The Zavos Organization

 

 

The House Subcommittee on Oversight and Investigation
Wednesday, March 28, 2001,
Rayburn House Office Building, Room 2123

Hearing on Issues Raised by Human Cloning Research

Click here for a transcipt of the Hearing (TXT file, 628K)
Click here for a transcipt of the Hearing (PDF file, 556K)


 

Dr Zavos at Congressional Hearing
Dr. 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)

Testimony before the House Subcommittee on Oversight and Investigation; Hearing on Issues Raised by Human Cloning Research

Copyright © 2001 Dr. P. Zavos.
Reproduced by the Zavos Organization with permission from Dr. Zavos.

Report Author:
Professor, Dr. Panayiotis Zavos, Ed.S., Ph.D.,
Director of the Andrology Institute of America,
Associate Director of the Kentucky Center for Reproductive Medicine & IVF
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
Website: www.zavos.org
E-mail: zavos@zavos.org

Wednesday, March 28, 2001
Room 2123 Rayburn Office Building
Washington D.C.

Contents

Introduction

Over 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).

Recently, 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.

I 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.

It 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.

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Current events in the ART market

With 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:

1. A child, (yesterday if possible), and

2. A healthy child.

These 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 medicine.

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Current status of Animal Cloning

A 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 are effective.

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Current status of Human Cloning

Although 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:

1. Who should develop this technology, and

2. 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 effort.

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Who should develop this technology?

The 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.

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What quality controls are necessary?

As 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.

On 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).

For this committee's benefit, I would like to make the following comments before I proceed further:

1. 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 this Congress.

2. "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.

3. 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.

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Closing Remarks

Those 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 technology.

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Copyright © 2001 Dr. P. Zavos.


EXHIBIT 1

PANAYIOTIS MICHAEL ZAVOS, Ed.S., Ph.D.

A SHORT BIOGRAPHY

Dr ZavosBorn 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.

Dr. 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.

Dr. 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 groups.

Dr. 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 and exchanges.

Dr. 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.

Dr. 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:

  1. Founder, The Zavos Organization, www.zavos.org
  2. 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
  3. Founder, Director and Chief Andrologist of the Andrology Institute of America, www.aia-zavos.com
  4. Founder and Executive Director of the Home Fertility Network, www.homefertility.com
  5. Founder, Semen Tests, for "Online" Semen Analysis, www.sementests.com
  6. Co-Founder and Associate Director of the Greek-American Andrology Institute of Athens, Greece
  7. Professor Emeritus of Reproductive Physiology-Andrology at the University of Kentucky, in Lexington, KY, USA
  8. Honorary Professor, China Academy of Science

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EXHIBIT 2

COMMITTEE OF SCIENTISTS FOR SAFE AND RESPONSIBLE THERAPEUTIC HUMAN CLONING

By Dr. Panayiotis Zavos et al
(e-mail: drz@aia-zavos.com)


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.


Report Authors:

A collaborative effort of developmental biologists and infertility specialists, lead by:

Professor, Dr. Panayiotis Zavos, Ed.S., Ph.D.,
Director of the Andrology Institute of America,
Associate Director of the Kentucky Center for Reproductive Medicine & IVF
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
Website: www.zavos.org
E-mail: zavos@zavos.org

Contents

1. Introduction

"...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."

(The Supreme Court of the United States of America (1971) Eisenstadt v. Baird. 405 U.S. 438)

A 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 [1]. 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.

2. The possible developmental abnormalities following human SCNT

Mammalian 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 conception.

First natural sexual reproduction shall be examined:

"1 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 abnormalities include:

Heart defects; single ventricle, truncus arteriosus, tetralogy of fallot, transposition of the great vessels.

Gastro-intestinal and respiratory defects; small intestine atresia, tracheal-esophageal fistula, colorectal/anal atresis, cleft lip/palate, general structural respiratory defects.

Genito-urinary defects; urinary tract obstruction, renal agenesis/dysgenesis.

Musculoskeletal defects; gastroschisis, omphalocele, lower and upper limb reduction defect, diaphragmatic hernia.

Central nervous system (and other) defects; spina bifida, down sydrome, cerebral palsy.

(Waitzman et al 1994)

The 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).

Using results and evidence from past mammalian research, it is possible to calculate the overall rate of developmental abnormality following SCNT.

The 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 [2]). 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):

Mammalian (bovine and porcine) SCNT results:

Dr. Wells et al 1999.

Bovine SCNT; NT micro-injection of 552 oocytes.

152 grade 1 and 2 blastocysts obtained (27.5% to G1 and G2).

100 G1 and G2 blastocysts transferred, with 10 resulting births.

1.8% overall efficiency (post-parturition offspring from oocytes injected).

Abnormalities 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)

Porcine SCNT; 483 nuclear transfers.

Independent experiments concluded that 8% (15/192) of nuclear transplants developed to G1 blastocyst. Please note that for porcine IVF this rate is 23%.

Of the transfers, four live offspring resulted.

0.83% overall efficiency (postparturition offspring from oocytes fused). This low rate is due to the difficulties in initiating porcine pregancies (Polge et al 1966)

Abnormalities 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.

(Betthauser et al 2000)

Dr. Polejaeva et al 2000 (utilizing a novel "Double NT" technique. Combined results from seven experiments). Porcine SCNT.

Oocytes injected: 1869.

Blastocysts obtained and transferred: 401 (21.5% efficiency).

Live offspring obtained: 5.

"0.27%" 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.

Abnormalities?

"All 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."

(Polejaeva et al 2000). Please note that Alan Colman contributed to the aforementioned research.

The 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).

It 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 utilized.

The 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 [3]. Screened SCNT is the only way certain infertile individuals can have a biologically related child.

"within 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."

(Lifchez 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 [4]. 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 [5].

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 [6]. 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:

It 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.

Chromosomal 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.

The 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 5.3.2.1). Common epigenetic mechanisms for "turning genes off" include linker histone acetylation, methyl-CpG binding proteins and Polycomb group proteins (Kikyo et al 2000).

Histones 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 [7].

4.3. Incompletely reprogrammed (imprinted) gene expression.

Genomic 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 [8]. 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 unscreened embryos).

5. The prenatal screening procedures requires to detect these abnormalities.

5.1. Overview of screening

The "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).

Prenatal 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

"PGD 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."[9]

As 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.

Preimplantation 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.

"Studies 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."

(Elder et al 2000)

One 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

The 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).

This 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 [10].

It 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 embryo.

This 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

To 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 [11]. 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 5.3.2.2.

5.3. Postimplantation screening.

5.3.1. PND chromosomal screening

Chromosomal screening following implantation.

The 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

5.3.2.1. Reprogrammed gene expression pattern screening.

Oligonucleotide and cDNA gene chips can be utilized to assay the mRNA expression pattern of the sampled tissue [12]. 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 [13]. 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).

Other 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 [14]. 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).

5.3.2.2. Secondary imprinted gene screening.

Igf2r is only one of over 30 imprinted mammalian genes [15]. 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

A 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.

Serum screening

Early 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 [16]. The detection rate with combined serum screening for specific chromosomal abnormalities can be as high as 70%.

Ultrasound

Ultrasound 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.

Chromosomal 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".

Standard methods for obtaining postimplantation fetal tissue samples

Chorionic 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).

The 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

No 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.

Past 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 pregnancy.

6.2. Probability of success (per cycle).

"To 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%"

(Elder et al 2000)

With 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:

1. The use of enucleated donor oocytes would significantly increase the probability of success (and also the cost) of this therapeutic human SCNT.

2. 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.

3. Another method of increasing the probability of success involves "embryo splitting" [17]. Although not yet a standard clinical procedure, this protocol has been considered ethically sound by the American Society for Reproductive Medicine [18].

The 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.

This 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" [19] 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%).

To 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).

Although below 3%, this is still a risk of developmental abnormality. Whether this minimal risk is acceptable or not, is the decision of the infertile patient/couple.

6.4. Recommendation.

In 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.

This report calculates [20] that properly screened therapeutic human cloning is safer [21] 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 related child.

(P. Zavos and R. Moorgate et al. 2001)

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8.Footnotes

[1] Announced at: www.aia-zavos.com

[2] 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]

[3] "...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).

[4] Please refer to general IVF and embryology textbooks such as Elder et al 2000

[5] Research by Professor J. B. Gurdon in the 1960's and 1970's (Review; Gurdon 1986)

[6] From Lee Silver's 1998 book: "Remaking Eden: How genetic engineering and cloning will transform the American family." ISBN: 0380792435

[7] Personal communication with members of Dr. A. Surani's lab.

[8] 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.

[9] OHSU Fertility Consultants 2000

[10] Online Resource: www.ich.ucl.ac.uk

[11] Personal communication with Dr. S. Simonson

[12] Online Resource: www.gene-chips.com

[13] Online Resource: www.protocol-online.net

[14] Personal communication with Dr. S. Simonson

[15] Online Resource: www.geneimprint.com

[16] Online Resource: www.genetests.org

[17] 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.

[18] Online Resource: www.asrm.org

[19] Online Resource: www.ich.ucl.ac.uk

[20] 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.

[21] "Safety" being defined as the risk of developmental abnormality in the post parturition offspring.

Copyright © 2001:
P. Zavos (drz@aia-zavos.com) and
R. Moorgate (rogermoorgate@hotmail.com).

All rights reserved.

Final draft submitted: March 25th 2001


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