THE PLACENTA AS A CONTRIBUTOR TO ... M. Bertolini and G.B. Anderson

M. Bertolini and G.B. Anderson
Department of Animal Science, University of California, Davis, CA 95616
Unusually high birth weights frequently occur in calves born from cultured bovine embryos. The
mechanism(s) through which in vitro manipulations during early cleavage are translated to enhanced
fetal growth is (are) incompletely understood. Accelerated growth is primarily prenatal, and the
placenta of an in vitro-derived conceptus could account for abnormal fetal growth. Results from a
systematic comparison of placental morphology and function in bovine concepti produced in vitro
versus in vivo are discussed.
Q 2001 by Elsevier Science Inc.
Key words: large calves, embryo manipulation, placental membranes
More than a decade ago, rumors circulated that some calves developing from embryos produced
by nuclear transfer procedures, using blastomeres as nuclear donors, were excessively large at birth.
Speakers at a 1992 satellite symposium of the International Embryo Transfer Society (IETS)
confirmed these rumors (17). Several years later, our laboratory reported that calves with
excessively high birth weights had developed from embryos cultured in vitro from fertilization to the
blastocyst stage (2), an observation reported earlier for cultured sheep embryos (22). Abnormally
high growth was also reported in 7-mo fetuses developing from oocytes having undergone IVM,
IVF, and IVC (7). Despite early skepticism by some researchers that in vitro embryo manipulations
were responsible for development of large calves, skepticism fed by the circumstance that some
laboratories using some culture systems had not observed large calves from cultured embryos, in
vitro manipulation of bovine cleavage-stage embryos is now widely accepted to produce the “large
offspring syndrome’: in some calves. Ironically, as described by Barnes (1). the first nuclear transfer
calves with abnormally high birth weights were produced using donor cells from in vivo-derived
embryos, recipient cytoplasm from in vivo-matured oocytes, and in vivo culture to the blastocyst
stage in a sheep oviduct.
The observation that in vitro manipulation of cleavage-stage bovine embryos can lead to aberrant
fetal growth is of both practical and biological significance. “Large calves” frequently present
delivery an&or survival problems that raise questions of animal welfare. Embryo manipulation
procedures that produce large calves will find only limited practical field use until the large calf
problems are solved. Large calves also raise fascinating questions of fundamental biology. For
example, why is it that bovine embryos can be split into halves, frozen in liquid nitrogen, or
subjected to numerous other insults and still develop to term normally, but in vitro attempts to mimic
in vivo conditions for early development can result in abnormal post-embryo transfer development to
0 2001 Elsevier Science
87, 2002
PII: SOO93-691
front matter
The large offspring syndrome has been the subject of numerous recent reviews (9) including a
symposium paper at the 200 1 IETS meeting (11) and a satellite symposium at the 2000 IETS meeting
(6). During 2001 the popular press even announced, based on a scientific publication with more
restrained conclusions (26), that the gene producing large offspring had been identified. What new is
there to report?
Molecular analysis of tissues from large fetuses resulting from nuclear transfer and from in vitro
embryo production has led to the suggestion that imprinted genes are involved in the large offspring
syndrome. Blondin et al. (4) reported that in vitro embryo production increases IGF-2 mRNA
expression in Day 70 fetal liver and decreases expression in skeletal muscle. Young et al. (26) found
similar steady-state transcript levels of IGF-2 mRNA in liver, kidney, and muscle but reduced
methylation and expression of the IGF-2 receptor gene of control and “large” in vitro-produced (IVP)
Day 125 sheep fetuses; however, reduced methylation was not seen consistently across all large
fetuses. These authors proposed that cleavage-stage embryos are vulnerable to epigenetic alterations
of imprinted genes and that unfavorable (i.e., unphysiological) IVC conditions lead to faulty gene
The physiological link between individual gene expression and the large-calf phenotype is not
always clear. Moreover, phenotypes and expression of the large calf syndrome are variable. For
example, in addition to high birth weights, some IVP calves suffer perinatal health problems that
may or may not be associated with birth weight. The degree to which these symptoms occur, similar
to the range in birth weights, is variable and unpredictable.
What do we know about birth weights and other characteristics of IVP calves? Despite the
attention paid to large calves, most IVP calves are normal. The distribution of birth weights is
shifted to the right, meaning that both the mean birth weight and the upper end of the range are
higher (15) an outcome also described for lambs born from cultured embryos (23). Importantly, the
excessive growth appears to be a prenatal problem; once born, with a few exceptions (16) growth
rates seem to normalize (24). The latter observation is particularly intriguing, because it implies that
conditions for expression of the large-calf phenotype exist during the prenatal but not postnatal
period. In seeking a physiological explanation for a problem that is primarily prenatal, altered
placental function would provide a logical role. The remainder of this paper will focus on the
potential role of the placenta in promoting excessive growth in IVP calves.
Evidence for Placental Abnormalities in IVP Calves
The earliest characterizations of IVP calves included mention of abnormal placental morphology
or function. Hasler et al. (13) and van Wagtendonk-de Leeuw et al. (20,21) reported an increased
incidence of hydrallantois in IVP calves, a problem typically associated with placental (versus fetal)
abnormalities. Farin and Farin (7) reported that IVP embryos develop fewer placentomes than do
normal, in vivo concepti, but, in another experiment conducted by Farin et al. (8), the number of
placentomes in Day 70 IVP concepti was not different from in vivo concepti. The same laboratory
(10) further studied Day 222 IVP concepti and reported heavier placentas in association with larger
fetuses and reduced caruncular surface area and villous density.
From results of a study conducted on nuclear transfer calves during the perinatal period, Garry et
al. (12) proposed that some observed conditions affecting calf survival and well-being could be due
to placental dysfunction. Stice et al. (19) reported failure of cotyledonary development in nucleartransfer concepti, and Hill et al. (14) later proposed that the primary cause of pregnancy losses in
bovine nuclear transfer pregnancies is faulty placental development.
These authors further
speculated that an abnormal placenta could affect development of an otherwise normal IVP fetus.
Most recently, DeSousa et al. (5) concluded that ovine nuclear transfer concepti suffer both fetal and
placental deficiencies, including fetal liver enlargement, dermal hemorrhage, and lack of placental
vascular development reflected in reduced or absent cotyledonary structures.
The retarded
development of cloned fetuses was strikingly associated with avascular or hypovascular placentas,
similar to the observations of Hill et al. (14).
Evidence Contrary to Placental Involvement in Large Calves
Although aberrant placental development has been reported in IVP concepti, some researchers
have not found an association between abnormal placentas and excessive growth. Sinclair et al. (18)
reported that, in Day 125 ovine fetuses developing from cultured embryos, increased fetal weight
was not the consequence of larger placentas. These and other data led Young et al. (25) to conclude,
“Thus, the overgrowth appears to be driven by the fetus and not the placenta, although placental
function, rather than size, may be affected.”
New Evidence of Placental Dysfunction in IVP Bovine Embryos
We conducted a series of experiments aimed at measuring placental morphology and function in
IVP versus typical embryo transfer calves. Oocytes were collected at slaughter from beef females for
IVM, IVF, and IVC. Embryos were co-cultured to the blastocyst stage with oviductal cells in
medium containing serum. In vivo-produced blastocysts were collected from superovulated,
inseminated beef females of similar breeding to oocyte donors. In vivo and IVP Day 7 blastocysts
were transferred nonsurgically to the uteri of synchronized recipients.
In one experiment, IVP and control embryos were allowed to develop in a physiologically
synchronous uterine environment to Day 16, after which they were retrieved from the uterus for
physical and molecular analysis. Distinct patterns were observed in Day 7 versus Day 16 IVP
embryos; specifically, Day 7 IVP embryos compared with in vivo embryos displayed differences in
gene expression that became less distinct by Day 16. Morphologically, Day 16 IVP concepti
presented smaller embryonic discs and a trend toward shorter trophoblastic lengths than controls.
In another experiment, pregnancies for IVP and in vivo control embryos were diagnosed on Day
30 of gestation, and recipients were either slaughtered on Days 90 and 180 of gestation or allowed to
carry the pregnancies to term. On Days 90 and 180, placental and fetal morphology and placental
function (substrate permeability, placental hormones, and proteins) were studied. Sixty minutes
before slaughter, a saline solution containing 10% D-xylose, a marker for substrates using the
glucose transporter system, was infused intravenously into each recipient. On Day 90 of gestation,
fetal D-glucose concentrations were lower (P <0.05) in IVP pregnancies, which presented fetuses
with greater crown-rump lengths and organ weights (P <0.05) and a tendency for greater total
placentome gross surface area (P =0.07). Physical traits such as fetal, liver, heart and ptacentome
weights correlated with one another and were greater in IVP pregnancies on Day 180 (P <0.05),
except for a trend toward greater fetal cro~vn-rump length (P =0.06). Physical traits also correlated
with total D-glucose and D-xylose accumulated in the allantoic and amniotic fluid and total fluids.
These results suggest differences in substrate transport between IVP and control pregnancies;
enhanced substrate transport to IVP fetuses might be expected to support greater fetal growth during
late pregnancy.
For the group of recipients allowed to carry pregnancies to term, fetal and placental development
during pregnancy and after delivery was examined. Ultrasound examinations were performed
weekly with images from embryos/fetuses and placentomes (amniotic vesicle and crown-rump
length, foreleg and humeral length, diameter and surface area of eye socket, heart rate, diameter and
thickness of placentomes) recorded. After birth, calf birth weights and several physical traits were
recorded. Our results revealed significant differences in embryonic, fetal, and placentome
development between IVP and control pregnancies at different stages of development. Following the
same developmental pattern seen on Day 16, IVP embryos and early fetuses were smaller than in
vivo controls, based on ultrasonographic examinations (Figure 1). Day 180 IVP pregnancies
sustained larger concepti, which coincided with increased substrate transport from the maternal
circulation to the fetus and associated fluids.
Figure 1. Sagittal views of in vivo- (left: 45.7 mm) and in vitro-derived fetuses (right: 35.0 mm)
on Day 51 of gestation (scale bars = 10 mm).
Birth weights oflVP calves were 33% higher (P <0.05) than controls, but gestation lengths were
similar. Linear measurements (e.g., crown-rump length, heart girth circumference, femoral length,
height at shoulders) for each calf increased linearly relative to birth weight, i.e., calf body size was
proportional to body mass in both IVP and in vivo-derived calves.
The most dramatic morphological observation related to the range in size of placentomes and
term cotyledons in the IVP group. Differences existed in the distributions of placentomes and
:otyledonary diameters, thicknesses, and surface areas in the fetal horn between IVP and control
pregnancies. Placentomes immediately surrounding the fetus, in the mid region of the fetal horn,
were longer and thinner from mid to late first trimester of IVP pregnancies. Placentome size
distribution, weight, and surface area in the fetal horn differed between groups on Days 90 and 180
of gestation. The presence of enlarged placentomes and cotyledons on the fetal membranes of IVP
concepti was associated with a larger gross surface area in the fetal horn on Days 90 and 180 and at
term. Fetal membranes from IVP calves contained 21% fewer cotyledons (P >0.05) than calves in
the control group but had a larger cotyledonary surface area in the fetal horn, as a result of a different
distribution of cotyledonary size (P <0.0001). The largest cotyledons measured 13 and 21 cm in
diameter in the fetal horn for control and IVP calves, respectively. Approximately 13% of
cotyledons in the fetal horn were larger than 13 cm in diameter in IVP calves. Cotyledonary surface
area was greater in IVP than control calves, which likely was linked to the presence of giant
cotyledons in IVP concepti (Figure 2).
Figure 2. Fetal membranes of in vivo- (left) and in vitro-derived (right) calves in the umbilical cord
area (scale bars = 20 cm; numbers on the cotyledons are the individual diameters in cm)
Concentrations oflGF- 1 and IGF-2 and their binding proteins were also measured in maternal and
fetal plasma and amniotic and allantoic fluids. Maternal plasma concentrations of IGF-1 in IVP
pregnancies were elevated throughout gestation; the highest levels occurred in the first trimester of
pregnancy, coinciding with the period of growth retardation in IVP concepti. Components of the
IGF system were differentially regulated, according to gestation period (Day 90 or 180), fluid type
(maternal or fetal plasma, amniotic or allantoic fluid), and origin of conceptus (IVP versus control
embryos). Increases in fetal weight resulting from IVP embryos appeared to be associated with
increases in concentrations oflGF- 1 and IGF-2 in the maternal circulation and alterations in the IGF
binding proteins within amniotic and allantoic fluids that are evident at or before Day 90 of
Significant embryonic and fetal losses (27/49) occurred between Days 30 and 65 of gestation in
the IVP group. The majority of losses took place between Days 30 and 44 (17/27). The in vivo
group presented lower losses (11/51), which occurred predominantly dispersed throughout the first
trimester, between Days 30 and 90 of gestation. Although we have not systematically analyzed the
physiological explanations for this high loss rate in IVP pregnancies, it is intriguingly suggestive to
speculate that faulty placental development may be linked to early losses and growth retardation, as
reported by DeSouza et al. (5) and Hill et al. (14) for cloned sheep and cattle.
Placenta: Important or Not in Producing Large Calves?
Our results presented in the previous section strongly suggest that morphological and functional
differences between IVP and in vivo-derived pregnancies could affect fetal growth and calf birth
weight. How are these results reconciled with other reports of large IVP calves in the absence of
placental defects? Certainly in some previous studies in which large calves were reported, including
our own (2), placental membranes were neither collected nor examined. Other studies in which
placental anomalies were not identified involved examination of placentas during pregnancy but not
at term. Moreover, just as all IVP calves are not large, variations in phenotypes might be expected
with regard to placental normalcy in IVP concepti. Placental and fetal development in IVP concepti
might be viewed as a spectrum instead of in discreet categories. Finally, similarities and differences
might be expected in IVP concepti resulting from IVM/IVF/IVC and those from nuclear transfer; it
has yet to be determined whether large calves and associated anomalies resulting from IVC and those
from nuclear transfer result from the same developmental perturbations or hold different positions
along the same spectrum.
1. Barnes, FL. The effects of the early uterine environment on the subsequent development of
embryo and fetus. Theriogenology 2000;53:649-658.
2. Behboodi, E, Anderson, GB, BonDurant, RH, Cargill, SL, Kreuscher, BR, Medrano, JF, Murray,
JD. Birth of large calves that development from in vitro-derived bovine embryos.
Theriogenology 1995;44:227-232.
3. Bertolini M, Famula TR, Anderson GB. Appearance of giant cotyledons in the Large Calf
Syndrome. Proceedings, Western Section, American Society of Animal Science 2000,5 1:65-68.
4. Blondin P, Farin PW, Crosier AE, Alexander JE, Farin CE. In vitro production of embryo alters
levels of insulin-like growth factor-II messenger ribonucleic acid in bovine fetuses 63 days after
transfer. Biol Reprod 2000;62:384-389.
5. De Sousa PA, King T, Harkness L, Young LE, Walker SK, Wilmut I. Evaluation of gestational
deficiencies in cloned sheep fetuses and placentae. Biol Reprod 2001;65:23-30.
6. Dieleman, SJ. Proceedings of the satellite symposium of the International Embryo Transfer
Society, Maastricht, The Netherlands. Theriogenology 2000;53:658.
7. Farin PW, Farin CE. Transfer of bovine embryos produced in vivo or in vitro: survival and fetal
development. Biol Reprod 1995;52:676-682.
8. Farin CE, Farin PW, Mungal SA. Maternal insulin-like growth factor-I (IGF-I) and development
of in vivo-and in vitro-derived bovine fetuses. Theriogenology 1997;47-3 18 abst.
9. Farin CE, Farin PW, Blondin P, Crosier AE. Fetal development of manipulated embryos:
Possible association with uterine function. 1999 Biennial Symposium on Animal Reproduction.
Proceedings, Am. Sot. Anim. Sci., Available at:
10. Farin PW, Stockburger EM, Rodrigues KF, Crosier AE, Blondin P, Alexander JE, Farin CE.
Placental morphology following transfer of bovine embryos produced in vivo or in vitro.
Theriogenology 2000;53:474 abst.
11. Farin PW, Crosier AE, Farin CE. Influence of in vitro systems on embryo survival and fetal
development in cattle. Theriogenology 2001 ;55:151-170.
12. Garry FB, Adams R, McCann JP, Odde KG. Postnatal characteristics of calves produced by
nuclear transfer cloning. Theriogenology 1996;45:141-152.
13. Hasler JF, Henderson WB, Hurtgen PJ, Jin ZQ, McCauley AD, Mower SA, Neely B, Shuey LS,
Stokes JE, Trimmer SA. Production, freezing and transfer of bovine IVF embryos and
subsequent calving results. Theriogenology 1995;43:141-152.
14. Hill JR, Burghardt RC, Jones K, Long CR, Looney CR, Shin T, Spencer TE, Thompson JA,
Winger QA, Westhusin ME. Evidence of placental abnormality as a major cause of mortality in
first-trimester somatic cell cloned bovine fetuses. Biol Reprod 2000;63:1787-1794.
15. Kruip ThAM, den Daas JHG. In vitro produced and cloned embryos: Effects on pregnancy,
parturition and offspring. Theriogenology 1997;47:43-52.
16. McEvoy TG, Sinclair KD, Broadbent PJ, Goodhand KL, Robinson JJ. Post-natal growth and
development of Simmental calves derived from in vivo or in vitro embryos. Reprod Fertil Dev
1998; 10:459-64.
17. Seidel, GE Jr. Proceedings Symposium on Cloning Mammals by Nuclear Transplantation. 1992.
Colorado State University, Fort Collins, CO.
18. Sinclair KD, McEvoy TG, Carolan C, Maxfield EK, Maltin CA, Young LE, Wilmut I, Robinson
J J, Broadbent PJ. Conceptus growth and development following in vitro culture of ovine
embryos in media supplemented with bovine sera. Theriogenology 1998;49:218 abst.
19. Stice SL, Strelchenko NS, Keefer CL, Matthews L. Pluripotent bovine embryonic cell lines direct
embryonic development following nuclear transfer. Biol Reprod 1996;54:100-110.
20. Van Wagtendonk-de Leeuw AM, Aerts BJG, den Daas JHG. Abnormal offspring following in
vitro production of bovine preimplantation embryos: A field study. Theriogenology 1998;49:883894.
21. Van Wagtendonk-de Leeuw AM, Mullaart E, de Roos APW, Merton JS, den Daas JHG, Kemp
B, de Ruigh L. Effects of different reproduction techniques: AI, MOET, or IVP, on health and
welfare of bovine offspring. Theriogenology 2000;53:575-597.
22. Walker SK, Heard TM, Seamark RF. In vitro culture of sheep embryos without co-culture:
success and perspectives. Theriogenology 1992;37:111-126.
23. Walker SK, Hartwich KM, Seamark RF. The production of unusually large offspring following
embryo manipulation: concepts and challenges. Theriogenology 1996;45:111-120.
24. Wilson JM, Williams JD, Bondioli KR, Looney CR, Westhusin ME, McCalla, DF. Comparison
of birthweight and growth characteristics of bovine calves produced by nuclear transfer
(cloning), embryo transfer and natural mating. Anim. Reprod. Sci 1995;38:73-83.
25. Young LE, Sinclair KD, Wilmut I. Large offspring syndrome in cattle and sheep. Rev Reprod
26. Young, LE, Fernandes K, McEvoy TG, Butterwith, SC, Gutierrez, CG, Carolan C, Broadbent PJ,
Robinson, J J, Wilmut I, Sinclair K. Epigenetic change in IGF2R is associated with fetal
overgrowth after sheep embryo culture. Nature Genetics 2001 ;27:153-154.