Newborn surgery

1. Newborn Surgery

2. To Veena, Abir, Anita and Niki for their love and patience

3. Newborn Surgery Second Edition Edited by Prem Puri MS FRCS FRCS (Ed) FACS Newman Clinical Research Professor, University College Dublin Consultant Paediatric Surgeon, Our Ladys Hospital for Sick Children and National Childrens Hospital, Dublin, Ireland Director of Research, Childrens Research Centre, Our Ladys Hospital for Sick Children, Dublin, Ireland A member of the Hodder Headline Group LONDON

4. First published in Great Britain in 1996 by Butterworth-Heinemann Ltd This edition published in 2003 by Arnold, a member of the Hodder Headline Group, 338 Euston Road, London NW1 3BH http://www.arnoldpublishers.com Distributed in the United States of America by Oxford University Press Inc. 198 Madison Avenue, New York, NY 10016 Oxford is a registered trademark of Oxford University Press 2003 Arnold All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronically or mechanically, including photocopying, recording or any information storage or retrieval system, without either prior permission in writing from the publisher or a licence permitting restricted copying. In the United Kingdom such licences are issued by the Copyright Licensing Agency: 90 Tottenham Court Road, London W1T 4LP Whilst the advice and information in this book are believed to be true and accurate at the date of going to press, neither the author[s] nor the publisher can accept any legal responsibility or liability for any errors or omissions that may be made. In particular (but without limiting the generality of the preceding disclaimer) every effort has been made to check drug dosages; however it is still possible that errors have been missed. Furthermore, dosage schedules are constantly being revised and new side effects recognized. For these reasons the reader is strongly urged to consult the drug companies printed instructions before administering any of the drugs recommended in this book. British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress ISBN 0 340 76144 X (hb) 2 3 4 5 6 7 8 9 10 Publisher: Joanna Koster Development Editor: Michael Lax Production Editor: James Rabson Production Controller: Bryan Eccleshall Cover Design: Stewart Larking Typeset in Great Britain by Phoenix Photosetting, Chatham, Kent Printed and bound in Great Britain by CPI Bath

5. Contents Preface xi Contributors xiii PART 1 GENERAL 1 1 Embryology of malformations 3 Dietrich Kluth, Wolfgang Lambrecht and Christoph Bhrer 2 Prenatal diagnosis of surgical diseases 15 Tippi C. MacKenzie and N. Scott Adzick 3 Fetal and birth trauma 27 Prem Puri 4 Transport of the surgical neonate 39 Prem Puri and Diane De Caluw 5 Preoperative assessment 45 Prem Puri and Diane De Caluw 6 Anesthesia 59 Declan Warde 7 Postoperative management 71 Desmond Bohn 8 Fluid and electrolyte balance in the newborn 89 Winifred A. Gorman 9 Nutrition 103 Agostino Pierro 10 Vascular access in the newborn 121 Juda Z. Jona 11 Radiology in the newborn 131 Noel S. Blake 12 Immune system of the newborn 139 Denis J. Reen 13 Hematological problems in the neonate 147 Owen P. Smith 14 Genetics in neonatal surgical practice 157 Andrew Green 15 Ethical considerations in newborn surgery 173 Jacqueline J. Glover and Donna A. Caniano 16 Minimally invasive neonatal surgery 183 Ashley Vernon, Timothy Kane and Keith E. Georgeson

6. 17 Fetal surgery 189 Jyoji Yoshizawa, Loureno Sbragia and Michael R. Harrison PART 2 HEAD AND NECK 199 18 Choanal atresia in the newborn 201 Francesco Cozzi and Denis A. Cozzi 19 Pierre Robin sequence 207 Evelyn H. Dykes 20 Macroglossia 215 George G. Youngson 21 Tracheostomy in infants 219 Thom E. Lobe 22 Miscellaneous conditions of the neck and oral cavity 227 Anies Mahomed PART 3 CHEST 237 23 Congenital thoracic deformities 239 Robert C. Shamberger 24 Mediastinal masses in the newborn 247 Steven J. Shochat 25 Subglottic stenosis 253 Felix Schier 26 Tracheomalacia 259 I. Vinograd and R. M. Filler 27 Vascular rings 267 Ehud Deviri and Morris J. Levy 28 Pulmonary air leaks 277 Prem Puri 29 Chylothorax and other pleural effusions in neonates 283 Richard G. Azizkhan 30 Congenital malformations of the lung 295 Horace P. Lo and Keith T. Oldham 31 Congenital diaphragmatic hernia 309 Tina Granholm, Craig T. Albanese and Michael R. Harrison 32 Extracorporeal membrane oxygenation for neonatal respiratory failure 317 Eugene S. Kim and Charles J. H. Stolar 33 Bronchoscopy in the newborn 329 John D. Russell PART 4 ESOPHAGUS 335 34 Esophageal atresia and tracheo-esophageal stula 337 Paul D. Losty and Colin T. Baillie 35 Congenital esophageal stenosis 353 Shintaro Amae, Masaki Nio, Yutaka Hayashi and Ryoji Ohi vi Contents

7. 36 Esophageal duplication cysts 359 Leela Kapila, H. W. Holliday 37 Esophageal perforation in the newborn 365 Hirikati S. Nagaraj 38 Gastro-esophageal reux 369 Victor E. Boston PART 5 GASTROINTESTINAL 381 39 Pyloric atresia and prepyloric antral diaphragm 383 Vincenzo Jasonni 40 Hypertrophic pyloric stenosis 389 Prem Puri and Ganapathy Lakshmanadass 41 Gastric volvulus 399 Mark D. Stringer 42 Gastric perforation 405 Robert K. Minkes 43 Gastrostomy 411 Michael W. L. Gauderer 44 Duodenal obstruction 423 Yechiel Sweed 45 Malrotation 435 Lewis Spitz 46 Persistent hyperinsulinemic hypoglycemia of infancy 441 Lewis Spitz 47 Jejuno-ileal atresia and stenosis 445 Heinz Rode and A. J. W. Millar 48 Colonic and rectal atresias 457 Tomas Wester 49 Meconium ileus 465 Edward Kiely 50 Meconium peritonitis 471 Jose Boix-Ochoa and J. Lloret 51 Duplications of the alimentary tract 479 Prem Puri 52 Mesenteric and omental cysts 489 Daniel L. Mollitt 53 Neonatal ascites 497 Prem Puri 54 Necrotizing enterocolitis 501 Ann M. Kosloske 55 Hirschsprungs disease 513 Prem Puri 56 Anorectal anomalies 535 Alberto Pea 57 Congenital segmental dilatation of the intestine 553 Hiroo Takehara and Hiroki Ishibashi Contents vii

8. 58 Intussusception 557 Spencer W. Beasley 59 Inguinal hernia 561 Juan A. Tovar 60 Short bowel syndrome and surgical techniques for the baby with short intestines 569 Michael E. Hllwarth PART 6 LIVER AND BILIARY TRACT 577 61 Biliary atresia 579 Ken Kimura 62 Congenital biliary dilatation (choledochal cyst) 589 Takeshi Miyano and Atsuyuki Yamataka 63 Hepatic cysts and abscesses 597 David A. Partrick and Frederick M. Karrer PART 7 ANTERIOR ABDOMINAL WALL DEFECTS 603 64 Omphalocele and gastroschisis 605 Steven W. Bruch and Jacob C. Langer 65 Omphalomesenteric duct remnants 615 David A. Lloyd 66 Bladder exstrophy: considerations and management of the newborn patient 619 Fernando A. Ferrer and John P. Gearhart 67 Cloacal exstrophy 629 Jonathan I. Groner and Moritz M. Ziegler 68 Prune belly syndrome 637 Prem Puri and Hideshi Miyakita 69 Conjoined twins 643 Harry Applebaum PART 8 TUMORS 649 70 Epidemiology and genetic associations of neonatal tumors 651 Sam W. Moore and Jack Plaschkes 71 Hemangiomas and vascular malformations 663 Prem Puri and Laszlo Nemeth 72 Congenital nevi 675 Bruce S. Bauer and Julia Corcoran 73 Lymphatic malformations (cystic hygroma) 687 Jacob C. Langer and Vito Forte 74 Cervical teratomas 697 Michael W. L. Gauderer 75 Sacrococcygeal teratoma 703 Kevin C. Pringle 76 Nasal tumors 715 Alfred Lamesch and Peter Lamesch viii Contents

9. 77 Neuroblastoma 721 Raymond J. Fitzgerald 78 Soft-tissue sarcoma 733 David A. Lloyd 79 Hepatic tumors 739 Yoshiaki Tsuchida and Norio Suzuki 80 Congenital mesoblastic nephroma and Wilms tumor 747 Robert Carachi 81 Neonatal ovarian tumors 751 Jean Gaudin PART 9 SPINA BIFIDA AND HYDROCHEPHALUS 759 82 Spina bida and encephalocele 761 Prem Puri and Rajendra Surana 83 Hydrocephalus 775 Raymond J. Fitzgerald PART 10 GENITOURINARY 785 84 Imaging of the renal tract in the neonate 787 Isky Gordon 85 Management of antenatally detected hydronephrosis 793 Jack S. Elder 86 Multicystic dysplastic kidney 809 David F. M. Thomas and Azad S. Najmaldin 87 Upper urinary tract obstructions 817 Prem Puri and Boris Chertin 88 Duplication anomalies 831 Prem Puri and Hideshi Miyakita 89 Vesico-ureteric reux 837 Prem Puri 90 Ureteroceles in the newborn 845 Peter Frey, Mario Mendoza-Sagaon and Blaise J. Meyrat 91 Congenital posterior urethral obstruction 855 Reisuke Imaji, Daniel Moon and Paddy A. Dewan 92 Neuropathic bladder 867 Paddy A. Dewan, Paul D. Anderson and Gunnar Aksnes 93 Hydrometrocolpos 875 Devendra Gupta 94 Intersex 883 Ronald J. Sharp 95 Male genital anomalies 903 John M. Hutson 96 Neonatal testicular torsion 909 David M. Burge Contents ix

10. PART 11 LONG-TERM OUTCOMES IN NEWBORN SURGERY 913 97 Long-term outcomes in newborn surgery 915 Mark D. Stringer Index 925 x Contents

11. The 2nd edition of Newborn Surgery has been extensively revised. Many new chapters have been added to take account of the recent developments in the care of the newborn with congenital malformations. This edition which comprises 97 chapters by 121 contributors from all ve continents of the world, provides an authoritative, comprehensive and complete account of the various surgical conditions in the newborn. Each chapter is written by the current leading expert(s) in their respective elds. Newborn Surgery in the 21st century demands of its practitioners detailed knowledge and understanding of the complexities of congenital anomalies as well as the highest standards of operative techniques. In this textbook great emphasis continues to be placed on providing a comprehensive description of operative techniques of each individual congenital condition in the newborn. The book is intended for trainees in paediatric surgery, established paediatric surgeons, general surgeons with an interest in paediatric surgery as well as neonatologists and paediatricians seeking more detailed information on newborn surgical conditions. I wish to thank most sincerely all the contributors for the outstanding work they have done for the production of this innovative textbook. I also wish to express my gratitude to Mrs Karen Alfred and Ms Ann Brennan for their secretarial help and to the staff of Arnold for their help during the preparation and publication of this book. I am thankful to the Childrens Medical & Research Foundation, Our Ladys Hospital for Sick Children, Dublin for their support. Prem Puri 2003 Preface to the Second Edition

12. During the last three decades, newborn surgery has developed from an obscure subspeciality to an essential component of every major academic paediatric surgical department throughout both the developed and the developing world. Major advances in perinatal diagnosis, imaging, neonatal resuscitation, intensive care and operative techniques have radically altered the management of newborns with congenital malformations. Embryological studies have provided new valuable insights into the development of malformations, while improvements in prenatal diagnosis are having a signi- cant impact on approaches to management. Monitoring techniques for the sick neonate pre- and postoperatively have become more sophisticated and there is now greater emphasis on physiological aspects of the surgical newborn as well as their nutritional and immune status. This book provides a comprehensive compendium of all these aspects as a prelude to an extensive description of surgical conditions in the newborn. Modern-day newborn surgery demands detailed knowledge of the complexities of newborn problems. Research developments, laboratory diagnosis, imaging and innovative surgical techniques are all part of the challenge facing surgeons dealing with congenital conditions in the newborn. In this book, a comprehensive description of operative techniques of each individual condition is presented. Each contributor was selected to provide an authoritative, comprehensive and complete account of their respective topics. The book, comprising 90 chapters, is intended primarily for trainees in paediatric surgery, established paediatric surgeons, general surgeons with an interest in paediatric surgery and neonatologists. I am most grateful to all contributors for their willing- ness to contribute chapters at considerable cost of time and effort. I am indebted to Mr Maurice De Cogan for artwork, Mr Dave Cullen for photography and Ms Ann Brennan and Ms Deirdre ODriscoll for skilful secretarial help. I am thankful to the Childrens Research Centre, Our Ladys Hospital for Sick Children, for their support. Finally, I wish to thank the editorial staff, particularly Ms Susan Devlin, of Butterworth-Heinemann for their help during the preparation and publication of this book. Prem Puri Preface to the First Edition

13. N. Scott Adzick MD Professor of Surgery Surgeon-in-Chief Department of Surgery The Center for Fetal Diagnosis and Treatment Childrens Hospital of Philadelphia Philadelphia, USA Gunnar Aksnes MD PhD Consultant Paediatric Surgeon Department of Paediatric Surgery Ulleval University Hospital Oslo, Norway Craig T. Albanese MD Professor of Surgery Chief, Division of Pediatric Surgery Stanford University Medical Center Palo Alto California, USA Shintaro Amae MD Lecturer Division of Pediatric Surgery Tohoku University School of Medicine Sendai, Japan Paul D. Anderson MBBS Urology Research Fellow Urology Unit Royal Childrens Hospital Melbourne, Australia Harry Applebaum MD Head, Division of Pediatric Surgery Department of Surgery Kaiser Permanente Medical Center Los Angeles California, USA Richard G. Azizkhan MD Surgeon-in-Chief Lester Martin Chair of Pediatric Surgery Cincinnati Childrens Hospital Professor of Surgery and Pediatrics University of Cincinnati School of Medicine Cincinnati Ohio, USA Colin T. Baillie MBChB DCH ChM FRCS(Paeds) Consultant Paediatric Surgeon Royal Liverpool Childrens Hospital (Alder Hey) Liverpool, UK Bruce S. Bauer MD FACS FAAP Professor & Head Division of Pediatric Plastic Surgery Childrens Memorial Hospital Division of Plastic Surgery McGraw Medical School of Northwestern University Chicago Illinois, USA Spencer W. Beasley MBChB (Otago) MS (Melb) FRACS Professor of Paediatric Surgery Paediatric Surgeon and Urologist Department of Paediatric Surgery Christchurch Hospital Christchurch, New Zealand Noel S. Blake FRCR FFRRCSI Consultant Radiologist Our Ladys Hospital for Sick Children Dublin, Ireland Desmond Bohn MB FRCPC MRCP(UK) FFARCS Associate Chief Department of Critical Care Medicine The Hospital for Sick Children Toronto, Canada Jose Boix-Ochoa MD Chairman of Pediatric Surgery Professor of Pediatric Surgery Autonomous University of Barcelona Hospital Materno-Infantil Vall dHebron Barcelona, Spain Victor E. Boston MD FRCS(Ed) FRCSI FRCS (Eng) Consultant Paediatric Surgeon Royal Belfast Hospital for Sick Children Honorary Senior Lecturer Department of Surgery Queens University Belfast, UK Contributors

14. LCDR Steven W. Bruch MC USNR Staff Pediatric Surgeon Naval Medical Center Portsmouth, USA Christoph Bhrer MD Consultant Paediatrician Department of Neonatology Campus-Virchow-Klinikum Medical Faculty Charite Humboldt University Berlin, Germany David Burge FRCS FRCPCH Consultant Paediatric Surgeon Wessex Regional Centre for Paediatric Surgery Southampton, UK Diane De Caluw MD Consultant Paediatric Surgeon Department of Paediatric Surgery Chelsea and Westminster Hospital London, UK Donna A. Caniano MD Surgeon-in-Chief Department of Pediatric Surgery Childrens Hospital Ohio, USA Robert Carachi MD FRCS Head of Department Department of Surgical Paediatrics Royal Hospital for Sick Children Glasgow, UK Boris Chertin MD Consultant Pediatric Urologist Department of Urology Shane Zedek Medical Center Jerusalem, Israel Julia Corcoran MD FACS FAAP Attending Surgeon Division of Pediatric Plastic Surgery Childrens Memorial Hospital Division of Plastic Surgery McGraw Medical School of Northwestern University Chicago Illinois, USA Denis A. Cozzi MD Consultant Pediatric Surgeon Department of Pediatric Surgery University of Rome Rome, Italy Francesco Cozzi MD Associate Professor and Head of Pediatric Surgery Department of Pediatric Surgery University of Rome Rome, Italy Ehud Deviri MD MSurg Consultant Cardiothoracic Surgeon Department of Cardiothoracic Surgery Hadassah University Hospital Hebrew University Jerusalem, Israel Paddy A. Dewan PhD MD MS MmedSc FRCS FRACS Paediatric Urologist Royal Childrens Hospital Melbourne, Australia Evelyn H. Dykes MBChB FRCS (Paeds) Senior Lecturer in Paediatric Surgery Kings College London, UK Jack S. Elder MD Director Division of Pediatric Urology Rainbow Babies & Childrens Hospital Professor of Urology & Pediatrics Case Western Reserve University School of Medicine Cleveland Ohio, USA Fernando A. Ferrer MD Assistant Professor of Pediatric Urology Connecticut Childrens Hospital Hartford Connecticut, USA R. M. Filler MD FRCS(C) Professor and Surgeon-in-Chief Hospital of Sick Children Professor of Pediatrics University of Toronto Ontario, Canada Raymond J. Fitzgerald MA MB FRCSI FRCS FRACS (Paed Surg) FRCS (Ed) Ad. hom Associate Professor in Paediatric Surgery Trinity College Consultant Paediatric Surgeon Childrens Hospital and Our Ladys Hospital for Sick Children Dublin, Ireland Vito Forte MD FRCSC Paediatric Otolaryngologist Hospital for Sick Children Associate Professor of Otolaryngology University of Toronto Toronto, Canada Peter Frey MD BSc PD FMH Consultant Pediatric Surgeon Department of Pediatric Surgery Centre Hospitalier Universitaire Vaudois (CHUV) Lausanne, Switzerland xiv Contributors

15. Michael W. L. Gauderer MD FACS, FAAP Professor of Surgery University of South Carolina School of Medicine Chief, Department of Pediatric Surgery Childrens Hospital Greenville Hospital System Greenville South Carolina, USA Jean Gaudin MD Paediatric Surgeon Department of Paediatric Surgery Hpital St Louis La Rochelle, France John P. Gearhart MD Professor & Director Division of Pediatric Urology James Buchanan Brady Urological Institute Johns Hopkins Hospital Baltimore Maryland, USA Keith E. Georgeson MD Professor and Director Division of Pediatric Surgery Childrens Hospital of Alabama Birmingham Alabama, USA Jacqueline J. Glover PhD Associate Professor Center for Health Ethics and Law West Virginia University College of Medicine and Childrens Hospital Morgantown West Virginia, USA Isky Gordon FRCR Consultant Radiologist Great Ormond Street Hospital for Children Honorary Senior Lecturer Institute for Child Health London, UK Winifred A. Gorman BSc FRCPI FAAP Consultant Paediatrician Department of Neonatology National Maternity Hospital Dublin, Ireland Tina Granholm MD PhD Associate Professor Department of Pediatric Surgery Astrid Lindgren Childrens Hospital Director of Postgraduate Studies Department of Woman and Child Health Karolinska Hospital Karolinska Institute Stockholm, Sweden Andrew Green MB, PhD, FRCPI, FFPath(RCPI) Director National Centre for Medical Genetics Our Ladys Hospital for Sick Children Dublin, Ireland Jonathan I. Groner MD Assistant Professor Department of Surgery Childrens Hospital Columbus Ohio State University Columbus Ohio, USA Devendra Gupta MS MCH Professor of Paediatric Surgery All India Institute of Medical Sciences New Delhi, India Michael R. Harrison MD Professor of Surgery Pediatrics and Obstetrics Gynecology and Reproductive Sciences Director, Fetal Treatment Center Chief, Division of Pediatric Surgery University of California` San Francisco California, USA Yutaka Hayashi MD Professor Division of Pediatric Oncology Tohoku University School of Medicine Sendai, Japan Howard W. Holliday FRCS Consultant Paediatric Surgeon Derbyshire Childrens Hospital Derbyshire, UK Michael E. Hllwarth MD Professor & Head Department of Paediatric Surgery University of Graz Medical School Graz, Austria John M. Hutson BS MD(Monash), MD(Melb) FRACS Professor & Director Russell Howard Department of General Surgery Royal Childrens Hospital F Douglas Stephens Surgical Research Laboratory Murdoch Childrens Research Institute Melbourne, Australia Reisuke Imaji MD PhD Clinical Research Fellow Urology Unit Royal Childrens Hospital Department of Paediatrics University of Melbourne Murdoch Childrens Research Institute Melbourne, Australia Contributors xv

16. Hiroki Ishibashi MD Pediatric Surgeon Department of Digestive and Pediatric Surgery University of Tokushima Tokushima, Japan Vincenzo Jasonni MD Professor and Director School of Pediatric Surgery Istituto Scientico G Gaslini University of Genoa Genoa, Italy Juda Z. Jona MD FACS FAAP(S) Chief Division of Pediatric Surgery Evanston Northwestern Healthcare Evanston Illinois, USA Timothy Kane MD Chief Clinical Fellow Division of Pediatric Surgery Childrens Hospital of Alabama Birmingham Alabama, USA Leela Kapila OBE FRCS Consultant Paediatric Surgeon Department of Paediatric Surgery Queens Medical Centre Nottingham, UK Frederick M. Karrer MD Associate Professor of Surgery & Pediatrics & Head Division of Pediatric Surgery University of Colorado Health Sciences Center Surgical Director Pediatric Liver Transplantation Department of Pediatric Surgery The Childrens Hospital Denver Colorado, USA Edward Kiely FRCSI FRCS FRCPCH Consultant Paediatric Surgeon Hospital for Sick Children Great Ormond Street London, UK Eugene S. Kim MD Chief Resident Division of Pediatric Surgery College of Physicians and Surgeons Columbia University Childrens Hospital of New York New York Presbyterian Hospital New York, USA Ken Kimura MD Professor of Surgery and Pediatrics Department of Surgery University of Iowa Hospitals & Clinics Iowa City Iowa, USA Dietrich Kluth MD PhD Paediatric Surgeon Department of Paediatric Surgery University Hospital Hamburg Hamburg, Germany Ann M. Kosloske MD MPH Professor of Surgery and Pediatrics Texas Technical University Health Science Center Lubbock Texas, USA Ganapathy Lakshmanadass MS MChFRCS Senior Registrar in Paediatric Surgery Department of Paediatric Surgery National Childrens Hospital Dublin, Ireland Wolfgang Lambrecht MD Surgeon-in-Chief Department of Paediatric Surgery Eppendorf University Hospital Hamburg, Germany Alfred Lamesch MD FACS Emeritus Professor Universit Libre de Bruxelles Surgeon-in-Chief Emeritus Department of Paediatric Surgery Luxembourg Hospital Center Honorary Member of the Acadmie Royale de Mdecine Belgium Peter Lamesch MD FACS Professor of Surgery Department of Abdominal, Transplant & Vascular Surgery University of Leipzig Leipzig, Germany Jacob C. Langer MD FRCSC Chief, Paediatric General Surgery Hospital for Sick Children Toronto, Canada Morris J. Levy MD Professor of Surgery Department of Thoracic and Cardiovascular Surgery Sackler School of Medicine Tel Aviv University Tel Aviv, Israel David A. Lloyd MChir FRCS FCS(SA) Professor of Paediatric Surgery Institute of Child Health Royal Liverpool Childrens Hospital (Alder Hey) Liverpool, UK xvi Contributors

17. J. Lloret MD Pediatric Surgeon Neonatal and Oncological Unit Hospital Materno-Infantil Vall dHebron Barcelona, Spain Horace P. Lo MD Senior Resident Department of Surgery Medical College of Wisconsin Milwaukee Wisconsin, USA Thom E. Lobe MD Chairman, Section of Pediatric Surgery University of Tennessee Memphis Tennessee, USA Paul D. Losty MD FRCSI FRCS(Eng) FRCS(Ed) FRCS(Paed) Reader & Honorary Consultant Paediatric Surgeon Department of Paediatric Surgery Royal Liverpool Childrens Hospital (Alder Hey) and The University of Liverpool Liverpool, UK Tippi C. MacKenzie MD Fetal Surgery Research Fellow The Center for Fetal Diagnosis and Treatment Childrens Hospital of Philadelphia Philadelphia, USA Anies Mahomed MBBCH FCS(SA) FRCS(Glas.Ed) FRCS(Paeds) Consultant Paediatric Surgeon Department of Paediatric Surgery Royal Aberdeen Childrens Hospital Aberdeen, UK Mario Mendoza-Sagaon MD Senior Registrar Department of Pediatric Surgery CHUV Lausanne, Switzerland Blaise J. Meyrat MD Consultant Paediatric Urologist and Surgeon Department of Pediatric Surgery CHUV Lausanne, Switzerland A. J. W. Millar FRCS (Eng) (Edin) FRACS DCH Associate Professor Department of Paediatric Surgery University of Cape Town Senior Surgeon Red Cross War Memorial Childrens Hospital Cape Town, South Africa Robert K. Minkes MD PhD Associate Professor of Surgery Chief, Section of Pediatric Surgery Louisiana State University Heath Sciences Center Childrens Hospital of New Orleans Louisiana, USA Hideshi Miyakita MD Consultant Paediatric Urologist Tokai University School of Medicine Kanagawa, Japan Takeshi Miyano MD, PhD, FAAP(Hon), FACS, FAPSA(Hon) Director of Juntendo University Hospital Professor and Head Department of Pediatric Surgery Juntendo University Scholl of Medicine Tokyo, Japan Daniel L. Mollitt MD Professor and Chief Division of Pediatric Surgery University of Florida Health Scince Center Jacksonville Florida, USA Daniel Moon MB, BS Urology Research Fellow Kids Urology Research Unit Royal Childrens Hospital Melbourne, Australia Sam W. Moore MBChB FRCS MD Professor & Head Department of Paediatric Surgery Faculty of Medicine University of Stellenbosch Tygerberg, South Africa Hirikati S. Nagaraj MD Associate Professor of Surgery Kosair Childrens Hospital University of Louisville Chief, General and Thoracic Surgery Kentucky, USA Azad S. Najmaldin MB ChB MS FRCSEd FRCS Consultant Paediatric Surgeon & Urologist St Jamess University Hospital Leeds, UK Laszlo Nemeth MD Consultant Paediatric Surgeon University of Szeged Szeged, Hungary Masaki Nio MD Associate Professor of Pediatric Surgery Senior Lecturer Division of Pediatric Surgery Tohoku University School of Medicine Sendai, Japan Contributors xvii

18. Ryoji Ohi MD Professor and Chief Division of Pediatric Surgery Tohoku University School of Medicine Sendai, Japan Keith T. Oldham MD Professor and Chief Division of Pediatric Surgery Vice Chairman Department of Surgery Medical College of Wisconsin Milwaukee Wisconsin, USA David A. Partrick MD Assistant Professor in Surgery and Pediatrics University of Colorado Health Sciences Center Director of Surgical Endoscopy The Childrens Hospital Denver Colorado, USA Alberto Pea MD FACS FAAP Professor & Chief Division of Pediatric Surgery Albert Einstein College of Medicine Schneider Childrens Hospital New Hyde Park New York, USA Agostino Pierro MD FRCS FAAP Professor of Paediatric Surgery Institute of Child Health and Great Ormond Street Hospital London, UK Jack Plaschkes MD FRCS Department of Paediatric Surgery Faculty of Medicine University of Stellenbosch Tygerberg, South Africa Kevin C. Pringle MB ChB FRACS Professor of Paediatric Surgery & Head Department of Obstetrics & Gynaecology Wellington School of Medicine and Health Sciences University of Otago Wellington, New Zealand Prem Puri MS FRCS FRCS(Ed) FACS Newman Clinical Research Professor University College, Dublin Consultant Paediatric Surgeon, Our Ladys Hospital for Sick Children and National Childrens Hospital, Dublin Director of Research, Childrens Research Centre, Dublin, Ireland Denis Reen MSc PhD Adjunct Professor in Medicine, University College, Dublin Professor, The Childrens Research Centre Our Ladys Hospital for Sick Children Dublin, Ireland Heinz Rode Mmed(Chir) FCS(SA) FRCSEd Charles F M Saint Professor of Paediatric Surgery Department of Paediatric Surgery Red Cross Childrens Hospital Rondebosch, South Africa John D. Russell FRCSI, FRCS(ORL) Consultant Paediatric Otolaryngologist Our Ladys Hospital for Sick Children Dublin, Ireland Loureno Sbragia MD PhD Postdoctoral Research Scholar Fetal Treatment Center Division of Pediatric Surgery University of California San Francisco, USA Robert C. Shamberger MD Professor of Surgery Harvard Medical School Chief of Surgery (Interim) Department of Paediatric Surgery Childrens Hospital Boston Massachusetts, USA Ronald J. Sharp MD Director of Surgery Childrens Mercy Hospital Kansas City Missouri, USA Felix Schier MD Head of Department Department of Paediatric Surgery University Medical Centre Jena, Germany Steven J. Shochat MD Surgeon-in-Chief & Chairman Department of Surgery St Jude Childrens Research Hospital Memphis Tennessee, USA Owen P. Smith MA MB BA Mod (Biochem), FRCPCH FRCPI, FRCPLon, FRCPEdin, FRCPGlasg, FRCPath Consultant Paediatric Haematologist Our Ladys Hospital for Sick Children, and St Jamess Hospital, Dublin Senior Lecturer in Haematology, Trinity College Dublin, Ireland xviii Contributors

19. Lewis Spitz MB ChB PhD MD(Hon), FRCS(Edin), FRCS(Eng), FAAP(Hon), FRCPCH Nufeld Professor of Paediatric Surgery Institute of Child Health University College London and Great Ormond Street Hospital London, UK Charles J. H. Stolar MD Professor of Surgery and Pediatrics Division of Pediatric Surgery College of Physicians and Surgeons Columbia University Director of Pediatric Surgery Childrens Hospital of New York New York Presbyterian Hospital New York, USA Mark D. Stringer BSc MS FRCS FRCS(Paed) FRCP FRCPCH Consultant Paediatric Surgeon Childrens Liver & GI Unit St Jamess University Hospital Leeds, UK Rajendra Surana MS FRCS(Paed) Consultant Paediatric Surgeon Welsh Centre for Paediatric Surgery University Hospital of Wales Cardiff, UK Norio Suzuki MD Chief of Surgery Department of Surgery Gunma Childrens Medical Center Gunma, Japan Yechiel Sweed MD Senior Lecturer in Surgery Rappaport School of Medicine The Technion Haifa Head Pediatric Surgery Western Galilee Hospital Nahariya, Israel Hiroo Takehara MD Associate Professor and Chief of Pediatric Surgeons Department of Digestive and Pediatric Surgery University of Tokushima Tokushima, Japan David F.M. Thomas FRCP FRCS Consultant Paediatric Urologist Reader in Paediatric Surgery Leeds Teaching Hospitals University of Leeds Leeds, UK Juan A. Tovar MD Professor of Surgery Department of Surgery Hospital Infantil La Paz Madrid, Spain Yoshiaki Tsuchida MD PhD FACS Director Department of Surgery Gunma Childrens Medical Center Gunma, Japan Ashley Vernon MD Research Fellow Division of Pediatric Surgery Childrens Hospital of Alabama Birmingham Alabama, USA I. Vinograd MD Head Department of Pediatric Surgery DANA Childrens Hospital Tel-Aviv, Israel Declan Warde MB BCH FFARCSI Consultant Anaesthetist Department of Anesthesia The Childrens Hospital Dublin, Ireland Tomas Wester MD PhD Consultant Paediatric Surgeon Department of Paediatric Surgery University Childrens Hospital Uppsala, Sweden Atsuyuki Yamataka MD Associate Professor of Pediatric Surgery Department of Pediatric Surgery Juntendo University School of Medicine Tokyo, Japan Jyoji Yoshizawa MD PhD Assistant Professor of Surgery Fetal Treatment Center University of California San Francisco, USA George G. Youngson PhD FRCS Honorary Professor of Paediatric Surgery Department of Paediatric Surgery Royal Aberdeen Childrens Hospital Aberdeen, UK Moritz M. Ziegler MD Robert E Gross Professor of Surgery Harvard Medical School Surgeon-in-Chief Childrens Hospital Boston Maryland, USA Contributors xix

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21. 1 General

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23. 1 Embryology of malformations DIETRICH KLUTH, WOLFGANG LAMBRECHT AND CHRISTOPH BHRER INTRODUCTION Approximately 3% of human newborns present with congenital malformations.1 Without surgical intervention, one-third of these infants would die since their malformations are not compatible with sustained life outside the uterus.1,2 In gures, this means that in a country such as Germany, nearly 6000 children are born every year with a life-threatening malformation. Due to the development of prenatal diagnostic procedures, advanced surgical techniques, and intensive postoperative care, most infants with otherwise fatal malformations can be rescued by an operation in the neonatal period. However, morbidity remains high in some of these children2 with the necessity of repeated operations and hospitalizations despite a successful primary operation. This may also be the fate of many children with non-life-threatening malformations such as hypospadias or cleft palate. Mortality is still high in newborns with certain malformations such as congenital diaphragmatic hernias or severe combined defects. As a consequence, congenital malformations today are the main cause of death in the neonatal period. In the USA, 21% of neonatal mortality can be related to congenital malformations.3 These gures probably do not reect a real increase of the actual incidence of congenital malformation. The observed mortality shift might rather be due to improved intensive care medicine in todays western world countries where neonates (even those with birth defects) have a better chance of survival. On the other hand, this statistical shift indicates that knowledge about congenital malformations lags behind the progress clinical research has made in the surrounding elds. Efforts are needed to close the gap and learn more about baby killer No. 1. Identication of teratogens will help to reduce the incidence of malformations when exposure can be avoided, and pathogenetic studies might aid in designing therapeutic measures. Both treatment and prevention critically depend on basic embryological research. DEFINITION OF THE TERM MALFORMATION After birth neonates can present with a broad spectrum of deviations from normal morphology. This extends from minor variations of normal morphology without any clinical signicance to maximal organ defects with extreme functional decits of the malformed organs or of the whole organism. The degree of functional disorder is decisive when dealing with the question of whether a variation of normal morphology has to be viewed as a dangerous malformation requiring surgical correction. This means that functional disturbance is essential when using the term malformation. Inborn deviations can be detri- mental, neutral, or even benecial, otherwise evolution- ary progress could not take place. An example of a benecial deviation is the longevity syndrome of people with abnormally low serum cholesterol levels. Abnor- malities with little or no functional disturbance might still require surgical correction when patients are in danger of social stigmatization. Coronal or glandular hypospadias might serve as an example for this condition. ETIOLOGY OF CONGENITAL MALFORMATIONS In most cases, the etiology of congenital malformations remains unclear. Possible etiological factors are listed in Table 1.1. In about 20% of cases genetic factors (gene mutation and chromosomal disorders) can be identied.1,2,4 In 10% an environmental origin can be demonstrated.1,2 In 70% the factors responsible remain obscure. Table 1.1 Etiology of congenital malformations Genetic disorders 20% Environmental factors 10% Unknown etiology 70%

24. Environmental factors A large number of agents are known which might interfere with the normal development of organ systems during embryogenesis.1,4 The underlying mechanisms of this interference is poorly understood in most cases. Characteristically, during organogenesis, different organs of the embryo show distinct periods of greatest sensitivity to the action of the teratogen. These phases of greatest sensitivity are called the teratogenetic period of determination.5 The typical patterns of some syndromes can be explained by an overlap of these phases during embryological development. In 1983, Shepard2 published a catalog of suspected teratogenic agents. Over 900 agents are known to produce congenital anomalies in experimental animals. In 30 evidence for teratogenic action in humans could be demonstrated. Teratogenic agents can be divided into four groups (Table 1.2). The teratogenic potential of virus infections,1 especially rubella and herpes, and that of radiation1 has been clearly established. Maternal metabolic defects and lack of essential nutritives can be teratogenic. After a vitamin A-free diet6 and riboavin-free diet7 various congenital malformations were observed in rats and mice. Among these were diaphragmatic hernias, isolated esophageal atresias, and isolated tracheo-esophageal stulas. Similarly, inappropriate administration of hormones can be associated with intrauterine dysplasias.8 Industrial and pharmaceutical chemicals such as tetrachlor-diphenyl-dioxin (TCDD) or thalidomide have inicted tragedies by their teratogenic action. When thalidomide was prescribed to women in the early 1960s as a safe sleeping medication, numerous children were born with dysmelic deformities.4,9,10 In addition, atresias of the esophagus, the duodenum, and the anus were observed in some children.9 The data collected suggest that teratogenic agents do not cause new patterns of malformations but rather mimic sporadic birth defects. This had posed problems in identifying thalidomide as the responsible agent. It appears likely that among those 70% congenital malformations with unclear etiology a considerable percentage might be precipitated by as yet unidentied environmental factors. In a rat model, the herbicide nitrofen (2.4-dichloro-phenyl-p-nitrophenyl ether) has been shown to induce congenital diaphragmatic hernias, cardiac abnormalities and hydronephrosis.1115 In 1978, Thompson et al. described the teratogenicity of the anti-cancer drug adriamycin in rats and rabbits.16 More recently, Diez-Pardo et al.17 re- described this model with emphasis to its potentials as a model for foregut anomalies. Today, the adriamycin model is generally described as a model for the VACTERL-association (V=vertebral, A=anorectal, C=cardiac, T=tracheal, E=esophageal, R=renal, L=limb).18,19 Thus, classic malformations such as atresias of the esophagus and the intestinal tract, intestinal duplications and others can be mimicked by teratogens in animal models. Genetic factors Approximately 20% of congenital malformations are of genetic origin. Most surgically correctable malformations are associated with chromosomal disorders, e.g. trisomy 21,13, or 18, or are of multifactorial inheritance20 with a small risk of recurrence. The assumption of multifactorial inheritance results from the fact that with nearly all major anomalies familiar occurrences had been observed.1 In animals inheritance has also been found for some malformations.2124 EMBRYOLOGY OF MALFORMATIONS Disturbances of normal embryological processes will result in malformations of organs. This was rst shown by Spemann25 in 1901 by experimentally producing supernumary organs in the triton embryo after establishing close contact between excised parts of triton eggs and other parts of the same egg. Spemann and Mangold5 coined the term induction to describe this observation. They found that certain parts of the embryo obviously were able to control embryonic development of other parts. These controlling parts were called organizers.5 The process of inuence itself was called induction. It was believed by many scientists in the eld that induction could serve as the overall principle of hierarchical control of embryonic development. Ensuing investigations, however, made modications necessary, which nally resulted in a very complex model of organizers and inductors. The nature of inductive substances remained obscure and attempts to isolate inductive substances, meanwhile called morphogenes, were unsuccessful.26 Interestingly, not only live cells could induce development in certain experiments but also dead and denaturated material.5 A process essential for the formation of early embryonic organs is the invagination of epithelial sheets. This invagination is preceded by a thickening of the 4 Embryology of malformations Table 1.2 Teratogenic agents in congenital malformations Physical agents Radiation, heat, mechanical factors Infectious agents Viruses, treponemes, parasites Chemical, drug, Thalidomide, nitrofen, environmental agents hormones, vitamin deciencies Maternal, genetic Chromosomal disorders, factors multifactorial inheritance After Nadler.1

25. epithelial sheet,27 a process known as placode formation. The thickening itself is caused by elongation of individual cells of the placode. This process can be studied in detail in epithelial morphogenesis.28 The same sequence of developmental events has been observed in the formation of the neural plate, in the formation of the otic and lens placode and in the development of most epitheliomesenchymal organs including lung, thyroid gland and pancreas. From these observations it can be concluded that most epithelial cells behave uniformly in the early phase of embryonic development. Today it is generally accepted, that early embryonic organs are especially sensitive for alterations. Therefore researchers are more and more interested to understand the formation of early embryonic organs. In 1985 ETTERSOHN.29 stated that most invagi- nations are the results of mechanical forces that are local in origin. He focussed on three possible mechanisms which might lead to placode formation and subsequent invagination: 1 Change of cell shape by cell adhesion 2 Microlament-mediated change of cell shape 3 Cell growth and division. In the following part, we will discuss some aspects of these mechanisms. A teratological method used to determine the function of cell adhesion molecules in vivo during embryogenesis has been reported recently.30 Mouse hybridoma cells producing monoclonal antibodies against the avian integrin complex were grafted into 2- or 3-day-old chick embryos. Depending on the site of engraftment, local muscle agenesis was observed. This is an example that the immunologic immaturity of the embryo can be exploited to study the contribution of cell attachment molecules to organ development in a functional fashion. A number of monoclonal antibodies directed against cell attachment molecules of various species have become available over the last 8 years, and the structure of the binding molecules has been elucidated biochemically and by cDNA cloning. Functionally, adhesion molecules may be grouped into three families: Cell adhesion molecules (CAMs), which mediate specic and mostly transient cell recognition of other cells, substrate adhesion molecules (SAMs), necessary for attachment to extracellular matrix proteins, and cell-junctional molecules (CJMs), found in tight and gap junctions. Whereas CJMs apparently play an important role for metabolic signalling within established tissues, CAMs and SAMs are necessary for the formation of histologically distinct structures and directed migration of single cells. Among CAMs and SAMs, at least three families have been identied biochemically: integrins,31 members of the immunoglobulin superfamiliy, and LEC-CAMS.32 Integrins are hetero- dimeric molecules consisting of a larger chain, which is associated with a smaller chain in a calcium-dependent way. Usually, one given chain might be found in association with various chains but promiscuity of chains has been described recently. Functionally, members of the integrin family present as SAMs (adhesion to vitronectin, collagen, bronectin, complement components, or other intercellular matrix proteins) or CAMs (direct adhesion to other cells via correspond- ing cell surface target molecules). For example, cells bearing the integrin LFA-1 on their cell surface bind to cells expressing ICAM-1 or ICAM-2, both of which are members of the immunoglobulin superfamily.33,34 Other members of the immunoglobulin superfamily which are known to be important during morphogenesis include L-CAM35 (liver cell adhesion molecule) and N-CAM36,37 (neural cell adhesion molecule). Both show homophilic aggregation, that is, N-CAM serves as a target structure for N-CAM, and L-CAM serves as a target structure for L-CAM, but there is no cross-reactivity. In developing feather placodes in avian embryos, L-CAM and N-CAM are mutually exclusive expressed on epidermal or mesodermal cells, respectively. When the placodes are incubated with antibodies to L-CAM, primarily only epidermal cell-to-cell contact is disturbed.38 However, the structure of the surrounding mesoderm is altered subsequently, suggesting an inductive signal loop between epidermal and mesodermal cells. A third group of adhesion molecules has been termed LEC-CAMs to indicate that their extracellular part consists of a lectin domain, an epidermal growth factor-like domain, and a complement regulatory protein repeat domain. The lectin domain is presumed to contain the active center; binding mediated by the murine homolog to the leuko- cyte adhesion molecule 1 (LAM-1)39 can be blocked by mannose-6-phosphate or its polymers.40 Lectin- dependent organ formation should be accessible experimentally by administration of the respective carbo- hydrates but few if any data have been reported so far. Cell shape is mainly maintained by microtubules forming the cellular cytoskeleton. In addition, con- tractile elements exist such as actin, which are essential for cell movement, the so-called microlaments. These structures are thought to be essential for the process of placode formation and invagination.41 Microlament- mediated change of cell shape is based on the idea that actin laments could alter the shape of cells by contraction. Most of these laments are found at the apex of epithelial cells. Contraction of these laments in each individual cell of a cell layer would result in an increasing infolding of the whole cell layer,41,42 nally resulting in invagination. It is a disadvantage of this model, however, that there is no apparent reason why apical constriction should be proceeded by cell elongation.29 Cell proliferation is probably an essential factor in the morphogenesis of epithelio-mesenchymal organs.22 During morphogenesis of these organs repeated invagination can be observed, which might be dependent upon cell proliferation.43 The way in which epithelial cell growth and proliferation is controlled in the embryo is Embryology of malformations 5

26. not clear. However, it is believed that the surrounding mesenchyme might regulate the timing and location of invagination of the epithelial layer. Goldin and Opperman44 proposed that epidermal growth factor (EGF) might be excreted by mesenchymal cells, which would stimulate epithelial cell proliferation and repeated invagination. When agarose pellets impregnated with EGF were cultured alongside 5-day embryonic chick tracheal epithelium, supernumerary buds were induced to form at those sites. EGF and the related peptide transforming growth factor- (TGF) have been shown to lead to precocious eyelid opening when injected into newborn mice.45 Thus, complex changes of late-stage organ development can be induced by physiological stimuli in the laboratory. Interestingly, EGF is a mitogen for many epithelial cells in vitro without affecting most mesenchymal cells. A large variety of cells have been demonstrated to display the receptor for EGF/TGF on their cell surface, which is encoded by the cellular proto- oncogene c-erbB. Structural alterations of this receptor are known to result in uncontrolled proliferation and ultimately malignant transformation. When secreted locally, EGF might provide physically associated cells with appropriate on- and off-signals required for the formation of complex organs. Other polypeptides, such as platelet-derived growth factor (PDGF) or transforming growth factor- (TGF) appear to function in an antagonistic way in that they stimulate rather the proliferation of mesenchymal cells.46,47 In dened experimental situations, TGF has been shown to be a mitogen for osteoblasts while being a potent inhibitor of the proliferation of epithelial and endothelial cells at the same time. Embryonic broblasts, however, are also inhibited by TGF.48 TGF is a powerful chemotactic agent for broblasts and enhances the production of both collagen and bronectin by these cells. There is, however, little data available concerning the involvement of these factors during normal and pathologic development of the embryo. Future investigations using such powerful approaches as in situ hybridization with cloned genes, preparation of transgenic animals, and direct administration of the recombinant proteins to various parts of the embryo might shed some light on signalling pathways mediated by soluble cytokines. The surrounding mesenchyme might limit the epithelial bud to expand49 forcing the epithelial sheet to fold in characteristic patterns. If a growing cell layer is restricted from lateral expansion, mitotic pressure by dividing cells will result in elongation of cells and then invagination of the crowded cell sheet. This does not necessarily imply that cells divide more rapidly in the region of invagination than in the surrounding areas. The main effect is caused by restriction of lateral expansion.50,51 In the early anlage of the thymus, cell proliferation counts are actually lower in the thymus anlage than in the surrounding epithelium.52 Steding50 and Jacob51 have shown experimentally that restriction of lateral expansion might be responsible for thickening and subsequent invagination of epithelial sheets. In their experiments, restriction of lateral expansion was caused by a tiny silver ring placed on the epithelium of chick embryos. EXAMPLES OF PATHOLOGICAL EMBRYOLOGY The focus of our research has been the embryology of foregut, anorectal and diaphragmatic malformations. We studied the normal development of all embryonic organs involved by scanning electron microscopy (SEM).5359 In addition, we employed two rodent animal models to study malformations of the anorectum and the diaphragm. Pathogenetic concepts concerning these malformations were controversial in the past due to lack of detailed data. EMBRYOLOGY OF FOREGUT MALFORMATIONS The differentiation of the primitive foregut into the ventral trachea and dorsal esophagus is thought to be the result of a process of septation.60 It is guessed that lateral ridges appear in the lateral walls of the foregut, which fuse in midline in a caudo-cranial direction thus forming the tracheo-esophageal septum. This theory of septation has been described in detail by Rosenthal and Smith.6162 However, others6364 were not able to verify the importance of the tracheo-esophageal septum for the differentiation of the foregut. They instead proposed individually that the respiratory tract develops simply by further growth of the lung bud in a caudal direction. Using scanning electron microscopy (SEM), we studied the development of the foregut in chick embryos.53,54 In this study, we were unable to demonstrate the formation of a tracheo-esophageal septum (Fig. 1.1). A sequence of SEM photographs of staged chick embryos suggests that differentiation of the primitive foregut is best explained by a process of reduction of size of a foregut region called tracheo-esophageal space (Fig. 1.2). This reduction is caused by a system of folds that develops in the primitive foregut. They approach each other but do not fuse (Fig. 1.2). Based on these observations, the development of the malformation can be explained by disorders either of the formation of the folds or of their developmental movements: 1 Atresia of the esophagus with stula (Fig. 1.3a): The dorsal fold of the foregut bends too far ventrally.As a result the descent of the larynx is blocked. Therefore the tracheo-esophageal space remains partly undivided and lies in a ventral position. Due to this ventral position it differentiates into trachea. 6 Embryology of malformations

27. 2 Atresia of the trachea with stula (Fig. 1.3b): The foregut is deformed on its ventral side. The developmental movements of the folds are disturbed and the tracheo-esophageal space is dislocated in a dorsal direction. Therefore it differentiates into esophagus. 3 Laryngo-tracheo-esophageal clefts (Fig. 1.3c): Faulty growth of the folds results in the persistence of the primitive tracheo-esophageal space. Recently it has been shown that esophageal atresias and tracheo-esophageal stulas can be induced by maternal application of adriamycin into the peritoneal cavity of pregnant rats.16,17 The dosage may vary between 1.5 mg to 2.0 mg/kg depending on the number of days it will be given. In most reports the most promising dosage is 1.75 mg/kg given on days 69 of pregnancy. The adriamycin model has been intensively studied over the last couple of years, resulting in more than 30 reports between 1997 and 2001.65 It could be demonstrated that in this model not only foregut malformations but also atypical patterns of malformation can be observed which are usually summarized under the term VATER or VACTERL association.18,19 Therefore, this model is not only promising for the studies of foregut anomalies but also for anomalies of the hind- and mid-gut. DEVELOPMENT OF THE DIAPHRAGM In the past, several theories were proposed to explain the appearance of postero-lateral diaphragmatic defects: 1 Defects caused by improper development of the pleuro-peritoneal membrane66,67 Development of the diaphragm 7 Figure 1.1 SEM photograph of the inner layer of foregut epithelium in a chick embryo (approx. 3.5 days old). View from cranial. Between trachea (tr) on bottom and esophagus (es) on top, the tip of the tracheo-esophageal fold (tef) is recognizable. Lateral ridges or signs of fusion are not found45,46 Figure 1.2 Summarizing sketch of foregut development. The tracheo-esophageal space (tes) is reduced in size by developmental movements of folds (indicated by arrows) (es, esophagus; la, anlage of larynx; br, bronchus; tr, trachea). Short arrow marks tip of tracheo-esophageal fold (tef) (compare Figure 1.1) Figure 1.3 Sketch of formal pathogenesis of typical foregut malformations (see text for details): (a) atresia of esophagus with stula; (b) atresia of trachea with stula; (c) laryngotracheo-esophageal cleft. Arrows indicate sites of possible deformation of the developing foregut

28. 2 Failure of muscularization of the lumbocostal trigone and pleuro-peritoneal canal, resulting in a weak part of the diaphragm66,68 3 Pushing of intestine through postero-lateral part (foramen of Bochdalek) of the diaphragm69 4 Premature return of the intestines into the abdominal cavity with the canal still open66,68 5 Abnormal persistence of lung in the pleuro- peritoneal canal, preventing proper closure of the canal70 6 Abnormal development of the early lung and posthepatic mesenchyme, causing non-closure of pleuro-peritoneal canals.15 Of these theories, failure of the pleuro-peritoneal membrane to meet the transverse septum is the most popular hypothesis to explain diaphragmatic herniation. However, using SEM techniques,55 we could not demonstrate the importance of the pleuro-peritoneal membrane for the closure of the so-called pleuro-peritoneal canals (Fig. 1.4). As stated earlier, most authors assume that delayed or inhibited closure of the diaphragm will result in a diaphragmatic defect that is wide enough to allow herniation of gut into the fetal thoracic cavity. However, this assumption is not the result of appropriate embryological observations but rather the result of interpreta- tions of anatomical/pathological ndings. In a series of normal staged embryos we measured the width of the pleuro-peritoneal openings and the transverse diameter of gut loops.54 On the basis of these measurements we estimated that a single embryonic gut loop requires at least an opening of 450 size to herniate into the fetal pleural cavity. However, in none of our embryos the observed pleuro-peritoneal openings were of appropriate dimensions. This means that delayed or inhibited closure of the pleuro-peritoneal canal cannot result in a diaphragmatic defect of sufcient size. Herniation of gut through these openings is therefore impossible. Thus the proposed theory about the pathogenetic mechanisms of congenital diaphragmatic hernia (CDH) development lacks any embryological evidence. Furthermore the proposed timing of this process is highly questionable.57 Recently, an animal model for diaphragmatic hernia has been developed1115 using nitrofen as noxious substance. In these experiments CDHs were produced in a reasonably high percentage of newborns.12,13 Most diaphragmatic hernias were associated with lung hypoplasias. Using electron microscopy, our group5659 used this model to give a detailed description of the development of the diaphragmatic defect. Our results are as follows: Timing of diaphragmatic defect appearance Iritani15 was the rst to notice that nitrofen-induced diaphragmatic hernias in mice are not caused by an improper closure of the pleuro-peritoneal openings but rather the result of a defective development of the so- called post-hepatic mesenchymal plate (PHMP). In our study in rats, clear evidence of disturbed development of the diaphragmatic anlage was seen on day 13 (left side) and day 14 (right side, Fig. 1.5).56,59 In all embryos 8 Embryology of malformations Figure 1.4 SEM photograph of right pleural sac in a rat embryo (approx. 16.5 days old). View from cranial. The so- called pleuro-peritoneal canal (PPC) is nearly closed. Small arrows point at the margin of PPC. In the depth of the abdomen the right adrenals (ad) are seen. Large arrows point at margins of the so-called pleuro-peritoneal membrane. Its contribution to the closure of the canal is minimal47 (es, esophagus) Figure 1.5 Cranial view of the pleural sacs in a rat embryo after exposition to nitrofen on day 11 of pregnancy. The embryo is approx. 15 days old. Note the big defect of the right diaphragmatic primordium. Small black arrows point at margins of the defect, which leaves parts of the liver (li) uncoated. On the left, the diaphragmatic anlage is normal. Note the low position of the cranial border of the pleuro-peritoneal opening on this side (white arrows). (ad, adrenals; di, anlage of diaphragm)

29. affected, the PHMP was too short. This age group is equivalent to 45-week-old human embryos.56 Location of diaphragmatic defect In our SEM study, the observed defects were localized in the PHMP (Fig. 1.5). We identied two distinct types of defects: (1) large dorsal defects and (2) small central defects.56 Large defects extended into the region of the pleuro-peritoneal openings. In these cases, the closure of the pleuro-peritoneal openings was usually impaired by the massive ingrowth of liver (Figs 1.6 & 1.7). If the defects were small, they were consistently isolated from the pleuro-peritoneal openings closing normally at the 16th or 17th day of gestation. Thus, in our embryos with CDH, the region of the diaphragmatic defect was a distinct entity and was separated from that part of the diaphragm where the pleuro-peritonealcanals are localized. We conclude therefore that the pleuro-peritoneal openings are not the precursors of the diaphragmatic defect. Why lungs are hypoplastic Soon after the onset of the defect in the 14-day-old embryo, liver grows through the diaphragmatic defect into the thoracic cavity (Fig. 1.6). This indicates that from this time on the available thoracic space is reduced for the lung and further lung growth hampered. In the following stages, up to two-thirds of the thoracic cavity can be occupied by liver (Fig. 1.7). Herniated gut was found in our embryos and fetuses only in late stages of development (21 days and newborns). In all of these, the lungs were already hypoplastic, when the bowel entered the thoracic cavity.53 Based on these observations, we conclude that the early ingrowth of the liver through the diaphragmatic defect is the crucial step in the pathogenesis of lung hypoplasia in CDH. This indicates that growth impairment is not the result of lung compression in the fetus but rather the result of growth competition in the embryo: the liver that grows faster than the lung reduces the aviable thoracic space. If the remaining space is too small, pulmonary hypoplasia will result. DEVELOPMENT OF THE CLOACA In the literature several theories have been put forward to explain the differentiation of the cloaca into the dorsal anorectum and the ventral sinus urogenitalis. To many authors this differentiation is caused by a septum which develops cranially then caudally and thus divides the cloaca in a frontal plane. Disorders in this process of differentiation are thought to be the cause of cloacal anomalies such as persistent cloaca and anorectal malformations. However, there is no agreement on the mechanisms of the septational process. While some authors71,72 believe that the descent of a single fold separates the urogenital part from the rectal part by ingrowth of mesenchyme from cranial, others73 think that lateral ridges appear in the lumen of the cloaca, which progressively fuse along the midline and thus form the septum. In a recent paper74 the process of septation had been questioned altogether. Using SEM techniques, our group studied cloacal development in rat and sd-mice embryos. The sd-mouse Development of the cloaca 9 Figure 1.6 Liver (li) protrudes through diaphragmatic defect. Arrows point to the margin of the defect (di, diaphragmatic anlage). Rat embryo (approx. 16 days old), nitrofen exposition on day 11 of pregnancy Figure 1.7 SEM photograph of a right pleural sac in a rat embryo after nitrofen exposure on day 11 of pregnancy. The embryo is approximately 15.5 days old. Note the big defect of the right dorsal diaphragm (large arrows). The closure of the pleuro-peritoneal canal (PPC) is impaired by the ingrowths of liver (small arrows). Li1 = liver growing through PPC. Li1 + Li2 = liver growing through the defect of the diaphragm

30. is a spontaneous mutation of the house mouse characterized by having a short tail (Fig. 1.8). Homo- zygous or heterozygous offspring of these mice show skeletal, urogenital and anorectal malformations.18 Therefore these animals are ideal in the study of the development of anorectal malformations. Normal cloacal embryology (rat) As in the foregut of chick embryos, signs of median fusion of lateral cloacal parts could not be demonstrated during normal cloacal development in the rat. However, in contradiction to vdPUTTE,74 the current authors think that downgrowth of the urorectal fold takes place, although it is probably not responsible for the formation of cloacal malformations. Abnormal cloacal embryology (sd-mouse) Cloacal malformations are caused by improper development of the early anlage of the cloacal membrane as demonstrated in sd-mice embryos.75,76 Our studies of abnormal cloacal development in sd- mice had the following results: 1 The basis of the pathogenesis of anorectal malformations is too short a cloacal membrane 2 The anlage of the cloacal membrane is too short and results in a maldeveloped anlage of the cloaca, which is undeveloped in its dorsal part (Fig. 1.9) 3 The caudal movement of the urorectal fold is impaired by the malformed cloaca. Thus the hindgut remains in abnormal contact with the cloaca. This opening is true ectopic and will develop into the recto-urogenital stula (Fig. 1.10). HYPOSPADIAS Many investigators7780 believe that the urethra develops by fusion of the paired urethral folds following the disintegration of the urogenital membrane. Impairment of this process is thought to result in the different forms 10 Embryology of malformations Figure 1.8 Characteristic short tail (arrow) of sd-mouse embryo (approx. 13 days old) (ll, left lower limb; ge, genital tuberculum, abnormal) Figure 1.9 Malformed cloaca of sd-mouse embryo (approx. 11 days old). The surrounding mesenchyme is removed by microdissection. View on the basal layer of the cloacal entoderm. The cloaca has lost its contact to the ectoderm of the genitals (white arrow). The dorsal part of the cloaca is missing (black arrow). Tailgut (tg) and hindgut (hg) are hypoplastic. This malformed cloaca developed because the anlage of the cloacal membrane was too short in early embryogenesis (see text for details) (cc, rest of cloaca; u, urachus, rudimentary) Figure 1.10 Malformed cloaca of sd-mouse, embryo (approx. 13 days old). Urachus (u) and rectum (re) nearly normal (cl, ventral part of cloaca with short cloacal membrane). The dorsal part of the cloaca is missing (long white arrows). Short white arrow points to the region of the future stula

31. of hypospadia80 However, in our study of normal cloacal development,81 we were puzzled by the fact that disintegration of the urogenital part of the cloacal membrane could not be observed in rat embryos (Fig. 1.11). This nding caused us to call in question the generally assumed concepts of hypospadia formation. Instead we found that: 1 The urethra is always present as a hollow organ during embryogenesis of rats and that it is always in contact with the tip of the genitals, and that 2 An initially double urethral anlage exists. The differentiation in female and male urethra starts in rats of 18.5 days old. On the other hand, we found no evidence for: The disintegration of the urogenital cloacal membrane, and A fusion of lateral portions within the perineum. In our opinion, more than one embryological mech- anism is at play in the formation of the hypospadias complex. The moderate degrees, such as the penile and glandular forms, represent a developmental arrest of the genitalia (Fig. 1.12). They take their origin from a situation comparable to the 20-day-old embryo. Consequently the penis, not the urethra is the primary organ of the malformation. Perineal and scrotal hypospadias are different from the type discussed previously. Pronounced signs of feminization in these forms suggest that we are dealing with a female-type urethra. Origin of this malformation complex is an undifferentiated stage as may be seen in the 18.5-day-old rat embryo. CONCLUSION Despite the long history of experimental embryology, we know very little about etiology and pathogenesis of congenital malformations. For decades, hypotheses were abundant while few data existed to support them. The tremendous progress of neighboring biological sciences is now providing powerful tools for researchers in the eld, such as recombinant DNA and hybridoma tech- nology. Future investigations will monitor closely how genes are switched on and off during embryogenesis and determine the relation of spatial and temporal disturbances to ensuing malformations. Target structures of chemical or viral teratogens within the embryonic cells await identication. Finally, improved understanding of growth coordination in utero will extend to related areas such as wound healing and proliferation of cancer cells. REFERENCES 1. Nadler HL. Teratology. In: Welch KJ, Randolph JG, Ravitch MM, ONeill JA, Rowe MJ. Pediatric Surgery. 4th edn (eds), Year Book Medical Publishers: Chicago: Year Book Medical Publishers 1986: 1113. 2. Shepard TH. Catalogue of Teratogenic Agents. 4th edn. Baltimore: Johns Hopkins Press, 1983. 3. United States National Center for Health Statistics. Monthly Vital Statistics Report, Vol. 31. No. 5. Birth, marriages, divorces, and deaths for May 1982. Hyattsville, MD: Public Health Service, 1982:110. 4. McCredie J, Loewenthal J. Pathogenesis of congenital malformations. Am J Surg 1978; 135:2937. 5. Spemann, Mangold, cited by Starck D. Stuttgart: Embryologie, Thieme, 1975: 13563. References 11 Figure 1.11 Genitals of a normal female rat embryo (approx. 18.5 days old) (gl, glans). Arrow points to future opening of the female urethra. No signs of disintegration of the cloacal membrane Figure 1.12 Genitals of a normal male rat embryo (approx. 20 days old) (gl, glans; pf, preputial fold; sc, scrotum). Arrow points to the raphe up to this stage; disintegration of the urogenital part of the cloacal membrane was not seen. Note similarity with clinical picture of hypospadia!

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Nature 1988; 331:6247. 34. Staunton DE, Dustin L, Springer TA. Functional cloning of ICAM-2, a cell adhesion ligand for LFA-1 homologous to ICAM-1. Nature 1989; 339:614. 35. Gallin WJ, Sorkin C, Edelman GM et al. Structure of the gene for the liver cell adhesion molecule, L-CAM. Proc Natl Acad Sci USA 1987; 84:280812. 36. Edelman GM. Morphoregulatory molecules. Biochemistry 1988; 27:353343. 37. Rutishauser U, Acheson A, Hall AK et al. The neural cell adhesion molecule (NCAM) as a regulator of cell-cell interactions. Science 1988; 240:537. 38. Edelman GM. Topobiology. Sci Amer 1989; May: 4452. 39. Tedder TF, Isaacs CM, Ernst TJ et al. Isolation and chromosomal localisation of cDNAs encoding a novel human lymphocyte cell surface molecule, LAM-1. J Exp Med 1989; 170:12333. 40. Yednock TA, Rosen D. Lymphocyte homing. Adv Immunol 1989; 44:31378. 41. Spooner BS. Microlaments, microtubules, and extracellular materials in morphogenesis. BioScience 1975; 25:44051. 42. Baker PC, Schroeder TE. Cytoplasmatic laments and morphogenetic movement in the amphibian neural tube. Devl Biol 1967; 15:43250. 43. Alescio T, DiMichele M. Relationship of epithelial growth to mitotic rate in mouse embryonic lung developing in vitro. Embryol Exp Morphol 1968; 19:22737. 44. Goldin GV, Opperman LA. Induction of super-numerary tracheal buds and the stimulation of DNA synthesis in the embryonic chick lung and trachea by epidermal growth factor. J Embryol Exp Morphol 1980; 60:23543. 12 Embryology of malformations

33. 45. Smith JM, Sporn MB, Roberts AB et al. Human transforming growth factor-alpha causes precocious eyelid opening in newborn mice. Nature 1985; 315:51516. 46. Sporn MB, Roberts AB, Wakeeld LM et al. Transforming growth factor-beta: biological function and chemical structure. Science 1986; 233:53234. 47. Sporn MB, Roberts AB, Wakeeld LM et al. Some recent advances in the chemistry and biology of transforming growth factor-beta. J Cell Biol 1987; 105:103945. 48. Anzano MA, Roberts AB, Sporn MB. Anchor-age- independent growth of primary rat embryo cells is induced by platelet-derived growth factor and inhibited by type-beta transforming growth factor. J Cell Physiol 1986; 126:31218. 49. Nogawa H. Determination of the curvature of epithelial cell mass by mesenchyme in branching morphogenesis of mouse salivary gland. J Embryol Exp Morphol 1983; 73:22132. 50. Steding G. Ursachen der embryonalen Epithelverdickung. Acta Anat 1967; 68:3767. 51. Jacob HJ. Experimente zur Entstehung entodermaler Organanlagen. Untersuchungen an explantierten Hhnerembryonen. Anat Anzeiger 1971; 128:2718. 52. Smuts MS, Hilfer SR, Searls RL. Patterns of cellular proliferation during thyroid organogenesis. J Embryol Exp Morphol 1978; 48:26986. 53. Kluth D, Steding G, Seidl W. The embryology of foregut malformations. J Pediatr Surg 1987; 22:38993. 54. Kluth D, Habenicht R. The embryology of usual and unusual types of oesophageal atresia. Pediatr Surg Int 1987; 1:2237. 55. Kluth D, Petersen C, Zimmermann HJ et al. The embryology of congenital diaphragmatic hernia. In: Puri P, editor. Congenital Diaphragmatic Hernia: Modern Problems in Pediatrics, Vol. 24, Basel: Karger, 1989: 721. 56. Kluth D, Tenbrinck R, v. Ekesparre M et al. The natural history of congenital diaphragmatic hernia in pulmonary hypoplasia in the embryo. J Pediatr Surg 1993; 28:45663. 57. Kluth D, Tander B, v. Ekesparre M et al. Congenital diaphragmatic hernia: the impact of embryological studies. Pediatr Surg Int 1995; 10:1622. 58. Kluth D, Losty PD, Schnitzer JJ, Lambrecht W, Donahoe PK. Toward understanding the developmental anatomy of congenital diaphragmatic hernia. Clin Perinatol 1996; 23:65569. 59. Kluth D, Keijzer R, Hertl M, Tibboel D. Embryology of congenital diaphragmatic hernia. Semin Pediatr Surg 1996; 5:22433. 60. His W. Zur Bildungsgeschichte der Lungen beim menschlichen Embryo. Arch Anat Entwickl Gesch 1887; 89106. 61. Rosenthal AH. Congenital atresia of the esophagus with tracheo esophageal stula: report of eight cases. Arch Pathol 1931; 12:75672. 62. Smith EL. The early development of the trachea and the esophagus in relation to atresia of the esophagus and tracheo-oesophageal stula. Contrib Embyol Carneg Inst 1957; 36:4157. 63. Zaw Tun HA. The tracheo-esophageal septum fact or fantasy? Acta Anat 1982; 114:121. 64. ORahilly R, Muller F. Chevalier Jackson Lecture. Respiratory and alimentary relations in staged human embryos. New embyrological data and congenital anomalies. Ann Otol Rhinol Laryngol 1984; 93:4219. 65. Medline recherch: http://www.ncbi.nlm.nih.gov/PubMed/. 66. Gray SW, Skandalakis JE. Embryology for Surgeons. Philadelphia: Saunders, 1972: 35985. 67. Grosser 0, Ortmann R. Grundri der Entwicklungs- geschichte des Menschen. 7th edn. Berlin: Springer, 1970: 1247. 68. Holder RM, Ashcraft KW. Congenital diaphragmatic hernia. In: Ravitch MM, Welch KJ, Benson CD, Aberdeen E, Randolph JG, editors. Pediatric Surgery. 3rd edn. Vol. 1. (eds), Chicago: Year Book Medical Publishers, 1979: 43245. 69. Bremer JL. The diaphragm and diaphragmatic hernia. Arch Pathol 1943; 36:53949. 70. Gattone VH II, Morse DE. A scanning electron microscopic study on the pathogenesis of the posterolateral diaphragmatic hernia. J Submicrosc Cytol 1982; 14:48390. 71. Tourneux F. Sur le premiers developpements du cloaque du tubercle genitale et de lanus chez lembryon moutons, avec quelques remarques concernant le developpement des glandes prostatiques. J Anat Physiol 1888; 24:50317. 72. DeVries P, Friedland GW. The staged sequential development of the anus and rectum in human embryos and fetuses. J Pediatr Surg 1974; 9:75569. 73. Retterer E. Sur lorigin et de levolution de la region ano- gnitale des mammiferes. J Anat Physiol 1890; 26:126216. 74. vd Putte SCJ. Normal and abnormal development of the anorectum. J Pediatr Surg 1986; 21:43440. 75. Kluth D, Hillen M, Lambrecht W. The principles of normal and abnormal hindgut development. J Pediatr Surg 1995; 30:11437. 76. Kluth D, Lambrecht W. Current concepts in the embryology of anorectal malformations. Semin Pediatr Surg 1997; 6:1806. 77. Felix W. Die Entwicklung der Harn-und Geschlechtsorgane. In: Keibel F, Mall FP, editors. Handbuch der Entwicklungsgeschichte des Menschen. Vol. 2. Leipzig: Hirzel, 1911: 925. 78. Spaulding MH. The development of the external genitalia in the human embryo. Contrib Embryol Carneg 1921; 13:6788. 79. Glenister TW. A correlation of the normal and abnormal development of the penile urethra and of the intraabdominal wall. J Urol 1958; 30:11726. References 13

34. 80. Gray SW, Skandalakis JE. Embryology for Surgeons. Philadelphia: Saunders, 1972: 595631. 81. Kluth D, Lambrecht W, Reich P. Pathogenesis hypospadias more questions than answers. J Pediatr Surg 1988; 23:10951101. 14 Embryology of malformations

35. 2 Prenatal diagnosis of surgical diseases TIPPI C. MACKENZIE AND N. SCOTT ADZICK INTRODUCTION Prenatal diagnosis has undergone an explosion of growth in the past decade. The primary impetus for this rapid expansion has come from the widespread use of prenatal ultrasonography. Most correctable malformations that can be diagnosed in utero are best managed by appropriate medical and surgical therapy after maternal transport and planned delivery at term. Prenatal diagnosis may inuence the timing (Box 2.1) or mode (Box 2.2) of delivery, and in some cases may lead to elective termination of the pregnancy. In rare cases, various forms of in utero therapy may be possible (Table 2.1). Prenatal diagnosis has dened a hidden mortality for some lesions such as congenital diaphragmatic hernia, bilateral hydronephrosis, sacrococcygeal teratoma, and cystic hygroma. These lesions, when rst evaluated and treated postnatally demonstrate a favorable selection bias. The most severely affected fetuses often die in utero or immediately after birth, before an accurate diagnosis has been made. Consequently, such a condition detected prenatally may have a worse prognosis than the same condition diagnosed after delivery.1 The perinatal management of the patients involves many different medical disciplines, including obstetricians, sono- graphers, neonatologists, geneticists, pediatric surgeons, and pediatricians. It is essential that the affected family be managed using a team approach, and that information and experience be exchanged freely. In this chapter we will discuss the prenatal diagnosis of neonatal surgical lesions. First, a brief summary of the diagnostic methods currently available will be given. Then a review of prenatal diagnosis by organ system will be presented. DIAGNOSTIC METHODS Ultrasound Ultrasound testing has become a routine part of the prenatal evaluation of almost all pregnancies. It is especially important to perform ultrasound for pregnancies with maternal risk factors (e.g. age over 35 years, diabetes, Box 2.1 Defects that may lead to induced preterm delivery Obstructive hydronephrosis Gastroschisis or ruptured omphalocele Intestinal ischemia and necrosis secondary to volvulus, meconium ileus, etc. Sacrococcygeal teratoma with hydrops Box 2.2 Defects that may require cesarian delivery Myelomeningocele Gastroschisis Large sacrococcygeal teratoma Giant neck masses (EXIT procedure) Table 2.1 Diseases amenable to fetal surgical intervention in selected cases Malformation Effect on development In utero treatment Congenital diaphragmatic hernia Pulmonary hypoplasia, respiratory failure Tracheal occlusion CCAM or BPS Pulmonary hypoplasia, hydrops Thoracoamniotic shunting, lobectomy Sacrococcygeal teratoma Massive arteriovenous shunting, placentomegaly, hydrops Excision Urethral obstruction Hydronephrosis, lung hypoplasia Vesicoamniotic shunting Myelomeningocele Damage to spinal cord, paralysis Closure of defect

36. previous child with anatomic or chromosomal abnormality) and if there is an elevation in maternal serum alphafetoprotein (MSAFP). Most defects can be reliably diagnosed in the late rst or early second trimester by a skilled sonographer. More recently, nuchal translucency measurements have emerged as an independent marker of chromosomal abnormalities, with a sensitivity of about 60%.2 This abnormality may be detected on trans- vaginal ultrasound at 1015 weeks gestation, thus providing an early test for high-risk pregnancies. Nuchal cord thickening may also be a marker for congenital heart disease3 and may be a valuable initial screen to detect high-risk fetuses for referral for fetal echocardio- graphy. It is important to remember that sonography is operator dependent; the scope and reliability of the information obtained is directly proportional to the skill and experience of the sonographer. Magnetic resonance imaging Until recently, the long acquisition times required for magnetic resonance imaging (MRI) were not conducive to fetal imaging because fetal movements resulted in poor quality images. Obtaining adequate images with the traditional spin-echo techniques required fetal sedation or paralysis.4 With the development of ultrafast scanning techniques, the artifacts caused by fetal motion have almost been eliminated.5 This technique is now an important part of prenatal evaluation of fetuses referred to our institution and has greatly enhanced our ability to diagnose and treat fetal malformations. Amniocentesis The rst report of the culture and karyotyping of fetal cells from amniocentesis was by Steele and Berg in 1966.6 Since then, it has become the gold standard for detecting fetal chromosomal abnormalities by karyotyping. It is usually performed at 1516 weeks gestation and involves a very low risk of fetal injury or loss. Attempts at early amniocentesis (at 1112 weeks gestation) have been complicated by a higher pregnancy loss, increased risk of iatrogenic fetal deformities and increased postamnio- centesis leakage rate.7 For this reason, the most reliable method for rst trimester diagnosis remains chorionic villus sampling. Chorionic villus sampling Chorionic villus sampling (CVS) may be performed at 1014 weeks gestation and involves the biopsy of the chorion frondosum, the precursor for the placenta. Either a transcervical or transabdominal approach may be used, both under ultrasound guidance. The cells obtained may be subjected to a variety of tests including karyotype, genetic probes, or enzyme analysis. Due to the high mitotic rate of the chorionic villus cells, results for karyotyping may be obtained in less than 24 hours. Disadvantages include diagnostic errors due to maternal decidual contamination or genetic mosaicism of the trophoblastic layer of the placenta. When preformed by experienced operators, the pregnancy loss rate is equivalent to that of second trimester amniocentesis.8 BIOCHEMICAL MARKERS Maternal blood and amniotic uid can be screened for the presence of various biochemical markers that indicate fetal disease. About two-thirds of women in the USA currently undergo screening for Down syndrome and other chromosomal abnormalities with the triple test, which includes measuring serum alphafetoprotein with human chorionic gonadotropin and unconjugated estriol.9 This screening is performed in the early second trimester, and the detection rate for Down syndrome is 69%, with a 5% false-positive test.10 A positive result on the serum screening test indicates a need for chromosome analysis by amniocentesis. Percutaneous umbilical blood sampling Obtaining umbilical venous blood can also be used to determine the karyotype and diagnose various metabolic and hematological disorders. The percutaneous umbilical blood sampling (PUBS) procedure is performed at around 18 weeks gestation under ultrasound guidance. Karyotype results may be obtained within 2448 hours. In various large series, the mortality from the procedure has been reported to be 12%, with increasing mortality rates with long procedure times and multiple punctures.1113 Fetal cells in the maternal circulation Since the advent of uorescence-activated cell sorting (FACS), there has been growing interest and progress in detecting circulating fetal cells in maternal blood for diagnostic purposes.14 The cell type most successfully used in this endeavor is the fetal nucleated red blood cell, since these are abundant in the rst trimester fetal circulation. These cells may be separated from maternal nucleated red blood cells by staining for CD71 or fetal and embryonic hemoglobins.15 Genetic analysis may then be performed using polymerase chain reaction (PCR) or uorescence in situ hybridization (FISH) for chromosome-specic probes. Although the test currently has a low sensitivity (4050%),15 the false- positive rate is negligible, which is an advantage over the 5% false-positive rate of the conventional triple screen. 16 Prenatal diagnosis of surgical diseases

37. PRENATAL DIAGNOSIS OF SPECIFIC SURGICAL LESIONS Neck masses Fetal airway obstruction could be a result of extrinsic compression of the airway by lesions such as cervical teratoma or cystic hygroma, or intrinsic defects in the airway such as congenital high airway obstruction syndrome. Although large congenital neck masses causing airway obstruction previously carried an enormous perinatal mortality16 the advent of the ex utero intra- partum treatment (EXIT) procedure17,18 has improved their outcome by providing a means of controlling the airway during delivery and converting an airway emergency into an elective procedure (Fig. 2.1). Cystic hygroma diagnosed in utero is a severe diffuse lymphatic abnormality which is frequently associated with hydrops, polyhydramnios, and other abnormalities.19 Chromosomal abnormalities are very common (62% overall), the most common being Turners syndrome.20 There are two groups of prenatally diagnosed cervical lymphangiomas: those diagnosed in the second trimester (usually in the posterior triangle of the neck, have a high incidence of associated abnormalities, and carry a very poor prognosis),21 and those diagnosed later in gestation (most often isolated lesions and generally do not lead to hydrops). Hydrops is an ominous nding in fetuses with cystic hygroma,16 as is the presence of aneuploidy and septations in the mass.22 However, fetuses with normal karyotype, non-septated masses, and no evidence of hydrops may have a good prognosis.23 Therefore, it is important to monitor the fetus for development of hydrops by serial evaluations. Teratomas are asymmetrical lesions which are frequently unilateral, with well-dened margins. They may also be multiloculated, irregular masses with solid and cystic components. Most teratomas contain calcica- tions. MRI is a very useful adjunct to ultrasound in evaluating giant neck masses. We have used it successfully for showing the relationship of the mass to the airway in preparation for an EXIT procedure.24 T1-weighted images may help differentiate teratomas from lymphangiomas.25 The EXIT procedure, originally designed for removal of tracheal clips,17 has proven to be life-saving for many fetuses with giant neck masses.18 This procedure involves performing a maternal hysterotomy and obtaining control of the fetal airway while the fetus remains on placental support. In order to prevent uterine con- tractions during the procedure, the mother is given inhalational anesthetic and tocolytics, warm saline is infused through a level I device, and only the head and shoulders of the fetus are delivered. After attaching a pulse oximeter to the fetal hand to monitor heart rate and oxygen saturation, direct laryngoscopy and, if possible, endotracheal intubation is performed. If the airway cannot be secured in this way, a rigid broncho- scope is inserted to determine the anatomy. If secure airway establishm

Newborn surgery

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