7 57.1 41.8 72.4 16.6 35.0 85.0 60.0
24 h 7 53.7 31.6 75.8 25.0 21.0 90.0 60.0
48 h 7 40.1 22.0 58.3 19.6 21.0 70.0 30.0
A statistically significant drop of FiO2 in group A1 was observed between zero and first hour of treatment (P ¼ 0.0323).

Table 4 Fraction of inspired oxygen in group A2

FiO2 (%) N Mean value 95% confidence interval +95% confidence interval Standard deviation Minimum value Maximum value Median

0h 6 95.0 82.1 107.9 12.2 70.0 100.0 100.0

1h 6 93.3 82.5 104.2 10.3 80.0 100.0 100.0
2h 6 96.8 88.1 105.2 8.3 80.0 100.0 100.0
4h 6 89.2 72.8 105.6 15.6 60.0 100.0 95.0
24 h 6 64.0 36.5 91.6 22.2 40.0 100.0 60.0
48 h 6 46.0 5.9 86.1 32.3 21.0 100.0 35.0
After 48 h of treatment, the drop in FiO2 that was observed was not statistically significant (P>0.05).

The initial values of PaO2 in groups A1 and A2 did not differ
statistically. A comparative analysis of both groups demonstrated
that in group A1 the increase in PaO2 was statistically significantly
higher in comparison with group A2 after 1 h of treatment
(P ¼ 0.0105). After several hours, the difference was still noted;
however, the value was not statistically significant. After 2, 4, 24
and 48 h, there was a smaller difference in the value of PaO2 in
groups A1 and A2 (Figure 3).
Fraction of inspired oxygen
The mean value of the initial (0 h) fraction of inspired oxygen
before treatment initiation was 95.7% (±11.3) in group A1 and
95.0% (±12.2) in group A2. In group A1, a drop in this parameter
to 79.4% (±23.2) was achieved after 1 h, to 67.1% (±20.0) after
2 h, to 57.1% (±16.6) after 4 h, and after 24 h this parameter was
maintained at a similar level of 53.7% (±23.2). After 48 h, a drop
of FiO2 to 40.1% (±19.6) was observed (Table 3).
In group A2, before treatment initiation (0 h) the value of FiO2
was 95.0% (±12.2), and after 1 h of treatment a drop in this
parameter was achieved (to 93.3% (±10.3)), and after 2 h the
level was 96.8% (±8.3%). A drop to 89.2% (±15.6) was observed
after 4 h, and to 64.0% (±22.2) after 24 h and to 46.0% (±32.3)
after 48 h (Table 4).
The difference in the initial values of FiO2 in groups A1 and A2
was not statistically significant. When comparing both groups, it
was observed that after the first hour of treatment, the drop of FiO2
in group A1 was faster than it was in group A2. After several hours,
a difference that was statistically insignificant was still noted. In
group A1, a drop of FiO2 was statistically significantly higher than
it was in comparison with group A2 after 2 h (P ¼ 0.0130) and

Journal of Perinatology
 Treatment of MAS with PPHN using combined therapy
J Gadzinowski et al
S61

120
P =0.0130
P =0.0113
100

80
FiO2 A1

%
60
FiO2 A2
40

20

0
0h 1h 2h 4h 24h 48h

Figure 4 Fraction of inspired oxygen in groups A1 and A2.

Table 5 Mean airway pressure in group A1

MAP (cm H2O) N Mean value 95% confidence interval +95% confidence interval Standard deviation Minimum value Maximum value Median

0h 7 14.3 10.5 18.2 4.2 8.1 20.6 13.6

1h 7 11.5 8.1 14.9 3.7 8.2 17.4 9.2
2h 7 9.8 6.0 13.4 4.0 4.6 15.6 10.8
4h 7 6.6 1.8 11.5 5.2 1.4 17.5 4.8
24 h 7 4.1 0.8 7.4 3.6 1.4 11.7 3.1
48 h 7 5.0 0.7 10.7 6.1 1.4 18.6 2.6
In this group of patients, a drop in MAP that was statistically significant took place between 0 and 24 h of treatment (P ¼ 0.0114).

Table 6 Mean airway pressure in group A2

MAP (cm H2O) N Mean value 95% confidence interval +95% confidence interval Standard deviation Minimum value Maximum value Median

0h 6 13.8 5.1 22.5 8.3 6.6 29.3 13.0

1h 6 13.2 8.0 18.4 5.0 7.8 21.7 12.5
2h 6 12.0 6.6 17.4 5.2 6.2 20.7 12.2
4h 6 10.9 6.8 14.9 3.9 7.2 17.3 10.5
24 h 6 8.0 3.4 12.6 3.7 4.3 13.9 7.2
48 h 6 4.2 0.8 7.6 2.8 2.2 8.9 3.5
In this group, the drop in MAP during 48 h of treatment was statistically significant between the 1st and 48th hour of treatment (P ¼ 0.0008).

after 4 h of treatment (P ¼ 0.0113). After 24 and 48 h, a smaller
difference in the value of FiO2 was observed in groups A1 and A2
(Figure 4).
MAP
The initial (0 h) MAP before treatment initiation was 14.3 cm H2O
(±4.2) in group A1, and 13.8 cm H2O (±8.3) in group A2.
A drop in this parameter was observed after 1 h of treatment in
group A1 to 11.5 cm H2O (±3.7) with a further drop in this group
after 2 h to 9.8 cm H2O (±4.0) and after 4 h to 6.6 cm H2O
(±5.2). A further drop was observed after 24 h to 4.1 cm H2O
(±3.6). After 48 h, a further but small increase to 5.0 cm H2O
(±6.1) was observed (Table 5).
In group A2, before treatment initiation (0 h), the value of MAP
was 13.8 cm H2O (±8.28). After 1 h of treatment, no drop in this
parameter was observed and it was 13.2 cm H2O (±5.0). After 2 h
of treatment, a drop to 12.0 cm H2O (±5.2) was observed. A
further drop was observed after 4 h to 10.9 cm H2O (±3.9) and
after 24 h to 8.0 cm H2O (±3.7). This value decreased to 4.2 cm
H2O (±2.8) after 48 h (Table 6).
The difference between the initial value of MAP in
groups A1 and A2 was not statistically significant. When
comparing both groups, it was noticed that after the first hour
of treatment, the drop in MAP was more rapid in group A1 than in
group A2. After 1 and 2 h, the difference was still noted; however,
it was statistically insignificant. In group A1, a drop in
MAP was statistically significantly higher in comparison with
group A2 after 24 h of treatment (P ¼ 0.0423) (Figure 5).
After 48 h, the values of MAP in groups A1 and A2 were
similar.

Journal of Perinatology
 Treatment of MAS with PPHN using combined therapy
J Gadzinowski et al
S62

16
14
12
10 P =0.0423
cmH2O

MAP A1
8 MAP A2
6
4
2
0
0h 1h 2h 4h 24h 48h

Figure 5 Mean airway pressure in group A.

Table 7 Oxygenation index in group A1

OI N Mean value 95% confidence interval +95% confidence interval Standard deviation Minimum value Maximum value Median

0h 7 29.8 18.3 41.4 12.5 21.1 56.6 25.0

1h 7 11.2 0.3 22.7 12.5 3.8 39.0 6.9
2h 7 10.0 4.9 15.1 5.5 3.8 18.1 10.0
4h 7 6.3 0.4 12.3 6.4 1.2 19.6 3.,9
24 h 7 2.7 0.7 4.8 2.2 0.4 6.7 2.8
48 h 7 5.0 3.4 13.5 9.1 0.4 25.5 1.7
A statistically significant drop took place between 0 and 1 h (P ¼ 0.0003), 0 and 4 h (P ¼ 0.0398) and 0 and 48 h of treatment (P ¼ 0.0001).

Table 8 Oxygenation index in group A2

OI N Mean value 95% confidence interval +95% confidence interval Standard deviation Minimum value Maximum value Median

0h 6 32.4 6.1 58.6 25.0 11.0 80.0 24.3

1h 6 28.8 16.1 41.4 12.0 11.0 41.2 33.9
2h 6 26.8 6.8 46.7 19.0 8.9 59.5 18.0
4h 6 22.2 2.9 41.5 18.4 4.8 53.0 18.2
24 h 6 10.4 0.4 20.5 8.1 1.9 22.1 9.0
48 h 6 5.7 5.6 17.0 9.1 0.7 22.0 1.8
In group A2, a drop in OI between 0 and 48 h of treatment was statistically significant (P ¼ 0.0011).

Oxygenation index was noted that after 1 h of treatment a drop in OI in group A1

The mean initial (0 h) value of the oxygenation index before
treatment initiation was 29.8 (±12.5) in group A1 and 32.4
(±25.0) in group A2. In group A1, this value decreased after
several hours and was 11.2 (±12.5) after 1 h, 10.0 (±5.5) after
2 h and 6.3 (±6.4) after 4 h. After 24 h, this value decreased to
2.7 (±2.2), and after 48 h to 5.0 (±9.1) (Table 7).
In group A2, a drop in this parameter was observed after 1 h to
28.8 (±12.1), after 2 h to 26.8 (±19.0), after 4 h to 22.2
(±18.4), after 24 h to 10.4 (±8.1), and after 48 h a drop to 5.7
(±9.1) (Table 8).
The difference in the initial value of OI in groups A1 and A2 was
not statistically significant. When comparing both groups, it
was statistically significantly faster than it was in group A2
(P ¼ 0.0222). In group A1, a drop in OI was also statistically
significantly higher when compared with group A2 after 2 h
(P ¼ 0.0455), 4 h (P ¼ 0.0455) and 24 h of treatment
(P ¼ 0.0423). After 48 h, the values of OI in groups A1 and A2
were similar (Figure 6).
Alveolar–arterial oxygen gradient
The initial values of AaDO2 before treatment initiation (0 h) were
575.6 mm Hg (±91.0) for group A1 and 589.5 mm Hg (±95.8)
for group A2. In group A1, a drop in these values was observed after
1 h of treatment to 423.7 mm Hg (±184.3), after 2 h to

Journal of Perinatology
 Treatment of MAS with PPHN using combined therapy
J Gadzinowski et al
S63

35
P =0.0222
30 P =0.0455
25
P =0.0455
20 OI A1
P =0.0423
15 OI A2

10
5
0
0h 1h 2h 4h 24h 48h

Figure 6 Oxygenation index in group A.

P = 0.0036
700 P = 0.0106
600
500
mmHg

400 AaDO2 A1
AaDO2 A2
300
200
100
0
0h 1h 2h 4h 24h 48h

Figure 7 Alveolar–arterial oxygen gradient in group A.

364.2 mm Hg (±128.8), after 4 h to 305.3 mm Hg (±122.9), Duration of iNO treatment

after 24 h to 261.0 mm Hg (±160.9), and after 48 h to
178.3 (±144.0).
A drop in AaDO2 during treatment was statistically significant
between 0 and 24 h (P ¼ 0.0024) and between 0 and 48 h of
treatment (P ¼ 0.0021).
In group A2, after 1 h the value was 575.3 mm Hg (±88.2).
After 2 h, an increase to 581.2 mm Hg (±71.2) in this parameter
was observed. A drop was noted after 12 h to 506 mm Hg
(±112.6), with a further drop after 24 h to 352.3 mm Hg
(±177.5) and after 48 h to 226.0 mm Hg (±239.8).
A drop in AaDO2 during the time of the treatment was statistically
significant between 0 and the 48th hour of treatment (P ¼ 0.0017).
The difference between the initial values of AaDO2 in groups A1
and A2 was not statistically significant. When comparing both groups,
it was noted that after 1 h of treatment a drop in AaDO2 was more
rapid in group A1 in than in group A2. Subsequently, the difference
was statistically significant (P ¼ 0.0036). After 4 h, a drop of AaDO2
in group A1 was also statistically significantly higher in comparison
with group A2 (P ¼ 0.0106). After 24 and 48 h, a smaller difference
was observed between the value of AaDO2 in groups A1 and A2. These
differences were statistically insignificant (Figure 7).
Duration of mechanical ventilation
In group A1, the mean time on mechanical ventilation was
6.6 days (±2.6), and in group A2 it was 7.3 days (±1.7).
This difference was not statistically significant (P>0.05).
The mean duration of iNO treatment was 2.9 days (±1.5) in
group A1. In group A2, this value was higher and the duration was
4 days (±1). This difference was not statistically significant
(P>0.05).
Length of hospitalization
In group A1, the length of hospitalization was 16.4 days (±5.4)
and in group A2 it was 19.8 days (±2.9). This difference was not
statistically significant (P>0.05).
Complications
Air leaks. The incidence of pneumothorax was analyzed. In
group A1, no cases of pneumothorax were noted. This complication
was observed in two newborns from group A2. This value was not
statistically significant (P>0.05).
Deaths. There were no deaths in group A1. There were two cases
of deaths in group A2. This difference was not statistically
significant (P>0.05).
Discussion
Meconium in the airways and alveoli leads to inactivation of
endogenously and exogenously replaced surfactant. Therefore, we
hypothesized that SLL would improve the clearance of meconium
from the airways while replacing the surfactant.7 Earlier studies on

Journal of Perinatology
 Treatment of MAS with PPHN using combined therapy
J Gadzinowski et al
S64
the use of SLL in newborn piglets were published by Paranka et al.8
A solution of Survanta was used for lung lavage. Following this
treatment, a rapid improvement in oxygenation was observed.
Similar experimental studies on the use of SLL in the treatment of
MAS were conducted at different centers by Dargaville et al.9 and
Ogawa and co-workers.10–12
The first clinical studies on SLL were conducted by
Ogawa10 and Lam and Yeung.13 Ogawa administered as a lavage a
surfactant solution with a concentration of 12 mg of phospholipids
in 1 ml of physiological salt. Earlier, Ohama et al.11 attempted to
prove that a higher concentration is more effective. The volume of
the solution was 5 ml per kg of body weight. A significant drop in
OI and AaDO2 was achieved after 4 h of treatment.
Another attempt to define the most optimal dose of surfactant
for lavage was undertaken by Chang et al.14 In group I, the lungs
were washed with a surfactant solution with a concentration of
5 mg ml1 of physiological salt. In group II, lavage was conducted
with a surfactant solution with a concentration of 10 mg ml1. A
significant increase in PaO2 was observed within 24 h. It was noted
that both the first and the second doses of surfactant were effective.
The next phase of study was a multi-center study by Ogawa10
conducted in 11 centers. A comparative analysis of the group with
SLL against the group without SLL demonstrated that a drop in the
oxygenation parameter and an increase in the oxygen pressure
after 180 min were significantly higher in the SLL group than in
the control group.
Interesting results of clinical trials were presented by Lam
and Yeung.13,15 Survanta, a natural surfactant solution with a
concentration of 5 mg phospholipids per ml of saline and a
quantity of 15 ml per kg of body weight, was administered as a
lavage. The results of the trial demonstrated that the average values
of OI, MAP, AaDO2 and FiO2 were significantly and statistically
lower after 48 h of treatment.
Similar trial results were published in 2003 by Maroszyńska
et al.16,17
The inclusion criteria used in our study did not differ much
from the ones proposed by other researchers. Some of the
researchers quoted above considered the level of OI X20 as the
only inclusion criterion.18 In our study, the criteria used were
similar to those used by Lam and Yeung.13,15 The only difference
was in the time for enrollment in the trial, which was extended to
24 h (average time to include newborns transferred from a level II
unit). We did not use OI and MAP as the inclusion criteria; only
the value of FiO2X40% was used.
A more extended time for the inclusion in the studyFup to
120 h was proposed only by Maroszyńska et al.16,17 The majority of
the quoted experimental and clinical trials as well as our own
study studied the use of natural surfactant solutions in SLL. Earlier,
Meister et al.19 compared the natural and the synthetic surfactants.
They proved that there are no statistically significant differences in
the results achieved after administrations of either type of
Journal of Perinatology
surfactant in SLL. Studies on these subjects were also published by
Wiswell et al.20 Their trial was focused on the use of Surfaxin, a
new generation synthetic surfactant solution. The trial group
comprised 22 newborns and the control group included 15
newborns. The inclusion criteria were similar to the ones quoted
above. A statistically significantly faster drop of OI after SLL was
observed in comparison with the control group.
The dose of surfactant used for lavage is still a subject for
discussion. The smallest dose (8 ml per kg of body weight) of
lavage solution was proposed by Wiswell et al.21 Others have
proposed a dose of 10 ml solution per kg of body weight.9,14 The
most frequently suggested dose is a higher doseFfrom 15 15 to
20 ml kg1.10,17 In his earlier experimental trials, Ogawa10
attempted to define the most optimal dose of the lavage solution
and he came to the conclusion that a dose of 10 ml kg1 is most
effective.
Similarly, the concentration used in the solution is also a
subject of trials. Wiswell et al.21 proposed the lowest concentration
of the drugF2.5 mg ml1 of physiological salt. The majority of
researchers propose a dose from 4 23 to 5 7,8,15,16 or 6 mg ml1.12
Only Ogawa in their subsequent clinical studies proposed a higher
dose of 12 mg ml1. On the other hand, Chang et al.14 proved that
a dose of 5 mg ml1 is as effective as a dose of 10 mg ml1. In our
trial, a concentration of 5 mg ml1 at a dose of 15 ml per kg of
body weight was used.
All of the aforementioned clinical trials on the use of SLL in
MAS used saline as control groups. None compared bolus
treatments with SLL. Meister et al.19 conducted experimental trials
on a rabbit model in which they compared the results of surfactant
with bolus surfactant treatment. They also studied the differences
in natural and synthetic surfactant treatments. A significant
increase in PaO2 was observed in both natural and synthetic
surfactants, SLL, in comparison with groups treated with bolus
surfactant and a control group. The increase was observed after
4 h of treatment.
The group of patients with the most severe clinical course of
MAS includes cases complicated by PPHN. In the majority of cases,
studies on the use of iNO in the treatment of PPHN in term
neonates with MAS confirm its effectiveness in oxygenation
improvement.2,24 It was observed that, 30 min after the
administration of NO, there was a visible oxygenation improvement
manifested by an increase in PaO2 and a drop in OI and AaDO2.
When conducting independent randomized trials, Chen and Kao
observed a significant drop in OI after iNO treatment of PPHN in
MAS. Chen et al.1 noted improvement in 30 min of iNO, whereas
Kao et al. observed a statistically significant drop in OI after 60 min
of iNO.25 The effectiveness of iNO in the treatment of PPHN in MAS
was also proved by Gupta et al.26 A clinical trial on the use of iNO
in PPHN in MAS was also conducted by Hwang et al.27 After 1 h of
iNO treatment, they observed a statistically significant drop of OI
and MAP.

The neonates presented in our study who were diagnosed by
an echocardiographic examination with PPHN in the course of a
severe MAS, were treated with surfactant prior to the administration
of iNO. In this group of patients, the intention was to assess the
combined therapy as well as to answer the question whether it was
more effective to use SLL followed by bolus surfactant or surfactant
only prior to the administration of iNO. The initial dose of NO was
20 p.p.m. The treatment was conducted in the first 24 h of the
newborn’s life. The average time of treatment initiation was
9.7±7.4 h in group A1 and 11±6.4 h in group A2. Oxygenation,
ventilation and other selected clinical parameters were assessed.
Rais-Bachrami et al.28 attained a faster improvement in
oxygenation after surfactant administration followed by iNO. After
20 min, they lowered the dose of NO and after 15 min they stopped
the dose of iNO.
The administration of bolus surfactant followed by iNO (group
A2) had a positive influence on the improvement in oxygenation.
However, in a majority of cases, these results were not statistically
significant. An increase in PaO2 was observed only after 2 h of
treatment, and a further increase was noted within 48 h; however,
this was not statistically significant. A similar tendency was
observed in the values of FiO2 and OI. Within 48 h, a gradual
decrease in these parameters was observed; however, this drop was
not statistically significant either. AaDO2 declined as well. A
statistically significant drop was noted after 24 h and then after
48 h of treatment as well as in the time range between the
12th and 24th hour of treatment. A drop in MAP was observed
only after 4 h of treatment and a further drop was noted until the
48th hour of treatment; however, this drop was not statistically
significant. Substantially better results were observed in cases when,
prior to iNO, SLL was used followed by one dose of bolus surfactant.
There is a lack of information regarding the effectiveness of this
therapy. Investigators have used solely iNO treatment in severe
cases of MAS complicated by PPHN. A drop in OI and an increase
in PaO2 were observed after 30 min,24 after 60 min8 and after 24 h
of iNO treatment29 in different studies.
In our studies, a significant increase in PaO2 was observed in
the group with SLL after 1 h. In the SLL group, a drop in OI and
FiO2 was statistically significant after 24 and 48 h. When we
compared SLL with the group without SLL, we noticed statistically
significant differences much sooner, that is, after the first, second
and fourth hours.
The drop in AaDO2 in the SLL group was substantial after 2 and
4 h, with a further drop after 24 and 48 h. MAP also declined
significantly in the SLL group after 24 h. However, when
comparing the two groups we observed a significant difference only
after 48 h. These results are similar to others who did not use SLL
but were treated with only iNO. Therefore, we cannot conclude with
certainty whether the use of SLL increases the effectiveness of iNO.
Similar to our findings, others did not find statistically
significant differences in the length of time on IMV, number of
Treatment of MAS with PPHN using combined therapy
J Gadzinowski et al
S65
complications and deaths when in comparison with the group that
was not treated with iNO. Similarly, in our trial no statistically
significant differences were observed in the length of mechanical
ventilation and hospitalization. In the group with SLL, bolus
surfactant and iNO, as well as no air leaks or deaths, were
encountered. In the group with bolus surfactant and iNO, there
were 2/6 cases of pneumothorax and 2/6 deaths. However, this
difference was not statistically significant. In the first case,
pneumothorax was one of the causes of death. In the second case
which was complicated by pneumothorax, the patient survived. The
other death was due to a sudden stop in heart activity in the course
of a severe circulatory failure.
During our study, there was no attempt to answer the important
question whether the use of bolus surfactant along with SLL will
prevent the development of PPHN and whether it may decrease the
need for iNO use. Chang et al.14 attempted to address this question.
They conducted clinical studies on neonates with MAS in whom OI
was X20. Prior to iNO use, the airways were washed with a
surfactant solution with a concentration of 5 mg ml1 or
10 mg ml1 of physiological salt. A statistically significant increase
in PaO2 was observed within 24 h; 5 of 12 neonates required iNO
treatment.
Therefore, the results of the trials conducted by researchers quoted
in this paper as well as by our own trial indicate that, in severe cases of
MAS complicated by PPHN, the administration of bolus surfactant or
SLL followed by bolus surfactant prior to iNO treatment improves
oxygenation of the patient and may lead to a decrease both in the
severity of PPHN and in the duration of iNO treatment. However, no
significant decrease in the duration of IMV, hospitalization or in the
number of complications and deaths was observed.
Conclusions
(1) The combined therapy: SLL followed by bolus surfactant, in
combination with iNO in the treatment of meconium
aspiration syndrome accompanied by PPHN, showed
significant improvement in oxygenation as well as a decrease
in mechanical ventilation parameters.
(2) However, SLL followed by bolus surfactant as well as combined
therapy: SLL followed by bolus surfactant combined with
iNO did not exert significant influence over the duration of
mechanical ventilation, iNO treatment as well as the length
of hospitalization and the number of complications.
(3) On account of small number of the patients, these studies do
not have enough power to choose a particular combination of
therapies. There is a need for larger series to answer these
questions.
Disclosure
J Gadzinowski has received consulting fees from Abbott. The remaining authors
have declared no financial interests.
Journal of Perinatology
 Treatment of MAS with PPHN using combined therapy
J Gadzinowski et al
S66
References
1 Park KH, Bae CW, Chung SJ. In vitro effect of meconium on the physical surface
properties and morphology of oxygenous pulmonary surfactant. J Korean Med Sci
1996; 11: 429–436.
2 Gadzinowski J, Moœcicka A, Kowalska K. Zespół aspiracji smółkiFwspółczesne
metody leczenia. Kl Perinat i Ginekol 1998; 1: 153–160.
3 Alekszewicz-Baranowska J, Nadciœnienie płucne W. Echokardiografia wad
wrodzonych serca. In: Alekszewicz-Baranowska J (Pod red). Wyd Med MAKmed:
Gdañsk 2000; 165–169.
4 Kawalec W. Diagnostyka nieinwazyjna ukadu kra żenia. W: Kardiologia okresu
‘ administration of dilute surfactant in a piglet model of meconium aspiration. Lung
noworodkowego. Kawalec W (Pod red). PZWL Biblioteka Pediatry: Warszawa 1998;
38–50.
5 Kawalec W, Wołoszczuk-Ge˛bicka B (eds). kra żenia przejœciowe i przetrwałe
‘
nadciœnienie płucne. PZWL Biblioteka Pediatry: Warszawa 1998; 73–84.
6 Rydlewska-Sadowska W. Echokardiografia kliniczna. BIK Warszawa 1991; 11: 135–136.
7 Kurtis PS. Meconium aspiration. Pediatrics 2000; 106: 867.
8 Paranka MS, Walsh WF, Stancombe BB. Surfactant lavage in a piglet model of
meconium aspiration syndrome. Pediatr Res 1992; 31: 625–628.
9 Dargaville PA, Mills JF, Headley BM, Chan Y, Coleman L, Loughnan PM et al.
Therapeutic lung lavage in the piglet model of meconium aspiration syndrome.
Am J Respir Crit Care Med 2003; 168: 456–463.
10 Ogawa Y. Bronchial lavage with surfactant solution for the treatment of meconium
aspiration syndrome. In: Lucey J (ed) Conference Paper at Ross Special Conference
Hot Topics in Neonatology 1997; 259–264.
11 Ohama Y, Itakura Y, Koyama N, Eguchi H, Ogawa Y. Effect of surfactant lavage
in a rabbit model of meconium aspiration syndrome. Acta Paediatr Jpn 1994;
36: 236.
12 Ohama Y, Ogawa Y. Treatment of meconium aspiration syndrome with surfactant
lavage in an experimental rabbit model. Pediatr Pulmonol 1999; 28: 18–23.
13 Lam BC, Yeung CY. Surfactant lavage for meconium aspiration syndrome. In: Lucey J
(ed) Conference paper at Ross Special Conference Hot Topics in Neonatology,
Washington 1997; 252–256.
14 Chang HY, Hsu CH, Kao HA, Hung HY, Chang JH, Peng CC et al. Treatment of severe
meconium aspiration syndrome with dilute surfactant lavage. J Formos Med Assoc
2003; 102: 326–330.
15 Lam BC, Yeung CY. Surfactant lavage for meconium aspiration syndrome: a pilot
study. Pediatrics 1999; 103(5 Part 1): 1014–1018.
Journal of Perinatology
16 Maroszyńska I, Stoiñska B, Czop J, Dygas T, Gadzinowski J. Surfactant lavage as an
effective treatment in newborn with meconium aspiration syndrome. Arch Perinat Med
2003; 2: 15–21.
17 Maroszyńska I, Wilkowski J, Czop J, Dygas T. Bronchoalveolar lavage with saline
surfactant solution in the treatment of neonates with severe respiratory failure due to
MAS, PIE or BPD. Med Sci Monit 2003; 9(Suppl 5): 22–28.
18 Davey AM, Becker JC, Davis JC. Meconium aspiration syndrome. physiological
and inflammatory changes in a newborn piglet model. Pediatr Pulmonol 1993;
16: 300.
19 Meister J, Balaraman V, Ramirez M, Uyehara CF, Killeen J, Ku T et al. Lavage
2004; 182: 227–240.
20 Wiswell TE, Peabody SS, Davis JM, Slayter MV, Bent RC, Merritt TA. Surfactant therapy
and high frequency jet ventilation in management of a piglet model of meconium
aspiration syndrome. Pediatr Res 1994; 36: 494–500.
21 Wiswell TE, Rebecca C, Bent M. Meconium staining and the meconium aspiration
syndrome. Pediatr Clin North Am 1993; 40: 955–977.
22 Castellheim A, Lindenskov PH, Pharo A, Aamodt G, Saugstad OD, Mollnes TE.
Meconium aspiration syndrome induces complement-associated systemic inflammatory
response in newborn piglets. Scand J Immunol 2005; 61: 217–225.
23 Ting P, Brady JP. Tracheal suction in meconium aspiration. Am J Obstet Gynecol
1975; 122: 767.
24 Marini G, Berefield ES, Carlo W. The role of nitric oxide in the treatment of neonatal
pulmonary hypertension. Curr Opin Pediatr 1996; 8: 118.
25 Ko SY, Chang YS, Park WS. Clinical response to inhaled nitric oxide in persistent
pulmonary hypertension of the newborn. J Korean Med Sci 1998; 13: 500–506.
26 Gupta V, Bhatia BD, Mishra OP. Meconium stained amniotic fluid antenatal
intrapartum and neonatal attributes. Indian Pediatr 1996; 33: 293.
27 Hwang SJ, Lee KH, Hwang JH. Factors affecting the response to inhaled nitric oxide
therapy in persistent pulmonary hypertension of the newborn infants. Yonsei Med J
2004; 45: 49–55.
28 Rais-Bachrami K, Rivera O, Seale WR, Short BL. Effect of nitric oxide and high-
frequency oscillatory ventilation in meconium aspiration syndrome. Pediatr Crit Care
Med 2000; 1: 66–160.
29 Gupta A, Rastogi S, Sahni R, Bhutada A, Bateman D, Rastogi D et al. Inhaled
nitric oxide and gentle ventilation in the treatment of pulmonary hypertension
of the newbornFa single-center, 5-year experience. J Perinatol 2002; 22:
435–441.
 Journal of Perinatology (2008) 28, S67–S71
r 2008 Nature Publishing Group All rights reserved. 0743-8346/08 $30
www.nature.com/jp
REVIEW
Nitric oxide and beyond: new insights and therapies for
pulmonary hypertension
RH Steinhorn
Department of Pediatrics, Division of Neonatology, Children’s Memorial Hospital, Northwestern University’s Feinberg School of
Medicine, Chicago, IL, USA
Persistent pulmonary hypertension of the newborn (PPHN) contributes
significantly to the morbidity and mortality associated with meconium
aspiration syndrome. This review article discusses new insights into the
vascular abnormalities that are associated with PPHN, including the recent
recognition of the importance of oxidant stress in its pathogenesis. Recent
data are presented showing that treatment with high oxygen concentrations
may increase production of oxygen free radicals. The rationale for the use of
inhaled nitric oxide, and strategies for enhancing nitric oxide signaling are
discussed. Finally, the rationale for new treatment approaches is reviewed,
including inhibition of cyclic guanosine monophosphate-specific
phosphodiesterases and scavengers of reactive oxygen species.
Journal of Perinatology (2008) 28, S67–S71; doi:10.1038/jp.2008.158
Introduction
Neonatal respiratory failure affects 2% of all live births and is
responsible for a substantial proportion of neonatal mortality.
Although preterm infants are at higher risk of respiratory failure,
term and near-term infants account for one-third of the cases.1
A better understanding of the pathophysiology of hypoxemic
respiratory failure is needed to develop more specific and effective
therapies. This review will focus on recent progress in our
understanding of pulmonary hypertension, which is commonly
associated with the severe respiratory failure that accompanies
meconium aspiration syndrome (MAS).
Pathophysiology of PPHN
Shortly after birth, the fetus normally undergoes a rapid
cardiopulmonary transition to meet the new demands of
extrauterine life. However, if pulmonary vascular resistance does
not fall, pulmonary blood flow cannot increase, and the result is
hypoxemic respiratory failure or persistent pulmonary hypertension
of the newborn (PPHN). The incidence of severe PPHN is estimated
at 0.2% of live-born term infants. MAS is the most common cause
Correspondence: Dr RH Steinhorn, Department of Pediatrics, Division of Neonatology,
Children’s Memorial Hospital, Northwestern University’s Feinberg School of Medicine, 2300
Children’s Plaza, Neonatology, Box 45, Chicago, IL 60614, USA.
E-mail: r-steinhorn@northwestern.edu
of PPHN, and when present, it can contribute significantly to its
morbidity and mortality. Newborns with hypoxemic respiratory
failure and/or PPHN are at risk for numerous complications
including death, neurological injury and other morbidities.
Persistent pulmonary hypertension of the newborn can
generally be characterized as one of three types: (1) the abnormally
constricted pulmonary vasculature due to lung parenchymal
diseases, such as MAS, respiratory distress syndrome or pneumonia;
(2) the lung with normal parenchyma and remodeled pulmonary
vasculature, also known as idiopathic PPHN; or (3) the hypoplastic
vasculature as seen in congenital diaphragmatic hernia. The most
common cause of PPHN is MAS. Infants with MAS will typically fall
into the first or second categories, and the most severe cases are
probably affected by both parenchymal and vascular disease.
Understanding the pathophysiology of the abnormally
remodeled pulmonary vasculature is of utmost importance in
directing therapy. As it is not feasible to study the remodeling
process in the human infant, much of our current understanding
is derived from animal models. One cause of idiopathic PPHN is
constriction of the fetal ductus arteriosus in utero because of
exposure to nonsteroidal anti-inflammatory drugs during the third
trimester. Ductal constriction or ligation can be surgically
performed in utero in lambs, leading to rapid antenatal
remodeling of the pulmonary vasculature. Findings in PPHN lambs
are similar to those observed in human infants, including
increased fetal pulmonary artery pressure, pulmonary vascular
remodeling and profound hypoxemia after birth.
On the basis of work from animal models, there is strong
evidence that disruptions of the nitric oxide (NO)-cyclic guanosine
monophosphate (cGMP), prostacyclin-cyclic adenosine
monophosphate (cAMP) and endothelin signaling pathways play
an important role in the vascular abnormalities associated with
PPHN.2 The NO-cGMP pathway has been a topic of particularly
intense investigation over the past decade. Decreased expression
and activity of endothelial NO synthase have been documented in
the PPHN lamb model,3 and decreased endothelial NO synthase
expression has also been reported in umbilical venous endothelial
cell cultures from human infants with meconium staining who
develop PPHN.4 These important findings were rapidly followed by
 Nitric oxide and beyond
RH Steinhorn
S68
clinical testing of inhaled NO (iNO) as a therapy for hypoxemic
respiratory failure and PPHN.
Inhaled nitric oxide
The primary aim of PPHN therapy is selective pulmonary
vasodilatation. Inhaled NO appears to be well suited for this effect:
it is a rapid and potent vasodilator, and because NO is a small gas
molecule, it can be delivered through a ventilator directly to
airspaces approximating the pulmonary vascular bed. Once in the
blood stream, NO binds avidly to hemoglobin, limiting its systemic
vascular activity and increasing its selectivity for the pulmonary
circulation.
Large placebo-controlled trials provided clear evidence that iNO
significantly decreases the need for extracorporeal membrane
oxygenation (ECMO) support in newborns with PPHN.5,6 However,
it is important to note that up to 40% of infants will not improve
oxygenation or maintain a response to iNO, and iNO did not
reduce mortality or length of hospitalization. In addition, follow-up
studies for 12 to 24 months indicate that iNO does not significantly
alter the incidence of chronic lung disease or neurodevelopmental
impairment.7,8 This is an interesting and important observation
that may indicate that the underlying disease is associated with
early neurological injury. Finally, Konduri et al.9,10 determined
that starting iNO earlier in the disease course (for an oxygenation
index of 15 to 25) did not decrease the incidence of ECMO and/or
death or improve other patient outcomes, including the incidence
of neurodevelopmental impairment.
Following the introduction of high-frequency ventilation (HFV),
surfactant and iNO in the early 1990s, the patient demographic of
neonatal support with ECMO has changed. Data from the large
registry maintained by the Extracorporeal Life Support
Organization indicate that the use of these therapies has increased
steadily over the past 10 years, accompanied by a greater than 40%
reduction in the number of neonates cannulated for ECMO.
However, as overall ECMO survival has diminished over the same
time period, some physicians have speculated that these new
treatment modalities may delay ECMO cannulation and have a
negative effect on mortality and morbidity in those infants that
continue to require extracorporeal support. Therefore, we recently
examined data from the Extracorporeal Life Support Organization
registry between 1996 and 2003. We found that NO, HFV and
surfactant use were not associated with any adverse outcomes
during ECMO, including increased hours on ECMO or
increased time to extubation.11 Furthermore, both surfactant and
NO use were associated with lower ECMO mortality, and
NO use was associated with a decreased risk of cardiac
arrest before cannulation. As ECMO is a proven therapy for
severe respiratory failure, it is reassuring that these new
therapies have not had a negative impact on the most severely
affected infants.
Journal of Perinatology
AA
L-Arginine COX-1
PGH2 Endothelial
NOS
PGIS Cell
NO PGI2
Guanylate Cyclase Adenylate Cyclase
GTP ATP Smooth
cGMP cAMP Muscle
5´-GMP
AMP
Cell
PDE5 PDE3
VASODILATION
Sildenafil Milrinone
Figure 1 Nitric oxide (NO) and prostacyclin signaling pathways in regulation of
pulmonary vascular tone. NO is synthesized by NO synthase (NOS) from the
terminal nitrogen of L-arginine. NO stimulates soluble guanylate cyclase (sGC) to
increase intracellular cGMP. PGH2 is an arachidonic acid (AA) metabolite formed
by cyclooxygenase (COX-1) and prostacyclin synthase (PGIS) in the vascular
endothelium. Prostacyclin (PGI2) stimulates adenylate cyclase in vascular smooth
muscle cells, which increases intracellular cAMP. Both cGMP and cAMP indirectly
decrease free cytosolic calcium, resulting in smooth muscle relaxation. Specific
phosphodiesterases hydrolyze cGMP and cAMP, thus regulating the intensity and
duration of their vascular effects. Inhibition of these phosphodiesterases with
agents such as sildenafil and milrinone may enhance pulmonary vasodilation.
New insights into PPHN pathophysiology
As NO is not universally effective, there has been considerable
interest in better understanding the biochemical pathways that
regulate pulmonary vasoconstriction and remodeling in PPHN. For
instance, NO mediates vasodilatation by stimulating soluble
guanylate cyclase in vascular smooth muscle cells, which then
converts guanosine triphosphate to cGMP (Figure 1). cGMP is the
central and critical second messenger that regulates contractility of
the smooth muscle cell by modulating the activity of
cGMP-dependent kinases, phosphodiesterases and ion channels. The
cGMP-dependent or type 5 phosphodiesterase is potentially
important, as it can downregulate cGMP concentrations by
degrading cGMP to the inactive 50 -GMP. Therefore, there are critical
points in the pathway downstream of NO production that serve as
attractive targets for manipulating cellular cGMP concentrations.
For example, expression and activity of soluble guanylate cyclase are
decreased in the abnormally remodeled pulmonary vessels of the
PPHN lamb model, which could potentially diminish responses to
both endogenous and exogenous NO. This finding would indicate
that new compounds that directly stimulate sGC at an NO-
independent but heme-dependent site may be helpful, a hypothesis
that appears to be promising in preclinical testing.12 Another
potential cause for low cGMP concentrations would be increased
expression and/or activity of cGMP-specific phosphodiesterases,
which could then be manipulated through use of specific inhibitors.
Several new lines of evidence now indicate that oxidant stress is
important in the pathogenesis of PPHN. An increase in reactive
 Nitric oxide and beyond
RH Steinhorn
S69

SOD H2O2
O2
Arachidonic Acid
O2.- and PUFAs

Isoprostanes

Contractility

Peroxynitrite

NO

Pulmonary Artery

Alveolus

Figure 2 Hypothesized role of reactive oxygen species (ROS) and their metabolites in mediating increased pulmonary arterial contractility following exposure to 100%
oxygen. Exposure to high oxygen concentrations results in the formation of superoxide anions (O 2 ). Superoxide anions combine with nitric oxide (NO) to form
peroxynitrite, a potent pulmonary vasoconstrictor. Superoxide dismutase (SOD) enzyme converts superoxide anions to hydrogen peroxide (H2O2), also a pulmonary
vasoconstrictor. When membrane lipids (arachidonic acid and polyunsaturated fatty acids (PUFA)) are exposed to ROS, such as superoxide anions and hydrogen peroxide
or peroxynitrite, a variety of isoprostanes are formed. Isoprostanes are known constrictors of pulmonary arteries. Adapted from Lakshminrusimha et al.16 with permission.

oxygen species (ROS) such as superoxide and hydrogen peroxide in
the smooth muscle and adventitia of pulmonary arteries has been
demonstrated in the PPHN lamb model.13,14 Possible sources of
elevated concentrations of ROS include increased expression and
activity of NADPH oxidase and a reduction in superoxide dismutase
(SOD) activity. PPHN lambs also demonstrate diminished binding
of the chaperone protein, heat shock protein 90, to endothelial NO
synthase.15 Decreased heat shock protein 90–endothelial NO
synthase interactions lead to an ‘uncoupling’ of NOS activity,
which results in decreased synthesis of NO and increased
superoxide production. Once present in the lung, elevated
concentrations of ROS are believed to play a role in vascular
smooth muscle cell proliferation in PPHN, as well as abnormal
vascular reactivity.
Finally, current therapeutic practices may have an effect on
pulmonary vascular reactivity and remodeling. In particular, the
use of oxygen has recently become controversial in numerous
settings. While oxygen is a pulmonary vasodilator, the extreme
hyperoxia routinely used in PPHN management may be toxic to
the developing lung by the formation of ROS. Although hyperoxic
ventilation is a mainstay of the treatment of PPHN, we know
surprisingly little about what oxygen concentrations will maximize
benefits and minimize risk. Superoxide may react with arachidonic
acid to increase concentrations of isoprostanes and may also
combine with NO to form peroxynitrite. Both are potent oxidants
with the potential to produce vasoconstriction, cytotoxicity and
damage to surfactant proteins and lipids (Figure 2). New data
indicate that even brief periods of ventilation with 100% O2
(30 min) are sufficient to increase reactivity of pulmonary vessels
in normal lambs16 and to diminish the response of the lung to
endogenous and exogenous NO.17
Alternative and emerging pulmonary vasodilators
An improved knowledge of the biochemical abnormalities
responsible for refractory PPHN is leading to a growing list of
promising new therapeutic strategies. Many investigators are
seeking to enhance cGMP-mediated vasodilation through the use of
cGMP-specific phosphodiesterase inhibitors, direct soluble
guanylate cyclase activators, scavengers of ROS as well as
manipulation of the cAMP pathway.
Similar to cGMP, cAMP also stimulates vasodilatation
(Figure 1). One potential approach that might take advantage of
this mechanism is using milrinone to inhibit PDE3, the
phosphodiesterase that metabolizes cAMP. Milrinone has been
shown to decrease pulmonary artery pressure and resistance and to
act additively with iNO in animal studies. Recent reports indicate
that it may decrease rebound pulmonary hypertension after
discontinuation of iNO and may enhance pulmonary vasodilation
of infants refractory to iNO.18 Prostacyclin (PGI2) stimulates

Journal of Perinatology
 Nitric oxide and beyond
RH Steinhorn
S70

Figure 3 Superoxide dismutase (SOD) and nitric oxide (NO) improve the arterial-to-alveolar oxygen (a/A) ratio to a similar degree. The a/A ratio of four groups of
lambs with persistent pulmonary hypertension of the newborn (PPHN) ventilated with 100% oxygen alone (100% O2), 100% oxygen with rhSOD 5 mg kg1 administered
at birth (100% O2 SOD), 100% oxygen with rhSOD 5 mg kg1 administered at 4 h of life (100% O2 4 h delay SOD) and 100% oxygen and 20 p.p.m. of inhaled NO
(iNO; 100%O2 NO). PPHN lambs ventilated with 100% oxygen were critically ill, and two of these lambs died at approximately 10 and 16 h of age (shown by arrows).
The dashed line beyond these points represents mean±s.e.m. for the remaining lambs. *P<0.05 compared with 100% oxygen-alone group. Adapted from
Lakshminrusimha et al.25 with permission.

membrane bound adenylate cyclase, increases cAMP and inhibits
pulmonary artery smooth muscle cell proliferation in vitro.
Although the use of systemic infusions of PGI2 may be limited by
systemic hypotension, inhaled PGI2 has been shown to have
vasodilator effects limited to the pulmonary circulation. Reports in
children have been positive, but to date there have been few reports
of inhaled PGI2 use in neonates with PPHN.19 It is most likely that
further investigations will focus on preparations specifically
designed for inhalation, such as iloprost.
There has been particular interest in the inhibition of
cGMP-metabolizing phosphodiesterase activity, which would
theoretically increase cGMP concentrations and result in
pulmonary vasodilation and/or increased efficacy of iNO
(Figure 1). On the basis of clinical trials, sildenafil, a potent and
highly specific phosphodiesterase inhibitor, has recently been
approved by the Food and Drugs Administration for the treatment
of pulmonary hypertension in adults. In lambs with experimental
pulmonary hypertension, both enteric and aerosolized sildenafil
dilate the pulmonary vasculature and augment the pulmonary
vascular response to iNO. Intravenous sildenafil was found to be a
selective pulmonary vasodilator with efficacy equivalent to inhaled
NO in a piglet model of meconium aspiration, although
hypotension and worsening oxygenation resulted when it was used
in combination with iNO.20,21 Sildenafil may attenuate rebound
pulmonary hypertension after withdrawal of iNO in newborn and
pediatric patients.22 Use of sildenafil in PPHN has been limited by
its availability only as an enteric form, although a recent report
indicates that it improved oxygenation and survival in
human infants with PPHN compared with placebo.23 A pilot
trial of intravenous sildenafil was recently conducted in newborns
with pulmonary hypertension, and data analysis is nearing
completion.
New laboratory studies indicate that scavengers of ROS such as
SOD may augment responsiveness to iNO. As described above,
increased production of superoxide is noted in experimental models
of PPHN. As iNO is usually delivered with high concentrations of
oxygen, there is the potential for enhanced production of additional
oxidants such as peroxynitrite. Superoxide dismutase scavenges
and converts superoxide radical to hydrogen peroxide, which is
subsequently converted to water by the enzyme catalase.
Administration of recombinant human SOD (rhSOD) has been
tested in preterm infants without adverse effects and with trends
toward decreased pulmonary morbidity. In lambs with pulmonary
hypertension, rhSOD was found to dilate the pulmonary circulation
and enhance responsiveness to inhaled NO.24 A recent study
examined the effects of rhSOD on oxygenation over a 24-h period
in ventilated PPHN lambs.25 The results showed that a single dose
of rhSOD by itself improved oxygenation to a degree that was
similar to iNO (Figure 3). Furthermore, rhSOD treatment appeared
to block formation of oxidants such as peroxynitrite and
isoprostanes. Thus, an antioxidant therapeutic approach may
have multiple beneficial effects: scavenging superoxide may
make both endogenous and inhaled NO more available to
stimulate vasodilatation and may also reduce oxidative
stress and limit lung injury. It is hoped that human trials will
begin soon.
Disclosure
RH Steinhorn has received consulting fees from Ikaria. Grant support from the
NIH (HL 54705) supported the research presented in the paper.

Journal of Perinatology

References
1 Angus DC, Linde-Swirble WT, Clermont G, Griffin MF, Clark RH. Epidemiology of
neonatal respiratory failure in the United States. Am J Resp Crit Care Med 2001; 164:
1154–1160.
2 Farrow KN, Fliman P, Steinhorn RH. The diseases treated with ECMO: focus on PPHN.
Semin Perinatol 2005; 29: 8–14.
3 Shaul PW, Yuhanna IS, German Z, Chen Z, Steinhorn RH, Morin III FC.
Pulmonary endothelial NO synthase gene expression is decreased in fetal lambs
with pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol 1997; 272:
L1005–L1012.
4 Villaneuva ME, Zaher FM, Svinarich DM, Konduri GG. Decreased gene expression of
endothelial nitric oxide synthase in newborns with persistent pulmonary hypertension.
Pediatr Res 1998; 44: 338–343.
5 Neonatal Inhaled Nitric Oxide Study Group. Inhaled nitric oxide in full-term and
nearly full-term infants with hypoxic respiratory failure. New Engl J Med 1997; 336:
597–604.
6 Clark RH, Kueser TJ, Walker MW, Southgate WM, Huckaby JL, Perez JA et al. Low dose
nitric oxide therapy for persistent pulmonary hypertension of the newborn. N Engl J
Med 2000; 342: 469–474.
7 Neonatal Inhaled Nitric Oxide Study Group. Inhaled nitric oxide in term and near-
term infants: neurodevelopmental follow-up of the neonatal inhaled nitric oxide study
group (NINOS). J Pediatr 2000; 136: 611–617.
8 Clark RH, Huckaby JL, Kueser TJ, Walker MW, Southgate WM, Perez JA et al. Low-dose
nitric oxide therapy for persistent pulmonary hypertension: 1-year follow-up.
J Perinatol 2003; 23: 300–303.
9 Konduri GG, Solimani A, Sokol GM, Singer J, Ehrenkranz RA, Singhal N et al. A
randomized trial of early versus standard inhaled nitric oxide therapy in term and
near-term newborn infants with hypoxic respiratory failure. Pediatrics 2004; 113:
559–564.
10 Konduri GG, Vohr B, Robertson C, Sokol GM, Solimano A, Singer J et al. Early inhaled
nitric oxide therapy for term and near-term newborn infants with hypoxic respiratory
failure: neurodevelopmental follow-up. J Pediatr 2007; 150: 235–240.
11 Fliman PJ, deRegnier RA, Kinsella JP, Reynolds M, Rankin LL, Steinhorn RH. Neonatal
extracorporeal life support: impact of new therapies on survival. J Pediatr 2006; 148:
595–599.
12 Deruelle P, Grover TR, Abman SH. Pulmonary vascular effects of nitric oxide-cGMP
augmentation in a model of chronic pulmonary hypertension in fetal and neonatal
sheep. Am J Physiol Lung Cell Mol Physiol 2005; 289: L798–L806.
Nitric oxide and beyond
RH Steinhorn
S71
13 Brennan LA, Steinhorn RH, Wedgwood S, Mata-Greenwood E, Roark EA, Russell JA
et al. Increased superoxide generation is associated with pulmonary hypertension in
fetal lambs. A role for NADPH oxidase. Circ Res 2003; 92: 683–691.
14 Wedgwood S, Steinhorn RH, Bunderson M, Wilham J, Lakshminrusimha S, Brennan LA
et al. Increased hydrogen peroxide downregulates soluble guanylate cyclase in the
lungs of lambs with persistent pulmonary hypertension of the newborn. Am J Physiol
Lung Cell Mol Physiol 2005; 289: L660–L666.
15 Konduri GG, Ou J, Shi Y, Pritchard Jr KA. Decreased association of HSP90 impairs
endothelial nitric oxide synthase in fetal lambs with persistent pulmonary
hypertension. Am J Physiol Heart Circ Physiol 2003; 285: H204–H211.
16 Lakshminrusimha S, Russell JA, Steinhorn RH, Ryan RM, Gugino SF, Morin III FC
et al. Pulmonary arterial contractility in neonatal lambs increases with 100% oxygen
resuscitation. Pediatr Res 2006; 59: 137–141.
17 Lakshminrusimha S, Russell JA, Steinhorn RH, Swartz DD, Ryan RM, Gugino SF et al.
Pulmonary hemodynamics in neonatal lambs resuscitated with 21, 50, and 100%
oxygen. Pediatr Res 2007; 62(3): 313–318.
18 McNamara PJ, Laique F, Muang-In S, Whyte HE. Milrinone improves oxygenation in
neonates with severe persistent pulmonary hypertension of the newborn. J Crit Care
2006; 21: 217–222.
19 Kelly LK, Porta NF, Goodman DM, Carroll CL, Steinhorn RH. Inhaled prostacyclin for
term infants with persistent pulmonary hypertension refractory to inhaled nitric oxide.
J Pediatr 2002; 141: 830–832.
20 Shekerdemian L, Ravn H, Penny D. Intravenous sildenafil lowers pulmonary vascular
resistance in a model of neonatal pulmonary hypertension. Am J Resp Crit Care Med
2002; 165: 1098–2002.
21 Shekerdemian LS, Ravn HB, Penny DJ. Interaction between inhaled nitric oxide and
intravenous sildenafil in a porcine model of meconium aspiration syndrome. Pediatr
Res 2004; 55: 413–418.
22 Atz AM, Wessel DL. Sildenafil ameliorates effects of inhaled nitric oxide withdrawal.
Anesthesiology 1999; 91: 307–310.
23 Baquero H, Soliz A, Neira F, Venegas ME, Sola A. Oral sildenafil in infants with
persistent pulmonary hypertension of the newborn: a pilot randomized blinded study.
Pediatrics 2006; 117: 1077–1083.
24 Steinhorn RH, Albert G, Swartz DD, Russell JA, Levine CR, Davis JM. Recombinant
human superoxide dismutase enhances the effect of inhaled nitric oxide in persistent
pulmonary hypertension. Am Rev Resp Crit Care Med 2001; 164: 834–839.
25 Lakshminrusimha S, Russell JA, Wedgwood S, Gugino SF, Kazzaz JA, Davis JM et al.
Superoxide dismutase improves oxygenation and reduces oxidation in neonatal
pulmonary hypertension. Am J Respir Crit Care Med 2006; 174: 1370–1377.
Journal of Perinatology
 Journal of Perinatology (2008) 28, S72–S78
r 2008 Nature Publishing Group All rights reserved. 0743-8346/08 $30
www.nature.com/jp
REVIEW
Pharmacotherapy for meconium aspiration
A Asad and R Bhat
Division of Neonatology, Department of Pediatrics, University of Illinois at Medical Center, Chicago, IL, USA
In this article we have attempted to review the current pharmacological
treatment options for infants with meconium aspiration syndrome with or
without persistent pulmonary hypertension. These treatments include
ventilatory support, surfactant treatment and inhaled nitric oxide (INO), in
addition to older and newer pharmacological treatments. These include
sedatives, muscle relaxants, alkali infusion, antibiotics and the newer
vasodilators. Many aspects of treatment, including ventilatory care,
surfactant treatment and the use of INO, are reviewed in great detail in this
issue. On the other hand, many newer pharmacological modalities of
treatment described here have not been evaluated with randomized control
trials. We have given an overview of these emerging therapies.
Journal of Perinatology (2008) 28, S72–S78; doi:10.1038/jp.2008.160
Introduction
Meconium aspiration syndrome (MAS) is defined as the presence of
respiratory distress and hypoxemia associated with the presence of
meconium during or just before delivery. Meconium-stained
amniotic fluid is a relatively common occurrence seen in
approximately 10 to 15% of deliveries and approximately 5% of
these develop true MAS.1 The most severe cases require assisted
ventilation for greater than 48 h and are often associated with
persistent pulmonary hypertension. Several recent publications and
other reviews in this issue have described in detail the
pathophysiology and different aspects of the management. Our goal
is to review the pharmacological management of MAS.
Treatment strategies: the past, present and future
Various treatment modalities including intrapartum (perineal) and
post-delivery oral suctioning were the routine until recently but two
recent randomized controlled trials (RCTs) have shown that none
of these techniques have decreased the incidence of MAS.2,3 These
approaches are well covered by different authors in this special
issue. We will focus primarily on different treatments in the
management of infants admitted to neonatal intensive care unit
Correspondence: Dr R Bhat, Department of Pediatrics, M/C 856, University of Illinois at
Chicago Medical Center, 840 S Wood Street, Chicago, IL 60612, USA.
E-mail: ramabhat@uic.edu
with moderate-to-severe MAS. Figure 1 shows the schema of our
presentation.
The treatment options can be divided into general or specific.
The general treatment options are the optimization of ventilation,
sedation and alkalanization. The more specific pharmacological
therapies are aimed at reducing hypoxia with the use of pulmonary
vasodilators (inhaled nitric oxide (INO), oral sildenafil and
bosentan), and the lung injury by anti-inflammatory agents,
surfactant lavage or replacement.
General management
In all newborns with evidence of severe respiratory distress
handling is minimized to prevent worsening of gas exchange due
to agitation. Sedation and analgesia are used frequently in infants
with MAS and persistent pulmonary hypertension to alleviate pain
and discomfort that may lead to hypoxia and right-to-left shunting.
Opioids particularly morphine or fentanyl with midazolam are
frequently used across the world (77 to 100%). Major side effects of
these medications include hypotension, urinary retention,
development of tolerance and withdrawal symptoms. Opioids can
be given either intermittently or as a continuous drip.
Pharmacokinetics and metabolism of these drugs are well
studied4,5 but their impact on long-term outcome has not been
reported in term infants. Infact, no RCTs are available in term
infants to support its routine use.
Muscle relaxants
Prior to 2000, depolarizing muscle relaxants were widely used
along with opioids to decrease agitation and subsequent hypoxic
episodes in ventilated infants. Pancuronium and Vecuronium
(0.1 mg kg
1 h
1) are the two most commonly used muscle
relaxants in neonatal intensive care unit. The benefits of
neuromuscular blockade include improved oxygenation, decreased
oxygen consumption and decreased accidental extubations.
However, a prospective multicenter observational study by
Walsh-Sukys et al.6 showed considerable variations in muscle
relaxant use between centers (33 to 98%). Use of muscle relaxants
was also associated with increased mortality (Odds ratio: 1.95,
confidence interval: 0.74, 5.18). In their series, 41% (total
N ¼ 385) of the cases had MAS as the primary diagnosis. The side

General
• Sedation Analgesia
• Muscle Relaxants
• Alkalinization
Infection & Inflammation Pharmacotherapy
of
• Antibiotics MAS
• Anti-inflammatory agents
Pulmonary care
• Surfactant treatment
• Surfactant lavage
• Ventilator support
Figure 1 Management of MAS.
effects of nondepolarizing agents include cardiovascular effects
from histamine release, though this is less common in neonates
and also tachycardia (vagolytic effect). Neuromuscular blockade
can also mask seizures especially in infants depressed at birth. No
randomized control studies are available at this time.
Neonatologists are urged to consider the risks and benefits of
neuromuscular blockade prior to their use.
Alkalinization
Increasing the pH from 7.45 to 7.5 either by hyperventilation or by
sodium bicarbonate infusion has been shown to produce
pulmonary vasodilatation both in animal models and human
newborns.7,8 However, both hyperventilation and sodium
bicarbonate infusion should be used with caution in newborns due
to their adverse effects on cerebral and coronary circulation.9,10
Sodium bicarbonate infusion can increase intracellular acidosis
and worsen myocardial perfusion and cardiac output.
Hyperventilation may also increase barotrauma and volutrauma,
and increase the risk for chronic lung disease.11 Hypocarbia below
25 mm Hg can also result in hearing loss secondary to injury to
hair cells.10 Walsh-Sukys et al.6 reported an increased need for
extracoporeal membrane oxygenation (ECMO) and oxygen at
28 days in patients treated with sodium bicarbonate. Neither
hyperventilation nor alkali infusion have been vigorously tested in
a RCT. Fortunately their use seems to be on the decline since the
availability of INO.
Initial management has also traditionally included inotropic
support to assist cardiac function against suprasystemic pulmonary
pressures. Once again, there is no randomized study to support this
practice. Drummond et al.8 were able to show in a case series that
dopamine infusion along with tolazoline and hyperventilation
helped reduce pulmonary pressure. The combination of tolazoline
and dopamine had a variable and unpredictable effect on
Pharmacotherapy for MAS
A Asad and R Bhat
S73
Pulmonary hypertension
• Vasodilators
• Hemodynamic stabilizers
oxygenation. It is noted that these drugs were not studied
independently of each other. Hypotension is frequently secondary to
heavy sedation and the use of high mean airway pressure.
Dopamine and dobutamine are the two most frequently used
inotropes in neonates. High doses of dopamine
(>10 mg kg
1 min
1) on its own can also contribute to
pulmonary vasoconstriction.12
Pulmonary vasodilators
Regulation of pulmonary vascular tone is a dynamic process and
depends on the balance between various endogenous constrictors
(endothelin and thrombaxane) and dilators (nitric oxide (NO) and
prostaglandins). Majority of these substances are produced by the
pulmonary endothelium. In addition, arterial oxygen and carbon
dioxide tension, and lung volume play a major role in the
regulation of pulmonary vascular tone. Pulmonary hypertension in
patients with MAS is secondary to hypoxia, acidosis, release of the
inflammatory mediators and alveolar atelectasis. Thus the
pulmonary hypertension of MAS should ideally be treated with
adequate ventilation and pulmonary vasodilators. The problem
until recently was the inability to find such a truly selective
pulmonary vasodilator. We will briefly review the past and present
pulmonary vasodilators used in MAS. Table 1 shows dosages of
various pulmonary vasodilators used in pulmonary hypertension.
Tolazoline
Tolazoline is a nonspecific vasodilator, which had been used for
the treatment of pulmonary hypertension at least two decades
before the introduction of INO. Lee and Hox31 showed that
tolazoline led to pulmonary vasodilatation even in the absence of
endothelium. Whereas Curtis et al.32 showed in the animal model
that the vasodilatation produced was independent of NO
production. Its mechanism of action is directly on the vascular
Journal of Perinatology
 Pharmacotherapy for MAS
A Asad and R Bhat
S74

Table 1 Pulmonary vasodilators Several of these agents are being used in adults with severe
Tolazoline IV 0.5–2 mg kg
1 13,14
pulmonary arterial hypertension. Milrinone, dipyridamole,
(Not available in USA) NEB 1–2.5 mg kg
1 15 zaprinast and sildenafil (Viagra) have all been used in newborns
but the number of patients treated so far are few and no large
Magnesium sulfate IV 200 mg kg
1 bolus control studies are available to date.
20–150 mg kg
1 h
1 16
Diltiazem IV 1–2 q12 to q6 h17 Milrinone. Milrinone is a PDE-3-specific inhibitor and is one of
the earliest drugs studied in newborns. It acts synergistically with
PDE inhibitors INO inducing pulmonary vasodilatation, but in addition it is also a
Dipyridamole IV 0.3–0.6 mg kg
1 18– 21 systemic vasodilator and has positive myocardial inotropic activity.
Esoprostenol/ IV 2–5 ng kg
1 min
1 increments of It is frequently used in newborns, pediatric and adult patients
prostacyclin 2–5 ng kg
1 q15 min22–24
following cardiac surgery. Inhaled milrinone has been tried in
ET 50 ng kg
1 can be repeated 2  25
adults with pulmonary hypertension undergoing cardiac surgery.
Milrinone IV Term
Loading 75 mcg kg
1  60 min
In a recent case report, milrinone was successfully used in four
Maintenance 0.5–0.75 mcg kg
1 min
1 infants who failed to respond to INO.35 Pharmacokinetics of
<30 GA milrinone have not been studied in newborns. Milrinone may have
Loading 0.75 mcg kg
1 min
1 over 3 h limited role with the advent of more specific PDE-5 inhibitors, for
Maintenance 0.2 mcg kg
1 min
1 26 example, sildenafil.
Sildenafil PO 0.25–1 mg kg
1 27,28
Tezosentan IV 5 mg h
1 for 30 min, 1 mg h
1 for 24–72 h29 Dipyridamole. Dipyridamole is a selective PDE-5 inhibitor. In
Adenosine UVC 25–50 mg kg
1 min
1 30 experimental models it has been shown to augment and prolong
Abbreviations: ET, endotracheal; IV, intravenous; GA, gestational age; NEB, nebulized; the response of NO in post-cardiac surgery patients.36 However,
PO, per oral; UVC, through umbilical vein catheter. augmentation response is not consistent and in some pediatric
See the text for details on each drug. studies decrease in pulmonary vascular resistance of >20% is noted
in only half the treated cases. Dipyridamole has been shown to help
smooth muscle through direct a-adrenergic antagonistic action. in attenuating the rebound phenomenon seen after discontinuation
Tolazoline can be given intravenously as well as by intratracheal of INO therapy in pediatric patients.18,19 Dosage of dipyridamole
route.33,34 There is still no consensus on the dose of tolazoline at ranged from 0.3 to 0.6 mg kg
1.18–21
which pulmonary vasodilatation can be maximized while eliminating
the severe systemic hypotension. Severe systemic hypotension is the Zaprinast. Zaprinast is a more selective inhibitor of PDE-5 and
most common and severe adverse effect. The drug is currently not was one of the original inhibitors studied along with dipyridamole.
available in the United States and is not recommended for the Intravenous administration showed, in a fetal lamb model, to
treatment of MAS with pulmonary hypertension. decrease pulmonary arterial resistance as well as a potentiation of
INO inhaled NO-mediated pulmonary vasodilatation.36–38 The concern
An ideal pulmonary vasodilator should (a) decrease pulmonary with intravenous administration was that at moderate-to-high
vascular resistance but not systemic vascular resistance, (b) rapidly doses systemic vasodilatation was noted. Aerosolized drug therapy
relieve hypoxia, acidosis and vasoconstriction, (c) have no or has been shown to have less systemic side effects, preferential
minimal systemic effects and (d) be rapidly metabolized or cleared delivery to better ventilated areas of the lungs and a potentiation of
from the body. INO is a good example of such a drug. Indications the INO effect.39,40
and dose of INO are discussed elsewhere in this issue.
Sildenafil. The most specific PDE-5 inhibitor currently available
Phosphodiesterase inhibitors is sildenafil. It is becoming increasingly popular in the treatment
Phosphodiesterases (PDEs) are a family of enzymes that hydrolyze of pulmonary hypertension, especially in post-cardiac surgery
cyclic nucleotides and regulate their intracellular level. Both cyclic cases.41 Initial success has been noted in some pediatric cases in
adenosine monophosphate and cyclic guanosine monophosphate attenuation of INO-rebound pulmonary hypertension.42 Baquero
play a crucial role in cell signaling. They are classified based on et al.,27 in a proof-of-concept, randomized masked study showed
their substrate specificities, pharmacological properties and an improvement in oxygenation index and pulse oxygen saturation
distribution in the body. PDE inhibitors block the hydrolysis of within 6 to 30 h, and significant increase in survival rate
cyclic nucleotides and thus increase intracellular accumulation of (85%, 6/7 infants) in those randomized to receive oral sildenafil.
cyclic adenosine monophosphate or cyclic guanosine monophos- In the placebo group only 1/6 (16%) survived. The dose of
phate. A brief review of the PDEs’ inhibitors is given below. sildenafil was 1 mg kg
1 every 6 h till oxygenation index decreased

Journal of Perinatology

to <20. Pharmacokinetics and bioavailability of orally
administered sildenafil has not been studied so far in neonates.
A recent report by Martell et al.43 have shown a significant drop in
pulmonary pressures within 2 min following intratracheal
administration of 0.75 or 1.5 mg kg
1 dose of sildenafil in piglets
with meconium aspiration. Inotropic support was needed to
maintain systemic blood pressure. A multicenter study with a large
sample size is needed to assess the safety and efficacy of
intravenous, oral or intratracheal sildenafil with or without INO.
Oral sildenafil can be a lifesaving drug in the developing
countries to treat pulmonary hypertension and also for infants with
severe hypoxia and respiratory failure in community hospitals prior
to their transfer to a tertiary care center. Both dipyridamole and
sildenafil have been approved for clinical use in adults with
pulmonary hypertension.
Prostaglandins and prostacyclins
It has been known that both the fetal and neonatal pulmonary
vascular bed is sensitive to the arachidonic acid metabolites.
Prostaglandin E1 (PGE1) has been in clinical use for more than
three decades for maintaining ductal patency, but it is a weak
pulmonary vasodilator when compared with prostacyclin (PGI2).44
PGI2 has been in use to treat adult pulmonary hypertension for
several years and is the first drug to be approved for clinical use. It
is a potent pulmonary and systemic vasodilator, and its action is
mediated through increased cyclic adenosine monophosphate. It
has antiproliferative and antiplatelet adhesion effects. The major
drawback of PGI2 is the need for long-term intravenous infusion
and its very short half-life. Various synthetic analogs of PGI2,
namely treprostinil, iloprost and beraprost, are in clinical use in
adults because of their longer half-life and ease of administration,
either orally or as aerosol and subcutaneous drip. Aerosolized PGI2
has been tried in newborns who failed to respond to INO.45 Another
prostaglandin, PGD2 has been evaluated in newborns with
pulmonary hypertension but is yet to show any clinical benefit.46
Endothelin antagonists
Endothelins are vasoconstrictors derived from the endothelial cells.
Of the two major types of endothelins, ET-1 mediates
vasoconstriction and smooth muscle cell proliferation through
ET-A receptors. ET-A receptors have been found on smooth muscle
cells in pulmonary arteries.47 Endothelins are involved in
maintaining the pulmonary vascular tone in the neonatal period.
Recent studies have reported elevated ET-1 level48 and an
upregulation of ET-1 gene expression in the pulmonary
vasculature following MAS.22,49 In adults with pulmonary
hypertension, the combined ET-A and ET-B receptor antagonist
bosentan has been used successfully.50,51 In pediatric and neonatal
populations information is limited to case reports. The drug is
currently available only in oral formulation, which may hinder its
use in critically ill newborns. Bosentan is approved by the FDA for
Pharmacotherapy for MAS
A Asad and R Bhat
S75
pulmonary hypertension in adults. Side effects of the drug include
abnormal liver enzymes. Second-generation ET-A blockers such as
tezosentan and sitaxsentan are being studied both in animal
models and in adults with pulmonary hypertension and/or heart
failure at this time.29,52
Adenosine
ATP is a purine nucleotide and well-known dilator of systemic and
pulmonary vasculature in both fetal and neonatal vessels.53 ATP
causes vasodilatation by acting on endothelial A2 adenosine
receptors and subsequent release of NO. Its use in a meconium
lung was studied by Kappa et al.53 in the porcine model and he
showed that at low doses it was able to cause an amelioration of
the pulmonary hypertension. However, withdrawal of ATP led to
significant rebound and even an overshoot phenomenon.
From a randomized placebo-controlled trial of 18 infants,
Konduri et al.30 showed an improvement in oxygenation only in
4/9 (45%) term infants with persistent pulmonary hypertension.
The dosage used was 25 to 50 mg kg
1 min
1 and no systemic side
effects were observed. It did not decrease the need for ECMO,
bronchopulmonary dysplasia or mortality. Adenosine does have a
short half-life and rapid metabolism, therefore it should be given
through the umbilical venous line directly to right atrium to reach
the pulmonary artery.
Magnesium sulfate
Magnesium at high doses is a smooth muscle relaxant and
vasodilator. It acts by antagonizing calcium entry in to smooth
muscle cells. Wu et al. have shown that it also suppresses
catecholamine release and alters metabolism of prostaglandins and
responsiveness of smooth muscles to vasopressors.54 Owing to its
varying modes and site of action, it is a relatively nonspecific
vasodilator with a significant risk for systemic hypotension.55,56
However, in clinical trials in children who have failed conventional
therapies, significant improvement has been shown in both
oxygenation and oxygenation index without significant
hypotension.16,56 Magnesium sulfate also has sedative and
antithrombotic activities. In third world countries where INO is not
easily available, this multifaceted drug can be used to alleviate
hypoxia. RCTs have not been done so far.
Future pulmonary vasodilators
Table 2 shows some of the newer pulmonary vasodilators currently
undergoing investigation.
Table 2 Emerging pulmonary vasodilators
1 Superoxide dismutase
2 Vasoactive intestinal peptide
3 Adrenomedulline
4 Arginine
Journal of Perinatology
 Pharmacotherapy for MAS
A Asad and R Bhat
S76
Arginine
L-arginine infusion is being studied as an adjunct for NO as well as
for its potential use in the weaning process. L-arginine is a required
substrate for NO synthesis and under oxidative stress conditions it
increases the activity of nitric oxide synthase.57 It also helps to
preserve endogenous nitric oxide synthase activity thus has
potential for helping in the weaning of NO. RCTs of L-arginine in
persistent pulmonary hypertension are still lacking.
Superoxide dismutase
Superoxide dismuatse is an endogenous scavenger and potent
anti-inflammatory and anti-oxidative enzyme. There is now a
recombinant version (rhSOD) that is commercially available. The
rhSOD is being studied as an adjunct with INO therapy.
Steinhorn et al.58 initially showed how single dose administration
of rhSOD in a lamb model with and without INO led to selective
pulmonary vasodilatation. In view of these anti-inflammatory
and pulmonary vasodilator effects, rhSOD appears to be an exciting
new addition to treat pulmonary hypertension in MAS.
Other drugs
Surfactant. Meconium contents are highly viscous and consist of
desquamated epithelial cells and inspissated intestinal secretions.
The large glycoproteins in meconium cause increased
adhesiveness.59 Rubin et al.60 in 1996 reported that the mucociliary
transportability of meconium was lower than that of sputum
clearance in cystic fibrosis patients. Meconium constituents
especially the fatty acids and other soluble proteins and bilirubin
are direct inhibitors of surfactant.61,62 Two small randomized
control studies have reported significant improvement in
oxygenation and decrease in barotrauma with surfactant
treatment.63,64 Readers are referred to excellent reviews on
surfactant therapy and lavage in meconium aspiration in this
issue.
Antibiotics
The use of antibiotics in MAS has been controversial. Initial
use was advocated as it was believed that stress caused the passage
of meconium and the most likely reason for perinatal stress
was an infection, hence the need to treat with antibiotics. This
was substantiated in 1967 when Bryan65 showed that sterile
meconium was never fatal on its own, but when given
intratracheally along with Escherichia coli it reduced the number
of organisms needed to cause death. A more recent in vitro study
by Eidelman et al.66 showed once again that clear amniotic fluid
was inhibitory for bacterial growth but in the presence of
meconium b-Streptococci grew at much faster rate. It is still
controversial whether every infant with meconium aspiration
needs antibiotic therapy. Few prospective RCTs are available from
outside the United States for comparison of antibiotic use. In a
prospective RCT of non-ventilated infants Lin et al.67 showed that
Journal of Perinatology
antibiotic treatment did not alter duration of tachypnea,
oxygen support and the need for nasal continuous positive airway
pressure between the treated and untreated infants. Of the
306 infants, 2-month follow-up data were available only for
259 infants. None of these infants required ventilator support and
none had perinatal risk factors. The use of antibiotics gave no
advantage in terms of oxygen supplementation, severity of
respiratory distress or mortality. Two other prospective studies from
Shankar et al.68 and Krishnan et al.69 from India also concluded
that routine post-natal antibiotic therapy is of no benefit. These
studies do support the current view that routine antibiotic therapy
in MAS does not alter clinical course. Currently in our unit only
infants admitted with MAS requiring ventilatory support or infants
admitted with risk factors (prolonged rupture of membranes,
chorioamnionitis or positive antenatal group B beta streptococcus
screen) receive antibiotics after obtaining initial cultures.
Anti-inflammatory agents
Steroids. Although the initial phase of deterioration in the lung
mechanics is because of the mechanical obstruction and surfactant
inactivation, the persistence of the dysfunction is thought to be
because of activation of the inflammatory cascade. Chemical
pneumonitis is the second cardinal finding in MAS. Khan et al.70
in 2002 showed histological and biochemical evidence of
meconium-induced airway dysfunction in a murine model. Our
own animal studies in newborn rabbits following tracheal
instillation of meconium showed significant increase in several
cytokines namely TNFa, IL-8 and IL-1b in tracheal aspirate.71
This was associated with significant apoptosis.72 As inflammation is
one of the major findings in MAS; several investigators have
attempted to treat MAS with steroids. Steroids are potent anti-
inflammatory medications. Glucocorticoids, particularly
dexamethasone, has been shown to improve oxygenation and lung
function in animal models of MAS73 and in at least one
uncontrolled study of human newborns with MAS.74 A recent
Cochrane review75 found two small RCTs (a 3rd study was
unpublished) and the meta-analysis of the data showed no
differences in mortality, chronic lung disease and length of stay.
The major weaknesses in these studies were small sample size and
lack of long-term follow-up. Owing to the lack of consistent
short-term benefit as well as recent concerns about its long-term
outcome, dexamethasone cannot be recommended at this time as
an anti-inflammatory agent in MAS.
In summary the recent advances in pharmacological treatment
namely INO and surfactant has certainly decreased the need for
ECMO in MAS infants. However there is a definite need for further
multicenter studies to evaluate some of the specific and nonspecific
therapies discussed in this review. As MAS is a major cause of
mortality and morbidity in the developing countries, studies
focusing on prevention and early treatment should be continued to
alleviate this tragedy.

Acknowledgments
We thank Dr Dharmapuri Vidyasagar for his many contributions on the
pathophysiology and treatment of MAS during the last 30 years.
Disclosure
R Bhat has lectured while on a speakers bureau. A Asad has declared no
financial interests.
References
1 Wiswell TM. Advances in treatment of the meconium aspiration syndrome. Acta
Paediatr Suppl 2001; 436: 28–30.
2 Wiswell TE. Meconium in the Delivery Room Trial Group: delivery room management
of the apparently vigorous meconium-stained neonate: results of the multicenter
collaborative trial. Pediatrics 2000; 105: 1–7.
3 Linder N, Aranda JV, Tsur M. Need for endotracheal intubation and suction in
meconium-stained neonates. J Pediatr 1988; 112: 613–615.
4 Bhat R, Chari G, Gulati A, Aldana O, Velamati R, Bhargava H. Pharmacokinetics of
Morphine in preterm and term neonates. J Pediatr 1990; 117: 477–481.
5 Bhat R, Abu-Harb M, Chari G, Gulati A. Morphine metabolism in acutely ill preterm
newborn infants. J Pediatr 1992; 120: 795–799.
6 Walsh-Sukys MC, Tyson JE, Wright LL, Bauer CR, Korones SB, Stevenson DK et al.
Persistent pulmonary hypertension of the newborn in the era before nitric oxide:
practice variation and outcomes. Pediatrics 2000; 105: 14–20.
7 Lyrene RK, Welch KA, Godoy G, Philips III JB. Alkalosis attenuates hypoxic pulmonary
vasoconstriction in neonatal lambs. Pediatr Res 1985; 19: 1268–1271.
8 Drummond WH, Gregory GA, Heymann MA, Phibbs RA. The independent effects of
hyperventilation, tolazoline, and dopamine in infants with persistent pulmonary
hypertension. J Pediatr 1981; 98: 608–611.
9 Hansen NB, Brubakk AM, Bratlid D, Oh W, Stonestreet BS. The effects of variations in
PaCO2 on brain blood flow and cardiac output in the newborn piglet. Pediatr Res
1984; 18(11): 1132–1136.
10 Ambalavanan N, Carlo WA. Hypocapnia and hypercapnia in respiratory management of
newborn infants. Clin Perinatol 2001; 28(3): 517–531. Review.
11 Greenough A, Khetriwal B. Pulmonary hypertension in the newborn. Paediatr Respir
Rev 2005; 6(2): 111–116.
12 Booker PD, Evans C, Franks R. Comparison of the haemodynamic effects of dopamine
and dobutamine in young children undergoing cardiac surgery. Br J Anaesth 1995;
74(4): 419–423.
13 Stevenson DK, Kasting DS, Darnall Jr RA, Ariagno RL, Johnson JD, Malachowski N et al.
Refractory hypoxemia associated with neonatal pulmonary diseases: the use and
limitations of tolazoline. J Pediatr 1979; 95: 595–599.
14 Nuntnarumit P, Korones SB, Yang W, Bada HS. Efficacy and safety of tolazoline for
treatment of severe hypoxemia in extremely preterm infants. Pediatrics 2002; 109(5):
852–856.
15 Parida SK, Baker S, Kuhn R, Desai N, Pauly TH. Endotracheal tolazoline
administration in neonates with persistent pulmonary hypertension. J Perinatol 1997;
17: 461–464.
16 Tolsa JF, Cotting J, Sekarski N, Payot M, Micheli JL, Calame A. Magensium Sulphate as
an alternative and safe treatment for persistent pulmonary hypertension of the
newborn. Arch Dis Child Fetal Neonatal Ed 1995; 72: F184–F187.
17 Islam S, Masiakos P, Schnitzer JJ, Doody DP, Ryan DP. Diltiazem reduces pulmonary
arterial pressures in recurrent pulmonary hypertension associated with pulmonary
hypoplasia. J Pediatr Surg 1999; 34: 712–714.
18 Al-Alaiyan S, Al-Omran A, Dyer D. The use of phoshodiestersae inhibitor( dipyridamole)
to wean from inhaled nitric oxide. Intensive care med 1996; 22: 1093–1095.
Pharmacotherapy for MAS
A Asad and R Bhat
S77
19 Ivy DD, Kinsella JP, Ziegler JW, Abman SH. Dipyridamole attenuates rebound
pulmonary hypertension after inhaled nitric oxide withdrawl in postoperative
congenital heart disease. J Thorac Cardiovasc Surg 1998; 115: 875–882.
20 Kinsella JP, Toricelli F, Ziegler JW, Ivy DD, Abman SH. Dipyridamole augmentation of
response to nitric oxide. Lancet 1995; 346: 647–648.
21 Travadi JN, Patole SK. Phosphodiesterase inhibitors for persistent pulmonary
hypertension of the newborn: a review. Pediatr Pulmonol 2003; 36(6): 529–535.
22 Shekerdamien LS, Penny DJ, Ryhammer PK, Reader JA, Ravn HB. EndothelinFa
receptor blockade and inhaled nitric oxide in a porcine model of meconium aspiration
syndrome. Pediatr Res 2004; 56: 353–358.
23 Schranz D, Zepp F, Iversen S, Wippermann C, Huth R, Zimmer B et al. Effects of
tolazoline and prostacyclin on pulmonary hypertension in infants after cardiac surgery.
Crit Care Med 1992; 20(9): 1243–1249.
24 Bush A, Busst CM, Knight WB, Shinebourne EA. Comparison of the haemodynamic
effects of epoprostenol (prostacyclin) and tolazoline. Br Heart J 1988; 60(2): 141.
25 De Jaeger AP, den Anker JN. Endotracheal instillation of prostacyclin in preterm infants
with persistent pulmonary hypertension. Eur Respir J 1998; 12: 932–934.
26 Paradisis M, Jiang X, Mclachlan AJ, Evans N, Kluckow M, Osborn D. Population
pharmacokinetics and dosing regimen of milrinone in preterm infants. Arch Dis Child
Fetal Neonatal Ed 2007; 92: F204–F209.
27 Baquero H, Soliz A, Neira F, Venegas ME, Sola A. Oral sildenafil in infants with
persistent pulmonary hypertension of the newborn: a pilot randomized blinded study.
Pediatrics 2006; 117: 1077–1083.
28 Erickson S, Reyes J, Bohn D, Adatia I. Sildenafil (Viagra) in childhood and neonatal
pulmonary hypertension. J Am Coll Cardiol 2002; 39(Suppl 2): 402–402(1).
29 McMurray JJV, Teerlink JR, Cotter G, Bourge RC, Cleland JG, Jondeau G et al. Effects of
Tezosentan on symptoms and clinical outcomes in patients with acute heart failure.
JAMA 2007; 298(17): 2009–2019.
30 Konduri GG, Garcia DC, Kazzi NJ, Shankaran S. Adenosine infusion improves
oxygenation in term infants with respiratory failure. Pediatrics 1996; 97: 295–300.
31 Lee TS, Hou X. Dual vasoactive effects of tolazoline on rabbit pulmonary arteries.
J Cardiothoc Vasc Anesth 1996; 10: 364–367.
32 Curtis J, Palacino JJ, O’Neill JT. Production of pulmonary vasodilation by tolazoline,
independent of nitric oxide production in neonatal lambs. J Pediatr 1996; 128:
118–124.
33 Paret G, Eyal O, Mayan H, Ben-Abraham R, Vardi A, Manisterski Y et al.
Pharmacokinetics of endobronchial tolazoline administration in dogs. Am J Perinatol
1999; 16: 1–6.
34 Welch JC, Bridosn JM, Gibbs JL. Endotracheal tolazoline for severe persistent pulmonary
hypertension of the newborn. Br Heart J 1995; 73: 99–100.
35 Bassler D, Choong K, McNamara P, Kirpalani H. Neonatal persistent pulmonary
hypertension treated with milrinone: four case reports. Biol Neonate 2006; 89: 1–5.
36 Ziegler JW, Ivy DD, Fox JJ, Kinsella JP, Clarke WR, Abman SH et al Dipyridamole, a
cGMP phosphodiesterase inhibitor, causes pulmonary vasodilation in the ovine fetus.
Am J Physiol 1995; 269: 473–479.
37 Thusu KG, Morin III FC, Russell JA, Steinhorn RH. The cGMP phosphodiesterase
inhibitor zaprinast enhances the effect of nitric oxide. Am J Respir Crit Care Med 1995;
152: 1605–1610.
38 Ichinose F, Adrie C, Hurford WE, Zapol WM. Prolonged pulmonary vasodilator
action of inhaled nitric oxide by Zaprinast in awake lambs. J Appl Physiol 1995; 78:
1288–1295.
39 Ichinose F, Adrie C, Hurford WE, Bloch KD, Zapol WM. Selective pulmonary
vasodilation induced by aerosolized zaprinast. Anesthesiology 1998; 88: 410–416.
40 Newman SP. Aerosol deposition considerations in inhalation therapy. Chest 1985;
88(2 Suppl): 152S–160S. Review.
41 Stocker C, Penny DJ, Brizard CP, Cochrane AD, Soto R, Shekerdemian LS. Intravenous
sildenafil and inhaled nitric oxide: a randomised trial in infants after cardiac surgery.
Intensive Care Med 2003; 29(11): 1996–2003.
42 Atz AM, Wessel DL. Sildenafil ameliorates effects of inhaled nitric oxide withdrawl.
Anesthesiology 1999; 91: 307–310.
Journal of Perinatology
 Pharmacotherapy for MAS
A Asad and R Bhat
S78
43 Martell M, Blasina F, Silvera F, Tellechea S, Godoy C, Vaamonde L. Intratracheal sildenafil
in the newborn with pulmonary hypertension. Pediatrics 2007; 119: 215–216.
44 Tamura M, Kurumatani H, Matsushita T. Comparative effects of beraprost, a stable
analogue of prostacyclin, with PGE(1), nitroglycerin and nifedipine on canine model
of vasoconstrictive pulmonary hypertension. Prostaglandins Leukot Essent Fatty Acids
2001; 64(3): 197–202.
45 Bindl L, Fahnenstich H, Peukert U. Aerosolised prostacyclin for pulmonary hypertension
in neonates. Arch Dis Child Fetal Neonatal Ed 1994; 71(3): F214–F216.
46 Soifer SJ, Clyman RI, Heymann MA. Effects of prostaglandin D2 on pulmonary arterial
pressure and oxygenation in newborn infants with persistent pulmonary hypertension.
J Pediatr 1998; 112: 774–777.
47 Buchan KW, Magnusson KW, Rabe KF, Summer MJ, Watts IS. Characterization of the
endothelin receptor mediating contraction of human pulmonary artery using BQ123
and Ro46-2005. Eur J Pharmacol 1994; 260: 221–226.
48 Kuo C, Chen J. Effect of meconium aspiration on plasma endothelin-1 level and
pulmonary hemodynamics in a piglet model. Biol Neonate 1999; 76: 228–234.
49 Kyo CY. EndothelinFa receptor antagonist prevents neonatal pulmonary hypertension
in meconium aspiration in piglets. J Formos Med Assoc 2001; 100: 420–423.
50 Rubin LJ, Badesch DB, Barst RJ, Galie N, Black CM, Keogh A et al. Bosentan therapy for
pulmonary arterial hypertension. N Eng J Med 2002; 346: 896–903.
51 Sitbon O, Badesch DB, Channick RN, Frost A, Robbins IM, Simonneau G et al. Effects
of dual endothelin receptor antagonist bosentan in patients with pulmonary arterial
hypertension: a 1-year follow-up study. Chest 2003; 124: 247–254.
52 Geiger R, Pajk W, Neu N, Maier S, Kleinsasser A, Fratz S et al. Tezosentan decreases
pulmonary artery pressure and improves survival rate in an animal model of
meconium aspiration. Pediatr Res 2006; 59: 147–150.
53 Kappa P, Jahnukainen T, Grinlund J, Rautanen M, Halkola L, Välimäki I. Adenosine
triphosphate treatment for meconium aspiration-induced pulmonary hypertension in
pigs. Acta Physiol Scand 1997; 160(3): 283–289.
54 Wu TJ, Teng RJ, Tsou KI. Persistent pulmonary hypertension of the newborn treated
with magnesium sulfate in premature neonates. Pediatrics 1995; 96: 472–474.
55 Patole SK, Finer NN. Experimental and clinical effects of magnesium infusion in the
treatment of neonatal pulmonary hypertension. Magnes Res 1995; 8(4): 373–388.
Review.
56 Abu-Osaba YK, Galal O, Mansara K, Rejjal A. Treatment of severe persistent
pulmonary hypertension of the newborn with magnesium sulphate. Arch Dis Child 1992;
67: 31–35.
57 Palmer RM, Rees DD, Ashton DS, Moncada S. L-arginine is the physiological precursor
for the formation of nitric oxide in endothelium-dependent relaxation. Biochem
Biophys Res Commun 1988; 153(3): 1251–1256.
58 Steinhorn RH, Albert G, Swartz DD, Russell JA, Levine CR, Davis JM. Recombinant
human superoxide dismutase enhances the effect of inhaled nitric oxide in persistent
pulmonary hypertension. Am J Respir Crit Care Med 2001; 164(5): 834–839.
Journal of Perinatology
59 Lam BCC, Yeung CY. Surfactant lavage for meconium aspiration syndrome: a pilot
study. Pediatrics 1999; 103: 1014–1018.
60 Rubin BK, Tomkiewicz RP, Patrinos ME, Easa D. The surface and transport
properties of meconium and reconstituted meconium solutions. Pediatr Res 1996;
40: 834–838.
61 Sun B, Curstedt T, Robertson B. Surfactant inhibition in experimental meconium
aspiration syndrome. Acta Pediatr 1993; 82: 182–189.
62 al-Mateen KB, Dailey K, Grimes MM, Gutcher GR. Improved oxygenation with
exogenous surfactant administration in experimental meconium aspiration syndrome.
Pediatr Pulmonol 1994; 17(2): 75–80.
63 Dargaville PA, South M, McDougall PN. Surfactant and surfactant inhibitors in
meconium aspiration syndrome. J Pediatr 2001; 138: 113–115.
64 Findlay RD, Taeusch HW, Walther FJ. Surfactant replacement therapy for meconium
aspiration syndrome. Pediatrics 1996; 97: 48–52.
65 Bryan CS. Enhancement of bacterial infection by meconium. Johns Hopkins Med J
1967; 121: 9–13.
66 Eidelman AI, Nevet A, Rudensky B, Rabinowitz R, Hammerman C, Raveh D et al. The
effect of meconium staining of amniotic fluid on the growth of Escherichia coli and
group B Streptococcus. J Perinatol 2002; 22: 467–471.
67 Lin HC, Su BH, Tsai CH, Lin TW, Yeh TF. Role of antibiotics in management of
non-ventilated cases of meconium aspiration syndrome without risk factors for
infection. Biol Neonate 2005; 87: 51–55.
68 Shankar V, Paul VK, Deorari AK, Singh M. Do neonates with meconium aspiration
syndrome require antibiotics? Indian J Pediatr 1995; 62: 327–331.
69 Krishnan L, Nasruddin, Prabhakar P, Bhaskaranand N. Routine antibiotic cover for
newborns intubated for aspirating meconium: is it necessary? Indian Pediatr 1995;
32(5): 529–531.
70 Khan AM, Elidemir O, Epstein CE, Lally KP, Xue H, Blackburn M et al. Meconium
aspiration produces airway hyperresponsiveness and eosinophilic inflammation in a
murine model. Am J Physiol Lung Cell Moll Physiol 2002; 283: L785–L790.
71 Zagariya A, Bhat R, Navale S, Vidyasagar D. Cytokine expression in meconium-induced
lungs. Indian J Pediatr 2004; 71(3): 195–201.
72 Zagariya A, Bhat R, Uhal B, Navale S, Freidine M, Vidyasagar D. Cell death and lung
cell histology in meconium aspirated newborn rabbit lung. Eur J Pediatr 2000; 159:
819–826.
73 Khan AM, Shabarek FM, Kutchback JW, Lally KP. Effects of dexamethasone on
meconium aspiration syndrome in newborn piglets. Pediatr Res 1999; 46(2):
179–183.
74 da Costa DE, Nair AK, Pai MG, Al Khusaiby SM. Steroids in full term infants with
respiratory failure and pulmonary hypertension due to meconium aspiration syndrome.
Eur J Pediatr 2001; 160: 150–153.
75 Ward M, Sinn J. Steroid therapy for meconium aspiration syndrome in newborn
infants. Cochrane Database Syste Rev 2003; (4): CD003485. Review.
 Journal of Perinatology (2008) 28, S79–S83
r 2008 Nature Publishing Group All rights reserved. 0743-8346/08 $30
www.nature.com/jp
REVIEW
Extracorporeal membrane oxygenation: use in meconium
aspiration syndrome
BL Short
Division of Neonatology, Department of Pediatrics, Children’s National Medical Center, The George Washington School of Medicine,
Washington, DC, USA
Extracorporeal membrane oxygenation (ECMO) has been successful as a
rescue therapy for infants with respiratory failure with some diagnoses such
as meconium aspiration syndrome (MAS) having a survival rate of more
than 94%. New therapies have allowed many infants who would have
required ECMO to be kept off ECMO, but at what cost. The survival rate for
the neonatal ECMO patient has dropped over the years, whereas the time of
ECMO has increased, indicating that the new therapies are keeping the less
ill infants off ECMO. The major cause of non-survival in this population
remains intraventricular hemorrhage. The primary risk factors related to
this are thought to be pre-ECMO events, such as hypoxia and/or ischemia
either prenatally or post-delivery. ECMO events that may complicate this are
heparinization that is required while on ECMO and concern for the effect of
shear stress and blood flow pattern changes created by the ECMO pump with
venoarterial ECMO, although these changes are not seen in venovenous
ECMO, the more common form of ECMO. Newer low-resistant microporous
artificial lungs and miniaturized pumping systems may allow ECMO to be
performed using less blood and safer equipment. The smaller low-resistant
artificial lungs provide the ability to consider giving extracorporeal
life support using only this membrane with flow provided by an
arterial–venous shunt, thus eliminating the pumping system all together.
Trials are ongoing in adults and, if effective, may direct further research
into using this technique in newborns where the umbilical artery and vein
could be used as the arterial–venous shunt.
Journal of Perinatology (2008) 28, S79–S83; doi:10.1038/jp.2008.152
Introduction
Extracorporeal membrane oxygenation (ECMO) is the use of a
modified cardiopulmonary bypass circuit to supply respiratory
and/or cardiac support for patients in cardiorespiratory failure.1
The ECMO circuit supplies an artificial lung and an artificial
pump, which allow the patient to be supported for both respiratory
and cardiac needs. On account of this, many describe ECMO
Correspondence: Dr BL Short, Division of Neonatology, Department of Pediatrics, Children’s
National Medical Center, The George Washington School of Medicine, 111 Michigan Ave.,
NW, Washington, DC 20010, USA.
E-mail: bshort@cnmc.org
therapy as a ‘time-buying’ procedure. If you give extracorporeally
respiratory and cardiac support to the level, the patient can be
placed on minimal ventilatory and pressor therapy over time, and a
percentage of the patients will have recovery of lung and/or cardiac
function. As all patients have to be systemically heparinized to be
on an ECMO circuit, the known vasodilatory effect of heparin on
the pulmonary vessels may assist in the reduction of pulmonary
vascular resistance in patients placed on ECMO.2
In the mid-1960s, ECMO use was directed at the premature
infant with the hopes that ECMO would provide the ‘artificial’
placenta for these infants.3,4 Although the procedure provided
sufficient cardiorespiratory support for these infants, all infants
died secondary to the high intracranial hemorrhage (ICH) rate in
the premature population. This was thought to be related to the
immature brain of these infants with an already high rate of ICH
and to the heparinization required for the procedure. The first
newborn survivor in 1976 was an infant with meconium aspiration
syndrome (MAS), turning the focus of treatment to the term
infant.5 Why would MAS be a good disease to treat with a therapy
such as ECMO? Figure 1 depicts the complex pathophysiology
associated with MAS, with a combination of obstructive lung
disease, parenchymal disease caused by inflammation and
persistent pulmonary hypertension. Ventilation alone, either
conventional or high-frequency ventilation, of the severe cases may
not be able to break the cycle associated with severe hypoxia and
persistent pulmonary hypertension, thus making ECMO an ideal
therapy for this complex disease. While on ECMO, the ventilator
can be placed at lung rest, allowing gentle ventilation, time for the
body to absorb the meconium and time for the pulmonary
hypertension to resolve. Figure 2 from the Extracorporeal Life
Support Organization database shows the changing patterns of use
in ECMO, with the MAS infant ranging from 45 to now 35% of the
neonatal patients treated with ECMO. The survival rate for MAS
infants compared with infants with other disease states is shown in
Figure 3. Infants with MAS, who do not survive ECMO, do not do so
because of a major intracranial complication, not because of
irreversibility of the lung disease. These findings indicate that
infants with severe MAS need to be referred to an ECMO center
 ECMO use in MAS
BL Short
S80

prior to meeting conventional ECMO criteria to reduce the risk for
pre-ECMO cardiovascular instability making transfer difficult.
Most intracranial bleeds are thought to be secondary to hypoxic/
ischemic insults prior to ECMO that would, in a non-heparinized
patient, result in an infarction, but in the heparinized ECMO
population results in a hemorrhage and/or hemorrhagic
infarction.6,7 Studies are now indicating that ECMO conducted
using venoarterial (VA) ECMO, in which non-pulsatile flow is
created with the ECMO pump, may also place the brain at risk.7–11
This risk is minimal in venovenous (VV) ECMO, the more common
form of ECMO for the infant with MAS.
As new therapies have become available, such as the use of
high-frequency ventilation, surfactant use in term infants, and
Meconium Aspiration
Mechanical Chemical
Obstruction Inflammation
Air Trapping
Atelectasis
inhaled nitric oxide, the need for ECMO has reduced.1,12 –15
Figure 4 shows the reduction in the number of infants being placed
on ECMO over time. Also depicted in this figure is the reduction in
the survival rate of the ECMO patient over time. Although the cause
of this reduction is not clear, it may be assumed that as increasing
numbers are rescued by other therapies, the severity and acuity of
the patients going on to ECMO has increased. This finding differs
from those of Fliman et al.,15 who only looked at cases from 1996
to 2003. This difference in survival data is probably related to the
time period studied, with the late 1980s, as depicted in Figure 4,
representing the highest survival rate prior to most new
interventions. Figure 5 may support the theory of sicker patients
now being placed on ECMO. This figure shows that as the number
of ECMO patients has decreased, the time on ECMO has increased,
indicating that the severity of lung disease in infants going on
ECMO is greater, that is, the less severe are being rescued by other
therapies. Table 1 compares the pre-ECMO ventilator and blood gas
100
94
90
84
80 78
75
Percent Survival

70
63
Uneven Intrapulmonary 60
Ventilation Shunting 52
50
40
Air Leaks Hypoxia 30
PPHN 20
10
0
MAS PPHN CDH SEPSIS HMD Other

Figure 1 Mechanical obstruction and chemical inflammation are the hallmark Figure 3 Survival data by diagnoses for extracorporeal membrane oxygenation
for meconium aspiration syndrome (MAS), leading to respiratory failure and the (ECMO) patients (data from the Extracorporeal Life Support (ECLS) database,
development of pulmonary hypertension in the MAS patient. January 2007 report, n ¼ 21 258 patients).

100% Others
Sepsis
80% RDS
PPHN/PFC
60% MAS
CDH

40%

20%

0%
19 7
88

19 9
19 0
91

19 2
19 3
94

19 5
96

19 7
19 8
99

20 0
20 1
20 2
20 3
20 4
05
8

8
9

9
9

9
9

0
0
0
0
0
19

19

19

19

20
<=

Figure 2 This figure shows the major diagnoses treated with extracorporeal membrane oxygenation (ECMO). Although the numbers are reduced, the meconium
aspiration syndrome (MAS) population continues to be the largest group of patients treated with ECMO (data from the Extracorporeal Life Support Organization database
from 1987 to 2005).

Journal of Perinatology
 ECMO use in MAS
BL Short
S81

1600 100
HFOV
Surfactant 90
1400 Cases % Survival

80
1200
70
1000 iNO
60

Survival (%)
# Cases

800 50

40
600
30
400
20
200
10

0 0
86 87 88 89 90 91 92 93 94 95 96 97 98 99 '00 '01 '02 '03 '04 '05 '06
Year

Figure 4 There has been a reduction in patients requiring extracorporeal membrane oxygenation (ECMO) (black bar) related to new therapies as depicted in the
graph. Survival (line) has decreased over this same time period (data from the Extracorporeal Life Support Organization database January summary report, n ¼ 21 258
patients; iNO, inhaled nitric oxide).

250 100
HFOV Pump Time % Survival
Surfactant 90
iNO
200 80

70
Pump Time (hours)

Survival (%)
150 60

50

100 40

30

50 20

10

0 0
86 87 88 89 90 91 92 93 94 95 96 97 98 99 '00 '01 '02 '03 '04 '05 '06
Year

Figure 5 As survival (line) has decreased, time on extracorporeal membrane oxygenation (ECMO) has increased over the years (black bar) (data from the
Extracorporeal Life Support Organization database January summary report, n ¼ 21 258 patients). iNO, inhaled nitric oxide.

data for the top three diagnoses going on to ECMO. It is interesting
that the MAS infant has an average mean airway pressure of 16
compared with the congenital diaphragmatic infant with a mean
airway pressure of 15 mm Hg, indicating the severity of the lung
disease in these infants.
The risk for cerebral injury in the ECMO population ranges
from 5 to 12%, with severe hypoxia, birth weight and sepsis
representing the highest risk factors.6 Although the pre-ECMO
events are thought to be the primary factors leading to ICH, data
from animal models simulating clinical ECMO have shown an
altered cerebral autoregulation pattern in animals exposed to VA
ECMO both with and without prior exposure to hypoxia.7–11
Figure 6 depicts the changes in cerebral blood flow with lower
cerebral perfusion pressure (lower end of the autoregulatory curve).
In the newborn lamb model, 2 h of exposure to severe hypoxia
altered cerebral autoregulation, but when the animals were
recovered by placing them on ECMO, the alteration in
autoregulation was markedly worse. Further studies using isolated

Journal of Perinatology
 ECMO use in MAS
BL Short
S82

Table 1 Pre-ECMO ventilator and blood gas data (data from the ECLS 20
Control
organization neonatal database, summary from 1997–2007) VV ECMO
VA ECMO
MAS PPHN CDH 10

Age on ECMO (days) 2.00 2.86 2.27

0
Time on ECMO (hours) 138 159 266

% Change
BW (kg) 3.0 3.15 2.89
GA 38 37 37 -10
Apgar (1/5 min) 4/6 6/7 4/6
FIO2 0.96 0.95 0.95 -20
PIP/PEEP 34/3 32/3 32/3
*
MAP 16 14 15 *
-30
Pre-pH 7.00 7.18 7.08 *
Pre-pCO2 44 44 58
Pre-pO2 45 43 39 -40
Pre-sats (%) 65 61 56 1µM 10µM 100µM
Ach Concentration
Abbreviations: BW, birth weight; CDH, congenital diaphragmatic hernia;
ECLS, extracorporeal life support; ECMO, extracorporeal membrane oxygenation; Figure 7 Isolated vessel chamber studies on lamb middle cerebral arteries taken
FIO2, fractional inspired oxygenation; GA, gestational age; MAP, mean airway pressure; from animals exposed to no extracorporeal membrane oxygenation (ECMO)
MAS, meconium aspiration syndrome; PEEP, peep end expiratory pressure; PIP, peep (controls), venoarterial (VA) or venovenous (VV) ECMO are shown in this figure.
inspiratory pressure; PPHN, persistent pulmonary hypertension; Pre-sat, pre-oxygen Vessels were exposed to acetylcholine (Ach) and monitored for dilation. Percent
saturation.
change in vessel diameter is noted on the Y axis.11,12
70
60 VA ECMO may have an alteration of cerebral vascular reactivity
CBF (ml / 100 g / min)

50 resulting in altered autoregulation of the cerebral circulation. The

40
30
20
10
0 is higher in the VA ECMO population. Many centers still use VA
CPP<25 CPP 25–39 CPP 40–55 Baseline
Cerebral perfusion pressure (mm Hg)
Control Hypoxia Hypoxia-ECMO
Figure 6 Lamb studies of cerebral autoregulation after prolonged hypoxia and
recovery using extracorporeal membrane oxygenation (ECMO) therapy. Control
animals (circle) autoregulated down to cerebral perfusion pressures of
<25 mm Hg, whereas animals exposed to hypoxia (square) and recovered with
increasing fractional inspired oxygen (FIO2) could only autoregulate down to
cerebral perfusion pressure (CPP) of 25 to 39. If animals were exposed to hypoxia
and then recovered with ECMO, the alteration would be much worse with loss of
autoregulation at a CPP of 40 to 55 mm Hg.7
arterial vascular chamber techniques have shown that cerebral
vessels taken from animals exposed to VA ECMO have a marked
alteration in reactivity and that this reactivity is associated with an
altered production of nitric oxide (see Figure 7).10–12 This
alteration is thought to be secondary to the shear stress changes
caused by the non-pulsatile VA ECMO circuit, because this
alteration is seen only in the VA ECMO-exposed animals and not to
animals exposed to VV ECMO, in which the pumping chamber is
the native heart with normal pulsatile blood flow patterns
(see Figure 7).10–12 These findings indicate that patients placed on
cerebral circulation becomes pressure-passive under these
conditions, making hypertension and hypotension risks for either
cerebral ischemia (infarction) or cerebral overperfusion (cerebral
hemorrhage). There has been no randomized comparison of VA
versus VV ECMO patients to determine whether the incidence of ICH
ECMO primarily, but when they use VV ECMO, it is in the less
severe infants, making a comparison of the rate of ICH in these
populations difficult. A recent study by Radhakrishnan et al.,16
reviewing data from the Extracorporeal Life Support Organization’s
registry on the outcome and complication rate in the MAS
population treated with ECMO, found that infants with MAS were
treated with VV ECMO 55% of the time compared with 29% for the
non-MAS patient. They also found that the MAS population had a
significantly higher survival rate and lower number of
complications per patient versus the non-MAS patient. They
concluded that, with the lower mortality and morbidity, it was time
to consider relaxing the criteria for the MAS patient and ECMO use.
This may be a consideration when using VV ECMO, in which
potential cerebral alterations do not exist and ligation of the carotid
artery is not a concern. It may be time to consider a randomized
trial looking at morbidity instead of mortality in this population.
Although MAS is the most common neonatal respiratory failure
diagnoses going on in ECMO, the increasing use of ECMO is now
in neonatal and pediatric patients with heart failure post-surgery
for congenital heart disease.17 With this increase, there has been
the introduction of newer devices for cardiovascular support

Journal of Perinatology

including various types of ventricular assist devices. Artificial heart
support for bridge to transplant available for the adult population
is now available for the newborn and pediatric population in
Europe. Research and development of similar devices is ongoing in
the United States. The field of conventional ECMO is also
changing, with newer equipment, including low-resistant
microporous oxygenators and centrifugal pumps that will allow the
procedure to be performed with smaller and safer systems.18 Newer
low-resistant microporous membrane lungs have the ability of
allowing ECMO to be conducted without a pump.19–21 On account
of the low resistance of these membranes, gas transfer can be
achieved through an arterial–venous shunt through these
membranes. These techniques are presently used in clinical trials
in the adult population in Europe and have shown good success
with survival rates as high as 70% is some studies.19–21 If the
results of these studies show improvement in outcome, the use of a
non-pump-driven circuit with potentially less complications and
lower blood prime may allow for the consideration of studies using
extracorporeal support for early intervention instead as a rescue
therapy. These trials would be designed to look at reduction in the
lung injury, time of ventilation and reduction in hospital length of
stay. To date, this technique has not been used in newborns, but the
umbilical vessels make this an attractive approach. The MAS
population would be perfect candidates to consider for such a study.
Disclosure
The author has declared no financial interests.
References
1 Rais-Bahrami K, VanMeurs K. ECMO for neonatal respiratory failure. Semin Perinatol
2005; 29: 15–23.
2 Thompson BT, Spence CR, Janssens SP, Joseph PM, Hales CA. Inhibition of hypoxic
pulmonary hypertension by heparins of differing in vitro antiproliferative potency. Am
J Respir Crit Care Med 1994; 149(6): 1512–1517.
3 White JJ, Risemberg H, Andrews HG, Mazur D, Haller Jr JA. Prolonged respiratory support
in newborn infants with a membrane oxygenator. Surgery 1971; 70: 288–296.
4 Dorson WJ, Baker E, Cohen MI, Meyer B, Molthan M, Trump A et al. A perfusion
system for infants. ASAIO Trans 1969; 15: 155–160.
ECMO use in MAS
BL Short
S83
5 Bartlett RH, Gazzaniga AB, Jefferies R, Huxtable RF, Haiduc NJ, Fong SW et al.
Extracorporeal membrane oxygenation (ECMO) cardiopulmonary support in infancy.
Trans Am Soc Artif Intern Organs 1976; 22: 80–88.
6 Bulas D, Glass P. Neonatal ECMO: neuroimaging and neurodevelopmental outcome.
Semin Perinatol 2005; 29: 58–65.
7 Short BL. The effect of extracorporeal life support on the brain: a focus on ECMO.
Semin Perinatol 2005; 29: 45–50.
8 Short BL, Walker LK, Gleason CA, Bender KS, Traystman RJ. Effect of extracorporeal
membrane oxygenation on cerebral blood flow and cerebral oxygen metabolism in
newborn seep. Pediatr Res 1990; 28: 50–53.
9 Short BL, Walker LK, Bender KS, Traystman RJ. Impairment of cerebral autoregulation
during extracorporeal membrane oxygenation in newborn lambs. Pediatri Res 1993;
33: 289–294.
10 Ingyinn M, Lee J, Short BL, Viswanathan M. Venoarterial extracorporeal membrane
oxygenation (VA ECMO) impairs basal nitric oxide production in cerebral arteries of
newborn lambs. Pediatr Crit Care Med 2000; 1: 161–165.
11 Ingyinn I, Short BL, Rais-Bahrami K, Viswanathan M. Altered cerebrovascular
responses after exposure to venoarterial extracorporeal membrane oxygenation: role of
the nitric oxide pathway. Pediatr Crit Care Med 2006; 7(4): 368–373.
12 The Neonatal Inhaled Nitric Oxide Group. Inhaled nitric oxide in full-term and
near-term infants in respiratory failure. N Engl J of Med 1997; 336: 597–604.
13 Clark RH, Kueser TJ, Walker MW, Southgate WM, Huckaby JL, Perez JA et al. Low-dose
nitric oxide therapy for persistent pulmonary hypertension of the newborn. N Engl J
Med 2000; 342: 469–474.
14 Walker LK, Short BL, Traystman RH. Impairment of cerebral autoregulation during
venovenous extracorporeal membrane oxygenation in the newborn lamb. Crit Care
Med 1996; 24: 2001–2006.
15 Fliman PJ, DeRegnier RA, Kinsella JP, Reynolds M, Rankin LL, Steinhorn RH. Neonatal
extracorporeal life support: impact of new therapies on survival. J of Pediatr 2006;
148: 595–599.
16 Radhakrishnan RS, Lally PA, Cox CS. ECMO for meconium aspiration syndrome:
support for relaxed entry criteria. ASAIO J 2007; 53: 489–491.
17 Hines MH. ECMO and congenital heart disease. Semin Perinatol 2005; 29: 34–39.
18 Bartlett RH. Extracorporeal life support: history and new directions. Semin Perinatol
2005; 29: 2–7.
19 Wales T. Clinical experience with the iLA membrane ventilator pumpless
extracorporeal lung-assist device (review). Expert Rev Med Devices 2997; 4(3):
297–305.
20 Bein T, Weber F, Philipp A, Prasser C, Pfeifer M, Schmid FX et al. A new pumpless
extracorporeal interventional lung assist in critical hypoxemia/hypercapnia. Crit Care
Med 2006; 34(5): 1371–1377.
21 Liebold A, Reng CM, Philipp A, Pfeifer M, Birnbaum DE. Pumplesss extracorporeal
lung assist-experience with the first 20 cases. Eur J Cardiothorac Surg 2000; 17(5):
608–613.
Journal of Perinatology
 Journal of Perinatology (2008) 28, S84–S92
r 2008 Nature Publishing Group All rights reserved. 0743-8346/08 $30
www.nature.com/jp

REVIEW
Role of iNO in the modulation of pulmonary vascular resistance
A Bin-Nun and MD Schreiber
Department of Pediatrics, Section of Neonatology, The University of Chicago, Chicago, IL, USA

endothelial cells that relaxed blood vessels, endothelium-derived

Inhaled nitric oxide (iNO) has quickly become a standard therapy for term
and near-term infants with hypoxic respiratory failure and persistent
pulmonary hypertension. Its effect on the lung is believed to be through the
stimulation of soluble guanylyl cyclase and the increased production of
cyclic guanosine 30 ,50 -monophosphate (cGMP). However, in addition to
pulmonary vasodilation and a decrease in pulmonary vascular resistance,
nitric oxide (NO) shows several additional potential beneficial effects on the
lung. This article reviews NO mechanisms of action, early clinical trial of
iNO and clinical aspects for the use of iNO in acute respiratory failure of the
term and near-tem neonates.
Journal of Perinatology (2008) 28, S84–S92; doi:10.1038/jp.2008.161
relaxing factor. Ignarro et al.,2 in 1987, help solve the riddle with
compelling evidence supporting NO as the active signaling
component of endothelium-derived relaxing factor. With
Moncada’s work in 1987,3 also showing that NO is the active
component of endothelium-derived relaxing factor, NO has also
become appreciated as an important neurotransmitter. In 1996, by
allowing NO gas to bubble through a tissue preparation containing
guanylylcyclase, Ferid Murad4 showed an increase in the
production of cyclic guanosine 30 ,50 -monophosphate (cGMP), thus
identifying the mechanism of action of this important molecule.
For their pioneering work, Drs Furchgott, Ignarro and Murad
received the 1998 Nobel Prize for Medicine.

Introduction: nitric oxide

Nitric oxide (NO) is a colorless, odorless gas. Earlier, NO was Pulmonary vasodilatation: mechanism of action
considered only as the toxic product of internal combustion Nitric oxide activates soluble guanylyl cyclase, leading to the
engines, burning cigarettes and lightening storms. The activation of cGMP-dependent protein kinase. In turn,
atmospheric concentration of NO is 10 to 500 p.p.b. (parts per cGMP-dependent protein kinase decreases the sensitivity of myosin
billion) in general, 1.5 p.p.m. (parts per million) in heavy traffic to calcium-induced contraction and lowers the intracellular
and 1000 p.p.m. in tobacco smoke. Human airways produce NO; calcium concentration by activating calcium-sensitive potassium
within the nasal mucosa, NO concentrations reach 25 to 64 p.p.b. channels and inhibiting the release of calcium from the
NO concentrations are considerably lower more distal, reaching sarcoplasmic reticulum. The degradation of cGMP back to
1 to 6 p.p.b. in the mouth, trachea and distal airways. guanosine triphosphate is regulated under the control of a specific
In spite of its Food and Drug Administration (FDA) approval in
December 1999, NO continues to be confused with other
compounds. Some are listed in Table 1 along with their chemical Table 1 NO impersonators
formula and bioactivity. The ability of NO to exist and exert Name Symbol Bioactivity
biological activity in several states strengthens its position as a
Nitrous oxide N2O Anesthetic (‘laughing gas’)
perfect second messenger (Table 2). .
Nitrogen dioxide NO2 Toxic free radical
Nitrate NO
3 Oxidation end product (70%)
Abbreviation: NO, nitric oxide.
Historical perspective
In 1980, Furchgott and Zawadzki1 described how intact blood Table 2 NO equivalents
vessels, possessing intact endothelium and smooth muscle, dilated Name Symbol
to stimuli that induced vasoconstriction when the endothelium was
disturbed. He termed this substance, produced in vascular Nitric oxide NO
.
Nitroxyl anion NO
Correspondence: Dr MD Schreiber, The University of Chicago Comer Children’s Hospital, Nitrosonium NO+
MC8000, 5721 S Maryland Avenue, Chicago, IL 60637, USA.
E-mail: mschreiber@peds.bsd.uchciago.edu Abbreviation: NO, nitric oxide.
 iNO and PVR
A Bin-Nun and MD Schreiber
S85

Blood vessel

Endothelial cells

Vascular smooth muscle cells

Phosphodiesterase Inhibition by sildenafil

type 5 and zaprinast Inositol 1,4,5,
Soluble guanylyl -triphosphate
Nitric oxide cyclase

GTP
Activation Ca2+
cGMP
cGMP-dependent Inhibition
protein kinase of calcium release
Activation
Vascular
smooth muscle Decreased Sarcoplasmic
cell sensitivity of myosin reticulum
Inhibition

Phosphory- Myosin light-chain L-type calcium

lated myosin phosphatases Myosin channel
(contraction) (relaxation) Activation
Ca2+
K+

Calcium-sensitive
potassium channel

Figure 1 Regulation of the relaxation of vascular smooth muscle by nitric oxide (NO).5

type 5 phosphodiesterase.5 Inhaled nitric oxide (iNO) is the first
virtually selective pulmonary vasodilator in contrast to other
medications. Tolazoline, nitroprusside or prostaglandins E1 and D2,
which also decrease pulmonary vascular resistance, but as non-
selective vasodilating compounds, often has profound systemic
effects (Figures 1 and 2).
The biochemical fates of iNO at the alveolar-capillary
membrane: small amounts of nitrogen dioxide (NO2) may be
formed if iNO mixes with high concentrations of oxygen (O2) in
the air space. Depending on the milieu of the lung parenchyma,
NO may react with reactive oxygen species to form reactive nitrogen
species such as peroxynitrite. In the vascular space, dissolved NO is
scavenged by oxyhemoglobin (forming methemoglobin and
nitrate) and, to a lesser extent, plasma proteins (for example,
forming nitrosothiols, which are stable intravascular sources of NO
activity).5
The fate of intravascular NO is dose-dependent: Under normal
conditions, NO produced by endothelial NO synthase with arginine
as a substrate diffuses into smooth muscle to activate soluble
guanylyl cyclase (sGC) to form cGMP and thus maintain basal
vascular tone. Intravascular destruction of NO by intraerythrocytic
hemoglobin is limited by diffusional barriers, including the
red-cell-free layer adjacent to the endothelium and factors
in and around the erythrocyte membrane that slow NO transit
(Figure 3). However, sufficient NO is consumed to limit the
activity of NO to the local environment. When NO is administered
by inhalation, high intravascular NO concentrations promote
NO reactions, with erythrocyte hemoglobin and plasma proteins
that can either protect it, thereby promoting systemic
vasodilatation, or destroy it. During hemolysis or the infusion of
hemoglobin-based blood substitutes, cell-free ferrous hemoglobin
in the plasma rapidly destroys NO by being oxidized to
methemoglobin and nitrate ions. Cell-free hemoglobin may
diffuse into extravascular spaces. Limited NO bioavailability under
these conditions promotes systemic vasoconstriction and organ
dysfunctions.6

Journal of Perinatology
 iNO and PVR
A Bin-Nun and MD Schreiber
S86

Air space Release of

O2 NO2 reactive
Type II
oxygen species
alveolar cell
Nitric oxide
Type I
alveolar cell Formation of
reactive nitrogen
species

Inactivation by
hemoglobin
Red cell Formation of
S-nitrosothiols

Plasma proteins
Leukocyte

Vascular space Endothelial cell

Figure 2 Biochemical fates of inhaled nitric oxide (iNO) at the alveolar–capillary membrane.5

Pharmacology Physiology Pathology

Blood vessel NO concentration

-cys93 S NO


NO+ Erythrocytes Diffusion
  barrier (plasma)
II NO2–


Fe NO
 
–


Fe II – O2 + NO•

Fe III + NO3
   
pO2 pO2
pH pH
H+
NO•

Plasma SNO NO• Xanthine NO

oxidoreductase NO• synthase
Arginine NO•
NO Citrulline
synthase
NO•
Smooth muscle cells
sGC Endothelial cells

Blood vessel

Figure 3 A model of the interactions of iNO (NO) with erythrocytes and cell-free hemoglobin in an arterial blood vessel.6

Early clinical trials vasodilation. Pulmonary hypertension resumed within minutes of

ceasing NO inhalation. While inhaling NO, there was an improved
In 1991, Frostell et al.7 showed in a lamb model that iNO (5 to oxygenation and cardiac output and decreased pulmonary artery
80 p.p.m.) can reverse pulmonary hypertension, without systemic pressure and pulmonary vascular resistance.

Journal of Perinatology

Figure 4 Change of preductal (J) and postductal (&) oxygen saturation
(SpO2) in infants with persistent pulmonary hypertension of the newborn (PPHN)
before and during inhalation of 80 p.p.m. NO at FiO2 0.9 wP<0.05.
The first human study with iNO was conducted in eight adults
with pulmonary hypertension.8 Pulmonary vascular resistance fell
with iNO in all patients. This was not associated with a decrease in
systemic vascular resistance, in contrast to treatment with
intravenous prostacyclin.
Roberts et al.9 and Kinsella et al.10 described in 1992 the first
use of iNO for the treatment of persistent pulmonary hypertension
of the newborn (PPHN). Roberts treated six newborn infants with
PPHN with up to 80 p.p.m. of iNO. In all six patients, preductal
oxygen saturation increased; in five infants, postductal oxygen
saturation and oxygen tensions also increased (Figure 4).
Inhalation of NO did not cause systemic hypotension.
Kinsella used a lower dose (10 to 20 p.p.m.) in nine newborn
infants with PPHN. All showed a rapid improvement in
oxygenation without a reduction in systemic blood pressure
(Figure 5). This study was the first to show the sustained effect of
low-dose iNO in maintaining adequate oxygenation.
Larger randomized controlled studies examining different
strategies of treatment with iNO in acute severe PPHN soon
followed these pilot studies. The NINOS trial enrolled 235 term and
near-term newborn infants with hypoxic respiratory failure,
randomized to receive either iNO (20 p.p.m.) or a standard medical
treatment (FiO2 100%). Infants receiving iNO were significantly less
likely to be treated with extracorporeal membrane oxygenation
(ECMO).11
In another study,12 Roberts randomized 58 newborn infants
with PPHN to either iNO (80 p.p.m.) or placebo: in 53% of infants
treated with iNO, oxygenation improved as compared with 7% in
the control group. The use of ECMO was again significantly
decreased in the iNO group (40 vs 71%). In the final randomized,
controlled trial, which led to the FDA approval, Clark et al. studied
a low-dose iNO therapy (20 p.p.m. in the first 24 h and then
iNO and PVR
A Bin-Nun and MD Schreiber
S87
Figure 5 Serial changes in arterial/alveolar oxygen tension ratio (a/AO2) for
six patients over 24 h of NO inhalation.10
5 p.p.m. for up to 4 days) in 248 infants who were randomized to
iNO or placebo: ECMO treatment was again significantly lower in
the iNO group (38 vs 64%). In addition, this was the first study to
show in term infants a reduction in the risk for chronic lung
disease (7 vs 20%).13 Because ECMO was used as a rescue therapy,
none of these studies showed a change in mortality.
Endogenously released and exogenously iNO may influence
many facets of the developing perinatal lung, including the lung
parenchyma, bronchi and vascular structures.14 In a fetal or
preterm lung during the transition from a saccular to an alveolar
stage (25 to 28 weeks of gestation), endogenous NO is released
primarily from epithelial and endothelial cells that contain NO
synthase, and it is implicated in the structural and functional
aspects of the development of pulmonary vasculature and airway
smooth muscle. NO also contributes to growth of the lung
parenchyma and to extracellular matrix deposition and may
modulate surfactant and inflammation in the developing lung.
The implications of supplementation with iNO in preterm infants
are15 beyond the scope of this review.
Potential beneficial effects of iNO
Thus, given the multiple mechanisms of action exerted by NO on
the lung, its benefit may be mediated in several ways. Several are
listed here:
(1) Improved V/Q matching: Hypoxic pulmonary
vasoconstriction minimizes ventilation–perfusion
mismatching in the presence of abnormal ventilation. iNO
improves oxygenation by increasing blood flow to ventilated
lung units.5
(2) Improved PVR/SVR ratio: This results in a decrease in R to L
shunting at the ductal and foramen level and an improvement
in oxygenation.
(3) Enhancement of pulmonary surfactant activity: Neonatal
animal models showed that low-dose iNO decreased or
prevented the O2-induced detrimental effects on alveolar
Journal of Perinatology
 iNO and PVR
A Bin-Nun and MD Schreiber
S88
surfactant, improved the apparent ability of hydrophobic
surfactant proteins to promote low-surface tension and improved
lung volume by a process unrelated to surfactant function.15
(4) Prevention of neomuscularization in patients with PPHN:
In a model of injured pup lungs, iNO protected against
pulmonary remodeling induced by lung injury by mechanisms
that are independent of pulmonary tone, inflammation or
thrombosis.16
(5) Decreased reperfusion injury: A neonatal animal model of
lung ischemia–reperfusion showed that iNO can prevent
microvascular injury, endothelial dysfunction and pulmonary
neutrophil accumulation.16
(6) NO replacement: NO, produced by endothelial NO synthase, is
critically involved in the cardiopulmonary transition from fetal
to neonatal life. Endothelial NO synthase protein expression
and mRNA were decreased by a half and two-thirds,
respectively, in an animal model of pulmonary hypertension
(Figure 6).17
Clinical considerations
Hypoxic respiratory failure of the term near-term infant:
Hypoxemia of the near-term infant is caused by complex
pathopysiological pathways that have a common end point of
reduced perfusion of the lungs. In some patients, one of these
mechanisms dominates but in others several mechanisms play
along, contributing to the severe clinical picture.
 Patent ductus arteriosus and/or patent foramen ovale with right-
to-left extrapulmonary shunting.
 V/Q mismatching in the lungs: as in meconium aspiration
syndrome. In some segments of the lungs, there is reduced
ventilation and right-to-left intrapulmonary shunting, and other
Figure 6 Pulmonary endothelial NO synthase gene expression is decreased in
fetal lambs with pulmonary hypertension.17 *P<0.05.
Journal of Perinatology
segments are hyperinflated, increasing the dead space. In a
heterogeneous, patchy, a form of lung disease, iNO may improve
the V/Q matching by dilating the vasculature bed around the
ventilated segments. In a homogenous parenchymal lung
disease, the response to iNO may be low: treatment of the
underlying lung disease and better recruitment of the atelectatic
lung may improve the response to iNO.
 Left ventricular dysfunction.
It is important to evaluate and treat each of these factors when
treating the hypoxemic infants.
Echocardiography. Echocardiography is crucial in the
evaluation and management of the hypoxemic (blue) baby. The
study will measure evidence for PPHN: existence of patent ductus
arteriosus and patent foramen ovale in the direction of the shunt,
and the pressure at the right ventricle. PPHN can be also shown
by the evidence of right-to left-extrapulmonary shunting, by a
difference between the preductal and the postductal O2 saturation.
Currently, the best way to distinguish between a congenital
heart lesion, which is dependent on right-to-left shunting (for
example, interrupted aortic arch, total anomalous pulmonary
venous return and hypoplastic left heart syndrome) in which
treatment with iNO may not be indicated, other anatomical
variations or a normal heart with maladaptation to the
extrauterine circulation is with echocardiography.
Echocardiography is also important to evaluate the cardiac
performance and assess ventricular function.
Timing of treatment. Large-scale randomized studies support
the efficacy of iNO in term and near-term infants (>34 weeks of
gestation) in the first 2 weeks of life.18 iNO has also been used
post-ECMO. iNO has also been used in the treatment of late
pulmonary hypertension complicating chronic lung disease with
some reported success,19,20 although much of the data are
anecdotal. The treatment of the premature infant is still under
investigation and is beyond the scope of this review.
Indication for treatment. Most of the studies that tested the
efficacy of iNO in acute respiratory failure used enrolled infants
with oxygenation index (OI) of 25 or above.18 Most evidence
supports the use of iNO to reduce the need for ECMO and/or death.
Some studies that used less-stringent entry criteria showed
improved oxygenation but did not reduce ECMO/death when
compared with the more ‘traditional’ criteria.21,22
Dose. The first clinical trials used higher initial doses of
80 p.p.m. Subsequent studies changed the practice to an initial
dose of 20 p.p.m. A review of the iNO treatment of 476 neonates
revealed that starting at higher doses does not improve the major
outcomes (ECMO and death), and there was no evidence that
using higher dose leads to a more rapid improvement of the

hypoxemia. Using higher does of iNO (>22 p.p.m.) led to an
increase in methemoglobin levels. This may decrease the oxygen-
carrying capacity by hemoglobin. Using lower doses of iNO
(<18 p.p.m.) achieved the same response of PaO2 as the higher
doses, and there were no differences in the adverse events.23
Risks and complications
Methemoglobinemia. The incidence of methemoglobinemia has
been low in clinical trials, occurring more commonly in neonates
and with high inhaled doses, but usually is tolerated well.
Left ventricular dysfunction
Inhaled NO may have adverse hemodynamic effects in patients
with significant preexisting left ventricular dysfunction. Studies
with iNO in adults with preexisting, severe left ventricular
dysfunction showed an increase in the left ventricular end-diastolic
pressure.24,25 This increase in end-diastolic volume resulting from
an iNO-induced increased pulmonary perfusion may not be
tolerated by the poorly compliant left ventricle resulting in
worsened, rather than improved, oxygenation.
Withdrawal. A randomized control study of iNO also examined
the safety of its withdrawal in 155 term infants with pulmonary
hypertension. For infants responding to the treatment, a
dose–response relationship between the iNO dose and decrease in
PaO2 after discontinuing iNO was found (Figure 7). A reduction in
iNO to 1 p.p.m. before discontinuation of the drug seems to
minimize the observed decrease in PaO2. For infants failing
treatment, discontinuation of iNO could pose a life-threatening
reduction in oxygenation (Figure 8).26
Figure 7 Changes in PaO2 (no ventilator changes), 30 min after cessation of
inhaled NO treatment gas (masked) in patients with persistent pulmonary
hypertension of the newborn (PPHN) treated successfully.26
iNO and PVR
A Bin-Nun and MD Schreiber
S89
A possible explanation for this phenomenon might be through
an increase in the concentration of superoxide and peroxynitrite
when treating with iNO. In an animal model, reactive oxygen
species scavenging ameliorated alterations in endogenous NO
signaling during iNO therapy (Figure 9).27
Direct toxicity. At high inspired levels, NO is directly toxic to
tissues and can cause methemoglobinemia, formation of NO2,
pulmonary edema, alveolar hemorrhage and hypoxemia. At low
inspired levels of iNO (20 p.p.m.) NO2 formation is minimal.28
Bleeding tendency. iNO is associated with increased bleeding
time and inhibition of platelet aggregation in adults.29,30 A
Cochrane review of randomized studies of iNO in the
near-term infants did not find an association between the use
of iNO and bleeding events.18
New information
The new clinical data emerging in the literature concentrate on the
safety of iNO: A study in premature infants dropped the concerns
that iNO might have a deleterious effect on surfactant proteins and
function. This study showed a transient improvement of surfactant
function after iNO treatment,31 and that administration of iNO does
not increase inflammatory markers in the alveolar fluid.32
Neurodevelopmental outcome. Concerns raised regarding the
long-term complications of long-term use of iNO. In a study that
examined the effects of iNO in premature infants, the infants in the
study group had improved neurodevelopmental outcomes at 2 years
of age as compared with the placebo group.33 A follow-up study at
18 to 24 months of age of the infants participating in the NINOS
study showed that early iNO therapy (OI: 15 to 25) as compared
Figure 8 Percent change in oxygenation on withdrawal of placebo or NO study
gas (5, 20, or 80 p.p.m.) in persistent pulmonary hypertension of the newborn
(PPHN) patients declared treatment failures.26
Journal of Perinatology
 iNO and PVR
A Bin-Nun and MD Schreiber
S90

Figure 9 Reactive oxygen species scavenging. Lung tissue nitric oxide synthase (NOS) activity and endothelial NOS (eNOS) protein levels after inhaled NO (NO) therapy
in control lambs. (a) Relative NOS activity is decreased after 24 h of inhaled NO. (b) Lung tissue eNOS protein levels, as determined by western blot analysis, are not
changed after 24 h of inhaled NO.27 *P<0.05.

with late therapy (OI>25) for respiratory failure in term and 70

Placebo
Change in 6-min walking distance (m)

near-term infants was not associated with an increase in 60 20 mg of sildenafil

40 mg of sildenafil
neurodevelopmental impairment or hearing loss.21 This new data 50 80 mg of sildenafil
suggest that iNO might be beneficial for the developing brain. 40

Congenital diaphragmatic hernia. The pulmonary artery
hypertension in congenital diaphragmatic hernia is, in part,
secondary to underdevelopment and anatomical changes of the
pulmonary vasculature (that is, maladaptation). In addition, left
ventricular dysfunction and surfactant dysfunction may contribute
to hypoxemia. These may lead to a different approach in treating
the hypoxemic respiratory failure in these patients. Two studies that
randomized infants with congenital diaphragmatic hernia to iNO
or placebo13,34 failed to show improved outcome in terms of
reduced use of ECMO or death.
Non-invasive low-dose iNO, delivered via nasal canula at the
time of tracheal extubation, may benefit a subset of infants with
congenital diaphragmatic hernia who cannot be weaned from iNO
after surgical correction.35 In this subset of patients, pulmonary
vascular hyperactivity persisted in spite of parenchymal
improvement. The role of iNO is likely far more complicated than
simply vasodilation alone, as the NO/cGMP pathway is also
involved in alveolar growth and lung angiogenesis.36
Future directions and research consideration
Unfortunately, not all infants with PPHN improve after the
initiation of iNO therapy and refractory PPHN is common. Other
pharmacologic modalities are important. On the basis of the
regulation pathways that control the production of the NO–cGMP
cascade, and other physiologic mechanisms that induce pulmonary
vasoconstriction, the following pharmacological strategies might be
useful in the prevention or amelioration of PPHN. Some of these
have been studied in humans (adults or pediatric population) and
some are still experimental, shown to be effective in animal studies.
30
20
10
0
–10
–20
0 4 8 12
Week
Figure 10 Mean changes from baseline, with 95% confidence intervals, in the 6-
min walking distance at week 12 in the placebo and sildenafil groups.38
cGMP-specific phosphodiesterase inhibitors (sildenafil)
The secondary messenger of NO, cGMP, is inactivated
predominantly by PDE5 (phosphodiesterase type 5). Sildenafil, an
inhibitor of PDE5, is a selective pulmonary vasodilator, partially
because PDE5 is highly expressed in the lung.5
Amelioration effects of iNO withdrawal. Rebound pulmonary
hypertension caused by withdrawal of iNO can be markedly
attenuated by increasing cGMP with sildenafil.37
Therapy for pulmonary hypertension. A randomized
placebo-controlled study in 278 adult patients with symptomatic
pulmonary arterial hypertension, treatment with oral sildenafil
showed an improvement in exercise capacity, WHO functional class
and hemodynamics (Figure 10).38
A model for meconium aspiration in piglets tested the efficacy of
the intravenous form of sildenafil. Sildenafil completely reversed
the increase in pulmonary vascular resistance, and was shown to

Journal of Perinatology

be a highly effective pulmonary vasodilator, which is at least as
effective as iNO.39
The effectiveness of the aerosolized form of sildenafil was tested
in an animal model of PPHN. Sildenafil selectively decreased the
pulmonary artery pressure. When Sildenafil was inhaled while
simultaneously breathing NO, the pulmonary artery pressure
decreased even further.40
Human studies followed the animal studies for PPHN. Six
patients with PPHN were randomized to enteral sildenafil and
compared with seven patients who received placebo. Oral sildenafil
improved OI in infants with severe PPHN, which suggests that oral
sildenafil may be effective in the treatment of PPHN.41
Soluble guanylate cyclase activators
BAY 41-2272 is a novel direct activator of soluble guanylate cyclase.
This compound was tested in an animal model for PPHN. The
effectiveness of BAY 41-2272-induced pulmonary vasodilation
increased during the development of pulmonary hypertension in a
response that was greater than sildenafil. It also augmented the
pulmonary vasodilation induced by iNO.42
PGI2 analogs
Iloprost is a stable analog of prostacyclin. Its mechanism of action
is through a different signaling pathway, involving cAMP. The use
of the inhaled formulation has been approved by the FDA for the
use in adults. When given as the inhaled formulation, iloprost is a
selective pulmonary vasodilator. A first study of the use of inhaled
iloprost for children with pulmonary artery hypertension has been
recently published, reporting sustained functional improvement in
some children, although occasionally induced
bronchoconstriction.43 There are several anecdotal reports for the
use of inhaled iloprost for refractory cases of PPHN,44 most
reporting improved oxygenation after the initiation of therapy. No
adverse events were reported.
Endothelin-1 receptor antagonists (bosentan, sitaxsentan)
Endothelin-1 is a potent vasoconstrictor, and has been shown to
participate in the regulation of the pulmonary vascular tone.45 A
Cochrane review of this class of drugs in conjunction with
conventional therapy has shown that it can improve the clinical
outcomes in adults with idiopathic pulmonary artery
hypertension.46 Almost no experience with ERAs exists in the
neonatal population.
Rho-kinase inhibitors (fasudil, Y-27632)
Rho-kinase inactivates myosin light-chain phosphatase and
promotes vasoconstriction. The inhalation of rho-kinase inhibitor
in an animal model achieved pulmonary vasodilation.47
Superoxide dismutase
Hyperventilation and the formation of reactive oxygen species can
inactivate iNO. In an animal model, intratracheal recombinant
iNO and PVR
A Bin-Nun and MD Schreiber
S91
human superoxide dismutase, with or without iNO, increased
oxygenation and reduced pulmonary vasoconstriction.48
Expanding the pharmacologic options and the combination of
several of these might improve the response to iNO and the clinical
outcome of this devastating disease.
Summary
Since its discovery two decades ago, NO found its way to the arsenal
of therapeutic approaches available to the neonatologist in the
treatment of PPHN, a disease with high morbidity and mortality.
Vast research, both in animal models and human, increased our
understanding of the features of this gas and its benefits and
downsides. iNO was found to be the first potent ‘fairly’ selective
pulmonary vasodilator. NO has multiple mechanisms of actions
that may benefit newborn infants with diseases associated with
meconium aspiration and persistent pulmonary hypertension.
Understanding the mechanism of action of iNO through the cGMP
pathway led to the introduction of other medications in this
pathway: in addition to increasing cGMP concentration through
NO-induced stimulation, inhibiting cGMP metabolism through
phosphodiesterase inhibition, by sildenafil, for example, may
provide additional benefit. The combination of drugs affecting
through this pathway and other pathways might have a synergistic
effect, increasing the rate of success in the treatment of PPHN.
Disclosure
MD Schreiber has received lecture fees, consulting fees, and grant support from
Ikaria. A Bin-Nun has declared no financial interests.
Duality of interest
Dr Schreiber has received grant support and honoraria from iNO Therapeutics/
IKARIA.
References
1 Furchgott RF, Zawadzki JV. The obligatory role of endothelial cells in the relaxation of
arterial smooth muscle by acetylcholine. Nature 1980; 288(5789): 373–376.
2 Ignarro LJ, Buga GM, Wood KS, Byrns RE, Chaudhuri G. Endothelium-derived relaxing
factor produced and released from artery and vein is nitric oxide. Proc Natl Acad Sci
USA 1987; 84(24): 9265–9269.
3 Palmer RM, Ferrige AG, Moncada S. Nitric oxide release accounts for the biological
activity of endothelium-derived relaxing factor. Nature 1987; 327(6122): 524–526.
4 Murad F. The 1996 Albert Lasker Medical Research Awards. Signal transduction using
nitric oxide and cyclic guanosine monophosphate. JAMA 1996; 276(14): 1189–1192.
5 Griffiths MJ, Evans TW. Inhaled nitric oxide therapy in adults. N Engl J Med 2005;
353(25): 2683–2695.
6 Schechter AN, Gladwin MT. Hemoglobin and the paracrine and endocrine functions of
nitric oxide. N Engl J Med 2003; 348(15): 1483–1485.
7 Frostell C, Fratacci MD, Wain JC, Jones R, Zapol WM. Inhaled nitric oxide. A selective
pulmonary vasodilator reversing hypoxic pulmonary vasoconstriction. Circulation
1991; 83(6): 2038–2047.
8 Pepke-Zaba J, Higenbottam TW, Dinh-Xuan AT, Stone D, Wallwork J. Inhaled nitric
oxide as a cause of selective pulmonary vasodilatation in pulmonary hypertension.
Lancet 1991; 338(8776): 1173–1174.
Journal of Perinatology
 iNO and PVR
A Bin-Nun and MD Schreiber
S92
9 Roberts JD, Polaner DM, Lang P, Zapol WM. Inhaled nitric oxide in persistent
pulmonary hypertension of the newborn. Lancet 1992; 340(8823): 818–819.
10 Kinsella JP, Neish SR, Shaffer E, Abman SH. Low-dose inhalation nitric oxide in
persistent pulmonary hypertension of the newborn. Lancet 1992; 340(8823): 819–820.
11 The Neonatal Inhaled Nitric Oxide Study Group. Inhaled nitric oxide in full-term and
nearly full-term infants with hypoxic respiratory failure.. N Engl J Med 1997; 336(9):
597–604.
12 Roberts Jr JD, Fineman JR, Morin III FC, Shaul PW, Rimar S, Schreiber MD et al.
Inhaled nitric oxide and persistent pulmonary hypertension of the newborn. The
Inhaled Nitric Oxide Study Group. N Engl J Med 1997; 336(9): 605–610.
13 Clark RH, Kueser TJ, Walker MW, Southgate WM, Huckaby JL, Perez JA et al. Low-dose
nitric oxide therapy for persistent pulmonary hypertension of the newborn. Clinical
Inhaled Nitric Oxide Research Group. N Engl J Med 2000; 342(7): 469–474.
14 Martin RJ, Walsh MC. Inhaled nitric oxide for preterm infantsFwho benefits? N Engl
J Med 2005; 353(1): 82–84.
15 Roberts Jr JD, Chiche JD, Weimann J, Steudel W, Zapol WM, Bloch KD. Nitric oxide
inhalation decreases pulmonary artery remodeling in the injured lungs of rat pups.
Circ Res 2000; 87(2): 140–145.
16 Barbotin-Larrieu F, Mazmanian M, Baudet B, Detruit H, Chapelier A, Libert JM et al.
Prevention of ischemia–reperfusion lung injury by inhaled nitric oxide in neonatal
piglets. J Appl Physiol 1996; 80(3): 782–788.
17 Shaul PW, Yuhanna IS, German Z, Chen Z, Steinhorn RH, Morin III FC. Pulmonary
endothelial NO synthase gene expression is decreased in fetal lambs with pulmonary
hypertension. Am J Physiol 1997; 272(5 Part 1): L1005–L1012.
18 Finer NN, Barrington KJ. Nitric oxide for respiratory failure in infants born at or near
term. Cochrane Database Syst Rev 2006; (4): CD000399.
19 Clark PL, Ekekezie II, Kaftan HA, Castor CA, Truog WE. Safety and efficacy of
nitric oxide in chronic lung disease. Arch Dis Child Fetal Neonatal Ed 2002; 86(1):
F41–F45.
20 Banks BA, Seri I, Ischiropoulos H, Merrill J, Rychik J, Ballard RA. Changes in
oxygenation with inhaled nitric oxide in severe bronchopulmonary dysplasia.
Pediatrics 1999; 103(3): 610–618.
21 Konduri GG, Vohr B, Robertson C, Sokol GM, Solimano A, Singer J et al. Early inhaled
nitric oxide therapy for term and near-term newborn infants with hypoxic respiratory
failure: neurodevelopmental follow-up. J Pediatr 2007; 150(3): 235–240, 240 e1.
22 Davidson D, Barefield ES, Kattwinkel J, Dudell G, Damask M, Straube R et al. Inhaled
nitric oxide for the early treatment of persistent pulmonary hypertension of the term
newborn: a randomized, double-masked, placebo-controlled, dose–response, multi-
center study. The I-NO/PPHN Study Group. Pediatrics 1998; 101(3 Part 1): 325–334.
23 Guthrie SO, Walsh WF, Auten K, Clark RH. Initial dosing of inhaled nitric oxide in
infants with hypoxic respiratory failure. J Perinatol 2004; 24(5): 290–294.
24 Loh E, Stamler JS, Hare JM, Loscalzo J, Colucci WS. Cardiovascular effects of inhaled
nitric oxide in patients with left ventricular dysfunction. Circulation 1994; 90(6):
2780–2785.
25 Semigran MJ, Cockrill BA, Kacmarek R, Thompson BT, Zapol WM, Dec GW et al.
Hemodynamic effects of inhaled nitric oxide in heart failure. J Am Coll Cardiol 1994;
24(4): 982–988.
26 Davidson D, Barefield ES, Kattwinkel J, Dudell G, Damask M, Straube R et al. Safety of
withdrawing inhaled nitric oxide therapy in persistent pulmonary hypertension of the
newborn. Pediatrics 1999; 104(2 Part 1): 231–236.
27 Oishi P, Grobe A, Benavidez E, Ovadia B, Harmon C, Ross GA et al. Inhaled nitric oxide
induced NOS inhibition and rebound pulmonary hypertension: a role for superoxide
and peroxynitrite in the intact lamb. Am J Physiol Lung Cell Mol Physiol 2006;
290(2): L359–L366.
28 Kirmse M, Hess D, Fujino Y, Kacmarek RM, Hurford WE. Delivery of inhaled
nitric oxide using the Ohmeda INOvent Delivery System. Chest 1998; 113(6):
1650–1657.
Journal of Perinatology
29 Hogman M, Frostell C, Arnberg H, Hedenstierna G. Bleeding time prolongation and NO
inhalation. Lancet 1993; 341(8861): 1664–1665.
30 Samama CM, Diaby M, Fellahi JL, Mdhafar A, Eyraud D, Arock M et al. Inhibition of
platelet aggregation by inhaled nitric oxide in patients with acute respiratory distress
syndrome. Anesthesiology 1995; 83(1): 56–65.
31 Ballard PL, Merrill JD, Truog WE, Godinez RI, Godinez MH, McDevitt TM et al.
Surfactant function and composition in premature infants treated with inhaled nitric
oxide. Pediatrics 2007; 120(2): 346–353.
32 Truog WE, Ballard PL, Norberg M, Golombek S, Savani RC, Merrill JD et al.
Inflammatory markers and mediators in tracheal fluid of premature infants treated
with inhaled nitric oxide. Pediatrics 2007; 119(4): 670–678.
33 Mestan KK, Marks JD, Hecox K, Huo D, Schreiber MD. Neurodevelopmental
outcomes of premature infants treated with inhaled nitric oxide. N Engl J Med 2005;
353(1): 23–32.
34 The Neonatal Inhaled Nitric Oxide Study Group (NINOS). Inhaled nitric oxide and
hypoxic respiratory failure in infants with congenital diaphragmatic hernia.. Pediatrics
1997; 99(6): 838–845.
35 Kinsella JP, Parker TA, Ivy DD, Abman SH. Noninvasive delivery of inhaled nitric oxide
therapy for late pulmonary hypertension in newborn infants with congenital
diaphragmatic hernia. J Pediatr 2003; 142(4): 397–401.
36 Ladha F, Bonnet S, Eaton F, Hashimoto K, Korbutt G, Thebaud B. Sildenafil improves
alveolar growth and pulmonary hypertension in hyperoxia-induced lung injury. Am J
Respir Crit Care Med 2005; 172(6): 750–756.
37 Atz AM, Wessel DL. Sildenafil ameliorates effects of inhaled nitric oxide withdrawal.
Anesthesiology 1999; 91(1): 307–310.
38 Galie N, Ghofrani HA, Torbicki A, Barst RJ, Rubin LJ, Badesch D et al. Sildenafil
citrate therapy for pulmonary arterial hypertension. N Engl J Med 2005; 353(20):
2148–2157.
39 Shekerdemian LS, Ravn HB, Penny DJ. Intravenous sildenafil lowers pulmonary
vascular resistance in a model of neonatal pulmonary hypertension. Am J Respir Crit
Care Med 2002; 165(8): 1098–1102.
40 Ichinose F, Erana-Garcia J, Hromi J, Raveh Y, Jones R, Krim L et al. Nebulized
sildenafil is a selective pulmonary vasodilator in lambs with acute pulmonary
hypertension. Crit Care Med 2001; 29(5): 1000–1005.
41 Baquero H, Soliz A, Neira F, Venegas ME, Sola A. Oral sildenafil in infants with
persistent pulmonary hypertension of the newborn: a pilot randomized blinded study.
Pediatrics 2006; 117(4): 1077–1083.
42 Deruelle P, Grover TR, Abman SH. Pulmonary vascular effects of nitric oxide–cGMP
augmentation in a model of chronic pulmonary hypertension in fetal and neonatal
sheep. Am J Physiol Lung Cell Mol Physiol 2005; 289(5): L798–L806.
43 Ivy DD, Doran AK, Smith KJ, Mallory Jr GB, Beghetti M, Barst RJ et al. Short- and long-
term effects of inhaled iloprost therapy in children with pulmonary arterial
hypertension. J Am Coll Cardiol 2008; 51(2): 161–169.
44 Kelly LK, Porta NF, Goodman DM, Carroll CL, Steinhorn RH. Inhaled prostacyclin for
term infants with persistent pulmonary hypertension refractory to inhaled nitric oxide.
J Pediatr 2002; 141(6): 830–832.
45 Ivy DD, Parker TA, Ziegler JW, Galan HL, Kinsella JP, Tuder RM et al. Prolonged
endothelin A receptor blockade attenuates chronic pulmonary hypertension in the ovine
fetus. J Clin Invest 1997; 99(6): 1179–1186.
46 Liu C, Chen J. Endothelin receptor antagonists for pulmonary arterial hypertension.
Cochrane Database Syst Rev 2006; 3: CD004434.
47 Nagaoka T, Fagan KA, Gebb SA, Morris KG, Suzuki T, Shimokawa H et al. Inhaled Rho
kinase inhibitors are potent and selective vasodilators in rat pulmonary hypertension.
Am J Respir Crit Care Med 2005; 171(5): 494–499.
48 Lakshminrusimha S, Russell JA, Wedgwood S, Gugino SF, Kazzaz JA, Davis JM et al.
Superoxide dismutase improves oxygenation and reduces oxidation in neonatal
pulmonary hypertension. Am J Respir Crit Care Med 2006; 174(12): 1370–1377.
 Journal of Perinatology (2008) 28, S93–S101
r 2008 Nature Publishing Group All rights reserved. 0743-8346/08 $30
www.nature.com/jp

REVIEW
Neurodevelopmental outcome of infants with meconium
aspiration syndrome: report of a study and literature review
N Beligere and R Rao
Department of Pediatrics, University of Illinois at Chicago Medical Center, Chicago, IL, USA

when MDI and PDI scores were >85 to 110; mildly delayed when scores
There is a paucity of information on long-term outcome of infants who
were >70 to 84; and severely delayed if the scores were <69. In addition,
have suffered from meconium aspiration syndrome (MAS) in the neonatal
neurological evaluation also confirmed the disability. The report is based on
period. We analyzed long-term developmental outcome data of 35 infants
the final analysis of 29 infants. Data of six infants were not included in the
who were admitted to the neonatal intensive care unit (NICU) at the
final analysis because of incomplete information. The mean BW of the
University of Illinois Hospital at Chicago (UICMC) with a diagnosis of MAS,
infants was 3269±671 g; mean gestational age was 39.5±3.1 weeks. The
and we reviewed the literature pertinent to the subject. The objective of the
median APGAR score at 10 was 4, and at 50 was 6. Out of 29, 11 (38%)
study was to assess the neurodevelopment status of MAS infants and
infants were normal. Out of 29, 2 infants (7%) had cerebral palsy (CP) and
compare the possible effects of different variables that are known to affect
4 (14%) had severe delay at 12 months of age. Out of 29, 2 who were
the later developmental outcome. The variables included mode of delivery,
neurologically disabled had PDI <69. Out of 29, 12 (41%) had mild delay
APGAR score, cord pH, mode of treatment, and neurological findings during
in speech. No statistical difference in neurodevelopment was found in
the course of NICU. The infants were enrolled in the developmental follow-
infants born vaginally or by C-section. Our findings show poor outcome (CP
up program (DFUP) after discharge from the nursery for assessment of
and global delay) in 21% of infants who suffered MAS, even though the
long-term developmental status and neurodevelopmental outcome. In order
majority of the infants (26/29) responded to conventional ventilator support
to assess the impact of the treatment on long-term outcome and compare
alone. No difference was found in the outcome of infants between NSVD vs
our findings with previously published reports, we also reviewed the
C-section delivery. These findings suggest that infants with the diagnosis of
previously published literature on neurodevelopment outcome of infants
MAS manifest later neurodevelopmental delays, even if they respond well to
treated for MAS (with different modalities) during the last three decades.
conventional treatment. This abstract was presented at the Society for
Total of 35 infants with a diagnosis of MAS admitted to the NICU at UICMC
Pediatric Research Annual Meeting, 2000.
were followed in the DFUP clinic for 3 years during January 1999 to
September 2001. The medical records of these infants were reviewed for the
Journal of Perinatology (2008) 28, S93–S101; doi:10.1038/jp.2008.154
mode of delivery, APGAR score, birth weight (BW), gestational age, mode of
treatment during the neonatal period, and neurodevelopment status. 19/35
(54%) infants were delivered vaginally, 16/35 (46%) by cesarean section
(C-section). All were treated in the delivery room using the standard Introduction
resuscitation protocol. Following initial resuscitation, all except three Meconium aspiration syndrome (MAS) is a clinical entity that
required intubation and ventilation for varying duration. One infant contributes to the high incidence of mortality and morbidity among
required inhaled nitric oxide therapy, and two required extracorporeal term pregnancies.1 The prevalence and mortality in MAS is noted to
membrane oxygenation treatment. Subsequent to discharge, the infants be steadily decreasing with newer and more efficient modalities of
were evaluated in the clinic at 2 months of age, and then every 4 months treatment.1,2 With steady improvement in survival, the amount of
up to 3 years. The developmental assessment of mental development index morbidity among the survivors has not changed. There is a great
(MDI), psychomotor development index (PDI), and behavior rating scale need for follow-up data on the neurodevelopmental outcome of this
(BRS) were obtained using the Bayley II infant motor scale, and group of infants. The purpose of this study was to report our findings
neurodevelopment evaluation was performed using the Amiel–Tison of neurodevelopmental outcome of infants with the diagnosis of MAS
technique. Speech evaluation was performed in infants >18 months using in the neonatal period and review the literature on this supplement.
the Rossetti Infant–Toddler language scale. Infants were considered normal

Correspondence: Dr N Beligere, Division of Neonatology, Department of Pediatrics, University Methods

of Illinois at Chicago Medical Center, 840 South Wood Street, M/C 808, Chicago, IL 60612,
USA. We reviewed the medical records of 35 infants with the primary
E-mail: beligere@uic.edu diagnosis of MAS followed in the developmental follow-up program
 Neurodevelopmental outcome of infants with MAS
N Beligere and R Rao
S94
(DFUP) clinic during January 1999 to September 2001 at the
University of Illinois Hospital at Chicago (UICMC). Information
was collected using the specific diagnostic code for MAS ICD-907
criteria recorded in the obstetric and neonatal intensive care units
(NICUs). The criteria for the diagnosis of MAS (ICD-907) include:
history of meconium-stained amniotic fluid before delivery, the
presence of meconium below the vocal cords at the time of birth in
infants >37 weeks of gestation, evidence of respiratory distress, and
radiological evidence of aspiration pneumonitis. Many of these
infants also developed persistent pulmonary hypertension (PPHN).
The medical records of the infants who met the above criteria were
selected for the retrospective analysis of perinatal and neonatal
events. The study was approved by the internal review board at the
University of Illinois at Chicago.
Obstetrical and NICU registry and medical records were reviewed
for maternal complications of pregnancy, such as diabetes,
hypertension, toxemia, and chorioamnionitis. The presence of
meconium-stained amniotic fluid, consistency of meconium, and
presence of amnioinfusion were also ascertained. The presence of
fetal distress and the mode of delivery were also noted. The infant’s
condition in the delivery room and any interventions were
reviewed. The following data were retrieved from the infants chart:
APGAR score at 1 and 5 min, presence of meconium below the
vocal cords, method of resuscitation, presence of respiratory distress
at birth, the requirement of the use of supplemental oxygen, and
ventilator support. The data of cord blood gases and blood gas
analysis at the time of admission to the NICU were also noted. The
gestational age was determined using fetal ultrasound assessment
or Ballard score. Birth weight (BW), length, and head
circumference on admission to the NICU were recorded.
Developmental assessment
During the period of 1999 to 2001, 75 infants were diagnosed as
having meconium-stained amniotic fluid at the time of delivery. Of
these, only 40 infants who developed respiratory distress and met
the criteria for MAS were admitted to the NICU. Parents of all
infants who were admitted to NICU with a diagnosis of MAS were
counseled by the clinic nurse and enrolled for developmental
follow-up subsequent to discharge. Only 35 infants attended the
follow-up clinic for varying periods of time during the 3-year study
period. Among this group, medical records of six infants’ did not
have complete information; hence, they were not included in the
final analysis of this study. The final report includes information
on 29 infants.
Developmental evaluation was conducted at 2, 4, 8, 12, 18, 24,
30, and 36 months. Each infant had at least five visits during this
period. These evaluations consisted of neurodevelopment
assessment by an experienced developmental pediatrician (NB)
using the Amiel–Tison neurodevelopment scale and neurological
examination.3 During these visits, complete interim history,
immunization status, and physical exams including
Journal of Perinatology
anthropometric measurements were recorded. Infants were
independently evaluated using the Bayley II infant motor
developmental scale4 by a certified pediatric occupational
therapist, physical therapist, and the developmental specialist.
Infants were scored based on their performance on the Bayley II
infant motor scale. Scores of mental development index
(MDI), psychomotor development index (PDI), and behavior
rating scale (BRS) were also used as criteria for eligibility to enroll
in the early intervention program (EIP). Acquisition of
language was evaluated by the certified pediatric speech pathologist
after the age of 18 months using the Rossetti infant–Toddler
language scale.5
The degree of disability was further classified using one or more
of the following criteria. When the PDI <2 s.d. below the mean of
p69, the baby’s condition was classified as cerebral palsy (CP)
with neurological evidence of spasticity. When the MDI <2 s.d.
below the mean of p69 for evidence of mental disability, the baby
was considered as cognitive delay. Mild delay was considered when
the scores of PDI or MDI were X70–84, with clinical evidence of
hypotonia (gross motor delay or fine motor) and/or cognitive
delay. Speech delay was considered when expressive or receptive
function of communication was delayed according to the Rossetti
infant–Toddler language scale.5 Diagnoses of speech delay or
hearing impairment were assigned according to the disability. The
children with mild and severe delays were enrolled in the state-
supported EIP between 4 and 6 months of age. The EIP includes
speech therapy, physical or occupational therapy, and
developmental therapy as needed.
Analysis and results
Maternal data
Complete maternal data were available in all 29 infants who had
complete follow-up data. Total of 16 of these infants were delivered
by C-section for fetal distress. 13 infants were delivered by normal
spontaneous vaginal delivery. Pregnancy complications included
diabetes in 4 patients who were on insulin. Pregnancy induced
hypertension in 3, fetal decelaration in 4, chorioamnionitis
in 3, premature rupture of membranes in 3 maternal drug use
in 6, sickle-cell disease in 4, fever of unknown origin in 3, and
presence of prolapsed cord in 3. Umbilical cord pH was available
in 16/18 inborn infants and in 2/11 outborn infants. It was noted
8/18 infants had cord pH>7.2 and in two infants cord pH was
6.75 and 7.10.
Neonatal data
Table 1 shows the perinatal data of infants in the study. Total of 11
outborn infants were transferred to UICMC within 12 h after birth.
Method of resuscitation was not available. Mean admission age was
6 h after birth APGAR score ranged from 0 to 7 at 1 min with mean
of 4, and at 5 min APGAR score ranged from 4 to 9 with mean of 6.

Table 1 Demographics and birth weight gestational age
Inborn 18
Outborn 11
Gestation age (weeks) 39.5±3.1
Birth weight (g) 3269±671
APGAR score
10 Range 0–7 median of 4
50 Range 0–9 median of 6
We were unable to get the fetal heart rate pattern in many of the
infants therefore not included in our data. Thick meconium fluid
at the time of delivery in 14 infants and meconium was present
below the vocal cord in four infants. Mean gestational age was
39.5±3.1 weeks. All infants were resuscitated in the delivery room,
in accordance with the standard NRP protocol.6 All infants were
admitted to the NICU, all required assisted ventilation. Duration of
the ventilatory support varied between 2 and 12 days with a mean
of 6.4 days. Only one infant was treated with inhaled nitric oxide
(INO) and high frequency ventilaton who had severe metabolic
acidosis and pulmonary hypertension. Two infants required
extracorporeal membrane oxygenation (ECMO) for 12 to 24 days
who did not respond to mechanical ventilation.
The neurological findings in the neonatal period showed:
fourteen infants developed seizure activity during the first
week of life. All had electroencephalography (EEG) recordings.
Six of the EEGs demonstrated abnormal foci in the temporal
and parietal areas. Thirteen infants had magnetic resonance
imaging (MRI) scans during the neonatal course. Eleven infants
had normal MRI, and two infants demonstrated cerebral
hemorrhage.
Developmental outcome data
Table 2 shows developmental outcome at 36 months. In 38%
(11/29) of infants, the developmental status at 3 years of age was
normal. 41% (12/29) had mild deficits, which consisted of mild
hypotonia or mild speech delay. 7% (2/29) had CP. 14% (4/29)
had severe global delay. None required oral or gastric tube feeding.
All had normal nutritional status. 19/29 infants (65%) were
enrolled in the EIP. Other 10 required anticipatory development
counseling.
The growth and development of these infants at 3 years
were appropriate for age, with the exception of one infant who
had suffered from birth asphyxia resulting in CP. Two infants
required phenobarbital for control of seizure even at 2 years
of age.
Of the 13 infants delivered by C-section, only 5 were normal,
4 had mild delay, and 4 had severe delay. 16 infants were
delivered by NSVD: 6/16 (37.5%) were normal, 8/16 (50%)
had mild delay, and 2/16 (12.5%) had severe delay.
Neurodevelopmental outcome of infants with MAS
N Beligere and R Rao
S95
Table 2 Developmental outcome
Normal 11 38%
Abnormal findings
Mild defectsa 12 41%
Cerebral palsy 2 7%
Severe global delay 4 14%
Entry in early intervention program (EIP) 19 65%
a
Includes mild speech delay or mild hypotonicity without any motor or cognitive abnormality.
Neurodevelopmental outcome did not differ whether they were born
vaginally or by C-section.
Discussion
MAS is a distinct clinical syndrome that occurs in term infants.
Severe meconium aspiration occurs in one in 500 deliveries: that
is, in only 0.2% of all births.1,7 Intrauterine fetal distress and fetal
asphyxia in utero before birth have been recognized as stimulating
and enhancing the intestinal peristalsis of the infant, resulting in
relaxation of the anal sphincter and passage of meconium in
utero.8,9 Previous reports8,10 have described the pathophysiology of
MAS. The hypoxic distress of the fetus and gasping in utero have
been speculated as the cause of the meconium aspiration during
labor and at birth. Aspiration of thick meconium may occur during
the respiratory effort of the first breath. This leads to obstruction of
airways, resulting in profound hypoxia. In addition, meconium
may lead to chemical pneumonitis, parenchymal lung damage,
and inactivation of surfactant. Other complications such as
pulmonary hypertension or pulmonary atelectasis with or without
pneumothorax have been reported. Severe hypoxia may cause
brain injury and hypoxic ischemic encephalopathy.
Since the recognition of these issues, both obstetricians and
neonatologists have intensified their efforts to decrease the
incidence of MAS using new interventions. The obstetric preventive
interventions include amnioinfusion in the presence of thick or
thin meconium-stained amniotic fluid11 before birth of the infant,
and suctioning of the oropharynx at the perineum before the
delivery of the shoulders.12,13 The neonatal interventions include
tracheal suctioning at birth in the presence of meconium-stained
amniotic fluid12 to prevent meconium aspiration.
Although the survival rate of MAS has greatly improved over the
last three decades,2 the long-term morbidities among survivors
have been a major concern. We undertook a study to analyze the
long-term outcome of infants treated for MAS in our NICU and
compare the same with published reports. However, comparisons of
our findings with the published studies are constrained for several
reasons. First, the treatment modalities differed. Second, the
primary objective of most reports was to study ‘the treatment effect’
rather than the ‘effect of MAS’ on long-term outcome. The third
Journal of Perinatology
 Neurodevelopmental outcome of infants with MAS
N Beligere and R Rao
S96
issue was that the population included in follow-up studies was the
sum total of varied diagnoses (MAS, CDH, PPHN, sepsis, and
pneumonia), making it difficult to assess the independent effect of
MAS alone on the outcome. Finally, the methods of developmental
assessment were not uniform and comparable. We attempt here to
summarize the uniformity of evaluation and follow-up methods
and findings of the published reports of long-term studies.
We try to provide some perspective on our follow-up study, its
importance, and its relation to the current literature. Review of the
literature during the past 30 years has shown that there were
three major strategies of treatment and outcome reports.
They included the use of mechanical ventilation in the 1970s,
additional ECMO treatment in the 1980s, and INO treatment
in the 1990s. The outcome results of the infants treated with
these different modalities are summarized in Table 3 and
discussed below.
Studies in which the primary treatment of MAS was
conventional ventilation alone
In the 1970s, the treatment of MAS infants who developed acute
respiratory failure consisted of providing early ventilatory support.10
The survival was low. The clinical course10 during the immediate
neonatal period and complications of MAS included hypoxic injury
of brain, pulmonary atelectasis, air leak syndrome, and other
systemic insults.1 During 1978 to 2000, there were 10 other
studies14–16 published in this category, and they reported long-
term outcome of infants with the primary diagnosis of MAS. In two
of these studies,14,15 MAS infants were treated with conventional
ventilation alone. Marshall et al.14 in a retrospective study in 1978
identified 13 of the 17 infants (78%) with MAS who survived
following conventional ventilator therapy. Follow-up of 11 of the
survivors showed that 2/11 had developmental delay (18%). Both
had seizures. Brett et al.15 in 1981 followed 9 of their 11 survivors
of hyperventilation using conventional mechanical ventilation
therapy. Seven of the survivors had MAS. The range of MDI was 89
to 130. They concluded that hyperventilation was not associated
with adverse outcome. Here, the emphasis was on the efficacy of
treatment rather than the MAS. Walsh and Suky16 compared the
infants treated with conventional ventilation with the ECMO
survivors. 90% of the survivors of the ECMO-treated group had
primary diagnosis of MAS. The reported morbidity in the
ECMO-treated group was twice that of conventional ventilation
treated group (55 vs 28%). The reported high morbidity among the
long-time survivors, Macrocephaly developed in 24% of the ECMO
group. They also found 4/17(28%) of the conventionally treated
group had severe neurodevelopmental disabilities, compared with
the 20/38 (26%) of the ECMO-treated group.
Outcome of infants following ECMO treatment in the 1980s
Bartlett et al.17 used ECMO in the management of infants with
MAS who failed to respond to conventional therapy. The rest of the
Journal of Perinatology
eight studies18–25 reported following outcome with ECMO
treatment. Four of them18–20,25 showed adverse outcome. Three
studies21–23 showed that, although MAS infants had higher
disabilities compared with the normal infants, they were better
than infants with diaphragmatic hernia (CDH) and sepsis. Ikle
et al.24 showed in their study no significant difference in the
intelligence quotient (IQ) of children at school age, in the ECMO-
treated group, except the children with diagnosis of MAS had lower
IQ. These studies indicate that infants with the primary diagnosis
of MAS have poor neurodevelopmental outcome. Within the ECMO-
treated group, the MAS group had a lower incidence of disability
than the CDH and sepsis group, suggesting that the presence of
sepsis will increase the neurological insult in comparison to MAS.
The ECMO trials reported long-term outcome results.16,26–29
These studies included both MAS and PPHN infants. However,
separation of the MAS group was not possible. Overall, the studies
showed improved survival following ECMO without concomitant
increase in morbidities. There were a few differences among the
studies. Vaucher et al.26 on the other hand reported higher
morbidity in the conventional group compared with the ECMO
group (25 vs 6% of CP). They also reported that abnormalities
found in neuroimaging predicted later clinical neurodevelopmental
abnormalities. The UK Collaborative27 study showed no
concomitant increase in morbidities in the ECMO group. Rais-
Bahrami et al.28 similarly showed that the neurodevelopmental
outcome in the ‘near-miss ECMO’ group (who were sick enough to
be on ECMO but were not) had higher handicaps (25 vs 16%) at
follow-up than those treated with ECMO.
Glass et al.29 is the only report that compared the long-term
outcome of MAS infants treated with ECMO with healthy controls.
These data showed that MAS infants had significantly higher
neurodevelopmental delays compared with controls. Most infants
undergoing ECMO were in the MAS group. It follows that MAS is
associated with higher adverse neurodevelopmental sequelae than
the normal population and that these disabilities did not decrease
with different treatment modalities, as demonstrated in most of the
studies discussed above.
Blackwell et al.30 showed that there were no differences in the
occurrence of seizures between infants with MAS and cord pH<7.20
and those with cord pH>7.20, suggesting that there was a
pre-existing injury or that a nonhypoxic mechanism is
involved in infants with severe MAS. Casey et al.31 showed that pH
change from birth to immediate neonatal period predicted
neonatal morbidity. Infants whose both cord pH and subsequent
2 h arterial pH were <7.20 had a higher incidence of seizures.
Total of 55% of infants in this group had associated meconium
aspiration, compared with only 36% in infants who had cord
pH>7.20. In our study, only 2/29 (6.9%) had severe
neurodevelopment impairment; neither required ECMO treatment.
These infants experienced severe intrauterine hypoxia, as noted
by their cord pH.
 Neurodevelopmental outcome of infants with MAS
N Beligere and R Rao
S97

Table 3 Outcome of infants with MAS treated with conventional ventilation

Reference Author(s) Number of cases Outcome Comments

no.

14 Marshall R et al., Study period, 1974; records Outcome at 1 year: four (23.5%) died of Significant psychomotor retardation and
1978 of 1 year respiratory failure cognitive delay among the survivors
Follow-up MAS: 11 infants Two had PFC by cardiac catheterization,
two had significant psychomotor
retardation at 8 and 13 months. CP 11.9%,
both had seizures and one had
microcephaly <5%

15 Brett C et al., Study period March 1977–1979,
1981 number of cases followed 9, MAS: 7,
RDS: 1, sepsis: 1, age at follow-up
16 months
Hyper ventilated 64.4±18.6
Outcome at 16 months: seven infants were Although short-term follow-up reported,
normal neurodevelopment, one had severe preliminary observation assures normal
delay, six had normal neurological exam, development outcome with prolonged
one infant had growth retardation hyperventilation

Outcome of ECMO trials

16 Walsh-Sukys MC Study period 1987–1998 Outcome at 20 months Disability among CV was 4 (24%) and in
et al., 1994 Cases followed: 61 ECMO: 2 CP, 2 sesures, disability 20 ECMO 20 (26%)
For 20 months of both CV group, and and 1 deaf. CV: 2 disability, No difference between the two groups
ECMO group 1 CP for blind followed. 24% ECMO group had
macrocephaly at 20 months
18 Towne BH et al., Study period 1973–1980 Outcome at 12 months: 9/11 MAS infants ECMOFlife saving without increasing
1985 Cases followed: 18 were normal; 1 had developmental delay, morbidity
MAS: 14/18 seizures; and 1 had hemiperisis
11 of MAS cases follow-up

19 Andrews AF et al., Study period March Outcome mental function: normal EEG in Majority of survivors of neonates with
1986 1981–September 1983 71%, abnormal in 14, one long-time high risk of respiratory failure can be
Cases followed: 14 with PPHN treatment expected to have near normal growth and
MAS: 5, PPHN: 13, CDH: 1, RDS: 3, Normal outcome in seven (50%), five with development
others: 4 delay and failure to thrive, three with CP,
three had microcephally, and dilated
ventricles
20 Hofkosh D et al., Study period 1990–1993 in 67 cases At 6–30 months outcome by MDI/PDI: Out of entire cohort, normal in 43 (64%),
1991 from 6 months to 10 years. 6–30 normal: 27, abnormal: 4, suspect: 16. abnormal 7 (11%), suspect: 17 (25%).
months: 47 cases; 2.5–4.11 years: 10 2.5–4.11 years, IQ test normal: 7, ECMO did not increase the morbidity;
cases; 5–10 yearsFschool age: abnormal: 2, CP: 2. School age 5–10 years. comparison of control conventional
10 cases IQ normal: 9, abnormal CP: 1 (10%), ventilation group is absent
Diagnosis MAS: 33, CDH: 13, RDS: 8, sensory neural hearing loss: 21%, seizures:
PPHN: 10, others: 3 4, with abnormal outcome
21 Wilden SR et al., Study period January 1990–1992 At 24 months ECMO: GP MDI <85, PDI Overall ECMO group 23% significant
1994 Cases followed: 22 <73, abnormal expressive and receptive impairment
Diagnosis language in 48%. Hearing loss in four
MAS: 10, RDS: 7, PPHN: 3, (15%), visual impairment in one, and
sepsis: 6, CDH: 8 abnormal MRI in nine infants
22 Bernbaum J et al., Study period 1990–1993 Follow-up 6–12 months BPD and feeding difficulties, hypotonicity
1995 Cases followed: 60 Neurologic morbidity in four MAS, and in were greater in CDH, compared to MAS
MAS: 21, CDH: 19, sepsis: 7 five CDH. Hearing loss in two MAS,
hypotonicity in 8% of MAS, and low
cognitive scores in CDH. Total of 68% of
CDH had the worst outcome, with BPD and
feeding difficulty at 12 months

Journal of Perinatology
 Neurodevelopmental outcome of infants with MAS
N Beligere and R Rao
S98

Table 3 Continued
Reference Author(s) Number of cases Outcome Comments
no.

23 Robertson C et al., Study period 1989–1992 In ECMO: GP disability: 6, hearing loss: 3, The infants with sepsis and respiratory
1995 Outcome at 2 years of age severe cognitive disability: 2, seizures: 1, distress had worse outcome compared to
Cases followed: 40 visual loss: 3, expressive language delay: 7, MAS alone
ECMO: 30, CDH: 8, MAS: 19, PPHN: 3, moderate cognitive delay: 2. MDI were lower
sepsis: 4 in sepsis group, with mean MDI 91.8 (9.5)

24 Ikle L et al., Study period 1988–1991
1999 Cases followed: 17 for 5–8 years;
studied IQ at school age
Diagnosis PPHN with ECMO, MAS: 9,
sepsis: 3, HMD: 2, others: 3
Outcome of 17 MAS: full-scale IQ varied High percentage of ECMO survivors had
from 90 to 115, performance IQ, 81–121, discrepancies between the PIQ and VIQ.
verbal IQ, 89–118; 15 children in MAS with PIQ had higher correlation to the number
low functioning had low average IQ of hours the infants were on ECMO

25 Nield TA et al., Study period 1987–January 1993 Outcome MAS: major neurologic sequelae in No significant difference was found in
2000 Cases followed: 130 for 3.5 years 20%, including MR and CP, functioning functional or neurologic status between
MAS: 63, CDH: 39, PPHN: 4, sepsis: 29, disability in 17%, 24% at risk for disability, the different diagnostic groups
RDS: 8, cardiac: 5 normal function in 59%

26 Vaucher Y et al., Study period 1987–1993
1996 Comparative study, conventional HFV
ventilation to ECMO, CV: 53, ECMO:
138, MAS: 97, PPHN: 19, RDS: 46,
sepsis: 28, studied CLD, and MRI
Neuromotor outcome at age CLD had lower ECMO survivors had less CLD than non-
MDI, PDI scores <84, 27% had CP, ECMO survivors
neuroimaging abnormalities. Severe in 11%,
moderate in 9%, mild in 24%

27 Benett CE et al., Neurodevelopmental outcome at 1. Death (1) Mortality 30/93 (32%) in ECMO and
2001 1 year of age in ECMO 2. Severe disability DQ <50 using Griffiths 54/92 (59%) in non-ECMO
Infants compared with those treated mental developmental scale (2) 10/62 (16%) in ECMO and 5/37
with conventional therapy (99 (13.5%) in non-ECMO had tone changes
survivors) (3) ECMO improves survival with no
increase in morbidity

28 Rais-Bahrami K Study period for 11 years Outcome tested for IQ at 5 years of age. Major handicap was cognitive deficits at 5
et al., 2000 Cases followed: ECMO: 76, near miss FSIQ <0.70 as cognitive disability found in years, and risk for academic school
ECMO: 20 12% of ECMO group, 11% in near miss failures
ECMO GP; MAS: 47, PPHN: 16, ECMO, half of them had MR CP. Sensory
RDS: 13 neural hearing loss academic achievement
Near miss ECMO group; MAS: 11, test, using V IQ, PIQ, FSIQ, academic school
PPHN: 6, RDS: 3 failure, ECMO (38%), near miss ECMO (37%)
29 Glass P et al., Study period, 1984–1988: 103 post-ECMO Parent interview at 5 years, major disability ECMO patient had lower threshold for
1995 compared with 37 healthy controls in 17%, 13 had MR, 4 seizures, 5 severe seizure, even as adults. Learning disability
Diagnosis motor delay, 3 with SNHL and poor academic achievement, high
MAS: 48 FSIQ (96 vs 115), VIQ (96 vs 115), rate of behavioral problems, and high risk
PPHN: 12 PIQ (96 vs 110) of school failures
CDH: 6, number of cases
followed ¼ 42
Outcome of INO trials
35 Rosenberg A et al., Study period 1992–1995 At 1 year, 6 (11.8%) had major handicap, Neurodevelopmental outcomes are similar
1997 Cases followed: 51 with PPHN, 61% MDI, PDI <69, mild disability in 7 (13.7%). to initial INO-treated group
followed up to 1 year, 33% followed to At 2 years, 4 (12.1%) had
2 years, MAS: 23, CDH: 13, PPHN: all neurodevelopmental disability, MDI, PDI
sepsis: 22, RDS: 8, other: 2 <69, 4 infants had MDI, PDI <84, 3 had
sensory neural hearing loss

Journal of Perinatology
 Neurodevelopmental outcome of infants with MAS
N Beligere and R Rao
S99

Table 3 Continued
Reference Author(s) Number of cases Outcome Comments
no.

36 NINOS, 2000 Study period Outcome at 24 months, cerebral palsy in There was difference between the control
Cases followed: 173 for 18–24 months 30%, with MDI, PDI <70, bilateral group and INO-treated group
blindness in 12%, 6 had CP in INO group,
SNHL: 10 (12%) in INO 34.5% of the INO
group had at least one disability

37 Ellington M et al., Study period September 1992–1997 Outcome INO and CV group: any morbidity: ECMO-treated group had slightly higher
2001 Satisfaction survey of families of 34% (35%) disability CP: 9% (20%), morbidity compared with controls
children treated with INO between 1 hearing, speech: 11% (28%), visual
and 4 years. Cases followed: 60, INO impaired: 12% (3%), DQ: 70 0% (16%)
treated: 42, CV: 40
38 Lipkin PH et al., Study period 1994–1996 INO group: 13% had major disability, No significant difference between the INO
2002 Cases followed: 129, INO treated: 94, hearing loss in 14%. Cognitive delay: 30%, and control. Moderately severe PPHN
control: 35, diagnosis: PPHN only rehospitalization: 22%; INO had 8% CP, and within 24 h after birth had higher rate of
CD in 8%, s.d. in 10%, compared with disability
control had 6% CP, and 6% CD, 6% s.d.

39 Ichiba H et al., Study period 1996–1998 total 15 of Outcome at 3 years of age, one infant with 2/18 (9%) had neurodevelopmental
2003 INO treated follow-up to 3 years CP, one with mid disability, five with disability
MAS: 11, CDH: 1, pneumonia: 1 RDS: reactive airway disease, eight with early
1, hypoplastic lung: 1 response to INO treatment had normal
Only diagnosis PPHN, response to INO development compared with two late
treatment classified as early, late responders
and poor
Abbreviations: BPB, broncho pulmonary dysplasia, CLD, chronic lung disease; CP, cerebral palsy; CV, conventional ventilation; ECMO, extracorporeal membrane oxygenation;
FSIQ, full scale intelligent quotient; HMD, hyaline membrane disease; INO, inhaled nitric oxide; IQ, intelligence quotient; MAS, meconium aspiration syndrome; MDI, mental
development index; PFC, persistant fetal circulation; PIQ, performant intelligent quotient; PPHN, persistent pulmonary hypertension; RDS, respiratory distress syndrome;
VIQ, verbal intelligent quotient.

Outcome of infants treated with INO fundamental to the pathogenesis of neurodevelopment

In 1990, INO was introduced to treat severe PPHN associated
with MAS.32,33 Recently, Rosenberg critically reviewed the long-
term outcomes of MAS and PPHN infants treated with INO
therapy.34 He concluded that, overall, INO was effective in treating
PPHN in MAS. He observed that all the four studies under review35–
38
reported significant medical and neurodevelopmental sequelae.
Ichiba et al.39 later reported that the outcome of INO-treated
infants depended on their response to treatment. Infants who
responded early had zero sequelae, compared with those who
responded late, who had 11% disabilities. Rosenberg et al.
reported35 one-year survival of INO-treated infants with severe
morbidities. He also reported the outcome of infants who survived
the randomized trial of INO treatment with confirmed diagnosis of
PPHN. These infants were followed for 1 to 2 years. At 1 year of age,
6/51 (11.8%) were found to have severe neurological disability as
measured by a Bayley II MDI or PDI score of <69, abnormal
neurological findings, and poor weight gain. He concluded that,
regardless of the various postnatal treatment strategies, the
predisposing factors and underlying factors leading to MAS are
handicap among the survivors. The NINOS study36 reported no
difference in the outcome between INO-treated infants of the larger
group and the control group of infants at 2 years of age. 6/84 INO-
treated infants and 7/87 of the controls had CP. 29.9% of the
controls and 34.5% of the INO-treated infants had at least one
disability, which was defined as CP. Sensory neural hearing loss
was observed in 12% of infants. Lipkin et al.38 reported higher
incidence of neurodevelopment disability (CP): 21 and 19% with
sensory neural hearing loss. This largest group of survivors from
the INO-treated group was also found to have 20%
neurodevelopmental disability in the follow-up. However, the
resulting morbidity was much higher than noted in our
current group.
All the four reports35–38 following the treatment with INO
claimed improvement in the survival but identified significant
neurodevelopment morbidity among the survivors. The NICHD
collaborative reports36,40 on infants treated with INO who were
followed up at 24 months showed no difference in disabilities
between the control and the INO-treated groups.

Journal of Perinatology
 Neurodevelopmental outcome of infants with MAS
N Beligere and R Rao
S100
Summary
We report follow-up of 29 MAS infants, almost all of whom were
treated only with conventional ventilation. Only one infant was
treated with INO, and two other infants required ECMO. The
infants were followed up to 3 years of age. The findings of this
study showed that, in spite of a milder clinical course of illness,
severe delay was noted in 21% of the infants. Our study shows that
infants with MAS, in spite of adequate respiratory support, have the
potential to develop later neurodevelopment deficits. In our study,
we analyzed the importance of cord pH to determine the factors
that predispose to eventual morbidity. We were able to identify cord
pH<6.9 in two infants who also developed CP. There were no
differences in the outcome by mode of delivery. As reviewed above,
previous follow-up studies show that although different treatment
modalities, improved the survival, did not improve the outcome
among MAS infants.
Conclusion
Although various degrees of neurologic abnormalities are found in
infants who survived MAS in the neonatal period, establishing the
causation of neurological insult has been a difficult problem. The
above findings suggest poor outcome in MAS, regardless of the
mode of delivery (NSVD vs C-section) or mode of treatment (ECMO
vs INO). Most infants are asymptomatic at the time of discharge
from the nursery. However, on later follow-up, 10–20% of infants
demonstrated developmental disabilities. The findings above
suggest that there are yet other unidentified events that occur
antecedent to delivery of the infant. The presence of meconium at
birth may be simply an associated event.41 The retrospective
descriptive study has limitations because of the limited number of
patients available. A multicenter trial involving a large number of
cohorts of infants with meconium-stained amniotic fluid as well as
MAS is very much needed to better understand the mechanisms of
development of neurodevelopmental disabilities in these infants.
Acknowledgments
This study was partially supported by Perinatal Program Grant, IDHS #53789004.
Disclosure
The authors have declared no financial interests.
References
1 Cleary GM, Wiswell TE. Meconium stained amniotic fluid and the MAS: an update. Ped
Clin North Am 1998; 45(3): 511–525.
2 Sriram W, Khoshnood B, Singh JK, Msieh HL, Lee KS. Racial disparity in meconium
stained amniotic fluid and MAS in the US, 1989–2000. Obstet Gynecol 2003; 102(6):
1262–1267.
Journal of Perinatology
3 Amiel-Tison C, Grenier A. Neurological Assessment During First Year of Life. Oxford
Press: New York, 1979.
4 Bayley NM. Bayley Scales of Infant Development. New York: 2nd edn, Psychological
Corporation: San Antonio, 1963.
5 Rossetti L. Infant Toddler Language Scale. East Moline Illinois Linguisystems. Rosetti
Louis: Illinois, 1990.
6 Bloom RS, Cropley C. Textbook of Neonatal Resuscitation, 4th edn, Elk. Grove
Village, IL: American Heart Association, American Academy of Pediatrics, 2000.
7 Berkus MD, Langen O, Samueloff A, Xenakis EM, Field NT, Ridgway LE. Meconium
stained amniotic fluid: increased risk for adverse neonatal outcome. Obstet Gynecol
1994; 84: 115.
8 Wiswell TE, Bent RC. Meconium staining and meconium aspiration syndrome. Ped
Clin North Am 1993; 40: 957.
9 Bascik RD. Meconium aspiration syndrome. Pediatr Clin North Am 1977; 24: 467.
10 Vidyasagar D, Yeh T, Harris V, Pildes R. Assisted ventilation in infants with meconium
aspiration syndrome. Pediatrics 1975; 56: 208.
11 Pierce J, Gaudier FL, Sanchez R. Intrapartum amnioinfusion for meconium
stained fluid: meta analysis of prospective clinical trials. Obstet Gynecol 2000; 95:
1051–1056.
12 Ting P, Brady JP. Tracheal suction in meconium aspiration. Am J Obst Gynecol 1975;
122: 767–771.
13 Carson BS, Loosey RW, Bowes Jr WA, Simmons MA. Combined obstetric and pediatric
approach to prevent meconium aspiration syndrome. Am J Obstet Gynecol 1976; 126:
712–715.
14 Marshall R, Tyrala E, McAlister W, Sheehan M. MAS neonatal and follow-up study. Am
J Obstet Gynecol 1978; 131(6): 672–676.
15 Brett C, Dekle M, Leonard CM, Clark C, Snederman S, Roth R et al. Developmental
follow-up of hyperventilated neonates: preliminary observations. Pediatrics 1981;
68(4): 588–591.
16 Walsh-Sukys MC, Bauer RE, Cornell DJ, Friedman HG, Stork EK, Hack M. Severe
respiratory failure in neonates: mortality and morbidity rates and neurodevelopmental
outcomes. J Pediatr 1994; 125(1): 104–110.
17 Bartlett RH, Roloff DW, Connell RG, Andrews AT, Dillon PW, Zwischenberger JB.
Extracorporeal circulation in neonatal respiratory failure: a prospective randomized
study. Pediatrics 1985; 76: 479–487.
18 Towne BH, Lott IT, Hicks DA, Healey T. Long term follow-up of infants
and children treated with ECMO: a preliminary report. J Pediatr Surg 1985; 20(4):
410–414.
19 Andrews AF, Nixon CA, Cilley RE, Roloff DW, Bentlett DH. One to three year outcomes
for 14 neonatal survivors of ECMO. Pediatrics 1986; 78(4): 692–698.
20 Hofkosh D, Thompson AE, Nozza RJ, Kemp SS, Bowen A, Feldman HM. Ten years of
extracorporeal membrane oxygenation: neurodevelopmental outcome. Pediatrics 1991;
87(4): 549–555.
21 Wilden SR, Landry SH, Zwischenberger JB. Prospective, controlled study of
developmental outcome in survivors of extracorporeal membrane oxygenation: the first
24 months. Pediatrics 1994; 93(3): 404–408.
22 Bernbaum J, Schwartz IP, Gerdes M, D’Agostino JA, Coburn CE, Polin RA. Survivors of
extracorporeal membrane oxygenation at 1 year of age: the relationship of primary
diagnosis with health and neurodevelopmental sequelae. Pediatrics 1995; 96
(5 Part 1): 907–913.
23 Robertson C, Finer N, Sauve RS, Whitfield MF, Belgaumkar TK, Synnes AR et al.
Neurodevelopmental outcome after neonatal extracorporeal membrane oxygenation.
CMAJ-JAMC 1995; 152(12): 1981–1988.
24 Ikle L, Ikle DN, Moreland SG, Fashaw LM, Waas N, Rosenberg AR. Survivors of
neonatal extracorporeal membrane oxygenation at school age: unusual findings on
intelligence testing. Dev Med Child Neurol 1999; 41: 307–310.
25 Nield TA, Langenbacher D, Poulsen MK, Platzker AC. Neurodevelopmental outcome at
3.5 years of age in children treated with extracorporeal life support: relationship to
primary diagnosis. J Pediatr 2000; 136(3): 338–344.

26 Vaucher Y, Dudell GG, Bejar R, Gist K. Predictors of early childhood outcome
in candidates for extracorporeal membrane oxygenation. J Pediatr 1996; 128(1):
109–117.
27 Bennett CE, Johnson A, Field DJ, Elbourne D. UK Collaboratie ECMO Trial Group. UK
collaborative randomized trial of neonatal ECMO, follow-up at 4 years of age. Lancet
2001; 357(9262): 1094–1096.
28 Rais-Bahrami K, Wayner AF, Coffman C, Glass P, Short BL. Neurodevelopmental
outcome in ECMO v/s Near ECMO patients at 5 years of age. Clin Pediatr 2000; 39(3):
145–152.
29 Glass P, Wagner AE, Papero PH, Rajasingham SR, Civitello LA, Kjaer MS et al.
Neurodevelopmental status at age five years of neonates treated with extracorporeal
membrane oxygenation. J Pediatr 1995; 127(3): 447–457.
30 Blackwell SC, Modenhauer J, Hassan SS, Redman ME, Refuerzo JS,
Berry SM et al. Meconium aspiration syndrome in term neonates with normal
acid base status at delivery: is it different? Am J Obstet Gynecol 2001; 184(7):
1422–1425.
31 Casey BM, Goldaber KG, McIntire DD, Leveno KJ. Outcomes among term infants when
two-hour postnatal pH is compared with pH at delivery. Am J Obstet Gynecol 2001;
184(3): 447–450.
32 Roberts JD, Polaner DM, Lang P, Zapor WM. Inhaled nitric oxide in PPHN of the
newborn. Lancet 1992; 340: 818–819.
33 Kinsella JP, Neish SR, Shaffer F, Abman SH. Low dose inhalational inhaled INO in
PPHN of the newborn. Lancet 1992; 340: 819–820.
Neurodevelopmental outcome of infants with MAS
N Beligere and R Rao
S101
34 Rosenberg A. Outcome in term infants treated with inhaled nitric oxide. J Pediatr 2002;
140(3): 284–287.
35 Rosenberg A, Kennaugh J, Moreland SG. Longitudinal follow-up of a cohort of
newborn infants treated with inhaled nitric oxide for persistent pulmonary
hypertension. J Pediatr 1997; 131(1): 70–75.
36 The Neonatal Inhaled Nitric Oxide Study Group Journal of Pediatrics. Neonatal inhale.
Group inhaled nitric oxide. Neurodevelopmental follow-up of NINOS. Pediatrics 2000;
136(5): 611–617.
37 Ellington M, O’Reilly D, Allred EN, McCormick MC, Wessel DL, Kourembanas S. Child
health status, neurodevelopmental outcome, and parental satisfaction in a randomized,
controlled trial of nitric oxide for persistent pulmonary hypertension of the newborn.
Am Acad Pediatr 2001; 107(6): 1351–1356.
38 Lipkin PH, Davidson D, Spivak L, Straube R, Rhines J, Chang CT. Neurodevelopmental
and medical outcomes of persistent pulmonary hypertension in term newborns treated
with nitric oxide. J Pediatr 2002; 140(3): 306–310.
39 Ichiba H, Matsunami S, Itoh F, Ueda T, Ohsasa Y, Yamano T. Three-year follow-
up of term and near term infants with inhaled nitric oxide. Pediatr Int 2003; 45(3):
290–293.
40 Neonatal Inhaled Nitric Oxide Study Group. Inhaled nitric oxide in full-term
and nearly term infants with hypoxic respiratory failure. N Engl J Med 1997; 336:
597–604.
41 Ghidini A, Spong CY. Severe meconium aspiration syndrome is not caused by
aspiration of meconium. Am J Obstet Gynecol 2001; 185(4): 931–938.
Journal of Perinatology
 Journal of Perinatology (2008) 28, S102–S107
r 2008 Nature Publishing Group All rights reserved. 0743-8346/08 $30
www.nature.com/jp
REVIEW
Studies of meconium-induced lung injury: inflammatory
cytokine expression and apoptosis
D Vidyasagar and A Zagariya
Division of Neonatology, Department of Pediatrics, The University of Illinois at Chicago, Chicago, IL, USA
To review current literature related to cellular mechanisms of meconium-
induced lung injury (MILI). Review of published experimental in vitro and
in vivo MAS studies using human and animal lung cells. We found that
meconium induces expression of cytokines and angiotensin II (ANG II)-
induced apoptotic process in the lung cells. We postulate that inflammatory
cytokines induce ANG II expression, which causes apoptotic cell death after
binding to its AT1 receptors. We also demonstrated expression of serpins
associated with meconium instillation into the lungs. Serpins are proteins
that inhibit cellular proteases and elastases. Expression of serpins may be an
attempt to recover lung from these injurious effects. In summary our
studies show that whereas meconium induces inflammatory cytokines and
subsequent cell apoptosis, the lung cells also try to protect themselves by
inducing serpins. The balance of these interactions will determine the
residual damage. We believe these new findings are very important in
understanding of MILI.
Journal of Perinatology (2008) 28, S102–S107; doi:10.1038/jp.2008.153
Introduction
Meconium aspiration syndrome (MAS) is one of the major clinical
problems encountered in the neonatal period. It is an
inflammatory newborn lung disease that frequently leads to severe
respiratory failure with a potential fatal outcome in term and
post-term infants.1,2 Aspiration of meconium is known to cause
airway obstruction, damage of airways and surfactant inactivation.
Meconium also causes chemical pneumonitis.1–3 In the newborn,
the major clinical manifestations include problems of oxygenation
and respiratory failure associated with increased pulmonary
vascular reactivity. It has been shown that persistent pulmonary
hypotension is a hallmark of severe MAS. Number of investigators
has extensively studied these aspects of MAS.
In addition to the above-described derangements meconium
also induces intense inflammatory reaction in the airways and
alveoli. However not much has been explored in this field. There is
emerging evidence from different animal models that intense
Correspondence: Dr D Vidyasagar, Division of Neonatology, Department of Pediatrics, The
University of Illinois at Chicago, 840 South Wood Street, Chicago, IL 60517, USA.
E-mail: dsagar@uic.edu
inflammatory reaction may be central to the initial progression of
MAS and associated severe pulmonary hypertension.3–5
Investigators using animal models have shown the presence of
inflammatory-induced cell death through apoptotic process in
meconium-instilled lungs.6,7 There is increased interest to better
understand complex mechanisms of inducing meconium-induced
lung cell apoptosis. The purpose of this paper is to review the
literature and better understands the mechanisms of
meconium-induced lung injury (MILI).
The evidence for meconium-induced inflammation and
apoptosis comes from different investigators. These studies can be
classified into two categories: one from human studies and the
other from experimental animal models. Table 1 shows selected
papers in this regard. In general, these findings showed that
meconium in both human and animal models produces
inflammatory reaction, which is followed by cascade of events
leading to lung epithelial cell apoptosis. Findings of these studies
are summarized below.
Human studies
There are very few human studies (Table 1). De Beaufort et al.8
were the earliest investigators who studied in vitro response of
human neutrophils to MAS. They showed that there was
overexpression of interleukin 8 (IL-8) in neutrophils exposed to
meconium. They also showed anti-IL-8 antibodies inhibited
neutrophils migration. These studies suggested that MAS in infants
may induce neutrophils migration and overexpression of IL-8 lead
to inflammation.
Uotila et al.9 studied meconium exposure to monocytes in
culture and showed increased expression of syclogexynase 2
expression. Jones et al.4 demonstrated increased production of
inflammatory cytokines in preterm infants with MAS. Castelheim10
showed compliment activation in human monocytes culture of
MAS model.
Animal studies
Investigators have studied meconium-induced inflammation using
different animal models (Table 1). Davey et al.,11 in piglets, showed
 Inflammatory cytokine expression and apoptosis
D Vidyasagar and A Zagariya
S103

Table 1 Human and animals models of MAS

Human models of MAS

Srinivasan and Vidyasagar2
Jones et al.4
de Beaufort et al.8
Uotila and Kaapa9
Castellheim et al.10
Newborn infants Increase in bile acids, lipids, polysaccharides, in vivo and in vitro
Preterm and term newborns Production of TNFa, IL1b, and IL8 is regulated by IL-10; IL-10 not detected in preterm infants with
or without MAS
Newborn infants Overexpression of IL-8 in MAS in vitro; increased migration of neotrophils; anti-IL-8 antibodies
inhibit neutrophil migration
Human monocytes In culture increase of cycloxygenase-2 expression in vitro
Human monocytes in culture Complement activation in MAS

Animal models of MAS

Tyler et al.3 Animal models Surfactant inactivation, chemical pneumonitis, airway obstruction, direct toxic damage of animal
lung tissues
Zagariya et al.6 Rabbit pups Intense inflammatory reactions involved in meconium-induced injury in vivo; expression of
endothelin 1 in MAS
Davey et al.11 Piglets Acute decrease in gas exchange and dynamic lung compliance; increase of total lung protein;
inhibition of surfactant
Holopainen et al.12 Piglets Inflammation, airway epithelium targeted, necrotic changes, phospholipase 2 activity increased.
Mollnes et al.13 Animal models Complement activation in MAS
Khan et al.14 Mice Increase of IL-13 expression in MAS in vivo; lymphocytic and eosinophilic inflammation
Zagariya et al.15 Rabbit pups Phospholipase A2 activates apoptosis, influx of T-lymphocytes and macrophages in vivo; increase of
TNFa, IL-1b, IL-6 and IL-8 in MAS in vivo
Zagariya et al.16 Rabbit pups Cell death by apoptosis, influx of neutrophils and macrophages in vivo
Tessler et al17 Newborn infants Reactive oxygen species in vitro
Abbreviations: IL, interleukin; MAS, meconium aspiration syndrome; TNF, tumor-necrosis factor.

acute decrease in gas exchange and dynamic lung compliance,
increase of total lung protein and inhibition of surfactant.
Holopainen12 made extensive observations in piglets exposed to
meconium. They demonstrated severe inflammation and airway
epithelial damage, necrotic changes associated with increased
phospholipase 2 activities. Mollnes also showed an increased
compliment activation following experimental MAS in piglets.13
Khan14 showed that MAS increases IL-13 expression along with
lymphocytic and iosinophilic inflammation. Tyler3 in animal model
showed that MAS leads to surfactant inactivation and pneumonitis.
In our own laboratory, using rabbit pups,6,15,16 we have shown
that lung exposure to meconium leads to intense inflammatory
reaction with increased expression of lymphocytes, macrophages
and neutrophils. Experimental studies showed that inflammatory
response and apoptotic epithelial cell death is important feature of
meconium-induced newborn lung injury. Our studies differ from
previously published studies because of the methodology used. In
our studies we used rabbit pups with no previous hypoxia. Another
major distinction from other studies is that we did not use whole
meconium, because whole meconium contains many components
that can cause lung injury. Instead, we used debris-free meconium
to avoid mechanical obstruction of airways. One gram of fresh
newborn meconium (1 g) was homogenized on ice with 9 ml of
0.9% NaCl (pH 7.2) to a final concentration of 10% (weight/
volume) and was spun down at 1000 g for 20 min (at 4 1C) to
separate supernatant and meconium debris. Supernatant was
filtered using 0.45 mm filter paper (Millipore Co., Bedford, MA,
USA). The cell-free filtered solution instilled into the lungs.
Following instillation, rabbits were allowed to breathe
spontaneously in room air with no additional oxygen. No
respiratory support was provided to pups following instillation of
meconium. Animals were sacrificed at 0, 2, 4, 8 and 24 h after
meconium instillation. The findings of other studies including ours
are described below.
Inflammatory responses to meconium
The first response to meconium injury is the release of active
inflammatory cells, the most important of which are neutrophils
and macrophages.8,9,16 Especially, neutrophils are dramatically
increased in meconium-instilled lungs compared to saline-instilled
lungs. Macrophages are also increased, but not as dramatically as
neutrophils. Tessler recently suggested that meconium also induced
free oxygen radicals.17 Now we describe the pathways of induction
of inflammatory response following cell influx.

Journal of Perinatology
 Inflammatory cytokine expression and apoptosis
D Vidyasagar and A Zagariya
S104
Neutrophil influx
Aspirated meconium produces a vigorous but transient leukocytic
inflammatory reaction in the newborn lungs, but the mechanisms
of the neutrophil influx are still unclear.10 Meconium itself may
stimulate neutrophil chemotaxis and it supposedly induces the
lung inflammatory cells to produce proinflammatory chemotactic
cytokines.4,8,10,18 Ex vivo studies using a piglet model show that
moderate and high concentrations of aspirated meconium rapidly
activate circulating neutrophils.19 Experimental study in piglet
model suggests that meconium-induced inflammation is localized
in the meconium-contaminated areas of the lungs with no
generalized inflammatory injury.20 Correspondingly, local
accumulation of neutrophils and macrophages and production of
IL-1b and tumor-necrosis factor-a (TNFa) cytokines have been
observed.2,20 Current experimental data suggests that
intrapulmonary accumulation of activated neutrophils, similar to
various forms of acute injury in adult lungs21 could aggravate the
hypoxic respiratory failure. Myeloperoxidase activity is usually used
as an important indicator of neutrophil accumulation.22 The
chemotaxis is related to the presence of powerful chemotactic role
of IL-8.
In a study by Ruiz et al.23 addition of polyclonal
immunoglobulin G (IgG) activated the mononuclear cells to
express IL-8 cytokine production, which could promote
intrapulmonary accumulation and infiltration of neutrophils.
Other studies have shown that intravenous IgG may increase of
neutrophil migration to the sites of inflammation via unknown
mechanisms.21 Sterile meconium increased migration of
neutrophils obtained from neonates in comparison with random
migration in vitro studies. These studies show that meconium
induces chemotaxis of inflammatory cells especially neutrophils.8
These above studies showed the neutrophil response to meconium.
Cytokine release and their effect
In experimental models of MAS, an intense pulmonary
inflammatory reaction with influx of polymorphonuclear
leukocytes, T-lymphocytes, monocytes and macrophages is
demonstrated within a few hours.10,12,16 It is further apparent that
intrapulmonary meconium stimulates the lung inflammatory cells
to express inflammatory cytokines, such as TNF-a, IL-1b, IL-6 and
IL-8, and may thereby propagate development of parenchyma cell
injury in the exposed animal and human lungs.8,10,16 A novel
signaling pathway involved in MAS is the role of IL-13. This
molecule has been shown to be markedly elevated in a murine
model of MAS and could be a mediator of persistent airway
dysfunction and remodeling in survivors of MAS.14 In fact,
dramatic initiation of the pulmonary inflammatory response to
meconium aspiration is highlighted by immediate appearance of
the lung pro-inflammatory cytokine expression in rabbit models,
with a maximum at 8 to 14 h after the meconium insult.16 As low
levels of anti-inflammatory IL-10 is simultaneously found in these
Journal of Perinatology
lungs,4,16 the resulting cytokine imbalance may regulate the
susceptibility of the newborn lung to meconium-induced injury.
Complement activation
Various investigators10,13,24 using animal and human cells showed
that human meconium is a potent activator of complement, an
important mediator of inflammatory tissue damage. Meconium
activated the alternate complement pathway by increase of
alternative convertase C3bBbp.10 Meconium can also induce
systemic complement activation, paralleling the increase in lung
dysfunctions.
Phospholipase activation
Local release of phospholipid-degrading enzyme, phospholipase A2,
may further play an exacerbating role in the pathogenesis of acute
inflammatory lung injury after meconium aspiration in a human
model. As showed by Kaapa,12 experimental studies in animal
models indicate that this proinflammatory activity is also contained
in human meconium itself. The existing data on human and
animal models thus indicate that constituents of aspirated
meconium, including cytokines, phospholipases, bile acids, lipids
and polysaccharides, may trigger a local inflammatory reaction
supposedly through stimulation of lung inflammatory cells to
produce proinflammatory cytokines and prostaglandins.8,25
Moreover, recent data demonstrate that activation of lung tissue
phospholipase A2, in meconium injury, may mediate cellular
apoptosis, as a noninflammatory mechanism of programmed cell
death, in porcine meconium aspiration.26
Meconium-induced apoptosis
Apoptosis of cells exposed to meconium was one of the interesting
findings of our experimental studies. Apoptosis is a programmed
cell death, which does not result in inflammatory tissue injury, but
rather is a cell clearance mechanism promoting resolution of
inflammation.27 Normal rate of apoptosis is beneficial to the host
organ. However, increased apoptosis may clearly cause harm for
the tissue. Recent literature including data from our laboratory
demonstrate that apoptosis may play an important role in acute
lung injury of newborn.12,16 As was demonstrated by several
techniques, meconium-induced apoptosis is localized preferably in
lung and its airway epithelial cells.12,16 These data demonstrate
that meconium-induced lung cell apoptosis leads to damage and
detachment of lung airway or epithelial cells. It is a very important
process as airway epithelium plays a very important role in
maintenance of cell viability and normal lung functions. In spite a
several existing theories, exact mechanism of meconium-induced
lung cell apoptosis is presently unknown.
Usually apoptosis is seen immediately after instillation of
meconium into the lungs. Apoptosis occurs under the influence of
different substances present in meconium. Apoptotic cells are

140
120
% of Caspase Activity
100
80
60
40
20
0 by human meconium in vitro,9 may modulate NO-mediated
0 2 4 8 24
Figure 1 Expression of caspase 3 at 0, 2, 4, 8 and 24 h after meconium
instillation into 2-week-old rabbit lungs.
smaller in size; they appear shrunk, their cytoplasm and nuclei are
condensed. Examination of the lung lavage cells demonstrated that
apoptosis usually is accompanied with inflammatory reactions,
extensive cell death. The apoptotic cells are identified by measuring
caspase 3 expression.28 Maximal expression of caspase 3 was also
seen at 8 h after meconium instillation (Figure 1).
ISEL labeling is another method of identifying the apoptotic
cells.16 Using ISEL labeling, we found a fragmented DNA in
numerous airways and alveolar epithelial nuclei.16 ISEL-positive
cells were seen specifically in meconium-instilled lungs but not in
saline-instilled lungs. ISEL-positive sells surround the bronchioles
and gradually expanded to the entire lung field.
Apoptosis destroys many important cells in the lung airway or
alveolar epithelium. In worst cases, such destruction may reach up
to 50% of airway epithelial cells. The mechanism of
meconium-induced epithelial cell apoptosis and their detachment
presently remains unknown. Also unknown are mediating
mechanisms of apoptotic cell death, which we will describe below.
Oxygen radicals in apoptosis
Increased release of reactive oxygen species from the animal
pulmonary inflammatory cells may be associated with apoptotic
cell death in the lung.29 Neutrophil influx following meconium
exposure may also activate alveolar macrophages to produce
oxygen radicals, which may, at least in part, explain the
proapoptotic effect of meconium in animal and human lungs.17,19
Nitric oxide and apoptosis
Although the bulk of evidence indicates that nitric oxide (NO) may
induce apoptosis in different cell lines, including macrophages, it
may have a dual effect. It is interesting to note that inhaled smaller
quantities of NO may also inhibit apoptosis.30,31,32 Inhalation of
NO (10 p.p.m.), in addition to having a beneficial effect on
hemodynamics and oxygenation in newborn lungs, also
significantly inhibited cell death by apoptosis. The effect of a
Inflammatory cytokine expression and apoptosis
D Vidyasagar and A Zagariya
S105
smaller amount (1 p.p.m.) of NO inhalation was similar but did
not reach statistical significance.30 Apoptotic cell death was induced
by exogenous NO in rat type II pneumocytes in vitro32 but not in
cultured human cells of pulmonary epithelial orendothelial
origin.33 Although the inhibitory effect of NO is still unclear,
interaction of NO with oxygen-reactive species may eliminate the
adverse affects of these toxic radicals or, alternatively, upregulate
protective genes that attenuate apoptosis.32 Furthermore, increased
cycloxygenase-2 expression, shown to be stimulated in monocytes
apoptosis in macrophages.32
Role of ANG II and its receptors in apoptosis
Pulmonary angiotensin II (ANG II) has been recently proposed to
contribute to receptor-mediated lung epithelial apoptosis in vivo
and in vitro, but in neonatal lung injuries in vivo this mechanism
is still unclear.34 We demonstrated using a rabbit model of MAS
that apoptosis occurs via cytokine-induced angiotensinogen gene
expression, conversion of ANG I to ANG II, and binding of ANG II
to its AT1 receptors.34–36 This conversion requires the presence of
ANG-converting enzyme, ACE.34,36 In animal models captopril may
also decrease the apoptosis process in alveolar epithelium
induced by FAS, bleomycin, plant alkaloid monocrotaline, or
g-irradiation.34–37 Consequently, it was hypothesized that
meconium-induced apoptosis could be prevented by suppression of
ANG I conversion to ANG II by captopril pretreatment or by
blocking of its AT1 receptors. Recent data from our laboratory
indicate that captopril and inhibition of AT receptor action may
diminish pulmonary epithelial apoptosis in meconium lungs.28,37
Additional data from our laboratory show that cytokine-treated
rabbit lung and human lung airway epithelial cell line A549
demonstrate a similar rate of lung apoptosis, compared to
meconium-instilled lungs.35,38 From the above discussion it may be
postulated that intrapulmonary meconium induces influx of
inflammatory cells in the lungs. These cells express inflammatory
cytokines, involved in expression of ANG I, which is immediately
processed to ANG II by ACE. Lung cell damage and apoptosis occur
when ANG II binds to its AT1 but not AT2 receptors (Figure 1). It
is, however, not clear how the above-discussed mechanisms are
initiated.
Protective role of serpin in apoptosis
During our studies to explain the mechanisms of apoptosis, we
isolated a protein from tracheal aspirates. We found that this
protein to be a novel serine proteinase inhibitor, with an apparent
molecular mass of 50 kDa, which was identified to be e
a1-antitripsin.39 Serpins are known inhibitors of proteases and
apoptosis. We hypothesize that serpin may attenuate pulmonary
inflammation and improve surfactant function after meconium
Journal of Perinatology
 Inflammatory cytokine expression and apoptosis
D Vidyasagar and A Zagariya
S106
MECONIUM-INDUCED LUNG INJURY
Meconium
LUNG
Alveolar & Airway
Responce
Neutrophil Endothelial
Macrophages
Influx Cells
Cytokines Endothelin-l Cytokines
Lung Injury
Inflammation Apoptosis
Histological
Changes
Figure 2 Meconium-induced lung injury.
aspiration. a1-antitripsin is a member of the proteinase inhibitor
(serpin) superfamily and inhibitor of neutrophil elastase.
a1-Antitripsin is synthesized by epithelial cells, macrophages,
monocytes and neutrophils. Deficiency in a1-antitripsin leads to
exposure of lungs to uncontrolled proteolytic attack from
neutrophil elastase or other damaging factors culminating in lung
destruction and cell apoptosis. We hypothesize that accumulation
of a1-antitripsin in the lungs serves as a predisposed protection
against MILI.
Unified concept of MILI
Taking into consideration of all the above findings we propose a
unified concept of the mechanism of MILI. The construct of the
hypothesis is shown in Figure 2. It is proposed that meconium
aspiration by the newborn besides causing airway obstruction
causes local inflammation. The inflammatory reaction leads to
cellular influx of neutrophils and macrophages. Both release
inflammatory cytokines, which in turn cause inflammation and set
the process of apoptosis. The end result depends on the extent of
inflammation and degree of apoptosis. Moderate apoptosis may
leave no permanent damage. Severe apoptosis initiates fibrotic
reaction. These results may become perceptible on histological
examination of the tissues.
The sequence of events in the epithelial cells is different.
Epithelial cells express ANG II and their AT1 and AT2 receptors.
Both participate in the process of cell apoptotic cell death.
It is also to be noted that meconium can induce serpin
expression, which plays a protective role against injury and
apoptosis which outcomes depends from the ration of proteases and
serpins. If this ration is large a large damage is observed; if small,
it is minimal.
Journal of Perinatology
Conceptual model of MILI mechanisms
Meconium
Lung
Epithelial Cells
Inflammatory Responce
Neutrophils and
Macrophages
+ –
Cytokines APOPTOSIS Serpin
Figure 3 Cytokines and serpin in meconium-induced apoptosis.
Summary
In summary, meconium aspiration induces a rapid and intensive
inflammatory reaction and lung injury within the contaminated
areas of the newborn lungs and produces simultaneously lung cell
death by apoptosis. In addition, based on our work, we believe that
meconium-induced lung cell apoptosis is also induced by
accumulation of ANG II and binding to its AT1 receptors. TNFa or
IL-8 cytokines may be involved in processing ANG II and thus is
also involved in increasing meconium-induced cell apoptosis.
We also believe that degree of apoptosis is dependent on the
production of proteases and serpins in the cells. Serpins inactivate
meconium-induced proteases thus prevent lung damage. In
summary, we propose that meconium induces cytokine expression
and lung cell apoptosis. At the same time expression of serpins in
response to meconium may negate the effect of proteases induced
by meconium (Figure 3).
The significance and contribution of these lung injury processes
to the clinical manifestation of MAS, however, needs to be further
investigated. Better understanding of meconium-induced
inflammatory injury and apoptosis may lead to a development of
new therapeutic interventions of MAS.
Acknowledgments
We acknowledge the grant support of Thrasher Foundation (ID no. 02823-6) for
these studies. We also thank staff of Michael Reese Animal Laboratory,
Dr Shankar Rao Navale, Dr Rama Bhat, Dr Gopal Chari, Dr Bruce Uhal, Dr Pekka
Kaapa, Dr Charles Rosenfeld and summer students for everyday assistance
in experimental work, collecting data and other support of this work.
Disclosure
The authors have declared no financial interests.

References
1 Vidyasagar D, Yeh TF, Harris MD, Pildes RS. Assisted ventilation in infants with
meconium aspiration syndrome. Pediatrics 1975; 56(2): 208–213.
2 Shrinivasan HB, Vidyasagar D. Meconium aspiration syndrome: current concepts and
management. Compr Ther 1999; 25(2): 82–89.
3 Tyler DC, Murphy J, Cheney FW. Mechanical and chemical damage to lung tissue
caused by meconium aspiration. Pediatrics 1978; 62: 454–459.
4 Jones CA, Cayabyab RG, Kwong KY, Stotts C, Wong B, Hamdan H et al. Undetectable
interleukin (IL)-10 and persistent IL-8 expression early in hyaline membrane disease:
a possible development basis for the predisposition to chronic lung inflammation in
preterm infants. Pediatr Res 1996; 39: 966–975.
5 Holopainen R, Soukka H, Kaapa P. Meconium aspiration induced a
concentration-dependent pulmonary hypertension in newborn piglets.
Pediatr Pulmonol 1998; 25: 107–113.
6 Zagariya A, Doherty J, Bhat R, Navale S, McMillan M, Uhal B et al. Elevated
immunoreactive level of ET-1 levels in newborn rabbit lungs after meconium
aspiration. Pediatr Crit Care Med 2002; 3(3): 297–302.
7 Soukka H, Viinikka L, Kaapa P. Involvement of thromboxane A2 prostacyclyn in the
early pulmonary hypertension after porcine meconium aspiration. Pediatr Res 1998;
44(6): 838–842.
8 de Beaufort AJ, Pelikan DM, Elferink JG, Berger HM. Effect of interleukin 8 in
meconium on in-vitro neutrophil chemotaxis. Lancet 1998; 352(9122): 102–105.
9 Uotila PJ, Kaapa PO. Cyclooxygenase-2 expression in human monocytes stimulated by
meconium. Lancet 1998; 351: 978.
10 Castellheim A, Lindenskov PH, Pharo A, Fung M, Saugstad OD, Mollnes TE. Meconium
is a potent activator of complement in human serum and in piglets. Pediatr Res 2004;
55(2): 310–318.
11 Davey AM, Becker JD, Davies JM. Meconium aspiration syndrome: physiological and
inflammatory changes in the newborn piglet model. Pediatr Pulmonol 1993; 16:
101–108.
12 Holopainen R, Aho H, Laine J, Peuravuori H, Soukka H, Kaapa P. Human meconium
has high phospholipase A2 activity and induced cellular injury and apoptosis in piglets
lungs. Pediatr Res 1999; 46: 626–632.
13 Mollnes TE, Song WC, Lambris JD. Complement in inflammatory tissue damage and
disease. Trends Immunol 2002; 23(2): 61–64.
14 Khan AM, Elidemir O, Epstein CE, Lally KP, Xue H, Blackburn M et al. Meconium
aspiration produces airway hyperresponsiveness and eosinophilic inflammation in a
murine model. Am J Physiol Lung Cell Mol Physiol 2002; 283(4): L785–L790.
15 Zagariya A, Bhat R, Navale S, Vidyasagar D. Cytokine expression in meconium-induced
lungs. Indian J Pediatr 2004; 71(3): 195–201.
16 Zagariya A, Bhat R, Uhal B, Navale S, Freidine M, Vidyasagar D. Cell death and lung
cell histology in meconium aspirated newborn rabbit lung. Eur J Pediatr 2000;
159(11): 819–826.
17 Tessler R, Pan J, Fiori HH, Belik J. Human meconium has a pulmonary vascular and
airway smooth muscle relaxant effect. Pediatr. Res 2008; 64(1): 24–28.
18 Kojima T, Hattori K, Fujuwara T, Takedatsu-Sasai M, Kobayashi Y. Meconium-induced lung
injury mediated by activation of alveolar macrophages. Life Sci 1994; 54: 1559–1562.
19 Soukka HR, Ahotupa M, Ruutu M, Kaapa PO. Meconium stimulates neutrophil
oxidative burst. Am J Perinatol 2002; 19(5): 279–284.
20 Korhonen K, Soukka H, Halkola L, Peuravuori H, Aho H, Pulkki K et al. Meconium
induces only localized inflammatory lung injury in piglets. Pediatr Res 2003; 54(2):
192–197.
Inflammatory cytokine expression and apoptosis
D Vidyasagar and A Zagariya
S107
21 Fijishima S, Aikawa N. Neutrophil-mediated tissue injury and its modulation. Intensive
Care Med 1995; 21: 277–285.
22 Holopainen R, Soukka H, Halkola L, Kaapa P. Intravenous immunoglobulin G
attenuates pulmonary hypertension but induces local neutrophil influx in meconium
aspiration in piglets. Biol Neonate 2005; 87: 221–228.
23 Ruiz de Souza V, Carreno MP, Kaveri SV, Ledur A, Sadeghi H, Cavaillon JM et al.
Selective induction of interleukin-1 receptor antagonist and interleukin-8 in human
monocytes by normal polyspecific IgG (intravenous immunoglobulin). Eur J
Immunol 1995; 25: 1267–1273.
24 Baggiolini M, Loetcher P. Chemokines in inflammation and immunity. Immunol
Today 2000; 21: 418–420.
25 Lopez A, Bildfell R. Pulmonary inflammation associated with aspirated meconium and
epithelial cells in calves. Vet Pathol 1992; 29: 104–111.
26 Kaapa P. Meconium aspiration syndrome: a role for phospholipase A2 in the
pathogenesis? Acta Paediatr 2001; 90: 365–367.
27 Bardales RH, Xie SS, Schaefer RF, Hsu SM. Apoptosis is a major pathway responsible for
the resolution of type II pneumocytes in acute lung injury. Am J Pathol 1996; 149:
845–852.
28 Zagariya A, Bhat R, Chari G, Uhal B, Navale S, Vidyasagar D. Apoptosis
of airway epithelial cells in response to meconium. Life Sci 2005; 76(16):
1849–1858.
29 Kazzas JA, Xu J, Palaia TA, Mantel L, Fein AM, Horowitz S. Cellular
oxygenotoxicity. Oxidant injury without apoptosis. J Biol Chem 1996; 271:
15182–15186.
30 Gaboury J, Woodman RC, Granger DN, Reinhardt P, Kubes P. Nitric oxide prevents
leukocyte adherence: roleof superoxyde. Am J Physiol 1993; 265(3 pt 2): H862–H867.
31 Brune B, von Knethen A, Sanday KB. Nitric oxide and its role in apoptosis. Eur J
Pharmacol 1998; 351: 261–272.
32 Janssen YMW, Matalon S, Mossman BT. Differential induction of c-fos, c-jun, and
apoptosis in lung epithelial cells exposed to ROS or RNS. Am J Physiol 1997;
273(4 Pt 1): L789–L796.
33 Narula P, Xu J, Kazzaz A, Robbins CG, Davis JM, Horowitz S. Synergistic cytotoxicity
from nitric oxide and hyperoxia in cultured lung cells. Am J Physiol 1998; 274:
L411–L416.
34 Wang R, Zagariya A, Ang E, Ibarra-Sunga O, Uhal B. FAS-induced apoptosis of alveolar
epithelial cells require angiotensinogen II generation and receptor interaction. Am J
Physiol Lung Cell Mol Physiol 1999; 277: L1245–L1250.
35 Wang R, Alam G, Zagariya A, Gidea C, Pinillos H, Lalude O et al. Apoptosis of lung
epithelial cells in response to TNFa requires Angiotensin II generation de novo. J Cell
Physiol 2000; 185: 253–259.
36 Wang R, Zagariya A, Ibarra-Sunga O, Gidea C, Ang E, Deshmukh S et al.
Angiotensin II induced apoptosis in human and rat alveolar epithelial cells. Am J
Physiol 1999; 276: L885–L889.
37 Zagariya A, Uhal B, Navale S, Vidyasagar D. Inhibition of Meconium Induced Lung
Cell Apoptosis by Captopril. Pediatric Academic Societies and American Academy of
Pediatrics Joint Meeting, Boston, MA 2000, Abstract.
38 Zagariya A, Bhat R, Uhal B, Navale S, Vidyasagar D. Captopril Inhibits Meconium
Induced Cytokines Expression and Lung Cell Apoptosis, Pediatric Academic Societies
Meeting, Philadelphia, PA 2001, Abstract.
39 Zagariya AM, Bhat R, Zhabotynsky E, Chari G, Navale S, Xu Q et al. Characterization of
serine/cysteine protease inhibitor alpha1-antitripsin from meconium-instilled rabbit
lungs. J Cell Biochem 2005; 96(1): 137–144.
Journal of Perinatology
 Journal of Perinatology (2008), S108–S112
r 2008 Nature Publishing Group All rights reserved. 0743-8346/08 $30
www.nature.com/jp
REVIEW
Angiotensin II in apoptotic lung injury: potential role in
meconium aspiration syndrome
BD Uhal and A Abdul-Hafez
Department of Physiology, Michigan State University, East Lansing, MI, USA
Meconium aspiration injures a number of cell types in the lung, most
notably airway and alveolar epithelial lining cells. Recent data show that at
least some of the cell death induced by meconium occurs by apoptosis, and
therefore has the potential for pharmacologic inhibition through the use of
apoptosis blockers or other strategies. Related work in adult animal
models of lung injury has shown that apoptosis of lung epithelial cells
induces a local (that is, entirely lung tissue specific) renin-angiotensin
system (RASL). Furthermore, this inducible RASL is required for the apoptotic
response and affects other adjacent cell types through the release of angiotensin
II and related peptides. This manuscript reviews the published data supporting
this viewpoint as well as more recent works that suggest the involvement of a
RASL in the perinatal lung damage associated with meconium aspiration
syndrome (MAS). The implications of these findings regarding their potential
for the clinical management of MAS are also discussed.
Journal of Perinatology (2008) 28, S108–S112; doi:10.1038/jp.2008.149
General mechanisms in apoptosis
Apoptosis, sometimes referred to as programmed cell death (PCD)
or ‘cell suicide’, is a metabolically active process of cellular
dismantling. Apoptosis avoids the inflammation often associated
with necrotic cell death because cellular contents are often not
released from apoptotic cells as they are from necrotic cells.
A variety of cellular signaling pathways regulates apoptosis, which
is characterized morphologically by nuclear fragmentation,
cleavage of chromosomal DNA into internucleosomal fragments,
packaging of cellular debris into ‘apoptotic bodies’ without plasma
membrane breakdown and exposure of surface molecules targeting
the cell remnants for phagocytosis.1,2
Major signaling pathways leading to apoptosis involve the
activation of cystein-dependent, aspartate-directed proteases termed
caspases. Depending on the type of stimuli and/or cell type, the
apoptotic cascade can follow either an intrinsic pathway, involving
Correspondence: Dr BD Uhal, Department of Physiology, Michigan State University,
3185 Biomedical and Physical Sciences Building, East Lansing, MI 48824, USA.
E-mail: uhal@msu.edu
apoptogenic mitochondrial proteins such as cytochrome c, or an
extrinsic pathway that follows activation of a death receptor. The
receptor is usually activated by its extracellular ligand, such as the
binding of Fas ligand (FasL) to Fas (APO-1 or CD95) or tumor
necrosis factor-a (TNF-a) binding to TNF-a receptor. On activation,
initiator caspases (caspase-2, -8, -9, and -10) propagate death signals
by activating downstream effector caspases (caspase-3, -6, and -7) in
a cascade-like manner. Activated effector caspases mediate the release
of caspase-activated DNase (CAD) from the complex with its
endogenous inhibitor (ICAD) through a proteolytic cleavage. The
activated CAD then cleaves genomic DNA between nucleosomes to
generate DNA fragments, which are multiples of 180 to 200 bp in
length. These fragments can form the ‘DNA ladder’ pattern seen on
elecrophoresis, a frequently used marker of apoptosis.
Caspase-independent mechanisms of apoptosis are mediated by
mitochondrial effectors; in this pathway, poly(ADP-ribose)
polymerase-1-mediated DNA damage is signaled to mitochondria.
This leads to release and translocation of apoptosis-inducing factor
(AIF) from the mitochondria to the nucleus. AIF enhances
endonuclease activity through its cooperation with endonuclease G
or cyclophilin A, which leads to large-scale DNA fragmentation and
chromatin condensation.
In contrast to apoptosis, which requires energy in the form of
ATP, necrosis is the end result of a bioenergetic catastrophe
resulting from ATP depletion to a level incompatible with cell
survival. Cells that die by necrosis frequently exhibit changes in
plasma membrane and nuclear morphology but not the organized
chromatin condensation or DNA laddering that are characteristic of
apoptosis.1 Necrosis-like PCD, or autophagy, is also considered as
alternative PCD that is caspase independent. Autophagy features
degradation of cellular components within the intact dying cell in
autophagic vacuoles that undergoes phagocytosis. Autophagic cells
exhibit vacuolization, degradation of cytoplasmic contents and
slight chromatin condensation; the molecular mechanisms
regulating autophagy are now being elucidated.2,3
Mechanisms of apoptosis specific to the lungs
The topic of apoptosis and its regulation in the lungs has been
reviewed recently;4 this section will briefly summarize apoptosis

mechanisms specific to acute respiratory distress syndrome (ARDS),
chronic obstructive pulmonary disease (COPD) and pulmonary
fibrosis (PF) that may be relevant to meconium aspiration
syndrome (MAS).
Apoptosis of epithelial cells, neutrophils and endothelial cells are
all thought to play roles in ARDS. The Fas-FasL system is thought
to play a role in epithelial apoptosis in ARDS on the basis that
bronchoalveolar lavage (BAL) fluids obtained from ARDS patients
contain soluble Fas and FasL and induce apoptosis in lung
epithelial cell line.5–8 Involvement of BAX, a homolog of Bcl-2 and
its likely proapoptotic effect on type II pneumocytes was suggested
in studies of diffuse alveolar damage.9,10 Neutrophils were found to
accumulate at inflammatory sites in the lung, in pulmonary
edema fluid and BAL fluid from patients with ARDS.11 This
accumulation is due in part to delayed neutrophil apoptosis,; in
addition, apoptosis of epithelial cells is enhanced at the same
site.12–14 BAL fluids from patients with ARDS had elevated levels of
granulocyte/macrophage colony-stimulating factor that inhibits
neutophil apoptosis through phosphoinositide 3-kinase and ERK
pathway15,16 and stabilizes the factor Mcl-1 that is crucial for the
delay of apoptosis initiated by antiapoptotic factors.17,18 BAL fluids
from patients with ARDS was also cytotoxic to lung microvascular
endothelial cells due to the presence of TNF-a and angiostatin, an
inhibitor of angiogenesis in vivo.19,20 Furthermore, apoptosis
signal-regulating kinase (ASK)-1 was involved in apoptosis of
pulmonary vascular endothelial cells challenged with H2O2.21
Given the known roles of H2O2 generated by activated neutrophils
and other phagocytes, this suggests the possibility that inhibitors of
ASK-1 and its signaling pathway might have potential benefit in
the management of ARDS.
Cell death by apoptosis is likely an important contributor to the
pathogenesis of COPD. Apoptosis of both inflammatory and alveolar
wall cells in emphysema was reported by several research
groups.22–25 Induction of apoptosis by active caspase-3 instillation
in mice caused architectural changes in the lungs similar to those
observed in human emphysema.26 Oxidant stress, such as that
resulting from cigarette smoke, results in the killing of lung cells
by both apoptosis and necrosis. Glutathione S-transferase was
shown to have a protective effect on tobacco smoke-induced
apoptosis.27,28
Other studies have implicated endogenous growth factors and
cytokines in COPD-associated apoptosis. One example is vascular
endothelial growth factor (VGEF); decreased VGEF expression or
blockade of VGEF receptors caused emphysematous changes in
lung architecture.29,30 Another group of endogenous mediators
believed to play role in apoptosis of parenchymal cells of the lung
in COPD is TNF-a, FasL and Fas. However, it is unclear at present
whether these molecules are elevated or contribute to the severity of
COPD31–33 and more work is needed to clarify these issues.
Recent studies support the notion that apoptosis contributes to
the pathogenesis of lung fibrosis as well as to its resolution. Many
Apoptosis and angiotensin II in meconium aspiration
BD Uhal and A Abdul-Hafez
S109
studies strongly suggest a role of epithelial apoptosis as a key
profibrotic event in lung fibrogenesis. Apoptosis of alveolar
epithelial cells (AECs) is found in patients with idiopathic PF (IPF)
and in animal models of the disease.34–36 The epithelial apoptosis
colocalizes with myofibroblasts where collagen deposition is
severe.37 Blockade of apoptosis by either the angiotensin-converting
enzyme (ACE) inhibitor captopril, caspase inhibition or the forced
expression of p21 in lung epithelial cells exerted antifibrotic effects
in animal models.36,38,39 Together, these data suggested that the
fibrotic response is secondary to the apoptotic death of epithelial
cells in the lung. The death receptor Fas was found to be expressed
in AECs within the lungs of IPF patients and circulating levels of its
ligand FasL correlated with disease activity.40–42 Similar
observations were observed in animal models.43,44
Activation of Fas-induced apoptosis of bronchial and AECs is
sufficient to initiate a fibrotic response in animal models.43
However, development of bronchiolar and alveolar epithelial
apoptosis and fibrosis in the lungs of Fas-null mice was similar to
wild-type mice,45,46 which suggests that other pathways different
from Fas can initiate epithelial apoptosis and facilitate fibrogenesis.
Other works have suggested the involvement of c-Jun N-terminal
kinase and p38, members of the mitogen-activated protein kinases
family pathways in the signaling of epithelial apoptosis.46,47
Finally, several lines of evidence have shown that the angiotensin
system plays a critical role in AECs apoptosis and in lung
fibrogenesis, and these will be discussed next.
The endocrine RAS versus local tissue RASes
In the classical or ‘endocrine’ RAS described many years ago, the
58 kDa precursor protein angiotensinogen (AGT) is synthesized
primarily by the liver and is secreted into the circulation, where it
is proteolytically cleaved by the kidney-derived aspartyl protease
renin to yield the 10 amino-acid peptide angiotensin I (ANGI).
ANGI travels through the vasculature and on entry into the
pulmonary vascular bed, is cleaved by the dipeptidyl
carboxypeptidase ACE to yield the eight amino-acid peptide
angiotensin II (ANGII). ANGII then travels through the blood and
elicits a number of endocrine functions including the release of
aldosterone from the adrenals and contraction of vascular smooth
muscle throughout a variety of vascular beds.48
More recently described local RASes, which occur in virtually
ever organ and tissue in which their presence has been evaluated,
are classified as ‘extrinsic’ or ‘intrinsic’ depending on the origin of
RAS system components. In extrinsic local RASes (RASL[ext]), such
as is believed to occur in the heart,49 one or more of the RAS
components are synthesized locally but others are derived from the
endocrine RAS. In intrinsic local RASes (RASL[int]), all of the
components of a functional RAS are synthesized locally. In many
local organ systems the term ‘RAS’ is a misnomer because often
renin is not expressed locally but other aspartyl proteases such as
Journal of Perinatology
 Apoptosis and angiotensin II in meconium aspiration
BD Uhal and A Abdul-Hafez
S110
cathepsin D can accomplish the same proteolytic conversion of AGT
to ANGI; moreover, other dipeptidyl carboxypeptidases such as
chymase can cleave ANGI to ANGII. For this reason, many tissues
have ‘ACE-independent’ pathways of ANGII formation that may not
be affected by specific ACE inhibitors. Regardless, most tissues
exhibit some degree of expression of the major ANGII receptor
subtypes AT1 and AT2, either at baseline or after injury, which
often upregulates one or both of these receptors.50
The role of RASes in lung injury and fibrogenesis
A wealth of literature supports the ability of ACE inhibitors or ANG
receptor blockers to attentuate experimental lung cell death and
fibrogenesis. The prototype ACE inhibitors captopril or lisinopril
attenuated experimental lung fibrosis induced by monocrotaline,51
bleomycin52 or paraquat;53 the ACE inhibitors also were found to
be potent inhibitors of bleomycin- or TNF-a-induced apoptosis of
lung epithelial cells.54,55 The ANG receptor AT1 antagonists
losartan or candesartan have been shown to prevent lung fibrosis
in response to radiation56 or bleomycin,57,58 and also blocked
apoptosis of lung alveolar epithelial cells in response to
bleomycin57 or the antiarrhythmic agent amiodarone.59 Given the
central role of epithelial cells death in lung injury in a variety of
disease states, attention as been focused on defining the role of
ANGII in apoptosis of lung alveolar epithelials (AECs).
In cell culture studies, ANGII itself was found to be a potent
inducer of apoptosis in both the human AEC cell line A549 and in
primary AECs isolated from adult rats.60 The EC50s for
ANGII-induced apoptosis in these cells (50 and 10 nM, respectively)
are far above the concentrations of ANGII found in the circulation
(B1 to 10 pM), even in severe hypertension,48 which provides an
explanation of why circulating ANGII does not kill AECs as it passes
through the lungs. Although the cultured AECs expressed both the
AT1 and AT2 ANG receptor subtypes, only AT1-selective antagonists
could prevent ANGII-induced apoptosis.61
Subsequent studies found that when cultured AECs are exposed
to other inducers of apoptosis, they begin to synthesize their own
ANGII de novo, that is, from the precursor AGT. The apoptosis
inducers Fas ligand,62 TNF-a,55 bleomycin54 and amiodarone63 all
induce expression of the gene for AGT in AECs at the level of AGT
transcription, and cause a significant increase in the steady-state
level of AGT mRNA. Moreover, AECs express all the enzymes
necessary for conversion of AGT to the mature peptide ANGII,
including ACE62 and the aspartyl protease cathepsin D.64 Thus, the
alveolar epithelial cell expresses its own intrinsic local RAS in
response to a variety of apoptosis inducers.
Further work showed that the expression of this local RAS and
the generation of ANGII by AECs are required for AEC apoptosis in
response to FAS ligand, TNF-a, bleomycin or amiodarone. In all
those cases, AEC apoptosis in response to the inducer could be
blocked by either ANG receptor blockers,55,62 ACE inhibitors54,55 or
Journal of Perinatology
by antisense oligonucleotides against AGT mRNA.54,55,62 Therefore,
not only do AECs produce ANGII in response to apoptosis inducers,
but the ANGII production is an essential component of the
apoptotic response and blockade of ANGII production or receptor
interaction can block the cell death. Recent studies of human lung
biopsies from patients with IPF have found double labeling of ANG
peptides and markers of apoptosis in AECs;65 these data support the
notion that ANG peptide synthesis occurs in apoptotic AECs in the
intact injured human lung as well as in animal models and
cultured cells.
The role of RAS in MAS and potential for
pharmacologic control
Apoptotic AECs have been observed in several models of
experimental MAS.66,67 Interest therefore was turned to the
possibility that AECs dying in response to meconium might also
require the local intrinsic RAS for their death and might be
prevented from dying by ACE inhibitors of ANG receptor blockers.
Several lines of investigation support the theory that meconium
does indeed induce expression of the local RAS that might be
amenable to pharmacologic blockade. Pretreatment of 2-week-old
rabbit pups with the ACE inhibitor captopril before
intratracheal instillation of human meconium prevented both
meconium-induced upregulation of interleukin (IL)-6 and IL-8
and also reduced lung epithelial cell apoptosis detected by DNA
fragmentation assay.68 The captopril also appeared to reduced
meconium-induced upregulation of AGT mRNA.
In a similar study of rats, Lukkarinen et al.69 showed that
pretreatement of rats with the nonselective ANG receptor blocker
saralasin, given intraperitoneally before meconium instillation,
prevented AEC apoptosis detected by both DNA fragmentation assay
and electron microscopy. The meconium increase AGT mRNA in
the lungs, suggesting apoptosis-induced upregulation of AGT in
AECs. The saralasin also reduced meconium-induced upregulation
of endothelial monocyte-activating polypeptide (EMAP-II) and total
lung neutrophil accumulation, but had little effect on the
increased wet/dry weight ratio induced by the meconium. The
saralasin also had a small but statistically insignificant ability to
reverse altered arterial blood gases.
Although not statistically significant, this latter result suggests
that further refinement of dosing or route of administration might
offer improvement of lung function, especially if more potent and
selective ANGII antagonists (AT1 receptor-selective) or intratracheal
administrations are explored. Regardless, all the data taken
together strongly suggest that meconium, like other inducers of
AEC apoptosis and lung injury, elicits the expression of the local
RAS in AECs and that the RAS is intimately involved in their
demise. Given the many agents already in clinical use for the
manipulation of the RAS, it is proposed that further exploration of
antagonists of the RAS and creative approaches to their application

(for example, aerosols) may offer clinically useful adjuncts to the
current pharmacotherapy of MAS in the future.
Disclosure
BD Uhal has received consulting fees from Actelion Pharmaceuticals. A Abdul-Hafez has
declared no financial interests.
References
1 Edinger AL, Thompson CB. Death by design: apoptosis, necrosis and autophagy. Curr
Opin Cell Biol 2004; 16(6): 663–669.
2 Fink SL, Cookson BT. Apoptosis, pyroptosis, and necrosis: mechanistic description of
dead and dying eukaryotic cells. Infect Immun 2005; 73(4): 1907–1916.
3 Krantic S, Mechawar N, Reix S, Quirion R. Apoptosis-inducing factor: a matter of
neuron life and death. Prog Neurobiol 2007; 81(3): 179–196.
4 Li X, Shu R, Filippatos G, Uhal BD. Apoptosis in lung injury and remodeling. J Applied
Physiol. Invited Review, 2004; 97(4): 1535–1542.
5 Albertine KH, Soulier MF, Wang Z, Ishizaka A, Hashimoto S, Zimmerman GA et al. Fas
and fas ligand are up-regulated in pulmonary edema fluid and lung tissue of
patients with acute lung injury and the acute respiratory distress syndrome. Am
J Pathol 2002; 161: 1783–1796.
6 Hodge SJ, Hodge GL, Reynolds PN, Scicchitano R, Holmes M. Increased production of
TGF-beta and apoptosis of T lymphocytes isolated from peripheral blood in COPD. Am
J Physiol Lung Cell Mol Physiol 2003; 285(2): L492–L499.
7 Matute-Bello G, Liles WC, Steinberg KP, Kiener PA, Mongovin S, Chi EY et al. Soluble
Fas ligand induces epithelial cell apoptosis in humans with acute lung injury (ARDS).
J Immunol 1999; 163: 2217–2225.
8 Ware LB, Matthay MA. Alveolar fluid clearance is impaired in the majority of patients
with acute lung injury and the acute respiratory distress syndrome. Am J Respir Crit
Care Med 2001; 163: 1376–1383.
9 Guinee D, Brambilla E, Fleming M, Hayashi T, Rahn M, Koss M et al. The potential
role of BAX and BCL-2 expression in diffuse alveolar damage. Am J Pathol 1997; 151:
999–1007.
10 Husain KD, Stromberg PE, Javadi P, Buchman TG, Karl IE, Hotchkiss RS et al. Bcl-2
inhibits gut epithelial apoptosis induced by acute lung injury in mice but has no effect
on survival. Shock 2003; 20: 437–443.
11 Pittet JF, Mackersie RC, Martin TR, Matthay MA. Biological markers of acute lung
injury: prognostic and pathogenetic significance. Am J Respir Crit Care Med 1997;
155: 1187–1205.
12 Goodman ER, Stricker P, Velavicius M, Fonseca R, Kleinstein E, Lavery R et al. Role of
granulocyte-macrophage colony-stimulating factor and its receptor in the genesis of
acute respiratory distress syndrome through an effect on neutrophil apoptosis. Arch
Surg 1999; 134: 1049–1054.
13 Matute-Bello G, Liles WC, Radella II F, Steinberg KP, Ruzinski JT, Hudson LD et al.
Modulation of neutrophil apoptosis by granulocyte colony-stimulating factor and
granulocyte/macrophage colony-stimulating factor during the course of acute
respiratory distress syndrome. Crit Care Med 2000; 28: 1–7.
14 Parsey MV, Kaneko D, Shenkar R, Abraham E. Neutrophil apoptosis in the lung after
hemorrhage or endotoxemia: apoptosis and migration are independent of IL-1beta.
Clin Immunol 1999; 91: 219–225.
15 Matute-Bello G, Liles WC, Radella II F, Steinberg KP, Ruzinski JT, Hudson LD et al.
Modulation of neutrophil apoptosis by granulocyte colony-stimulating factor and
granulocyte/macrophage colony-stimulating factor during the course of acute
respiratory distress syndrome. Crit Care Med 2000; 28: 1–7.
16 Klein JB, Rane MJ, Scherzer JA, Coxon PY, Kettritz R, Mathiesen JM et al.
Granulocyte-macrophage colony-stimulating factor delays neutrophil constitutive
Apoptosis and angiotensin II in meconium aspiration
BD Uhal and A Abdul-Hafez
S111
apoptosis through phosphoinositide 3-kinase and extracellular signal-regulated kinase
pathways. J Immunol 2000; 164: 4286–4291.
17 Leuenroth SJ, Grutkoski PS, Ayala A, Simms HH. The loss of Mcl-1 expression in
human polymorphonuclear leukocytes promotes apoptosis. J Leukoc Biol 2000; 68:
158–166.
18 Derouet M, Thomas L, Cross A, Moots RJ, Edwards SW. GM-CSF signalling and
proteasome inhibition delay neutrophil apoptosis by increasing the stability of Mcl-1.
J Biol Chem 2004; 279(26): 26915–26921.
19 Hamacher J, Lucas R, Lijnen HR, Buschke S, Dunant Y, Wendel A et al. Tumor necrosis
factor-alpha and angiostatin are mediators of endothelial cytotoxicity in
bronchoalveolar lavages of patients with acute respiratory distress syndrome. Am J
Respir Crit Care Med 2002; 166: 651–656.
20 Luca R, Lijnen HR, Suffredini AF, Pepper MS, Steinberg KP, Martin TR et al. Increased
angiostatin levels in bronchoalveolar lavage fluids from ARDS patients and from
human volunteers after lung instillation of endotoxin. Thromb Haemost 2002; 87:
966–971.
21 Machino T, Hashimoto S, Maruoka S, Gon Y, Hayashi S, Mizumura K et al. Apoptosis
signal-regulating kinase 1-mediated signaling pathway regulates hydrogen
peroxide-induced apoptosis in human pulmonary vascular endothelial cells.
Crit Care Med 2003; 31: 2776–2781.
22 Kasahara Y, Tuder RM, Cool CD, Voelkel NF. Expression of 15-lipoxygenase and
evidence for apoptosis in the lungs from patients with COPD. Chest 2000;
117(5 Suppl 1): 260S.
23 Kelly MG, Elborn JS, Kelly MG, Brown V, Ennis M. Measuring granulocyte apoptosis in
airway inflammation. Thorax 2002; 57(4): 376; author reply 376. 30: Imai K,
D’Armiento J. Differential gene expression of sFRP-1 and apoptosis in pulmonary
emphysema. Chest. 2002;121(3 Suppl):7S.
24 Majo J, Ghezzo H, Cosio MG. Lymphocyte population and apoptosis in the
lungs of smokers and their relation to emphysema. Eur Respir J 2001; 17(5):
946–953.
25 Yokohori N, Aoshiba K, Nagai A, Respiratory Failure Research Group in Japan.
Increased levels of cell death and proliferation in alveolar wall cells in patients with
pulmonary emphysema. Chest 2004; 125(2): 626–632.
26 Aoshiba K, Yokohori N, Nagai A. Alveolar wall apoptosis causes lung destruction and
emphysematous changes. Am J Respir Cell Mol Biol 2003; 28: 555–562.
27 Ishii T, Fujishiro M, Masuda M, Nakajima J, Teramoto S, Ouchi Y et al. Depletion of
glutathione S-transferase P1 induces apoptosis in human lung fibroblasts. Exp Lung
Res 2003; 29(7): 523–536.
28 Ishii T, Matsuse T, Igarashi H, Masuda M, Teramoto S, Ouchi Y. Tobacco smoke
reduces viability in human lung fibroblasts: protective effect of glutathione
S-transferase P1. Am J Physiol Lung Cell Mol Physiol 2001; 280(6):
L1189–L1195.
29 Tsao PN, Su YN, Li H, Huang PH, Chien CT, Lai YL et al. Overexpression of placenta
growth factor contributes to the pathogenesis of pulmonary emphysema. Am J Respir
Crit Care Med 2004; 169(4): 505–511.
30 Kasahara Y, Tuder RM, Taraseviciene-Stewart L, Le Cras TD, Abman S, Hirth PK et al.
Inhibition of VEGF receptors causes lung cell apoptosis and emphysema. J Clin Invest
2000; 106(11): 1311–1319.
31 Lucey EC, Keane J, Kuang PP, Snider GL, Goldstein RH. Severity of elastase-induced
emphysema is decreased in tumor necrosis factor-alpha and interleukin-1beta
receptor-deficient mice. Lab Invest 2002; 82(1): 79–85.
32 Takabatake N, Nakamura H, Inoue S, Terashita K, Yuki H, Kato S et al. Circulating
levels of soluble Fas ligand and soluble Fas in patients with chronic obstructive
pulmonary disease. Respir Med 2000; 94(12): 1215–1220.
33 Yasuda N, Gotoh K, Minatoguchi S, Asano K, Nishigaki K, Nomura M et al. An increase
of soluble Fas, an inhibitor of apoptosis, associated with progression of COPD. Respir
Med 1998; 92(8): 993–999.
34 Kuwano K, Kunitake R, Kawasaki M. p21 and p53 expression in association with DNA
strand breaks in idiopathic pulmonary fibrosis. Am J Respir Crit Care Med 1996; 154:
477–483.
Journal of Perinatology
 Apoptosis and angiotensin II in meconium aspiration
BD Uhal and A Abdul-Hafez
S112
35 Hagimoto N, Kuwano K, Nomoto Y, Kunitake R, Hara N. Apoptosis and expression of
Fas/Fas ligand mRNA in bleomycin-induced pulmonary fibrosis in mice. Am J Respir
Cell Mol Biol 1997; 16: 91–101.
36 Wang R, Ibarra-Sunga O, Verlinski L, Pick R, Uhal BD. Abrogation of
bleomycin-induced epithelial apoptosis and lung fibrosis by captopril or by a caspase
inhibitor. Am J Physiol Lung Cell Mol Physiol 2000; 279: L143–L151.
37 Uhal BD, Joshi I, Hughes WF, Ramos C, Pardo A, Selman M. AEC death adjacent to
underlying myofibroblasts in advanced fibrotic human lung. Am J Physiol 1998; 275:
L1192–L1199.
38 Kuwano K, Kunitake R, Maeyama T, Hagimoto N, Kawasaki M, Matsuba T et al.
Attenuation of bleomycin-induced pneumopathy in mice by a caspase inhibitor. Am J
Physiol Lung Cell Mol Physiol 2001; 280: L316–L325.
39 Inoshima I, Kuwano K, Hamada N, Hagimoto N, Yoshimi M, Maeyama T et al.
Anti-monocyte chemoattractant protein-1 gene therapy attenuates pulmonary
fibrosis in mice. Am J Physiol Lung Cell Mol Physiol 2004; 286:
L1038–L1044.
40 Domagala-Kulawik J, Droszcz P, Kraszewska I, Chazan R. Expression of Fas antigen in
the cells from bronchoalveolar lavage fluid (BALF). Folia Histochem Cytobiol 2000;
38: 185–188.
41 Kazufumi M, Sonoko N, Masanori K, Takateru I, Akira O. Expression of bcl-2 protein
and APO-1 (Fas antigen) in the lung tissue from patients with idiopathic pulmonary
fibrosis. Microsc Res Tech 1997; 38: 480–487.
42 Kuwano K, Maeyama T, Inoshima I, Ninomiya K, Hagimoto N, Yoshimi M et al.
Increased circulating levels of soluble Fas ligand are correlated with disease activity in
patients with fibrosing lung diseases. Respirology 2002; 7: 15–21.
43 Hagimoto N, Kuwano K, Miyazaki H. Induction of apoptosis and pulmonary fibrosis
in mice in response to ligation of FAS antigen. Am J Respir Cell Mol Biol 1997; 17:
272–278.
44 Hagimoto N, Kuwano K, Nomoto Y, Kunitake R, Hara N. Apoptosis and expression of
Fas/Fas ligand mRNA in bleomycin-induced pulmonary fibrosis in mice. Am J Respir
Cell Mol Biol 1997; 16: 91–101.
45 Aoshiba K, Yasui S, Tamaoki J, Nagai A. The Fas/Fas-ligand system is not required for
bleomycin-induced pulmonary fibrosis in mice. Am J Respir Crit Care Med 2000; 162:
695–700.
46 Matsuoka H, Arai T, Mori M, Goya S, Kida H, Morishita H et al. p38 MAPK inhibitor,
FR-167653, ameliorates murine bleomycin-induced pulmonary fibrosis. Am J Physiol
Lung Cell Mol Physiol 2022; 283: L103–L112.
47 Yoshida K, Kuwano K, Hagimoto N, Watanabe K, Matsuba T, Fujita M et al. MAP
kinase activation and apoptosis in lung tissues from patients with idiopathic
pulmonary fibrosis. J Pathol 2002; 198: 388–396.
48 Filippatos G, Tilak M, Pinillos H, Uhal BD. Regulation of apoptosis by
angiotensin II in the heart and lungs (Invited Review). Int J Mol Med 2001; 7(3):
273–280.
49 Von Lutterotti N, Catanzaro DF, Sealey JE, Laragh JH. Renin is not synthesized by
cardiac and extrarenal vascular tissues. A review of experimental evidence. Circulation
1994; 89: 458–470.
50 Atlas SS. The renin angiotensin system revisited: classical and nonclassical pathways of
angiotensin formation. Mount Sinai J Med 1998; 65: 87–96.
51 Molteni A, Ward W, Ts’ao C, Solliday N, Dunne M. Monocrotaline-induced pulmonary
fibrosis in rats: amelioration by captopril and penicillamine. Proc Soc Exp Biol Med
1985; 180: 112–120.
Journal of Perinatology
52 Wang R, Ibarra-Sunga O, Pick R, Uhal BD. Abrogation of bleomycin-induced epithelial
apoptosis and lung fibrosis by captopril or by a caspase inhibitor. Am J Physiol Lung
Cell Mol Physiol 2000; 279: L143–L151.
53 Mohammadi-Karakani A. Lisinopril ameliorates paraquat-induced lung fibrosis. Clin
Chim Acta 2006; 367(1–2): 170–174.
54 Li X, Zhang H, Soledad-Conrad V, Zhuang J, Uhal BD. Bleomycin-induced apoptosis of
alveolar epithelial cells requires angiotensin synthesis de novo. Am J Physiol 2003;
284(3): L501–L507.
55 Wang R, Alam G, Zagariya A, Gidea G, Pinillos H, Lalude O et al. Apoptosis of lung
epithelial cells in response to TNF-alpha requires angiotensin II generation de novo.
J Cell Physiol 2000; 185(2): 253–259.
56 Molteni A, Moulder JE, Cohen EF, Ward WF, Fish BL, Taylor JM et al. Control of
radiation-induced pneumopathy and lung fibrosis by angiotensin-converting enzyme
inhibitors and an ANG II type 1 receptor blocker. Int J Radiat Biol 2000; 76: 523–532.
57 Li X, Rayford H, Uhal BD. Essential roles for angiotensin receptor AT1a in bleomycin-
induced apoptosis and lung fibrosis in mice. Am J Pathol 2003; 163(6): 2523–2530.
58 Otsuka M, Takahashi H, Shiratori M, Chiba H, Abe S. Reduction of bleomycin induced
lung fibrosis by candesartan cilexetil, an angiotensin II type 1 receptor antagonist.
Thorax 2004; 59(1): 31–38.
59 Uhal BD, Wang R, Laukka J, Zhuang J, Soledad-Conrad V, Filippatos G. Inhibition of
amiodarone-induced lung fibrosis but not alveolitis by angiotensin system antagonists.
Pharmacol Toxicol 2003; 92: 81–87.
60 Wang R, Zagariya A, Ibarra-Sunga O, Gidea C, Ang E, Deshmukh S et al. Angiotensin
II induces apoptosis in human and rat alveolar epithelial cells. Am J Physiol 1999;
276: L885–L889.
61 Papp M, Li X, Zhuang J, Wang R, Uhal BD. Angiotensin receptor subtype AT1
mediates alveolar epithelial cell apoptosis in response to ANGII. Am J Physiol 2002;
282: L713–L718.
62 Wang R, Zagariya A, Ang E, Ibarra-Sunga E, Uhal BD. Fas-induced apoptosis of
alveolar epithelial cells requires angiotensin II generation and receptor interaction. Am
J Physiol 1999; 277: L1245–L1250.
63 Uhal B, Zhang H, Abdul-Hafez A, Shu R, Uhal BD. Amiodarone induces
angiotensinogen gene expression in lung alveolar epithelial cells through AP-1. Basic
Clin Pharmacol Toxicol 2007; 100: 59–66.
64 Li X, Rayford H, Shu R, Zhuang J, Uhal BD. Essential role for cathepsin D in
bleomycin-induced apoptosis of alveolar epithelial cells. Am J Physiol 2004; 287(1):
L46–L51.
65 Li X, Molina-Molina M, Abdul-Hafez A, Ramirez J, Serrano-Mollar A, Xaubet A et al.
Extravascular sources of lung angiotensin peptide synthesis in idiopathic pulmonary
fibrosis. Am J Physiol Lung Cell Mol Physiol 2006; 291(5): L887–L895.
66 Lu MP, Du LZ, Gu WZ, Chen XX. Nitric oxide inhalation inhibits inducible nitric oxide
synthase but not nitrotyrosine formation and cell apoptosis in rat lungs with
meconium-induced injury. Acta Pharmacol Sin 2005; 26(9): 1123–1129.
67 Zagariya A, Bhat R, Chari G, Uhal B, Navale S, Vidyasagar D. Apoptosis of airway
epithelial cells in response to meconium. Life Sci 2005; 76(16): 1849–1858.
68 Zagariya A, Bhat R, Navale S, Chari G, Vidyasagar D. Inhibition of meconium-induced
cytokine expression and cell apoptosis by pretreatment with captopril. Pediatrics 2006;
117(5): 1722–1727.
69 Lukkarinen H, Laine J, Lehtonen J, Zagariya A, Vidyasagar D, Aho H et al.
Angiotensin II receptor blockade inhibits pneumocyte apoptosis in experimental
meconium aspiration. Pediatr Res 2004; 55(2): 326–333.
 Journal of Perinatology (2008) 28, S113–S115
r 2008 Nature Publishing Group All rights reserved. 0743-8346/08 $30
www.nature.com/jp

REVIEW
Toxic effects of different meconium fractions on lung function:
new therapeutic strategies for meconium aspiration syndrome?
OD Saugstad1,2, PA Tølløfsrud1,2, P Lindenskov1,2 and CA Drevon1,2
1
Department of Pediatric Research and Pediatrics, Rikshospitalet Medical Center, University of Oslo, Oslo, Norway and
2
Department of Nutrition, Institute of Basic Medical Sciences, Medical Faculty, University of Oslo, Oslo, Norway

bile acids of meconium are cholic, hydroxycholic and

To review and summarize experimental data examining the effects of
different fractions of meconium, and to test the effect of albumin on
meconium aspiration both as prophylactic and rescue treatment. Newborn
piglets 2 to 5 days of age were made hypoxic and then instilled meconium
or fractions of meconium intratracheally. Meconium-added albumin and
albumin instilled after meconium were also tested. Lung function and
inflammatory cytokines were measured. Both the lipid- and water-soluble
fractions induce inflammation in the lungs with elevation of inflammatory
cytokines. When meconium was mixed with albumin, the inflammatory
effects of meconium were significantly ameliorated. Rescue therapy with
intratracheal albumin 5 min after the meconium aspiration syndrome was
induced also improved lung function. These results indicate that at least
part of the symptoms seen in the meconium aspiration syndrome could be
prevented by blocking the active substances of meconium such as bile acids
and free fatty acids.
Journal of Perinatology (2008) 28, S113–S115; doi:10.1038/jp.2008.151
Introduction
Aspiration of meconium in the airways of the newborn infant leads
to plugging and subsequently to inflammatory changes, surfactant
inhibition, respiratory failure and pulmonary hypertension. There
have been three approaches to moderate these effects: (1) dilute
meconium (amnioinfusion, lavage), (2) improve/‘strengthen’
surfactant by adding for instance polymers and (3) inhibit harmful
effects of meconium per se.
Meconium is a complex mixture containing 75 to 80% water.
The other chemical components can be divided into lipid- and
water-soluble material. The lipid includes free fatty acids (FFA),
triglycerides and cholesterol. The FFA include mainly palmitic,
stearic and oleic acids. The water-soluble components include bile
acids, bilirubin, dietary fibers and inorganic molecules. The main
Correspondence: Dr OD Saugstad, Pediatrisk Forskningsinstitutt, Rikshospitalet, Oslo 0027,
Norway.
E-mail: o.d.saugstad@medisin.uio.no
chenodeoxycholic acids.1–3
These different subfractions of meconium are leading to reduced
activity of surfactant and inflammation therefore promoting
atelectasis and impaired gas exchange. Both hydrophilic and
hydrophobic components of meconium reduce surfactant function.
Furthermore, the lipid fraction has a stronger inhibitory effect on
surfactant function than the water-soluble subfraction.4–7 It is well
known that bile salts may cause inflammation8,9 and that FFA,
such as oleic acid, induce severe lung failure when administered
intravenously.10
We have tested the effects of different meconium subfractions on
pulmonary function in an animal model of newborn piglets.
Because albumin is able to bind FFA, we also examined whether
albumin added to meconium alleviates the clinical symptoms of
meconium aspiration. In case such an inhibitory effect was found,
perhaps this also could be used as rescue therapy. We therefore
developed a rescue model instilling albumin intratracheally
following meconium aspiration. In this article we summarize and
review some of our previous results.
Methods
We used newborn piglets undergoing hypoxia and meconium
aspiration followed by resuscitation.11 Human meconium was
collected from healthy term newborns and diluted in distilled
water, and rotavaporated dry. It was sterilized by g-irradiation and
diluted with sterile water to 110 mg dry weight per ml. Hypoxia
with FiO2 of 0.08 was induced in newborn piglets 2 to 5 days of
age until they were close to collapse. The piglets were then
hand-ventilated for 30 s and put back on ventilator with rate
increased from 30 to 60 per min, and peak inspiratory pressure
increased to 5 Cm H2O. Tidal volume was kept constant at 10 to
15 ml kg 1 and paCO2 4.5 to 6 kPa (34 to 45 mm Hg).
In the first study 3 ml kg 1 body weight of meconium was
instilled intratracheally giving an oxygenation index (OI) of 6 to 7
indicating a mild meconium aspiration syndrome (MAS). In this
 Toxic effects of meconium fractions on lung
OD Saugstad et al
S114
study meconium was also mixed with albumin (30% albumin in
stoichiometric relation to FFA estimated to be 1.4 ml kg 1) before
intratracheal in administration of the mixture.12,13
In the second study 3 ml kg 1 body weight of either meconium,
lipid- or water-soluble meconium fractions was instilled
intratracheally in separate groups.14
In the third study a moderate and severe model was developed. A
moderate MAS was developed by instilling meconium of 4 ml kg 1
body weight, giving an OI of approximately 12. A severe MAS was
induced by instilling 5 ml meconium mixture per kg leading to a
typical OI of 25. Five min after meconium had been instilled, 3 ml
of 30% albumin was instilled intratracheally.15
The animals in all studies were resuscitated for 8 h with room
air or supplemental O2 to achieve a SaO2 of 85%.
Results
Effect of albumin
Animals receiving albumin mixed with meconium compared with
meconium alone had a significantly reduced OI as well as
ventilation index. OI was approximately 50% in animals given
meconium mixed with albumin compared with those given
meconium. The need for oxygen and mean airway pressure on the
ventilator was also significantly reduced and compliance increased
in animals given the combination of meconium and albumin.12
The inflammatory cytokine interleukin (IL)-8 in tracheal
aspirate, 8 h after reoxygenation was started, was in mean fivefold
higher (P<0.05) in animals given meconium alone (93 pg ml 1)
as compared to those given meconium mixed with albumin
(18 pg ml 1) before intratracheal instillation.13
Effect of meconium fractions
Both the lipid- and water-soluble fractions of meconium induced
inflammatory changes, however, more in the lipid extract than the
water extract group. At 8 h after instillation of meconium, IL-8 in
tracheal aspirate was threefold higher in the lipid extract group than
in the animals given the water-soluble fractions of meconium. Intact
meconium had more severe effects on the lungs than the lipid- and
water-soluble fractions of meconium separately.14
Effect of rescue therapy with albumin
Rescue therapy with a low-dose intratracheal albumin
(stoichiometric binding of FFA) did not affect OI or IL-8 in tracheal
aspirate when measured at 8 h. However, a significantly attenuated
decrease in lung compliance was found.15
Discussion
In these studies we found that albumin inhibits clinical effects of
meconium and that the lipid fraction is more harmful than the
water fraction. Rescue therapy with albumin increases compliance
Journal of Perinatology
but does not seem to have significant clinical effects in the doses
and administration tested out.
Both the lipid- and water-soluble fractions of meconium induce
pulmonary inflammation and injury, however, less than
meconium alone. The lipid fraction was responsible for most of
the cytokine activation assessed by IL-8 in tracheal aspirate.
However, the water-soluble fraction also had some inflammatory
properties.
We chose albumin as a protein with high capacity to bind FFA
in spite of the fact it is known that albumin also inhibits surfactant
function. However, in our animal model, and probably in patients
with MAS as well, surfactant function is already abolished.
Albumin added to meconium before intratracheal instillation,
almost completely blocked the negative effects of meconium.
Albumin seems to prevent development of MAS when added to
meconium. However, when albumin is given as rescue therapy the
effect is less clear. In another of our studies rescue therapy by
lavaging the lungs with excess albumin gave worse outcome than
meconium alone (submitted). Rescue therapy with albumin in low
doses and low volume therefore had a small but significant and
positive effect on compliance, however, larger doses and volumes
seem to have a detrimental effect on pulmonary function.
The optimal albumin dose, timing and mode of administration
therefore are unknown. Other inhibitors of both the lipid- and
water-soluble fractions of meconium should be searched for and
tested aiming at developing an efficient rescue therapy. Several
such inhibitors as polymers and anti-inflammatory agents16,17
have been described already. Furthermore, meconium-induced
apoptosis may be inhibited by angiotensin-converting enzyme
inhibitors, which may reduce injury.18,19
Meconium is a potent complement activator and complement
inhibition may represent a future therapeutic principle in MAS.20
In the future all the principles discussed above may be combined to
develop a powerful therapy for MAS. Given the available presented
data, there does not appear to be a robust argument for the use of
albumin in the human infant with meconium aspiration, let alone
suggest that there may be ‘an optimum dose’, timing and mode of
administration.
Acknowledgments
These studies have been supported by the Norwegian Council for Research.
Disclosure
The authors have declared no financial interests.
References
1 Terasaka D, Clark DA, Singh BN, Rokahr J. Free fatty acids of human meconium.
Biol Neonate 1986; 50: 16–20.

2 Harries JT. Meconium in health and disease. Br Med Bull 1978; 34: 75–78.
3 Moses D, Holm BA, Spitale P, Liu MY, Enhorning G. Inhibition of pulmonary
surfactant function by meconium. Am J Obstet Gynecol 1991; 164: 477–481.
4 Sun B, Curstedt T, Robertson B. Surfactant inhibition in experimental meconium
aspiration. Acta Paediatr 1993; 82: 182–189.
5 Schrama AJ, de Beaufort AJ, Sukul YR, Jansen SM, Poorthuis BJ, Berger HM.
Phospholipase A2 is present in meconium and inhibits the activity of pulmonary
surfactant: an in vitro study. Acta Paediatr 2001; 90: 412–416.
6 Hall SB, Lu RZ, Venkitaraman AR, Hyde RW, Notter RH. Inhibition of pulmonary
surfactant by oleic acid: mechanisms and characteristics. J Appl Physiol 1992; 72:
1708–1716.
7 Wang Z, Notter RH. Additivity of protein and nonprotein inhibitors of lung surfactant
activity. Am J Respir Crit Care Med 1998; 158: 28–35.
8 Henderson RD, Fung K, Cullen JB, Milne EN, Marryatt G. Bile aspiration: an
experimental study in rabbits. Can J Surg 1975; 18: 64–69.
9 Zecca E, Costa S, Lauriola V, Vento G, Papacci P, Romagnoli C. Bile acid pneumonia: a
‘new’ form of neonatal respiratory distress syndrome? Pediatrics 2004; 114: 269–272.
10 Trawoger R, Cereda M, Kolobow T. A standardized method of oleic acid infusion in
experimental acute respiratory failure. Scand J Clin Lab Invest 2001; 61: 75–81.
11 Tllfsrud PA, Solås AB, Saugstad OD. Newborn piglets with meconium aspiration
resuscitated with room air or 100% oxygen. Pediatr Res 2001; 50: 423–429.
12 Tllfsrud PA, Medb S, Solås AB, Drevon CA, Saugstad OD. Albumin mixed with
meconium attenuates pulmonary dysfunction in a newborn piglet model with
meconium aspiration. Pediatr Res 2002; 52: 545–553.
Toxic effects of meconium fractions on lung
OD Saugstad et al
S115
13 Tllfsrud PA, Medb S, Solås AB, Robertson B, Speer CP, Seidenspinner S et al.
Intratracheal albumin reduces interleukin-8 in tracheobronchial aspirates in piglets
after meconium aspiration. J Perinat Med 2004; 32: 78–83.
14 Tllfsrud PA, Lindenskov PH, Drevon CA, Speer CP, Seidenspinner S,
Saugstad OD. Comparison of pulmonary and inflammatory effects of lipid- and water-
soluble components in meconium in newborn piglets. Biol Neonate 2003; 84:
330–337.
15 Lindenskov PH, Castellheim A, Aamodt G, Saugstad OD. Meconium induced IL-8
production and intratracheal albumin alleviated lung injury in newborn pigs. Pediatr
Res 2005; 57: 371–377.
16 Lu KW, Goerke J, Clements JA, Taeusch HW. Hyaluronan reduces surfactant
inhibition and improves rat lung function after meconium injury. Pediatr Res 2005;
58: 206–210.
17 Korhonen K, Kiuru A, Svedstrom E, Kaapa P. Pentoxifylline reduces regional
inflammatory and ventilatory disturbances in meconium-exposed piglet lungs. Pediatr
Res 2004; 56: 901–906.
18 Zagariya A, Bhat R, Chari G, Uhal B, Navale S, Vidyasagar D. Apoptosis of airway
epithelial cells in response to meconium. Life Sci 2005; 76: 1849–1858.
19 Zagariya A, Bhat R, Navale S, Chari G, Vidyasagar D. Inhibition of meconium-induced
cytokine expression and cell apoptosis by pretreatment with captopril. Pediatrics 2006;
117: 1722–1727.
20 Castellheim A, Lindenskov PH, Pharo A, Fung M, Saugstad OD, Mollnes TE. Meconium
is a potent activator of complement in human serum and in piglets. Pediatr Res 2004;
55: 310–318.
Journal of Perinatology
 Journal of Perinatology (2008) 28, S116–S119
r 2008 Nature Publishing Group All rights reserved. 0743-8346/08 $30
www.nature.com/jp
REVIEW
The role of complement in meconium aspiration syndrome
TE Mollnes1, A Castellheim1,2, PHH Lindenskov1,2, B Salvesen1,2 and OD Saugstad2
1
Institute of Immunology, Rikshospitalet, Oslo, Norway and 2Department of Pediatric Research, Rikshospitalet, Medical Faculty,
University of Oslo, Oslo, Norway
The complement system is part of the host defense with a number of
biological effects, most of which contribute to the inflammatory reaction by
activation of cells like leukocytes and endothelial cells. An intact
complement system is required for protection against infection and for
maintaining internal inflammatory homeostasis. However, the system is a
double-edged sword as improperly or uncontrolled activation is
disadvantageous and potentially harmful for the host. Meconium aspiration
syndrome (MAS) is associated with a local inflammatory reaction in the
lungs, frequently described as a chemical pneumonitis. Cytokines,
arachidonic acid metabolites and reactive oxygen species are involved in
this reaction. We have recently documented that meconium is a potent
activator of complement in vitro and in an experimental piglet model of
MAS, the latter presenting with an inflammatory profile closely resembling
systemic inflammatory response syndrome. We postulate that complement
activation may contribute to the pathogenesis of MAS.
Journal of Perinatology (2008) 28, S116–S119; doi:10.1038/jp.2008.148
Inflammatory mediators in MAS
The pathophysiology of meconium aspiration syndrome (MAS) is
complex. Several mechanisms may be operative in the development
of lung injury in MAS, including mechanical obstruction,1
inactivation of surfactant,2 pulmonary hypertension3–6 and lung
inflammation.7 Although the controversies still remain in the
literature as to what extent meconium per se leads to MAS,8,9
increasing evidence indicates that the inflammation induced by
meconium is an essential part of the pathophysiology.7,10 The
assumed chemical pneumonitis caused by meconium was first
documented in rabbits by infiltration of polymorphonuclear
leukocytes in alveolar septa within 6 h after instillation of
meconium,11 and a later study documented increased counts and
chemotactic activity of neutrophils in lung lavage fluid from pigs.12
Leukocytes are important sources for three main inflammatory
Correspondence: Dr TE Mollnes, Institute of Immunology, Rikshospitalet, Oslo NO-0027,
Norway.
E-mail: t.e.mollnes@medisin.uio.no
branches that are induced or activated by meconium: cytokines,
arachidonic acid metabolites and reactive oxygen species.
In vitro meconium has been shown to trigger peritoneal
macrophages to produce a proinflammatory tumor necrosis
factor-a (TNF-a) response13 and in animals instilled with human
meconium interleukin (IL)-6, IL-8, TNF-a and IL-1b have been
detected in cells harvested from lung lavage within 8 h after
meconium instillation.14 In another animal studies with the same
observation time, TNF-a15 and IL-8 have been detected in
bronchoalveolar lavage fluid16,17 whereas IL-5 and IL-13 were
elevated at day 7 in mice.18 Furthermore, meconium may
stimulate chemotaxis, the latter effect claimed to be because of IL-8
present in meconium.19 Finally, the levels of IL-1b, IL-6, IL-8 and
TNF-a have been compared in sterile meconium vs meconium
contaminated with bacteria and in spite of variation among
samples the median concentrations did not differ.20 The
transcription factor NF-kB, known to induce a number of
proinflammatory cytokines, was shown to be activated by
meconium in rat alveolar macrophages.21
Human meconium is shown to contain phospholipase A2,
thereby capable of directly damaging alveolar cells.22 Meconium
was found to increase the production of arachidonic acid
metabolites by phospholipase A223 and elevated catalytic activity of
phospholipase A2 was found both in tissue and lavage fluid.24
In addition, meconium has been shown capable of upregulating
cyclooxygenase-2 mRNA expression in rat lungs, a process
that is inhibited by dexamethasone, but not indomethacin25
and enhance the release of thromboxane-A2 in human airway
epithelial cells.26 Finally, apoptosis associated with
meconium22,27,28 could be inhibited by angiotensin II receptor
blockade.27
The effect of meconium on oxidative burst (release of reactive
oxygen species) has been difficult to interpret. Although one study
showed that meconium stimulated alveolar macrophages to
produce superoxide anion,29 another study showed that light and
very light meconium inhibited neutrophil oxidative burst.30
Recently, it has been showed that meconium has an inhibitory
effect on neutrophils in low concentrations (0.2 mg ml 1) but in
higher concentrations (1 and 2 mg ml 1) stimulates neutrophil
oxygen radical production progressively.31 A possible role for

complement in meconium-induced oxidative burst has been
recently postulated (see below).
The complement system
Complement is one of the plasma cascade systems and represents a
main part of fluid-phase innate immunity.32 It consists of more
than 30 proteins acting together in a specific manner with the
principal function to protect the host against invading organisms.
A simplified cartoon of the complement system is shown in
Figure 1. There are three initial pathways of activation.
The classical pathway (upper left) is activated either by
natural or elicited antibodies binding to antigen, or by direct,
antibody-independent activation, for example, by C-reactive
protein. C1q triggers the serine proteases C1r and C1s, the latter
cleaving C4 to C4b, which exposes a specific binding site for C2.
C1s then cleaves C2 and the resulting C3 convertase C4b2a cleaves
C3 to C3b to form the C5 convertase C4b2a3b. Splitting of C5 to the
highly potent anaphylatoxin C5a and the C6-binding fragment C5b
is the last enzymatic step in the cascade. Activation of the lectin
pathway (upper middle) is initiated by mannose-binding lectin
(MBL) recognizing mannose on bacteria, by immunoglobulin A
and probably by structures exposed by damaged endothelium. MBL
is homologous to C1q and triggers the MBL-associated serine
proteases (MASPs), of which three forms (MASP1, MASP2 and
MASP3) have been described. Further lectin pathway activation is
virtually identical to classical pathway activation forming the same
C3 and C5 convertases. The alternative pathway (upper right)
activation differs from the classical and lectin pathways by a
physiological, low-graded spontaneous hydrolysis of the internal
thiol ester of the C3 molecule thereby binding factor B, which is
then cleaved by factor D. The C3 convertase then formed cleaves C3
to C3a and C3b, the latter binds factor B forming C3bB, which is
Figure 1 Schematic illustration of the complement system. Activation may
occur through three initial routes: the classical, the lectin and the alterative
pathways. All three pathways converge at the level of C3 and proceed through the
terminal pathway. Biologic active fragments are released when native components
are activated, like the potent anaphylatoxin C5a when C5 is cleaved. To keep the
system under strict control, a number of inhibitor and regulatory proteins are
present, as illustrated with dotted boxes and lines.
Complement and meconium
TE Mollnes et al
S117
cleaved by factor D and the alternative pathway C3 convertase,
C3bBb, is formed leading to further cleavage of C3. C5 is then
activated in the same manner as for the classical and lectin
pathway C5 convertase.
Thus, all three pathways converge at C3 and the terminal
pathway (lower part of Figure 1) proceeds in the same way
irrespective of the initial pathway activation, by assembly of C7 to
C5b-6, forming an amphiphilic complex able to insert into a lipid
membrane. One C5b-7 moiety binds one C8 and one or several C9
molecules, creating a physical pore penetrating the membrane
(C5b-9(m) or membrane attack complex, leading to
transmembrane leakage and subsequent cell activation, or more
infrequently to lysis (lower right). If the activation occurs in the
fluid phase and there is no membrane present, the C5b-7 complex
binds to vitronectin and clusterin (fluid-phase regulators of the
terminal pathway) and thus retains hydrophilic properties. Final
assembly of a soluble C5b-9 (SC5b-9), the second form of the
terminal complement complex (TCC), occurs by binding of C8 and
C9. Soluble TCC is a particularly useful indicator of complement
activation and an assay for use in humans, baboons and pigs has
been developed.33
Complement activation is strictly regulated by inhibitory
proteins of which some are illustrated in Figure 1 (boxes with
broken lines). The biological potency of this system is substantial
and when activated out of control it may break down essential
homeostatic mechanisms, for example, as seen in irreversible septic
shock.34 Thus, it is crucially important to keep the system under
control by the regulatory proteins. C1 inhibitor regulates the initial
classical and lectin pathways. Carboxypeptidase N inactivates the
anaphylatoxins C5a, C3a and C4a, of which C5a is particularly
potent. Factor I cleaves and inactivates C4b and C3b and uses
C4b-binding protein (C4BP) as cofactor in the classical/lectin
pathway and factor H in the alternative pathway. The membrane
regulators complement receptor 1 (CR1; CD35), membrane
cofactor protein (MCP; CD46) and decay-accelerating factor
(DAF; CD55) regulate complement activation by either acting as
cofactors for factor I-mediated cleavage of C4b and C3b (CR1 and
MCP), or accelerating the decay of the biomolecular C3 and C5
convertases (CR1 and DAF). CD59, also a membrane regulator,
prevents the binding of C9 to the C5b-8 complex in the terminal
pathway.
The main biological effect of complement activation is
induction of an inflammatory reaction. Many of the activation
products are involved in this process, but C5a is particularly
important. Innumerable inflammatory mediators are induced by
C5a, when it reacts with its receptor (C5aR). Most of the
inflammatory branches can be triggered by C5a (Figure 2),
illustrating that complement has a key function as an upstream
mediator of a broad spectrum of inflammatory reactions. Thus,
activated complement is a double-edged sword with undesired
effects in many conditions. Various reagents with potential
Journal of Perinatology
 Complement and meconium
TE Mollnes et al
S118
Figure 2 Biological effects of C5a. The potent anaphylatoxin C5a released
during complement activation induces a number inflammatory mediators of
which several are released to the fluid phase whereas others are expressed on the
surface of cells. The fluid-phase mediators may then act directly as inflammatory
agents or may trigger secondary effects through receptor binding. The surface
proteins induced by C5a include adhesion molecules, leading to trafficking of cells
through interaction between endothelial cells and leukocytes.
therapeutic applications have been recently developed to target
complement activation and function.35
MAS and the complement system
In the first study of a possible interaction of complement and MAS
it was documented that meconium in vitro is a highly potent
activator of the complement system.36 Adding meconium to human
umbilical serum induced a substantial increase in TCC formation,
which was found to be mediated mainly through the alternative
activation pathway. As judged by their relative amounts in whole
meconium, the lipid and water fractions contributed equally to this
activation. However, corrected for the lower amount of the lipid
fractions compared with the water fractions in normal meconium,
the lipid fraction per weight was more potent.
In a whole-blood model established for studying the interaction
of the complement system with the inflammatory network,37 we
studied the role of complement in meconium-induced leukocyte
activation as measured by oxidative burst and expression of the
surface activation markers CD11b and L-selectin.38 Meconium
induced a marked oxidative burst that was reduced by 60 to 70%
when adding the alternative complement pathway inhibitor
anti-factor D. Furthermore, meconium induced a substantial
increase in CD11b and decrease in L-selectin, typical for leukocyte
activation, which was virtually abolished (95 to 100%) by adding
anti-factor D. Importantly, inhibition of complement using a
specific C5aR antagonist gave the same inhibition of both oxidative
burst and expression of CD11b and L-selectin as anti-factor D,
implying that C5a was solely responsible for the
meconium-induced complement-dependent leukocyte activation.
On the basis of these in vitro experiments we proceeded with
in vivo studies using a well-established piglet model of MAS.39 Here
Journal of Perinatology
we demonstrated that complement is activated systemically, as
detected by a substantial increase in plasma TCC. The increase in
TCC correlated positively with oxygenation and ventilatory indexes
and negatively with lung compliance. Finally, the mortality of the
piglets correlated with the increase in TCC, indicating that the
breakdown in homeostatic mechanisms may be associated with
uncontrolled complement activation. These observations led us to
further investigate the role of complement in experimental MAS.40
In this study we found that experimental MAS piglets reflected an
inflammatory response, measured by cytokines and complement,
closely resembling the systemic inflammatory response syndrome
(SIRS). We therefore propose that MAS is associated with a general
whole-body SIRS-like inflammatory reaction. It remains, however,
to be shown whether complement activation actually contributes to
the pathophysiology of clinical MAS and if inhibition of
complement may attenuate inflammation and severity in MAS.
Lately, we have focused our research on the two main branches
of innate immunity: the fluid-phase complement system and the
cellular Toll-like receptor (TLR) system, the latter with focus on
the CD14/MD2/TLR4 pathway. Thus, we studied the effect of
meconium on these two branches as well as the effect of specific
inhibition of both complement and CD14. Interestingly, we found
that inhibition of both pathways efficiently attenuated the
inflammatory reaction induced by meconium (B Salvesen et al.,
manuscript submitted).
Addendum
To the best of our knowledge we are the first and so far the only
group that has studied the interaction between complement and
MAS. How comes that these studies were initiated? The answer is
simple: an incidental meeting in a PhD dissertation a few years ago
by two scientists (first and last authors of this paper), one an expert
on complement and the other on MASFboth of them at that time
virtually devoid of knowledge on the other’s research fieldFled to
an interesting discussion. First author asked: ‘what is at present
your main research’? After getting the answer he replied ‘what is
really MAS’? Having got a brief introduction to MAS, the last author
asked: ‘but what is your main focus’? After answering, last author
replied: ‘but what is actually complement’? Two completely
different scientific worlds met and recognized that we had to
proceed on a novel trackFdoes meconium as such activate the
complement system and is complement activated in experimental
MAS? As there were no reports of this topic in the literature we
initiated studies by mutually utilizing well-established models to
study complement activation and MAS, respectively. Lessons to
learn are that scientific groups to a greater extent should discuss
possible interacting mechanisms of pathophysiology when they
meet, and we should all accept that homeostasis in human biology
is more complex than seen just from our own window,
consequently leading to collaboration to obtain scientific progress.

Disclosure
The authors have declared no financial interests.
References
1 Tran N, Lowe C, Sivieri EM, Shaffer TH. Sequential effects of acute meconium
obstruction on pulmonary function. Pediatr Res 1980; 14: 34–38.
2 Clark DA, Nieman GF, Thompson JE, Paskanik AM, Rokhar JE, Bredenberg CE.
Surfactant displacement by meconium free fatty acids: an alternative explanation for
atelectasis in meconium aspiration syndrome. J Pediatr 1987; 110: 765–770.
3 Holopainen R, Soukka H, Halkola L, Kaapa P. Meconium aspiration induces a
concentration-dependent pulmonary hypertensive response in newborn piglets. Pediatr
Pulmonol 1998; 25: 107–113.
4 Murphy JD, Vawter GF, Reid LM. Pulmonary vascular disease in fatal meconium
aspiration. J Pediatr 1984; 104: 758–762.
5 Perlman EJ, Moore GW, Hutchins GM. The pulmonary vasculature in meconium
aspiration. Hum Pathol 1989; 20: 701–706.
6 Aaltonen M, Soukka H, Halkola L, Jalonen J, Holopainen IE, Kero P et al. Asphyxia
aggravates systemic hypotension but not pulmonary hypertension in piglets with
meconium aspiration. Pediatr Res 2003; 53: 473–478.
7 Soukka H, Rautanen M, Halkola L, Kero P, Kaapa P. Meconium aspiration induces
ARDS-like pulmonary response in lungs of ten-week-old pigs. Pediatr Pulmonol 1997;
23: 205–211.
8 Ghidini A, Spong CY. Severe meconium aspiration syndrome is not caused by
aspiration of meconium. Am J Obstet Gynecol 2001; 185: 931–938.
9 Katz VL, Bowes Jr WA. Meconium aspiration syndrome: reflections on a murky subject.
Am J Obstet Gynecol 1992; 166: 171–183.
10 Cleary GM, Wiswell TE. Meconium-stained amniotic fluid and the meconium
aspiration syndrome. An update. Pediatr Clin North Am 1998; 45: 511–529.
11 Tyler DC, Murphy J, Cheney FW. Mechanical and chemical damage to lung tissue
caused by meconium aspiration. Pediatrics 1978; 62: 454–459.
12 Davey AM, Becker JD, Davis JM. Meconium aspiration syndrome: physiological and
inflammatory changes in a newborn piglet model. Pediatr Pulmonol 1993; 16: 101–108.
13 Lally KP, Mehall JR, Xue H, Thompson J. Meconium stimulates a pro-inflammatory
response in peritoneal macrophages: implications for meconium peritonitis. J Pediatr
Surg 1999; 34: 214–217.
14 Zagariya A, Bhat R, Navale S, Vidyasagar D. Cytokine expression in meconium-induced
lungs. Indian J Pediatr 2004; 71: 195–201.
15 Korhonen K, Kiuru A, Svedstrom E, Kaapa P. Pentoxifylline reduces regional
inflammatory and ventilatory disturbances in meconium-exposed piglet lungs.
Pediatr Res 2004; 56: 901–906.
16 Tollofsrud PA, Medbo S, Solas AB, Robertson B, Speer CP, Seidenspinner S et al.
Intratracheal albumin reduces IL-8 in tracheobronchial aspirates in piglets after
meconium aspiration. J Perinat Med 2004; 32: 78–83.
17 Lindenskov PH, Castellheim A, Aamodt G, Saugstad OD. Meconium induced IL-8
production and intratracheal albumin alleviated lung injury in newborn pigs. Pediatr
Res 2005; 57: 371–377.
18 Khan AM, Elidemir O, Epstein CE, Lally KP, Xue H, Blackburn M et al. Meconium
aspiration produces airway hyperresponsiveness and eosinophilic inflammation in a
murine model. Am J Physiol Lung Cell Mol Physiol 2002; 283: L785–L790.
19 de Beaufort AJ, Pelikan DM, Elferink JG, Berger HM. Effect of interleukin 8 in
meconium on in-vitro neutrophil chemotaxis. Lancet 1998; 352: 102–105.
20 de Beaufort AJ, Bakker AC, van Tol MJ, Poorthuis BJ, Schrama AJ, Berger HM.
Meconium is a source of pro-inflammatory substances and can induce
cytokine production in cultured A549 epithelial cells. Pediatr Res 2003; 54:
491–495.
Complement and meconium
TE Mollnes et al
S119
21 Li YH, Yan ZQ, Brauner A, Tullus K. Meconium induces expression of inducible NO
synthase and activation of NF-kappaB in rat alveolar macrophages. Pediatr Res 2001;
49: 820–825.
22 Holopainen R, Aho H, Laine J, Peuravuori H, Soukka H, Kaapa P. Human meconium
has high phospholipase A2 activity and induces cellular injury and apoptosis in piglet
lungs. Pediatr Res 1999; 46: 626–632.
23 Schrama AJ, de Beaufort AJ, Sukul YR, Jansen SM, Poorthuis BJ, Berger HM.
Phospholipase A2 is present in meconium and inhibits the activity of pulmonary
surfactant: an in vitro study. Acta Paediatr 2001; 90: 412–416.
24 Korhonen K, Soukka H, Halkola L, Peuravuori H, Aho H, Pulkki K et al. Meconium
induces only localized inflammatory lung injury in piglets. Pediatr Res 2003; 54:
192–197.
25 Kytola J, Kaapa P, Uotila P. Meconium aspiration stimulates cyclooxygenase-2
and nitric oxide synthase-2 expression in rat lungs. Pediatr Res 2003; 53:
731–736.
26 Khan AM, Lally KP, Larsen GL, Colasurdo GN. Enhanced release of thromboxane A(2)
after exposure of human airway epithelial cells to meconium. Pediatr Pulmonol 2002;
33: 111–116.
27 Lukkarinen H, Laine J, Lehtonen J, Zagariya A, Vidyasagar D, Aho H et al. Angiotensin
II receptor blockade inhibits pneumocyte apoptosis in experimental meconium
aspiration. Pediatr Res 2004; 55: 326–333.
28 Zagariya A, Bhat R, Uhal B, Navale S, Freidine M, Vidyasagar D. Cell death and lung
cell histology in meconium aspirated newborn rabbit lung. Eur J Pediatr 2000; 159:
819–826.
29 Kojima T, Hattori K, Fujiwara T, Sasai-Takedatsu M, Kobayashi Y. Meconium-induced
lung injury mediated by activation of alveolar macrophages. Life Sci 1994; 54:
1559–1562.
30 Clark P, Duff P. Inhibition of neutrophil oxidative burst and phagocytosis by
meconium. Am J Obstet Gynecol 1995; 173: 1301–1305.
31 Soukka HR, Ahotupa M, Ruutu M, Kaapa PO. Meconium stimulates neutrophil
oxidative burst. Am J Perinatol 2002; 19: 279–284.
32 Mollnes TE, Song WC, Lambris JD. Complement in inflammatory tissue damage and
disease. Trends Immunol 2002; 23: 61–64.
33 Mollnes TE, Redl H, Hogasen K, Bengtsson A, Garred P, Speilberg L et al. Complement
activation in septic baboons detected by neoepitope-specific assays for C3b/iC3b/C3c,
C5a and the terminal C5b-9 complement complex (TCC). Clin Exp Immunol 1993;
91: 295–300.
34 Brandtzaeg P, Mollnes TE, Kierulf P. Complement activation and endotoxin levels in
systemic meningococcal disease. J Infect Dis 1989; 160: 58–65.
35 Mollnes TE, Kirschfink M. Strategies of therapeutic complement inhibition. Mol
Immunol 2006; 43: 107–121.
36 Castellheim A, Lindenskov PH, Pharo A, Fung M, Saugstad OD, Mollnes TE. Meconium
is a potent activator of complement in human serum and in piglets. Pediatr Res 2004;
55: 310–318.
37 Mollnes TE, Brekke OL, Fung M, Fure H, Christiansen D, Bergseth G et al. Essential
role of the C5a receptor in E coli-induced oxidative burst and phagocytosis revealed by
a novel lepirudin-based human whole blood model of inflammation. Blood 2002; 100:
1869–1877.
38 Castellheim A, Pharo A, Fung M, Saugstad OD, Mollnes TE. Complement C5a is
a key mediator of meconium-induced neutrophil activation. Pediatr Res 2004; 57:
242–247.
39 Lindenskov PH, Castellheim A, Aamodt G, Saugstad OD, Mollnes TE. Complement
activation reflects severity of meconium aspiration syndrome in newborn pigs. Pediatr
Res 2004; 56: 810–817.
40 Castellheim A, Lindenskov PH, Pharo A, Aamodt G, Saugstad OD, Mollnes TE.
Meconium aspiration syndrome induces complement-associated systemic inflammatory
response in newborn piglets. Scand J Immunol 2005; 61: 217–225.
Journal of Perinatology
 Journal of Perinatology (2008) 28, S120–S122
r 2008 Nature Publishing Group All rights reserved. 0743-8346/08 $30
www.nature.com/jp
REVIEW
Phospholipase A2 in meconium-induced lung injury
P Kääpä and H Soukka
Department of Pediatrics, Research Centre of Applied and Preventive Cardiovascular Medicine, University of Turku, Turku, Finland
Although the triggering mechanisms of tissue inflammation and injury in
meconium-contaminated lungs are still unclear, there is increasing
evidence to suggest a central role for phospholipase A2’s (PLA2). In fact,
elevated PLA2 activities together with high enzyme concentrations, especially
the amount of pancreatic (group I) secretory PLA2 (PLA2-I), have been
detected in human meconium and in meconium-contaminated lungs.
Recent data from our laboratory further indicate that human pancreatic
PLA2, introduced in high amounts within aspirated particulate meconium,
is a potent inducer of lung tissue inflammatory injury. Our finding of
elevated human PLA2-I concentrations in plasma during the first hours
after intratracheal meconium administration in newborn piglets further
suggests that intrapulmonary aspiration of meconium could also have
systemic inflammatory and injurious effects. This, however, remains to be
studied in further detail.
Journal of Perinatology (2008) 28, S120–S122; doi:10.1038/jp.2008.147
Aspiration of meconium is known to initiate a complex cascade of
pulmonary processes, resulting in progressively increasing
pulmonary vascular tone and a rapidly developing inflammatory
tissue injury with concomitant surfactant dysfunction in the
affected lungs.1,2 Although the critical role of pulmonary
inflammation in the course of neonatal meconium aspiration
syndrome (MAS) has been emphasized, the triggering
mechanisms and the manifold interplay of the injurious mediators
in MAS are still poorly identified. There is yet evidence that
human meconium may induce production of proinflammatory
agents within the lungs, and that some proinflammatory
components of meconium itself, such as cytokines and
phospholipase A2 (PLA2), may additionally contribute to intense
inflammation and injury in the meconium-contaminated lung
tissue.3,4 PLA2 represents a family of ubiquitous enzymes that
through cleavage of membrane phospholipids release arachidonic
acid, the precursor of proinflammatory eicosanoid.5,6 Although
Correspondence: Dr P Kääpä, Department of Pediatrics and Research Centre of Applied and
Preventive Cardiovascular Medicine, University of Turku, Kiinamyllynkatu 10, Turku,
FIN 20520, Finland.
E-mail: pekka.kaapa@utu.fi
PLA2 may occur as cytosolic (cPLA2) and secretory (sPLA2)
types, increasing attention has been paid to the role of secretory
PLA2 as an acute phase protein in inflammatory conditions.7
In this paper, the present understanding of the role of secretory
PLA2 in acute lung injury, specifically when induced by meconium
aspiration, is delineated. A new line of basic and clinical
research of MAS, namely the possible contribution of meconium-
introduced secretory PLA2 to systemic inflammation, is
also highlighted.
PLA2 and acute lung injury
Several lines of clinical and experimental evidence implicate that
excess pulmonary PLA2 activity, through generation of potent
vasoactive and proinflammatory lysophospholipids and
eicosanoids,5,6 may contribute to the progression of various
inflammatory lung disorders.5–9 In agreement, high blood and
pulmonary enzyme activity, correlating with the disease severity,
has been found in acute respiratory distress in adults.10 Although
high PLA2 activity in inflammatory lung injuries is commonly
connected with increased release of group II secretory PLA2
(PLA2-II) from macrophages and platelets,6,7,11 group I PLA2
(PLA2-I), secreted by the pancreas, is also known to be expressed
and to modulate cellular function in human lungs.12 Mammalian
PLA2-I is in fact able to inactivate pulmonary surfactant concentra-
tion-dependently through hydrolysis of phosphatidylcholine.13
PLA2-I may additionally, independent of the enzyme catalytic
activity, stimulate through specific membrane receptor action
neutrophil function and cytokine and eicosanoid production from
pulmonary cells.7,14 Moreover, experimental investigations have
shown that exogenously administered PLA2-I may induce, most
likely through increased pulmonary thromboxane A2 synthesis,
receptor-mediated contractile responses in the airways.15 It is
therefore apparent that PLA2 activity associated with upregulation
of PLA2-I may be important in the pathogenesis of acute
inflammatory lung injury. Still, variations in the tissue expressions
of different PLA2 enzyme types in the insulted lungs may also have
consequences in the clinical course and therapeutic approaches
(for example, use of specific PLA2 inhibitors) of acute lung injuries
from various origin.6,7

The role of PLA2 in meconium-induced lung injury
Earlier studies in our laboratory have demonstrated that human
meconium has very high catalytic PLA2 activity, mainly (>90%)
due to high concentration of human pancreatic PLA2-I.4 In line
with this finding, tissue PLA2 activity and concentration of human
pancreatic PLA2 in the meconium-contaminated lungs are
concentration-dependently elevated and correlate directly with the
severity of the meconium-induced lung injury.4,16,17 In these
studies, promotion of lung edema formation and neutrophil influx
through insufflated human PLA2-I, either in soluble form or within
meconium, further substantiates the central role of pulmonary
PLA2-I in the development of lung injury after meconium
aspiration.16,17 High PLA2 activity in meconium, mainly due to
secretory PLA2-I, is additionally shown to be associated with
decreased surfactant biophysical activity in the affected lungs.18
This inactivation could be explained by enzymatic hydrolysis of
dipalmitoyl-phosphatidylcholine, the major lipid constituent of
surfactant, but also the produced lyso-phosphatidylcholine may
have an inhibitory role.19 Although some experimental data
suggest that type I secretory PLA2 may induce type II PLA2
expression,20 challenge of the lungs with high pancreatic PLA2
activity within aspirated meconium does not seem to influence
pulmonary PLA2-II production.16,17 It is thus conceivable that
exposure of the lungs meconium with high pancreatic PLA2
activity, specifically through aspiration of thick particulate
meconium, may markedly participate in propagation of the
ensuing pulmonary failure.17 Yet, the possible involvement of other
phospholipases in the development of meconium-induced lung
injury remains to be investigated.
Although the above considerations may potentially offer new
ways to treat infants with MAS, most of the current therapies of
MAS are symptomatic, and their clinical effects have been
unsatisfactory and often conflicting.1 Accordingly, our earlier data
indicate that early administration of dexamethasone, known to
inhibit stimulated PLA2 synthesis,21 does not reduce lung PLA2
activity or inflammation in experimental meconium aspiration.22
Alike, some of our preliminary data show that mepacrine, an
unspecific PLA2 inhibitor, does not decrease PLA2 activity or prevent
inflammatory injury, but rather tends to elevate tissue enzyme
activity in the meconium-contaminated lungs (P. Kääpä,
unpublished observation). Clearly, more investigations are needed
before the pathophysiological relevance of PLA2 in neonatal MAS is
revealed.
The role of PLA2 in systemic inflammatory reaction
after meconium aspiration
Recent investigations have suggested that the inflammatory injury
processes found in MAS may not be restricted to the lungs, but may
also result in systemic inflammatory reaction and possible sequelae
PLA2 in meconium-injured lungs
P Kääpä and H Soukka
S121
Figure 1 Serum human pancreatic phospholipase A2 (PLA2) concentrations at
baseline (BL) and during 6 h after meconium aspiration in newborn piglets
(n ¼ 5). Mean (s.d.); *P<0.05 vs BL.
in extrapulmonary organs.23 Intrapulmonary meconium exposure
is indeed demonstrated to result in systemic complement
activation, cytokine release, and activation of circulating
neutrophils with production of a variety of biologically active
mediators, including reactive oxygen radicals.1,23 In line with these
findings, our recent studies indicate that pancreatic PLA2,
contained in high amount in meconium and thereby introduced
into the lungs, may be absorbed, at least to some extent, into the
pulmonary circulation.4,17 In fact, we were able to demonstrate
that human pancreatic PLA2 concentrations in plasma are elevated
during the first hours after intratracheal human meconium
administration in newborn piglets (Figure 1). Similar PLA2
activation and circulatory release have been recognized in clinical
disorders that promote systemic inflammation.24 Alike, markedly
elevated lung and blood PLA2 activity, correlating with the disease
severity, has been found in adults with acute respiratory distress.10
The systemic inflammatory reaction with systemic release of
mediators, such as PLA2 and cytokines, is supposed to ultimately
lead to extrapulmonary organ dysfunction and injury.24 Recent
experimental data from our laboratory in fact indicate that severe
meconium aspiration itself, without any complicating incidents,
may result in brain tissue injury, specifically in the hippocampus.25
Nevertheless, it still remains undetermined how often and at what
intensity systemic inflammation occurs in connection with
meconium aspiration and what is its significance for the outcome
of infants with severe MAS.
Conclusions
Taken together, there is a bulk of evidence indicating that
intrapulmonary aspirated meconium, specifically in thick
particulate form, challenges the lungs with high human pancreatic
PLA2 concentration and activity, and may thereby contribute to
pulmonary inflammatory perturbations, systemic inflammatory
reactions and eventually distant organ damage. These findings
Journal of Perinatology
 PLA2 in meconium-injured lungs
P Kääpä and H Soukka
S122
may be amenable to development of new modes of more specific
therapeutic approaches.
Acknowledgments
This study was supported by the Foundation for Pediatric Research, the Sigrid
Juselius Foundation, and the Paulo Foundation, Finland.
Disclosure
The authors have declared no financial interests.
References
1 Wiswell TE. Meconium staining and the meconium aspiration syndrome. In:
Stevenson D, Benitz WE, Sunshine P (eds). Fetal and Neonatal Brain Injury.
Mechanisms, Management and the Risks of Practice. Cambridge University Press:
Cambridge, UK, 2003, pp 612–635.
2 Davey AM, Becker JD, Davis JM. Meconium asiration syndrome: physiological and
inflammatory changes in a newborn piglet model. Pediatr Pulmonol 1993; 16:
101–108.
3 de Beaufort AJ, Pelikan DMV, Elferink JGR, Berger HM. Effect of interleukin 8 in
meconium on in-vitro neutrophil chemotaxis. Lancet 1998; 352: 102–105.
4 Holopainen R, Aho H, Laine J, Peuravuori H, Soukka H, Kääpä P. Human meconium
has high phospholipase A2 activity and induces cellular injury and apoptosis in piglet
lungs. Pediatr Res 1999; 46: 626–632.
5 Dennis EA. Diversity of group types, regulation, and function of phospholipase A2.
J Biol Chem 1994; 269: 13057–13060.
6 Valentin E, Ghomashchi F, Gelb MH, Lazdunski M, Lambeau G. On the diversity of
secreted phospholipases A2. J Biol Chem 1999; 274: 31195–31202.
7 Triggiani M, Granata F, Frattini A, Marone G. Activation of human inflammatory cells
by secreted phospholipases A2. Biochim Biophys Acta 2006; 1761(11): 1289–1300.
8 Niewoehner DE, Rice K, Duane P, Sinha AA, Gebhard R, Wangensteen D. Induction of
alveolar epithelial injury by phospholipase A2. J Appl Physiol 1989; 66: 261–267.
9 Vadas P, Pruzanski W. Role of secretory phospholipases A2 in the pathobiology of
disease. Lab Invest 1986; 55: 391–404.
10 Kim DK, Fukuda T, Thompson BT, Cockrill B, Hales C, Bonventre JV. Bronchoalveolar
lavage fluid phospholipase A2 activities are increased in human adult respiratory
distress syndrome. Am J Physiol 1995; 269: L109–L118.
11 Arbibe L, Koumanov K, Vial D, Rougeot C, Faure G, Havet N et al. Generation of
lyso-phospholipids from surfactant in acute lung injury is mediated by type-II
Journal of Perinatology
phospholipase A2 and inhibited by a direct surfactant protein A-phospholipase A2
protein interaction. J Clin Invest 1998; 102: 1152–1160.
12 Seilhamer JJ, Randall TL, Yamanaka M, Johnson LK. Pancreatic phospholipase A2:
isolation of the human gene and cDNA from porcine pancreas and human lung. DNA
1986; 5: 519–527.
13 Hite RD, Seeds MC, Jacinto RB, Balasubramanian R, Waite M, Bass D. Hydrolysis of
surfactant-associated phosphatidylcholine by mammalian secretory phospholipase A2.
Am J Physiol Lung Cell Mol Physiol 1998; 275: L740–L747.
14 Granata F, Petraroli A, Boilard E, Bezzine S, Bollinger J, Del Vecchio L et al. Activation
of cytokine production by secreted phospholipase A2 in human lung macrophages
expressing the M-type receptor. J Immunol 2005; 174: 464–474.
15 Sommers CD, Bobbitt JL, Bemis KG, Snyder DW. Porcine pancreatic phospholipase
A2-induced contractions of guinea pig lung pleural strips. Eur J Pharmacol 1992;
216: 87–96.
16 Korhonen K, Soukka H, Halkola L, Peuravuori H, Aho H, Pulkki K et al. Meconium
induces only localized inflammatory lung injury in piglets. Pediatr Res 2003; 54(2):
192–197.
17 Sippola T, Aho H, Peuravuori H, Lukkarinen H, Gunn J, Kaapa P. Pancreatic
phospholipase A2 contributes to lung injury in experimental meconium aspiration.
Pediatr Res 2006; 59(5): 641–645.
18 Schrama AJJ, de Beaufort AJ, Sukul YRM, Jansen SM, Poorthuis BJHM, Berger HM.
Phospholipase A2 is present in meconium and inhibits the activity of pulmonary
surfactant: an in vitro study. Acta Paediatr 2001; 90: 412–416.
19 Holm BA, Keicher L, Liu M, Sokolowski J, Enhorning G. Inhibition of pulmonary
surfactant function by phospholipases. J Appl Physiol 1991; 71: 317–321.
20 Kishino J, Ohara O, Nomura K, Kramer RM, Arita H. Pancreatic-type phospholipase A2
induces Group II phospholipase A2 expression and prostaglandin biosynthesis in rat
mesangial cells. J Biol Chem 1994; 269: 5092–5098.
21 Hoeck WG, Ramesha CS, Chang DJ, Fan N, Heller RA. Cytoplasmic
phopholipase A2 activity and gene expression are simulated by tumor necrosis
factor: dexamethasone blocks the induced synthesis. Proc Natl Acad Sci USA 1993; 90:
4475–4479.
22 Holopainen R, Laine J, Halkola L, Aho H, Kääpä P. Dexamethasone treatment
attenuates pulmonary injury in piglet meconium aspiration. Pediatr Res 2001;
49: 1–7.
23 Lindenskov PH, Castellheim A, Aamodt G, Saugstad OD, Mollnes TE. Complement
activation reflects severity of meconium aspiration syndrome in newborn pigs. Pediatr
Res 2004; 56: 810–817.
24 Anderson BO, Moore EE, Banerjee A. Phospholipase A2 regulates critical inflammatory
mediators of multiple organ failure. J Surg Res 1994; 56: 199–205.
25 Aaltonen M, Soukka H, Halkola L, Jalonen J, Holopainen IE, Kääpä PO. Meconium
aspiration induces oxidative injury in the hippocampus of newborn piglets. Early Hum
Dev 2005; 81(5): 439–447.
 Journal of Perinatology (2008) 28, S123–S126
r 2008 Nature Publishing Group All rights reserved. 0743-8346/08 $30
www.nature.com/jp
REVIEW
Meconium-induced release of nitric oxide in rabbit alveolar cells
R Fontanilla, A Zagariya and D Vidyasagar
Division of Neonatology, Department of Pediatrics, University of Illinois at Chicago Medical Center, Chicago, IL, USA
Previous studies have shown meconium-induced lung injury occurs
throughout release of inflammatory cytokines. The exact mechanism of
cytokine-induced apoptosis is not known. In this study we hypothesized that
meconium-induced apoptosis in the lungs is mediated through the
production of inducible nitric oxide (NO). We studied two groups of
newborn rabbit pups: one group was instilled with meconium and other
with normal saline. We measured precursors of NO in lung lavage from
both groups of rabbits and NO levels were calculated accordingly. The levels
of NO and NO-derivatives increased significantly in both groups. However
NO expression in meconium group 2 h after meconium instillation was
significantly higher than in saline-instilled group suggesting NO production
plays a role in meconium-induced inflammation.
Journal of Perinatology (2008) 28, S123–S126; doi:10.1038/jp.2008.170
Introduction
Meconium aspiration syndrome (MAS) is a major cause of
morbidity and mortality in neonates. It is characterized by initial
obstruction of the airway resulting in ventilation–perfusion
mismatch, hypoxemia and increased vascular resistance. MAS
effects 1 to 3% of both term and post-term infants. Earlier studies
have shown that MAS includes accumulation of inflammatory
cells, release of inflammatory cytokines and massive cell death in
experimental models. The pathogenesis is still unclear, although
the role of bioactive mediators such as nitric oxide (NO),
pro-inflammatory cytokines, prostaglandins, thromboxane, reactive
oxygen species has been proposed in sepsis-induced lung injury.1
Nitric oxide is a biological signaling and effector molecule
involved in many biologic functions including maintaining blood
pressure, modulating neural transmissions and host defense
mechanisms.2 It is catalyzed by enzymes of the NO synthase (NOS)
family. It is generated through the reaction of L-arginine,
nicotinamide adenine dinucleotide phosphate oxidase and O2 to
NO and citrulline. Reactions of NO with oxygen in aqueous
solution produce a relatively unreactive nitrate and nitrite ion as
an end product. Two of the three isoforms are constitutive and the
Correspondence: Dr D Vidyasagar, Professor of Pediatrics, University of Illinois at Chicago
Medical Center, 840 South Wood Street, M/C 856, Chicago, IL 60612, USA.
E-mail: dsagar@uic.edu
third is inducible, Ca-independent NO (iNOS). It has been
shown in the lungs of septic murine to have high levels of NO
produced by iNOS.3–6 NO is also a known regulator of apoptosis
and has been shown to inhibit the activity of many caspases both
in vivo and in vitro conditions in endotoxin-induced lung injury.7
High levels of NO can react with superoxide to produce a highly
oxidative peroxynitrate. We have reported in an earlier study
apoptosis and necrosis occurring after meconium instillation in rabbit
lungs using DNA staining and detection of DNA fragments by in situ
end labeling.8
Nitric oxide is an important inflammatory mediator. In MAS, an
intense inflammatory reaction occurs and Li et al. (2001) reported
an increase in rat alveolar macrophage cell line (ATCC8383) when
exposed to meconium. They also found iNOS expression leading to
increased production of NO.9,10 The purpose of the study is to
measure the NO production in meconium-induced injury in rabbit
alveolar cells.
Hypothesis
Meconium-induced apoptosis in the lungs is mediated through the
production of inducible NO. NO production is a major intermediary
step in meconium-induced lung cell apoptosis.
Methodology
Animal model (in vivo)
The care and handling of the animals were in accordance with
National Institutes of Health guidelines. The Animal Care and USE
Committee of Michael Reese Hospital (Chicago, IL, USA) approved
the experimental protocol. Pregnant rabbits were housed for 3 days
before the experiments with the mother in stainless steel rabbit
cages. Mothers were given regular Purina rabbit chow (Scientific
Animal Feed Co., Arlington Heights, IL, USA). After delivery, pups
were used in the study.
Two groups, 2-week-old New Zealand white rabbit pups, were
used in the study. Ten rabbits per group: group 1, saline-instilled
rabbits and group 2, meconium-instilled rabbits. Tracheotomy was
performed under sedation with intraperitoneal ketamine
(10 mg kg 1) and xylazine (1 mg kg 1). Tracheal instillation of
 Meconium-induced release of NO in alveolar cells
R Fontanilla et al
S124
10% meconium solution (1.2 ml kg 1) or 0.9% NaCl (1.2 ml kg 1)
was made. Skin incision was closed with a 4-0 nylon suture, and
rabbits were allowed to breathe spontaneously in the room air
without assisted ventilation or supplemental oxygen. At set point
(0, 2, 4 and 8 h), the animals were euthanized by intraperitoneal
injection of Nembutal (100 mg kg 1) for studies.
Meconium preparation
A total of 15 first-pass human meconium samples were obtained
from term, healthy neonates. Fresh newborn infant meconium of
1 g was homogenized on ice in a blender with 9 ml of 0.9% NaCl to
a 10% (w/v) final concentration and was spun down at 5000 r.p.m.
for 20 min (4 1C) to separate the supernatant and pellet. The
supernatant was filtered by a glass filter followed by sterilization by
0.2 mm filter (both filters from Millipore Co., Bedford, MA, USA)
and was used for instillation in the lungs of 2-week-old newborn
rabbit pups.
Reagents
ELISA kits were obtained from R&D System Co. (Minneapolis, MI,
USA). All other materials are obtained from different sources and
were of reagent grade.
In vivo animal model experiments
The lungs were isolated and homogenized in saline and
centrifuged at 5000 r.p.m. for 5 min. The supernatant was filtered
before measuring NO. Filtration eliminates nucleophiles,
antioxidants and compounds containing sulfhydryl groups such as
cysteine and glutathione, which may interfere with color formation
in Griess reaction.
Studies of NO using ELISA
NO has a very short half-life (<10 s), which makes it difficult to
measure directly. However, NO is metabolized to nitrate and nitrite.
Quantification of these anions can be used indirectly to determine
the amount of NO originally present. There is a direct correlation
between the nitrate and nitrite levels and NO. Nitrates and nitrites
were measured using a commercially available ELISA kit
(R&D System Co.). The assay is based on colorimetric detection
of nitrite as an azo dye product of Griess reaction. The Griess
reaction is a two-step diazotization reaction in which acidified NO2
produces a nitrosating agent, which reacts with sulfanilic
acid to produce a diazonium ion. This ion is then coupled to
N-1-napthylethylenediamine to form the chromophoric
azo-derivative, which absorbs light between 540 and 570 nm
and is quantitated by spectrophotometer.
Data analysis
Data from the experiments were reported as the mean±s.d. Data
were analyzed using analysis of variance and unpaired Student’s
t-test. A P<0.05 was considered to be significant.
Journal of Perinatology
Result and analysis
NO derivative (nitrite) production in meconium-instilled lungs was
significantly high (P<0.05) at 2 h after meconium instillation
compared with saline instillation. The levels of nitrite also
increased at 4 and 8 h after meconium instillation, but these values
were not significantly different compared with saline-instilled
lungs. Nitrites in saline-instilled lungs show low levels at 2 h and
increased levels at 4 and 8 h comparable with meconium-instilled
pups. This shows that meconium initiates lung injury faster than
saline and that both meconium and saline induced NO production.
The NO is released in response to the external injury factors such
as meconium or saline (Table 1 and Figure 1)
NO is a biological signaling and effector molecule involved in
many biologic functions including maintaining blood pressure,
modulating neural transmissions and host defense mechanisms. It
is a free radical, which makes it react very readily with superoxide
to form peroxynitrite. Peroxynitrite is a highly oxidative species that
is capable of nitrating tyrosine residues of numerous proteins lead
to the production of nitrotyrosine.11 High levels of nitrotyrosine
formation have been shown to be involved in acute lung injury by
way of increasing vascular permeability of endothelial cells,
inhibiting leukocyte adhesion, degrading carbohydrates, inhibiting
lipid peroxidation and cleaving DNA through nitration and
oxidation.11,12 The excess amount of NO required to form
peroxynitrite to produce apoptosis is not known. What has been
shown in this research is that within 2 h after instillation,
meconium or saline enhances the production of NO. Khan et al.10
Table 1 NO production 2, 4 and 8 h after meconium instillation.
Levels on NO after meconium of saline instillation
2 h* 4h 8h
Meconium 43.5±12.3 mmol l 1 52.5±5 mmol l 1 55.59±26.3 mmol l 1
Saline 28.24±9.8 mmol l 1 52.28±26.3 mmol l 1
48.9±12.7 mmol l 1
*P<0.05.
Meconium
60 Saline
50
40
30
20
10
0
2h 4h 8h
Figure 1 NO production at 2, 4, 8 h after meconium and saline instillation in
rabbit lungs. Measured using ELISA technique.

Figure 2 Mechanism of meconium-induced lung injury.
showed the production of NO from monolayers of A549 cells
immediately after incubating the cells with meconium.
The instillation of meconium or saline itself does not possess
any significant neutrophil chemotaxis13 but could stimulate the
production of the pro-inflammatory cytokine and upregulate the
production of the inducible form of iNOS, which produces large
amount of NO.4,14 –16 iNOS is produced in many cells including
alveolar cells in response to infection, inflammation and other
circumstances when the immune system is activated. The other
form is neuronal NOS from nerve cells and endothelial cells.2 The
inflammatory damage in this research marked by increased levels
of NO lasted for 8 h. A study carried out Holopainen et al.,17 in the
ultrastructural analysis of porcine lungs, 6 h after instillation of
meconium, showed a significant amount of alveolar exudates and
edematous alveolar epithelium and endothelium. The increase in
the production of NO at 4 and 8 h could have been due to the
worsening inflammatory process and increasing apoptosis in the
alveolar cells. This inflammatory reaction causes an increased
pulmonary vascular permeability producing the exudates and
eventually inactivation of the surfactant and decreased pulmonary
compliance.16,18 Earlier studies also showed in vitro, those
neutrophils and plasma proteins in the alveoli inactivate
surfactant.13 This may also explain why meconium can inactivate
a surfactant. Meconium enhancing the production of NO and the
inflammatory reactions may play an important part in the
pathogenesis of MAS.
Meconium aspiration may also cause apoptosis in alveolar cells.
Apoptosis is characterized by loss of cell function and decrease in
cell size and its morphology.19 Zagariya et al.8 showed apoptosis in
the airway epithelium 8 h after meconium instillation using
ISEL-DNA end labeling. The apoptosis reported from an earlier
study coincide with an increase in the production of NO
in this study up to 8 h. This might play a crucial role in
Meconium-induced release of NO in alveolar cells
R Fontanilla et al
S125
meconium-induced apoptosis. Apoptosis is induced by two main
pathway: (1) mitochondrial pathway, which involves cytochrome c
release from the mitochondria, and the activation of caspase-9,
which then cleaves and activates caspases 3, 6 and 7; and (2) the
death domain receptor pathway that involves the activation of Fas
or tumor necrosis factor receptors followed by the activation of
caspase-8 and subsequent activation of other caspases.5,7 The
long-lasting overproduction of NO acts as a modulator of apoptosis,
activating the caspase family through the release of cytochrome c
into the cytosol.20,21 Once in the cytosol, cytochrome c interacts
with Apaf-1 (apoptotic protease-activating factor 1), leading to the
activation of caspase-9 and subsequent caspases, thereby leading to
apoptosis. The activated caspases lead to cleavage of the nuclear
lamin and breakdown of the nucleus through caspase-3.22 In our
study, the meconium-induced NO production may represent a key
mechanism for the apoptosis associated with meconium aspiration
syndrome.
Despite the significant advances in management and diagnosis,
MAS still causes an important morbidity and mortality in neonates.
Earlier studies by Holopainen et al.17 showed that NO inhalation
controls the rise in the pulmonary artery pressure, improves
arterial oxygenation and prevents further increase in epithelial
apoptosis, but does not protect against early inflammatory damage
caused by meconium. Steroids have been used to inhibit the
inflammatory response and can downregulate the
pro-inflammatory cytokine production in vitro. Li et al.9 have
shown that steroids also inhibits meconium-stimulated NO
production and suggested that steroids could be used in the
treatment of MAS.
In conclusion, our findings show that meconium is a potent
inducer of NO production in the alveolar and airway cells
(Figure 2), resulting in inflammation and apoptosis, which could
result in MAS.
Journal of Perinatology
 Meconium-induced release of NO in alveolar cells
R Fontanilla et al
S126
Significance of the research
This study might be essential in understanding the role of
endogenous NO in the pathophysiology of MAS and might represent
a key point in future therapeutic application.
Disclosure
The authors have declared no financial interests.
References
1 Hibbs JB, Taintor RR, Vavrin Z. Nitric oxide: a cytotoxic activated macrophage effector
molecule. Biochem Biophys Res Commun 1987; 157: 87–94.
2 Grisham MB, Jour d’Heuil D, Wink OA. Nitric oxide I. Physiological chemistry of nitric
oxide and its metabolites: implication in inflammation. Am J Physiol 1999; 276:
G315–G321.
3 Kristoff AS, Goldrberg P, Laubauch V, Hussan SN. Role of inducible nitric oxide
synthase in endotoxin acute lung injury. Am J Respir Crit Care Med 1998; 158:
1883–1889.
4 Mehta S. The effects of nitric oxide in acute lung injury. Vasc Pharmacol 2005; 43:
390–403.
5 Rudkowski J, Barreiro E, Harfouche R, Goldberg P, Kishta O, D’orleans-Juste P et al.
Role of iNOS and nNOS in sepsis induced pulmonary apoptosis. Am J Physiol Lung
Cell Mol Physiol 2004; 286: L793–L800.
6 Farley KS, Wang LF, Rasavi HM, Law C, Rohan M, McCormack DG et al. Effects of
macrophage inducible nitric oxide synthase in murine septic lung injury. Am J Physiol
Lung Cell Mol Physiol 2006; 290: L1164–L1172.
7 Chung HT, Pae HO, Choi BM, Billiar TR, Keim YM. Nitric oxide as a bioregulator of
apoptosis. Biochem Biophys Res Commun 2001; 282: 1075–1079.
8 Zagariya A, Bhat R, Uhal B, Navale S, Vidyasagar D. Cell death and lung cell histology
in meconium aspirated newborn rabbit lung. Eur J Pediatr 2000; 159: 819–826.
Journal of Perinatology
9 Li YH, Yan ZQ, Braunner A, Tullus K. Meconium induces expression of inducible NO
synthase and activation of NF-(kappa)B in rat alveolar macrophages. Pediatr Res
2001; 49: 820–825.
10 Khan AM, Lally KP, Elidemir O, Colosurdo GN. Meconium enhances the release of
nitric oxide in human epithelial cells. Biol Neonate 2002; 81: 99–104.
11 Kooy NW, Royall JA, Ye YZ, Kelly DR, Beckmann JS. Evidence for in vivo peroxynitrate
production in human lung injury. Am J Respir Crit Care Med 1995; 151:
1250–1254.
12 Haddad IY, Pataki G, Hu P, Galliani C, Beckman JS, Matalon S. Quantitation of
nitrotyrosine levels in lung sections of patients and animals with acute lung injury.
J Clin Invest 1994; 94: 2407–2413.
13 Davey AM, Becker JO, Davis JM. Meconium aspiration syndrome: physiological and
inflammatory change in newborn piglet model. Pediat Pulmonol 1993; 16: 101–108.
14 Speyer CL, Neff TA, Warner RL, Guo RF, Sarna JV, Riedemann NC et al. Regulatory
effects of iNOS an acute lung inflammatory response in mice. AM J Pathol 2003; 163:
2319–2328.
15 Zagariya A, Bhat R, Uhal B, Chari G, Vidyasagar D. Apoptosis of airway epithelial cells
in response to meconium. Life Sci 2005; 76: 1849–1858.
16 Wiswell TE, Bent RC. Meconium staining and MAS. Pediatr Clin North Am 1993; 40:
955–981.
17 Holopainen R, Aho H, Laine J, Halkola J, Kaapa P. Nitric oxide inhalation inhibits
pulmonary apoptosis but not inflammatory injury in porcine meconium aspiration.
Acta Paediatr 1999; 88: 1147–1155.
18 Tyler DC, Murphy J, Cheng FW. Mechanical and chemical damage to lung tissue
caused by meconium aspiration. Pediatrics 1978; 62: 454–457.
19 Arends MJ, Weyllie AN. Apoptosis: mechanism and roles in pathology. Int Rev Exp
Pathol 1991; 68: 251–306.
20 Choi BM, Pae HO, Jan SI, Kim YM, Chung HT. Nitric oxide as pro-apoptotic as well as
anti-apoptotic modulator. J Biochem Mol Biol 2002; 35: 116–126.
21 Boyd CS, Cadenas E. Nitric oxide and cell signaling pathways in
mitochondrial-dependent apoptosis. Biol Chem 2002; 383: 411–423.
22 Pandya N, Jain S, Santani D. Apoptosis: a friend or a foe? Internet J Pharmacol ISSN
1993; 2: 1531–2976.
 Journal of Perinatology (2008) 28, S127–S135
r 2008 Nature Publishing Group All rights reserved. 0743-8346/08 $30
www.nature.com/jp
REVIEW
Intracellular and extracellular serpins modulate lung disease
DJ Askew and GA Silverman
UPMC Newborn Medicine Program, Children’s Hospital of Pittsburgh and Magee-Womens Research Institute, University of Pittsburgh
School of Medicine, Pittsburgh, PA, USA
An imbalance between peptidases and their inhibitors leads to pulmonary
disease. Imbalances occur in the adult and the neonate at risk for a specific
set of lung pathologies. Serpins (serine peptidase inhibitors) make up the
major source of antipeptidase activity in the lung. The purpose of this
review is to describe the serpin mechanism of inhibition, their roles in the
normal and pathological lung and their potential as therapeutic agents.
Journal of Perinatology (2008) 28, S127–S135; doi:10.1038/jp.2008.150
Introduction
The lung functions in the face of many physical challenges:
exposure to oxygen and environmental toxins, airborn pathogens,
continuous expansion and compression while breathing and
maintenance of a delicate interface enabling gas exchange with the
body’s vascular system. As a result, tissue damage, inflammation,
repair and remodeling are constant. These processes, from the
induction of apoptosis and necrosis in acute injury to the defense
mechanisms of inflammatory cells, coagulation and fibrinolysis, to
extracellular matrix degradation and cell migration, are all
peptidase driven. Peptidase inhibitors are required to regulate these
processes and neutralize peptidases upon completion of their
intended roles. Most pulmonary diseases are associated with an
imbalance between peptidase and peptidase inhibitor activity
(Table 1). This holds true in the neonate at risk for a specific set of
dysfunctional lung pathologies, including meconium aspiration
syndrome (MAS), respiratory distress syndrome (RDS) and
bronchopulmonary dysplasia (BPD). The balance between
peptidase and antipeptidase activities appears critical in the
development and progression of these diseases of premature and
full-term newborns. Serpins (serine peptidase inhibitors) make up
the major source of peptidase inhibitors in the lung. Others
peptidase inhibitors include Kunitz, Kazal and Bowman-Birk
protein families. Unlike the other peptidase inhibitors, serpin
Correspondence: Dr DJ Askew, Magee-Womens Research Institute, 204 Craft Avenue,
Room 210, Pittsburgh, PA 15213, USA.
E-mail: rsidja@mwri.magee.edu
inhibition occurs at a 1:1 stoichiometry. Importantly, peptidase
inhibition by serpins is irreversible. The purpose of this review
is to describe the general mechanism and regulation of serpin
function in the lung. Critical roles in the normal and
pathological lung, and serpins as potential therapeutic agents are
examined.
Serpins are found in animals, plants, prokaryotes and viruses.
They are distinguished from other peptidase inhibitors because
their unique suicide substrate-like mechanism of inhibition
(reviewed in Gettins1,2). A total of 37 serpin genes in humans are
distributed within 9 clades (A–I). Clade A has 13 members
including SERPINA1 (a1-antitrypsin, a1AT) and SERPINA5
(protein C inhibitor, PCI) that are secreted into the circulation.
Clade B is made up of 13 serpins that are primarily intracellular.
The remaining eleven serpins are dispersed across clades C–I and
are secreted into the fluid phase. The tertiary structure of serpins is
highly conserved and consists of three b-sheets (A–B), 7 to 9
a-helices (A–I) and extended reactive site loop (RSL) that acts as
the bait for peptidase targets. The structure is critical to the unique
suicide-substrate-like mechanism of peptidase inhibition.3
Inhibitory serpins exist in a metastable state resembling a loaded
mousetrap. Upon peptidase binding to the RSL, hydrolysis of the
P1–P10 peptide bond releases the RSL and allows the serpin to
undergo a rapid conformational transition. With the peptidase
covalently bound to the P1 residue, the RSL is inserted into b-sheet
A as strand 4 and the peptidase is trapped in an inactive complex
with the serpin.
This mechanism of inhibition has a side effect resulting in
clinical diseases collectively referred to as serpinopathies.4,5
Mutations that cause structural instability, specifically the opening
of b-sheet A, can lead to the formation of serpin polymers. Under
conditions of high serpin concentration, such as the endoplasmic
reticulum of cells in the liver, the RSL of one molecule is inserted
into b-sheet A of another. The prototype of this disease mechanism
is the genetic deficiency of a1AT.6 The two most common alleles,
a1AT*S and a1AT*Z, result in formation of a1AT polymers in the
liver and significantly diminished a1AT plasma levels. The
consequence of this is twofold for the individual: hepatocyte cell
death leading to cirrhosis,7 and predisposition to emphysema,
asthma and additional respiratory diseases.8 Other serpins with
 Serpins in pulmonary disease
DJ Askew and GA Silverman
S128

naturally occurring alleles shown to develop polymers and
subsequent serpinopathies are C1 inhibitor, antithrombin,
a1-antichymotrypsin (ACT), heparin cofactor II and neuroserpin.
a1AT is the major source of protection against
proteolytic damage in the lung
a1AT (SERPINA1) is the major peptidase inhibitor in plasma and
provides the main source of antipeptidase activity in lung. a1AT is
a 52 kDa glycoprotein produced in and secreted from the liver, as
well as bronchial epithelial cells (BECs). Amino-acid residues
Met-Ser at the P1–P10 position within the RSL make a1AT
a potent inhibitor of neutrophil elastase (NE), cathepsin G (catG)
and proteinase-3 (Table 1).9 In the lung, NE is capable of causing
extensive damage because of its proteolytic activity against
structural components collagen and elastin. During inflammation
large amounts of this peptidase are delivered to the lung.
Infiltrating neutrophils express catG on the cell surface and release
peptidases including NE into the airway space. The primary role of
a1AT is to maintain a local balance between peptidase activities
required for inflammatory cell function and to protect the lung
against peptidase-mediated tissue damage.10
a1AT deficiency is one of the most common inherited defects in
Caucasians. Mutant alleles a1AT*S (Glu264Val) and a1AT*Z
(Glu342Lys) account for most a1AT deficiencies, with a1AT*Z the
more deleterious. Approximately 4% of northern Europeans carry
the Z allele and approximately 1 in 2000 is homozygous
(a1AT*ZZ) whereas 1 in 1000 is heterozygous for the two mutant
alleles (a1AT*SZ). Individuals homozygous for the more common
S allele (a1AT*SS) exhibit an B40% decrease in a1AT plasma
levels. The a1AT*ZZ homozygous genotype results in a deficit of
B85%. Individuals with this phenotype, or heterozygous for the
two mutant alleles, are at risk for developing diseases associated
with excess elastase activity such as emphysema.

Table 1 Serpins and their proposed functions in the lung

Serpin Peptidase targets Function Pulmonary diseases

SERPINA1 Neutrophil elastase, cathepsin G, proteinase-3 Protect the lung against elastase activity Empysema
a1-antitrypsin Chronic pulmonary obstructive
disease
SERPINA5 Activated protein C, thrombin-thrombomodulin Activate coagulation
Peptidase C
inhibitor
Thrombin, factors Xa, XIa Suppress coagulation
Urokinse-type plasminogen activator, tissue-type Suppress fibrinolysis
plasminogen activator

SERPINC1 Thrombin, factors IXa, Xa, XIa, XIIa, kallikrein Suppress coagulation Sepsis/ALI
Antithrombin III

SERPINE1 Urokinse-type plasminogen activator, tissue-type Suppress fibrinolysis ALI

Plasminogen plasminogen activator Idiopathic pulmonary fibrosis
activator ARDS
inhibitor-1 Asthma

SERPINB1 Neutrophil elastase, cathepsin G, proteinase-3 Protect the lung against elastase activity

SERPINB2 Urokinse-type plasminogen activator, tissue-type Protect against cell death

Plasminogen plasminogen activator
activator
inhibitor-2

SERPINB3 Cathepsins K, L, S, V Protection of cells against cytosolic lysosomal peptidases,

Inhibit cell death
SERPINB4 Cathepsin G, mast cell proteinase Protect against cell death
SERPINB6 Cathepsin G Protect cells from granule peptidases
SERPINB9 Granzyme B Protect cytotoxic lymphocytes/maintain granzyme B granules
SERPINB10 Trypsin, thrombin Protect against cell death
SERPINB12 Trypsin
SERPINB13 Cathepsin K, L Protect against cell death

Journal of Perinatology

a1AT deficiency: COPD and emphysema
The proposed mechanism in the development of chronic
obstructive pulmonary disease (COPD), including emphysema, is
an imbalance of elastase and anti-elastase activity in the lung
(reviewed in Elias et al.11). a1AT*ZZ and a1AT*SZ individuals are
at a greater risk of developing COPD. For a1AT*ZZ individuals,
plasma a1AT*Z levels of 15% translate into elastase-inhibitory
activities in the lung far below this. First, protein encoded by the
a1AT*Z allele exhibits an association rate with NE approximately
fivefold slower than normal a1AT. Second, a1AT*Z protein
maintains its inherent instability and continues to form polymers
as it diffuses into the lung, leading to the detection of polymers in
lungs of a1AT*ZZ individuals, and further loss of anti-elastase
activity.12,13 Finally, it was observed that a1AT*ZZ polymers served
as a chemoattractant for human neutrophils.14 This translates into
increased inflammation and NE peptidase in conjunction with
decreased a1AT function.
COPD development is largely influenced by environmental
factors, of which the most common is tobacco smoke. It has been
proposed that the P1-Met residue in the RSL renders a1AT sensitive
to inactivation by oxidation from tobacco smoke exposure and the
reactive oxygen burst released by neutrophils (reviewed in
Carrell10). Accordingly, a1AT*ZZ individuals who smoke exhibit a
rapid onset of emphysema, often by 30 years of age, and death by
the age of 50.15 However, most a1AT*ZZ individuals who avoid
smoking live normal length lives with minimal complications.
Replacement therapy for a1AT deficiency, with intravenous a1AT
from pooled human plasma, results in longer survival. Data
compiled by the National Heart, Lung and Blood Institute indicate
that a1AT serum levels and lung function are improved with a1AT
replacement therapy.16 However, plasma is limited and therapy
expensive. Gene therapy is an alternative toward which much
progress been made in animal models.17,18 Synthetic small
molecule peptidase inhibitors are also being developed as a tool to
counteract imbalances in peptidase activity (reviewed in Chughtai
and O’Riordan19). On the basis of nature of a1AT deficit caused by
polymerization, another therapeutic strategy is to prevent
polymerization and facilitate a1AT secretion from the liver. This
may be accomplished using small molecules interacting with a1AT
directly or by enhanced chaperone function.20–22
Cystic fibrosis and a1AT
Cystic fibrosis (CF) presents another scenario where lung pathology
and disease progression is associated with increased elastase
activity. However, unlike a1AT deficiency, CF patients express
normal amounts of a1AT. Decreased fluidity of mucus in the CF
airway impairs mucociliary clearance, obstructs normal diffusion
of innate immune components, as well as a1AT, and creates
localized environments bacteria may colonize. Chronic infection
Serpins in pulmonary disease
DJ Askew and GA Silverman
S129
recruits an excess of migrating and activated neutrophils that
release their serine peptidases to the cell surface or directly into the
airway space. The elevated peptidase levels overwhelm the available
neutralizing activity of a1AT. CF progression is associated with
exacerbations, frequently caused by increased bacterial load or viral
infection (reviewed in Goss and Burns23). Inflammation is central
to this event, including increases in interleukin (IL)-8, IL-6, IL-1b,
tumor necrosis factor-a (TNFa), leukotriene B4 (LKTB4) and free
NE. Exacerbations lead to airway remodeling and decreased lung
function.
a1AT replacement therapy to combat NE activity in CF lungs
has been available for over two decades, however, there is little
statistical data derived from clinical trials relating to its
effectiveness.24,25 Two recent papers described promising results
with inhaled a1AT in CF patients. Both studies found decreased
inflammation associated with lowered cytokines and neutrophil cell
numbers.26,27 Griese et al. compared peripheral lung versus the
bronchial deposition of aerosolized prolastin (a1AT purified from
plasma; Bayer Corporation, Clayton, NC, USA). Although no
difference was observed between the two sites of treatment
deposition, both groups receiving 4 weeks of daily prolastin
exhibited significant decreases in IL-8, TNFa, IL-1b protein and
LKTB4 concentrations in sputum. In a second study, 4 weeks of
treatment with recombinant a1AT (ra1AT) made in sheep
produced significant decreases in neutrophil infiltration, and
reduced complexes between NE and endogenous a1AT, suggesting
that ra1AT was effective in neutralizing endogenous NE.27
Favorable results have also been collected from nebulized a1AT
therapy studies in animal models.28 Together these results
suggested that a1AT aerosol treatment would benefit CF patients.
However, it will be important to examine in detail the mechanism
by which a1AT inhibited inflammation and cytokine production.
a1AT and lung disease in the newborn
The premature infants lacking surfactant synthesis develop RDS.
A direct result of underdeveloped lungs and respiratory system,
therapy includes oxygen and mechanical ventilation, and
surfactant replacement. Even with improved oxygen saturation
monitoring and ventilation, unavoidable acute lung injury (ALI)
adds to the severity of RDS. Infants born prematurely frequently go
on to develop BPD. Prematurity is the primary risk factor for BPD,
followed by oxygen toxicity and ventilation induced lung damage
(reviewed in Jobe and Ikegami29 and Chess et al.30). The ‘new’
BPD pathology consists of arrested alveolar development, resulting
in decreased total alveoli, and abnormal vasculature localization
observed in the lung periphery. Inflammatory cytokines have been
associated with accelerated lung maturation.29 Serine peptidases,
released from inflammatory cells, caused epithelial cell injury and
lung remodeling that is associated with RDS and BPD.31 In the
neonate the balance between elastase and elastase inhibitor activity
Journal of Perinatology
 Serpins in pulmonary disease
DJ Askew and GA Silverman
S130
has been associated with lung injury and progression to BPD.32 In
addition to a1AT, low molecular mass inhibitor secretory leukocyte
peptidase inhibitor (SLPI) was found to be important in
neutralizing NE in tracheal aspirates of infants born prematurely.33
Watterberg et al.34 found that SLPI increases in the tracheal lavage
of RDS, whereas neonates going on to develop BPD had a
significantly higher elastase to elastase-inhibitor ratio. Sveger
et al.35 also reported significant correlations with BPD development
and low levels of inhibitors a1AT and ACT (SERPINA3) in
tracheobronchial aspirate of preterm infants at 3 to 4 days of age,
and low SLPI at 7 to 8 days of age. Two trials assessed the effect of
a1AT therapy in preterm infants on recovery from RDS and
development of BPD.36,37 A meta-analysis of the two trials found
that although a trend for reduced risk of oxygen dependency after
28 days existed, no significant difference was observed between
groups receiving a1AT therapy and placebo for risk of BPD or
long-term neurodevelopmental abnormalities.38
Similar to endotoxin exposure, meconium aspiration leads to a
rapid induction of cytokines, inflammation, decreased surfactant
function, hypoxemia, pulmonary hypertension and excessive cell
death in the airway of the newborn. In animal models, all of these
events take place within 2 to 4 h of exposure to dilute human
meconium.39–42 Following the initial inflammatory response, MAS
patients frequently undergo further pulmonary distress associated
with oxygen toxicity upon intubation and mechanical ventilation.
Zagariya et al.43 hypothesized that a1AT in the lungs of neonates
may attenuate meconium aspiration-induced lung injury. As has
been observed in a1AT deficiency and CF patients, the absence of
adequate anti-elastase activity associated with a1AT resulted in
extensive pulmonary damage. Treatment with supplemental a1AT
activity would seem to be a promising approach to arrest
elastase-dependent lung damage following meconium aspiration.
Plasminogen activator inhibitor-1
Plasminogen activator inhibitor-1 (PAI-1, SERPINE1) is an
inhibitor of the two plasminogen activators (PAs), urokinase-type
plasminogen activator (uPA) and tissue-type plasminogen activator
(tPA). uPA is expressed in specific tissues, whereas tPA functions as
a soluble protein in the vascular system. The balance between PA
and PAI-1 activities determines local fibrinolysis (Table 1). Diseases
of acute inflammation, such as ALI and acute respiratory distress
(ARDS) (reviewed in Ware and Matthay44), and fibrotic diseases
like idiopathic pulmonary fibrosis (IPF)45 share a common
pathology of fibrin deposition in the alveolar compartment. To
determine the best approach for treatment and prevention of these
related diseases, it is important to identify the source of imbalanced
fibrinolysis resulting in fibrin deposition. As the main inhibitor of
fibrinolysis, PAI-1 is expressed by several cell types found in the
lung and is modulated by inflammatory cytokines and tissue
damage. Elevated levels of PAI-1 were observed in alveolar
Journal of Perinatology
macrophages from patients with ARDS46 and IPF.45 An
investigation of PAI-1, uPA and tPA levels in preterm infants with
RDS also identified elevated ratios of PAI-1 to uPA protein in
tracheal aspirate fluid.47 Elevated PAI-1 in BAL fluid occurred in
bacterial pneumonia.48,49 Excessive PAI-1 levels were observed in
ARDS and severe pneumonia cases requiring mechanical
ventilation.50 These findings supported the hypothesis that PAI-1 is
a critical regulator of fibrin deposition in the alveolar space in
response to ALI. Further support for this theory was gained from
animal models of lung injury, including bacterial endotoxin,51 and
bleomycin52 models of ALI. These studies established PAI-1, its
activity and its regulation, as a critical target for therapies treating
the family of diseases displaying poor lung function associated with
fibrin deposition including ALI, ARDS and COPD.
PAI-1 has been identified as a potential mediator of host
defense. In mouse lung infection models, PAI-1 deficiency did not
affect the outcome of infection by Gram-positive Streptococcus
pneumonia,53 but was found to be critical in defense against
Gram-negative Klebsialla pneumonia.54 PAI-1-deficient animals
exhibited increased mortality at 24 and 48 h, coinciding with
increased bacterial dissemination, and elevated fibrinolysis. These
results may be attributed to the ascribed function of PAI-1 in
regulating fibrinolysis or a potential role of PAI-1 in mediating
neutrophil recruitment signals, as was demonstrated in vitro.55 In
either case, these results suggest that PAI-1 function may prove to
be a useful alternative target in the battle against bacterial
pathogens.
Antithrombin III
Antithrombin III (SERPINC1, ATIII) regulates coagulation by
inhibiting activated serine peptidases. ATIII can inhibit serine
peptidases thrombin, factors IXa, Xa, XIa, XIIa and kallikrein
(Table 1).56 The peptidase inhibitor activity of ATIII is positively
regulated by its cofactor heparin binding to the D helix. This
induces a conformational switch putting the RSL in a more
favorable position for serine peptidase binding.57 Heparin
molecules longer than 26 residues further accelerate peptidase
inhibition through interactions with the peptidase. Defects in ATIII
function primarily result in complications relating to thrombosis.
However, a protective role of ATIII has been experimentally
demonstrated in animal models of sepsis58 and ischemia
reperfusion of grafted lungs.59 Recombinant ATIII (rATIII) at
elevated levels resulted in reduced to complete inhibition of
pulmonary vascular permeability associated with endotoxin
treatment.58 In a lung transplant model, lungs were stored for 28 h
with normal saline, then transplanted into dogs receiving ATIII or
vehicle alone. Transplant animals treated with ATIII exhibited no
change in O2 partial pressure, alveolar–arterial O2 difference or
pulmonary vascular resistance for up to 3 h.59 These encouraging

results suggested that ATIII might be used to treat sepsis-related
lung dysfunction, ARDS and ALI resulting from ischemia.
In humans, plasma ATIII levels normally decline with sepsis
severity, and correlate with high mortality rate.60 Small clinical
trials suggested improved survival rates in patients receiving
rATIII.61–64 Eisele et al.62 reported that rATIII therapy was
associated with decreased lung dysfunction. However, Waydas
et al.63 found no difference in the duration of organ failure.
A single large trial of 2314 patients with severe sepsis produced
inconsistent results.65 One group in this trial, receiving ATIII but
not heparin, exhibited an increased 90-day survival rate compared
with placebo. It was also observed that new pulmonary dysfunction
was decreased in the group receiving rATIII therapy. Unfortunately,
no critical trials have been performed to date specifically with ALI
or ARDS patients to test ATIII or heparin therapy. Overall,
improvement in fibrin deposition and lung function observed with
ATIII in animal models of sepsis and ALI is promising but is
unconfirmed in humans.
Peptidase C inhibitor
Peptidase C inhibitor (PCI, SERPINA5) has a broad peptidase
inhibitor profile through which it modulates both the coagulation
and fibrinolysis systems (Table 1) (reviewed in Church et al.56 and
Geiger66). PCI in plasma is the major inhibitor of the
anticoagulant peptidase activated protein C (APC). PCI also inhibits
the thrombin–thrombomodulin complex,67 kallikrein, factors Xa,
XIa and thrombin. PCI may downregulate fibrinolysis by inhibiting
plasmin activator peptidases uPA and tPA. Like ATIII, the
interaction between PCI and many of its target peptidases is
modulated by heparin and other glycosaminoglycans.68–70 The
interaction between PCI and target peptidase kallikrein is inhibited
by glycosaminoglycans. In humans, PCI is a plasma protein,
present at a concentration of B100 nM and a half-life of B23 h.71
PCI is found in many other body fluids and secretions, and in a
wide range of tissues. The broad peptidase-inhibitory profile and
widely distributed expression has made it difficult to assign specific
biological functions to this serpin.
To determine the role of this serpin, PCI function was studied in
the mouse. Expression of mouse PCI was limited to the male and
female reproductive systems.72 Homozygous-null PCI-knockout
mouse appeared normal except male sterility was observed.72
Limited endogenous PCI in the mouse allowed a unique approach
to model human PCI pulmonary function. Hayashi et al.73
expressed hPCI in mice from a transgene consisting of the human
PCI gene contained within 25 kb of human genomic DNA. hPCI
protein in these transgenic (Tg) animals was found to be an active
inhibitor of APC, and expressed in a pattern similar to humans.
This hPCI Tg animal system may be used as a tool to further
explore PCI function in the lung under physiological and
pathological conditions, as well as to test the therapeutic effect of
Serpins in pulmonary disease
DJ Askew and GA Silverman
S131
human APC in vivo. Nishi et al.74 used the hPCI Tg mouse to
identify a role for PCI in pulmonary hypertension. Monocrotaline
treatment was used to specifically induce pulmonary hypertension.
A significant increase in right ventricular pressure was observed in
treated wild-type (WT) control mice compared to hPCI Tg animals.
BAL fluid levels of thrombin–antithrombin complex, monocyte
chemoattractant protein-1, platelet-derived growth factor and IL-13,
and the plasma level of TNFa were significantly increased in
treated WT mice compared to hPCI Tg animals.74 This study
established that PCI in the lung is protective against
monocrotaline-induce hypertension. Furthermore, it suggested that
PCI fulfills both anti-inflammatory and anticoagulant activities in
the lung.74,75 To take advantage of PCI therapeutically it will be
important to determine which activities as a serine peptidase
inhibitor are protective in specific physiological conditions.
Clinical studies on the role of PCI are limited. Examination of
58 patients with interstitial lung disease (ILD) associated with
diverse underlying pathologies discovered elevated PCI in the BAL
fluid of each of them.76 Groups with cryptogenic-organizing
pneumonia, collagen vascular disease (CVD-ILD) and sarcoidosis
exhibited elevated levels of PCI and thrombin-activatable
fibrinolysis inhibitor (TAFI), supporting the hypothesis that PCI
inhibition of APC results in elevated TAFI levels.77
The clade B serpins protect cells with an intracellular
antipeptidase shield
In humans there are 13 clade B genes that encode serine and
cysteine peptidase inhibitors. The intracellular serpins are expressed
in a wide range of tissues including lung, and target a wide range
of peptidases (Table 1). In the face of environmental insults such
as bacterial and viral infection, and excessive peptidase levels
associated with inflammation, the induction of cell death is a
common yet critical step in ALI. There is increasing evidence that
intracellular serpins function as a cytoprotective antipeptidase
shield, limiting damage by the misdirected peptidases as well as the
induction of necrosis and apoptosis (reviewed in Silverman et al.78
and Scott79). SERPINB1 inhibits elastases expressed by the
neutrophil.80 SERPINB2, B3, B4, B10 and B13 have been
implicated in blocking proapoptotic signals.81,82 SERPINB6 and B9
inhibit peptidases stored in the cytolytic granules including catG
and granzyme B (GzmB), respectively.83,84 SERPINB12 inhibits
trypsin and is expressed in the lung.85 Together, the clade B serpin
genes encode proteins with diverse antipeptidase activity, with
critical intracellular roles in protecting cells from damage, and
maintaining normal function in the lung.
SERPINB1 inhibits elastases in CF and BPD
SERPINB1 (monocyte/neutrophil elastase inhibitor) is an inhibitor
of NE, catG and neutrophil peptidase-3 (pr-3) (Table 1).80,86
Journal of Perinatology
 Serpins in pulmonary disease
DJ Askew and GA Silverman
S132
SERPINB1 protein was shown to exist at elevated levels and in a
complex with NE in the lavage of CF patients compared to normal
individuals.87 Recombinant human SERPINB1 (rSERPINB1) was
able to protect rat lungs against injury, including hemorrhage and
epithelial permeability, from the instillation of NE or CF patient
sputum preparations.88 rSERPINB1 was shown to inhibit
Surfactant-A degradation by the peptidase(s) in BAL fluid from CF
patients.89 Yasumatsu et al. probed SERPINB1 function in the
established baboon BPD model.90,91 As in humans, SERPINB1 was
localized to bronchial and glandular epithelial cells, as well as
mast cells, neutrophils and macrophages in the baboon lung.92
SERPINB1 protein in baboon lung tissue was found in high
molecular weight complexes with both NE and catG in BPD models
but not gestational controls.90 These results suggested that
SERPINB1 function in the newborn lung is critical as an
antipeptidase shield against elastases associated with inflammation
and responsible for lung injury.
Plasminogen activator inhibitor 2 blocks apoptosis
Plasminogen activator inhibitor 2 (SERPINB2, PAI-2) inhibits uPA
and tPA, and is expressed in macrophages and monocytes
(Table 1). Its primarily intracellular and nuclear localization
suggests, however, that PAI-2 has functions in addition to
regulating fibrinolysis (reviewed in Medcalf and Stasinopoulos93).
Several studies have demonstrated the ability of intracellular PAI-2
to inhibit apoptosis. TNFa-induced apoptosis was inhibited by
ectopic PAI-2 expression in HeLa and fibrosarcoma cells.94,95
Recently, PAI-2 was found to be required for macrophage survival
following pathogen activation of the toll-like receptor-4 apoptosis
pathway.96 On the basis of these studies, PAI-2 appears to be a
critical regulator of cell survival in cells of the host defense system
where it is expressed.
SERPINB3 and SERPINB4 are serine and cysteine
peptidase inhibitors
SERPINB3 and B4 are co-expressed in lung epithelium.97
SERPINB3 inhibits the lysosomal cysteine peptidases, cathepsin L
(catL), catK and catS.98 SERPINB4 inhibits the serine peptidases
catG and mast cell chymase (Table 1).99 Ectopic SERPINB3
expression was cytoprotective against TNFa and natural killer (NK)
cell-induced apoptosis,81 and both SERPINB3 and B4 have been
shown independently to protect cells against radiation.82 The
peptidase inhibitor activity of SERPINB3 and B4 may provide
cellular protection in the environment of the lung during ALI and
inflammation. SERPINB3 and B4 expression was induced in BECs
in asthmatics and by IL-4 and IL-13.100 SERPINB3 and B4 may
provide protection against neutrophil and mast cell peptidases as
well as localized bursts of reactive oxygen generated by neutrophils
used to destroy pathogens. Cells exposed to reactive oxygen
Journal of Perinatology
succumb to necrosis or apoptosis following lysosomal damage and
release of its cysteine peptidases into the cytosol (reviewed in
Lockshin and Zakeri101 and Guicciardi et al.102). Cysteine
peptidases are likely to be involved in development of BPD, as catK,
catL, and catS were shown to be elevated in a baboon model of
BPD.103 SERPINB3 is predicted to provide protection to cells against
cysteine peptidases. Maintenance of BEC by SERPINB3 and B4
would limit ALI and prevent epithelial permeability during
infection.
SERPINB9 inhibits granzyme B
SERPINB9 is the only known inhibitor of GzmB in humans
(Table 1).84 SERPINB9 is expressed in cytotoxic lymphocytes
(CTLs), dendritic cells (DCs) and NK cells of the monocyte and
lymphocyte lineages.104,105 Using a Serpinb9-deficient mouse
model, Zhang et al.106 demonstrated that Serpinb9 is required to
protect CTLs from ‘accidental death’ induced by residual GzmB in
the cytosol. Serpinb9 was found to be required for the maintenance
of GzmB-containing granule integrity in CTLs. Finally, in the
absence of Serpinb9, animals exhibited impaired clearance of
lymphocytic choriomeningitis virus.106 On the basis of these
findings and the localization of SERPINB9-positive monocytes and
DCs in lung tissue,107 it is proposed that this serpin is required for
normal host defense in the lung, particularly against bacterial and
viral pathogens requiring cell contact-mediated killing by the
immune system.
Summary and future directions
Lung function is dependent on several peptidase driven systems
including the innate and adaptive immune systems, coagulation,
fibrinolysis and tissue remodeling. As a result, pulmonary diseases
are commonly linked to excessive peptidase activity. Serpins are the
major regulators of peptidase activity in the lung. It follows that
understanding the biological function and regulation of serpins
will be critical in the development of therapies against pulmonary
disease.
Serpins occupy two niches in the lung, regulation of
extracellular peptidases and intracellular peptidases. The blood
plasma serpins a1AT, PAI-1, ATIII and PCI, function
extracellularly. Their localization within the lung makes them
primary targets for the development of therapeutic agents, from
simple replacement by inhalation, to recombinant gene transfer
and development of small molecule inhibitors that mimic their
activity. In the case of a1AT, this serpin inhibits a limited number
of elastolytic peptidases. In diseases associated with excess elastase
activity (empysema, CF), replacement therapy appears very
promising. In contrast, ATIII and PCI are multi-peptidase
inhibitors and their local active concentration in vivo is likely
critical to properly balancing the function of specific peptidase

targets. To modulate the activity of these serpins therapeutically, we
may take advantage of the endogenous mechanisms regulating
their activity, interactions with glycosaminoglycans and
conformational changes in structure. Alternatively, extensive
success has been met using recombinant APC, a PCI target
peptidase, in the treatment of sepsis and ALI.
The clade B serpins are mainly intracellular. They are predicted
to form an anti-peptidase shield, protecting cells against exogenous
and endogenous peptidase activity. This hypothesis is based upon
their intracellular localization and broad tissue distribution,
combined with the varied peptidase-inhibitory specificities of the
clade B members. In their absence, cellular injury would lead to
cellular stress and death. SERPINB9, the only inhibitor of GzmB in
humans, is required for CTL survival, and therefore host defense
against some viral infections. SERPINB3 and SERPINB4, expressed
in BEC, are poised to protect the bronchial airways against both
exogenous and endogenous serine and lysosomal cysteine
peptidases. In lung epithelium, this antipeptidase shield is very
important due to repeated exposure to neutrophil-derived serine
peptidases and oxidative stress accompanying inflammation in
response to pulmonary injury or infection. On the basis of the
ability to block cellular damage, including that caused by
lysosomal cysteine peptidases, and cell death, the intracellular
clade B serpins are powerful agents to target in the development of
therapeutics.
Disclosure
The authors have declared no financial interests.
References
1 Gettins PGW. Serpin structure, mechanism, and function. Chem Rev. 102; 2002;
4751–4804.
2 Gettins PGW. Mechanisms of serpin inhibition. In: Silverman GA, Lomas DA (eds).
Molecular and Cellular Aspects of the Serpinopathies and Disorders in Serpin
Activity. World Scientific Publishing: Tuck Link, Singapore, 2007, pp 67–100.
3 Huntington JA, Read RJ, Carrell RW. Structure of a serpin–protease complex shows
inhibition by deformation. Nature 2000; 407: 923–926.
4 Lomas DA, Evans DL, Finch JT, Carrell RW. The mechanism of Z alpha1-antitrypsan
accumulation in the liver. Nature 1992; 357: 605–607.
5 Lomas D. The serpinopathies and respiratory disease. In: Silverman GA, Lomas DA
(eds). Molecular and Cellular Aspects of the Serpinopathies and Disorders in Serpin
Activity. World Scientific Publishing: Tuck Link, Singapore, 2007, p 639.
6 Carrell RW, Lomas DA. Alpha1-antitrypsin deficiencyFa model for conformational
diseases. N Engl J Med 2002; 346: 45–53.
7 Eriksson S, Carlson J, Velez R. Risk of cirrhosis and primary liver cancer in
alpha 1-antitrypsin deficiency. N Engl J Med 1986; 324: 736–739.
8 Eden E, Mitchell D, Mehlman B, Khouli H, Nejat M, Grieco MH et al. Atopy, asthma,
and emphysema in patients with severe alpha-1-antitrypysin deficiency. Am J Respir
Crit Care Med 1997; 156: 68–74.
9 Travis J, Salvesen GS. Human plasma proteinase inhibitors. Annu Rev Biochem 1983;
52: 655–709.
Serpins in pulmonary disease
DJ Askew and GA Silverman
S133
10 Carrell RW. alpha 1-Antitrypsin: molecular pathology, leukocytes, and tissue damage.
J Clin Invest 1986; 78: 1427–1431.
11 Elias JA, Kang MJ, Crouthers K, Homer R, Lee CG. State of the art. mechanistic
heterogeneity in chronic obstructive pulmonary disease: insights from transgenic mice.
Proc Am Thorac Soc 2006; 3: 494–498.
12 Elliott PR, Bilton D, Lomas DA. Lung polymers in Z alpha1-antitrypsin deficiency-
related emphysema. Am J Respir Cell Mol Biol 1998; 18: 670–675.
13 Mahadeva R, Atkinson C, Li Z, Stewart S, Janciauskiene S, Kelley DG et al. Polymers of
Z alpha1-antitrypsin co-localize with neutrophils in emphysematous alveoli and are
chemotactic in vivo. Am J Pathol 2005; 166: 377–386.
14 Parmar JS, Mahadeva R, Reed BJ, Farahi N, Cadwallader KA, Keogan MT et al.
Polymers of alpha(1)-antitrypsin are chemotactic for human neutrophils: a new
paradigm for the pathogenesis of emphysema. Am J Respir Cell Mol Biol 2002; 26:
723–730.
15 Hodges JR, Millward-Sadler GH, Barbatis C, Wright R. Heterozygous MZ alpha 1-
antitrypsin deficiency in adults with chronic active hepatitis and cryptogenic cirrhosis.
N Engl J Med 1981; 304: 557–560.
16 National Heart, LaBI. A registry of patients with severe deficiency of alpha1-antitrypsin.
Chest 1994; 1223–1232.
17 Lu Y, Choi YK, Campbell-Thompson M, Li C, Tang Q, Crawford JM et al. Therapeutic
level of functional human alpha 1 antitrypsin (hAAT) secreted from murine
muscle transduced by adeno-associated virus (rAAV1) vector. J Gene Med 2006; 8:
730–735.
18 Flotte TR, Conlon TJ, Poirier A, Campbell-Thompson M, Byrne BJ. Preclinical
characterization of a recombinant adeno-associated virus type 1-pseudotyped vector
demonstrates dose-dependent injection site inflammation and dissemination of vector
genomes to distant sites. Hum Gene Ther 2007; 18: 245–256.
19 Chughtai B, O’Riordan TG. Potential role of inhibitors of neutrophil elastase in treating
diseases of the airway. J Aerosol Med 2004; 17: 289–298.
20 Schmidt BZ, Perlmutter DH. Grp78, Grp94, and Grp170 interact with alpha1-antitrypsin
mutants that are retained in the endoplasmic reticulum. Am J Physiol Gastrointest
Liver Physiol 2005; 289: G444–G455.
21 Burrows JA, Willis LK, Perlmutter DH. Chemical chaperones mediate increased secretion
of mutant alpha 1-antitrypsin (alpha 1-AT) Z: a potential pharmacological strategy for
prevention of liver injury and emphysema in alpha 1-AT deficiency. Proc Natl Acad Sci
USA 2000; 97: 1796–1801.
22 Teckman JH. Lack of effect of oral 4-phenylbutyrate on serum alpha-1-antitrypsin in
patients with alpha-1-antitrypsin deficiency: a preliminary study. J Pediatr
Gastroenterol Nutr 2004; 39: 34–37.
23 Goss CH, Burns JL. Exacerbations in cystic fibrosis. 1: epidemiology and pathogenesis.
[see comment]. Thorax 2007; 62: 360–367.
24 Brennan S. Revisiting alpha1-antitrypsin therapy in cystic fibrosis: can it still offer
promise? [comment]. Eur Respir J 2007; 29: 229–230.
25 McElvaney NG, Hubbard RC, Birrer P, Chernick MS, Caplan DB, Frank MM et al.
Aerosol alpha 1-antitrypsin treatment for cystic fibrosis. Lancet 1991; 337: 392–394.
26 Griese M, Latzin P, Kappler M, Weckerle K, Heinzlmaier T, Bernhardt T et al.
alpha1-Antitrypsin inhalation reduces airway inflammation in cystic fibrosis patients.
[see comment]. Eur Respir J 2007; 29: 240–250.
27 Martin SL, Downey D, Bilton D, Keogan MT, Edgar J, Elborn JS et al. Safety and
efficacy of recombinant alpha(1)-antitrypsin therapy in cystic fibrosis. Pediatr
Pulmonol 2006; 41: 177–183.
28 Cantin AM, Woods DE. Aerosolized prolastin suppresses bacterial proliferation in a
model of chronic Pseudomonas aeruginosa lung infection. Am J Respir Crit Care
Med 1999; 160: 1130–1135.
29 Jobe AH, Ikegami M. Prevention of bronchopulmonary dysplasia. Curr Opin Pediatr
2001; 13: 124–129.
30 Chess PR, D’Angio CT, Pryhuber GS, Maniscalco WM. Pathogenesis of broncho-
pulmonary dysplasia. Semin Perinatol 2006; 30: 171–178.
31 Merritt TA, Cochrane CG, Holcomb K, Bohl B, Hallman M, Strayer D et al. Elastase and
alpha 1-proteinase inhibitor activity in tracheal aspirates during respiratory distress
Journal of Perinatology
 Serpins in pulmonary disease
DJ Askew and GA Silverman
S134
syndrome. Role of inflammation in the pathogenesis of bronchopulmonary dysplasia.
J Clin Invest 1983; 72: 656–666.
32 Speer CP. Inflammation and bronchopulmonary dysplasia. Semin Neonatol 2003;
8: 29–38.
33 Sluis KB, Darlow BA, Vissers MC, Winterbourn CC. Proteinase–antiproteinase balance
in tracheal aspirates from neonates. Eur Respir J 1994; 7: 251–259.
34 Watterberg KL, Carmichael DF, Gerdes JS, Werner S, Backstrom C, Murphy S. Secretory
leukocyte protease inhibitor and lung inflammation in developing bronchopulmonary
dysplasia. J Pediatr 1994; 125: 264–269.
35 Sveger T, Ohlsson K, Polberger S, Noack G, Morse H, Laurin S. Tracheobronchial
aspirate fluid neutrophil lipocalin, elastase- and neutrophil protease-4-alpha1-
antitrypsin complexes, protease inhibitors and free proteolytic activity in respiratory
distress syndrome. Acta Paediatr 2002; 91: 934–937.
36 Dunn MS, Stiskal JA, O’Brien KK, Ito S, Cox DW, Kelly EN. alpha1-Proteinase inhibitor
(A1PI) therapy for the prevention of chronic lung disease (CLD) of prematurityFa
dose ranging study and meta-analysis with previous randomized clinical trial (RCT)
(Abstract). Pediatr Res 2000; 47: 397A.
37 Stiskal JA, Dunn MS, Shennan AT, O’Brien KK, Kelly EN, Koppel RI et al.
alpha1-Proteinase inhibitor therapy for the prevention of chronic lung disease of
prematurity: a randomized, controlled trial. Pediatrics 1998; 101: 89–94.
38 Shah P, Ohlsson A. Alpha-1 proteinase inhibitor (a1PI) for preventing chronic lung
disease in preterm infants. Cochrane Database Syst Rev 2001 CD002775.
39 Zagariya A, Bhat R, Uhal B, Navale S, Freidine M, Vidyasagar D. Cell death and lung cell
histology in meconium aspirated newborn rabbit lung. Eur J Pediatr 2000; 159: 819–826.
40 Zagariya A, Bhat R, Chari G, Uhal B, Navale S, Vidyasagar D. Apoptosis of airway
epithelial cells in response to meconium. Life Sci 2005; 76: 1849–1858.
41 Holopainen R, Aho H, Laine J, Peuravuori H, Soukka H, Kaapa P. Human meconium
has high phospholipase A2 activity and induces cellular injury and apoptosis in piglet
lungs. Pediatr Res 1999; 46: 626–632.
42 Holopainen R, Soukka H, Halkola L, Kaapa P. Meconium aspiration induces a
concentration-dependent pulmonary hypertensive response in newborn piglets. Pediatr
Pulmonol 1998; 25: 107–113.
43 Zagariya AM, Bhat R, Zhabotynsky E, Chari G, Navale S, Xu Q et al. Characterization of
serine/cysteine protease inhibitor alpha1-antitrypsin from meconium-instilled rabbit
lungs. J Cell Biochem 2005; 96: 137–144.
44 Ware LB, Matthay MA. Clinical practice. Acute pulmonary edema. N Engl J Med 2005;
353: 2788–2796.
45 Kotani I, Sato A, Hayakawa H, Urano T, Takada Y, Takada A. Increased procoagulant
and antifibrinolytic activities in the lungs with idiopathic pulmonary fibrosis. Thromb
Res 1995; 77: 493–504.
46 Chapman HA, Yang XL, Sailor LZ, Sugarbaker DJ. Developmental expression of
plasminogen activator inhibitor type 1 by human alveolar macrophages. Possible role
in lung injury. J Immunol 1990; 145: 3398–3405.
47 Cederqvist K, Siren V, Petaja J, Vaheri A, Haglund C, Andersson S. High concentrations
of plasminogen activator inhibitor-1 in lungs of preterm infants with respiratory
distress syndrome. Pediatrics 2006; 117: 1226–1234.
48 Choi G, Schultz MJ, van Till JW, Bresser P, van der Zee JS, Boermeester MA et al.
Disturbed alveolar fibrin turnover during pneumonia is restricted to the site of
infection. Eur Respir J 2004; 24: 786–789.
49 El-Solh AA, Okada M, Pietrantoni C, Aquilina A, Berbary E. Procoagulant and
fibrinolytic activity in ventilator-associated pneumonia: impact of inadequate
antimicrobial therapy. Intensive Care Med 2004; 30: 1914–1920.
50 Gunther A, Mosavi P, Heinemann S, Ruppert C, Muth H, Markart P et al. Alveolar
fibrin formation caused by enhanced procoagulant and depressed fibrinolytic capacities
in severe pneumonia. Comparison with the acute respiratory distress syndrome. Am J
Respir Crit Care Med 2000; 161: 454–462.
51 Wygrecka M, Markart P, Ruppert C, Kuchenbuch T, Fink L, Bohle RM et al.
Compartment- and cell-specific expression of coagulation and fibrinolysis factors in the
murine lung undergoing inhalational versus intravenous endotoxin application.
[see comment]. Thromb Haemost 2004; 92: 529–540.
Journal of Perinatology
52 Wygrecka M, Markart P, Ruppert C, Petri K, Preissner KT, Seeger W et al. Cellular
origin of procoagulant and (anti)-fibrinolytic factors in bleomycin-injured lungs. Eur
Respir J 2007; 29(6): 1105–1114.
53 Rijneveld AW, Florquin S, Bresser P, Levi M, De Waard V, Lijnen R et al. Plasminogen
activator inhibitor type-1 deficiency does not influence the outcome of murine
pneumococcal pneumonia. Blood 2003; 102: 934–939.
54 Renckens R, Roelofs JJ, Bonta PI, Florquin S, de Vries CJ, Levi M et al. Plasminogen
activator inhibitor type 1 is protective during severe Gram-negative pneumonia. Blood
2007; 109: 1593–1601.
55 Kwak SH, Wang XQ, He Q, Fang WF, Mitra S, Bdeir K et al. Plasminogen activator
inhibitor-1 potentiates LPS-induced neutrophil activation through a JNK-mediated
pathway. Thromb Haemost 2006; 95: 829–835.
56 Church F, Pike RN, Tollefsen DM, Buckle AM, Ciaccia AV, Olson ST. Regulation of
hemostasis by heparin-binding serpins. In: Silverman GA, Lomas DA (eds). Molecular
and Cellular Aspects of the Serpinopathies and Disorders in Serpin Activity. World
Scientific Publishing: Tuck Link, Singapore, 2007, pp 509–554.
57 Quinsey NS, Greedy AL, Bottomley SP, Whisstock JC, Pike RN. Antithrombin: in control
of coagulation. Int J Biochem Cell Biol 2004; 36: 386–389.
58 Uchiba M, Okajima K, Murakami K. Effects of various doses of antithrombin III on
endotoxin-induced endothelial cell injury and coagulation abnormalities in rats.
Thromb Res 1998; 89: 233–241.
59 Salvatierra A, Guerrero R, Rodriguez M, Alvarez A, Soriano F, Lopez-Pedrera R et al.
Antithrombin III prevents early pulmonary dysfunction after lung transplantation in
the dog. Circulation 2001; 104: 2975–2980.
60 Fourrier F, Chopin C, Goudemand J, Hendrycx S, Caron C, Rime A et al. Septic shock,
multiple organ failure, and disseminated intravascular coagulation. Compared patterns
of antithrombin III, protein C, and protein S deficiencies. [see comment]. Chest 1992;
101: 816–823.
61 Fourrier F, Jallot A, Leclerc L, Jourdain M, Racadot A, Chagnon JL et al. Sex steroid
hormones in circulatory shock, sepsis syndrome, and septic shock. Circ Shock 1994;
43: 171–178.
62 Eisele B, Lamy M, Thijs LG, Keinecke HO, Schuster HP, Matthias FR et al.
Antithrombin III in patients with severe sepsis. A randomized, placebo-controlled,
double-blind multicenter trial plus a meta-analysis on all randomized, placebo-
controlled, double-blind trials with antithrombin III in severe sepsis. [see comment].
Intensive Care Med 1998; 24: 663–672.
63 Waydhas C, Nast-Kolb D, Gippner-Steppert C, Trupka A, Pfundstein C, Schweiberer L
et al. High-dose antithrombin III treatment of severely injured patients: results of a
prospective study. J Trauma 1998; 45: 931–940.
64 Baudo F, Caimi TM, de Cataldo F, Ravizza A, Arlati S, Casella G et al. Antithrombin III
(ATIII) replacement therapy in patients with sepsis and/or postsurgical complications:
a controlled double-blind, randomized, multicenter study. [see comment]. Intensive
Care Med 1998; 24: 336–342.
65 Warren BL, Eid A, Singer P, Pillay SS, Carl P, Novak I et al. Caring for the critically ill
patient. High-dose antithrombin III in severe sepsis: a randomized controlled trial.
[see comment][erratum appears in JAMA 2002 Jan 9;287(2):192]. JAMA 2001; 286:
1869–1878.
66 Geiger M. Protein C inhibitor, a serpin with functions in- and outside vascular biology.
Thromb Haemost 2007; 97: 343–347.
67 Rezaie AR, Cooper ST, Church FC, Esmon CT. Protein C inhibitor is a potent inhibitor
of the thrombin–thrombomodulin complex. J Biol Chem 1995; 270: 25336–25339.
68 Pratt CW, Church FC. Heparin binding to protein C inhibitor. J Biol Chem 1992; 267:
8789–8794.
69 Ecke S, Geiger M, Binder BR. Glycosaminoglycans regulate the enzyme specificity of
protein C inhibitor. Ann NY Acad Sci 1992; 667: 84–86.
70 Pike RN, Buckle AM, le Bonniec BF, Church FC. Control of the coagulation system by
serpins. Getting by with a little help from glycosaminoglycans. FEBS J 2005; 272:
4842–4851.
71 Laurell M, Stenflo J, Carlson TH. Turnover of *I-protein C inhibitor and *I-alpha 1-
antitrypsin and their complexes with activated protein C. Blood 1990; 76: 2290–2295.

72 Uhrin P, Dewerchin M, Hilpert M, Chrenek P, Schofer C, Zechmeister-Machhart M
et al. Disruption of the protein C inhibitor gene results in impaired spermatogenesis
and male infertility. J Clin Invest 2000; 106: 1531–1539.
73 Hayashi T, Nishioka J, Kamada H, Asanuma K, Kondo H, Gabazza EC et al.
Characterization of a novel human protein C inhibitor (PCI) gene transgenic mouse
useful for studying the role of PCI in physiological and pathological conditions.
J Thromb Haemost 2004; 2: 949–961.
74 Nishii Y, Gabazza EC, Fujimoto H, Nakahara H, Takagi T, Bruno N et al. Protective
role of protein C inhibitor in monocrotaline-induced pulmonary hypertension. [see
comment]. J Thromb Haemost 2006; 4: 2331–2339.
75 Beaulieu LM, Church FC. Is protein C inhibitor antithrombotic and protective in
pulmonary hypertension? [comment]. J Thromb Haemost 2006; 4: 2327–2330.
76 Fujimoto H, Gabazza EC, Hataji O, Yuda H, D’Alessandro-Gabazza CN, Nakano M et al.
Thrombin-activatable fibrinolysis inhibitor and protein C inhibitor in interstitial lung
disease. [see comment]. Am J Respir Crit Care Med 2003; 167: 1687–1694.
77 Mosnier LO, Elisen MG, Bouma BN, Meijers JC. Protein C inhibitor regulates the
thrombin–thrombomodulin complex in the up- and down regulation of TAFI
activation. Thromb Haemost 2001; 86: 1057–1064.
78 Silverman GA, Whisstock JC, Askew DJ, Pak SC, Luke CJ, Cataltepe S et al. Human clade
B serpins (ov-serpins) belong to a cohort of evolutionarily dispersed intracellular
proteinase inhibitor clades that protect cells from promiscuous proteolysis. Cell Mol Life
Sci 2004; 61: 301–325.
79 Scott FL. Serpins, apoptosis and other aspects of cell death. In: Silverman, GA,
Lomas DA (eds). Molecular and Cellular Aspects of the Serpinopathies and
Disorders in Serpin Activity. World Scientific Publishing: Tuck Link, Singapore, 2007,
pp 301–336.
80 Remold-O’Donnell E. A fast-acting elastase inhibitor in human monocytes. J Exp Med
1985; 162: 2142–2155.
81 Suminami Y, Nagashima S, Vujanovic NL, Hirabayashi K, Kato H, Whiteside TL.
Inhibition of apoptosis in human tumour cells by the tumour-associated serpin, SCC
antigen-1. Br J Cancer 2000; 82: 981–989.
82 Murakami A, Suminami Y, Hirakawa H, Nawata S, Numa F, Kato H. Squamous cell
carcinoma antigen suppresses radiation-induced cell death. Br J Cancer 2001; 84:
851–858.
83 Scott FL, Hirst CE, Sun J, Bird CH, Bottomley SP, Bird PI. The intracellular serpin
proteinase inhibitor 6 is expressed in monocytes and granulocytes and is a potent
inhibitor of the azurophilic granule protease, cathepsin G. Blood 1999; 93:
2089–2097.
84 Sun J, Bird CH, Sutton V, McDonald L, Coughlin PB, De Jong TA et al. A cytosolic
granzyme B inhibitor related to the viral apoptotic regulator cytokine response
modifier A is present in cytotoxic lymphocytes. J Biol Chem 1996; 271:
27802–27809.
85 Askew YS, Pak SC, Luke CJ, Askew DJ, Cataltepe S, Mills DR et al. SERPINB12 is a novel
member of the human ov-serpin family that is widely expressed and inhibits
trypsin-like serine proteinases. J Biol Chem 2001; 276: 49320–49330.
86 Cooley J, Takayama TK, Shapiro SD, Schechter NM, Remold-O’Donnell E. The serpin
MNEI inhibits elastase-like and chymotrypsin-like serine proteases through efficient
reactions at two active sites. Biochemistry 2001; 40: 15762–15770.
87 Cooley JRF, Sontag MK, Osberg I, Accurso FJ, Remold-O’Donnell E. MNEI (monocyte/
neutrophil elastase inhibitor) is found at increased levels in cystic fibrosis lavage fluid
(Abstract). Pediatr Pulmonol 2001; 227.
88 Rees DD, Rogers RA, Cooley J, Mandle RJ, Kenney DM, Remold-O’Donnell E.
Recombinant human monocyte/neutrophil elastase inhibitor protects rat lungs against
injury from cystic fibrosis airway secretions. Am J Respir Cell Mol Biol 1999; 20:
69–78.
Serpins in pulmonary disease
DJ Askew and GA Silverman
S135
89 Rubio F, Cooley J, Accurso FJ, Remold-O’Donnell E. Linkage of neutrophil serine
proteases and decreased surfactant protein-A (SP-A) levels in inflammatory lung
disease. Thorax 2004; 59: 318–323.
90 Yasumatsu R, Altiok O, Benarafa C, Yasumatsu C, Bingol-Karakoc G, Remold-
O’Donnell E et al. SERPINB1 upregulation is associated with in vivo complex
formation with neutrophil elastase and cathepsin G in a baboon model of
bronchopulmonary dysplasia. Am J Physiol Lung Cell Mol Physiol 2006; 291:
L619–L627.
91 Coalson JJ, Winter VT, Siler-Khodr T, Yoder BA. Neonatal chronic lung disease in
extremely immature baboons. Am J Respir Crit Care Med 1999; 160: 1333–1346.
92 Zeng W, Remold-O’Donnell E. Human monocyte/neutrophil elastase inhibitor (MNEI)
is regulated by PU.1/Spi-1, Sp1, and NF-kappaB. J Cell Biochem 2000; 78: 519–532.
93 Medcalf RL, Stasinopoulos SJ. The undecided serpin. The ins and outs of plasminogen
activator inhibitor type 2. FEBS J 2005; 272: 4858–4867.
94 Kumar S, Baglioni C. Protection from tumor necrosis factor-mediated cytolysis by
overexpression of plasminogen activator inhibitor type-2. J Biol Chem 1991; 266:
20960–20964.
95 Dickinson JL, Bates EJ, Ferrante A, Antalis TM. Plasminogen activator inhibitor type 2
inhibits tumor necrosis factor alpha-induced apoptosis. Evidence for an alternate
biological function. J Biol Chem 1995; 270: 27894–27904.
96 Park JM, Greten FR, Wong A, Westrick RJ, Arthur JS, Otsu K et al. Signaling pathways
and genes that inhibit pathogen-induced macrophage apoptosisFCREB and
NF-kappaB as key regulators. Immunity 2005; 23: 319–329.
97 Cataltepe S, Gornstein ER, Schick C, Kamachi Y, Chatson K, Fries J et al. Co-expression
of the squamous cell carcinoma antigens 1 and 2 in normal adult human tissues and
squamous cell carcinomas. J Histochem Cytochem 2000; 48: 113–122.
98 Schick C, Pemberton PA, Shi GP, Kamachi Y, Cataltepe S, Bartuski AJ et al. Cross-class
inhibition of the cysteine proteinases cathepsins K, L, and S by the serpin squamous
cell carcinoma antigen 1: a kinetic analysis. Biochemistry 1998; 37: 5258–5266.
99 Schick C, Kamachi Y, Bartuski AJ, Cataltepe S, Schechter NM, Pemberton PA et al.
Squamous cell carcinoma antigen 2 is a novel serpin that inhibits the chymotrypsin-
like proteinases cathepsin G and mast cell chymase. J Biol Chem 1997; 272: 1849–
1855.
100 Yuyama N, Davies DE, Akaiwa M, Matsui K, Hamasaki Y, Suminami Y et al. Analysis
of novel disease-related genes in bronchial asthma. Cytokine 2002; 19: 287–296.
101 Lockshin RA, Zakeri Z. Caspase-independent cell deaths. Curr Opin Cell Biol 2002;
14: 727–733.
102 Guicciardi ME, Leist M, Gores GJ. Lysosomes in cell death. Oncogene 2004; 23:
2881–2890.
103 Altiok O, Yasumatsu R, Bingol-Karakoc G, Riese RJ, Stahlman MT, Dwyer W et al.
Imbalance between cysteine proteases and inhibitors in a baboon model of
bronchopulmonary dysplasia. Am J Respir Crit Care Med 2006; 173: 318–326.
104 Phillips T, Opferman JT, Shah R, Liu N, Froelich CJ, Ashton-Rickardt PG. A role for
the granzyme B inhibitor serine protease inhibitor 6 in CD8+ memory cell
homeostasis. J Immunol 2004; 173: 3801–3809.
105 Medema JP, Schuurhuis DH, Rea D, van Tongeren J, de Jong J, Bres SA et al.
Expression of the serpin serine protease inhibitor 6 protects dendritic cells from
cytotoxic T lymphocyte-induced apoptosis: differential modulation by T helper type 1
and type 2 cells. [see comment]. J Exp Med 2001; 194: 657–667.
106 Zhang M, Park SM, Wang Y, Shah R, Liu N, Murmann AE et al. Serine protease
inhibitor 6 protects cytotoxic T cells from self-inflicted injury by ensuring the integrity
of cytotoxic granules. Immunity 2006; 24: 451–461.
107 Bladergroen BA, Strik MC, Bovenschen N, van Berkum O, Scheffer GL, Meijer CJ et al.
The granzyme B inhibitor, protease inhibitor 9, is mainly expressed by dendritic cells
and at immune-privileged sites. J Immunol 2001; 166: 3218–3225.
Journal of Perinatology
 ‘Substance abuse
treatment linked with
prenatal visits improves
prenatal outcomes:
a new standard’
Journal of Perinatology presents
this retrospective cohort study
online FREE of charge. This article
evaluates the impact of Early Start,
an obstetric clinic-based prenatal
substance abuse treatment
program, for pregnant women and
their newborns.

An ‘Early Start’ may mean a healthy

beginning for both mother and child.

www.nature.com/jp

20518-04 JP hot article AJFP.indd 1 6/8/08 13:45:19

 Having trouble reaching
your target audience?
Take advantage of the highly targeted advertising opportunities VOLUME 16 | NO. 12 | JANUARY 2008
www.obesityjournal.org

available in this journal. a research journal

For more information, contact:

Alf Anderson | Advertising Manager

Nature Publishing Group
Complexity of inter-organ

25 First Street, Suite 104, Cambridge, MA 02141 communication

T 617.475.9231 F 617.494.4960 E media@nature.com OBESITY_COVER.indd 1

nature publishing group

7/31/07 3:11:57 PM

www.nature.com/obesity

13400-05OBYAdvertise_RJFP4C.indd 1 29/11/07 18:21:50

 Cutting-edge · High-impact · Ground-breaking

The official journal of The Obesity Society

Submit your research to Obesity today.

Obesity offers high-quality original research and presents new information in the areas of pharmacology,
nutrition medicine, genetics, adipogenesis, behavioral epidemiology, biophysics and lipid metabolism,
exercise and human physiology, nutritional epidemiology, phenotyping, fat cell physiology, aging,
neuroscience, transgenic models, metabolic syndrome, nutrition behavior, pediatric obesity, adipocyte cell
biology, and surgical interventions.

Publishing your paper in Obesity provides a wide range of benefits, including:

• Online submission – Fast and secure submission with Manuscript Central

• Timely review of articles
• Advance Online Publication (AOP) – Articles are available online in final format before they appear
in print and are immediately citable with their digital online identifier number (DOI number).
• Supplementary online material – Include additional supporting material, essential to your published
paper, online (e.g. multi-media presentations, tables).
• NPG’s ‘license to publish’ – NPG does not require authors to release copyright of their
articles. Instead, authors provide an exclusive license; you are free to reuse your paper VOLUME 16 | NO. 12 | JANUARY 2008
www.obesityjournal.org

in any of your future published work and to post a pdf version on your own website. a research journal

• Wide Exposure – Wide online dissemination of content via institutional access

and worldwide distribution of content, including free access, to institutions in
developing nations through HINARI and SciDevNet.

…and much more.

Complexity of inter-organ
communication

Visit the journal’s website today at www.nature.com/obesity for more information. OBESITY_COVER.indd 1
nature publishing group

7/31/07 3:11:57 PM

www.nature.com/obesity

13399-05OBYSubmit_RJFP4C.indd 1 6/12/07 17:32:10