TO CLONE OR
NOT TO CLONE?
WHOSE CHOICE IS IT
ANYWAY?
Professor Dr. Panos Zavos, Ed.S., Ph.D.
Professor Emeritus of Reproductive
Physiology/Andrology, University of Kentucky
Director, Andrology Institute of America
Associate Director, Kentucky Center for Reproductive
Medicine & IVF, Lexington, KY, USA
Executive Director, The Zavos Organization
Introduction

Human reproductive
cloning today continues
to preoccupy the
general public and its
critics in a very
controversial manner.
There is also some
public hostility directed
against it.
“Public hostility to human
reproductive cloning may be
based on an illogical transient
fear of a new technology”
The British Medical Association
Infertility is a disease
Today, infertility is a
disease that reaches
epidemic proportions
throughout the
developing World
In Vitro Fertilization (IVF)



Low sperm count
and/or motility
Variety of female
factors
Success rate ~ 33%
live birth/transfer
Did not overcome severe asthenospermia
Intra Cytoplasmic Sperm
Injection (ICSI)


If patient has low
sperm count or having
sperm with no motility
Success rate ~ 32%
live birth/transfer
Required presence of sperm in the ejaculate
Sperm and Oocyte Donation


No mature sperm present
No oocyte (egg) production
Offspring not genetically or biologically related
Quotes from childless patients
 “..we want a child
(yesterday, if possible)
and a healthy child.”
 “..do not want to have
another person’s sperm
or eggs.”
 “..what other options do
we have?”
 “…we want to have a
biological child of our
own.”
(Received via e-mail from patients)
How Therapeutic Cloning Works
1. First, the nucleus of a donor egg
is removed.
2. Then a whole somatic cell or the
nucleus of a whole somatic cell
from a patient is inserted.
3. The result is an egg with the
patient's genetic material.
4. The egg is then induced (“jump
start”) to divide and become an
embryo which grows into
several stem cells, all of which
are genetically identical to the
donor cell.
Sexual and Asexual Reproduction
No difference in the type of
oocyte (egg) used
Sexual and Asexual Reproduction
Fusion of male
and female
genetic
material
(pronuclei)
Electrofusion of somatic
cell that carries ONLY
the male of female
genetic material to the
“oocyte”
Sexual and Asexual Reproduction
No difference in the cell division
stages after fertilization
Cloning in Animals

Various species have
been used as
biological models for
this effort but
extensive research on
somatic cell nuclear
transfer (SCNT) has
been performed using
the bovine model.
Our Studies

In our studies we set out to examine the
ability of the bovine metaphase oocyte
cytoplasm to support mitotic cell cycles
under the direction of differentiated
somatic cell nuclei of human granulosa
cells and fibroblast cells in order to test the
efficiency of our SCNT techniques.

Hybrid embryos
Materials and Methods

Bovine oocytes
were randomly
treated either for
induction of
parthenogenesis or
for enucleation and
SCNT using either
human granulosa
cells or human
fibroblast cells.
Materials and Methods

Bovine oocytes
were enucleated
by aspiration of
the first polar
body and the
metaphase plate.
Materials and Methods

Human granulosa
cells and fibroblast
cells were
aspirated into a
micropipette.
Materials and Methods

One human
granulosa cell or
fibroblast cell was
injected into the
perivitelline space of
each of the
enucleated bovine
oocytes.
Injecting the cell
Cell placed subzonally
Materials and Methods


Treated oocytes were
evaluated for
evidence of cleavage
and embryonic
development daily.
Embryo quality was
assessed using similar
grading criteria to
those employed in
human IVF.
Results
Experiment 1
Success
rate1
CEI 3
1Number
Granulosa
cells
Controls 2
31.2%
(15/48)
46.8%
(36/77)
66%
Experiment 2
Fibroblast Controls 2
cells
24.2 %
(15/62)
56.8%
(21/37)
42%
of embryos developed from the total number of oocytes;
2Parthenogenetic development;
3CEI: Cloning Efficiency Index=Embryo success rate/ Parthenogenetic
success rate X100
Conclusions


Our results point out that
SCNT as applied in the current
study fusing human granulosa
cells or fibroblast cells can be
done.
The technique could be quite
sensitive and predictive for
similar SCNT attempts in
humans for therapeutic or
reproductive purposes.
First Human Cloned Embryo

This technology has
enabled us to create
the first human
cloned embryo for
reproductive
purposes.
Zavos PM: Human reproductive cloning: the time is near.
Reproductive BioMedicine Online 6, 397–398, 2003.
First Human Cloned Embryo


Nine human oocytes
were enucleated.
Fused with whole
human granulosa cells
via electrical stimulation
and activation.
First Human Cloned Embryo

The resulting
cloned embryo
reached the 8-10
cell stage and
cryopreserved for
future molecular
analysis.
ANNOUNCEMENT
The First Fresh Cloned Embryo
Transfer in Human
 We have produced and transferred the
first fresh cloned embryo into the
mother and we are awaiting for results
 The mother is a 35 year-old woman
 The embryo was properly
evaluated and transferred
at a 4-cell stage
 No pregnancy was established
Difficulties noted by
“Animal Cloners”
 Poor cloning response.
 Poor implantation and pregnancy ratio.
 Poor health of animals born.
Those difficulties are due to:
 Poorly designed experiments.
(few animals used with no definite objective)
 Poorly executed experiments.
(hit and miss type of research)
 Poorly approached experiments.
(done under non-sterile and uncontrolled
environments)
 Poorly understood and interpreted.
(when animals died, no clear view of their
cause of death)
SOME DONE FOR FAME AND FORTUNE BY
THE ANIMAL CLONERS!
Ethics Morality & Hypocrisy
LET US EXAMINE THE
FACTS AS THEY APPEAR
WITH THE ANIMAL
CLONERS!
Hypocrisy in Action
“Animal cloning is inefficient and
is likely to remain so for the
foreseeable future”
(by Wilmut & Jaenisch, Time, 2001)
A number of studies have already
demonstrated far higher rates of
development, as measured in the proportion
of live births to the number of embryos
transferred, and in some cases matching or
exceeding developmental rates seen in
human IVF.
Nuclear-cytoplasmic interaction and
development of goat embryos
reconstructed by nuclear
transplantation: production of goats
by serially cloning embryos
(Yong and Yuqiang; Biol. Reprod. 58: 266-269, 1998)
Embryos created*
141
Live births
45
Success Rate (%)
32%**
*Re-cloned goat embryos from a previous cloning
procedure (serially cloned embryos)
**Similar to Current Human IVF Success Rates
Eight calves cloned from somatic
cells of a single adult
(Kato et al, Science, 282: 2095-8, 1998)
Embryos created
10
Live births
8
Success Rate (%)
80%
Excerpts from the Congressional
Hearings on Human Cloning*
• “Dolly is not normal. Dolly is
overweight.” (Jaenisch, 2001)
• “Dolly may have subtle defects like
in the brain. Dolly, I believe, is not
normal. …. we have no tests to
check that.” (Jaenisch, 2001)
*Under oath at the Congressional Hearing on Human Cloning
Research, March 28, 2001
Excerpts from President Bush’s
talk on Human Cloning
• “Scientists wanting to do
human cloning are going to
create humans for spare parts
and I am against that”
(George W. Bush, President of the USA)
Excerpts from the Oxford Union
Debate
• “You should be ashamed of yourself
for wanting to clone a human being.
You are going to create human
monsters and you will fail.”
(Robert Winston, June 5, 2001)
Ironically, he said the exact same things
about Robert Edwards 26 years ago about
his efforts to create the first human being
via IVF. Today, Winston embraced IVF and
he is known in the UK as Mr. IVF.
That is hypocritical!!
Excerpts from the Oxford Union
Debate
• “You should be ashamed of
yourself for experimenting and
killing human embryos.”
(Harry Griffin, Roslyn Institute, June 5,
2001)
Today, Mr. Griffin is given a license by the
British Government and HFEA to kill
human embryos and extract stem cells.
That is highly hypocritical!!!
Human-Bovine Hybrid Embryos
Created


In order to avoid “killing
human embryos” we have
created the Human-Bovine
hybrid embryo model to
study various phenomena
that needed to be
evaluated during SCNT.
What has Mr. Griffin
done to avoid killing
human embryos?
Excerpts from the National
Academy of Sciences
• “Animal cloning is
inefficient and is likely to
remain so for the
foreseeable future”
(Ian Wilmut, Roslyn Institute, August 2001)
Today the success is tremendous!!
They continue however to misrepresent the
facts!
Excerpts from the National
Academy of Sciences
• “Cloning will never be perfected and
applied for human or animal
purposes. It is impossible to
reprogram 35,000 genes present in
the human genome and yield a
healthy human being.”
(Rudolph Jaenisch, MIT Professor, August 2001)
Today we are cloning cattle commercially
with 83% success rate; astonishing!!
Another “Expert’s” Opinion on
Human Cloning
“….in monkeys the removal of the egg
nucleus also removes what Schatten called
"molecular motors" that are responsible for
separating chromosomes during cell
division.
He explained. "The cells that result after
those cell divisions all have the wrong
number of chromosomes."
“We cannot do it in monkeys and
therefore it cannot be done in humans”
Humans may be easier to
clone than animals!
(August, 2001)
“This is the first concrete genetic data showing
that the cloning process could be less
complicated in humans than in sheep”
Keith Killian, Duke University Medical Center
“…our data show that you don’t necessarily have
these problems (with the large offspring
syndrome) in humans.” Randy Jirtle, Duke researcher
Response to Open Letter to
British News Editors by
“leading UK scientists”

“to reconsider the prominence given to
repeated claims by certain scientists that
they have cloned a human being,
including those made by Dr. Panos Zavos
last weekend.” (21st January 2004)
We have NEVER claimed to have
cloned a human being!
Response to Open Letter to
British News Editors by
“leading UK scientists”

“none of those involved have produced
a shred of evidence to substantiate their
assertions .” (21st January 2004)
We have published and continue to publish in
peer-reviewed scientific journals!
It appears that these “leading scientists” do
not do their homework nor READ and get
informed before they offer an opinion.
Publications




Zavos PM: Human reproductive cloning: the time is near.
Reproductive BioMedicine Online 6, 397–398, 2003.
Illmensee K, Levanduski M, Zavos PM: Development of an
interspecies-specific bioassay using the bovine oocyte model
to evaluate the potential of SCNT in humans. Journal of
Assisted Reproduction and Genetics, 2004 (Accepted, in
press, withdrawn due to leading UK scientists pressure).
Zavos PM, Illmensee K: First Embryo Transfer of a Cloned
Human Embryo. Middle East Fertility Society Journal
(Submitted for publication).
Zavos PM, Illmensee K: Human Reproductive Cloning: The
Post Mortem Effort. (Currently in preparation).
Scientific Presentations







The American Society for Reproductive Medicine, San Antonio,
Texas, October 11-15, 2003.
The Austrian Society of Reproductive Medicine and
Endocrinology, Bregenz, Austria, October 17-18, 2003.
The Middle East Fertility Society, Beirut, December 10-13, 2003.
The 12th World Congress on Human Reproduction, Venice, Italy,
March 14-16, 2005
The World DNA and Genome Day, Dalian, China, April 25-30,
2005.
The Indian International Conference on Update in Infertility,
Bangalore, India, April 25-May 1, 2005.
The American Society for Reproductive Medicine and the
Canadian Fertility and Andrology Society, Montreal, Quebec,
Canada, October 15-19, 2005 (Submitted).
“HFEA grants the first
therapeutic cloning license for
research”
(11 August 2004)
Is it an act of God?
HFEA purposes



Increasing knowledge about the development
of embryos
Increasing knowledge about serious disease
Enabling any such knowledge to be applied in
developing treatments for serious disease
After destroying the embryo and
its potential for life
Therapeutic vs Reproductive
Cloning in the UK

Human reproductive cloning is illegal in the
UK. As a result of the Human Reproductive
Cloning Act (2001) nobody in the UK is
allowed to use cell nuclear replacement, or
any other technique, to create a child.
But Therapeutic Cloning for Stem Cell
Research is allowed
Therapeutic Cloning & Stem Cell
Research vs Reproductive Cloning
 Stem cells from Living  Somatic (body) cells
Human Embryos
Inject into enucleated
 Killing &
oocytes
Dismembering
 Induce embryo
Human Embryos
development
 Grow stem cells in
 Used to create healthy
culture
babies for childless
 Used in treatment of
couples and treating
various diseases
infertility
The Future of Cloning
 Further elucidation of the molecular
mechanisms involved during the processes
of embryogenesis
 Careful tailoring of subsequently
developed culture conditions and
manipulation strategies
 Appropriate screening methods
will eventually allow infertile couples to
safely have healthy, genetically related
children through Somatic Cell Nuclear
Transfer (SCNT) technology
This technology should be
developed by scientists and
medical experts that:
 understand this type of work and the
seriousness for its development
 should be focused on carrying out this
project, and
 should work with leaders and
governments at the International level,
to ensure that this technology can be
made safe and be disseminated
properly
We intend to develop this
technology by:
 Selecting appropriate cell lines for SCNT
 Proper reprogramming the cell
lines in tissue culture
prior to SCNT
 Screening the cloned embryos
prior to embryo transfer
into recipients
 Monitoring the ongoing
pregnancies from the cloned embryos
Vilified, ridiculed, accused of
perverting nature….
But the cloning pioneers are in
good company
Sunday Herald, Glasgow, Scotland,
10/21/01
The Future of Reproductive
Cloning
“Cloning, too, will
probably come to be
accepted as a
reproductive tool if it
is carefully
controlled”
Professor Robert Edwards, 2001
Sunday Herald, Glasgow, Scotland, 10/21/01