Lacey Floyd
10/25/2013
Dr. Hagen
AN SC 431W
Large Offspring Syndrome: Phenotype, Cause, and Application
Large Calf Syndrome, or Large Offspring Syndrome (LOS), as it will be referred to within this
paper, is a condition that affects cattle, primarily those conceived by Assisted Reproductive Techniques,
or ART, such as in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT). It also affects sheep
but has been studied to a greater degree in cattle. The first and foremost characteristic of LOS is perhaps
the most obvious, greater than average birth weight. In Vitro Fertilization involves allowing sperm and
ovum to meet outside the female’s reproductive tract in a petri dish. Somatic cell nuclear transfer is
slightly more invasive and requires the nucleus to be removed from the ovum and replaced with the
nucleus from a donor cell which is often an adult fibroblast (Everts 2007). Large Offspring Syndrome is
more often associated with SCNT as it is a more invasive procedure that requires infiltration of the
embryo to replace the endogenous DNA with DNA from the cultured fibroblast cell. However, both
techniques have a certain percentage of LOS progeny. This syndrome is one of the main inefficiencies of
ART, and for that reason, it is often studied in order to improve upon the success rates of IVF and SCNT.
Humans are also subject to a similar syndrome, Beckwith-Wiedemann Syndrome, which can occur with
or without the use of ARTs. Both syndromes are caused by a loss of imprinting within the genome, which
results in a very specific phenotype within calves and humans.
Humans and calves are the only species that when conceived via ART are more likely to have a
phenotype consistent with LOS (Chen 2013). Symptoms include: increased weight at birth, an enlarged
umbilicus, skeletal defects, respiratory problems, a decreased suckling reflex, hypoglycemia, enlarged
organs, prolonged gestation, abortion, and fluid accumulation in the placenta. In combination, these
symptoms create varying degrees of viability in calves that are born alive. However, ART calves may not
survive to birth: ART pregnancies often result in early embryonic loss, abortion, or stillbirth. Less than
1% of embryos that are destined for cloning survive (Smith 2012). Despite this figure, regarding SCNT
specifically, IVF pregnancies often result in LOS as well which decreases the efficiency of the technique.
The fetus is, as with any pregnancy, only as good as the placenta, and with LOS, the placenta has specific
features that require explanation.
The overgrowth of the placenta actually takes place before the overgrowth of the fetus. This is
likely due to a compensatory mechanism for faulty placentomes. This compensatory mechanism is in
response to a low-functioning placenta which causes the placentomes to enlarge to allow for greater
nutrient and oxygen uptake (Everts 2007). This has prompted some scientists to consider the term “large
placenta syndrome.” Large Offspring Syndrome placentas also have a reduced number of placentomes
and great placental weight. In addition to this, an LOS placenta has reduced vasculature and increased
placentome weight. Perhaps the most striking characteristic of a placenta in association with this
syndrome is the proportion of fetal to maternal tissue. The fetal tissue was particularly more prominent
in the LOS placentas while the maternal portion was thin, suggesting a poor fetal-maternal connection.
Placental growth factors and cellular metabolism likely contribute to some of the abnormalities
associated with LOS. However, the placentas are histologically normal. Phenotypical differences were
not the only abnormality present in these placentas though. Epigenetic abnormalities also existed.
It should be noted that placental abnormalities occur independently of fetal abnormalities
despite commonly being in conjunction with them. As with fetal overgrowth, placental overgrowth is
also due to a change in the imprinting of certain genes within the placentomes. The genes in question
are: BCKDHB (pentose phosphate pathway), ECT2, KIAA1333, and WDR61. All of these genes were found
to have the lowest expression in placentas that were harvested from SCNT pregnancies, but IVF-derived
placentas were also affected. These genes affect various pathways within the placental tissue of SCNT
pregnancies including: oxidative phosphorylation, cell cycle, DNA damage checkpoint regulation, and
purine metabolism. Meanwhile, the main pathway affected in IVF-derived placentas was oxidative
phosphorylation. Accumulation of fluid within the fetus is also common and associated with placental
abnormalities. This condition is known as hydrops and occurs in approximately 30% of SCNT pregnancies
according to a study conducted University of Illinois. Hydrops pregnancies also show abnormal gene
expression, overexpression of: GRSF1, KLF11, and TXNDC12. GRSF1 encodes a binding factor that affects
the formation of the embryonic epithelium, KLF11 is involved in downregulating cell growth and
proliferation. TXNDC12 helps guard the fetus from oxidative stress. Interestingly, placentas that were
harvested from successful pregnancies had normal gene expression. Remarkably all of the issues
associated with LOS and ART pregnancies are due to slight changes in the epigenetics of these placentas
and their embryos.
Epigenetic reprogramming occurs within the oocyte or sperm during the period shortly
proceeding fertilization and shortly after fertilization. The reprogramming involved in imprinting often
deals with addition or removal of methyl groups. Paternal and maternal DNA are demethylated by
slightly different methods at different points in the process. Removal of methyl groups from paternal
DNA occurs immediately after fertilization. This differs from maternal DNA which is inactively
demethylated during the beginning stages of embryonic growth (Smith 2012). In typical pregnancies
specific genes regarding placental and fetal development are then remethylated accordingly.
Pregnancies resulting from ART fail to complete this vital step in fetal development. This failure is likely
due to embryo culture systems which include high concentrations of ammonia, higher activity of growth
factors in medium, presence of paracrine growth factor, and general embryonic manipulations. The
main genes that have been studied are: IGF2R, H19, KCNQ1OT1, CDKN1C, and PLAG1. The three most
notable of these are IGF2R, H19, and KCNQ1OT1. According to one paper, IGF2R and H19 expression and
methylation was regarded as normal in all liveborn SCNT-derived calves (Suteevun-Phermthai 2009).
However, spontaneously aborted calves in the same study were found to have varying degrees of
expression most of which were abnormally monoallelic, favoring either the paternal or maternal allele.
This caused slowed early embryonic growth. The typical expression is leaky meaning that neither allele is
favored. As for KCNQ1OT1, it is also expressed abnormally within ART produced calves. These particular
genes have been studied in greater depth due to their application to human medicine and the coinciding
fetal overgrowth syndrome, Beckwith-Wiedemann Syndrome.
Beckwith-Wiedemann Syndrome (BWS) is a very similar syndrome in people. Its symptoms are
hypoglycemia, excess amniotic fluid, enlarged internal organs, unequal growth between the
hemispheres of your body, cardiac malformations, and difficulty breathing (Chen 2012). Infants with
BWS are also predisposed to abdominal deformities and certain types of tumors. These symptoms tie
closely to those of LOS and, as such, piqued interest in the use of cattle as an animal model. However, it
is not a simple process to create an animal model. First, the normal patterns of methylation and
imprinting had to be determined for both humans and cattle, and these had to be compared to
determine their similarity. It was found in one study that the important genes, IGF2R, H19, and
KCNQ1OT1, and their methylation patterns were conserved between humans and cattle (Chen 2012). It
was then determined that these genes malfunctioned similarly during cases of LOS and BWS. This
research justifies the use of cattle as an animal model. Unlike Large Offspring Syndrome, BWS symptoms
and loss of imprinting are not due to embryo culture techniques as embryos used in human IVF are not
cultured. Therefore, the source of the loss of imprinting in BWS is even more elusive than that of LOS.
There were several other factors considered when using cattle as the model. These factors were: only
humans and cattle display fetal overgrowth syndromes due to use of ARTs, both typically carry singleton
pregnancies, and both have nine month gestation lengths (Chen 2013). Further research is still needed
to hone the use of cattle as an animal model for BWS due to an increase in the number of imprinted
genes found that contribute to the condition. There are other applications outside the realm of human
medicine however.
Calves produced from ART pregnancies sometimes are born with no adverse effects. These
calves still show some signs of LOS but manage to function as normal calves. Calves such as these have a
distinct advantage regarding feed efficiency and weight gain. Calves that were conceived using IVF have
higher growth rates and feed intake throughout their growth period. In addition to this, they are also
more feed efficient during the first month of life when compared to calves conceived using artificial
insemination (AI). This gain in efficiency was thought to be due to some difference in blood composition,
metabolism, or hormone levels. However, no differences were found between calves conceived with IVF
versus AI (Rérat 2005). Despite the lack of knowledge of the mechanism regarding this increase in feed
efficiency, it is encouraging to know that IVF calves can provide a competitive edge to the agricultural
sector if the concerns associated with ART is addressed. It is important to note that calves that are born
with difficulties associated with LOS require greater postnatal care to survive and, as such, are likely to
outweigh the benefits of the associated feed efficiency and weight gain. It will be necessary to do a
study on these animals as they age to determine if their health and viability continue beyond the first
several months of the life. As with any scientific pursuit, further research will help solidify ART as viable
options for improving production animal agriculture.
Large Offspring Syndrome is a multifaceted problem that spans from the cattle industry to
human medicine. It is still not fully understood why this syndrome occurs or why ART do not allow for
proper DNA modification. With further research on the genes responsible for LOS, Assisted Reproductive
Techniques will become much more efficient and thus be more affordable. Large Offspring Syndrome
has a broad economic impact for farmers because these animals show an increase in feed efficiency and
are often products of very expensive procedures such as IVF or SCNT. It will also allow for greater
genetic advancements within production animals. In addition to the benefits to the agricultural industry,
LOS research could potentially benefit human medicine as LOS has a remarkable resemblance to
Beckwith-Wiedemann Syndrome, a condition that affects humans. It has great scientific value for those
researching the cause of Beckwith-Wiedemann Syndrome as it could potentially serve as an animal
model for the syndrome. Additional knowledge of Large Offspring Syndrome could potentially benefit
both the agricultural industry as well as the field of human medicine. The loss of imprinting associated
with LOS may also carry over to other scientific research as well which makes understanding the cause,
phenotype, and applications all the more important.
Works Cited
Chen, Z., R. Rivera, K. Robbins, and K. Wells. 2012. Expression of KCNQ1OT1, CDKN1C, H19, and PLAGL1
and the methylation patterns at the KvDMR1 and H19/IGF2 imprinting control regions is
conserved between human and bovine. J. Biomed. Sci. 19:95.
Chen, Z., R. Rivera, K. Robbins, and K. Wells. 2013. Large offspring syndrome: A bovine model for the
human loss-of-imprinting overgrowth syndrome Beckwith-Wiedemann. Landes Bioscience.
8:591-601.
Constant, F., M. Guillomot, Y. Heyman, X. Vignon, P. Laigre, J.L. Servely, J.P. Renard, and P. ChavattePalmer. 2006. Large Offspring or Large Placenta Syndrome? Morphometric Analysis of Late
Gestation Bovine Placentomes from Somatic Nuclear Transfer Pregnancies Complicated by
Hydrallantois. Bio. of Repro. 75:122-130.
Everts, R., P. Chavatte-Palmer, A. Razzak, I. Hue, C. Green, R. Oliveira, X. Vignon, S. Rodriguez-Zas, X.
Cindy Tian, X. Yang, J.P. Renard, and H. Lewin. 2007. Aberrant gene expression patterns in
placentomes are associated with phenotypically normal and abnormal cattle cloned by somatic
cell nuclear transfer. Physio. Geno. 33:65-77.
Hayashi, M., T. Hirai, M. Hirayama, N. Hori, S. Horike, K. Matsuda, M. Nagai, T. Ozawa, and T. Tanaka.
2010. Aberrant CpG methylation of the imprinting control region KvDMR1 detected in assisted
reproductive technology-produced calves and pathogenesis of large offspring syndrome. Anim.
Repro. Sci. 122:303-312.
Rérat, M., Y. Zbinden, R. Saner, H. Hammon, and J.W. Blum. 2005. In Vitro Embryo Production: Growth
Performance, Feed Efficiency, and Hematological, Metabolic, and Endocrine Status in Calves. J.
Dairy Sci. 88:2579-2593.
Smith, L., J. Suzuki Jr., A. Goff, F. Filion, J. Therrien, B. Murphy, H. Kohan-Ghadr, R. Lefebvre, A. Brisville,
S. Buczinski, G. Fecteau, F. Perecin, and F. Meirelles. 2012. Developmental and Epigenetic
Anomalies in Cloned Cattle. Repro. in Domest. Anim. 47:107-114.
Suteevun-Phermthai, T., C.L. Curchoe, A.C. Evans, E. Boland, D. Rizos, T. Fair, P. Duffy, L.Y. Sung, F. Du, S.
Chaubal, J. Xu, T. Wechayant, X. Yang, P. Lonergan, R. Parnpai, and X.C. Tian. 2009. Allelic
switching of the imprinted IGF2R gene in cloned bovine fetuses and calves. Anim. Repro. Sci.
116:19- 27.