Thursday, January 16, 2003
Session 1: Early Embryonic Development: An Up-to-Date
John M. Opitz, M.D.,
Professor of Pediatrics, Human Genetics, and Obstetrics/Gynecology,
School of Medicine, University of Utah.
DR. OPITZ: Thank you, Dr. Kass, ladies
and gentlemen, Members of the Council, members of the audience.
I feel very privileged and happy to be here and to share with
you some data, facts, experiences and insights that I have
gathered over a half a century of working in the field of
development. And if you think that I don't quite look as old
as all that, I'd like to just recall from a personal perspective
that when I was a 15-year-old immigrant, newly arrived in
Iowa City, the music department where my uncle was a Professor
of Cello and Chamber Music was right next to the Zoology Department
and my uncle's next door neighbor had been Professor Witschi,
Emil Witschi, one of the great founding fathers of modern
developmental biology, particularly endocrine development.
So my second day in Iowa City, my uncle took me over to Dr.
Witschi's department at the age of 15. I became immediately
an animal caretaker, laboratory assistant and as of Day 1,
I was introduced to human embryology.
The very first question that Professor Witschi asked me is
well, John, and what is the biogenetic fundamental law? And
I said well, I don't know, sir. And right then and there he
told me about Haeckel's famous statement that ontogeny recapitulates
phylogeny and not only that, he was a historian and his intellectual
ancestry, in fact, went back to Johannes Mueller to the very
beginning of the 19th century and he had many primary documents
from all of those great men from Johannes Mueller to Haeckel
to Virchow (?) and of course, to his teachers.
So I come from a tradition primarily of European, specifically
German, morphology which accomplished a huge amount of work
during the 19th century in just establishing the facts of
embryogenesis, of thousands of different forms of life and
today, I'll focus primarily on human development, but remember
that I do so from an evolutionary perspective. And I will
not hesitate anywhere along the line to use animal homologies
if they serve to illustrate the point I am making.
I appreciate also the help of staff who were most kind this
morning to help me come to grips with the technology of this
Shortly before I left Salt Lake City, this was one of the
cartoons in the paper, so I represent the chap on the left,
namely that I'll try to represent the facts as they are or
I think they are.
Dr. Kass mentioned the word "mysterious" and while the process
of development has lost much of its mystery, I never on a
daily basis have lost my awe about the very process of development
and the coming into being of living individuals from the moment
of fertilization to the time of sexual maturity.
I'm guided by a few sentiments, namely the old one of Hippocrates,
I think is still correct, namely that description is infinite
and easy; an explanation is still limited and difficult, especially
in the field of developmental biology.
Goethe's statement that "we see only what we know" applied
to me most quintessentially when it was discovered recently
that one of the syndromes that I described over 30 years ago,
the so-called RSH or Smith-Lemli-Opitz syndrome, turned out
to be a simple inborn error of metabolism involving the synthesis
of cholesterol from its immediate proceedings, namely 7-dehydrocholesterol
and for decades we had known that these babies, these fetuses
have a low cholesterol level, but since cholesterol was demonized
as something bad, we never gave the matter any thought that
there might be a cause and effect relationship between the
low cholesterol level and the child's developmental abnormalities
and stunted growth and mental retardation. And then it turned
out, in fact, this was a simple inborn error of metabolism,
a defect in the synthesis of cholesterol. We again learned
Goethe's aphorism there and we learned something extremely
important about the earliest stages of human development,
namely, that cholesterol is not only desirable, but is absolutely
necessary for normal development.
The last statement of Goethe there, it's difficult to translate,
but what he implied when he formulated the concept of the
signs of morphology in 1796 and in the early 19th century
is that the study of form, both of embryos and of adults is
at the same time an intent to understand its coming into being.
Now we shouldn't put more into that statement than is actually
there. Even though this was set at a time of Lamarck and Transformism,
descent and of evolution were already very widespread and
widely debated. All right, so much for philosophy.
Let me again try to begin by defining life and I think there
exists a reasonable consensus amongst biologists on this definition,
namely that life consists of all of the self-contained units
of nature considered primarily of organic matter, autonomously
and I stress the autonomously capable of undergoing development,
reproduction and evolution. Note that this definition excludes
the viruses because they're not autonomously capable of undergoing
development and reproduction.
Now like all scholars, I suppose you've all got a stack of
books for Christmas and the one that I got was by Christian
De Duve, "Life Evolving," in which he gives the definition
of life which I find highly tautological, but which he defends
vigorously as being nontautological, namely that life is what
is common to all living beings. However, on the same page,
there is a sentiment there that I can subscribe to, namely,
that there is only one life and it's certainly probably axiomatic
that all living beings descend from a single ancestral form.
And I will come back to this point again and again during
Now then, we need a perhaps not an axiomatic, but an operational
definition of development, namely that it is the biologic
process that is the attainment of the mature form characteristic
of a species. Or to paraphrase from one of my earlier definitions,
the process which generates a sexually mature organism from
a fertilized ovum. And the reason I put the first line in
brackets is that in Goethe's original definition of morphology,
he included Das Tierreich, and the mineral kingdom as well,
and indeed, mineralogists and gemologists and crystallographers
speak of the development of crystals, etcetera, etcetera,
but strict to the term development refers only to these biological
Now in all sexually reproducing organisms, reproduction occurs
as part of a life cycle. And there is a continuum of life
cycles that takes us back to the very first organism here
on earth. Here, out of Scott Gilbert's wonderful textbook,
the sixth or seventh edition which will be off the press in
just a few days, that is the seventh edition, is the life
cycle of a frog. Notice the adult on the extreme left, generating
germ cells which lead them to fertilization. And in purple,
the purple spot at the bottom of the egg in the two-cell stage,
and in the eight-cell stage and so on, that is the germ plasm
which was already identified by August Weismann, almost 110
years or so ago, which provides a continuity of germinal information
and the separation of the germ line and the somatic cell line
during development. Then at the very bottom at 6 o'clock,
there is "birth" in the frog which is namely the hatching
out of the gelatinous ovum, the formation of the tadpole and
then the metamorphosis is the coming on land of the animal
after it has sprouted legs and lost its gills, before it loses
its tail and then becomes a sexually mature adult in the life
cycle of one year.
Now let us be sure we understand the fundamental distinction
between reproduction and sex. Reproduction refers to the propagation
of the species, whether this is by fission or by budding or
by runners as in plants whereas the term sex refers to propagation
by mating and genetic recombination. That is the exchange
of genetic material in a process called meiosis, an enormously
complex later edition of evolution to our ways of reproducing
and maintaining life during which the chromosome number, the
genetic constitution is doubled before it is reduced to a
haploid chromosome number.
Asexual reproduction results in clones and confers potential
somatic immortality. So the clone of amoebas that you have
in your petri dish on your laboratory bench goes back to the
very beginnings of the common ancestor of the amoeba and of
us. And it, of course, does not exclude extinction, but so
long as these organisms are capable of continuing to reproduce,
they have immortality. It was sex which introduced death into
multicellular life. At least to somatic death, but potential
continuity of life and I say potential because it isn't guaranteed
through the germ plasm.
Chris Wiley once put it very nicely in 1999 that the germ
cells are the stem cells of the species.
Let me perhaps illustrate what I mean in an example which
was also in Scott Gilbert's textbook. Let me go back to this
There's a wonderful organism that many of you studied also
in high school biology called volvox. And volvox is one of
the most instructive early forms of multicellular life. Basically
what it is is it's a colony of chlamydomonas or dinoflagellates.
They're banded together into this wonderful hollow sphere
which actually acts together as a unit. It ordinarily has
a half number of chromosomes. In other words, it is haploid
and reproduces asexually. However, when towards the end of
the season, things get dry in the pond and pond starts to
freeze over, then suddenly very powerful sexual induction
protein is produced, half of the organisms become male; half
of them become female. The females undergo oogenesis. The
males spermatogenesis. Sperm packets are released which float
toward the female, release the sperm and then fertilization
occurs to create a diploid organism. And at 3 o'clock on the
right hand side of the slide, you see the zygotes, these diploid
organisms which have a very, very tough shell and can survive
the winter and then when the spring rains come, meiosis occurs,
germination and reduction to the haploid state and a repetition
of the asexual cycle. And once that happens, the former adult
dies. So there's continuity through the germ line and death
of the somatic cell line.
Now when we speak of the relationship between sex and death,
we're referring to two phenomena here, namely the death of
the parents which are mostly somatic events, namely all of
those thing which will ultimately kill us through cancer or
stroke or hypertension or diabetes and so on, and the death
of embryos which are overwhelmingly germinal events. There's
an occasional child or adult that dies over a germinal event
like a teratocarcinoma or a gonadal blastoma or something
like it, whereas most embryos, as I'll explain in a second,
die due to germinal events.
There are now wonderful websites available to anybody who
has a interest in human embryology and in fact, many of the
patients that I see in the clinic have already consulted those.
Let me particularly point to the last one which is available
right here in town, just not very far from here at the Armed
Forces Institutes of Pathology. There's a National Museum
of Health and Medicine, which now has the Carnegie Collection
of human embryos and it was based on the Carnegie Collection
that the staging of human embryos was based. And this staging
is now universally used throughout the world and that website
is available for free.
The first one, no, the second one, "From Conception to Birth,
a Life Unfolds," by Alexander Tsiaras who incidentally is
an artist and he has beautiful 3-dimensional, multi-colored
images. The program is available from amazon.com for $30 as
a CD ROM.
All right, conventionally then, human development is subsumed
under four stages. Pregenesis, which are the events that occur
in the parents and basically represent a consummation of events
that occurred in the grandparents. Then there are two stages
of embryogenesis called blastogenesis and organogenesis and
then finally phenogenesis are the events that occur during
fetal life and post-natal life.
Pregenesis, also called progenesis or proontogenesis or the
German morphological term is [Vorentwicklung] is a complex
process which subsumes the establishment of the germinal tract
during parental ontogeny. The migration of the original primordial
germ cells which do not arise in the gonads, to the gonadal
ridges, ridge differentiation into ovaries and testes, and
only then can the production begin of egg cells and sperm
cells through the process called meiosis, recombination and
germ cell formation. And it ends with fertilization, syngamy
Here are diagrams which are directly readapted from my teacher/professor
who actually did the work here in the Carnegie Institution
on human embryos on the migration of primordial germ cells.
This is an illustration from Scott Gilbert's book, showing
the events in the mouse, whereby you can see at the very caudal
end of the embryo, on the right hand side of the embryo to
your left where it says "alimentary", the red spots in the
alimentary yolk sac, hindgut rudiments are the primordial
germ cells. And they then undergo a very circuitous, complex
route of migration from the hindgut into the gonadal ridges
where they will then induce the development of gonads and
ovaries. Very early, as these germ cells, as they migrate
are alkaline phosphatase positive and you can see on the bottom
left hand slide there, three germ cells alkaline phosphatase
positive, that's the dark stain in the wall of the hindgut
and on the right then you can see as they migrate into the
gonadal ridge. And that phenomenon of germ cell formation
and of migration into the gonads is an exceedingly ancient
phenomenon and is present already is Drosophila.
Now, here's an important point. The specification of germ
cells identity and the capability of germ cells to perpetuate
themselves and to be totipotent, not just pluripotent, but
totipotent, is conferred through a very complex cascade of
molecular transcription factors, the most important of which
up until recently was OCT-4, O-C-T-4, which is a nuclear transcription
factor, stained here on the top left hand side, a bright orange-red.
So here you can see the inner cell mass of the mouse embryo
and that there are a few cells already in the inner cell mass
that are probably destined to become primordial germ cells.
In the middle top panel, on the tail end of the embryo which
is on the bottom of the slide here, with LacZ reporter gene
construct, these cells which will become the primordial germ
cells are stained a very dark brown. And then on the top right
hand embryo, you can see the germ cells migrating, actively
like amoebas from posterior to anterior into the gonadal ridges.
And on the bottom right hand side then is what the original
oogonia looked like after they first established a varying
differentiation and the nuclear still shows some OCT-4 staining
and the spermatogonia on the left are beginning to lose it,
but the oogonia will continue to show OCT-4 expression. And
it is one of the reasons and causes of the many failures of
stem cell transformation in vitro that if OCT-4 is not re-expressed,
the construct will not have the potential to begin development
from the beginning.
All right, let's rapidly go through blastogenesis. It is
the process from the first cell division to the end of gastrulation.
In humans (or in the human system) stem, that's day 1 through
day 28. That is the first four weeks of development or the
first half of embryogenesis, ages 1 through 13.
During the first week then there's stages 1 through 4; stage
1, fertilization; stage 2, the first cleavage division; stage
3, the free blastocyst in uterus and I'll illustrate this
in a second; stage 4, the blastocyst's actions and begins
Now during stage 3, as the free blastocyst is in the uterus,
still in its vitelline membrane, during increasing cell division,
the volume of the zygote doesn't increase. And the reason
why this is is because of a process of compaction, had been
initially a very loose ball of cells. They then grow very
tightly together and I'll illustrate this to you with scanning
electron micrographic pictures, so that gap junctions can
develop between these blastomeres and the cell's cell communication
process so necessary for development can begin.
Now on the top picture here is the process of fertilization.
In the middle picture you see a scanning electronmicrograph,
the single sperm having just entered a mammalian ovum, but
the most important item is on the bottom panel. There are
those four images there show you the formation of the male
pronucleus and the female pronucleus and their fusion in the
middle or the middle left hand panel in what is the essence
of fertilization is not the fusion of the germ cells because
that need not necessarily lead to development, but the process
of karyogamy. That is the fusion of the male and the female
pronuclei so that the diploid number of chromosomes is reestablished,
each pronucleus having a half number of chromosomes and only
then can the spindle be set up for the first cell division
in the beginning of development.
On the top then you see stages 1 through 5 and on the bottom,
10 cell embryo, human embryo with a zona pellucida removed
on the left before compaction and on the right, 10 cells beginning
compaction. And you notice a tight, tight gap function between
these two arrows on the right hand side and then in
as you can see, the two little images within the uterus, the
blastocyst hatches. And it has to hatch before it can implant
on the uterine wall.
Stage 5, the embryo is fully implanted. Stage 6, the primary
villi appear and the primitive streak appears. Then here's
some beautiful images from Bill Larson's third edition of
his textbook on human embryology. Here, you can see the little
blastocysts implanting. And in blue is the hypoblast and in
yellow is the epiblast. So during the second week of development,
the human embryo then develops two layers. During the first
week, unilaminar; during the second week, bilaminar; during
the third week, it is trilaminar.
There's further progress in implantation. And then on the
bottom you can see the fully implanted human embryo with the
amniotic cavity being formed on the left and the primary yolk
sac being formed to the right. In blue is the epiblast on
top and the hypoblast in yellow on the bottom.
Here's the formation of the primary yolk sac and of the extra
embryonic mesoderm. Notice now the big, jelly-like space around
the embryo. This is the extra embryonic mesoderm and as it
cavitates on the bottom right hand panel there, that cavity
will form the chorion cavity.
The primary yolk sac is then shed and a secondary yolk sac
is formed. Again, from the hypoblast, so this is basically
then an endodermal structure.
During the third week then we see the formation of the trilaminar
embryo, the beginning of gastrulation and notochord formation.
At stage 8, the primitive pit, the neural plate, neural groove
forms; stage 9, formation of the caudal eminence, the first
somites, the neuromeres and the primitive heart, too.
Now a recent human embryological work has shown that the
dating of cardiogenesis, the formation of the heart which
is in every embryology textbook is probably too late, that
there is a beating heart tube present day 17 already. That's
an important to remember. It's not completely formed heart
yet, but there certainly is a beating heart to present. And
here you can see the human embryo no more than 1.5 or 2 millimeters
in size with the amnion cut open. You're looking upon the
embryo from top. You can see the primitive pit, the primitive
node, the primitive groove. In other words, the primitive
streak. And already, two structures are evident, one in front
and one behind, namely the buccopharyngeal membrane and the
cloacal membrane which are two regions of the embryo where
mesoderm never intercalates itself, between the future ectoderm
Notice also that this thing has a polarity and indeed the
polarity, the future polarity of the embryo is probably established
during the very earliest stages of cell division. There's
a front end. There's a rear end, in other words an AP axis
which automatically defines a right-left axis and there obviously
is a dorsal side and a ventral side that is a backside, a
topside and a belly side or an underside.
Now the quintessence of gastrulation is the establishment
of the three germ layers and all metazomes that undergo this
complex process of development undergo gastrulation. It's
a sine qua non of mitosome development. And what you see here,
on the top, left hand panel is at the primitive streak, subduction
occurring of the epiblast into the primitive streak, down
and underneath and initially replacing the hypoblast to become
the definitive endoderm and in red forming the mesoderm which
is between the former epiblast now, the ectoderm and the definite
And on the bottom hand panel, the red arrows then show the
migration of the mesoderm between the ectoderm and the endoderm
and again, notice the two areas in front and in back where
the mesoderm intercolates. The red arrow that points straight
from the primitive node makes the prechordal plate.
Now it has been shown that even before or right at the very
beginning of primitive streak formation, you can label the
surface epiblast very carefully the four stratas with peroxidase
or with dyes, or with oil droplets and so on and you can follow
the fate of the various regions of the epiblast as it is subducted
into the primitive streak. And you can identify the future
head process, the notochord, the endoderm, the mesoderm and
the surface epiderm that will form the neural tube later on.
Now during the four then, the end of blastogenesis, the neural
folds fuse and here you see an image of the neural folds fusing
from the top on down. They fuse initially in the middle, in
the middle of your back and then they sort of zipper towards
the end, and towards the tail end and the bulges, the symmetrical
bulges on either side of the neural tube are the so-called
somites which are mesodermal condensations which later on
help to give rise to the vertebrae.
At stage 11, the primordial germ cells migration in humans,
the cranial neural pore closes, the buccopharyngeal membrane
ruptures, the optic vesicles and pit are forming. At stage
12, the caudal, that is the tail end, pores close. There's
a cystic diverticulum that is a bladder diverticulum, pancreatic
bud. The urorectal septum is forming that will separate the
anterior bladder from the posterial cloaca. The upper limb
buds begin to appear and pharyngeal arches 3 and 4 and then
this on page 99 are the final stages of blastogenesis at the
end of which the embryo looks as it does on the right hand
You have the head and notice the change in shape of the embryo.
Initially, early during the formation it's straight and then
it becomes to curve into the C-shape curve. There are something
like 28 somites present, four branchial arches, the heart
is pumping, the nasal pit is obvious. The eyes you can begin
to see the eyes, the otic vesicle and so in essence you have
a reasonably fully formed embryo.
Now this image is better seen on the slide projector, if
you may please. It did not copy very well into the computer.
It's the frontispiece of my teacher's textbook on vertebrate
embryology. This is a Carnegie embryo it's not much
better which he serially sectioned and reconstructed.
Now this is not 28 days, but rather 30 days. So it's a little
bit later than the canonical end of blastogenesis and there
you can see in detail all of the structures, including the
primordial germ cells settled in the gonadal ridges in red.
That red streak is the dorsal aorta. There's the heart. You
can see the eyes. You can see the otic vesicle. So this is
what the human embryo looks like shortly at the end of blastogenesis.
DR. ROWLEY: And the size?
DR. OPITZ: The size is no more than about
3 to 4 millimeters. It's minute still, but nevertheless, it
is exceedingly complex already at that stage.
Now if you could go back, please, to the thank you.
All right. Now the second of embryogenesis, I'm going to just
summarize in this one slide. They are stages 14 through 23.
The length then from about anywhere between 4, 5, 6 millimeters
to 3 centimeters, that is 31 millimeters. It's the middle
to the end of embryogenesis proper and the end of organogenesis,
that is the end of the eighth week is what used to be called
in classical morphology and I think it is still appropriate
to do so, metamorphosis, namely the transition of life then
from the embryo to the fetus.
And there is a good reason for doing so because this is the
time when all marsupials are born, all kangaroos, opossums,
etcetera, etcetera, at the end of embryogenesis and they make
their way into the pouch and then continue their development
on the outside.
And the organogenesis is then characterized by two important
processes, namely the formation of organs and of histogenesis,
that is the formation of cells and tissues. And it was recognized
early during the 19th century already as Meiko once said in
1822, that's the das die form vor der Strunktur ensteht, namely,
the form arises before structure, that is the growth form
before the cellular specification. He said that even before
the cell theory and before he had a microscope available.
And the embryology textbooks towards the end of the 19th century
made this canonical point.
Now on the right hand panel then, it's a double panel out
of Carlson's textbook. On the top right hand, the small embryo
there then is what's is say there, 4 weeks? No, 8 weeks.
So the small embryo in the top middle, 8 weeks. This is what
the embryo looks like at the end of embryogenesis and thereafter
you see the progressive changes during fetal life.
The term phenogenesis has a double use. It refers to the
events during fetal life, namely from the 9th to the 38th
week of gestation which is equivalent to the 40th week of
pregnancy, right? And from the time the embryo is 3 centimeters
to when the fetus is 50 centimeters and that growth in length,
that 47 centimeter growth in length occurs mostly during the
second trimester from a weight of 8 grams at 8 weeks to an
average weight of 3,400 grams at birth and this weight is
gained mostly during the third trimester.
So therefore, the period of phenogenesis is one of tremendous
growth, progressive maturation towards post-natal adaptation
and the attainment of all of those quantitative traits which
constitute family resemblance and ethnic resemblance. So that's
a very important difference.
Embryogenesis is about the attainment of qualitative differences,
eyeballs, liver, kidneys, limbs and so on. And fetuses grow
and the anthropometric characteristics change on a daily basis.
They increase in length and weight and head circumference,
etcetera, etcetera, etcetera.
And post-natal adaptation then involves not just the cardiovascular
system, the closure of the ductus and of the endoventriculum
and endoatrium communications, but continued growth and maturation
to change body proportion from little toddler with a huge
head and a small body to a more adult proportion as portrayed
by Leonardo da Vinci and his Vitruvius cartoon and then finally
pubertal changes and adulthood, pregnancy, gestation, parenthood,
senescence and death which are normal stages of homogenesis.
Remember that even in old age, the nose and the ears and other
parts of our body continue to grow.
Now let me summarize briefly with perhaps some oversimplification,
the development defects of each of these stages of human development.
By far, the most common defects of human development are defects
of pregenesis and they mostly lead to the lethal chromosomal
imbalances, mostly trisomies, also monosomies, monosomy X.
In other words, Down's Syndrome, trisomy-21, trisomy-18, trisomy-13
and so on. These are defects of meiosis.
It is estimated by multiple sources and authors and has been
for decades that at the very beginning of life, of human development,
of conception, about 50 percent of all potential human beings
have a chromosome abnormality, mostly a lethal chromosome
abnormality. Chromosome abnormalities are the commonest cause
of death in humans. They kill at the very minimum two-thirds
of potential humans, more likely 80 to 90 percent and they
mostly do so through these lethal aneuploidies.
Now during fertilization, some triploids arise, that is,
individuals with a triple set of chromosome numbers and during
the first cell division, some cases of tetraploidy with a
quadruple number of chromosomes, 99 percent lethal disorders.
And the only reason that a few 18-trisomy syndrome and 13-trisomy
syndrome babies survive to birth is because of the phenomenon
of confined placental mosaicism whereby the placenta, which
is a smart organ, chunks out the extra chromosome, establishes
a normal cell line and it is the normal cell line that supports
the severely defected babies until birth so that they can
be born. Down's Syndrome doesn't do that. Down's Syndrome
is the only trisomy that does not involve confined placental
The defects of blastogenesis then, put parentheses around
that, are the gross, mostly lethal, not necessarily, but mostly
lethal malformations. And the multiple gross, lethal malformations,
there was an entity that used to be called "associations."
And I'll spell it in lower case letters, rather than with
The defects of organogenesis then are the later, milder,
usually single malformations such as a cleft palate, a cleft
lip, an extra finger, a hypospadia.
The defects of phenogenesis are intrauterine growth retardation
or for that matter overgrowth which is far less common than
growth retardation and minor anomalies, namely those
to some extent objective, but also quantitative differences
in multiple subtle facial structures which take away family
resemblance and which make the parents wonder when they look
at their baby, where did he come from? He doesn't look any
one of us and that is a red flag. This is one of the most
sensitive signs we have for the presence of a chromosomal
abnormality because chromosome abnormalities produce multiple,
multiple minor anomalies that take away family resemblance
so that the parents will then say well, he doesn't look like
any one of us. And so on.
The defects of histogenesis, I guess is important to mention.
They cause dysplasias. All the moles and birthmarks and so
on, but also some developmental tumors, teratomas and embryonal
Now could we see the next series of slides, please? They
show up much better than here then is a little Japanese
Down's Syndrome oh here. I can probably advance this.
A little Japanese Down's Syndrome child. It used to be thought
that the reason for the gravity of this condition is because
they had such terrible series major malformations. As a matter
of fact, most of the anomalies in Down's Syndrome are minor
anomalies. And you can still recognize the ethnic origin of
this child and you do sort of a minute point by point comparison
between say Caucasian and black and Mongolian children with
Down's Syndrome, they look more like each other than they
do to their brothers and sisters.
In a bad year, this occurs 1 out of 750 deliveries. In a
good year, 1 out of a 1,000 deliveries. It's a very common
condition. It used to be the commonest cause of developmental
disability that we saw in our university clinics. Now we don't
see these patients any more because the pediatricians take
care of these kids themselves.
Now here is a girl, one of the very first ones that David
Smith and I studied with Turner's Syndrome. This is an aneuploidy
in which the individual instead of having 46 XX chromosome
constitutions, got a 45 X chromosome constitution. In other
words, a sex chromosome is missing, neither an X nor a Y.
This is one monozygotic twin girls and when I first arrived
in Madison Irene Uchita and Walter Nams were able to demonstrate
that in Turner Syndrome there is an increased incidence of
monozygotic twinning. What they were not able to answer was
the question which is the chicken and which is the egg here?
Does the aneuploidy cause the twinning or does the twinning
cause the aneuploidy and to this day I don't know the answer,
but the association is unquestioned and I'll show you another
striking example of that, namely that in these aneuploidy
syndrome including Down's Syndrome and Kleinfelter's Syndrome,
there's an increase incidence of monozygotic twinning.
It's temperamental. Thanks, Chuck.
So let me illustrate just a little bit more. Also to illustrate
what the concept of Turner Syndrome means. These two little
girls, you can see they're Mennonite girls out of the Lancaster
County area, they were born at the same time. The one on the
right is obviously a bit of a runt and was brought into the
clinic because she is so short and I'll show you the growth
curve of these two girls in a second. And the little one showed
some signs of Turner Syndrome and when her chromosomes, she
turned out to be a mosaic of two cell lines; one, a normal
one, 46XX and a Turner Syndrome cell line 45X.
And they thought hm, are these two girls identical or are
they not identical? They took blood from the normal, bigger
sister also and she also had exactly the same mosaicism. She
was 46XX, 45X. So are they mosaics or are they chimeras? Then
a skin biopsy was done on the little girl. She was pure XO.
And a skin biopsy from the big girl, she was pure XX.
So what happens is that at the moment of monozygotic twinning
at the first cell division when these two blastomeres fell
apart and formed one formed one twin and one formed
the other twin, a chromosome, an X chromosome or Y chromosome
was probably an X chromosome, was lost out of the cell that
made the small girl and the other cell line was normal. And
due to placental vasculature connections in the single placenta
in these individuals, they exchanged blood cell lines and
they became chimeras. So they're not mosaics. They're chimeras.
They're grafted, the XX, the XO girl grafted her XO cells
into the XX girl and vice versa.
Next one, please, Chuck.
Here's the growth curve. You see the little one was way below
the third percentile before she started to be treated with
growth hormone and the growth curve of the normal girl was
between 3rd and 50th percentile.
Next, please. And when the DNA analysis was done, ignore
the two left hand lanes, those are control lanes, but the
right, the third and the fourth lane where they used probes
for chromosome 2, chromosome 17, chromosome 15 and chromosome
16, you can see these are identical twins. There are several
very important points to be made here about this case.
Now the defects of blastogenesis which is a common one, Dr.
Rowley will recognize this as the handiwork of Dr. Edith Potter
at the Chicago Line, the so-called Potter Syndrome or Potter
Sequence due to absence of the kidneys. Absence of the kidneys
means absence of amniotic fluid, hence these are cramped,
contracted and this is the so-called Potter Sequence. So it's
a very early defect of blastogenesis.
Otocephaly, already well-known by the early French teratologists
of the 19th century where there's a defect of the mandibular
arch, early defect of blastogenesis, an inviable defect.
Next, please. Sirenomeli, named after the mermaid, where
there's a single, apparently fused or undivided lower limb,
usually with severe genital-anal-renal abnormalities. As you
can see in the two upper panels also, radius abnormalities.
So multiple defects of blastogenesis or an association, a
lethal disorder. Next, please.
Here's a defect that Dr. Oscar Borin, a German co-worker
of mine and I have studied intensively, so called lumbosacral
agenesis where portions of the spinal cord and the vertebral
column are missing. In the one baby on the right, there were
only four cervical vertebrae missing and most spinal cord
below, and interestingly enough, even without a spinal cord,
there was normal late development here. And if these kids
don't die of pulmonary abnormalities because they lack pulmonary
chest power, they have normal intelligence. Severe defect
of blastogenesis. Next, please.
Anencephaly. Sometimes even with complete absence of the
brain and the spinal cord, nevertheless, normal hand and upper
limb and lower limb development, so no central nervous system
is required for limb development.
This fetus that Dr. Gilbert and I studied that came from
St. Vincent's Hospital in Green Bay, sort of the epiphysis
of everything that can go wrong during blastogenesis. The
only normal parts of this baby are the upper limbs. And you
can see that there is an anencephaly the entire face is cleft.
You can see the right half and the left half of the nose.
There was rudiment of an eye on the left. There were no normal
vertebrae at all. There was a single umbilical vessel, renal
abnormalities, diaphragmatic abnormalities and so on.
Nevertheless, this baby lived to 23 weeks of gestation. And
when the parents came in for counseling and that picture on
the chart and I've since then adopted this as a practice,
on my desk, the mother picked up the picture of this baby
and asked me is this my baby? And I said yes, that was your
baby and she clutched the picture to her chest and mourned
for three or four hours, tears running down, regardless of
how malformed and sometimes unrecognizable these products
of conception may be. They may be mourned as much as if a
normal baby had been struck by a car or died of leukemia,
Is that the last one, Chuck? The next one looks yes,
let's go on to this one here.
Now again, there's a very similar story. This is a quintessential
twinning defect of monozygotic twinning again, but here the
umbilical cords of the two twins are connected and so one
becomes a parasite on the other one and the other one that
is with the reverse perfusion, then begins to sort of rot
away because of loss of blood supply and so called acephalus
acardia anomaly which has been very well known since the beginning
of the 19th century.
And so as they lose their head and they lose their heart
because of the perfusion in the upper part of the body is
lost, finally then they lose their toes and their limbs and
so on and then ultimately they may just be left as a shapeless,
formless, lump of tissue which nevertheless still at times
at birth shows some signs of movement and again, the parents
may mourn this as the loss of my baby. And so the question
arises then, given that this baby and in this particular case
this baby was born, still moving at 23 weeks of gestation,
the parents preferred that even this inviable remnant of a
fetus be baptized and be given a name and that some meaning
be bestowed on its existence and its passing.
Next. Mine you, the co-twin was perfectly normal.
This is a photo that I took in the Virchow Museum in Berlin.
This is conjoined twinning. When the events of twinning occur
relatively late, notice their tubular columns here. And the
co-twin, sitting on the shoulder of the normal twin was anencephalic.
This is one of the few specimens that survived a direct hit
on that museum during the end of the second World War. Virchow
at one time had over 50,000 specimens and there are only a
few hundred of them left.
Next, please. I would like you to see also the Hensel twins.
Many of you may have seen those are those fixed slides,
Chuck. If we can't see the previous one, let's just stick
with this one.
Did any of you see there they are the documentary
about the Hensel twins on public television the other day?
It was fairly recently. If I were you, I would go. This is
a most dramatic kind of a story, that these two girls with
a single body and two heads and dramatically different personalities,
so different in fact, when they came home one day from school
and the dad was sitting there absentmindedly reading the newspaper,
one of the girls said, "Dad, we learned to swim today." And
the dad said, "Well, which one of you jumped into the swimming
pool first?" So you know, absentminded dad. Wonderful, wonderful
little girls. Now the question is one soul, two souls. The
Catholic Church in this hemisphere began to regulate or to
address this question already during the 16th century by ordering
that both be baptized. And in this particular case, one is
Abigail, the other is Britney and their anatomical arrangement
is as you can see here. They've got two heads.
Next one, please, Chuck. Thank you. There was an arm between
their shoulders which was removed. You can see two sets of
lungs, two hearts, a single liver, two guts down to the ileocecal
valve and a single pelvis, single anus, single external genitalia,
two legs and two arms. How these girls manage with two different
will powers and minds to coordinate, let me say swimming or
bicycle riding and so on is really a miracle for the neuroanatomists
Next, please. All right. Why don't you put a little piece
of paper over that and we'll come back to you.
If you could return to these let me see. I need to
go forward because I wanted to show you the scheme of twinning
which I took out of Ronald O'Reilly's textbook. There's a
wonderful scheme which summarizes twinning in I can't
see why I showed you these slides, these Kodachromes because
they really did not import very well on this disk, this program
at all. Sorry about that. But they take up a lot of memory,
so it takes some time to advance slides.
The dizygotic twinning in this connection is relatively uninteresting.
Monozygotic twinning has been very well studied for a long,
long time. And one can make a correlation between the time
when the twinning event occurred and the outcome, namely,
whether there are two individuals, two amnions, two chorions
and two placentas. This is an event most likely had occurred
during the first or the second cell divisions when the blastocysts
then parted and set up independent housekeeping.
The later during blastogenesis that the twinning event
occurs with the midline being developmentally highly unstable
kind of a landmark. The greater is the likelihood that you'll
end up with a set of conjoined twins. Now the greater is the
likelihood that you'll end up then with a single chorion,
finally with a single amnion and finally with a single placenta.
And the events can occur according to O'Reilly, not let me
show you this figure, a similar kind of an experience. This
is a fetus that I was privileged to study with colleagues
from San Jose, Costa Rica.
It's gone again. In any event, the baby that I was trying
to show you yes very good was a hemibaby.
It was a half a baby. It was the right half of a baby which
I suppose in order to survive up until 23 weeks and a weight
of 500 grams, formed into a donut like shape that you saw
there, so this diagram which incidentally will be in your
handouts, shows a hemibaby consisting of why don't we
just leave it at that, consisting of a half there it
is, of a half right baby.
And now the question there is half a soul or a whole soul?
In any event, the mother went ahead and named the baby anyhow
and the baby was buried. This is an exceeding rare anomaly
of blastogenesis. I presume that this is the defect of the
earliest stages of development, but I really don't know because
there is virtually no published precedent about this in the
All right, here's the diagram from Ronald O'Reilly and if
you look at the bottom below the dotted line, those are the
dizygotic twins. They're basically uninteresting. And starting
in the most right hand column from top on down, that has been
the twinning event occurs as late as 14 days. There's a time
scale on top of the illustration there. And you end up with
conjoined twins, as presumably happened in the Hensel twins.
And in the extreme left hand side then, if the two blastomeres
fall apart and set up independent housekeeping as in one,
two, three the first panel on the right, then you can
see, there are two independent separate intercellular
masses, separate amnion and separate chorion and separate
placenta. If the fission occurs in the inner cell mass, let
me say at Day 5, then within a single blastocyst cavity, you've
got two embryos. You've got two amnions, but a single chorion
and a single placenta. If it occurs as late as Day 8 or Day
9, then you've got you end up with a set of monozygotic
twins with a single amnion, a single chorion and a single
placenta. So the membrane and the placentation situation at
the time of birth is it gives us a good idea as to what
occurred and when it occurred. So these then are genetically
identical individuals and yet every mother can tell their
monozygotic twins apart on the basis of small physical differences,
personality quirks and differences, differences in voice and
so on and so forth, showing that even though they are genetically
identical all development is an epigenetic process that is
continuously modified by an interplay between environment
and genetic constitution.
All right, you've seen that baby. You've seen the Hensel
twins. Now my time is over, Dr. Kass, right? And I don't know
I should go on any further.
The reason I put in this maybe a few more minutes.
This ART business here because of a very interesting and I
think rather dramatic new development which I again, by chance,
picked up the mail as I was going to the airport, the last
issue of the American Journal of Human Genetics had an astounding
report in it by ART, the referred to assisted reproduction
techniques, which is widely practiced, not just in the United
States, but worldwide, affecting as it does 15 to 30 percent
of all couples being infertile; 37 to 70 million worldwide,
Now these are very gross estimates. In the United States in
1999, 1 out of 150 children were born, were conceived by ART
and since Louise Brown in 1978, about one million kids worldwide
have been born in some form of ART or another. And about 40
percent of all infertility, we deal with the male factor in
fertility. And the practice in ART then involves procedures
for the collection of eggs and sperm fertilization in vitro.
That is in a petri dish in the laboratory and then the embryo
transfer and then the question arises, do you transfer a single
one or several? The probability of implantation being relatively
low, so that people try to increase the probability of implantation
by putting in three or four. Do you put in very early cleavage
stages or do you put in a blastocyst and if you put in blastocyst,
there is a substantially increased risk of monozygotic twinning
Now the ART forms then and pardon this dreadful pun, but
that's how the specialists themselves refer to it, is still
the commonest is artificial insemination by donor. In vitro
fertilization first practiced Bob Edwards and Patrick Steptoe
in 1969 leading to the birth of Louise Brown in 1978. Then
GIFT, that is gamete intra fallopian transfer, this is mostly
the injection of sperm into the fallopian tube, allowing normal
fertilization occur. Let's say if the tubes are closed or
if there is a problem of sperm concentration. Then ZIFT refers
to the zygote, interfallopian transfer, I'm sorry, that's
a misspell it should be IVF, in vitro fertilization,
interfallopian transfer of a fertilized embryo and then this
dreadful acronym ICSI, pronounced "icksy" refers to intracytoplasmic
sperm injection and embryo transfer, a technique which arose
in 1992 and already the need for it and the technology of
it has way stripped our ability to understand the biology
that's behind it. And the reason I mention this and then I'll
sit down and shut up is because last year I saw a paper and
I think it was in the American Journal of Medical Genetics
that a child with the Angelman Syndrome had been born after
intracytoplasmic sperm injection and that immediately aroused
an alarm bell in my mind because if there is a cause and effect
relationship between this procedure and the child's condition,
then you might make the prediction that not so much Angelman's
Syndrome but the Wiedemann-Beckwith Syndrome would occur with
increased frequency in children conceived in this manner.
Now let me try and explain what these conditions are. The
Angelman Syndrome and the Wiedemann-Beckwith Syndrome are
two clinically radically different looking conditions. The
Angelman Syndrome involving acquired microcephaly, severe
mental retardation, seizures, usually no speech development
and a very characteristic kind of behavior. And what was discovered
in these children is that there's either a deletion of the
short arm of chromosome 15 or else an imprinting defect whereby
in an attacked chromosome, the gene expression on a short
arm of chromosome 15 was altered through abnormal imprinting,
depending on from which parent the gamete came, whether it
was paternal or maternal. And in fact, we now know that there
are several regions in the human genome which are differentially
imprinted, mostly turned off, hypermethylated or hypomethylated,
depending on whether they come from the mother or through
So the process of myosis then may confer an epigenetic modification
of the genome by regulating gene dosage, especially during
earlier stages of embryo development, whether this is the
trophectoderm development or the innercell mass development
by differential imprinting of genes.
The Angelman Syndrome and the Wiedemann-Beckwith Syndrome
are complementary syndromes due to imprinting defects of exactly
the same genes on the short arm of chromosome 15. And what
people at the National Cancer Institute here across town and
at the University of Washington-Seattle have found is that
5 percent of all bavbies conceived in this way have Veidemann-Baechler
Syndrome. And so the LOS Syndrome, the large offspring syndrome,
that's being described so many times in infants conceived
in this manner, now finally has an explanation.
These are large because they have Wiedemann-Beckwith Syndrome.
Wiedemann-Beckwith Syndrome babies are large and they have
infantile embryonal carcinomas. So there's an increased incidence
of all kinds of carcinomas and Dr. Rowley knows this a whole
lot better than I do that hepatoblastomas, rhabdomyosarcomas,
adrenal-cortical carcinomas, what else do you know, Janet?
Those are some.
So in other words, it may in spite of all the best
intentions here, this may contribute then to childhood morbidity
and mortality and to cancer, morbidity also. So our technology
got a little bit ahead of our understanding exactly what goes
on because during sperm injection the neural events that occur
during fertilization in the sperm capacitation and the dissolving
of the acrosome, the shedding of the midpiece, none of that
occurs and the process then of forming a male pronucleus is
dramatically radically altered and different than if you do
it. Now I don't know about the other 95 percent of the kids
since I don't see those very commonly, but at our university,
for example, this is practiced and I will be at pains to call
this article to the attention of colleagues and I'm not so
sure how this can be prevented. The need, in any event, is
enormous for this technology and many, many clinics who practice
this throughout the world as a matter of fact, without really
being fully aware of the consequences that this might engender.
Let me perhaps stop here so that you have time for discussion
and for questions and then we would perhaps carry on later
Thank you very much.
CHAIRMAN KASS: Thank you very, very much, Dr.
Opitz for a wonderful presentation. We do have at least 15 minutes
at this point to run a little over, but Dr. Gómez-Lobo,
DR. GÓMEZ-LOBO: This is a question
out of ignorance, of course. When does the developing human
organism acquire its genetic material? What I'm trying to
get at is this, is there any genetic material coming into
the embryo after syngamy.
DR. OPITZ: After karyogamy.
DR. GÓMEZ-LOBO: After karyogamy.
My second question, if I may has to do with karyogamy. Do
you have something like in fanciful, numerical estimate of
the possible combinations in karyogamy?
DR. OPITZ: The answer to your first question
is no. Although the maternal genetic contribution which comes
through the cytoplasm of the ovum is variable because of the
mitochondrial DNA. So the DNA content is not the same in every
zygote. It can vary dramatically and considerably depending
on what the mother contributes by way of mitochondrial DNA
in its cytoplasm.
With respect to your second question, you can combinatorily
account for variations in every one of the 23 pairs of chromosomes.
So the number of combinatorial permutations that you can get
out of a fertilization is astronomical, especially if there
has been exchange of genetic material, but in homologues,
so that in fact, except for monozygotic twins, the probability
of having two identical human beings, same parents, is much
Did that answer your question?
CHAIRMAN KASS: Robby George.
PROF. GEORGE: Doctor, thank you for your
wonderful presentation. Is an embryo of any mammalian species
something distinct in kind or nature from developed members
of the species in question or is the embryonic stage a stage
in the development of a determinant member of the species?
DR. OPITZ: It's a stage. So there is, in
other words, increasing potentiality, increasing valuation
towards birth, towards full maturity, but in humans, remember
that's a relatively arbitrary cut off point because 200 grams,
300, 400 gram babies may survive, born prematurely.
PROF. GEORGE: Is there any biological sense
now, any biological sense in which an embryonic or fetal cow,
let's say, is prebovine rather than bovine in nature?
DR. OPITZ: No, it's always bovine in nature
from the conception. That was the point already emphasized
by Von Baer in the 1820s, that even though the early embryonic
stages may look remarkably similar, if you look closely enough
from the very beginning we have the unique and distinctive
development path whereby increasingly you can tell the development
from one species to the next species and so on.
PROF. GEORGE: I was wondering how early
in embryonic development in humans can we detect the production
of an immunosuppressant that would prevent the rejection of
the embryo by the mother?
DR. OPITZ: That question I don't know.
I am not a biologist, but I do know that human gene expression
occurs very early already in first or second cell division.
So unique gene expressions occur very early during embryogenesis.
Now mind you, many of these gene expressions are generic
because the generic body plan that may say of mammals and
vertebrates, all is built exactly the same by using exactly
the same molecular machinery so that you have initially the
molecular expression patterns in very early zygotes and embryos
is more phylum-like, you know, Class, Order, Family, you know
genos-like and then later on as development proceeds, it becomes
more, more and more specific to the species and then finally
to the individual.
PROF. GEORGE: Thank you, Doctor.
CHAIRMAN KASS: Michael Sandel and then
PROF. SANDEL: Thank you. I have two questions
about the rate of natural embryo loss in human beings. The
first is what percent of fertilized eggs fail to implant or
are otherwise lost? And the second question is is it the case
that all of these lost embryos contain genetic defects that
would have prevented their normal development and birth?
DR. OPITZ: The answer to your first question
is that it is enormous. Estimates range all the way from 60
percent to 80 percent of the very earliest stages, cleavage
stages, for example, that are lost.
PROF. SANDEL: Sixty to 80 percent?
DR. OPITZ: Sixty to 80 percent. And one
of the objective ways of establishing the loss at least as
of the moment of implantation, well, even earlier, let's say
as of five days because the blastocyst begins to make a chorionic
gonadotrophin and with extremely sensitive assay methods,
you can detect the presence of gonadotrophins, let me say,
first around Day 7. That's the beta of human chorionic gonadotrophin.
And if you follow prospectively the cycles that has been done
on quite a few occasions in the Permanente study in Hawaii
and so on, a group of women, of nonfertility, who want to
conceive and you detect the first sign of pregnancy there
of human chorionic gonadotrophin, about 60 percent of those
pregnancies are lost.
It is independently corroborated by the fact that the monozygotic
twin conception rate at the very beginning is much, much higher
than the birth rate and then if you follow with amniocentesis,
the presence of the two sacs in about 80 percent of cases,the
second sac disappears, one of the sacs disappears.
CHAIRMAN KASS: The 60 percent then would
be of those that have at least reached the 7 days so that
you could trace the so there might be even greater loss
at the early cleavage stage, is that correct?
DR. OPITZ: That's correct. And the earlier
the stage of loss, the greater the rate of aneuploidy. There
exists sort of a standard, textbook formula whereby 60 percent
of spontaneous abortions have a chromosome abnormality. Six
percent of all stillbirths and 6/10ths percent of all live
born children. Now the latter figure is probably closer to
1 percent if you include some growth variants. So that's sort
of a rule of thumb.
In my own lab in Helena where I did all of the autopsies
on all pregnancy losses for 18 years, the rate of chromosome
abnormalities was a little bit higher.
PROF. SANDEL: So if we take the 7-day stage,
it's 60 percent. The 80 percent is if you go back to the moment
of fertilization. But if you take just starting at the 7 days,
there's 60 percent rate of natural loss. And of those 60 percent
that are lost from the 7-day stage, what percentage of those
have abnormalities or defects such that they wouldn't otherwise
be able to be born?
DR. OPITZ: I would say somewhere around
50 to 60 percent and mind you, many of these are empty sacs,
tiny, tiny stunted little embryos, but when you culture the
sacs you find a chromosome abnormality, even though the embryo
has vanished already.
PROF. SANDEL: So of the 60 percent that
are lost at the 7-day stage, 40 to 50 percent did not contain
defects or abnormalities, could have been born?
DR. OPITZ: Right.
PROF. SANDEL: And become babies.
DR. OPITZ: Your point is well taken, which
doesn't mean that the chromosome abnormality isn't there.
There's a wonderful lady, Dagmar Kalousek at the University
of British Columbia, who has studied this question very intensively
and published on it and incidentally the question that you
addressed is a reference to that in the bibliography which
is in your handout. Of course, this presentation will be a
handout in which I tediously enumerated all of those data
that are being published until recently.
And Dagmar Kalousek has shown that even the low chromosomes
are apparently normal for XX on structural abnormalities,
they may be abnormal. The commonest chromosome abnormality
in humans is chromosome trisomy-16 which you may detect at
chlorionic villi sampling and then at amniocentesis, it's
And what the embryo has done is it has chopped out the extra
chromosome out of the somatic cells, but in the process it
has a two-thirds probability of forming an isodisomic pair
whereby both homologues either from a mother or from the father
they look perfectly normal, but there's the defect.
And so it is even recommended that you do imprinting studies
on every pair of chromosomes, even in those that are apparently
normal and nowadays, with subtelomeric probes, we can even
discover additional things because if the embryo is grossly
abnormal, let's say it's a 10 millimeter embryo under the
dissecting microscope, the changes are that it is a chromosome
So the selection against chromosome abnormalities in humans
before birth is enormous, it's over 90 percent. I would say
probably even higher than that.
CHAIRMAN KASS: Janet Rowley?
DR. ROWLEY: Well, I think just to follow
on with this before I ask my own questions, what has been
learned by these kinds of studies is that nature is remarkably
effective in identifying its mistakes and in disposing of
those mistakes before they develop, they can't develop into
a normal fetus, so that this is really the I think,
one of the lessons that we've learned from this.
PROF. SANDEL: Janet, could I just interrupt
just to ask a point of clarification on this. Of the 60 percent
that are lost from the 7-day stage, they're not all mistakes,
are they? Some of them are, but you were saying they're not
DR. OPITZ: They may appear normal, but
almost by definition they can't be normal because they died.
There must be some reason for it.
Now it could be placental. It need not be intrinsic, but
remember, that a major portion of the placenta also is of
PROF. SANDEL: Sorry, Janet, go ahead.
DR. ROWLEY: No, I think that's one
of the things that you didn't touch on that's important, I
think, is the relationship of aneuploidy with age and we've
often focused on maternal age, but I think there's evidence
in some of the chromosome abnormalities that paternal meiotic
errors are also involved in this so that was my first question
to ask you to amplify on that.
And the other issue that I wanted to bring out is the importance
of environment and environmental influences on embryology
and I realize in one sense you can say this is a lecture in
itself, but we know, for example, that neural tube defects
are very high. Women in poor areas that don't get folic acid,
then a simple way to not take care of all of these, but to
diminish the frequency of spina bifida and other things is
just to make sure that the mothers get adequate nutrition.
In what we're learning about many of the defects that you
illustrated here, I wonder how many of those might also be
apparently of some consequence of environmental exposure.
DR. OPITZ: You raise three important points.
I guess one of the major impetus for the development of prenatal
diagnosis is this relationship that Dr. Rowley alluded to
between maternal age and nondisjunction or the presence of
chromosome abnormalities with women reproducing at the age
of 45, having almost a 1 out of 7 chance of having a chromosomally
abnormal baby. So there's a direct linear relationship between
maternal age and the presence or a chromosome abnormality.
It is also true as Dr. Rowley has pointed out, that there
is some relationship with paternal age, mostly in the occurrence
of new mutation, that is of gene changes, rather than of chromosome
changes, but some of the Robertsonian, I think, translocations
may also arise with increased paternal age.
The influence of the environment, it cannot be underestimated.
Even though amnio, chorion, placenta, you know, buffer the
fetus to some extent, nevertheless there's a very active circulation
from the mother to the baby and it is very important that
where we can prevent birth defects due to environmental causes,
we do so and one of the most rational and most effective defects,
not just for neural tube defects, but for other major defects
of blastogenesis, including congenital heart defects, has
been the introduction of the recommendation that every woman
wanting to conceive take at least 4 milligram of folic acid
per day in order to prevent these common defects.
Now in the state in which I worked for 18 years, Montana,
by far the commonest environmental defect that we saw was
the fetal alcohol syndrome which is 100 percent preventable.
And on certain reservations and I will footnote that statement,
we estimated that 60 to 70 percent of all kids born on the
reservation had fetal alcohol syndrome, Rosebud was a particular
example, but the Crowe Indian Reservation, the Northern Cheyenne
Reservation, the Blackfeet Reservation were similar, but in
our overall patient cohort that we were examining in Montana
before my wife and I left, my wife being a particular expert
in the fetal alcohol syndrome, 30 percent of them were non-native,
you know, Caucasian individuals, smoking. And particularly
the combination of smoking and drinking and it has a dramatic
effect on the placenta. So interuterine growth stunting occurs
very, very commonly and then there's a whole raft of medications
that are known to cause birth defects and environmental abnormalities
of the baby.
CHAIRMAN KASS: Could I ask these
are they're partly biological questions, but with a
certain quasi-philosophical edge to them, if you wouldn't
mind. One has to do with the question of individuation which
is one of the issues that comes up here and I ask how the
phenomenon of twinning, how that enters into what one regards
as the thing which gives rise to twins, whether one sees this
really as something whose individuation is yet undetermined
and only can be somehow guaranteed after the time of twinning
is past, or whether one sees twinning as some kind of a response
to some abnormal event in an individual to which this is then
somehow a reaction.
And second, I'm interested also in the question of wholeness
and both as a biological fact, but also for what for
its bearing on things like blastomere biopsy when one removes,
let's say, as many as two out of an eight cell stage and what
the implications are for the residual organism. In Dreisch's
famous experiments, as you know, one took half and yet one
was still able to produce a whole and I'm puzzled about that
here and if I might just add, I was very interested in the
difference between the stage of the early embryo when the
cells seemed to be in there, lacking the compaction and lacking
the intracellular connections and wondered whether from an
embryologist's point of view, that's somehow crucial as part
of the answer to the question about whether you have a whole
yet or whether you've got something other? I'm not sure the
questions were stated as well as I would like, but I think
you get the drift. I'm interested in the question of wholeness
and the question of individuation as a biologist sees these.
DR. OPITZ: Well, I think every fertilized
zygote has the potential of becoming monozygotic twin.
CHAIRMAN KASS: Everyone?
DR. OPITZ: Everyone. Just simply because
of the phenomenon that the midline which is established very
early, even has the phenomena of polarity before you can even
see a midline at the primitive streak and so on, the midline
is morphogenetically highly unstable and I once enumerated
all of the biological attributes, you know, of the midline
which would support this kind of a statement, with most of
the products of monozygotic twinning, then dying. That is
And the surviving co-twin, having a very high incidence of
additional midline anomalies, heart defects, vertebral abnormalities,
etcetera, etcetera. So this would seem to be an intrinsic
attribute of the midline.
Now your question then of wholeness is also well taken because
the it's beginning to be shown in mammalian embryogenesis
that already at the time of the first cell division the axes
for the polarity of the embryo are being set up. And that
removing, let me say 2 out of 8 or 2 out of 10 blastomeres
may perhaps disturb this, but interestingly enough, as in
the Roux versus Dreisch experiment, some individuals have
regulative development whereby they can heal and repair and
start over again as if nothing had happened and then sort
of redetermine the remaining eight blastomeres as if they
were whole and others like in the sea urchin, for example,
when you take those two away, then in fact, that part of the
body is missing. And so which is regulative kind of development.
My preference is to look at instead of wholeness as integrity,
developmental integrity and the individuation issue then developing
an impact after the risk of twinning has past, after 14 days.
But even if it did occur and you've got the Hensel twins,
you have two individuals in one body. And even the half baby,
you know, was a living human organism up until about 23 weeks
and 500 grams and it even had a beating heart.
CHAIRMAN KASS: Thank you. Frank Fukuyama
and then Bill and I think we will break.
PROF. FUKUYAMA: Are there any chromosomal
abnormalities that don't show up until a much later stage
of development, when the individual is an adult or by the
time they get to an adult, do you pretty much know? I'm thinking
of this, for example, just with IVF and some of the ART procedures
where the Louise Brown is still in her 20s. Is it possible
that things will show up at later stages that we simply don't
know about or is once you get past a certain age, you're pretty
much home free?
DR. OPITZ: It's possible, Dr. Fukuyama,
but unlikely. There have been some adults who started to reproduce
and all of a sudden miscarriage after miscarriage after miscarriage
and then you begin to investigate and you find that you've
got a chromosome abnormality, usually in a mosaic form.
And usually if development is abnormal on account of a chromosomal
imbalance of abnormality, you will see it at birth or shortly
thereafter. Or let me say during infancy or childhood, if
the individual then is a little bit slow, is not doing well
in school and is then brought into the clinic for evaluation,
so I think it is unlikely, but the point is well taken.
Let me maybe, if you don't mind rephrase the question, what
chromosomal abnormality should we be alert to as a concept
of these ART forms, and one of them is imprinting. It's an
imprinting defect and where the chromosomes look perfectly
normal, there's no deletion there, there's no X chromosome
or missing chromosome, but the genes are not expressed properly
at the right time and the right place because of faulty imprinting
and that can persist into adult life, into later childhood
and embryonal carcinoma.
CHAIRMAN KASS: Bill Hurlbut and then we'll
take a break.
DR. HURLBUT: I want
DR. ROWLEY: Can I just interrupt just because
I think it's important to clarify that when, in the instance
that Dr. Opitz gave of a woman having multiple miscarriages
due a chromosome abnormality, there are things such as gonadal
mosaicism where some of the oocytes developed in that woman
as he's already shown, the gonads in her are developing during
the well, before the 24th week of gestation. Those abnormalities
will only show up in the oocytes that she produces as an adult.
Then there are other meiotic errors where a gamete is chromosomally
abnormal, but that error in formation in that abnormal gamete
probably occurred just in the cycle or just before the release
of the oocyte. At least I think that's correct.
CHAIRMAN KASS: Bill Hurlbut, please.
DR. HURLBUT: I want to follow up on Leon's
questions and try to make some sense of the moral meaning
of these matters and if it's okay, I'd like to ask Professor
Opitz' opinions on the moral matters. Is that within the
CHAIRMAN KASS: I will reserve the right
to cut you off, if it seems to break the rules.
DR. HURLBUT: Okay, I ask this in the spirit
of really wanting to understand these issues myself and I
guess what's in my mind is why don't all the cells at the
various stages up through the blastocyst even until the formation
of the primitive streak, why don't each of the individual
cells go on to form a trajectory of distinct development?
In other words, there must be something binding them as an
integrated unit in the drive of the direction of the individual
maturity. Let's start with that, is that
DR. OPITZ: So what you're addressing is
the question of determination, progressive determination during
progressive differentiation. For the on-going developments,
the more determined is the developmental fate of these tissues.
Now we used to say once you've got a brain made, those brain
cells, you know, are terminally differentiated. When they
die, they can't ever be replaced and there's no etcetera.
Now, of course, through the work of Irv Weissman and many
others, we know that there are, in fact, even in the brain
there are stem cells left over which can rejuvenate and can
give rise to various kinds of cell lines in the brain, including
the supporting cells, the astrocytes, the oligodendra sites
and even neurons.
Now during the course of normal development, these matters
are constrained, phylogenetically constrained into only very,
very few specific outcomes. Now you can probably start over
again taking certain pieces or certain cells as has been done,
certainly successfully in a mouse and in other mammals and
so on, but the further on in your development, the more determined
is the outcome of the specific developmental process.
DR. HURLBUT: But you did say there is polarity,
even as early as cell division.
DR. OPITZ: And it is potential polarity
in the sense that if you don't disturb the system, you can
recognize the meridians and the anterior, posterior, right
and left and dorsal and ventral sides already during the earlier
stages of cell division. If you disturb the system and take
out, for example, those two blastocyst cells, the system can
re-equilibrate itself and can reestablish communication between
each other so that the remaining cells will say, all right,
we'll start over again or we'll re-equilibrate the system.
And this phenomenon of developmental, how shall I say, equilibration
or homeostasis was recognized very early during the 20th century
and Waddington called this canalization or buffering and the
earlier the stage of development, usually in mammals, the
greater is this buffering capacity to repair, to heal and
to reconstitute, also at the same time the greatest vulnerability
then towards major disasters happening like twinning, for
DR. HURLBUT: So this restitution of the
integrated process can take place sometimes in two trajectories
of development, if the disturbance is great enough, but there
is from the beginning a drive in the direction of a single
maturity and it's only when it's disrupted that it becomes
two? What I'm getting at here is you said that there are early
cell divisions. There seems to be gene expression. Is there
even differential gene expression fairly early like four to
eight-cell stage and could we see that as the development
of a single individual in which case some events may disturb
it which become then two individuals, but see what I'm
DR. OPITZ: Yes. I wouldn't say the development
of a single individual. I would just say, gene expression
pattern appropriate to the cell at that stage of development.
And then after the establishment of the basic body plan, that
is when you begin to see the establishment of the specification
of cell lineages, specific cell lineages and one of the last
few issues of Science had a wonderful article in
it on the specification of the germ line, for example. And
the interesting thing is that these you're starting
off with totipotent cells in the inner cell mass or a few
of the inner cell mass cells. And they then become part of
the somatic component of the posterior, the rear end of the
embryo, the allantois, the yolk sac, the hind body and so
on. And then they are respecified through the developmental
context, environment in which they happened to be developing
into germ cells.
And you can, in fact, take nowadays single cells and do microarray
genetic analysis of the gene expression patterns of these
single cells and this is how it was discovered that germ cells
in mammals, that is in mouse, certainly are respecified as
totipotent cells from the development of milieu in which they
were and were influenced. And it's mostly interferon which
does it and then suddenly the cells that have been respecified
begin to express two proteins which are unique primordial
DR. HURLBUT: One final question and Leon
may veto this question. We've had to struggle here with this
question of when there is intrinsic moral value in this developing
entity and the criteria that had been used in various deliberations
on this worldwide relate to primitive streak and so forth.
Usually, the principles being some kind of differentiation
which you seem to indicate is already taking place in its
primordial forms very early, the issue of twinning, which
you say is a fairly ambiguous issue, and then third, implantation.
I didn't ask about implantation, but my assumption from my
scientific understanding is that it's a difference of quantity,
if you will, not quality. There's already growth factors influencing
the developing embryo in the fallopian tube.
But I want to ask you your feeling about the moral meaning
of this, is there some sense in which before say 14 days there
is something of different moral meaning at 14 days?
DR. OPITZ: Bill, let me be I don't
mean to be a moral coward here, sidestep that issue, by not
addressing my or expressing my moral feelings about
the subject which I think is slightly besides the point because
I have a strong suspicion that everybody in this room has
got their own moral feelings and opinions on the subject,
but I do the point I want to make very strongly is this,
that there's a continuum in developmental potential to the
very moment of conception.
As a matter of fact, there's a continuum even into the germ
cells which ought to be treated with exactly the same respect
as the fertilized ovum, as the implanting ovum, as the developing
embryo, simply because germ cells, for example, are extraordinarily
vulnerable to teratogens, viruses, x-radiation, chemicals,
etcetera, etcetera, etcetera which in the long run, being
damaged in any one of these wanton and random kind of race
may harm humanity infinitely more than the loss of a trisomic
CHAIRMAN KASS: Dr. Opitz, thank you very
much for a lucid, illuminating and forthcoming presentation
and response to the questions.
We've run over to take advantage of Dr. Opitz' presence and
generosity. We're running probably 15 minutes behind.
Why don't we start at 5 after the hour. We'll steal 15 minutes
from our long lunch. Let's take a break and then go into the
(Off the record.)