Incidence of abnormal offspring from cloning and other assisted reproductive technologies.

arav2Hill

ARI

03 November 2013

07:44

Annu. Rev. Anim. Biosci. 2014.2. Downloaded from www.annualreviews.org by Lomonosov Moscow State University on 01/23/14. For personal use only.

Review in Advance first posted online on November 13, 2013. (Changes may still occur before final publication online and in print.)

Incidence of Abnormal Offspring from Cloning and Other Assisted Reproductive Technologies Jonathan R. Hill School of Veterinary Science, University of Queensland, St. Lucia, Queensland 4072, Australia; email: [email protected]

Annu. Rev. Anim. Biosci. 2014. 2:16.1–16.15

Keywords

The Annual Review of Animal Biosciences is online at animal.annualreviews.org

clone, in vitro fertilization, abnormalities, placenta, fetus

This article’s doi: 10.1146/annurev-animal-022513-114109

Abstract

Copyright © 2014 by Annual Reviews. All rights reserved

In animals produced by assisted reproductive technologies, two abnormal phenotypes have been characterized. Large offspring syndrome (LOS) occurs in offspring derived from in vitro cultured embryos, and the abnormal clone phenotype includes placental and fetal changes. LOS is readily apparent in ruminants, where a large calf or lamb derived from in vitro embryo production or cloning may weigh up to twice the expected body weight. The incidence of LOS varies widely between species. When similar embryo culture conditions are applied to nonruminant species, LOS either is not as dramatic or may even be unapparent. Coculture with serum and somatic cells was identified in the 1990s as a risk factor for abnormal development of ruminant pregnancies. Animals cloned from somatic cells may display a combination of fetal and placental abnormalities that are manifested at different stages of pregnancy and postnatally. In highly interventional technologies, such as nuclear transfer (cloning), the incidence of abnormal offspring continues to be a limiting factor to broader application of the technique. This review details the breadth of phenotypes found in nonviable pregnancies, together with the phenotypes of animals that survive the transition to extrauterine life. The focus is on animals produced using in vitro embryo culture and nuclear transfer in comparison to naturally occurring phenotypes.

16.1

Changes may still occur before final publication online and in print

arav2Hill

ARI

03 November 2013

07:44

INTRODUCTION


Although not every naturally conceived pregnancy results in live, normal offspring, when one thinks of abnormal offspring, cloned animals immediately come to mind. Dolly was the first somatic cell clone and a prominent case study of the successes and variables inherent in advanced reproductive techniques (1–5). As the first viable animal cloned from a somatic cell, Dolly was outwardly healthy, raised several lambs, and had an average ovine life span. Beyond this brief synopsis, what additional data and assurances are required to declare that other surviving clones are normal? And what are the boundaries of normal for cloned sheep, cattle, horses, dogs, or mice? What evidence is available that performance or behavioral traits are faithfully passed to cloned offspring? In agricultural animals, such as sheep, cattle, and pigs, economically important phenotypes are of most interest. These are related to production traits, such as longevity and reproduction, and saleable products, such as meat, milk, or fiber. Other traits include athletic ability in horses and behavioral traits in companion animals and rodents. Many of these have been assessed in the 17 years since Dolly’s birth, but many more could also be investigated (6). Although the focus is often on abnormalities observed in cloned animals, a lower incidence of very similar abnormalities can be seen in animals produced using in vitro embryo culture and, to a lesser extent, in naturally bred animals.

LARGE OFFSPRING SYNDROME The use of in vitro fertilization (IVF) and in vitro embryo production (IVP) in ruminant species was welcomed as another tool to enable rapid multiplication of genetically superior animals (7). The technique was not without cost, as it also induced undesirable pregnancy outcomes. Pregnancy rates were lower, embryonic losses higher, gestations prolonged, and birth weights increased (8, 9, 10, 11, 12). This was followed at birth by reduced vigor and viability of neonates. During the late 1980s and 1990s, several large-scale bovine embryo transfer programs were conducted to expand elite genetics using embryo cloning and/or in vitro embryo culture technologies. At the time, in vitro embryo culture techniques relied upon tissue culture media with added serum and coculture with somatic cells derived from buffalo rat liver or oviduct epithelium (10, 13, 14–17). An alternative was to culture early embryos in sheep oviducts (16). A characteristic of a significant proportion of those offspring was their large size, accompanied by changes in internal organs (18). This often occurred in conjunction with abnormal placentas and excess placental fluids during the latter half of pregnancy, when fetal growth is at its most rapid (18, 19). Gestational abnormalities such as embryonic and fetal losses, late-term abortions, prolonged gestation lengths, high birth weights, placental anomalies, and reduced neonatal survival have been described (10, 13, 19, 11, 12, 20). By the early 1990s, it was apparent that the culture medium, the use of coculture, and the addition of serum were major contributors to aberrant embryonic development and were probable causes for large offspring syndrome (LOS) (8, 18, 21, 22). The resultant high–birth weight lambs and calves prompted additional research into the effect of embryo conditions on early embryonic gene expression (23). The morphology of embryos cultured with serum showed an abundance of lipid droplets (8). After embryo transfer, those lipiddense embryos developed into fetuses with longer gestations and higher birth weights. Clearly, the incidence and severity of LOS is closely related to embryo culture conditions (18, 24–26). In addition to serum, specific components of culture media were identified that lead to fetal abnormalities. High ammonium levels in embryo culture medium resulted in exencephaly, 16.2

Hill



arav2Hill

ARI

03 November 2013

07:44

enlarged brain area, and retardation of the fore and hind limbs in mouse fetuses (27). The source of bovine serum albumin was also identified as a contributor to early fetal losses. Improved vascularization of the allantois arose from use of an alternate source of bovine serum albumin (28). The removal of serum and cessation of coculture with somatic cells resulted in similar pregnancy rates but lower rates of abnormal pregnancies (29–31). These experiments clearly illustrate that early perturbations in the embryo’s immediate environment can cause long-term changes in fetal development. In a large-scale analysis of IVF calves by Holland Genetics, embryos produced with coculture and serum developed a higher incidence of congenital abnormalities, such as hydrallantois and limb deformities (3.7% versus 0.6% for artificial insemination calves) (25). Perinatal mortality was also higher in calves derived from the coculture system compared with those obtained through multiple ovulation and embryo transfer (7.5% versus 4.6%, respectively). Persistent effects on heart size have arisen in bulls produced by IVF and culture. The hearts of bulls from in vitro–frozen embryos were heavier than those of bulls from in vivo–frozen embryos (32). Within the IVF cohort, calves classified as having LOS had larger hearts than normal-sized IVF and in vivo–produced calves (32). Following culture of ovine embryos, LOS is associated with loss of methylation of the maternal IGF2R allele (33). Ovine embryos may be more sensitive to culture conditions, because they undergo a prolonged period of active chromatin demethylation and remethylation (34). This may partly explain the more extreme phenotypes observed in ovine IVF programs, where offspring as large as five times normal weight have been recorded (19). In ovine pregnancies derived from coculture systems, the incidence of hydrallantois increased to 23% (22). The ovine embryo is also very sensitive to an altered in vivo (uterine) environment. In a uterine environment with excess substrate availability (induced by exogenous progesterone from days 1–6 after conception), acceleration of fetal growth rates results (35). Asynchronous transfer of day-3 embryos into a day-6 uterus also increased fetal growth rates (36). Oversized lambs were born to ewes that were fed an excess of nonprotein nitrogen (urea) for 40 days from day 21 of gestation (37). In vitro culture of mouse embryos also leads to methylation alterations at both maternal and paternal imprinted alleles (38). In mice, fetal overgrowth is accompanied by placental overgrowth (39–41). Nuclear transfer further alters imprinted gene expression and is associated with fetal and placental overgrowth (42). The fetuses displayed reduced expression of H19 and IGF-II, whereas their placentas showed increased expression of an IGF-II placenta-specific variant (P0). Abnormal bovine placental gene expression has also been observed (43), and previously, in embryonic stem cell–derived fetuses, similar fetal gene expression changes were observed (44). When microinjected into tetraploid embryos or used as donor cells for somatic cell nuclear transfer, some embryonic stem cell lines result in a high incidence of abnormalities, such as fetal oversize, fetal edema, placental hypertrophy, and perinatal death (45, 46). These abnormalities are similar to those seen in IGF knockout mice and also in ruminant fetuses with LOS (18, 47). This illustrates the variability of animals born to assisted reproductive technologies and provides a baseline to compare with abnormalities observed in cloned animals. The next section looks specifically at abnormalities arising from nuclear transfer. Abnormalities arising from the nuclear transfer process may also occur with, or be exacerbated by, placental and/or fetal abnormalities arising from the LOS (e.g., hydrallantois, fetal oversize) (48, 49).

NUCLEAR TRANSFER USING EMBRYONIC AND SOMATIC CELLS Production of a viable nuclear transfer embryo from a somatic cell nucleus requires an extraordinary change in donor cell gene expression. Irregularities in development during embryonic, fetal, www.annualreviews.org

Abnormal Offspring


16.3

arav2Hill

ARI

03 November 2013

07:44

and postnatal life illustrate the incomplete reprogramming process. Various published results across several species show a wide spectrum of physiology and anatomy in a population of clones, but many individuals have been classified as normal. Even between clones with the same genetics, phenotypic differences can be striking (15). Since the birth of the first cloned sheep in 1996, a variety of other species have been cloned, including mice, cattle, goats, pigs, dogs, horses, rabbits, and cats. Each species has required modifications to nuclear transfer protocols and has continued to display the inefficiencies observed in the initial publications by Wilmut et al. (1, 50) (see Figure 1).

Early Development in Naturally Conceived Pregnancies


In naturally conceived animals, there is a low but expected incidence of embryonic and fetal loss owing to abnormalities of the embryo or its placenta, alterations in maternal uterine environment, or fetomaternal interactions (51). The maternal environment, from the period of oocyte development through fertilization and pregnancy, can impact fetal and/or placental growth, gestation length, and subsequent offspring health (52–54). The underlying incidence of abnormal offspring in naturally conceived pregnancies arises from genetic or acquired congenital abnormalities. Examples in livestock and companion animals

Donor cell

• Species of donor cell • Cell origin and type • Cell lines selected for clonability • Cell treatments to reduce global methylation or specific gene expression

Neonatal management

• Precision obstetrical management • Specialist neonatal intensive care to assist the transition to extrauterine life • Cardiopulmonary support • Postnatal health monitoring and treatments

Nuclear transfer

• Oocyte quality • Oocyte activation method • Enhanced reprogramming (e.g., via epigenetic approaches) • Donor cell cycle synchronization

Embryo development

Embryo culture

• Culture medium selection • Somatic cell coculture • Addition of serum • Components of culture media (e.g., source of bovine serum albumen)

• Recipient quality and synchrony with embryo stage • Fetal monitoring and assessment of placental function • Corticosteroids to induce maturation of fetal lung and gastrointestinal tract

Figure 1 Technical, cell biological, and physiological factors to control and thus minimize the incidence of abnormal offspring.

16.4

Hill


arav2Hill

ARI

03 November 2013

07:44

include changes to the musculoskeletal (e.g., ankylosed joints, tendon flexure), central nervous (hydrocephalus), integument (schistosoma reflexus, subcutaneous edema, or anasarca), and hematopoietic (blood clotting deficiencies or autoimmune) systems (55).


Early Development in Cloned Pregnancies Cloned pregnancies show a high incidence of embryonic loss during the first half of pregnancy, particularly in the first trimester. First-trimester losses of greater than 50% are common for nuclear transfer pregnancies in cattle, sheep, and goats (56–58), whereas 2–4% of naturally conceived day-30 bovine pregnancies and 11% of in vitro–produced embryos would be expected to be lost by day 60 (13, 59, 60). Placental abnormalities have been implicated as both a cause and effect in pregnancy losses owing to cloning technique and the associated oocyte/embryo culture environments that also can result in the LOS (48, 56, 58). Many cloned embryos fail to negotiate the critical period of maternal recognition of pregnancy at the elongation stage (days 12–15 in cattle), during which 40% may fail (48). Generally, in mammalian pregnancies, early fetal losses may be due to abnormalities of the embryo or its placenta, alterations in maternal uterine environment, or fetomaternal interactions (51). In naturally conceived pregnancies, fetal abnormalities are a major cause of pregnancy loss. However, in cloned and in vitro–produced pregnancies, the placenta is implicated as a major contributor to pregnancy failure. Losses at approximately the time of placental attachment to the endometrium (day 30 in the bovine) are thought to be related to failure of vascularization and allantois development (31, 58). During the placental attachment period, fetomaternal interactions may also be altered by inappropriate expression of MHC-1 proteins (61). In a large study of 535 cloned bovine pregnancies, two-thirds aborted prior to day 60, and three-quarters of the original pregnancies were lost prior to birth (62). By days 90–110, signs of impaired embryo development, such as reduced crown-rump length, low heart rate, and large abdominal circumference, are consistent with poor viability (62). Bovine cloned pregnancies that are viable at day 60 have a high probability of progressing through the second trimester and into the final trimester. At that later stage, 17% may develop excessive fetal fluids (hydrops allantois), with resultant low viability (63). Although the preceding section focused on cloned bovine pregnancies, it is interesting to note that the same authors [Forsberg et al. (63)] who observed the 17% incidence of hydrops allantois did not observe any similar cases in their concurrent porcine cloning program, in which cloned piglets were born with normal birth weights.

Alterations to Placental Physiology and Function Normal placental development is critical for normal fetal growth and viability. Suboptimal intrauterine conditions may result in a range of fetal responses, such as fetal death and growth retardation, as well as more subtle effects, such as altered organ size (64). More subtle effects of an inadequate uterine environment may not be observed until adult life (65). The placenta is an active endocrine organ that produces progesterone and catalyzes production of estrogen from progesterone, as well as growth factors essential for normal growth and development (placental lactogen and placental growth hormone). Because the placenta is often categorized as abnormal in cloned pregnancies, it is of little surprise that final maturation of the fetus and of the mammary gland is often incomplete. Levels of pregnancy-associated hormones may give an indication of the viability of the fetoplacental unit. Abnormal progesterone concentrations may lead to abnormal growth of organs and the skeleton (20). Low progesterone concentrations have occurred in day-80 cloned pregnancies, www.annualreviews.org

Abnormal Offspring


16.5

ARI

03 November 2013

07:44

whereas estrone sulfate concentrations were normal (66). Estradiol was later found to increase from days 80–240 (66). High levels of estradiol were associated with enlarged placentomes, likely because of estrogenic effects of increased fluid retention (66). Maternal serum pregnancy-associated glycan levels [pregnancy-specific protein B (PSPb) or PSP60] were higher in the first half (first 150 days) of cloned pregnancies (49, 58, 66, 67). Higher levels of pregnancy-associated glycans are likely due to longer serum half-life, because there was no increase in numbers of their source cells (binucleate cells) or in their major products, pregnancyassociated glycans or bovine placental lactogen (bPL) (67). Somewhat surprisingly, bPL levels are not different in third-trimester cloned pregnancies, as high bPL gene expression was found in firsttrimester cloned pregnancies (68). Placental abnormalities vary according to the species being cloned and the gene pathways most affected. IGF-II is commonly affected, and the phenotypes of IGF-II knockout mice are closely correlated with abnormal phenotypes observed in cloned mice. IGF-II knockout mice have very small placentas, and overexpression of IGF-II results in increased body size and placental edema (69). These features are also observed in cloned mouse placentas, together with reduced development of the spongiotrophoblast layer (39, 56, 58, 70–72). Smaller extraembryonic membranes with reduced vascularization have also been observed in cloned porcine placentas (73, 74). In cloned ruminant pregnancies, a well-recognized characteristic associated with abnormal offspring is variability in placentome number at all stages of gestation. Some early pregnancies appear to fail owing to inadequate placental contact points, whereas others at term have an excess of placentome surface area (75, 76). However, a reduced number of enlarged placentomes may not necessarily be harmful to fetal viability if the total surface area for nutrient exchange remains within normal limits (77, 78). In later gestation and at term, cloned placentas are commonly larger or heavier than normal in cows and mice (41, 79–81). These placental abnormalities reduce fetal viability and increase the risk of perinatal mortality (48).


arav2Hill

Birth and the Neonatal Period A lack of spontaneous parturition and increased perinatal mortality were early observations of lambs and calves cloned from somatic cells (2, 3). Many clone-bearing pregnancies progress beyond their due date. This may not be a problem in itself and may suggest that clones require more time in utero for full maturation. However, prolonged gestations are usually associated with increased birth weight and increased risk of dystocia. Rapidly increasing fetal demands, possibly associated with inadequate nutrient supply from a suboptimal placenta, argue against letting the gestation go beyond term (82); thus, induction is commonly performed around the anticipated due date (79, 83). Chavatte-Palmer et al. (84) reported that at term, the endocrine system of cloned fetuses was not premature, as demonstrated by a normal response to exogenous adrenocorticotropic hormone (ACTH) (84). Despite this, neonatal care protocols for cloned pregnancies recognize that many neonates should be managed similarly to premature neonates and thus will benefit from intensive treatment to aid the transition to extrauterine life (82). Reports on perinatal survival vary, and it is informative to review the results from large groups of bovine clones. Heyman et al. (49) found 13% of cloned calves showed signs consistent with LOS at birth. Also, at the same laboratory (85), 58 clones had 74.6% survival to the first week after birth and 64.4% survival to 6 months. Causes of death in the first week were pulmonary (pulmonary hypertension) or musculoskeletal (weakness, limb deformities) abnormalities, thymic atrophy, renal changes, enlarged umbilicus, and edematous placenta (79, 85). 16.6

Hill



arav2Hill

ARI

03 November 2013

07:44

Bovine somatic cell cloning has been undertaken as a commercial operation for more than a decade, and several commercial operators have documented their results. Because these results involve large numbers of births derived from many different cell lines, they provide an insight into the overall efficiency of the nuclear transfer technique. Cyagra/Goyaike produced 388 calves (9% survival of transferred embryos) in five years, from 2000–2005. Of calves born, 72% survived beyond the first two days, and 58% survived past five months (62). Musculoskeletal (e.g., contracted tendons) and pulmonary abnormalities were observed, along with hyperthermia, enlarged umbilicus or patent urachus, weakness, and depression. Biochemical changes observed were higher lactate and glucose. Hill et al. (86) had previously observed high glucose levels (type 1 diabetes) in a surviving cloned calf. In Japan, a nationwide study found 69% of cloned calves survived beyond 24 h (compared with 93% of a naturally conceived cohort), and 45% survived to day 150 (compared with 88% of a naturally conceived cohort) (87). From days 150 to 300, the losses lessened but were still significantly higher than for conventionally bred animals (2.5% losses versus 0.5%), and no difference was found in subsequent survival to 720 days (0.5% versus 0.6%). In Canada, 22 of 25 cloned calves had neonatal respiratory dysfunction (hypoxia or a combination of hypoxia and hypercapnia), and the majority of those affected (16) survived with treatment. Other abnormalities noted were enlarged umbilical vessels, poor suckle, and generalized weakness (88). Ovine cloning programs continue to have a low efficiency of producing live, healthy offspring: In one 2006 report, half of the 28 pregnancies at day 30 were lost by day 90, and all of the remaining 14 fetuses died by day 4 after birth (89). Detailed observations from the Roslin Institute illustrate the wide variety of major structural defects observed in cloned lambs that died at or just after birth (90). Prominent systems affected were renal (hydronephrosis), cardiac (right ventricular hypertrophy), pulmonary (pulmonary hypertension), hepatic (few bile ducts), and musculoskeletal (contracted tendons). Pre- or perinatal abnormalities such as hydrallantois, fetal lesions (including omphalocele), ascites, cardiac enlargement, liver steatosis, and asynchronous growth of organs have been observed. Early pig and goat cloning programs have not shown any increase in birth abnormalities (91– 93). This may be due to chance, as these experiments did not produce large numbers of offspring; however, it is also possible that these species will consistently have reduced problems at birth. Although early swine cloning programs reported good neonatal survival, over half of the cloned piglets (22 of 40) produced by Park et al. (94) died suddenly owing to low birth weights, cardiopulmonary abnormalities, and meningitis. Estrada et al. (95) found significantly lower birth weights in a large group of 143 pig clones matched against over 1,000 controls. Within this group of cloned neonates, both the healthy and unhealthy clones were smaller than controls. The abnormal, unhealthy neonates were substantially lighter (94) than their healthy littermates. This larger trial backs early data that showed a nonsignificant tendency toward low birth weights in pig clones (94). As smaller placentas were also observed in early gestation in pig clones, the lower birth weight suggests intrauterine growth restriction caused by inadequate placental function (95). Depressed immune function has been observed in both bovine and porcine clones (96, 97). In the latter study in piglets, a reduced response to endotoxin challenge was observed. Large numbers of cloned mice have now been produced, and the majority of experiments show low rates of birth abnormalities and high neonatal survival (72, 98, 99). Murine experiments with low neonatal survival were related to donor cell lines (40). Inoue et al. (100) recently demonstrated a substantial improvement in the survival to term of cloned mouse embryos by altering expression of an X chromosome–linked gene, Xist. Xist has a role in the inactivation of the female X chromosome, and when knocked out, expression of other X-linked www.annualreviews.org

Abnormal Offspring


16.7

arav2Hill

ARI

03 November 2013

07:44

genes approached normal patterns (100). A reduction in expression of Xist via either gene knockout or temporary RNAi inhibition improved development to term by 10 times, to almost 15% (100, 101). The improvement caused by RNAi inhibition was observed in male but not in female embryos (102).

Postnatal Development


The first four surviving cloned sheep (Dolly plus three others of fetal cell origin) were apparently normal. Dolly went on to produce several litters of lambs that demonstrated apparently normal physiology. Additional testing by the Roslin Institute revealed subtle variations from normal, such as shorter telomere lengths and mitochondrial DNA derived exclusively from the enucleated donor oocyte (103, 104). Other groups examined bovine clones and found telomere lengths to be elongated (105) or normal (106, 107). In six generations of cloned mice, telomeres were no shorter and even appeared to elongate with successive generations (108). Dolly was well looked after and died at seven years of age—middle-aged for a sheep. Her death prompted consideration of how the cloning process contributed to her relatively early death. Although Dolly’s death was attributed to a viral disease endemic in her native Scotland, she did have other abnormalities that occur more often in clones than in the general population. For example, Dolly had arthritis, which may or may not have been due to her cloning origin, as cloned cattle have a higher incidence of musculoskeletal abnormalities (109). Dolly is the most famous and best-studied individual, although more robust conclusions can be drawn from larger studies using animals across techniques, cell lines, and time periods. Several years of meticulously collected data at AgResearch, New Zealand, concluded that the annual mortality rate (including humane euthanasia) in somatic cell–cloned cattle is higher than in control cattle. It is also higher than that seen in the progeny of cloned cows. The most common causes of euthanasia are musculoskeletal problems, such as contracted flexor tendons and lameness. Contracted tendons are generally associated with uterine crowding due to an oversized fetus. This may also be the case in cloned pregnancies, although contracted tendons may also occur in cloned calves of normal birth weights (109). Two large North American studies that included commercial cloning services provide overviews of the current status of the art of cloning. According to data from two years of cloned pregnancies using 34 genetically different cell lines, 20% of pregnancies survived to term, and the majority (75%) of these survived to adulthood. Although a wide variation in birth weights was observed, the subsequent growth rates, reproductive performance, and milk production were similar to those of non-cloned dairy cattle (110). In a second study from Cyagra/Goyaike that included five years of data, losses from birth to five months were 42% and were due to enlarged umbilical cords (37%), respiratory problems (19%), prolonged recumbency (20%), and contracted flexor tendons (21%) (62). Cows and bulls that survived to adulthood showed normal production characteristics. In farm animals, production-related data, such as growth rates, milk production, behavior, and fertility, appear to be within normal ranges (109–113). Milk and meat products from cloned animals were similar to naturally mated animals, though there were some subtle differences, such as delayed onset of puberty, muscle maturation, and higher blood-neutrophil counts (114). Hematological variations have been observed in prepubertal cloned calves, which had higher mean corpuscular volume but lower hemoglobin and hematocrit (85). Another group found 24 cloned adult cows to have normal hematology and biochemistry profiles (115). Body temperature and leptin were raised, although T4 (tetraiodothyronine) was reduced in the first two weeks. IGF-II was higher at birth but lower at two weeks (85). Others have observed high levels of IGFBP-2, with 16.8

Hill


arav2Hill

ARI

03 November 2013

07:44

lower IGF-1 and cortisol at birth (116). At birth, cloned lambs also have higher ACTH and cortisol levels (117). Cloned mice have been found to be overweight, which suggests that very detailed evaluations of cloned animals may reveal subtle abnormalities (118). High leptin levels occurred in obese cloned mice with a shortened life span. Reduced life span was observed in adult cloned mice owing to pneumonia and liver lesions from ten months to two years of age. There was also a delay in learning for young mice prior to weaning (which disappeared after weaning) (118). When those obese cloned mice were mated, the obese phenotype was not transmitted to their progeny.


CONCLUSIONS Embryos are exquisitely sensitive to their environment in utero, and the combination of embryo culture with the nuclear transfer process increases the chance of an altered phenotype. The wide variability in development of nuclear transfer and IVP pregnancies results in substantial gestational and perinatal loss, such that less than 10% of nuclear transfer cattle embryos transferred to surrogate cows will develop into viable calves. (39). The variability in changes observed in cloned offspring (both within and between genetic lines) suggests a high degree of randomness in the effect of the technique upon later phenotype (6). Fetal oversize occurs in cloned ruminants derived from both embryonic and somatic cells (8, 15, 119). It also occurs in animals produced by in vitro embryo culture, which suggests that culture conditions can exaggerate the effect of cloning on fetal size (8, 18). Whereas fetal oversize occurs in cloned cattle, sheep, and mice, the opposite (fetal undersize) occurs in cloned goats and pigs (95, 112). Other fetal abnormalities, such as pathology of the cardiopulmonary, urinary, or immune systems, have a low incidence in IVP fetuses and are more specific to cloned fetuses (1, 79, 96, 120). Because postnatal health is affected by in utero conditions, IVP or cloned offspring are also prone to later health issues. Imprinted genes are central to the physiological changes in cloned fetuses and their placentas (33). The nuclear transfer process alters the normal imprinting patterns expected during early development (121). Although this causes significant problems in utero and postnatally, expression of imprinted genes is generally normalized by maturity (122, 123). Careful investigations of abnormal offspring have enabled insights into embryonic development, fetomaternal interactions, and the impact of very early perturbations on later phenotypes.

DISCLOSURE STATEMENT The author is not aware of any affiliations, memberships, funding, or financial holdings that might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS I gratefully acknowledge the skills, knowledge, and dedication of colleagues at AgResearch, Cyagra-Goyaike, Cornell University, INRA Jouy-en-Josas Centre, Roslin Institute, and Texas A&M University. LITERATURE CITED 1. Wilmut I, Schnieke AE, McWhir J, Kind AJ, Campbell KH. 1997. Viable offspring derived from fetal and adult mammalian cells. Nature 385:810–13

www.annualreviews.org

Abnormal Offspring


16.9

ARI

03 November 2013

07:44

2. Schnieke AE, Kind AJ, Ritchie WA, Mycock K, Scott AR, et al. 1997. Human factor IX transgenic sheep produced by transfer of nuclei from transfected fetal fibroblasts. Science 278:2130–33 3. Wells DN, Misica PM, Day TA, Tervit HR. 1997. Production of cloned lambs from an established embryonic cell line: a comparison between in vivo- and in vitro-matured cytoplasts. Biol. Reprod. 57:385–93 4. Cibelli JB, Stice SL, Golueke PJ, Kane JJ, Jerry J, et al. 1998. Cloned transgenic calves produced from nonquiescent fetal fibroblasts. Science 280:1256–58 5. Ogonuki N, Inoue K, Yamamoto Y, Noguchi Y, Tanemura K, et al. 2002. Early death of mice cloned from somatic cells. Nat. Genet. 30:253–54 6. Wilmut I. 2006. Are there any normal clones? Methods Mol. Biol. 348:307–18 7. Tervit HR, Whittingham DG, Rowson LEA. 1972. Successful culture in vitro of sheep and cattle ova. J. Reprod. Fertil. 30:493–97 8. Thompson JG, Gardner DK, Pugh PA, McMillan WH, Tervit HR. 1995. Lamb birth weight is affected by culture system utilized during in vitro pre-elongation development of ovine embryos. Biol. Reprod. 53:1385–91 9. Walker SK, Heard TM, Seamark RF. 1992. In vitro culture of sheep embryos without co-culture: successes and perspectives. Theriogenology 37:111–26 10. Behboodi E, Anderson GB, BonDurant RH, Cargill SL, Kreuscher BR, et al. 1995. Birth of large calves that developed from in vitro-derived bovine embryos. Theriogenology 44:227–32 11. Kruip ThAM, den Daas JHG. 1997. In vitro produced and cloned embryos: effects on pregnancy, parturition and offspring. Theriogenology 47:43–52 12. Schmidt M, Greve T, Avery B, Beckers JF, Sulon J, Hansen HB. 1996. Pregnancies, calves and calf viability after transfer of in vitro produced bovine embryos. Theriogenology 46:527–39 13. Hasler JF, Henderson WB, Hurtgen PJ, Jin ZQ, McCauley AD, et al. 1995. Production, freezing and transfer of bovine IVF embryos and subsequent calving results. Theriogenology 43:141–52 14. Hasler JF. 2000. In vitro culture of bovine embryos in Ménézo’s B2 medium with or without coculture and serum: the normalcy of pregnancies and calves resulting from transferred embryos. Anim. Reprod. Sci. 60:81–91 15. Wilson JM, Williams JD, Bondioli KR, Looney CR, Westhusin ME, McCalla DF. 1995. Comparison of birth weight and growth characteristics of bovine calves produced by nuclear transfer (cloning), embryotransfer and natural mating. Anim. Reprod. Sci. 38:73–83 16. Willadsen SM, Janze RE, McAlister RJ, Shea BF, Hamilton G, McDermand D. 1991. The viability of late morulae and blastocysts produced by nuclear transplantation in cattle. Theriogenology 35:161–70 17. Trounson A, Pushett D, Maclellan LJ, Lewis I, Gardner DK. 1994. Current status of IVM/IVF and embryo culture in humans and farm animals. Theriogenology 41:57–66 18. Young LE, Sinclair KD, Wilmut I. 1998. Large offspring syndrome in cattle and sheep. Rev. Reprod. 3:155–63 19. Walker SK, Hartwich KM, Seamark RF. 1996. Production of unusually large offspring following embryo manipulation: concepts and challenges. Theriogenology 45:111–20 20. Farin PW, Farin CE. 1995. Transfer of bovine embryos produced in vivo or in vitro: survival and fetal development. Biol. Reprod. 52:676–82 21. Holm P, Walker SK, Seamark RF. 1996. Embryo viability, duration of gestation and birth weight in sheep after transfer of in vitro matured and in vitro fertilized zygotes cultured in vitro or in vivo. J. Reprod. Fertil. 107:175–81 22. Sinclair KD, McEvoy TG, Maxfield EK, Maltin CA, Young LE, et al. 1999. Aberrant fetal growth and development after in vitro culture of sheep zygotes. J. Reprod. Fertil. 116:177–86 23. Watson AJ, Watson PH, Arcellana-Panlilio M, Warnes D, Walker SK, et al. 1994. A growth factor phenotype map for ovine preimplantation development. Biol. Reprod. 50:725–33 24. van Wagtendonk-de Leeuw AM, Aerts BJG, den Daas JHG. 1998. Abnormal offspring following in vitro production of bovine preimplantation embryos: a field study. Theriogenology 49:883–94 25. van Wagtendonk-de Leeuw AM, Mullaart E, de Roos APW, Merton JS, den Daas JHG, et al. 2000. Effects of different reproduction techniques: AI, moet or IVP, on health and welfare of bovine offspring. Theriogenology 53:575–97


arav2Hill

16.10

Hill



arav2Hill

ARI

03 November 2013

07:44

26. Jacobsen H, Schmidt M, Holm P, Sangild PT, Vajta G, et al. 2000. Body dimensions and birth and organ weights of calves derived from in vitro produced embryos cultured with or without serum and oviduct epithelium cells. Theriogenology 53:1761–69 27. Lane M, Gardner DK. 1994. Increase in postimplantation development of cultured mouse embryos by amino acids and induction of fetal retardation and exencephaly by ammonium ions. J. Reprod. Fertil. 102:305–12 28. Peterson AJ, Lee RS. 2003. Improving successful pregnancies after embryo transfer. Theriogenology 59:687–97 29. Lane M, Gardner DK, Hasler MJ, Hasler JF. 2003. Use of G1 2/G2 2 media for commercial bovine embryo culture: equivalent development and pregnancy rates compared to co-culture. Theriogenology 60:407–19 30. Hasler JF. 2000. In-vitro production of cattle embryos: problems with pregnancies and parturition. Hum. Reprod. 15:47–58 31. Thompson JG, Peterson AJ. 2000. Bovine embryo culture in vitro: new developments and post-transfer consequences. Hum. Reprod. 15(Suppl. 5):59–67 32. McEvoy TG, Sinclair KD, Broadbent PJ, Goodhand KL, Robinson JJ. 1998. Post-natal growth and development of Simmental calves derived from in vivo or in vitro embryos. Reprod. Fertil. Dev. 10:459–64 33. Young LE, Fernandes K, McEvoy TG, Butterwith SC, Gutierrez CG, et al. 2001. Epigenetic change in IGF2R is associated with fetal overgrowth after sheep embryo culture. Nat. Genet. 27:153–54 34. Beaujean N, Taylor J, Gardner J, Wilmut I, Meehan R, Young L. 2004. Effect of limited DNA methylation reprogramming in the normal sheep embryo on somatic cell nuclear transfer. Biol. Reprod. 71:185–93 35. Kleemann DO, Walker SK, Seamark RF. 1994. Enhanced fetal growth in sheep administered progesterone during the first three days of pregnancy. J. Reprod. Fertil. 102:411–17 36. Sinclair KD, Dunne LD, Maxfield EK, Maltin CA, Young LE, et al. 1998. Fetal growth and development following temporary exposure of day 3 ovine embryos to an advanced uterine environment. Reprod. Fertil. Dev. 10:263–69 37. McEvoy TG, Robinson JJ, Aitken RP, Findlay PA, Robertson IS. 1997. Dietary excesses of urea influence the viability and metabolism of preimplantation sheep embryos and may affect fetal growth among survivors. Anim. Reprod. Sci. 47:71–90 38. Rivera RM, Stein P, Weaver JR, Mager J, Schultz RM, Bartolomei MS. 2008. Manipulations of mouse embryos prior to implantation result in aberrant expression of imprinted genes on day 9.5 of development. Hum. Mol. Genet. 17:1–14 39. Lee RS, Peterson AJ, Donnison MJ, Ravelich S, Ledgard AM, et al. 2004. Cloned cattle fetuses with the same nuclear genetics are more variable than contemporary half-siblings resulting from artificial insemination and exhibit fetal and placental growth deregulation even in the first trimester. Biol. Reprod. 70:1–11 40. Eggan K, Akutsu H, Loring J, Jackson-Grusby L, Klemm M, et al. 2001. Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo complementation. Proc. Natl. Acad. Sci. USA 98:6209–14 41. Constant F, Guillomot M, Heyman Y, Vignon X, Laigre P, et al. 2006. Large offspring or large placenta syndrome? Morphometric analysis of late gestation bovine placentomes from somatic nuclear transfer pregnancies complicated by hydrallantois. Biol. Reprod. 75:122–30 42. Ogawa H, Ono Y, Shimozawa N, Sotomaru Y, Katsuzawa Y, et al. 2003. Disruption of imprinting in cloned mouse fetuses from embryonic stem cells. Reproduction 126:549–57 43. Everts RE, Chavatte-Palmer P, Razzak A, Hue I, Green CA, et al. 2008. Aberrant gene expression patterns in placentomes are associated with phenotypically normal and abnormal cattle cloned by somatic cell nuclear transfer. Physiol. Genomics 33:65–77 44. Dean W, Bowden L, Aitchison A, Klose J, Moore T, et al. 1998. Altered imprinted gene methylation and expression in completely ES cell-derived mouse fetuses: association with aberrant phenotypes. Development 125:2273–82


Abnormal Offspring


16.11

ARI

03 November 2013

07:44

45. Wang ZQ, Kiefer F, Urbanek P, Wagner EF. 1997. Generation of completely embryonic stem cell-derived mutant mice using tetraploid blastocyst injection. Mech. Dev. 62:137–45 46. Shimozawa N, Ono Y, Kimoto S, Hioki K, Araki Y, et al. 2002. Abnormalities in cloned mice are not transmitted to the progeny. Genesis 34:203–7 47. Wang ZQ, Fung MR, Barlow DP, Wagner EF. 1994. Regulation of embryonic growth and lysosomal targeting by the imprinted Igf2/Mpr gene. Nature 372:464–67 48. Chavatte-Palmer P, Camous S, Jammes H, Le Cleac’h N, Guillomot M, Lee RSF. 2012. Review: Placental perturbations induce the developmental abnormalities often observed in bovine somatic cell nuclear transfer. Placenta 33(Suppl.):S99–104 49. Heyman Y, Chavatte-Palmer P, LeBourhis D, Camous S, Vignon X, Renard JP. 2002. Frequency and occurrence of late-gestation losses from cattle cloned embryos. Biol. Reprod. 66:6–13 50. Campbell KH, McWhir J, Ritchie WA, Wilmut I. 1996. Sheep cloned by nuclear transfer from a cultured cell line. Nature 380:64–66 51. Wilmut I, Sales DI, Ashworth CJ. 1986. Maternal and embryonic factors associated with prenatal loss in mammals. J. Reprod. Fertil. 76:851–64 52. Armitage JA, Khan IY, Taylor PD, Nathanielsz PW, Poston L. 2004. Developmental programming of the metabolic syndrome by maternal nutritional imbalance: How strong is the evidence from experimental models in mammals? J. Physiol. 561:355–77 53. Kwong WY, Osmond C, Fleming TP. 2004. Support for Barker hypothesis upheld in rat model of maternal undernutrition during the preimplantation period: application of integrated ’random effects’ statistical model. Reprod. Biomed. Online 8:574–76 54. McMillen IC, Robinson JS. 2005. Developmental origins of the metabolic syndrome: prediction, plasticity, and programming. Physiol. Rev. 85:571–633 55. Parkinson T. 2001. Arthur’s Veterinary Reproduction and Obstetrics. London: WB Saunders 56. De Sousa PA, King T, Harkness L, Young LE, Walker SK, Wilmut I. 2001. Evaluation of gestational deficiencies in cloned sheep fetuses and placentae. Biol. Reprod. 65:23–30 57. Baguisi A, Behboodi E, Melican DT, Pollock JS, Destrempes MM, et al. 1999. Production of goats by somatic cell nuclear transfer. Nat. Biotechnol. 17:456–61 58. Hill JR, Burghardt RC, Jones K, Long CR, Looney CR, et al. 2000. Evidence for placental abnormality as the major cause of mortality in first-trimester somatic cell cloned bovine fetuses. Biol. Reprod. 63:1787–94 59. Alexander BM, Johnson MS, Guardia RO, Van den Graaf WL, Senger PL, Sasser RG. 1995. Embryonic loss from 30 to 60 days post breeding and the effect of palpation per rectum on pregnancy. Theriogenology 43:551–56 60. Forar AL, Gay JM, Hancock DD, Gay CC. 1996. Fetal loss frequency in ten Holstein dairy herds. Theriogenology 45:1505–13 61. Hill JR, Schlafer DH, Fisher PJ, Davies CJ. 2002. Abnormal expression of trophoblast major histocompatibility complex class I antigens in cloned bovine pregnancies is associated with a pronounced endometrial lymphocytic response. Biol. Reprod. 67:55–63 62. Panarace M, Agüero JI, Garrote M, Jauregui G, Segovia A, et al. 2007. How healthy are clones and their progeny: 5 years of field experience. Theriogenology 67:142–51 63. Forsberg EJ, Strelchenko NS, Augenstein ML, Betthauser JM, Childs LA, et al. 2002. Production of cloned cattle from in vitro systems. Biol. Reprod. 67:327–33 64. Farin PW, Crosier AE, Farin CE. 2001. Influence of in vitro systems on embryo survival and fetal development in cattle. Theriogenology 55:151–70 65. Barker DJP. 2000. In utero programming of cardiovascular disease. Theriogenology 53:555–74 66. Kohan-Ghadr HR, Fecteau G, Smith LC, Murphy BD, Lefebvre RC. 2011. Endocrine profiles of somatic nuclear transfer-derived pregnancies in dairy cattle. Theriogenology 76:911–20 67. Constant F, Camous S, Chavatte-Palmer P, Heyman Y, de Sousa N, et al. 2011. Altered secretion of pregnancyassociated glycoproteins during gestation in bovine somatic clones. Theriogenology 76:1006–21 68. Hoffert KA, Batchelder CA, Bertolini M, Moyer AL, Famula TR, et al. 2005. Measures of maternal-fetal interaction in day-30 bovine pregnancies derived from nuclear transfer. Cloning Stem Cells 7:289–305


arav2Hill

16.12

Hill



arav2Hill

ARI

03 November 2013

07:44

69. Gardner RL, Squire S, Zaina S, Hills S, Graham CF. 1999. Insulin-like growth factor-2 regulation of conceptus composition: effects of the trophectoderm and inner cell mass genotypes in the mouse. Biol. Reprod. 60:190–95 70. Wakisaka-Saito N, Kohda T, Inoue K, Ogonuki N, Miki H, et al. 2006. Chorioallantoic placenta defects in cloned mice. Biochem. Biophys. Res. Commun. 349:106–14 71. Stice SL, Strelchenko NS, Keefer CL, Matthews L. 1996. Pluripotent bovine embryonic cell lines direct embryonic development following nuclear transfer. Biol. Reprod. 54:100–10 72. Wakayama T, Yanagimachi R. 1999. Cloning of male mice from adult tail-tip cells. Nat. Genet. 22:127–28 73. Chae JI, Lee KS, Kim DJ, Han YM, Lee DS, et al. 2009. Abnormal gene expression in extraembryonic tissue from cloned porcine embryos. Theriogenology 71:323–33 74. Whitworth KM, Prather RS. 2010. Somatic cell nuclear transfer efficiency: How can it be improved through nuclear remodeling and reprogramming? Mol. Reprod. Dev. 77:1001–15 75. Batchelder CA, Bertolini M, Mason JB, Moyer AL, Hoffert KA, et al. 2007. Perinatal physiology in cloned and normal calves: physical and clinical characteristics. Cloning Stem Cells 9:63–82 76. Bertolini M, Moyer AL, Mason JB, Batchelder CA, Hoffert KA, et al. 2004. Evidence of increased substrate availability to in vitro-derived bovine foetuses and association with accelerated conceptus growth. Reproduction 128:341–54 77. Bazer FW, Roberts RM, Basha SM, Zavy MT, Caton D, Barron DH. 1979. Method for obtaining ovine uterine secretions from unilaterally pregnant ewes. J. Anim. Sci. 49:1522–27 78. Hill JR, Edwards JF, Sawyer N, Blackwell C, Cibelli JB. 2001. Placental anomalies in a viable cloned calf. Cloning 3:83–88 79. Hill JR, Roussel AJ, Cibelli JB, Edwards JF, Hooper NL, et al. 1999. Clinical and pathologic features of cloned transgenic calves and fetuses (13 case studies). Theriogenology 51:1451–65 80. Tanaka S, Oda M, Toyoshima Y, Wakayama T, Tanaka M, et al. 2001. Placentomegaly in cloned mouse concepti caused by expansion of the spongiotrophoblast layer. Biol. Reprod. 65:1813–21 81. Kohan-Ghadr HR, Lefebvre RC, Fecteau G, Smith LC, Murphy BD, et al. 2008. Ultrasonographic and histological characterization of the placenta of somatic nuclear transfer-derived pregnancies in dairy cattle. Theriogenology 69:218–30 82. Hill JR, Chavatte-Palmer P. 2002. Pregnancy and neonatal care of cloned animals. In Principles of Cloning, ed. J Cibelli, R Lanza, KHS Campbell, MD West, 1:247–66. San Diego, CA: Academic 83. Wells DN. 2003. Cloning in livestock agriculture. Reproduction 61:131–50 84. Chavatte-Palmer P, Heyman Y, Richard C, Monget P, LeBourhis D, et al. 2002. Clinical, hormonal, and hematologic characteristics of bovine calves derived from nuclei from somatic cells. Biol. Reprod. 66:1596–603 85. Chavatte-Palmer P, Remy D, Cordonnier N, Richard C, Issenman H, et al. 2004. Health status of cloned cattle at different ages. Cloning Stem Cells 6:94–100 86. Hill JR, Winger QA, Long CR, Looney CR, Thompson JA, Westhusin ME. 2000. Development rates of male bovine nuclear transfer embryos derived from adult and fetal cells. Biol. Reprod. 62:1135–40 87. Watanabe S, Nagai T. 2009. Death losses due to stillbirth, neonatal death and diseases in cloned cattle derived from somatic cell nuclear transfer and their progeny: a result of nationwide survey in Japan. Anim. Sci. J. 80:233–38 88. Brisville AC, Fecteau G, Boysen S, Dorval P, Buczinski S, et al. 2011. Respiratory disease in neonatal cloned calves. J. Vet. Intern. Med. 25:373–79 89. Loi P, Clinton M, Vackova I, Fulka J Jr, Feil R, et al. 2006. Placental abnormalities associated with postnatal mortality in sheep somatic cell clones. Theriogenology 65:1110–21 90. Rhind SM, King TJ, Harkness LM, Bellamy C, Wallace W, et al. 2003. Cloned lambs—lessons from pathology. Nat. Biotechnol. 21:744–45 91. Keefer CL, Baldassarre H, Keyston R, Wang B, Bhatia B, et al. 2001. Generation of dwarf goat (Capra hircus) clones following nuclear transfer with transfected and nontransfected fetal fibroblasts and in vitro-matured oocytes. Biol. Reprod. 64:849–56 92. Polejaeva IA, Chen S, Vaught TD, Page RL, Mullins J, et al. 2000. Cloned pigs produced by nuclear transfer from adult somatic cells. Nature 407:86–90


Abnormal Offspring


16.13

ARI

03 November 2013

07:44

93. Betthauser J, Forsberg E, Augenstein M, Childs L, Eilertsen K, et al. 2000. Production of cloned pigs from in vitro systems. Nat. Biotechnol. 18:1055–59 94. Park M, Cho S, Lee S, Choi Y, Park J, et al. 2005. A rare and often unrecognized cerebromeningitis and hemodynamic disorder: a major cause of sudden death in somatic cell cloned piglets. Proteomics 5:1928–39 95. Estrada J, Sommer J, Collins B, Mir B, Martin A, et al. 2007. Swine generated by somatic cell nuclear transfer have increased incidence of intrauterine growth restriction (IUGR). Cloning Stem Cells 9:229–36 96. Renard JP, Chastant S, Chesne P, Richard C, Marchal J, et al. 1999. Lymphoid hypoplasia and somatic cloning. Lancet 353:1489–91 97. Carroll JA, Carter DB, Korte SW, Prather RS. 2005. Evaluation of the acute phase response in cloned pigs following a lipopolysaccharide challenge. Domest. Anim. Endocrinol. 29:564–72 98. Ogura A, Inoue K, Ogonuki N, Lee J, Kohda T, Ishino F. 2002. Phenotypic effects of somatic cell cloning in the mouse. Cloning Stem Cells 4:397–405 99. Wakayama T, Perry ACF, Zuccotti M, Johnson KR, Yanagimachi R. 1998. Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei. Nature 394:369–74 100. Inoue K, Kohda T, Sugimoto M, Sado T, Ogonuki N, et al. 2010. Impeding Xist expression from the active X chromosome improves mouse somatic cell nuclear transfer. Science 330:496–99 101. Matoba S, Inoue K, Kohda T, Sugimoto M, Mizutani E, et al. 2011. RNAi-mediated knockdown of Xist can rescue the impaired postimplantation development of cloned mouse embryos. Proc. Natl. Acad. Sci. USA 108:20621–26 102. Oikawa M, Matoba S, Inoue K, Kamimura S, Hirose M, et al. 2013. RNAi-mediated knockdown of Xist does not rescue the impaired development of female cloned mouse embryos. J. Reprod. Dev. 59:231–37 103. Shiels PG, Kind AJ, Campbell KH, Waddington D, Wilmut I, et al. 1999. Analysis of telomere lengths in cloned sheep. Nature 399:316–17 104. Evans MJ, Gurer C, Loike JD, Wilmut I, Schnieke AE, Schon EA. 1999. Mitochondrial DNA genotypes in nuclear transfer-derived cloned sheep. Nat. Genet. 23:90–93 105. Lanza RP, Cibelli JB, Blackwell C, Cristofalo VJ, Francis MK, et al. 2000. Extension of cell life-span and telomere length in animals cloned from senescent somatic cells. Science 288:665–69 106. Betts DH, Bordignon V, Hill JR, Winger Q, Westhusin ME, et al. 2001. Reprogramming of telomerase activity and rebuilding of telomere length in cloned cattle. Proc. Natl. Acad. Sci. USA 98:1077–82 107. Jiang L, Carter DB, Xu J, Yang X, Prather RS, Tian XC. 2004. Telomere lengths in cloned transgenic pigs. Biol. Reprod. 70:1589–93 108. Wakayama T, Shinkai Y, Tamashiro KL, Niida H, Blanchard DC, et al. 2000. Cloning of mice to six generations. Nature 407:318–19 109. Wells DN, Forsyth JT, McMillan V, Oback B. 2004. The health of somatic cell cloned cattle and their offspring. Cloning Stem Cells 6:101–10 110. Pace MM, Augenstein ML, Betthauser JM, Childs LA, Eilertsen KJ, et al. 2002. Ontogeny of cloned cattle to lactation. Biol. Reprod. 67:334–39 111. Enright BP, Taneja M, Schreiber D, Riesen J, Tian XC, et al. 2002. Reproductive characteristics of cloned heifers derived from adult somatic cells. Biol. Reprod. 66:291–96 112. Behboodi E, Ayres SL, Memili E, O’Coin M, Chen LH, et al. 2005. Health and reproductive profiles of malaria antigen-producing transgenic goats derived by somatic cell nuclear transfer. Cloning Stem Cells 7:107–18 113. Tecirlioglu RT, Cooney MA, Korfiatis NA, Hodgson R, Williamson M, et al. 2006. Semen and reproductive profiles of genetically identical cloned bulls. Theriogenology 65:1783–99 114. Heyman Y, Chavatte-Palmer P, Fromentin G, Berthelot V, Jurie C, et al. 2007. Quality and safety of bovine clones and their products. Animal 1:963–72 115. Lanza RP, Cibelli JB, Faber D, Sweeney RW, Henderson B, et al. 2001. Cloned cattle can be healthy and normal. Science 294:1893–94 116. Matsuzaki M, Shiga K. 2002. Endocrine characteristics of cloned calves. Cloning Stem Cells 4:261–67 117. Edwards L, Peura T, Hartwich K, Rudiger S, McMillen IC, Walker S. 2002. Postnatal growth and circulating ACTH and cortisol concentrations during the first month of life in cloned lambs. Endocrinology 143:3699–702


arav2Hill

16.14

Hill


arav2Hill

ARI

03 November 2013

07:44


118. Tamashiro KL, Wakayama T, Blanchard RJ, Blanchard DC, Yanagimachi R. 2000. Postnatal growth and behavioral development of mice cloned from adult cumulus cells. Biol. Reprod. 63:328–34 119. Garry FB, Adams R, McCann JP, Odde KG. 1996. Postnatal characteristics of calves produced by nuclear transfer cloning. Theriogenology 45:141–52 120. Wells DN, Misica PM, Tervit HR, Vivanco WH. 1998. Adult somatic cell nuclear transfer is used to preserve the last surviving cow of the Enderby Island cattle breed. Reprod. Fertil. Dev. 10:369–78 121. Smith LC, Suzuki J Jr, Goff AK, Filion F, Therrien J, et al. 2012. Developmental and epigenetic anomalies in cloned cattle. Reprod. Domest. Anim. 47(Suppl. 4):107–14 122. Yang L, Chavatte-Palmer P, Kubota C, O’Neill M, Hoagland T, et al. 2005. Expression of imprinted genes is aberrant in deceased newborn cloned calves and relatively normal in surviving adult clones. Mol. Reprod. Dev. 71:431–38 123. Tamashiro KL, Wakayama T, Akutsu H, Yamazaki Y, Lachey JL, et al. 2002. Cloned mice have an obese phenotype not transmitted to their offspring. Nat. Med. 8:262–67


Abnormal Offspring


16.15

From Embryos to Adults: A DOHaD Perspective on In Vitro Fertilization and Other Assisted Reproductive Technologies.

International committee for monitoring assisted reproductive technologies: world report on assisted reproductive technologies, 2007.

Birth defects and assisted reproductive technologies.

Intrapartum interventions for singleton pregnancies arising from assisted reproductive technologies.

Surgical treatment before assisted reproductive technologies.

Current and future assisted reproductive technologies for fish species.

Current and future assisted reproductive technologies for mammalian farm animals.

International Committee for Monitoring Assisted Reproductive Technologies world report: Assisted Reproductive Technology 2006.

Excellence in assisted reproductive technologies: clinical and laboratory perspectives.

Spermbots: potential impact for drug delivery and assisted reproductive technologies.

Assisted reproductive technologies: impact on fetal and neonatal outcomes.

Religious views regarding treatment of infertility by assisted reproductive technologies.

Endometriosis-Related Infertility: The Role of the Assisted Reproductive Technologies.

Fertility preservation in the age of assisted reproductive technologies.

Training in reproductive endocrinology and infertility and assisted reproductive technologies: options and worldwide needs.

Adherence compounds in embryo transfer media for assisted reproductive technologies.

Cardiovascular dysfunction in children conceived by assisted reproductive technologies.

DNA methylation and gene expression changes derived from assisted reproductive technologies can be decreased by reproductive fluids.

Association between serum folate and vitamin B-12 and outcomes of assisted reproductive technologies.

Effects of the use of assisted reproductive technologies and an obesogenic environment on resistance artery function and diabetes biomarkers in mice offspring.

Increased Length of Awareness of Assisted Reproductive Technologies Fosters Positive Attitudes and Acceptance among Women.

The impact of assisted reproductive technologies on the genome and epigenome of the newborn.

The effect of assisted reproductive technologies on gynecological cancer: report of our experiences and literature review.

Association of Clomiphene and Assisted Reproductive Technologies With the Risk of Neural Tube Defects.