Insulin-like growth factor-I and binding proteins 1, 2, and 3 in bovine nuclear transfer pregnancies

In cloned pregnancies, placental deficiencies, including increased placentome size, reduced placentome number, and increased accumulation of allantoic fluid, have been associated with low cloning efficiency. To assess differences in paracrine and endocrine growth regulation in cloned versus normal bovine placentomes and pregnancies, we have examined the expression of insulin-like growth factor (IGF)-I and -II and their binding proteins (IGFBP)-1 through -3 in placentomes of artificially inseminated (AI), in vitro-produced (IVP), and nuclear transfer (NT) pregnancies at Days 50, 100, and 150 of gestation. Fetal, maternal, and binucleate cell counts in representative placentomes were performed on Days 50-150 of gestation in all three groups. Increased numbers of fetal, maternal, and binucleate cells were present in NT placentomes at all stages of gestation examined. Immunolocalization studies showed that spatial and temporal patterns of expression of IGFBP-2 and -3 were markedly altered in the placentomes of NT pregnancies compared to AI/IVP controls. Concentrations of IGF-I in fetal plasma, as determined by RIA, were significantly higher (P = 0.001) in NT pregnancies (mean +/- SEM, 30.3 +/- 2.3 ng/ml) compared with AI (19.1 +/- 5.5 ng/ml) or IVP (24.2 +/- 2.5 ng/ml) pregnancies on Day 150 of gestation. Allantoic fluid levels of IGFBP-1 were also increased in NT pregnancies. These findings suggest that endocrine and paracrine perturbations of the IGF axis may modulate placental dysfunction in NT pregnancies. Furthermore, increased cell numbers in NT placentomes likely have significant implications for fetomaternal communication and may contribute to the placental overgrowth observed in the NT placentomes.     

Large offspring or large placenta syndrome? Morphometric analysis of late gestation bovine placentomes from somatic nuclear transfer pregnancies complicated by hydrallantois

Somatic nuclear transfer (NT) in cattle is often complicated by fetal oversize (i.e., large offspring syndrome), hydrallantois, and placentomegaly in late gestation. The aims of this work were to obtain data on the placentome structure in NT-recipient cows with hydrallantois (NTH) and to relate these with fetal and placental weights to better understand the abnormalities observed in NTH pregnancies during the third trimester. Pregnant cows were slaughtered between Gestation Days 180 and 280. The fetuses were weighed, and the placentomes were numbered and weighed. Placentomes were examined by histologic and stereological techniques. Macroscopic data showed that placental overgrowth preceded fetal overgrowth, and the ratio of the fetal to the total placentome weight in the NTH group was lower than that in controls after Gestation Day 220. This suggests that placental overgrowth is due to placental default rather than due to fetal overgrowth, as shown also by stereological analysis showing primary deregulation of the growth of cotyledonary tissues. Observed alterations, such as thinning of the maternal epithelium within placentomes and increased trophoblastic surface, could be secondary adaptations. Thus, placental growth deregulations would be due to modifications of the expression of placental factors. Various examples of placental deficiency were observed, suggesting that some fetal abnormalities observed in NTH calves, such as enlarged heart, enlarged umbilical cord, and abdominal ascites, are consequences of placental dysfunction. Therefore, the condition described by the term "large offspring syndrome" might better be described by "large placenta syndrome," because this syndrome affects an average of 50% of late-gestation NT pregnancies. No conclusion can be drawn from this work on apparently normal pregnancies.     

Clinical,hormonal, and hematologic characteristics of bovine calves derived from nuclei from somatic cells

Although healthy animals are born after nuclear transfer with somatic cells nuclei, the success of this procedure is generally poor (2%-10%) with high perinatal losses. Apparently normal surviving animals may have undiagnosed pathologies that could develop later in life. The gross pathology of 16 abnormal bovine fetuses produced by nuclear transfer (NT) and the clinical, endocrinologic (insulin-like growth factors I and II [IGF-I and IGF-II], IGF binding proteins, post-ACTH stimulation cortisol, leptin, glucose, and insulin levels), and biochemical characteristics of a group of 21 apparently normal cloned calves were compared with those of in vitro-produced (IVP) controls and controls resulting from artificial insemination. Oocytes used for NT or IVP were matured in vitro. NT to enucleated oocytes was performed using cultured adult or fetal skin cells. After culture, Day 7, grade 1-2 embryos were transferred (one per recipient). All placentas and fetuses from clones undergoing an abnormal pregnancy showed some degree of edema due to hydrops. Mean placentome number was lower and mean placentome weight was higher in clones than in controls (69.9 +/- 9.2 placentomes with a mean weight of 144.3 +/- 21.4 g in clones vs. 99 and 137 placentomes with a mean individual weight of 34.8 and 32.4 g in two IVP controls). Erythrocyte mean cell volume was higher at birth (P < 0.01), and body temperature and plasma leptin concentrations were higher and T4 levels were lower during the first 50 days and the first week (P < 0.05), respectively, in clones. Plasma IGF-II concentrations were higher at birth and lower at Day 15 in clones (P < 0.05). Therefore, apparently healthy cloned calves cannot be considered as physiologically normal animals until at least 50 days of age.     

Ultrasonographic and histological characterization of the placenta of somatic nuclear transfer-derived pregnancies in dairy cattle

The high incidence of pregnancy loss and prenatal morbidity and mortality in cloned animals may be due to placental insufficiency, thereby compromising fetal survival. Our objective was to characterize morphological changes in fetal membranes of cloned bovine pregnancies. Two groups of cows with cloned fetuses, produced by two cloning techniques, a commercial group (n=16) and a hand-made group (n=4), and control fetuses derived from traditional embryo transfer (n=6) or AI (n=6), were compared at various stages of gestation (Days 80, 120, 150, 180, 210, and 240; Day 0=estrus). Thickness and shape of the amniotic membrane, placentome shape and length, umbilical cord shape and diameter, and fetal fluid echodensities were assessed by ultrasonography, and the placenta was evaluated histologically. Only eight (40%) of cloned pregnancies reached term and seven calves (35%) were alive at birth. Both placentome length and umbilical cord diameter were larger (P<0.05) in clones than in normal fetuses at all stages of gestation. Amniotic membrane abnormalities (Day 120) including focal edema and the presence of a series of nodules were detected in 38% of the clones and were always accompanied by hyper-echodense spikes or irregularities (detected ultrasonographically) around the umbilical cord. Histopathology revealed degenerate inflammatory cells, edematous chorioallantoic membranes, and decreased epithelial thickness. We inferred that these morphological anomalies of placentomes compromised fetal development, and we concluded that ultrasonographic monitoring of pregnancies enabled characterization of changes in the placentae and may be useful to assess fetal well-being.     

Functional significance and cortisol dependence of the gross morphology of ovine placentomes during late gestation

The gross morphological appearance of ovine placentomes is known to alter in response to adverse intrauterine conditions that increase fetal cortisol exposure. The direct effects of fetal cortisol on the placentome morphology, however, remain unknown, nor is the functional significance of the different placentome types clear. The present study investigated the gross morphology of ovine placentomes in relation to placental nutrient delivery to sheep fetuses during late gestation and after experimental manipulation of the fetal cortisol concentration. As fetal cortisol levels rose naturally toward term, a significant decrease was observed in the proportion of the D-type placentomes that had the hemophagous zone everted over the bulk of the placentomal tissue. When the prepartum cortisol surge was prevented by fetal adrenalectomy, there were proportionately more everted C- and D-type placentomes and fewer A-type placentomes with the hemophagous zone inverted into the placentome compared with those of intact fetuses at term. Raising cortisol concentrations by infusion before term reduced the incidence of D-type placentomes and lowered the proportion of individually tagged placentomes that became more everted during the 10- to 15-day period between tagging and delivery. Cortisol, therefore, appears to prevent hemophagous zone eversion in ovine placentomes during late gestation. The distribution of placentome types appeared to have no effect on the net rates of placental delivery of glucose and oxygen to the fetus under normal conditions. When fetal cortisol levels were raised by exogenous infusion, however, placental delivery of glucose, but not oxygen, to the fetus, measured as umbilical uptake, was reduced to a greater extent in fetuses with a higher proportion of C- and D-type placentomes. The gross morphology of the ovine placentomes is, therefore, determined, at least in part, by the fetal cortisol concentration and may influence placental nutrient transfer when fetal cortisol concentrations are high during late gestation. These findings have important implications for the placental control of fetal growth and development, particularly during adverse intrauterine conditions.     

Implantation and placental development in somatic cell clone recipient cows

Successful somatic cloned animal production has been reported in various domesticated species, including cattle; however, it is associated with a high rate of pregnancy failure. The low cloning yield could possibly arise from either an abnormal and/or poorly developed placenta. In comparison to control cows, fewer placentomes were found in somatic cell nuclear recipient (NT) cows at day 60 of gestation, suggesting a retardation of fetal/placental growth in these animals. NT cows not only had fewer numbers of chorionic villi but also had poorly developed caruncles. Macroscopic examination revealed atypical development of the placentome in terms of shape and size. Histological disruption of chorionic villi and caruncular septum was found in NT cows. Of particular interest was that the expression of genes, as well as proteins in the placentome, was disparate between NT and artificially inseminated cows, especially placental lactogen (PL) and pregnancy-associated glycoprotein (PAG). In contrast, prolactin-related protein-1 (PRP-1) signals were comparable across cows, including NT cows carrying immotile fetuses. The expression of extracellular matrix degrading molecule, heparanase (HPA), in NT cows was divergent from that of control cows. Microarray data suggest that gene expression was disorientated in early stages of implantation in NT cows, but this was eliminated with progression of gestation. These findings strongly support a delay in trophoblast development during early stages of placentation in NT cows, and suggest that placental specific proteins, including PLs, PAGs, and HPA, are key indicators for the aberration of gestation and placental function in cows.     

Perturbations in the biochemical composition of fetal fluids are apparent in surviving bovine somatic cell nuclear transfer pregnancies in the first half of gestation

Amniotic and allantoic fluid volumes and composition change dynamically throughout gestation. Cattle that are pregnant with somatic cell nuclear transfer (NT) fetuses show a high incidence of abnormal fluid accumulation (particularly hydrallantois) and fetal mortality from approximately midgestation. To investigate fetal fluid homeostasis in these pregnancies, Na, K, Cl, urea, creatinine, Ca, Mg, total PO(4), glucose, fructose, lactate, total protein, and osmolalities were measured in amniotic and allantoic fluids collected at Days 50, 100, and 150 of gestation from NT pregnancies and those generated by the transfer of in vitro-produced embryos or by artificial insemination. Deviations in fetal fluid composition between NT and control pregnancies were apparent after placental and fetal organ development, even when no gross morphological abnormalities were observed. Individual NT fetuses were affected to varying degrees. Elevated allantoic Na was associated with lower K and increased allantoic fluid volume or edema of the fetal membranes. Total PO(4) levels in NT allantoic and amniotic fluid were elevated at Days 100 and 150. This was not accompanied by hypophosphatemia at Day 150, suggesting that PO(4) acquisition by NT fetuses was adequate but that its readsorption by the kidneys may be impaired. Excessive NT placental weight was associated with low allantoic glucose and fructose as well as high lactate levels. However, the fructogenic ability of the NT placenta appeared to be normal. The osmolality of the fetal fluids was maintained within a narrow range, suggesting that the regulation of fluid composition, but not osmolality, was impaired in NT pregnancies.     

Morphometric analysis of collagen in gestational and retained bovine placentomes

Bovine placentome collagen was quantified (P<0.01) at four gestational stages (90, 150, 210 and 270 d, n = 8 d ), at 2 h post partum without (n = 4) and at 2 and 12 h post partum with (n = 8) experimentally-induced placental retention. Placentome sections were fixed and stained for collagen. Fetal cotyledonary (FC) collagen volume fraction (V(V)) increased over days of gestation studied (V(V)=0.03+/-0.01, 0.06+/-0.01, 0.13+/-0.01 and 0.19+/-0.01). Fetal cotyledonary hydroxyproline (3.15+/-0.41, 4.55+/-0.41 and 7.04+/-0.41 mg/g) and FC protein (432.0+/-17.1, 479.9+/-17.1, 585.4+/-17.1 mg/g) increased over Days 90, 150 and 210 and were similar on Days 210 and 270. Fetal cotyledonary collagen V(V) and hydroxyproline did not differ between Day 270, retained and nonretained cotyledons. Protein concentration was higher in 2 h (578.1+/-18.5 mg/g) and 12 h (526.0+/-18.5 mg/g) retained versus nonretained (400.4+/-36.2 mg/g) cotyledons. Maternal caruncular (MC) collagen V(V) and protein concentration were higher on Days 90 and 150 than on Days 210 and 270. Maternal caruncular hydroxyproline was similar from Day 90 to 210 and increased from Day 210 to 270. Maternal caruncular collagen V(V), hydroxyproline and protein concentrations were similar on Day 270 and in 2 h and 12 h retained membrane caruncles. Gestational increases in placentome collagen occurred from FC sources. No difference in FC or MC collagen V(V) existed between Day 270, retained and nonretained placentomes.     

Evidence for placental abnormality as the major cause of mortality in first-trimester somatic cell cloned bovine fetuses

The production of cloned animals is, at present, an inefficient process. This study focused on the fetal losses that occur between Days 30-90 of gestation. Fetal and placental characteristics were studied from Days 30-90 of gestation using transrectal ultrasonography, maternal pregnancy specific protein b (PSPb) levels, and postslaughter collection of fetal tissue. Pregnancy rates at Day 30 were similar for recipient cows carrying nuclear transfer (NT) and control embryos (45% [54/120] vs. 58% [11/19]), although multiple NT embryos were often transferred into recipients. From Days 30-90, 82% of NT fetuses died, whereas all control pregnancies remained viable. Crown-rump (CR) length was less in those fetuses that were destined to die before Day 90, but no significant difference was found between the CR lengths of NT and control fetuses that survived to Day 90. Maternal PSPb levels at Days 30 and 50 of gestation were not predictive of fetal survival to Day 90. The placentas of six cloned and four control (in vivo or in vitro fertilized) bovine pregnancies were compared between Days 35 and 60 of gestation. Two cloned placentas showed rudimentary development, as indicated by flat, cuboidal trophoblastic epithelium and reduced vascularization, whereas two others possessed a reduced number of barely discernable cotyledonary areas. The remaining two cloned placentas were similar to the controls, although one contained hemorrhagic cotyledons. Poor viability of cloned fetuses during Days 35-60 was associated with either rudimentary or marginal chorioallantoic development. Our findings suggest that future research should focus on factors that promote placental and vascular growth and on fetomaternal interactions that promote placental attachment and villous formation.     

Fetal and placental weight relationships in the rat at days 13 and 17 of gestation

Mean fetal and placental weights were, respectively, 0.018 and 0.051 g on Day 13 and 0.376 and 0.250 g on Day 17. Fetal and placental weights within litters were weakly correlated on Day 13 (r = 0.322) but not on Day 17. Litter size was negatively correlated with placental weight on Day 17 (r = -0.485) but not with fetal weight. Male fetuses were heavier than female fetuses on Day 17 but their placentas were not significantly different. Fetuses and placentas were lighter at the ovarian end of the uterine horn on both days examined, revealing an early influence of local environmental factors on their growth.     

Local changes in uterine dimensions in the pregnant pig

Twenty gilts were used in a study to determine local utilization of space by fetuses in the uterus under crowded or roomy conditions. Between Days 2 and 4 after onset of estrus and mating, the uterus was ligated to leave one and one-half times as much space for embryos in one side of the uterus (roomy) as in the other (crowded). At a second surgery on Day 28, serosal sutures were placed to mark the placental boundaries and every third embryo in the crowded side was crushed. Gilts were slaughtered on Day 100 and measurements taken on the uterus and fetuses. Survival from Day 28 to Day 100 of fetuses that were not crushed was 57% in the roomy side of the uterus versus 40% in the crowded side (P = 0.42). No differences were found between crowded and roomy sides for fetal crown rump length, weight or placental surface area. The length of segment occupied on Day 28 by fetuses surviving to Day 100 in both crowded and roomy sides was greater than the mean length occupied by all embryos present on Day 28. The length of segment occupied by surviving fetuses increased by 9.4 ± 1.4 cm from Day 28 to Day 100, and the length of segment on Day 28 occupied by a fetus that was crushed decreased by 2.0 ± 0.8 cm. The internal vascular area of the uterus associated with a surviving fetus on Day 100 did not extend beyond the suture placed in the serosa on Day 28, indicating that the surviving fetuses did not make use of space vacated by nonsurviving littermates.     

Maternal undernutrition from early- to mid-gestation leads to growth retardation,cardiac ventricular hypertrophy,and increased liver weight in the fetal sheep

Early gestation is critical for placentomal growth, differentiation, and vascularization, as well as fetal organogenesis. The fetal origins of adult disease hypothesis proposes that alterations in fetal nutrition and endocrine status result in developmental adaptations that permanently change structure, physiology, and metabolism, thereby predisposing individuals to cardiovascular, metabolic, and endocrine disease in adult life. Multiparous ewes were fed to 50% (nutrient restricted) or 100% (control fed) of total digestible nutrients from Days 28 to 78 of gestation. All ewes were weighed weekly and diets adjusted for individual weight loss or gain. Ewes were killed on Day 78 of gestation and gravid uteri recovered. Fetal body and organ weights were determined, and numbers, morphologies, diameters, and weights of all placentomes were obtained. From Day 28 to Day 78, restricted ewes lost 7.4% of body weight, while control ewes gained 7.5%. Maternal and fetal blood glucose concentrations were reduced in restricted versus control pregnancies. Fetuses were markedly smaller in the restricted group than in the control group. Further, restricted fetuses exhibited greater right- and left-ventricular and liver weights per unit fetal weight than control fetuses. No treatment differences were observed in any gross placentomal measurement. However, caruncular vascularity was enhanced in conceptuses from nutrient-restricted ewes but only in twin pregnancies. While these alterations in fetal/placental development may be beneficial to early fetal survival in the face of a nutrient restriction, their effects later in gestation as well as in postnatal life need further investigation.     

Endocrine profiles of somatic nuclear transfer-derived pregnancies in dairy cattle

In cattle, several hormones and proteins are necessary for maintenance of a normal pregnancy that will result in a viable calf. Deviation from the normal cascade or expected profile of reproductive hormones and proteins may be associated with impairment of somatic nuclear transfer-derived pregnancies and the high rate of fetal loss. The objectives of this study were to characterize maternal plasma concentrations of pregnancy-specific protein B (PSPB), progesterone (P4), estrone sulphate (E₁S), and estradiol (E2) during the last two-thirds of pregnancy (cloned calves), and to determine associations with gestational abnormalities. Cows with cloned fetuses, produced by either commercial (N = 16) or zona-free (N = 4) cloning techniques, were compared with pregnant animals derived from traditional embryo transfer (N = 6) or AI (N = 6), at various stages of gestation (Days 80, 120, 150, 180, 210, and 240; Day 0 = estrus). Fetal well-being was monitored with ultrasonography throughout gestation. At Day 80, progesterone concentration was lower (P < 0.0001) in nuclear transfer (NT) recipients than in control groups. Mean estrone sulphate concentrations did not vary significantly between NT and control groups. At Day 150, pregnancy-specific protein B concentrations were elevated (P < 0.002) in NT cows. Estradiol concentration was higher in NT recipients than control cows throughout the study period.     

Immunolocalization of progesterone receptors in bovine placentomes throughout mid and late gestation and at parturition.

The corpus luteum is the main source of progesterone (P(4)) responsible for maintenance of gestation in cattle. So far it has not been possible to assign any biological role to placental P(4), which contributes only marginally and temporarily to peripheral maternal blood levels. In order to identify possible P(4) target cells within the placenta, placentomes from 150-, 220-, 240-, and 270-day-pregnant cows and from parturient cows (3 animals per group) were screened immunohistochemically for expression of the progesterone receptor (PR). During gestation, PR-positive staining was found exclusively in the nuclei of caruncular stromal cells (CSC; maternal part of the placentome) and of caruncular vascular pericytes. In placentomes from parturient cows, occasional positive nuclear staining was also observed in the walls of small caruncular arteries. The percentage of PR-positive CSC increased slightly from 51.8 +/- 2.6% on Day 150 to 56.2 +/- 5.6% at Day 270 (p < 0.05) and was 58.9 +/- 1.8% at parturition. These results suggest that in pregnant cattle, CSC are under the control of P(4) of placental rather than luteal origin. Thus, whereas luteal P(4) may regulate "coarse" systemic progestational functions in the maternal compartment in the classical hormonal manner, placental P(4) may act as a paracrine factor involved in the local regulation of caruncular growth, differentiation, and functions.     

Fetal and placental traits at day 35 of pregnancy in relation to the estrogen receptor genotype in pigs

Fetuses from gilts with estrogen receptor (ESR) genotype AA (AA-AA and AA-AB) and BB (BB-AB and BB-BB) were compared at Day 35/36 of pregnancy, to examine whether fetal ESR genotype nested within maternal ESR genotype would affect fetal traits. Furthermore the relation of fetal body weight and fetal heart weight to various placental traits were evaluated relative to ESR genotype. Fetal and placental weight and length, and implantation surface area were not affected by fetal ESR genotype nested within maternal ESR genotype. Fetal weight was related similarly to placental length, placental weight, and implantation surface area: up to a certain threshold value (40 cm, 40 g and 250 cm2, respectively), an increase in the trait was associated with an increase of fetal weight. Thereafter, fetal weight did not change anymore. Thus, at Day 35/36 of pregnancy porcine fetuses seem to have a maximum growth potential. The percentage of AA-AA fetuses that had not reached this maximum growth potential was larger than of the other three genotype combinations studied, and therefore a higher subsequent fetal mortality may be expected in this group. Hearts of AA-AB fetuses were significantly heavier than those of BB-AB and BB-BB fetuses and tended to be heavier than those of AA-AA fetuses. The reason for this hypertrophy is unclear, but might be related to a difference in placental vascularity. Heart weight of fetuses from BB gilts increased with fetal weight, while heart weights of fetuses from AA gilts did not. Heart weight increased with an increase of placental length and implantation surface area up to 51 cm and 437 cm2, respectively, and thereafter decreased again. For BB-AB fetuses a similar relation was found between heart weight and placental weight, while heart weight of the other three genotype combinations remained unaffected as placental weight increased. The fetus and placenta are continuously changing during early pregnancy, therefore different mechanisms may change the demands for cardiac output. However, keeping in mind that placental size and blood volume are relatively large, placental vascularity and vascular development may play a major role. Therefore, further research on heart size, placental size and vascularity, relative to ESR genotype, is recommended.     

Morphology and morphometry of in vivo- and in vitro-produced bovine concepti from early pregnancy to term and association with high birth weights

This study was designed to characterize conceptus development based on pre- and postnatal measurements of in vivo- and in vitro-derived bovine pregnancies. In vivo-produced embryos were obtained after superovulation, whereas in vitro-produced embryos were derived from established procedures for bovine IVM, IVF and IVC. Blastocysts were transferred to recipients to obtain pregnancies of single (in vivo/singleton or in vitro/singleton groups) or twin fetuses (in vitro/twins group). Ultrasonographic examinations were performed weekly, from Day 30 of gestation through term. Videotaped images were digitized, and still-frames were used for the measurement of conceptus traits. Calves and fetal membranes (FM) were examined and measured upon delivery. In vitro-produced fetuses were smaller than in vivo controls (P < 0.05) during early pregnancy (Day 37 to Day 58), but in vitro/singletons presented significantly higher weights at birth than in vivo/control and in vitro/twin calves (P < 0.05). From late first trimester of pregnancy (Day 72 to Day 93), placentomes surrounding in vitro-derived singleton fetuses were longer and thinner than controls (P < 0.05). At term, the presence of giant cotyledons in the fetal membranes in the in vitro group was associated with a larger cotyledonary surface area in the fetal horn (P < 0.05). The biphasic growth pattern seen in in vitro-produced pregnancies was characterized by conceptus growth retardation during early pregnancy, followed by changes in the development of the placental tissue. Resulting high birth weights may be a consequence of aberrant placental development due to the disruption of the placental restraint on fetal growth toward the end of pregnancy.     

Tissue-specific elevated genomic cytosine methylation levels are associated with an overgrowth phenotype of bovine fetuses derived by in vitro techniques

Epigenetic perturbations are assumed to be responsible for abnormalities observed in fetuses and offspring derived by in vitro techniques. We have designed an experiment with bovine Day 80 fetuses generated by somatic cell nuclear transfer (SCNT), in vitro fertilization (IVF), and artificial insemination (AI) to determine the relationship between fetal phenotype and genome-wide 5-methylcytosine (5mC) content. When compared with AI controls, SCNT and IVF fetuses displayed significantly increased body weight (61% and 28%), liver weight (100% and 36%), and thorax circumference (20% and 11%). A reduced crown-rump length:thorax circumference ratio (1.175 +/- 0.017 in SCNT and 1.292 +/- 0.018 in IVF vs. 1.390 +/- 0.018 in AI, P < 0.001 and P < 0.002) was the external hallmark of this disproportionate overgrowth phenotype. The SCNT fetuses showed significant hypermethylation of liver DNA in comparison with AI controls (3.46% +/- 0.08% vs. 3.17% +/- 0.09% 5mC, P < 0.03), and the cytosine methylation levels for IVF fetuses (3.34% +/- 0.09%) were, as observed for phenotypic parameters, intermediate to the other groups. Regressions of fetal body and liver weight and thorax circumference on 5mC content of liver DNA were positive (P < 0.073-0.079). Furthermore, a significant negative regression (P < 0.021) of the crown-rump length:thorax circumference ratio on liver 5mC was observed. The 5mC content of placental cotyledon DNA was 46% lower than in liver DNA (P < 0.0001) but did not differ among groups. These data are in striking contrast with the recently reported hypomethylation of DNA from SCNT fetuses and indicate that hypermethylation of fetal tissue, but not placenta, is linked to the overgrowth phenotype in bovine SCNT and IVF fetuses.