House Subcommittee on Oversight and Investigation
Wednesday, March 28, 2001,
Rayburn House Office Building, Room 2123
Hearing on Issues Raised by Human Cloning Research
here for a transcipt of the Hearing (TXT file, 628K)
here for a transcipt of the Hearing (PDF file, 556K)
Panos Michael Zavos, founder, Andrology Institute of America
testifies on Capitol Hill Wednesday, March 28, 2001 before
the House subcommittee on Oversight and Investigations hearing
on issues raised by human cloning research.
Wednesday March 28, 2001
(AP Photo/Stephen J. Boitano)
before the House Subcommittee on Oversight and Investigation; Hearing
on Issues Raised by Human Cloning Research
© 2001 Dr. P. Zavos.
Reproduced by the Zavos Organization with permission from Dr. Zavos.
Professor, Dr. Panayiotis Zavos, Ed.S., Ph.D.,
Director of the Andrology Institute of America,
Associate Director of the Kentucky Center for Reproductive Medicine
President and CEO of Zavos Diagnostic Laboratories, Inc..
Professor Emeritus of Reproductive Physiology & Andrology
University of Kentucky
P.O. Box 23777, Lexington, KY 40523 USA
March 28, 2001
Room 2123 Rayburn Office Building
the last 25 years I have been involved in the area of reproductive
physiology, andrology, and assisted reproductive medicine. I have
received extensive formal education by obtaining four College degrees
in Biology, Chemistry, general physiology and reproductive physiology.
I have also received extensive training in the areas of gamete physiology,
manipulation, cell culture and in-vitro gamete manipulation. I have
been involved in the development of various technologies and products
and I have published on those subjects quite extensively. I have
developed technologies in gamete culture and manipulation, cryopreservation
and others (See short biography; Exhibit 1).
I was involved with a scientific group in Yonago, Japan in the development
of ROSNI during which immature spermatozoa (spermatids) were harvested
from the testes of infertile men and their nuclei were transferred
into nucleated oocytes and electrofusion was applied and pregnancies
were achieved. This clinical service is available to infertile couples
all over the world today.
own several US patents and have developed products that are currently
in use in ART centers throughout the world. Both my wife, who is
an OB/GYN and REI board eligible (Director of KCRM and IVF) and
my self as the Director of the Andrology Institute of America, are
involved in the infertility market and we also own a company that
markets infertility products throughout the world. In my family,
we are totally dedicated towards the treatment of infertility and
we regard our patients as our primary target for offering them the
best infertility service available.
is because of our total dedication and belief in those principles
that I have decided along with Prof. Antinori to undertake the great
effort and to offer our infertility patients that have exhausted
all options available to them, to bear a biological child of their
own through the option of human therapeutic cloning.
events in the ART market
the advent of in-vitro fertilization (IVF) and all the other advanced
assisted reproductive technologies (ART), we are able today to perform
incredible maneuvers and offer infertility couples options that
can give them hope for having a healthy biologically related child.
Never before in the history of mankind have we been so fortunate
to treat the infertility epidemic so incredibly well, and with such
high probabilities for success in a safe and responsible manner.
We all know that when our infertile couple comes for a visit they
want two things:
A child, (yesterday if possible), and
A healthy child.
incredible developments in the ART market today are no pure accident
but rather the end result of various forces that came into play.
These forces and capabilities came about because of the abilities
and the freedom that scientists and clinicians have to develop such
efforts and work together in organized groups such as ASRM, ESHRE
MEFS and others throughout the world. I have been, and continue
to work, with such groups in a very energetic and positive fashion,
because it is essential that those efforts should continue towards
the development of safe and effective modalities for proper infertility
diagnosis and treatment. In all the years that both Prof. Antinori
and I have been involved in the diagnosis and treatment of both
male and female infertility, we have never been involved in taking
unnecessary risks. This same principle will remain in place as we
venture into the development of new frontiers in the infertility
status of Animal Cloning
variety of mammalian species have been cloned utilizing S.C.N.T.
(somatic cell nuclear transfer). These include sheep, cattle, mice,
goats, and pigs. As pre-implantation and pre-natal chromosomal and
genetic screening was not performed in any of the aforementioned
animal cloning experiments, a small but significant proportion of
the resulting offspring exhibited developmental abnormalities and/or
perinatal death. On the 9th of March 2001 our international consortium
of scientists announced that the intention to perform human S.C.N.T.
to allow infertile couples to have their own biological children.
To avoid the developmental abnormalities observed in the unscreened
animal experiments, we propose to conduct a variety of screening
protocols on the nuclear transplant embryos. Comprehensive screening,
although expensive, would ensure that only healthy developmentally
normal embryos would be conceived. This is a fundamental aspect
of our Consortium's proposal, as producing developmentally abnormal
human children is clearly not ethically acceptable. We have submitted
a report that reviews the scientific literature, results and protocols
regarding somatic cell nuclear transfer (S.C.N.T.) and contemporary
morphological, chromosomal and genetic screening procedures required
to accompany this procedure. (see Exhibit 2). It
is anticipated that the Consortium will utilize a range of screening
protocols similar to (if not the same as) those discussed in this
report. Only future research will elucidate which of these protocols
status of Human Cloning
no one has (as of yet) publicly claimed that a human clone has been
produced, the rumors are that the development of cloning technology
for application in humans may not be too far off. If one examines
other events by studying historical data, one can conclude that
the development of human cloning is inevitable. In a recent report
by 60 Minutes, during which a group of scientists and others participated,
it was concluded that the recent developments are in tune with these
trends. Human cloning is around the corner and (as I stated over
and over), when it comes to human cloning "the genie is out
of the bottle". The technology for cloning a human being exists
and it almost every high tech IVF laboratory across the world. They
are 55 such IVF labs in New York City alone. So the questions that
we should be answering today are:
Who should develop this technology, and
What quality controls will be necessary to be developed and/or applied
in order to make this technology safe, with minimal risks to those
using it and most importantly to those that will be born from such
should develop this technology?
human therapeutic cloning technology should be developed by a group
of scientists and medical experts that understand this type of work
and the seriousness for its development. Furthermore such teams
should be focused on this effort, and work with leaders and governments
to see that this technology can be made safe and be disseminated
properly. This technology (like others) can have negative ramifications
if it is not developed properly and it is allowed to end in the
hands of the exploiters and the "pushers". It is because
of those possible developments that our government (along with others)
should join in and participate in rational, constructive debate
and dialogue, and contribute something logical to say about its
development and dissemination, rather than taking the attitude that
"I don't want to play". I believe that our government
recent attitude with similar situations, has adopted the principle
of establishing a dialogue with hostile groups and governments throughout
the world, and it did pay off great dividends. This is not to imply
however that the CHTC is either hostile or has any hostile tendencies
towards anyone, or any government in the world.
quality controls are necessary?
stated before, during animal experimentation with cloning, no pre-implantation
or pre-natal chromosomal and genetic screening was performed. This
resulted in a small but significant proportion of the resulting
offspring exhibited developmental abnormalities and/or perinatal
death. This according to the CHTC principles, this is totally inhumane,
and irresponsible for those that carried those experiments and gave
the world this "horrible" picture and impression that
cloning can not be offered and made to be safe in humans.
the contrary, this Consortium in order to avoid the developmental
abnormalities observed in the unscreened animal experiments, wishes
to develop and apply a variety of screening protocols on the nuclear
transplant embryos that could ensure that only healthy developmentally
normal embryos would be transferred to produce only healthy children.
This is a fundamental aspect of our Consortium's proposal, as producing
developmentally abnormal human children is clearly not ethically
acceptable. The Consortium has developed such array of testing procedures,
and wishes to make them available to this Committee for review and
as part of this testimony (Exhibit 2).
this committee's benefit, I would like to make the following
comments before I proceed further:
Our Consortium (the Consortium for Human Therapeutic Cloning) has
no intentions of developing this technology within the continental
USA. I am saying this to you Mr. Chairman at this time so that this
Committee will not have to worry about this Consortium breaking
any rules, laws, or having to be legislated out of extinction by
"Name calling" is not on "our cards", and those
that participate in this activity, do so because they believe that
they are "better" medically, scientifically or ethically.
This serves no constructive purpose, and the public is not served
in any positive fashion at all by these actions.
We have received several offers by people to pay to have them cloned
to have their own biological child. Such offers are not accepted
by us because we have no technology to offer to anyone. It is still
at its experimental stage.
that believe that this technology should be banned, those would
not be the Neil Armstrongs that would fly us to the moon and walk
us on it. Those that say stop it, those would not be the Columbus's
that would take the bold step to discover America. Those that say
don't do it, they would definitely not be the Steptoes and the Edwards
that changed the world by their innovative technologies of IVF.
Ironically, Mr. Chairman, those that say don't do it, they may be
the ones, that enjoy the fruits of Professor Edwards and his team's
efforts by doing IVF. This is hypocritical and this has to stop.
We are talking, Mr. Chairman, about the development of a technology
that can help people. We are talking about the development of a
technology that can give an infertile and childless couple the right
to reproduce, have a biological child of their own, and above all
complete their biological "life cycle". This is a human
right and should not be taken away from people, because someone
or a group of people have doubts about its development. We have
no intentions to step over dead bodies or deformed babies to accomplish
this. We never did it in the past, and have no intentions of doing
it while we attempt to develop this revolutionary and yet magnificent
© 2001 Dr. P. Zavos.
MICHAEL ZAVOS, Ed.S., Ph.D.
February 23, 1944, in a small village of Tricomo in Famagusta, Cyprus,
Panayiotis Michael Zavos is the second youngest son of Michael and
Theodora Zavos. He comes from a very successful family, holding
numerous national and international companies and institutions.
He grew up in Tricomo, and attended the Agricultural Gymnasium of
Morphou (High school) in the city of Morphou. He worked at the Agricultural
Research Institute of Cyprus as a Research Assistant and served
as a Lieutenant in the Cypriot Army from 1963-1966. He immigrated
to the United States for University studies in 1966.
Panayiotis Zavos received his B.S. in Biology-Chemistry in 1970,
his M.S. in Biology-Physiology in 1972 and Education Specialist
in Science (Ed.S.) in 1976 from Emporia State University in Emporia,
Kansas. He earned his Ph.D. in Reproductive Physiology, Biochemistry
and Statistics in 1978 from the University of Minnesota in the Twin
Cities, Minnesota. He received the Distinguished Alumnus Award and
the Graduate Teaching Award from Emporia State University and the
Student Leadership Award from the University of Minnesota.
Zavos has a long career as a reproductive specialist and he has
devoted more than 25 years to academia and research. He is the chief
scientist in the development of several new and innovative technologies
in the animal and human reproductive areas with worldwide implications.
He has authored or coauthored more than 400 peer-review publications,
along with a number of solicited reviews, book chapters and popular
press releases. He has presented more than 300 abstracts and other
presentations at a large number of national, international and professional
scientific meetings all over the world. Dr. Zavos' studies and findings
have been reported in the local, national and international press.
He served as an ad hoc reviewer for the NIH and other scientific
Zavos is currently serving as a Board Member of the Middle East
Fertility Society, and is a past Board Member of the China Academy
of Science. He was awarded the first ever Honorary Professorship
by the Chinese Academy of Science awarded to an American by Chinese
Scientists. He has given plenary lectures nationally and internationally
at a number of Scientific Societies meetings, has been and continues
to be a visiting scientist for a number of international collaborations
Zavos has numerous scientific collaborations nationally and internationally
and his publications have appeared in eight languages. He is a member
of the American Society for Reproductive Medicine (ASRM), the American
Society of Andrology (ASA), the European Society for Human Reproduction
and Embryology (ESHRE), the Middle East Fertility Society ( MEFS),
the Japanese Fertility Society, the International Society of Cryobiology
Sigma XI, Gamma Sigma Delta and a number of other Scientific and
Professional Societies. He has served on a large number of
committees for the International Society of Cryobiology, ASRM, MEFS,
ESHRE and others.
Professor Zavos has received a great deal of media coverage both
within the scientific and reproductive arena and the mainstream
press for his many scientific accomplishments and pioneering ventures.
He has made many television and radio appearances including: NPR
Radio, 60 Minutes with CBS, Twenty-Twenty with ABC, Dateline NBC,
Face the Nation, BBC World, Tech TV, Nightline, Fox TV, World News
Tonight, Good Morning America ABC, The Early Show, CBS This Morning,
CNN News, CNN, CNN International, Reuters, HBO, The View with Barbara
Walters, National Geographic, Televisione svizzera (Swiss TV), Cyprus
Broadcasting Corporation, Antena TV of Cyprus and Greece, Tokyo
Broadcasting System International, NHK Television (Japan), Nippon
Television of Japan, TV Asahi (Japan), ZDF TV (Germany), Deutsche
Welle TV (Germany), Nine Network TV (Australia), National TV (Israel),
Live Talk with Sabine Christiansen (Germany) and a great deal of
other local and regional TV programs throughout the US, Canada and
Europe, too numerous to mention.
Zavos is recognized worldwide as a leading researcher and a strong
authority in the areas of male reproductive physiology, gamete physiology,
male infertility, Andrology and other ART procedures including the
development of in-vitro round spermatid manipulations (ROSI procedures).
Dr. Zavos is also recognized as an international authority on smoking
and its effects on human reproductive performance.
Dr. Zavos founded and serves on various companies as:
- Founder, The Zavos Organization, www.zavos.org
- President and CEO of Zavos Diagnostic Laboratories, Inc., a private
corporation that markets infertility products and technologies,
in the USA and worldwide, www.zdlinc.com
- Founder, Director and Chief Andrologist of the Andrology Institute
of America, www.aia-zavos.com
and Executive Director of the Home Fertility Network, www.homefertility.com
- Founder, Semen Tests, for "Online" Semen Analysis, www.sementests.com
and Associate Director of the Greek-American Andrology Institute
of Athens, Greece
- Professor Emeritus of Reproductive Physiology-Andrology at the
University of Kentucky, in Lexington, KY, USA
- Honorary Professor, China Academy of Science
OF SCIENTISTS FOR SAFE AND RESPONSIBLE THERAPEUTIC HUMAN CLONING
Dr. Panayiotis Zavos et al
The potential risk of developmental abnormalities to a human child
conceived through somatic cell nuclear transfer, and the pre and
post implantation morphological, chromosomal and genetic screening
protocols required to accompany this procedure.
collaborative effort of developmental biologists and infertility
specialists, lead by:
Dr. Panayiotis Zavos, Ed.S., Ph.D.,
Director of the Andrology Institute of America,
Associate Director of the Kentucky Center for Reproductive Medicine
President and CEO of Zavos Diagnostic Laboratories, Inc..
Professor Emeritus of Reproductive Physiology & Andrology
University of Kentucky
P.O. Box 23777, Lexington, KY 40523 USA
the [constitutional] right of privacy means anything, it is the
right of the individual, married or single, to be free from unwarranted
governmental intrusion into matters so fundamentally affecting a
person, as the decision whether to bear or beget a child."
Supreme Court of the United States of America (1971) Eisenstadt
v. Baird. 405 U.S. 438)
variety of mammalian species have been cloned utilizing SCNT (somatic
cell nuclear transfer). These include sheep (Campbell et al 1996,
Wilmut et al 1997), cattle (Cibelli et al 1998, Wells et al 1999),
mice (Wakayama et al 1998), goats (Baguisi et al 1999), and pigs
(Betthauser et al 2000, Polejaeva et al 2000, Onishi et al 2000).
As pre-implantation and pre-natal chromosomal and genetic screening
was not performed in any of the aforementioned animal cloning experiments,
the resulting offspring have exhibited an increased incidence of
developmental abnormalities and/or peri-natal death (Wilson et al
1995, Hassler et al 1995, Garry et al 1996, Campbell et al 1996,
Stice et al 1996, Wells et al 1997, Wilmut et al 1997, Kruip et
al 1997, Schnieke et al 1997, Cibelli et al 1998, van Wagtendonk
et al 1998, Wells et al 1999). On the 9th of March 2001 an international
consortium of scientists (lead by Dr. P Zavos, lead author of this
report) announced that they intended to perform human SCNT to allow
infertile couples have children . To avoid the developmental
abnormalities observed in the un-screened animal experiments, they
proposed to conduct a variety of screening protocols on the nuclear
transplant embryos. Comprehensive screening, although expensive
(Schulman JD et al 1996), would ensure that only healthy developmentally
normal embryos would undergo parturition. This is a fundamental
aspect of the Consortium's proposal, as producing developmentally
abnormal human children (at an incidence above that obtained from
natural sexual reproductive conception) is ethically contentious.
This report is a review of the scientific literature, results and
protocols regarding somatic cell nuclear transfer (SCNT) and contemporary
morphological, chromosomal and genetic screening procedures. The
principal objective of this report, is to examine if the calculable
rate of developmental abnormality following SCNT, can be reduced
(using screening), to either a equal or lower rate than obtained
from natural sexual reproductive conception.
The possible developmental abnormalities following human SCNT
somatic cell nuclear transfer has resulted in an increased incidence
of developmental abnormalities in resulting offspring. These abnormalities
have been well documented in ovine and bovine SCNT experiments.
It is possible to calculate the incidence of developmental abnormality
and/or neonatal death in mammalian newborns resulting from SCNT,
and then compare this figure to the incidence of developmental abnormality
and/or neonatal death observed from natural sexually conceived newborns.
The purpose of this report is to calculate if the range of screening
protocols discussed, will bring down the SCNT abnormality rate to
the same level (or below) the natural "base line" rate
of developmental abnormality. The "base line" rate being
that observed in the population following natural sexual reproductive
natural sexual reproduction shall be examined:
in 33 babies is born with structural birth defects - the leading
cause of infant death and childhood disability" (Waitzman et
al 1994). "Natural" developmental abnormalities cover
a large range of post parturition defects, that overlap significantly
with defects resulting from SCNT. The naturally produced developmental
defects; single ventricle, truncus arteriosus, tetralogy of fallot,
transposition of the great vessels.
and respiratory defects; small intestine atresia, tracheal-esophageal
fistula, colorectal/anal atresis, cleft lip/palate, general structural
defects; urinary tract obstruction, renal agenesis/dysgenesis.
defects; gastroschisis, omphalocele, lower and upper limb reduction
defect, diaphragmatic hernia.
nervous system (and other) defects; spina bifida, down sydrome,
et al 1994)
neo-natal mortality rate is between 0.33-1.6% due to "medical
complication or other birth defects", depending on the country
examined (Waitzman et al 1994). In fact this neonatal mortality
rate would be over ten fold higher without current medical knowledge
and intensive care facilities available in developed countries.
In the early 20th century the neonatal mortality rate in the USA
and UK was 4-5% (Hill 1999). In 1997, this had dropped to 0.4-0.5%
in the aforementioned countries. However, in regions/countries where
medical expertise and intensive care facilities are limited, the
rate is predictably higher. In Asia, the neonatal mortality rate
in 1997 was 3.2%, and it was 2.2% in latin America (Hill 1999).
It should be noted that the neo-natal mortality rate observed in
livestock (when less comprehensive medical treatment is administered)
is approximately 8% (Nash et al 1996). To summarize, in the developed
world, the incidence rate of developmental abnormality (following
natural sexual reproduction and conception) is approximately 3%
(Waitman et al 1994), and the rate of neonatal mortality (in specifically
the UK and USA) is approximately 0.45% (Hill 99).
results and evidence from past mammalian research, it is possible
to calculate the overall rate of developmental abnormality following
developmental abnormalities (that occur in a certain proportion
of SCNT post parturition offspring) can be summarized as: Spontaneous
abortion throughout pregnancy, high birth weight (AKA "L.O.S."
or Large Offspring Syndrome), perinatal death, abnormal placentome
development, shortened cellular telomere length, structural abnormalities
in heart and lungs, and general developmental abnormalities resulting
in poor extra-uterine adaptation (including weakened immune system
abnormalities and/or low metabolism resulting in abnormally high
postnatal weight gain) (Wilson et al 1995, Hassler et al 1995, Garry
et al 1996, Campbell et al 1996, Stice et al 1996, Wells et al 1997,
Wilmut et al 1997, Kruip et al 1997, Schnieke et al 1997, Cibelli
et al 1998, van Wagtendonk et al 1998, Wells et al 1999, Kolata
2001 ). It should be noted that as far as shortened telomeres
are concerned, recent evidence suggests that this causes no physiological
disadvantage (Vogel 2000) and the phenomenon has not been observed
(or telomeres have been specifically elongated) in recent mammalian
SCNT experiments (Tian et al 2000). It should also be noted, although
as of yet unpublished, that much of the bovine LOS problems appear
to be correlated with embryo culture conditions, rather than the
specific SCNT procedure. IVF techniques and embryo culture conditions
are significantly more advanced in human IVF, thus LOS may not be
a problem in human SCNT. The most recent published rate of developmental
abnormality and/or neonatal death following SCNT was 50% (Colman
2000). However, as will be discussed, contemporary SCNT results
(utilising novel techniques and occurring after publication of Dr.
Colman's paper), have reduced this figure considerably (Polejaeva
et al 2000, Betthauser J et al 2000). Even prior to publication
it was clear that the incidence was falling below "50%"
(Wells et al 1999):
(bovine and porcine) SCNT results:
Wells et al 1999.
SCNT; NT micro-injection of 552 oocytes.
grade 1 and 2 blastocysts obtained (27.5% to G1 and G2).
G1 and G2 blastocysts transferred, with 10 resulting births.
overall efficiency (post-parturition offspring from oocytes injected).
observed during late (third trimester) pregnancy consisted of seven
fetuses lost due to excessive accumulation of allantoic fluid. Of
the ten newborns, all ten survived (0% neonatal mortality) but four
had mild developmental abnormalities (basically they had formed
enlarged umbilical cords during gestation). One needed epinephrine/doxaphram
to stimulate its cardiac and respiratory systems. This calf "responded
well to treatment and was standing 40 minutes later". Birth,
post cesarean, weight range was 26.5-51 kg. The "developmental
abnormalities" really just comprised of an enlarged umbilical
cord (enlarged umbilical vessels, edematous membranes and greater
than usual allantoic fluid volume) "none of these abnormalities
appeared to compromise fetal development... After a few hours of
life, regular animal health tests showed that the calves were physiologically
healthy" (Wells et al 1999).
Dr. Betthauser et al 2000 (experimental results 1 and 2 combined)
SCNT; 483 nuclear transfers.
experiments concluded that 8% (15/192) of nuclear transplants developed
to G1 blastocyst. Please note that for porcine IVF this rate is
the transfers, four live offspring resulted.
overall efficiency (postparturition offspring from oocytes fused).
This low rate is due to the difficulties in initiating porcine pregancies
(Polge et al 1966)
during late pregnancy consisted of a single fetal loss. This fetus
had passed into a late gestational stage before development arrested.
This resulted in re-absorption not occurring. All live-born offspring
were physiologically healthy and developmentally normal.
et al 2000)
Polejaeva et al 2000 (utilizing a novel "Double NT" technique.
Combined results from seven experiments). Porcine SCNT.
obtained and transferred: 401 (21.5% efficiency).
offspring obtained: 5.
overall efficiency (post-parturition offspring from oocytes injected)
or 5% overall efficiency if results from experiment one are isolated
and examined. Please note that six recipients did not give birth,
thus reducing the efficiency significantly.
five pigs, now three months old, are extremely healthy, in contrast
to the (usual) 50% postnatal loss of nuclear transfer animals [Referring
to Colman's paper 2000]. It is tempting then to speculate that this
modified method may have general utility in other species, even
those where single nuclear transfer has been shown to work."
et al 2000). Please note that Alan Colman contributed to the aforementioned
omission of Dr. Onishi's SCNT results is due to fact that the group
did not utilize ultrasound. Thus, information on rate of pregnancy
establishment, and fetal loss at various stages was not available
(Onishi et al 2000).
is clear that contemporary evidence suggests that the rate of developmental
abnormality and/or perinatal loss is below the 50% rate quoted previously
(Colman 2000). This report suggests the actual calculable rate without
screening is now between 25-50%; erring towards the former. It should
be noted that the incidence of developmental abnormality in normal
sexual reproductively conceived newborns is 3% (Waitzman et al 1994).
A greater than 90% detection rate (from screening) is required to
reduce the 25-50% SCNT developmental abnormality rate to below the
3% natural baseline rate. This is possible (as will be discussed
in this report), providing that comprehensive multiple pre and post
implantation morphological, chromosomal and genetic screens are
reasons screening is not utilized in animal SCNT
While the previously listed developmental abnormalities are not
desirable in non-human mammalian SCNT experiments, and the incidence
is between 25-50%, the high cost of screening (in both the time
required and the financial aspect) has resulted in the high incidence
of post-natal abnormalities being accepted as the norm. In essence,
it is more cost effective to postnatally euthanize or allow late
gestational spontaneous abortion of these abnormal offspring, rather
than comprehensively screen out these abnormal embryos pre-implantation
and pre-natally. However, when considering applying SCNT to allow
infertile couples to have a genetically related child; morphological,
chromosomal and genetic screening becomes essential, regardless
of the cost. A 25-50% risk of developmentally abnormal human offspring
is (we would suggest) not ethically acceptable. While a less than
3% risk (the natural rate) of developmental abnormality following
SCNT, is (we would suggest) ethically acceptable. Comprehensive
pre-implantation and pre-natal genetic screening (or as close to
truly "comprehensive" as is possible), is critical for
reducing the risk of developmental abnormality (following SCNT),
to below the baseline natural 3% rate. This appears to be the only
acceptable route, as lack of screening will result in an unacceptably
high risk of developmental abnormalities (50%), but federal regulation
would violate the reproductive freedom of infertile couples (Eibert
1999) and would inevitably be unconstitutional . Screened SCNT
is the only way certain infertile individuals can have a biologically
the cluster of constitutionally protected choices that includes
the right to contraceptives, there must be included the
right to submit to a medical procedure that may bring about, rather
than prevent, pregnancy."
v. Hartigan, 735 F.Supp. 1361 N.D. ILL.),
3. The developmental efficiency of mammalian SCNT
The developmental efficiency of somatic cell nuclear transfer has slowly
improved over the past five years. Dr. Wilmut achieved a developmental
efficiency (recipient oocytes to offspring obtained) of 0.4% in
1996 (Wilmut et al 1997). Dr. Wakayama managed to improve this to
2.8% in subsequent murine research, by using microinjection rather
than electrofusion (and other factors) (Wakayama et al 1998). However,
the efficiency of development from adult mammalian somatic cells
has remained at around 2% since that time (Polejaeva et al 2000).
It is anticipated that for every one hundred nuclear transplant
embryos, only one or two embryos will result in healthy developmentally
normal offspring (Colman 2000). It should be emphasized that this
does not mean that 98% of the live-born offspring will be developmentally
abnormal, the vast majority of nuclear transplant zygotes do not
even get implanted into the uterus.
From the scientific literature over the past few years, an averaged developmental
efficiency (at each stage following nuclear transfer) can be calculated.
If one hundred recipient enucleated oocytes are utilized for SCNT,
approximately 90% can be discarded through morphological screening
as not being "Grade 1" embryos (Wilmut et al 1997, Wakayama
et al 1998, Elder et al 2000). Although it should be noted that
recent evidence and new "double nuclear transfer" techniques
may reduce the number of nuclear transplant embryos lost at this
stage (Polejaeva et al 2000). "90%" may sound excessively
high, but should be considered in context to natural sexual reproduction,
where on average 75% of the embryos are flushed or reabsorbed .
The embryos in this category have either not initiated cleavage,
or cleavage has become abnormal. There's a variety of factors believed
to contribute to this phenomenon: oocyte source and quality, methods
of embryo culture, donor cell type, imprinting, activation failure
and failure to reprogram the somatic nucleus (Polejaeva et al 2000).
It has also been postulated that un-synchronized cell cycles between
donor and recipient have also contributed to this high level of
abnormal cleavage (Campbell et al 1996). In this model, multiple
exposures to MCM proteins (due to lack of a nuclear envelope in
a high MPF environment) results in re-replication of DNA and thus
aneuploidy (Kearsey et al 1998). Also, certain methods of nuclear
transfer, such as electrofusion (Wilmut et al 1997), appear to be
less efficient than microinjection (Wakayama et al 1998), although
other factors significantly contribute. However, the a significant
proportion of abnormal cleavage (amphibian models suggest) is simply
due to incomplete reprogramming (Kikyo et al 2000) and remodeling
of the somatic cells' nucleus, and mitosis initiating before replication
has finished, and thus resulting in extensive chromosomal damage
If one hundred recipient enucleated oocytes undergo mammalian SCNT,
approximately 90 are discarded through morphological screening.
It should be noted that this loss of oocytes will substantially
increase the cost of human SCNT as a treatment for infertility,
as oocytes in the US can cost between $500 - $1000 each . Approximately
10% of the reconstructed zygotes will undergo normal cleavage, and
develop into morphologically normal (grade 1) blastocysts. Traditionally,
in animal cloning experiments, it has not been cost effective to
screen these morphologically normal blastulae for hidden chromosomal
and genetic abnormalities. Thus they have just been implanted into
a surrogate and left to develop. As these embryos have not been
screened, a certain proportion of developmental abnormalities, in
resulting post parturition offspring, has been inevitable.
Mammalian SCNT experiments suggest that, of the morphologically normal blastulae
transferred to uterus, 50% will implant and develop through early
gestation. Of these implanted embryos over 70% of the pregnancies
will be lost due to spontaneous re-absorption (Stice et al 1993,
Stice et al 1996, McMillan et al 1997, Cibelli et al 1998, Peura
et al 1998, Wells et al 1998). It has been reported that the majority
of this loss occurs during the first trimester (Stice et al 1996,
Wells et al 1998). Thus, if 10 morphologically normal embryos are
transferred to the uterus, only about 5 will actually implant, and
only one or two will actually reach parturition. Without screening,
out of the offspring that actually undergo parturition, this report
assumes that 25-50% will have developmental abnormalities and/or
result in perinatal death. The 50% rate (Colman 2000) is a conservatively
high assumed rate, and is considerably higher than the actual rate
of abnormality from non human mammalian SCNT experiments (Wilson
et al 1995, Hassler et al 1995, Garry et al 1996, Campbell et al
1996, Stice et al 1996, Wells et al 1997, Wilmut et al 1997, Kruip
et al 1997, Schnieke et al 1997, Cibelli et al 1998, van Wagtendonk
et al 1998, Wells et al 1999, Baguishi et al 1999, Betthauser et
al 2000, Polejaeva et al 2000). In this report we shall assume the
upper end of the range (50%). This conservatively high assumed abnormality
rate, reflects the significant degree of cautiousness the authors
of this report feel should be employed, when applying this mammalian
evidence to humans, where no human SCNT results are available. In
essence this report wishes to examine whether the screening protocols
are able to reduce the risk of developmental abnormality following
human SCNT to a reasonable level (below the 3% baseline), even when
a conservatively high abnormality incidence rate is assumed.
To restate the information; if one hundred enucleated oocytes are injected
with somatic cell nuclei, one would have to expect ninety to undergo
obviously morphologically abnormal cleavage. If the ten morphologically
normal embryos are transferred to the surrogate without being screened,
one could expect about five to actually implant, and two of those
implanted embryos to result in live births. The assumed probability
(from animal SCNT experiments) is that one of the live births will
be developmentally normal, while the other will be developmentally
abnormal. However, as previously mentioned, recent evidence suggests
this 50% post-parturition developmental abnormality rate is conservatively
high (Wells et al 1999, Betthauser et al 2000, Polejaeva et al 2000).
"Developmentally normal" is defined as not suffering from
any of the developmental abnormalities listed in Section 2, and
being within the typically accepted weight range from natural births.
To put the conservatively high abnormality rate into context with
a famous example; "Dolly" the sheep was produced from
277 fused couplets (reconstructed zygotes), of these zygotes, 29
(11.7%) were transferred to surrogate hosts, and only one developmentally
normal sheep was born (Dolly), there were no developmentally abnormal
offspring from those 277 oocytes (Wilmut et al 1997). When human
SCNT critics use this famous example to argue the safety issue of
mammalian cloning, they have inevitably chosen a poor example, as
one healthy sheep resulted from one established pregnancy is hardly
a safety issue. What they are really arguing is the low efficiency
of adult mammalian SCNT, 277 oocytes to produce only one offspring,
although even this argument is somewhat out of date, as with new
NT techniques, we would expect an efficiency of around 2%, rather
than the 0.4% (ratio of zygotes created to post parturition offspring)
obtained by Wilmut (Wilmut et al 1997). It is also possible that
other factors may increase the efficiency of human SCNT, as discussed
in Section 6.2.
To summarize, in this report we assume that without screening, mammalian
SCNT will produce a developmentally abnormal offspring for each
developmentally normal offspring produced. The actual incidence
of abnormality is significantly below this rate. Pre and post implantation
morphological, chromosomal and genetic screening will reduce the
incidence of post parturition abnormality. Section 6 of this report
examines the calculable residual risk of abnormality (utilizing
mammalian SCNT and PGD/PND evidence), following multiple pre and
post implantation screening of the human SCNT embryos. The report
intends to discuss the protocols and evidence, that suggests that
screening will reduce the "25-50%" abnormality rate to
below the natural baseline rate (3%).
4. The causes of these developmental abnormalities.
The causes of the abnormalities observed in mammalian SCNT post-parturition
offspring, fall into three categories. The three categories are:
is once again re-emphasized that significantly less than half of
the post-natal offspring are in this group, the other half are developmentally
normal and healthy (under the classification previously described
in Section 2).
4.1. Chromosomal damage.
damage and disruption can result from physical damage to the chromatin
during the nuclear transfer process, due to re-replication of DNA
(resulting in aneuploidy), or due to the fact that the reconstituted
zygote initiates mitosis before S-phase replication has finished
(this results in the partially replicated chromosomes being torn
during Anaphase). Most chromosomal damage is very obvious, and can
be screened out very simply. At a purely morphological level, cleavage
of the embryo is not normal and that embryo must be discarded. As
discussed in Section 3, this results in the elimination of approximately
90% of the nuclear transplant zygotes. Less severe chromosomal damage
is not observable at a morphological level, but can be comprehensively
screened for, using any of the ubiquitous PGD (pre-implantation
genetic diagnosis) and post-implantation PND (pre-natal diagnosis)
chromosome screening protocols.
4.2. Incompletely reprogrammed (non-imprinted) gene expression.
vast majority of mammalian genes are non-imprinted. These genes
are either expressed or not expressed in different tissues, at different
times, to different levels. The active genes (or expression pattern)
in a hematopoietic cell, is very different from that of a epithelial
cell, which is again very different from that of a early embryonic
cell. Advances in DNA diagnostic techniques allows gene expression
pattern to be observed to a high resolution (as is discussed in
Section 126.96.36.199). Common epigenetic mechanisms for "turning
genes off" include linker histone acetylation, methyl-CpG binding
proteins and Polycomb group proteins (Kikyo et al 2000).
cause the DNA to be wrapped up into tightly bound structures called
chromatin. When exposed to the egg cytoplasm, this structure begins
to decondense and reprogram. The reprogramming activity of the ooplasm
changes the gene expression pattern (from somatic to embryonic).
When insufficient reprogramming of gene expression occurs following
NT (nuclear transfer), developmental abnormalities arise. A common
phenomenon resulting from incomplete reprogramming is incorrectly
differentiated trophoblasts. This (it is believed) is one of the
reasons for only (on average) 50% of the NT blastocysts implanting
4.3. Incompletely reprogrammed (imprinted) gene expression.
imprinting is an epigenetic phenomenon which occurs in gametogenesis.
Genomic imprinting occurs when both maternal and paternal alleles
are present, but one allele is expressed while the other remains
inactive. Methylation is the mechanism by which genes are either
turned on of off in imprinting. Genomic imprinting is necessary
for development, and regulates growth and various other developmental
features of the embryo (Browder et al 1991). Genomic imprinting
is an important phenomenon for SCNT reprogramming, because has implications
in embryonic and extra-embryonic growth and development in mammals.
Many experiments have demonstrated this (Li et al 1992, 1993). While
PND allows the methylation state of a range of imprinted genes to
be determined, PGD should focus on the imprinted gene that is the
"indicator" of developmental problems when incorrectly
reprogrammed. This indicator imprinted gene is the Igf2r gene (or
its other highly conserved mammalian homologues). Igf2r (insulin
like growth factor 2 repressor) is expressed from a methylated maternal
allele. DNA methylation is a requirement for the expression of the
Igf2r gene. (Li et al 1993). A methylated maternal and non-methylated
paternal allele is required for normal Igf2r expression and therefore
normal development. If methylation is lost from the DMR2 (a differently
methylated region of intron 2) during the in vitro culture and NT
process, then Igf2r is underexpressed. Igf2 expression levels (usually
regulated by Igf2r) are therefore abnormally high. Igf2 controls
the size the fetus is allowed to grow to. If the fetal size is not
regulated, then the offspring may cause internal injuries by growing
to extremes of size. This phenomenon is called LOS (large offspring
syndrome) and has been observed in a significant percentage of ovine
and bovine clones (Young, Sinclair and Wilmut 1998). Unregulated
Igf2 expression has also been correlated with the other developmental
abnormalities observed in SCNT mammals (e.g. Abnormally large placental
development). There is a significant correlative link between LOS
(due to Igf2r demethylation) and the other developmental abnormalities
observed following certain mammalian SCNT cases (Young et al 2001).
It is therefore apparent that the methylation state of Igf2r is
an indicator of not just LOS but also of a range of other aberrant
developmental phenomena. It should be mentioned that LOS has always
been a problem in ovine and bovine IVF, but not in human IVF. There is no contemporary evidence suggesting that LOS would affect humans
. However, there is also no conclusive evidence that it will
not. For this reason (uncertainty) and the fact that LOS is correlated
to a range of other developmental abnormalities, the authors of
this report suggests that it should be screened for. To summarize,
PGD screening for the methylation state of Igf2r, significantly
reduces the risk of obtaining developmental abnormalities in the
non-affected transferred embryos (when compared to transferring
5. The prenatal screening procedures requires to detect these abnormalities.
5.1. Overview of screening
"screening" required to accompany human SCNT encompasses
the pre and post implantation morphological, chromosomal and genetic
diagnosis protocols, as well as additional screening protocols such
as maternal serum screening. All these protocols have just one objective:
to identify and "selectively remove" developmentally abnormal
embryos and fetus's as early as possible, so that only normal healthy
embryos develop to term. All of the screening protocols technically
come under the heading of prenatal diagnosis (PND).
diagnosis (PND) refers to all diagnostic screening of the embryo
or fetus prior to parturition (birth). Preimplantation genetic diagnosis
(PGD) is an early form of prenatal diagnosis, that is performed
prior to implantation of the blastocyst into the uterus. The advantage
of PGD is that it provides a rapid means for diagnosing which morphologically
normal blastocysts are actually chromosomally normal, and are correctly
imprinted at the Igf2r indicator gene loci. PGD significantly increases
the probability of implanting only chromosomally normal (euploid)
fully reprogramming blastocysts. The disadvantage of PGD is the
cost in both time and money. Infertility centers can charge approximately
$10,000 per cycle to perform the PGD procedure (Schulman JD et al
1996), although this charge does vary. However, it is actually PND
during the late first and early second trimester that should be
utilized to ensure the health and normality of the early fetus.
It is the recommendation of this report (as will be discussed later)
that both PGD and PND (in association with other screening methods)
are utilized to ensure that developmental abnormalities are not
present in fetuses by the third trimester (produced via human SCNT).
5.2. Preimplantation screening
5.2.1. Introduction and Morphological screening
combines the existing technology of IVF and micromanipulation (ICSI,
embryo biopsy) with molecular genetic techniques used in clinical
practice, and allows the selection of normal embryos for transfer
thereby reducing the possibility of establishing an abnormal pregnancy."
previously discussed, the vast majority (~90%) of nuclear transplant
zygotes will undergo morphologically abnormal cleavage, with a high
level of blastomere fragmentation. Even at an early stage, it is
usually clear that these early embryos will not develop into morphologically
normal blastulae. Preimplantation chromosomal and (Igf2r) genetic
screening should be performed on the remaining 10% of grade 1 embryos
(Elder et al 2000), that appear morphologically normal, and are
thus possible candidates for uterine transfer.
genetic diagnosis (PGD) of in vitro generated embryos was first
utilized at the end of the eighties. The first healthy pregnancy
was reported in 1990 (Handyside et al. 1990). The procedure involves
sampling cells from the nuclear transplant morula prior to compaction
(and obviously implantation). After the nuclear transplant zygotes
have developed for 3 days, they will have divided into a ball (morula)
of eight cells. Although technically a morula, this ball is ubiquitously
referred to as a blastocyst in the scientific literature, and will
thus be termed for the rest of this report. The eight cell embryo
is held against the blunt end of a pipette, while a fine needle
is used to make a small slit in the zona pellucida ("zona drilling"),
and two cells are aspirated by gentle suction (Black 1997). This
process is called an embryo biopsy, and should be performed before
compaction has initiated. The latest clinical results conclude that
correctly performed embryo biopsy for PGD does not harm the blastocyst
development or pregnancy rate.
examining the effect of embryo biopsy have shown that at the 8-cell
stage, removal of two cells was not detrimental to embryo metabolism
or development and is an efficient process with more than 90% of
the embryos surviving... 97% of embryo biopsies were successful."
et al 2000)
of the aspirated cells can be used to make an initial screen for
chromosomal abnormalities, the other cell can be used to make an
initial detection of whether the imprinted indicator gene (Igf2r)
has been properly reprogrammed to an embryonic epigenetic methylation
pattern. There's a two day period of time in which to complete these
pre-implantation screening protocols, as blastocyst implantation
should occur on day 5 (following nuclear transfer). Section 5.2.2
discusses PGD chromosomal screening, and Section 5.2.3 explains
PGD imprinted indicator gene (Igf2r) screening.
5.2.2. PGD chromosomal screening
preimplantation chromosomal screen can only be conducted on the
chromosomes of a single cell. This obviously means the number of
preliminary chromosomal screens is limited to just one at this stage.
Obviously an almost unlimited number of chromosomal and genetic
screens can be conducted post-implantation (prior to the third trimester).
report recommends that the initial PGD chromosome screening protocol
utilized, is fluorescent in situ hybridization (FISH). This technology
is ubiquitously used to identify euploidy. This traditional approach
to pre-implantation genetic diagnosis relies on fluorescent probes
hybridizing to their relevant chromosome. A relatively new technique
(developed by Dr. Wells) called CGH (comparative genome hybridization)
allows every chromosomes "overall genetic content" to
be assessed in detail (Wells et al 2000). Wells' method is based
on PCR (polymerase chain reaction). It involves amplifying all the
genes in the sampled cell, and then analyzing the product for imbalances
in genetic content indicative of chromosomal abnormalities. Wells'
protocol is not yet finalized as it is (at present) taking to long
to be used as a pre-implantation screening technique. However, it
is an ideal post implantation technique from CVS or amniocentesis
derived fetal tissue. Another example of WGA (whole genome amplification)
is PEP (Primer Extension Preamplification) which uses random oligos
to amplify at least 90% of the genome more than 30 times .
is possible to observe the structural integrity of about five chromosomes
to high resolution (per FISH screen). This means that the initial
PGD chromosomal screen is just an initial indicator screen, and
further karyotype, CGH and FISH screens (postimplanation) are required
to conclusively verify the correct chromosomal complement of the
report recommends that FISH is utilized at the pre-implantation
level, as the preliminary screen for chromosomal damage. Utilizing
this chromosomal screening procedure, a large percentage of the
morphologically normal, but chromosomally abnormal embryos can be
identified and discarded. It should be noted that most embryos with
extensive chromosomal abnormalities are not viable, and spontaneously
reabsorb or don't even implant (if transferred). The most viable
chromosomal abnormalities are balanced translocations or whole chromosome
aneuploidy (Turner's, Klinefelter's, Down's). FISH by itself is
not a comprehensive chromosomal screen, but by also conducting the
various PND chromosomal screens discussed in Section 5.3.1, an approximately
comprehensive chromosomal screen (overall) can be achieved.
5.2.3. Imprinting marker gene (Igf2r) screening
detect if reprogramming of the Igf2r imprinting indicator gene has
occurred. The cells genetic material is purified and exposed to
methylation dependent restriction enzymes. For example, MboI doesn't
cut DNA if the GATC sites are methylated, while Sau3AI will. These
enzymes will either cut or not cut the Igfr2 alleles, dependent
on the methylation state of a region in the gene called the DMR2
(differently methylated region). It has been proven that LOS offspring
completely lose the 70% methylation at the Mbo1/Sau3AI sites in
the DMR2 region (Young et al 2001). After restriction enzyme exposure,
RE (restriction enzyme) denaturation using phenol chloroform, and
residual methyl group removal, the Igf2r gene can be amplified by
PCR (polymerase chain reaction). The presence or absence of complete
stretches of Igf2r DNA illustrates whether the imprinted gene's
methylation pattern had been reprogrammed or not . If reprogramming
of this indicator gene has not occurred, this embryo will develop
abnormally and must be discarded. If the reprogramming has been
successful and methylation pattern of the Igf2r gene is correct,
then uterine implantation can continue (assuming that the cell's
chromosomal state has also been checked). As previously discussed,
there is a strong correlation between the methylation state of the
imprinted Igf2r indicator gene, and the developmental potential
of the embryo. If the indicator gene is correctly imprinted, the
probability (due to the correlative evidence) that the other imprinted
genes are also correctly imprinted, is significantly increased.
However, the correlation is not absolute, and a second round of screening for other imprinted genes (and re-screening for Igf2r)
during the early second trimester (when more fetal tissue is available),
allows a "safety net", a means of double checking. This
secondary screening is discussed in Section 188.8.131.52.
5.3. Postimplantation screening.
PND chromosomal screening
Genetic (imprinted and non-imprinted) screening
Additional pre-natal screening and fetal tissue sampling
5.3.3. discusses the methods for obtaining a post-implantation
fetal tissue sample. This tissue sample provides a sufficiently
large source of cells on which to conduct the comprehensive chromosomal
and genetic "secondary" screens, to ensure that any
developmentally abnormal embryos that were not identified pre-implantation,
are diagnosed as inviable prior to the third trimester. It should
be noted that unscreened implanted inviable embryos usually reabsorb
rather than result in stillbirth (Section 3).
5.3.1. PND chromosomal screening
screening following implantation.
first step in post-implantation chromosomal screening is karyotype
analysis. The fetal tissue sample is cultured and then exposed to
mitotic inhibitors. The metaphase chromosomes can then be elongated
and then treated with Giemsa (or a similar stain). Each chromosome
results with a specific banding pattern, chromosomal damage following
nuclear transfer can usually be detected at this stage. Occasionally
the karyotype will be inconclusive, and fluorescent in situ hybridization
(FISH) can be utilized to assist in elucidating the diagnosis. In
the FISH screening protocol, fluorescent chromosome specific DNA
probes are incubated with the karyotype (instead of Giemsa). Visualization
of a FISH karyotype set provides the most detailed (visual) information
of the state of the chromosomes following NT. Humans have 46 chromosomes
(23 pairs), thus multiple FISH screening is required to observe
the detailed status of the entire genome. FISH was the technique
recommended for PGD chromosomal screening (see Section 5.2.2). A
third technique called CGH (comparative genome hybridization) allows
even the smallest amount of chromosomal damage to be detected. This
is because chromosomal damage usually leads to an increase or decrease
in genetic material in different cells. FISH or Giemsa karyotyping
will detect the unlikely event of balanced chromosomal damage (which
CGH can not). Thus, the chromosomal screening techniques should
be utilized in combination. It should be noted that a full karyotype
(Giemsa staining all 46 chromosomes) can only be performed post-implantation.
Full visual karyotyping can only be performed on metaphase spreads
of cells (not possible with biopsied cells).
5.3.2 Genetic (imprinted and non-imprinted) screening
184.108.40.206. Reprogrammed gene expression pattern screening.
and cDNA gene chips can be utilized to assay the mRNA expression
pattern of the sampled tissue . If reprogramming of the somatic
nucleus was successful following SCNT, then the expression pattern
should revert from the adult tissue specific pattern, to an embryonic
pattern (Gurdon et al 1976, 1977, 1986). Other methods for screening
the expression pattern of the embryo include multiple mRNA amplification
RT-PCR (Rodriguez et al 1992) and multiple mRNA-RNase protection
assays . These various protocols are standard in molecular biology
laboratories around the world. They all basically detect which mRNA's
are being transcribed, and this information can be compared with
the expected expression pattern for the tissue type sampled at that
stage, and the original donor cell tissue expression pattern. If
the mRNA expression pattern from the sampled fetal tissue does not
match the expected naturally produced pattern from a naturally conceived
fetus (of the same developmental stage), then it can be concluded
that reprogramming has not been complete, and "selective removal/reduction"
of the inviable fetus is strongly recommended. It should be noted
that by the second trimester, the vast majority of embryos have
properly reprogrammed their non-imprinted genes. Those that do not
reprogram gene expression properly, rarely develop into a transferable
blastocyst, and the probability that they will implant into the
uterus is very much reduced (Gurdon 1999).
PCR based screening protocols (albeit focused on diagnosing single
gene disorders) include: single stranded conformational polymorphism
(SSCP), amplification refractory mutation system (ARMS) and heteroduplex
analysis PCR (Elder et al 2000) (multiplex, nested or fluorescent
PCR included). Research is currently underway to exploit these protocols
for vertebrate SCNT screening . There is an extensive list of
protocols that can be used to detect the mRNA expression pattern
of various tissues sampled from the developing embryo (Lewin 1994).
220.127.116.11. Secondary imprinted gene screening.
is only one of over 30 imprinted mammalian genes . Wide ranging
imprinted gene screening (via the same protocol as Igf2r) is required
as a "safety net" during post implantation development.
Fetal tissue samples are obtained via the methods discussed in Section
5.3.3, and the methylation state of the various imprinted genes
can be checked by the same method explained in Section 5.2.3. As
previously discussed, if the fetal imprinted genes are not corrected
reprogrammed, then "selective removal" of the developmentally
abnormal early fetus is a strongly recommended option. It is imperative
that no abnormal fetus is allowed to progress into the third trimester.
If the comprehensive chromosomal and genetic screening protocols
are adhered to, mostly developmentally normal embryos will be implanted,
and the vast majority that progress into the third trimester will
be developmentally normal. With modern medical knowledge, it is
possible to keep an early third trimester fetus alive in an extra-uterine
environment. Thus it becomes increasingly ethically contentious
to "selectively remove" a progressively more "viable"
(but developmentally abnormal) fetus after this stage of development.
Also, if the epigenetic state of the Igf2r gene, and other imprinted
genes, are not checked, and the embryo does develop to the third
trimester without undergoing spontaneous abortion/re-absorption,
there is a significant risk to the surrogate. A LOS affected fetus
can grow to an abnormally large size, and thus internal damage may
be caused to both the surrogate, and the spatially restricted fetus.
This situation is not acceptable to the human mother or child, and
pre-implantation and pre-natal screening (before the third trimester)
must be comprehensively conducted.
5.3.3. Additional pre-natal screening and fetal tissue sampling
variety of other pre-natal tests can also be utilized to ensure
development is proceeding correctly. These are standard in clinical
practice and include: Serum screening, ultrasound, and standard
chromosomal and genetic PND tests.
during the second trimester, markers of chromosomal abnormality
can be detected in maternal serum (if these abnormalities are in
fact present). Markers that have been used include: low maternal
serum alpha fetoprotein levels, low unconjugated oestriol concentration,
and high human chorionic gonadotrophin levels . The detection
rate with combined serum screening for specific chromosomal abnormalities
can be as high as 70%.
allows morphological abnormalities in fetal development to be detected
from the first trimester onwards. Classically, developmental abnormalities
such as cardiac malformations, duodenal atresia, hydrops, choroid
plexus cysts, nuuchal oedema, renal pyelectasis and omphalocoele
are all detected in this way. Ultrasound scanning is one of the
most essential post-implantation screens, that can be repeated indefinitely
to detect very minor developmental abnormalities. Even with minor
morphological anomalies, detection rates can be over 85%, and much
higher when Ultrasound is used in combination with other screening
protocols (Elder et al 2000). Some animal cloning groups did utilize
low resolution ultrasound, solely to determine if pregnancies had
in fact established.
and genetic prenatal diagnosis screening (post-implantation) has
a very high priority. Of the morphologically normal blastocysts,
only 20 - 40% are actually chromosomally and genetically normal.
PGD provides a reasonable probability of screening out developmentally
abnormal blastocysts. However, the number of screens is limited
by time and genetic material. PGD is only performed on one or two
cells, and there is only a two day period in which to perform the
protocols. In addition to this, PCR based diagnosis on single cells
is subject to allele dropout (in which one allele is preferentially
amplified) and contamination. Post-implantation PND has extensive
genetic material, months in which to perform comprehensive chromosomal
and genetic screening, and is not subject to the range of problems
PGD encounters. The only advantage of PGD is that it allows SCNT
blastocysts, which have a greater than 50% probability of being
developmentally abnormal, of being screened out (with a reasonable
efficiency) prior to implantation. Thus reducing the probability
and necessity of post implantation "selective removal".
methods for obtaining postimplantation fetal tissue samples
Villus Sampling (CVS) and amniocentesis are the most commonly employed
methods of collecting a sample of fetal tissue. Less common techniques
include fetal blood or tissue sampling, cordocentesis and PUBS (percutaneous
umbilical blood sampling). Both amniocentesis and CVS are performed
in the second trimester (there is a 1.5% chance of causing miscarriage
with CVS, this risk is reduced to 1% with amniocentesis).
fetal tissue sample must be screened for all three possible sources
of abnormality. This involves karyotyping for chromosomal abnormalities,
multiple screens to identify if gene expression patterns have properly
reprogrammed following NT (nuclear transfer), and screening the
methylation state of the imprinted human genes. If the fetus is
identified as developmentally abnormal, then the option of a second
trimester "selective removal" is strongly recommended.
PND is discussed in detail in Section 5.3.1. and Section 5.3.2.
6. Risks and Recommendation
reproductive human SCNT research or clinical results have been publicly
announced (as of the 21st March 2001). Thus calculations of risk
can only be abstract deductions, based on risk of developmental
abnormality derived from reproductive mammalian SCNT research, and
the published detection rates of the PGD and PND screening discussed
in this report.
6.1. Risk of multiple pregnancy.
mammalian nuclear transfer models suggest that the probability of
obtaining a multiple pregnancy (from the 10-15 oocytes released
during super-ovulation) is relatively low. However, if enucleated
donor eggs are utilized, then the correspondingly increased number
of transferable blastulae, will increase the probability of a multiple
6.2. Probability of success (per cycle).
date, published fertilization rates [from IVF] for most categories
of patients reach 67%, with a clinical pregnancy rate of 36% and
an ongoing/delivery rate of 28%"
et al 2000)
traditional IVF, it can take (on average) four cycles to achieve
a pregnancy (Elder et al 2000). It is not known how long it would
take to achieve a successful pregnancy with human SCNT. Mammalian
models (Section Three) suggest it would be significantly longer
than conventional IVF, and the screening protocols would further
reduce the chance of a pregnancy (albeit an developmentally abnormal
pregnancy) establishing. On fertility drugs a woman will typically
produce only ten to fifteen oocytes. Mammalian models predict only
1-2% of those oocytes will result in a healthy offspring through
SCNT (Colman 2000), then this evidence (on itself) suggests that
many cycles may be required. However, there are several facts which
may significantly increase the probability of success in humans:
The use of enucleated donor oocytes would significantly increase
the probability of success (and also the cost) of this therapeutic
The fact that we know a great deal more about the various aspects
of human reproduction via IVF, and various other ART's (assisted
reproduction technologies), coupled with the fact that the livestock
that have been cloned are "highly inbreed", should (theoretically)
result in efficiency greater than that predicted by animal models.
Another method of increasing the probability of success involves
"embryo splitting" . Although not yet a standard clinical
procedure, this protocol has been considered ethically sound by
the American Society for Reproductive Medicine .
unknown components, and relatively low probability of success of
this infertility treatment, must be conveyed to the infertile patient/couple.
True informed consent can only be obtained after all of the relevant
information is provided, and the unknown risks are explained.
6.3. Risk of developmental abnormality with and without screening.
report has assumed that without screening, for every developmental
normal SCNT derived post-parturition mammal, there will be a developmentally
abnormal offspring produced. The actual rate of abnormality in reality
is significant lower than this assumed rate (Section Two). The probability
of obtaining developmentally abnormal post parturition offspring
(with screening) is dependent on the detection rate (and ability)
of the screening protocols used. While the "Misdiagnosis from
each PGD test can be as high as 5% in some cases"  the
overall misdiagnosis level becomes mathematically negligible when
screening protocols are utilized in combination, both before and
after embryo implantation. This calculation assumes inclusion of
repeated ultrasound post-implantation screening, which (by itself)
has a developmental abnormality detection rate above 85%, and maternal
serum screening (detection rate greater than 70% by itself). It
is self evident that the risk of developmental abnormality following
human SCNT drops dramatically, as the combination of various screening
protocols approaches a genuinely comprehensive screen. A combination
of comprehensive screening protocols (discussed in Section 5), results
in a detection rate of developmental abnormality significantly in
excess of the 90% required to reduce the risk of human SCNT (from
"50%") to below the baseline rate (3%).
summarize, the risk will never be zero, but to put this minimal
risk into context: The risk of post parturition developmental abnormality
resulting from reproductive human SCNT (when using the comprehensive
screening protocols described in this report), is significantly
less than the 3% risk of developmental abnormality newborns are
exposed to, following conception from unscreened natural fertilization.
And substantially below the risk (5% to >40% range) when the
maternal age is greater than forty five (Creasy et al 1994).
below 3%, this is still a risk of developmental abnormality. Whether
this minimal risk is acceptable or not, is the decision of the infertile
light of the aforementioned evidence, literature and screening protocols,
it is the recommendation of this report that human somatic cell
nuclear transfer be permitted as a treatment for infertility; on
condition that informed patient consent is obtained, and nuclear
transplant embryos are comprehensively screened for morphological,
chromosomal and genetic abnormalities. In addition, the patient
should be made aware of the current relatively low probability of
success and risks involved. An infertile patient/couple may have
to undergo multiple cycles before obtaining a genetically and chromosomally
normal embryo, that passes the various screening protocol checks.
However, certain infertile individuals and couples have no other
option to conceive a genetically related child, and for them this
novel reproductive technology is considered a necessity.
6.5. Concluding Remark.
report calculates  that properly screened therapeutic human
cloning is safer  than natural sexual reproduction. To ban therapeutic
human cloning for infertile couples on safety grounds, suggests
that procreation via sexual reproduction should also be banned on
safety grounds. The latter situation is obviously ridiculous. Screened
therapeutic human cloning is "reasonably safe" and offers
infertile individuals/couples the choice of conceiving a biologically
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Announced at: www.aia-zavos.com
This news article by Gina Kolata, was published in the New York
Times on March the 25th 2001. The one novel aspect of this news
article was that a certain proportion of cloned mice had lower then
normal metabolisms, and thus had a predisposition to put on weight.
Without Professor Yanagimachi's actual results, the authors can
not make a informed decision as to the validity of these statements.
However, incomplete reprogramming may very possibly result in developmental
abnormalities that include reduced metabolism, which re-emphasizes
the requirement to screen nuclear transfer embryos with protocols
discussed in this report. The authors would also like to note that
"Dolly" the sheep also put on excess weight, whether from
overfeeding or a low metabolism is unclear; but when placed on a
diet she lost that excess weight. However, this does not detract
from the fact that comprehensive pre and post implantation morphological,
chromosomal and genetic screening, is fundamentally required to
reduce the incidence of developmental abnormalities to levels below
the baseline incidence rate of 3% (from procreation via natural
sexual reproduction). [R. Moorgate. March 25th 2001]
"...if the [constitutional] right of privacy means anything,
it is the right of the individual, married or single, to be free
from unwarranted governmental intrusion into matters so fundamentally
affecting a person, as the decision whether to bear or beget a child."
(Reference: The Supreme Court of the United States of America; EISENSTADT
V. BAIRD, 405 U.S. 438, 1971) also the Supreme Court have declared
that the right to "have offspring" was a fundamental constitutional
right (SKINNER V. OKLAHOMA, 316 U.S. 535, 1942).
Please refer to general IVF and embryology textbooks such as Elder
et al 2000
Research by Professor J. B. Gurdon in the 1960's and 1970's (Review;
From Lee Silver's 1998 book: "Remaking Eden: How genetic engineering
and cloning will transform the American family." ISBN: 0380792435
Personal communication with members of Dr. A. Surani's lab.
Many IVF cows and sheep have been affected with LOS, which illustrates
that much of the problem is with the culture conditions, rather
than the NT process itself.
OHSU Fertility Consultants 2000
Online Resource: www.ich.ucl.ac.uk
Personal communication with Dr. S. Simonson
Online Resource: www.gene-chips.com
Online Resource: www.protocol-online.net
Personal communication with Dr. S. Simonson
Online Resource: www.geneimprint.com
Online Resource: www.genetests.org
This procedure was performed by Dr. Jerry Hall in 1993, but a disciplinary
action by the university involved destroying all of her research
records and results.
Online Resource: www.asrm.org
Online Resource: www.ich.ucl.ac.uk
This is based on past mammalian somatic cell nuclear transfer results,
and the combined detection rates of the various screening protocols
discussed in this report. It should be noted for screened therapeutic
human cloning to be considered "safer" than unscreened
natural sexual reproduction, the combined detection rate for developmental
abnormality had to be over 90%. With comprehensive screening, this
is certainly the case.
"Safety" being defined as the risk of developmental abnormality
in the post parturition offspring.
P. Zavos (email@example.com)
R. Moorgate (firstname.lastname@example.org).
draft submitted: March 25th 2001