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Abnormal in utero development of cloned animals: implications for human cloning
C Blackwell Wissenschafts-Verlag 2002
Differentiation (2002) 69:174–178
COMMENTARY
Jonathan R. Hill
Abnormal in utero development of cloned animals: implications for human cloning
Accepted in revised form: 19 November 2001
Key words human cloning ¡ in utero development ¡ somatic cells nuclear transplantation
Hundreds of animals have been cloned from animal somatic cells since the first mammalian clone, Dolly, was presented to a stunned world press in February 1997 (Wilmut et al., 1997). Although the concept of cloning or nuclear transfer (NT) was successful in amphibians in the 1950s and again in cattle, sheep and pigs (using embryonic nuclei) over the past 2 decades, Dolly was the first mammal to be reproduced from a cell taken from an adult animal (somatic cell cloning). After Dolly, in 1998 came cloned mice and cattle (Cibelli et al., 1998; Kato et al., 1998; Wakayama et al., 1998). Over the next few years goats and pigs were cloned while the numbers of cloned mice and cattle continued to increase (Table 1) (Baguisi et al., 1999; Onishi et al., 2000; Polejaeva et al., 2000). Research into mammalian cloning is driven by both commercial and biomedical research goals. The most lucrative commercial application for cloning is through its combination with genetic modification (transgenics); (Schnieke et al., 1997). This greatly improves the efficiency of transgenic animal production with the goals of harvesting human therapeutic proteins from milk or organs for transplantation. Cloning to copy elite livestock has already been commercialized, and the application to endangered animal conservation is actively being investigated. From a purely scientific viewpoint, cloning appears to be an invaluable tool in studying the nuclear reprogramming process during the first few J. R. Hill Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York, 14853, USA e-mail: jrh35/cornell.edu Tel: π1 607 253 3091, Fax: π1 607 253 3531 U. S. Copyright Clearance Center Code Statement:
hours or days after the transfer of a somatic cell into an enucleated oocyte. As yet unsuccessful attempts have been made to clone rats, rabbits, dogs, cats, horses, and monkeys. This illustrates that the technique developed by Ian Wilmut and Keith Campbell is not immediately applicable to each species, most likely due to subtle biological variations between even closely related species such as the rat and mouse. In primates, cloning with somatic cells is not yet successful, although nuclear transfer using embryonic cells has produced live offspring (Meng et al., 1997). In each of the species where somatic cell cloning has been successful, it has also been very inefficient. Early first trimester pregnancy rates are less than 1/2 that normally expected. Immediately following initial positive diagnosis of pregnancy, extraordinarily high rates of embryonic loss occur, where up to 80 % of pregnancies miscarry by the second trimester. In late gestation, placental and fetal abnormalities occur at a much higher than normal rate, and finally lowered viability at birth is common. These major developmental abnormalities alert us to the inefficiency inherent in cloning mammals. Gestational and neonatal abnormalities are consistent with irregular expression and likely incomplete reprogramming of imprinted genes (Young and Fairburn, 2000; Jaenisch and Wilmut, 2001; Rideout et al., 2001). However, a casual perusal of the lay and scientific literature may give a confusing picture of the status of cloned animals. Some well publicized reports give a more negative view of mammalian cloning by focusing attention on abnormalities in telomere length, gene expression or methylation patterns. Others focus on success and proclaim the birth of apparently normal offspring. The practical outcome is that there are many cloned animals that behave and appear normal, while closer investigations have revealed that even some of these apparently normal animals are subtly different from one another
0301–4681/2002/6904–174 $ 15.00/0
175 Table 1 Survival of mammals cloned from somatic cells. The data excludes animals that died during gestation, but prior to parturition. Data was obtained from published peer reviewed journal articles Animal
Live born
Live after 1 week
Live at puberty (% of live born)
Sheep1 Mice2 Cattle3 Goats4 Pigs5
28 126 91 17 10
15
11 88 60 12 10
1 2 3
4 5
66 14 10
(39%) (70%) (66%) (70%) (100%)
Sheep (Schnieke et al., 1997; Wilmut et al., 1997; McCreath et al. 2000; Denning et al., 2001) Mice (Wakayama et al., 1998; Wakayama and Yanagimachi 1999, 2001; Ogura et al., 2000; Zhou et al., 2000) Cattle (Cibelli et al., 1998; Kato et al., 1998, 2000; Wells et al., 1998, 1999; Shiga et al., 1999; Hill et al., 2000; Kishi et al., 2000; Kubota et al., 2000; Lanza et al., 2000; Zakhartchenko et al., 1999; Heyman et al., 2002) Goats (Baguisi et al., 1999; Keefer et al., 2001; Reggio et al. 2001; Zou et al. 2001) Pigs (Betthauser et al., 2000; Onishi et al., 2000; Polejaeva et al., 2000)
and from the naturally produced population. Most of these differences appear to be due to epigenetic abnormalities acquired during nuclear reprogramming (Rideout et al., 2001). Whether these observed differences (such as telomere lengths or imprinted gene expression) have any bearing on health in later life remains to be seen. For those clones that apparently experienced a normal pregnancy and neonatal period, the outlook for a normal life appears good. What is of significant concern is that placental development and the intrauterine environment for many clones is suboptimal and this alone may impact on their health in later life. Recent proponents of human cloning have shown an amazing lack of understanding of many of the critical details presented in the literature and at scientific conferences. The major problem faced with cloning is that we are only beginning to understand what is going wrong and how to fix it (Jaenisch and Wilmut, 2001). Viable, apparently normal offspring appear to be produced by chance. Those involved in animal cloning are constantly exposed to the disappointment of watching apparently good quality embryos fail to thrive during pregnancy or following birth. This close familiarity with the darker side of cloning prompts astonishment at the misguided rhetoric that is accompanying proposed human cloning experiments. It is reckless to attempt to use cloning to reproduce humans. There is so much work yet to be done in testing the preimplantation embryos for likely genetic abnormalities. Even so, many of the genes likely to be contributing to failed development are not expressed at the preimplantion stage and so would not be discovered prior to transfer into a surrogate woman. Thus, to pro-
duce a viable baby would require the use of many surrogate women to provide the number of pregnancies for the chance result of one normal birth. These surrogate women would be treated like a flock of sheep, with less than 1 out of every 20 women implanted with cloned embryos producing a baby. The prospect of immediately producing cloned human embryos for transfer is akin to deliberately administering drugs during pregnancy that are known to cause a high rate of abortions and congenital abnormalities. It is simply not ethical to attempt to produce and transfer cloned human embryos when the same technique is known in animals to have a high risk of causing an abnormal fetus. Proponents of human cloning provide the retort that there can be no progress in improving the success rate of human cloning until the technique is performed in humans. However the traditional first step in evaluating a human medical procedure or drug is to first prove its safety in animals, then when safety and efficacy studies are positive, to proceed to initial clinical trials. When the many steps of the nuclear transfer process are studied carefully, it is little wonder that so many cloned embryos are destined to failure. Somatic cell cloned embryos are generated by combining enucleated oocytes with somatic cells. The enucleation is accomplished by aspirating the oocyte nucleus and first polar body with a glass micropipette. The nucleus from a differentiated cell, that has often been maintained for several weeks in cell culture, is then introduced into the enucleated oocyte by one of two methods. Direct injection of the cell nucleus is performed by first lysing the plasma membrane of the donor cell then isolating and injecting the nucleus with a Piezo pipette (Wakayama et al., 1998). Alternatively, the entire donor cell is placed adjacent to the oocyte cell membrane, then both cell membranes are fused with an electric pulse to allow mixing of the donor cell contents with the oocyte cytoplasm. The newly reconstructed embryo is stimulated to divide chemically or by electric pulse. The NT embryos are then either cultured in vitro prior to transfer or transferred immediately into a recipient animal. The oocyte and embryo is thus exposed to a variety of media and physical insults prior to transfer into a recipient female. It is therefore not surprising that the overall success rates for cloning, as measured by the proportion of live offspring that result from each reconstructed embryo, are low – around 1 %. Although many cloned embryos may develop to preimplantation stage, the vast majority will not result in a viable pregnancy. Initially a high proportion of cloned embryos divide normally and reach the blastocyst stage at the expected time. These embryos show similar development and morphology to in vitro fertilized embryos cultured under the same conditions. This observation of apparently normal early development provokes a unique feeling of amazement and awe at the capacity of the oocyte to redirect development of a somatic cell nucleus. However, the initial excitement at
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producing an apparently normal embryo, without using germ cell nuclei, is tempered by the subsequent unpredictable development of the NT embryos following implantation into surrogate uteri. As pregnancy progresses, placental and fetal abnormalities dramatically reduce the numbers of cloned fetuses that survive to term. It is clear that the apparently normal appearance of cloned embryos does not correlate with their subsequent viability. This lack of normal in utero development is of critical importance to improving NT efficiency, and the cause appears to lie in the nuclear reprogramming process following NT, the NT technique itself, and epigenetic damage from embryo culture conditions. In cloning, the donor nucleus was a somatic cell with a very different chromatin configuration and gene expression pattern to that of a sperm. The somatic cell nucleus must be rapidly reprogrammed to assume its new life of an embryonic cell. It is likely that this reprogramming is incomplete in many cloned embryos, leading to failed development and perhaps non-lethal abnormalities in surviving clones. At present, there is much attention being devoted to the study of early developmental genes in IVF and cloned embryos – many of which are imprinted genes. Gene expression studies have shown these NT embryos possess some subtle variations from controls. Differential display analysis that compared NT, IVF and in vivo produced blastocysts showed most but not all mRNAs to be reprogrammed in cloned embryos (De Sousa et al., 1999). Some genes are correctly expressed in cloned embryos, such as important metabolic enzymes (Winger et al., 2000). Telomerase is also correctly expressed in NT embryos, which have high telomerase activity despite being cloned from cells with low/non detectable levels of telomerase (Betts et al., 2001). The expression of genes shown to be important for implantation in individual NT embryos (IL6, FGF4, and FGFr2) showed variable profiles (Daniels et al., 2000). This abnormal gene expression would likely have an impact on the subsequent development of these embryos. Imprinted gene expression in clones derived from ES cell nuclei was consistently different from controls (Humpherys et al., 2001). There is also likely to be an added effect of embryo culture conditions on imprinted gene expression profiles (Khosla et al., 2001; Young et al., 2001). Once the cloned embryos leave the culture dish to progress in utero, abnormalities in development become apparent. In each animal species so far studied, less than 10 % of transferred embryos have survived to term. Despite the use of healthy, fertile synchronous females as recipients this survival rate is well below the 50 – 70 % level expected using either in vitro or in vivo fertilized embryos. Studies in cows and sheep have shown the most dramatic period of fetal loss to be at the time of placental attachment, from the first to the second month of gestation.
The placenta is commonly poorly developed and is likely to be the cause of high rate of fetal death (up to 80 %) during the first trimester of pregnancy. Bovine pregnancies cloned from embryonic cells (Stice et al., 1996) displayed a lack of placental development and attachment sites which likely caused a high rate of first trimester death observed in that study. More recently, in somatic cell cloned cattle, normal placental development also appears to be rare, as placental abnormalities occur at a high incidence in early and late term cloned fetuses (Hill et al., 1999; 2000; Wells et al., 1999; ChavattePalmer et al., 2000; De Sousa et al., 2001). Incomplete first trimester placental development has been documented in bovine and ovine NT embryos where a lack of placental vascularization and attachment sites are apparent (Hill et al., 2000; De Sousa et al., 2001). It is presumed that this inadequate placental development is the major cause of the high rate of first trimester death in cloned fetuses. In third trimester cloned bovine pregnancies, placental abnormalities, such as edema and hydroallantois, have occurred in up to 50 % of cows pregnant with cloned fetuses (Hill et al., 1999; Wells et al., 1999; ChavattePalmer et al., 2000). The number of placental attachment sites in the bovine (placentomes) may be reduced from normal by as much as 80 % which suggests that the completeness of placental development varies widely in cloned animals. The reduction in placentome numbers may not harm fetal viability if the total surface area for nutrient exchange remains constant due to increase in size of the remaining placentomes (Bazer et al., 1979). However, it appears that placental gas exchange capacity is significantly reduced as late gestation cloned fetuses have been found to be hypoxic (Garry et al., 1998). In mice, overdeveloped placentas has been reported in cloned mice with placentas up to twice normal weight (Wakayama and Yanagimachi, 1999, 2001; Ogura et al., 2000). Postnatal viability is markedly lower for many cloned animals (Wilmut et al., 1997; Kato et al., 1998; 2000; Hill et al., 1999; Renard et al., 1999; Kubota et al., 2000; Eggan et al., 2001). Neonatal viability has been shown to be compromised due to pulmonary immaturity (neonatal respiratory distress syndrome) complicated by persistent fetal circulation (pulmonary hypertension, right to left shunt) and disturbed placental function (Hill et al., 1999; Chavatte-Palmer et al., 2000). It appears that inadequate placental function impairs fetal and neonatal viability in cloned neonates. At birth, cloned calves and lambs commonly show signs of a stressful uterine environment (meconium staining, hypoxia). Placental reserve capacity is most likely limited due to inadequate development. Although cloned fetuses are seldom smaller than average, they share many attributes of fetuses suffering from placental insufficiency. Cloned neonates also appear to be incompletely prepared for the critical transition to breathing room air. Thus, at
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present, some cloned neonates are unable to survive despite intensive treatment. The question of premature aging of cloned animals has been illustrated by the variation in telomere lengths found in cloned sheep, cattle and mice. Telomere rebuilding was observed in cloned bovine fetuses and calves that were derived from cultured cells with shortened telomeres (Tian et al., 2000; Betts et al., 2001). These studies showed that telomeres were rebuilt to similar lengths as control animals, and another study even showed elongation of telomeres in cloned offspring (Lanza et al., 2000). This is in contrast to observations on cloned sheep whose telomeres were shortened (Shiels et al., 1999). It is apparent that fetal viability in cloned animals varies between experiments and between species, with cloned mice and goats displaying better post natal viability (Keefer et al., 2000; Wakayama and Yanagimachi, 2001). Whether these differences are due to technique, animal strain, or to placental type remains to be determined. Published work on cloned placentas is preliminary and mainly centered on morphological descriptions. Further evaluation using histological and molecular techniques should improve our understanding of the failure of normal placental development. As we define the physiology of these prenatal placental problems more carefully, outcomes should improve. Defining the origins of these abnormalities may also lead to prevention through alterations in the cloning technique. The ability to clone animals from somatic cells is a tremendous advance for biomedical research and for agriculture. The majority of cloned animals alive today are normal in appearance and behavior. Indeed a significant number of cattle, sheep, and goats have produced normal offspring, with many cloned transgenic animals now producing valuable proteins in their milk for pharmaceutical use. However, in those clones that lack normal in utero development, profound consequences on fetal and neonatal viability result. In surviving clones, this will have unknown effects on adult health, which is especially important to address before human cloning can be contemplated. The efficiency of producing cloned animals will continue to improve, but the industry is still too early on its learning curve to safely transfer the technology to those who prose to clone humans.
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Shiels, P.G., Kind, A.J., Campbell, K.H., Waddington, D., Wilmut, I., Colman, A. and Schnieke, A.E. (1999) Analysis of telomere lengths in cloned sheep. Nature 399:316–317. Shiga, K., Fujita, T., Hirose, K., Sasae, Y. and Nagai, T. (1999) Production of calves by transfer of nuclei from cultured somatic cells obtained from Japanese black bulls. Theriogenology 52:527–535. Stice, S.L., Strelchenko, N.S., Keefer, C.L. and Matthews, L. (1996) Pluripotent bovine embryonic cell lines direct embryonic development following nuclear transfer. Biol Reprod 54:100–110. Tian, X.C., Xu, J. and Yang, X. (2000) Normal telomere lengths found in cloned cattle. Nat Genet 26:272–273. Wakayama, T., Perry, A.C., Zuccotti, M., Johnson, K.R. and Yanagimachi, R. (1998) Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei. Nature 394:369–374. Wakayama, T. and Yanagimachi, R. (1999) Cloning of male mice from adult tail-tip cells. Nat Genet 22:127–128. Wakayama, T. and Yanagimachi, R. (2001) Mouse cloning with nucleus donor cells of different age and type. Mol Reprod Dev 58:376–383. Wells, D.N., Misica, P.M. and Tervit, H.R. (1999) Production of Cloned Calves Following Nuclear Transfer with Cultured Adult Mural Granulosa Cells. Biol Reprod 60:996–1005. Wells, D.N., Pavla, P.M., Tervit, H.R. and Vivanco, W.H. (1998) Adult somatic cell nuclear transfer is used to preserve the last surviving cow of the Enderby Island cattle breed. Reprod Fertil Dev 10:369–378. Wilmut, I., Schnieke, A.E., McWhir, J., Kind, K.L. and Campbell, K.H.S. (1997) Viable offspring derived from fetal and adult mammalian cells. Nature 385:810–813. Winger, Q.A., Hill, J.R., Shin, T., Watson, A.J., Kraemer, D.C. and Westhusin, M.E. (2000) Genetic reprogramming of lactate dehydrogenase, citrate synthase, and phosphofructokinase mRNA in bovine nuclear transfer embryos produced using bovine fibroblast cell nuclei. Mol Reprod Dev 56:458–464. Young, L.E. and Fairburn, H.R. (2000) Improving the safety of embryo technologies: possible role of genomic imprinting. Theriogenology 53:627–648. Young, L.E., Fernandes, K., McEvoy, T.G., Butterwith, S.C., Gutierrez, C.G., Carolan, C., Broadbent, P.J., Robinson, J.J., Wilmut, I. and Sinclair, K.D. (2001) Epigenetic change in IGF2R is associated with fetal overgrowth after sheep embryo culture. Nat Genet 27:153–154. Zakhartchenko, V., Alberio, R., Stojkovic, M., Prelle, K., Schernthaner, W., Stojkovic, P., Wenigerkind, H., Wanke, R., Duchler, M., Steinborn, R., Mueller, M., Brem, G. and Wolf, E. (1999) Adult cloning in cattle: potential of nuclei from a permanent cell line and from primary cultures. Mol Reprod Dev 54:264–272. Zhou, Q.I., Boulanger, L. and Renard, J.P. (2000) A simplified method for the reconstruction of fully competent mouse zygotes from adult somatic donor nuclei. Cloning 2:35–44. Zou, X., Chen, Y., Wang, Y., Luo, J., Zhang, Q., Yang, Y., Ju, H., Shen, Y., Lao, W., Xu, S. and Du, M. (2001) Production of Cloned Goats from Enucleated Oocytes Injected with Cumulus Cell Nuclei or Fused with Cumulus Cells. Cloning 3:31–37.
Differentiation (2002) 69:174–178
COMMENTARY
Jonathan R. Hill
Abnormal in utero development of cloned animals: implications for human cloning
Accepted in revised form: 19 November 2001
Key words human cloning ¡ in utero development ¡ somatic cells nuclear transplantation
Hundreds of animals have been cloned from animal somatic cells since the first mammalian clone, Dolly, was presented to a stunned world press in February 1997 (Wilmut et al., 1997). Although the concept of cloning or nuclear transfer (NT) was successful in amphibians in the 1950s and again in cattle, sheep and pigs (using embryonic nuclei) over the past 2 decades, Dolly was the first mammal to be reproduced from a cell taken from an adult animal (somatic cell cloning). After Dolly, in 1998 came cloned mice and cattle (Cibelli et al., 1998; Kato et al., 1998; Wakayama et al., 1998). Over the next few years goats and pigs were cloned while the numbers of cloned mice and cattle continued to increase (Table 1) (Baguisi et al., 1999; Onishi et al., 2000; Polejaeva et al., 2000). Research into mammalian cloning is driven by both commercial and biomedical research goals. The most lucrative commercial application for cloning is through its combination with genetic modification (transgenics); (Schnieke et al., 1997). This greatly improves the efficiency of transgenic animal production with the goals of harvesting human therapeutic proteins from milk or organs for transplantation. Cloning to copy elite livestock has already been commercialized, and the application to endangered animal conservation is actively being investigated. From a purely scientific viewpoint, cloning appears to be an invaluable tool in studying the nuclear reprogramming process during the first few J. R. Hill Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York, 14853, USA e-mail: jrh35/cornell.edu Tel: π1 607 253 3091, Fax: π1 607 253 3531 U. S. Copyright Clearance Center Code Statement:
hours or days after the transfer of a somatic cell into an enucleated oocyte. As yet unsuccessful attempts have been made to clone rats, rabbits, dogs, cats, horses, and monkeys. This illustrates that the technique developed by Ian Wilmut and Keith Campbell is not immediately applicable to each species, most likely due to subtle biological variations between even closely related species such as the rat and mouse. In primates, cloning with somatic cells is not yet successful, although nuclear transfer using embryonic cells has produced live offspring (Meng et al., 1997). In each of the species where somatic cell cloning has been successful, it has also been very inefficient. Early first trimester pregnancy rates are less than 1/2 that normally expected. Immediately following initial positive diagnosis of pregnancy, extraordinarily high rates of embryonic loss occur, where up to 80 % of pregnancies miscarry by the second trimester. In late gestation, placental and fetal abnormalities occur at a much higher than normal rate, and finally lowered viability at birth is common. These major developmental abnormalities alert us to the inefficiency inherent in cloning mammals. Gestational and neonatal abnormalities are consistent with irregular expression and likely incomplete reprogramming of imprinted genes (Young and Fairburn, 2000; Jaenisch and Wilmut, 2001; Rideout et al., 2001). However, a casual perusal of the lay and scientific literature may give a confusing picture of the status of cloned animals. Some well publicized reports give a more negative view of mammalian cloning by focusing attention on abnormalities in telomere length, gene expression or methylation patterns. Others focus on success and proclaim the birth of apparently normal offspring. The practical outcome is that there are many cloned animals that behave and appear normal, while closer investigations have revealed that even some of these apparently normal animals are subtly different from one another
0301–4681/2002/6904–174 $ 15.00/0
175 Table 1 Survival of mammals cloned from somatic cells. The data excludes animals that died during gestation, but prior to parturition. Data was obtained from published peer reviewed journal articles Animal
Live born
Live after 1 week
Live at puberty (% of live born)
Sheep1 Mice2 Cattle3 Goats4 Pigs5
28 126 91 17 10
15
11 88 60 12 10
1 2 3
4 5
66 14 10
(39%) (70%) (66%) (70%) (100%)
Sheep (Schnieke et al., 1997; Wilmut et al., 1997; McCreath et al. 2000; Denning et al., 2001) Mice (Wakayama et al., 1998; Wakayama and Yanagimachi 1999, 2001; Ogura et al., 2000; Zhou et al., 2000) Cattle (Cibelli et al., 1998; Kato et al., 1998, 2000; Wells et al., 1998, 1999; Shiga et al., 1999; Hill et al., 2000; Kishi et al., 2000; Kubota et al., 2000; Lanza et al., 2000; Zakhartchenko et al., 1999; Heyman et al., 2002) Goats (Baguisi et al., 1999; Keefer et al., 2001; Reggio et al. 2001; Zou et al. 2001) Pigs (Betthauser et al., 2000; Onishi et al., 2000; Polejaeva et al., 2000)
and from the naturally produced population. Most of these differences appear to be due to epigenetic abnormalities acquired during nuclear reprogramming (Rideout et al., 2001). Whether these observed differences (such as telomere lengths or imprinted gene expression) have any bearing on health in later life remains to be seen. For those clones that apparently experienced a normal pregnancy and neonatal period, the outlook for a normal life appears good. What is of significant concern is that placental development and the intrauterine environment for many clones is suboptimal and this alone may impact on their health in later life. Recent proponents of human cloning have shown an amazing lack of understanding of many of the critical details presented in the literature and at scientific conferences. The major problem faced with cloning is that we are only beginning to understand what is going wrong and how to fix it (Jaenisch and Wilmut, 2001). Viable, apparently normal offspring appear to be produced by chance. Those involved in animal cloning are constantly exposed to the disappointment of watching apparently good quality embryos fail to thrive during pregnancy or following birth. This close familiarity with the darker side of cloning prompts astonishment at the misguided rhetoric that is accompanying proposed human cloning experiments. It is reckless to attempt to use cloning to reproduce humans. There is so much work yet to be done in testing the preimplantation embryos for likely genetic abnormalities. Even so, many of the genes likely to be contributing to failed development are not expressed at the preimplantion stage and so would not be discovered prior to transfer into a surrogate woman. Thus, to pro-
duce a viable baby would require the use of many surrogate women to provide the number of pregnancies for the chance result of one normal birth. These surrogate women would be treated like a flock of sheep, with less than 1 out of every 20 women implanted with cloned embryos producing a baby. The prospect of immediately producing cloned human embryos for transfer is akin to deliberately administering drugs during pregnancy that are known to cause a high rate of abortions and congenital abnormalities. It is simply not ethical to attempt to produce and transfer cloned human embryos when the same technique is known in animals to have a high risk of causing an abnormal fetus. Proponents of human cloning provide the retort that there can be no progress in improving the success rate of human cloning until the technique is performed in humans. However the traditional first step in evaluating a human medical procedure or drug is to first prove its safety in animals, then when safety and efficacy studies are positive, to proceed to initial clinical trials. When the many steps of the nuclear transfer process are studied carefully, it is little wonder that so many cloned embryos are destined to failure. Somatic cell cloned embryos are generated by combining enucleated oocytes with somatic cells. The enucleation is accomplished by aspirating the oocyte nucleus and first polar body with a glass micropipette. The nucleus from a differentiated cell, that has often been maintained for several weeks in cell culture, is then introduced into the enucleated oocyte by one of two methods. Direct injection of the cell nucleus is performed by first lysing the plasma membrane of the donor cell then isolating and injecting the nucleus with a Piezo pipette (Wakayama et al., 1998). Alternatively, the entire donor cell is placed adjacent to the oocyte cell membrane, then both cell membranes are fused with an electric pulse to allow mixing of the donor cell contents with the oocyte cytoplasm. The newly reconstructed embryo is stimulated to divide chemically or by electric pulse. The NT embryos are then either cultured in vitro prior to transfer or transferred immediately into a recipient animal. The oocyte and embryo is thus exposed to a variety of media and physical insults prior to transfer into a recipient female. It is therefore not surprising that the overall success rates for cloning, as measured by the proportion of live offspring that result from each reconstructed embryo, are low – around 1 %. Although many cloned embryos may develop to preimplantation stage, the vast majority will not result in a viable pregnancy. Initially a high proportion of cloned embryos divide normally and reach the blastocyst stage at the expected time. These embryos show similar development and morphology to in vitro fertilized embryos cultured under the same conditions. This observation of apparently normal early development provokes a unique feeling of amazement and awe at the capacity of the oocyte to redirect development of a somatic cell nucleus. However, the initial excitement at
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producing an apparently normal embryo, without using germ cell nuclei, is tempered by the subsequent unpredictable development of the NT embryos following implantation into surrogate uteri. As pregnancy progresses, placental and fetal abnormalities dramatically reduce the numbers of cloned fetuses that survive to term. It is clear that the apparently normal appearance of cloned embryos does not correlate with their subsequent viability. This lack of normal in utero development is of critical importance to improving NT efficiency, and the cause appears to lie in the nuclear reprogramming process following NT, the NT technique itself, and epigenetic damage from embryo culture conditions. In cloning, the donor nucleus was a somatic cell with a very different chromatin configuration and gene expression pattern to that of a sperm. The somatic cell nucleus must be rapidly reprogrammed to assume its new life of an embryonic cell. It is likely that this reprogramming is incomplete in many cloned embryos, leading to failed development and perhaps non-lethal abnormalities in surviving clones. At present, there is much attention being devoted to the study of early developmental genes in IVF and cloned embryos – many of which are imprinted genes. Gene expression studies have shown these NT embryos possess some subtle variations from controls. Differential display analysis that compared NT, IVF and in vivo produced blastocysts showed most but not all mRNAs to be reprogrammed in cloned embryos (De Sousa et al., 1999). Some genes are correctly expressed in cloned embryos, such as important metabolic enzymes (Winger et al., 2000). Telomerase is also correctly expressed in NT embryos, which have high telomerase activity despite being cloned from cells with low/non detectable levels of telomerase (Betts et al., 2001). The expression of genes shown to be important for implantation in individual NT embryos (IL6, FGF4, and FGFr2) showed variable profiles (Daniels et al., 2000). This abnormal gene expression would likely have an impact on the subsequent development of these embryos. Imprinted gene expression in clones derived from ES cell nuclei was consistently different from controls (Humpherys et al., 2001). There is also likely to be an added effect of embryo culture conditions on imprinted gene expression profiles (Khosla et al., 2001; Young et al., 2001). Once the cloned embryos leave the culture dish to progress in utero, abnormalities in development become apparent. In each animal species so far studied, less than 10 % of transferred embryos have survived to term. Despite the use of healthy, fertile synchronous females as recipients this survival rate is well below the 50 – 70 % level expected using either in vitro or in vivo fertilized embryos. Studies in cows and sheep have shown the most dramatic period of fetal loss to be at the time of placental attachment, from the first to the second month of gestation.
The placenta is commonly poorly developed and is likely to be the cause of high rate of fetal death (up to 80 %) during the first trimester of pregnancy. Bovine pregnancies cloned from embryonic cells (Stice et al., 1996) displayed a lack of placental development and attachment sites which likely caused a high rate of first trimester death observed in that study. More recently, in somatic cell cloned cattle, normal placental development also appears to be rare, as placental abnormalities occur at a high incidence in early and late term cloned fetuses (Hill et al., 1999; 2000; Wells et al., 1999; ChavattePalmer et al., 2000; De Sousa et al., 2001). Incomplete first trimester placental development has been documented in bovine and ovine NT embryos where a lack of placental vascularization and attachment sites are apparent (Hill et al., 2000; De Sousa et al., 2001). It is presumed that this inadequate placental development is the major cause of the high rate of first trimester death in cloned fetuses. In third trimester cloned bovine pregnancies, placental abnormalities, such as edema and hydroallantois, have occurred in up to 50 % of cows pregnant with cloned fetuses (Hill et al., 1999; Wells et al., 1999; ChavattePalmer et al., 2000). The number of placental attachment sites in the bovine (placentomes) may be reduced from normal by as much as 80 % which suggests that the completeness of placental development varies widely in cloned animals. The reduction in placentome numbers may not harm fetal viability if the total surface area for nutrient exchange remains constant due to increase in size of the remaining placentomes (Bazer et al., 1979). However, it appears that placental gas exchange capacity is significantly reduced as late gestation cloned fetuses have been found to be hypoxic (Garry et al., 1998). In mice, overdeveloped placentas has been reported in cloned mice with placentas up to twice normal weight (Wakayama and Yanagimachi, 1999, 2001; Ogura et al., 2000). Postnatal viability is markedly lower for many cloned animals (Wilmut et al., 1997; Kato et al., 1998; 2000; Hill et al., 1999; Renard et al., 1999; Kubota et al., 2000; Eggan et al., 2001). Neonatal viability has been shown to be compromised due to pulmonary immaturity (neonatal respiratory distress syndrome) complicated by persistent fetal circulation (pulmonary hypertension, right to left shunt) and disturbed placental function (Hill et al., 1999; Chavatte-Palmer et al., 2000). It appears that inadequate placental function impairs fetal and neonatal viability in cloned neonates. At birth, cloned calves and lambs commonly show signs of a stressful uterine environment (meconium staining, hypoxia). Placental reserve capacity is most likely limited due to inadequate development. Although cloned fetuses are seldom smaller than average, they share many attributes of fetuses suffering from placental insufficiency. Cloned neonates also appear to be incompletely prepared for the critical transition to breathing room air. Thus, at
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present, some cloned neonates are unable to survive despite intensive treatment. The question of premature aging of cloned animals has been illustrated by the variation in telomere lengths found in cloned sheep, cattle and mice. Telomere rebuilding was observed in cloned bovine fetuses and calves that were derived from cultured cells with shortened telomeres (Tian et al., 2000; Betts et al., 2001). These studies showed that telomeres were rebuilt to similar lengths as control animals, and another study even showed elongation of telomeres in cloned offspring (Lanza et al., 2000). This is in contrast to observations on cloned sheep whose telomeres were shortened (Shiels et al., 1999). It is apparent that fetal viability in cloned animals varies between experiments and between species, with cloned mice and goats displaying better post natal viability (Keefer et al., 2000; Wakayama and Yanagimachi, 2001). Whether these differences are due to technique, animal strain, or to placental type remains to be determined. Published work on cloned placentas is preliminary and mainly centered on morphological descriptions. Further evaluation using histological and molecular techniques should improve our understanding of the failure of normal placental development. As we define the physiology of these prenatal placental problems more carefully, outcomes should improve. Defining the origins of these abnormalities may also lead to prevention through alterations in the cloning technique. The ability to clone animals from somatic cells is a tremendous advance for biomedical research and for agriculture. The majority of cloned animals alive today are normal in appearance and behavior. Indeed a significant number of cattle, sheep, and goats have produced normal offspring, with many cloned transgenic animals now producing valuable proteins in their milk for pharmaceutical use. However, in those clones that lack normal in utero development, profound consequences on fetal and neonatal viability result. In surviving clones, this will have unknown effects on adult health, which is especially important to address before human cloning can be contemplated. The efficiency of producing cloned animals will continue to improve, but the industry is still too early on its learning curve to safely transfer the technology to those who prose to clone humans.
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