Table Of ContentPreimplantation
Genetics
Preimplantation
Genetics
Edited by
Yury Verlinsky
and Anver Kuliev
Reproductive Genetics Institute
Chicago, Illinois
Plenum Press e New York and London
Library of Congress Cataloging-in-Publication Oata
International Symposium on Preimplantation Genetics (1st 1990
Chicago, 111.)
Preimplantation genetics / edited by Yury Verlinskyand Anver
Ku 1 iev.
p. cm.
"Proceedings of the First International Symposium on
Preimplantation Genetics, held September 14-19, 1990, in Chicago,
Illinois"--T.p. verso.
Includes bibliographical references and index.
1. Preimplantation genetic diagnosis--Congresses. I. Verlinsky,
Yury. II. Kuliev, Anver. III. T,tle.
[ONLM, 1. Embryology--congresses. 2. Genetic Techniques
-congresses. 3. Genetics, Medical--congresses. 4. Preimplantation
Phase--genetics--congresses. az 50 1614pl
RG628.3.P74157 1990
616' . 042--d c20
ONLM/OLC
for Library of Congress 91-24088
CIP
Proceedings of the First International Symposium on
Preimplantation Genetics, held September 14-19, 1990,
in Chicago, Illinois
ISBN-13: 978-1-4684-1353-3 e-ISBN-13: 978-1-4684-1351-9
DOI: 10.1007/978-1-4684-1351-9
© 1991 Plenum Press, New York
Softcover reprint of the hardcover I st edition 1991
ADivision of Plenum Publishing Corporation
233 Spring Street, New York, N.Y. 10013
All rights reserve d
No part of this book may be reproduced, stored in a retrieval system, or transmitted
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PREFACE
Yury Verlinskyand Anver Kuliev
Reproductive Genetics Institute, Illinois
Masonic Medical Center, 836 W. Wellington
chicago, IL 60657
Although introduction of a first trimester prenatal
diagnosis by chorionic viIIus sampling (CVS) has
considerably improved the possibility for prevention of
genetic diseases, it requires a selective abortion in case
of an affected fetus. Following the direction of an earlier
prenatal diagnosis and to avoid the need for abortion,
preimplantation genetic diagnosis has been initiated based
on polar body removal and pre-embryo biopsy.
The First International symposium on Preimplantation
Genetics, Chicago, September 17-19, 1990, was organized to
explore these important developments, to review the state of
knowledge in the field, and to address existing problems to
be solved for developing and improving current approaches
for preimplantation diagnosis of genetic disorders. A
growing interest in the subject was obvious from the wide
attendance of the meeting: over 250 scientists from 19
countries participated.
This was the first attempt to put together the advances in
different areas of basic and applied research relevant to
Preimplantation Genetic Diagnosis, with the
multidisciplinary scientific program including the sessions
on embryology, micromanipulation and biopsy, genetic
analysis of gametes and pre-embryos, IVF, gene expression
and gene therapy, and ethical and legal issues.
The deliberations of the Symposium presented in the above
mentioned sessions, which comprise the contents of correspond
ing sections of the Proceedings, open a newarea in medical
research based on the interaction of IVF and New Genetics.
One of the sections of the Proceedings is devoted to the
technical aspects of preconception and preimplantation
diagnosis and contains the Manuals prepared for the Workshop
on Preimplantation Genetic Analysis, held September 14-15,
1990, just prior to the First International Symposium on
Preimplantation Genetics.
v
Being held 12 years after P.S. Steptoe and R.G. Edwards
reported the birth of a baby following the transfer of a
human egg fertilized in vitro, 10 years after the First
International Congress on IVF, and 5 years after
introduction of polymerase chain reaction (PCR), the
symposium provided intriguing challenges to be resolved and
important new data of theoretieal and practical value in
developing preconception and preimplantation genetic
diagnosis. These are further steps in improving and
developing mare ethically acceptable approaches for
prevention and control of genetic disorders, obviating the
need for a selective abortian as an obligatory component in
preventive services at present.
vi
CONTENTS
Introduction • • . . • . . • • . . . • • • . • • . • . . . • 1
R.G. Edwards
PART I. EMBRYOLOGICAL ASPECTS OF PREIMPLANTATION GENETICS
Oocyte Development . . . . . 11
J. Schulman
Experimental Cytogenetics of Preimplantation Development. . 15
A.P. Dyban
Changing Physiological Systems in the Preimplantation
Embryo • . . • . . . . . • . . • . . . . . 25
J.D. Biggers
PART II. MICROMANIPULATION AND BIOPSY OF GAMETES
AND PREEMBRYOS
Biopsy of Human Gametes . . • • . . . . . . . . . . . . • . 39
Y. Verlinsky
Manipulation Techniques with Potential Use in AnimaI
Agriculture . . . . . . . . . • . . . . . . . . . . • 49
N.L. First
Enhancement of Fertilization and Hatching Using
Micromanipulation . . . . . . . • . • .. ..... 63
J. Cohen, M. Alikani, H. Malter, B. Talansky,
M. Tucker, S. Wiker and G. Wright
Biopsy of Human Cleavage Stage Embryos and
Sexing by DNA Amplification . . . . . . . . . . . • . 75
A.H. Handyside
Biopsy of Blastocyst . . . . • . . . . . . . . . . . . . .• 85
S. Carson
PART III. GENETIC ANALYSIS OF GAMETES AND PREEMBRYOS
Chromosomal Analysis of Human Spermatozoa . . . . . . . . . 91
R. Martin
vii
Chromosome Analysis of Oocytes and Embryos • . • • . • . . 103
M. Plachot
Preimplantation Diagnosis of Genetic Disease Using
Enzyme Assays • • • • • • . • • • • • • • • • • •• • 113
P. Braude
Single Sperm PCR Analysis-Implications for
Preimplantation Genetic Disease Diagnosis . • . • • • 121
N. Arnheim, H. Li, X. Cui and W. Navidi
Preimplantation Genetic Analysis Using PCR • • • • • • . . 131
C.M. Strom, G. Enriquez and S. Rechitsky
Co-amplification of X- and Y-Specific Sequences for
Sexing Preimplantation Human Embryos • • • •• • 139
E.H. Kontogianni, K. Hardyand A.H. Handyside
In Situ Hybridization of Blastomeres from
Embryo Biopsy • • • • • • • • • • • • • • . • • 147
J.A. Grifo, D.C. Ward and A. Boyle
PART IV. CLINICAL ASPECTS OF PREIMPLANTATION GENETICS
In Vitro Fertilization and Preimplantation
Diagnosis • • • • • . • • • • • •• . 155
A. Van Steirteghem, J. Liu, E. Van den Abbeel,
I. Liebaers and P. Devroey
Uterine Lavage for Preimplantation Genetic
Diagnosis • • • • • • • • . • • • • • • • • • • ••• 165
B. Brambati and L. Formigli
Assessing Loss Rates in the Preimplantation Stages
of Gestation • • • • • • • • • • • • • • • • • • • • 175
J.L. Simpson
Practical Considerations in Preimplantation
Diagnosis • • • • • • • • • • • • • • • • • ••••• 181
L. Jackson
PART V. GENE EXPRESSION IN PREIMPLANTATION DEVELOPMENT
AND GENE THERAPY
Evolutionary Analysis of Mammalian Homeobox Genes • • . • • 189
C. Kappen and F.H. Ruddle
Gene Expression in Preimplantation Embryos • • • • • • • • 203
R.P. Erickson
Somatic Gene Therapy • • • • • • • • • • • • 213
A. Bank
Strategies for Human Germ Line Gene Therapy • • • • • • •• 221
J. Gordon
viii
PART VI. ETHICAL AND LEGAL ASPECTS OF PREIMPLANTATION GENETICS
Ethical Issues in the Control of Genetic
Diseases . . . . . . • . . . . . . . . • • . . 233
A. Kuliev and B. Modell
Ethical and Selected Medical Aspects of
Preimplantation Genetic Diagnosis . . . . • . . . . . 245
A. Milunsky
Legal and Ethical Issues in Preimplantation
Genetic Diagnosis .•.•.......•.. . . 253
J. Robertson
PART VII. TECHNICAL ASPECTS OF PREIMPLANTATION GENETICS
Techniques for Micromanipulation and Biopsy of
Human Gametes and Preembryos . . .. •..... 273
Y. Verlinsky, J. Cieslak and S. Evsikov
Techniques for Microsurgical Fertilization . . . . . . . . 279
H.E. Malter, B.E. Talansky and J. Cohen
Method for Obtaining Human Sperm Chromosomes . . . . . . . 285
R. Martin, E. Ko and L. Barclay
Reliable Technique for Chromosomal Preparations from
Mammalian Oocytes, Preimplantation Embryos
and Isolated Blastomeres • • . . • • . . . • . . . . 293
A.P. Dyban
DNA Amplification of Y-Specific Repeat Sequences for
Sexing Preimplantation Human Embryos • . • . • 299
A.H. Handyside and E.H. Kontogianni
DNA Analysis of Gametes and Embryos: Analysis of
Delta-F508 Cystic Fibrosis Mutation in
Single Cells . . . •. .• • • • • . • . • 305
S. Rechitsky, G. Enriquez and C.M. Strom
Summary . . . • . • . • . . . . 313
J.L. Simpson
Index . . • • . . . . • . . . • . . . . . . . . . . 319
ix
INTRODUCTION
~obert G. Edwards
Bourn Hall Clinic
Bourn, Cambridge CB3 7TR, U.K.
The growth of the human embryo in vitro has been assessed
by several investigators using in vitro fertilization (IVF).
As expected, many of the processes of growth are similar to
those in animals. In some details, however, there are
aspects of development peculiar to human embryos which could
influence our approaches to the diagnosis of genetic disease
before implantation. The first polar body is large, but
apparently degenerates with in 24 hours and might lase some
of its chromosomes during this process. The second polar
body is smaller, nucleated and mare persistent and it can be
mistaken for abiastomere in a cleaving embryo.
It is now wide ly accepted that 95% of eggs fertilized in
vitro have two pronuclei, but approximately 5% have three
pronuclei, implying that they are dispermic (i.e. fertilized
by two spermatozoa) or digynic (a polar body is retained in
the egg in addition to the oocyte chromosomes). We do not
know if the same situation arises in vivo, but the available
evidence implies that 5% of these embryos will also be
triploid or complex haploids or mosaics, and this is a point
to bear in mind for preimplantation diagnosis. It will be
essential to check pronuclear histories of any embryo before
undertaking diagnostic procedures, or to en sure that the
diagnostic tests can cape with triploids. Second, many
spermatozoa are attached to the zona pellucida after
fertilization in vitro, although we are .once again un sure of
the situation arising with fertilization in vivo. The
importance of sperm bound to the zona, or partially
penetrated into it, concerns the possibility of
contaminating biopsies removed from the embryos, thus
leading to a false diagnosis. A few cumulus eelis also
remain attached to the zona pellucida after IVF, and could
be another source of contamination in diagnostic procedures
if ca re is not taken in collecting the biopsy.
The zona pellucida is an important membrane, and its
constituent glycoproteins have been characterized by
Preimplarnation Genetics, Edited by Y. Verlinskyand
A. Kuliev, Plenum Press, New York, 1991
Wasserman and his eolleagues. It ean be dissolved by aeids
or proteolytie enzymes, or small perforations ean be made in
it by these enzymes or by physieal methods to permit
spermatozoa to migrate into the perivitelline space or to
extraet biopsies from the embryo. It is apparently
essential in early growth to hold the individual blastomeres
together until eompaetion establishes an outer epithelial
like trophoblast tissue~ it might also be important to
proteet the embryo against the motherIs immune response.
The eleavage of human embryos is regular, with even-sized
blastomeres, and eompaetion takes place after the 8-eell
stage. Presumably the eelIs of eleaving embryos are
totipotent or multipotent, as in animal embryos, so the
death or loss of one or two of them for diagnosis would be
tolerated by the embryo and it would eontinue to develop
normally. Very few embryos seem to develop
parthenogenetieally, whieh is a boon for preimplantation
diagnosis, although fragmented blastomeres are found in many
embryos. This eould eause problems if these tissues are
mistaken for blastomeres - a possibility with large
fragments, beeause some have mieronuelei or no nueleus at
all.
The human blastoeyst at 5 days post-fertilization has
approximately 120 eelIs, most of them trophoblastie, and an
inner eelI mass that is usually distinet but ean be
diffusely spread around the blastoeoelie eavity. The
blastoeoelie eavity is distinet, and there are large
seeretary-like eelIs adjaeent to this eavity at the base of
the inner eelI mass. The mitotie rate is high, and the size
of individual eelIs approximates to that of somatie eelIs in
general. There are elearly enough eelIs to remove some for
diagnosis without jeopardizing the eontinued growth of the
embryo.
After day 5, the human blastoeyst expands, the zona
pellucida beeomes thinner and the embryo begins the proeess
of hatching. This is an aetive proeess: it makes a small
hole in the zona, presumably through the action of an enzyme
similar to I trypsin I deseribed by Wasserman, and then
gradually extends through it. These events oeeur on day
6-7 when approximately one-half of the blastoeyst is
extruded, while the other half is stiIl expanded and remains
within the zona pellucida, giving a "figure 8" appearanee.
Anomalies in hatehing due to an espeeially hardened zona
pellucida might explain the origin of human identieal twins.
The blastoeyst finally hatehes eompletely, leaving the zona
pellucida with some eelIs or debris in it, and then enlarges
rapidIyas the extraembryonie endoderm differentiates from
the inner eelI mass. It is possible that these stages of
growth might also be valuable for the diagnosis of genetie
disease, espeeially sinee there is no need to penetrate the
zona pellucida to obtain tissue, but the embryos are
probably too late in development to be handled easily or
give high rates of implantation.
The blastoeyst now begins to seerete HCG, attaehes to the
uterus, the amniotie eavity forms, and the extra-embryonie
mesoderm differentiates. These stages signal the onset of
pregnaney and of organ differentiation.
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