University Faculty for Life Letter of Concern Re the Weldon/Brownback Human Cloning Bills

Endnotes:

1  Keith Moore and T.V.N. Persaud, The Developing Human: Clinically Oriented Embryology (6th ed. only) (Philadelphia: W.B. Saunders Company, 1998), p. 18: "Human development is a continuous process that begins when an oocyte (ovum) from a female is fertilized by a sperm (or spermatozoon) from a male. (p. 2); ibid.: ... but the embryo begins to develop as soon as the oocyte is fertilized. (p. 2); ibid.: Zygote: this cell results from the union of an oocyte and a sperm. A zygote is the beginning of a new human being (i.e., an embryo). (p. 2); ibid.: Human development begins at fertilization, the process during which a male gamete or sperm ... unites with a female gamete or oocyte ... to form a single cell called a zygote. This highly specialized, totipotent cell marks the beginning of each of us as a unique individual."

William J. Larsen, Essentials of Human Embryology (New York: Churchill Livingstone, 1998), p. 17: "Human embryos begin development following the fusion of definitive male and female gametes during fertilization" (p. 1); ibid.: ... "These pronuclei fuse with each other to produce the single, diploid, 2N nucleus of the fertilized zygote. This moment of zygote formation may be taken as the beginning or zero time point of embryonic development."

Ronan O'Rahilly and Fabiola Muller, Human Embryology & Teratology (New York: Wiley-Liss, 1994), pp. 5, 19, 55: "Fertilization is an important landmark because, under ordinary circumstances, a new, genetically distinct human organism is thereby formed. (p. 5); ibid.: Fertilization is the procession of events that begins when a spermatozoon makes contact with a secondary oocyte or its investments ... (p. 19); ibid.: The zygote ... is a unicellular embryo." (p. 19); ibid: "The ill-defined and inaccurate term pre-embryo, which includes the embryonic disc, is said either to end with the appearance of the primitive streak or ... to include neurulation. The term is not used in this book." (p. 55). [Back]

2  Ronan O'Rahilly and Fabiola Muller, Human Embryology & Teratology (New York: Wiley-Liss, 1994), p. 55: ÒPrenatal life is conveniently divided into two phases: the embryonic and the fetal. The embryonic period proper during which the vast majority of the named structures of the body appear, occupies the first 8 postovulatory weeks. ... [T]he fetal period extends from 8 weeks to birth ...

Bruce M. Carlson, Human Embryology & Developmental Biology (St. Louis, MO: Mosby, 1999), p. 447: After the eighth week of pregnancy the period of organogenesis (embryonic period) is largely completed, and the fetal period begins." [Back]

3 Bruce M. Carlson, Human Embryology & Developmental Biology (St. Louis, MO: Mosby, 1999), p. 2: "Human pregnancy begins with the fusion of an egg and a sperm. ... finally, the fertilized egg, now properly called an embryo, must make its way into the uterus ...." [Back]

4 Ronan O'Rahilly and Fabiola Muller, Human Embryology & Teratology (New York: Wiley-Liss, 1994), p. 3. [Back]

5 See, for example, Prof. Dr. Mithhat Erenus, "Embryo Multiplication" http://www.hekim.net/~erenus/20002001/asistedreproduction/micromanipulation/embryo_multiplication.htm: "In such cases, patients may benefit from embryo multiplication, as discussed in the study by Massey and co-workers. ... Since each early embryonic cell is totipotent (i.e., has the ability to develop and produce a normal adult), embryo multiplication is technically possible. Experiments in this area began as early as 1894, when the totipotency of echinoderm embryonic cells was reported ... In humans, removal of less than half of the cells from an embryo have been documented. No adverse effects were reported when an eighth to a quarter of the blastomeres were removed from an embryo on day 3 after insemination. ... Further evidence supporting the viability and growth of partial human embryos is provided by cryopreservation. After thawing four-cell embryos, some cells may not survive, leaving one-, two-, or three-cell embryos. These partial embryos survive and go to term, but at a lower rate than whole embryos. ... Based on the results observed in lower order mammals, the critical period of development to ensure success in separating human blastomeres should be at the time of embryonic gene expression, which is reported in humans to be between the four- and eight-cell stages. .... The second potential method of embryo multiplication is blastocyst splitting. ... Embryo multiplication by nuclear transfer has been used in experimental cattle breeding programs. ... IVF clinics routinely replace multiple (three to four) embryos into the uterus to increase the chances of a successful pregnancy. For couples who have less than three quality embryos for transfer, blastomere separation could be of benefit." [Back]

6  Ronan O'Rahilly and Fabiola Muller, Human Embryology & Teratology (New York: Wiley-Liss, 1994), p. 20: "In vitro fertilization involves the removal of an oocyte from an ovary under negative pressure, its culture and fertilization, and transfer of the embryo to the uterus. Successful IVF (Steptoe and Edwards) began with oocyte recovery, in vitro fertilization and culture, transfer of an 8-cell embryo to the uterus, and the birth of a girl in 1978. The various types of new reproductive technology, however, have important ethical, legal, and social implications that are under constant discussion.

"IVF involves the following steps. Ovarian hyper-stimulation is achieved by hormonal administration, usually hMG to induce follicular growth and hCG to encourage ovulation, so that a number of ovarian follicles develop for retrieval of pre-ovulatory oocytes. Follicular development is monitored by biochemical procedures (serum estradiol level) and ultrasound (to determine follicular size and position). Pre-ovulatory follicles are aspirated through the abdominal wall (laparoscopy) by direct visualization of the ovaries, or through the vagina (transvaginal) or through the bladder (transvesical ultrasound-directed oocyte recovery). The aspirated follicular fluid is then examined for the presence of oocytes, which are cultured. Insemination by the addition of numerous spermatozoa in vitro may result in fertilization and early development of embryos. The embryos are then cultured and transferred (ET). This involves the placement by catheter of several 1- to 16-cell embryos or (preferably) blastocysts in the fundus of the uterus, where implantation may occur in a relatively small number of instances. A higher rate is obtained by transferring an embryo to a uterine tube (zygote intratubal transfer) rather than to the uterus, or (as an alternative to IVF) by transferring gametes to a uterine tube (gamete intratubal transfer) and allowing intratubal fertilization to occur."

Bruce Carlson, Human Embryology & Developmental Biology (St. Louis, MO: Mosby, 1999), 2nd ed., p. 35: "... The embryos are usually allowed to develop to the two- to eight-cell stage before they are considered ready to implant into the uterus."

Keith Moore and T.V.N. Persaud, The Developing Human: Clinically Oriented Embryology (Philadelphia: W.B. Saunders, 1998), 6th ed. only, p. 39: "Successful transfer of four- to eight-cell embryos and blastocysts to the uterus after thawing is now a common practice (Fugger et al., 1991) ... Embryos and blastocysts resulting from in vitro fertilization can be preserved for long periods by freezing them with a cryoprotectant (e.g., glycerol)." [Back]

7  Ronan O'Rahilly and Fabiola Muller, Human Embryology & Teratology (New York: Wiley-Liss, 1994, p. 23: " ... The embryo enters the uterine cavity after half a week, when probably at least 8-12 cells are present and when the endometrium is early in its secretory phase (which corresponds to the luteal phase of the ovarian cycle). Each cell (blastomere) is considered to be still totipotent (capable, on isolation, of forming a complete embryo), and separations of these early cells is believed to account for one-third of cases of monozygotic twinning."

Bruce Carlson, Human Embryology & Developmental Biology (St. Louis, MO: Mosby, 1999), 2nd ed., pp. 44-49: "Early mammalian embryogenesis is considered to be a highly regulative process. Regulation is the ability of an embryo or an organ primordium to produce a normal structure if parts have been removed or added. [Note at bottom of page: Opposed to regulative development is mosaic development, which is characterized by the inability to compensate for defects or to integrate extra cells into a unified whole. In a mosaic system, the fates of cells are rigidly determined, and removal of cells results in an embryo or a structure that is missing the components that the removed cells were destined to form. Most regulative systems have an increasing tendency to exhibit mosaic properties as development progresses]. At the cellular level, it means that the fates of cells in a regulative system are not irretrievably fixed and that the cells can still respond to environmental cues. Because the assignment of blastomeres into different cell lineages is one of the principal features of mammalian development, identifying the environmental factors that are involved is important.

"Of the experimental techniques used to demonstrate regulative properties of early embryos, the simplest is to separate the blastomeres of early cleavage-stage embryos ad determine whether each one can give rise to an entire embryo. This method has been used to demonstrate that single blastomeres, from two- and sometimes four-cell embryos can form normal embryos, ....". (p. 44)

"Fate mapping experiments are important in embryology because they allow one to follow the pathways along which a particular cell can differentiate. Fate mapping experiments, which involve different isozymes of the enzyme glucose phosphate isomerase, have shown that all blastomeres of an eight-cell mouse embryo remain totipotent; that is, they retain the ability to form any cell type in the body. Even at the 16-cell stage of cleavage, some blastomeres are capable of producing progeny that are found in both the inner cell mass and the trophoblastic lineage. (p. 45)

"Another means of demonstrating the regulative properties of early mammalian embryos is to dissociate mouse embryos into separate blastomeres and then to combine the blastomeres of two or three embryos. The combined blastomeres soon aggregate and reorganize to become a single large embryo, which then goes on to become a normal-appearing tetraparental or hexaparental mouse. By various techniques of making chimeric embryos, it is even possible to combine blastomeres to produce interspecies chimeras (e.g., a sheep-goat). (p. 45)

... "The relationship between the position of the blastomeres and their ultimate developmental fate was incorporated into the inside-outside hypothesis. The outer blastomeres ultimately differentiate into the trophoblast, whereas the inner blastomeres form the inner cell mass, from which the body of the embryo arises. Although this hypothesis has been supported by a variety of experiments, the mechanisms by which the blastomeres recognize their positions and then differentiate accordingly have remained elusive and are still little understood. If marked blastomeres from disaggregated embryos are placed on the outside of another early embryo, they typically contribute to the formation of the trophoblast. Conversely, if the same marked cells are introduced into the interior of the host embryo, they participate in formation of the inner cell mass. Outer cells in the early mammalian embryo are linked by tight and gap junctions ... Experiments of this type demonstrate that the developmental potential or potency (the types of cells that a precursor cell can form) of many cells is greater than their normal developmental fate (the types of cells that a precursor cell normally forms)." (p. 45)

... "Classic strategies for investigating developmental properties of embryos are (1) removing a part and determining the way the remainder of the embryo compensates for the loss (such experiments are called deletion experiments) and (2) adding a part and determining the way the embryo integrates the added material into its overall body plan (such experiments are called addition experiments). Although some deletion experiments have been done, the strategy of addition experiments has proved to be most fruitful in elucidating mechanisms controlling mammalian embryogenesis. (p. 46)

"Blastomere removal and addition experiments have convincingly demonstrated the regulative nature (i.e., the strong tendency for the system to be restored to wholeness) of early mammalian embryos. Such knowledge is important in understanding the reason exposure of early human embryos to unfavorable environmental influences typically results in either death or a normal embryo. (p. 46)

"One of the most powerful experimental techniques of the last two decades has been the injection of genetically or artificially labeled cells into the blastocyst cavity of a host embryo. This technique has been used to show that the added cells become normally integrated into the body of the host embryo, additional evidence of embryonic regulation. An equally powerful use of this technique has been in the study of cell lineages in the early embryo. By identifying the progeny of the injected marked cells, investigators have been able to determine the potency (the range of cell and tissue types that an embryonic cell or group of cells is capable of producing) of the donor cells." (p. 46)

"Some types of twinning represent a natural experiment that demonstrates the highly regulative nature of early human embryos, ...". (p. 48)

"Monozygotic twins and some triplets, on the other hand, are the product of one fertilized egg. They arise by the subdivision and splitting of a single embryo. Although monozygotic twins could ... arise by the splitting of a two-cell embryo, it is commonly accepted that most arise by the subdivision of the inner cell mass in a blastocyst. Because the majority of monozygotic twins are perfectly normal, the early human embryo can obviously be subdivided and each component regulated to form a normal embryo." (p. 49)

William J. Larsen, Essentials of Human Embryology (New York: Churchill Livingstone, 1998), p. 325: [Monozygotic twinning] "If the splitting occurred during cleavage -- for example, if the two blastomeres produced by the first cleavage division become separated -- the monozygotic twin blastomeres will implant separately, like dizygotic twin blastomeres, and will not share fetal membranes. Alternatively, if the twins are formed by splitting of the inner cell mass within the blastocyst, they will occupy the same chorion but will be enclosed by separate amnions and will use separate placentae, each placenta developing around the connecting stalk of its respective embryo. Finally, if the twins are formed by splitting of a bilaminar germ disc, they will occupy the same amnion." (p. 325)

Geoffrey Sher, Virginia Davis, and Jean Stoess, In Vitro Fertilization: The A.R.T. of Making Babies (copyright 1998 by authors; information by contacting Facts On File, Inc., 11 Penn Plaza, New York, NY 10001), pp. 20: "(2) the fertilized egg, which has not yet divided, is now known as a zygote; (3) the egg begins to divide and is now known as an embryo; at this point each blastomere, or cell, within the embryo is capable of developing into an identical embryo." [Back]

8  Bruce Carlson, Human Embryology & Developmental Biology (St. Louis, MO: Mosby, 1999), pp. 46-47; William J. Larsen, Human Embryology (New York: Churchill Livingstone, 1997), pp. 22-28; Benjamin Lewin (ed.), Genes III (New York: John Wiley & Sons, 1987), pp. 353-354. The use of germ-line gene "therapy" by U.S. scientists has recently been published, producing genetically altered human infants. See, Dr. David Whitehouse, "Genetically altered babies born: Mitochondria contain genes outside the cell's nucleus", BBC News Online, http://news.bbc.co.uk/hi/english/sci/tech/newsid_1312000/1312708.stm.

See also, Tom Strachan and Andrew Read, Human Molecular Genetics: Second Edition (New York: Wiley-Liss, 1999), pp. 539-541], "The ethics of human germ line therapy":

"All current gene therapy trials involve treatment for somatic tissues (somatic gene therapy). somatic gene therapy, in principle, has not raised many ethical concerns. Clearly, every effort must be made to ensure the safety of the patients, especially since the technologies being used for somatic gene therapy are still at an underdeveloped stage. However, confining the treatment to somatic cells means that the consequences of the treatment are restricted to the individual patient who has consented to this procedure. ... The same technology has the potential, of course, to alter phenotypic characters that are not associated with disease, such as height for instance. Such genetic enhancement, although not currently considered, can be expected to pose greater ethical problems; attempts to produce genetically enhanced animals have not been a success and in some cases have been spectacular failures (Gordon, 1999).

Germline gene therapy, involving the genetic modification of germline cells (e.g., in the early zygote), is considered to be entirely different. It has been successfully practiced on animals (e.g., to correct beta-thalassemia in mice). However, thus far, it has not been sanctioned for the treatment of human disorders, and approval is unlikely to be given in the near future, if ever.

Human germline gene therapy has not been practiced because of ethical concerns and limitations of the technology for germline manipulation. The lack of enthusiasm for the practice of germline gene therapy can be ascribed to three major reasons:

[a] The imperfect technology for genetic modification of the germline

Germline gene therapy requires modification of the genetic material of chromosomes, but vector systems for accomplishing this do not allow accurate control over the integration site or event. In somatic gene therapy, the only major concern about lack of control over the fate of the transferred genes is the prospect that one or more cells undergoes neoplastic transformation. However, in germline gene therapy, genetic modification has implications not just for a single cell: accidental insertion of an introduced gene or DNA fragment could result in a novel inherited pathogenic mutation.

[b] The questionable ethics of germline modification

Genetic modification of human germline cells may have consequences not just for the individual whose cells were originally altered, but also for all individuals who inherit the genetic modification in subsequent generations. Germline gene therapy would inevitably mean denial of the rights of these individuals to any choice about whether their genetic constitution should have been modified in the first place (Wivel and Walters, 1993). Some ethicists, however, have considered that the technology of germline modification will inevitably improve in the future to an acceptably high level and, provided there are adequate regulations and safeguards, there should then be no ethical objections (see, for example, Zimmerman, 1991). At a recent scientific research meeting in the USA some scientists have also come out in support of such a development (Wadman, 1998).

From the ethical point of view, an important consideration is to what extent technologies developed in an attempt to engineer the human germline could subsequently be used not to treat disease but in genetic enhancement. There are powerful arguments as to why germline gene therapy is pointless. There are serious concerns, therefore, that a hidden motive for germline gene therapy is to enable research to be done on germline manipulation with the ultimate aim of germline-based genetic enhancement. The latter could result in positive eugenics programs, whereby planned genetic modification of the germline could involve artificial selection for genes that are thought to confer advantageous traits.

The implications of human genetic enhancement are enormous. Future technological developments may make it possible to make very large alterations to the human germline by, for example, adding many novel genes using human artificial chromosomes (Grimes and Cooke, 1998). Some people consider that this could advance human evolution, possibly paving the way for a new species, homo sapientissimus. To have any impact on evolution, however, genetic enhancement would need to be operated on an unfeasibly large scale (Gordon, 1999).

Even if positive eugenics programs were judged to be acceptable in principle and genetic enhancement were to be practiced on a small scale, there are extremely serious ethical concerns. Who decides what traits are advantageous? Who decides how such programs will be carried out? Will the people selected to have their germlines altered be chosen on their ability to pay? How can we ensure that it will not lead to discrimination against individuals? Previous negative eugenics programs serve as a cautionary reminder. In the recent past, for example, there have been horrifying eugenics programs in Nazi Germany, and also in many states of the USA where compulsory sterilization of individuals adjudged to be feeble-minded was practiced well into the present century.

[c] The questionable need for germline gene therapy Germline genetic modification may be considered as a possible way of avoiding what would otherwise be the certain inheritance of a known harmful mutation. However, how often does this situation arise and how easy would it be to intervene? A 100% chance of inheriting a harmful mutation could most likely occur in two ways. One is when an affected woman is homoplasmic for a harmful mutation in the mitochondrial genome and wished to have a child. The trouble here is that, because of the multiple mitochondrial DNA molecules involved, gene therapy for such disorders is difficult to devise.

A second situation concerns inheritance of mutations in the nuclear genome. To have a 100% risk of inheriting a harmful mutation would require mating between a man and a woman both of whom have the same recessively inherited disease, an extremely rare occurrence. Instead, the vast majority of mutations in the nuclear genome are inherited with at most a 50% risk (for dominantly inherited disorders) or a 25% risk (for recessively inherited disorders). In vitro fertilization provides the most accessible way of modifying the germline. However, if the chance that any one zygote is normal is as high as 50 or 75%, gene transfer into an unscreened fertilized egg which may well be normal would be unacceptable: the procedure would inevitably carry some risk, even if the safety of the techniques for germline gene transfer improves markedly in the future. Thus, screening using sensitive PCR-based techniques would be required to identify a fertilized egg with the harmful mutation. Inevitably, the same procedure can be used to identify fertilized eggs that lack the harmful mutation. Since in vitro fertilization generally involves the production of several fertilized eggs, it would be much simpler to screen for normal eggs and select these for implantation, rather than to attempt genetic modification of fertilized eggs identified as carrying the harmful mutation." [Back]

9 Benjamin Lewin (ed.), Genes VII (New York: Oxford University Press, pp. 81-86, 174-175, 546; Tom Strachan and Andrew Read, Human Molecular Genetics: Second Edition (New York: Wiley-Liss, 1999), pp. 60, 140-141. [Back]

10  William J. Larsen, Essentials of Human Embryology (New York: Churchill Livingstone, 1998), p. 4: "like all normal somatic (non-germ- cells), the primordial germ cells contain 23 pairs of chromosomes, or a total of 46."

Bruce Carlson, Human Embryology & Developmental Biology (St. Louis, MO: Mosby, 1999), p.2: "In a mitotic division, each germ cell produces two diploid progeny that are genetically equal."

Tom Strachan and Andrew Read, Human Molecular Genetics: Second Edition (New York: Wiley-Liss, 1999), p. 28"A subset of the diploid body cells constitute the term line. These give rise to specialized diploid cells in the ovary and testis that can divide by meiosis to produce haploid gametes (sperm and egg). ... The other cells of the body, apart from the germline, are known as somatic cells ... most somatic cells are diploid ...".

Ronan O'Rahilly and Fabiola Muller, Human Embryology & Teratology (New York: Wiley-Liss, 1994), pp. 13-14: "Gametogenesis is the production of germ cells (gametes), i.e., spermatozoa and oocytes. These cells are produced in the gonads, i.e., the testes and ovaries respectively. The gametes are believed to arise by successive divisions from a distinct line of cells (the germ plasm), and the cells that are not directly concerned with gametogenesis are termed somatic. ... The reduction of chromosomal number from 46 (the diploid number) to 23 (the haploid number) is accomplished by a cellular division termed meiosis. ... Primordial germ cells ... are difficult to recognize in very young human embryos. Claims for them have been made as early as in the blastocyst, and they are believed to be segregated at latest by 2 weeks and possibly much earlier."

Keith Moore and T.V.N. Persaud, The Developing Human: Clinically Oriented Embryology, (Philadelphia, PA: W.B. Saunders Company, 1998), 6th ed. only, p. 18: "Meiosis is a special type of cell division that involves two meiotic cell divisions; it takes place in germ cells only. Diploid germ cells give rise to haploid gametes (sperms and oocytes). [Back]

11 Tom Strachan and Andrew Read, Human Molecular Genetics: Second Edition (New York: Wiley-Liss, 1999), p. 541: " There are powerful arguments as to why germline gene therapy is pointless. There are serious concerns, therefore, that a hidden motive for germline gene therapy is to enable research to be done on germline manipulation with the ultimate aim of germline-based genetic enhancement. The latter could result in positive eugenics programs, whereby planned genetic modification of the germline could involve artificial selection for genes that are thought to confer advantageous traits." [Back]

12  Back-breeding has been done "naturally" for centuries by farmers using plants and animals. See discussions of back-breeding in, e.g.: http://www.aces.edu/department/extcomm/publications/he/he-728/he-728.htm, HE-728, New June 1996. Jean Olds Weese, Extension Food Science Specialist, Associate Professor, Nutrition and Foods, Auburn University: "Farmers have been trying for years to improve crop yields from both plants and animals. This has been accomplished through cross-breeding in animals and grafting in plants. Through this selective mating the plant or animal receives the genes from another plant or animal that will improve a trait in that species. The problem with this is that along with this one good trait comes several traits that may not be so positive. This is because plants have more than 100,000 genes and with each gene comes a character trait. After cross-breeding two plants, the farmer must then go through back-breeding to keep the good traits and breed out the bad traits."

http://www.cmf.gc.ca/en/cf/1998/vol3/html/1998fca22348.p.en.html, president and Fellows of Harvard College v. Canada (Commissioner of Patents): "What is involved here is the insertion of the myc gene and the subsequent breeding, cross-breeding and back-breeding of a mouse. ... The inventors were able to "backcross" and in-breed in order to obtain offspring with more widely varying sites of the new myc gene. ... As described by the Commissioner, 'analysis of the DNA of the resulting transgenic offspring indicated that the injected oncogene was transmitted through the germline in a ratio consistent with Mendelian inheritance of single loci.' In other words, the transgene will be passed on to the offspring in accordance with the ordinary principles of inheritance."

http://www.biology.iupui.edu/biocourses/K338/338_14.html, Cogenic MHC Mouse Strains: "Syngenic (mice with all identical locus) Cogenic (mice with all identical locus, except a single genetic locus) Cogenic mice are isolated by inter-breeding, selection, back-breeding for at least 12 generations." [Back]

13 See, e.g., Albert R. Jonsen, The Birth of Bioethics (New York: Oxford University Press, 1998), pp. 342, esp. Chapter 4, "Commissioning Bioethics: The Government in Bioethics, 1974-1983; David J. Rothman, Strangers at the Bedside: A History of How Law and Bioethics Transformed Medical Decision Making (New York: BasicBooks/Perseus Books, L.L.D., 1991), esp. Chapter 9, "Commissioning Ethics"; Dianne Nutwell Irving, "What is 'bioethics'?", in Joseph W. Koterski (ed.), University Faculty For Life: Proceedings of the Conference 2000 (in press). [Back]

14 Albert R. Jonsen, The Birth of Bioethics, ibid., pp. 109-110. [Back]

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