Middle East Fertility Society Journal (2010) 15, 57–63

Middle East Fertility Society

Middle East Fertility Society Journal

www.mefsjournal.com
www.sciencedirect.com

REVIEW

Embryo splitting
Karl Illmensee *, Mike Levanduski

Genesis Fertility Center, Patras, Greece

Embryoserv Ltd., New York, NY, USA

Received 28 August 2009; accepted 3 December 2009

Available online 11 June 2010

This article is dedicated in memoriam to Dr. Anne McLaren, a pioneer in mammalian embryology.

KEYWORDS Abstract Mammalian embryo splitting has successfully been established in farm animals. Embryo
Embryo twinning; splitting is safely and efficiently used for assisted reproduction in several livestock species. In the
Blastomere biopsy; mouse, efficient embryo splitting as well as single blastomere cloning have been developed in this
Blastomere cloning; animal system. In nonhuman primates embryo splitting has resulted in several pregnancies. Human
Embryonic stem (ES) cells; embryo splitting has been reported recently. Microsurgical embryo splitting under Institutional
Assisted reproductive Review Board approval has been carried out to determine its efficiency for blastocyst development.
technologies (ART) Embryo splitting at the 6–8 cell stage provided a much higher developmental efficiency compared to
splitting at the 2–5 cell stage. Embryo splitting may be advantageous for providing additional
embryos to be cryopreserved and for patients with low response to hormonal stimulation in assisted
reproduction programs. Social and ethical issues concerning embryo splitting are included regard-
ing ethics committee guidelines. Prognostic perspectives are presented for human embryo splitting
in reproductive medicine.
Ó 2010 Middle East Fertility Society. Production and Hosting by Elsevier B.V. All rights reserved.

* Corresponding author at: Genesis Fertility Center, Via Filopoime-

nos 24, Patras 262 21, Greece. Tel.: +30 2610 270 166; fax: +30 2610
221 401.
E-mail address: illmensee@genesis.vg (K. Illmensee).

1110-5690 Ó 2010 Middle East Fertility Society. Production and

Hosting by Elsevier B.V. All rights reserved. Peer-review under
responsibility of Middle East Fertility Society.
doi:10.1016/j.mefs.2010.05.001

Production and hosting by Elsevier

58 K. Illmensee, M. Levanduski

Contents

1. Animal embryo splitting. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

2. Human embryo twinning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
3. Embryo cell cloning. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
4. Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

1. Animal embryo splitting mothers, developed to term (10). Moreover, transfer of half-
embryos derived from 2-cell embryo splitting gave rise to
In farm animals, embryo splitting has successfully been estab- healthy offspring very similar in size to control live-born mice,
lished for several live stock species. In sheep, 36% of embryos thus demonstrating that experimental embryo twinning did not
split as 2- and 4-cell embryos developed to term following affect normal adult development (11).
transfer to recipient females (1). In cattle, embryos split into Again in the mouse it was recently found that there is a high
blastomeres at the 4-cell stage could further develop to term success rate for obtaining twin blastocysts with good morphol-
giving rise to multiple monozygotic healthy calves (2). Bisected ogy derived from 2- and 4-cell embryos split by blastomere
or biopsied early bovine embryos gave pregnancy rates similar biopsy. Such developmental efficiency was reduced for 6-
to those obtained from intact control embryos. Embryo twin- and 8-cell embryo splitting (12). Can early embryos be split
ning was therefore proposed for suitable applications under several times and, if so, how often? Serial embryo splitting
field conditions (3). Furthermore, cryopreserved split bovine has not previously been reported in any mammalian species.
embryos after their time-separated thawing and intrauterine In this first report, the in vitro potential of mouse embryos
transfer gave rise to live-born monozygotic calves of different after one, two or three splittings was investigated with respect
ages (4). In the goat, monozygotic twin kids were produced to blastocyst development (13). First and second splitting of
from bisected early embryos (5). In addition, split embryos embryos has yielded high efficiency rates for blastocysts when
that were transferred to genetically identical females could de- compared with the third splitting which did not provide any
velop to term in allogeneic pregnancies, being genetically iden- beneficial advantage and, in fact, resulted in a significantly re-
tical twins to these foster females (6). In the pig, split embryos duced number of embryos. These data clearly document that
were capable of full-term development giving rise to healthy first and second embryo splitting increases the multiplication
twin piglets (7). In the horse, from split embryos created via of blastocysts and therefore increases the number of embryos
blastomere biopsy at the 2- or 8-cell stage healthy monozygotic available for potential transfer.
foals were delivered at term pregnancy (8). Although embryo splitting to create monozygotic twins and
From a historic perspective, embryo splitting was first multiples has been successfully reported for several mamma-
achieved in the mouse by investigating the developmental lian species, in nonhuman primates embryo splitting has given
potential of blastomeres from early preimplantation embryos inferior results without leading to twin babies. The splitting of
(9). Further studies in the mouse showed that 65% of half- rhesus monkey embryos at the 8-cell stage and following their
embryos, split at the 2-cell stage and transferred to foster intrauterine transfer resulted in one live-born monkey (14).
Embryo twinning in rhesus monkey has also been attempted
by blastomere separation at the 2- and 4-cell stage and has
led to two twin pregnancies but without giving birth to mono-
zygotic twins (15). Over the past 25 years, mammalian embryo
splitting for the creation of genomically identical twins or mul-
tiples has advanced to a variety of applications in veterinary
and human medicine (Fig. 1). Further progress in refining
techniques for embryo splitting has improved the success rates
for split embryo survival and live-born offspring.

2. Human embryo twinning

Human embryo splitting has so far been reported on geneti-

cally abnormal embryos only in abstract form (16). These em-
bryos were obtained from IVF cycles and were donated for
research. They were split at early cleavage stages, coated with
artificial zona pellucida (ZP) and cultured in vitro. However,
these split embryos were arrested in development after a few
cell divisions at the most. In a commentary referring to these
preliminary experiments and to embryo splitting in general,
Figure 1 Splitting of early embryos (twinning) for various the merits of these attempts were acknowledged for future
applications in veterinary and human reproductive medicine. applications in reproductive medicine (17). With regard to
Embryo splitting
Figure 2 Splitting of triploid human embryo at the 6-cell stage. (A) Biopsy pipette is inserted through artificial opening in zona pellucida
(ZP) and carefully positioned for blastomere removal. (B) Biopsied blastomeres are transferred to previously emptied ZP. Twin embryos
are cultured in vitro during preimplantation development.
human embryo splitting, the Ethics Committee of the Ameri-
can Society for Reproductive Medicine (ASRM) considered
favorably research on embryo splitting and stated in its report
‘‘since embryo splitting has the potential to improve the effi-
cacy of IVF treatments for infertility, research to investigate
the technique is ethically acceptable’’ (18). According to these
recommendations we have first established efficient blastomere
biopsy for embryo splitting in the mouse as animal model
(12,13). We then applied the newly developed biopsy technol-
ogy to human embryo splitting in order to evaluate their effi-
cacy at different early embryonic stages (splitting efficiency)
and to determine the best success rates of twin embryo devel-
opment up to the blastocyst stage under in vitro culture condi-
tions (developmental efficiency).
From ART programs, triploid embryos at early cleavage
stages were donated for our study. Patients have given their
written consent to provide these genetically abnormal embryos
that are usually discarded for our study approved by an inde-
pendent Institutional Review Board (IRB). From human trip-
loid embryos at the 2–5 cell and 6–8 cell stage, blastomeres
were biopsied following additional adaptations that were re-
quired for improving blastomere biopsies on human embryos
(19).
Prior to blastomere biopsy, acidified Tyrode solution was
locally applied to the zona pellucida (ZP) of embryos to pre-
Figure 3 Development of twin bastocysts derived from 6-cell
embryo splitting with good morphological quality during in vitro
culture. Both blastocysts show signs of hatching.
59
pare an opening for pipette penetration. Empty ZPs have been
obtained by previously removing all cellular material from
highly abnormal oocytes or embryos, thereby providing clean
ZPs for insertion of biopsied blastomeres. Half the number of
blastomeres from donor embryos were removed and inserted
into empty ZPs (Fig. 2). Transferred blastomeres were pushed
together to facilitate cellular contacts. Individual blastomeres
that remained separated after transfer showed a tendency for
independent development by creating separate embryo struc-
tures. It has been reported from studies on assisted embryo
hatching that due to different sizes of artificially created ZP
openings, different developmental aberrations have been doc-
umented such as artificial twinning or premature hatching (20).
Following embryo splitting, the resulting donor embryos
(those from which blastomeres were biopsied) and the recipient
embryos (those with biopsied blastomeres inserted into empty
ZPs) were removed from the biopsy medium and cultured
in vitro. As controls, non-split human triploid embryos were
treated similarly to the biopsied embryos and were cultured
under the same conditions.
In our study we investigated the efficacy of human trip-
loid embryo splitting at the 2–5 cell versus 6–8 cell stage.
Additionally, we compared the resulting twinning efficiency
with regard to morphological quality grade 1 and 2 versus
grade 3 and 4 of embryos used for splitting according to
standard IVF embryo morphology grading. Furthermore,
the developmental rates for experimentally split embryos
were compared to non-split control embryos (19). Embryos
used for splitting at the 6–8 cell stage provided a much high-
er developmental efficiency than embryos split at the 2–5 cell
stage. These results on optimal human embryo splitting at
the 6–8 cell stage are in contrast to our earlier findings on
optimal mouse embryo splitting at the 2–4 cell stage (12).
Species-specific variations in early embryogenesis could
account for these observed differences in embryo splitting
efficiency with respect to early embryonic cell stage.
Twin blastocysts of good morphological quality were
observed to develop and hatch in culture between 4 and
5 days after embryo splitting (Fig. 3). In general, split embryos
hatched in advance of untreated controls, suggesting an ‘‘as-
sisted hatching effect’’. We assume this was facilitated by the
ZP opening prepared and required for blastomere biopsy.
For ART it has been proposed that assisted embryo hatching
may provide a beneficial effect to facilitate embryo implanta-
tion in patients with advanced age, with repeated implantation
60 K. Illmensee, M. Levanduski

failures after several IVF and ICSI cycles, or with cryopre-
served-thawed embryo transfer cycles (21).
Ultimately, embryo splitting resulted in more developing
embryos in comparison to non-split control embryos. Our data
further suggest that for the selected embryo population of 6–8
cells and morphological quality of grade 1–2, embryo splitting
will produce more viable embryos than originally available be-
fore splitting (19).
The numerical increase of embryos obtained after splitting
would have obvious clinical advantages for patients enrolled in
ART programs (22). Embryo splitting in ART may be applica-
ble and considered for those patients termed as ‘‘low respond-
ers’’ with only a few oocytes being usually recovered after
hormonal stimulation. However, embryo splitting should only
be considered if the embryos are of high quality and reach the
6–8 cell stage. In this case embryo splitting may increase the
likelihood for obtaining a pregnancy since more embryos
could be made available for intrauterine transfer. Embryo
splitting would not be of practical benefit for patients with
poor quality embryos that do not develop to the 6–8 cell stage.
Relevant to this crucial transition during early embryonic
development, it was documented at the molecular level that
maternally derived mRNA stored during oogenesis is utilized
for protein synthesis from fertilisation to the first cleavage
stages. From thereon, a new wave of parental mRNA is syn-
thesized and is responsible for continuation of embryogenesis
(23,24). From these data on gene expression profiling of early
embryos it therefore seems reasonable to consider the 6-cell
stage as most optimal for embryo splitting.
For selected couples with few embryos of good quality pro-
duced during one IVF cycle, embryo splitting may provide addi-
tional embryos to be cryopreserved for another possible
transfer. This would reduce or postpone the need to subject
ART-enrolled women to another hormonally stimulated retrie-
val process and potentially increase the likelihood of a preg-
nancy. In a report on embryo splitting as a modality for
infertility treatment from the Ethics Committee of the American
Society for Reproductive Medicine, it has been stated that
‘‘splitting one embryo into two or more embryos could serve
the needs of infertile couples in several ways. As long as a couple
is fully informed of the risk of such an outcome, there would ap-
pear to be no major ethical objection’’ (18). Now, since we have
established safe and efficient blastomere biopsy procedures on
genetically abnormal triploid human embryos, for the future it
should be anticipated to apply such twinning technology on nor-
mal diploid human embryos in ART. Infertile patients should

Figure 4 Blastomere cloning from 4-cell mouse embryo. (A) Biopsied blastomeres are inserted into empty ZPs. (B) Blastocysts are
derived from individual blastomeres after in vitro culture. (C–F) Outgrowth and formation of cellular colonies with ES cells (inner cell
mass) and peripheral cells (trophoblast) from hatched blastocysts originating from single blastomeres are established during in vitro
culture.
Embryo splitting
not be denied access to modern techniques that could enhance
their ability to procreate their own offspring.
An increased number of embryos obtained after splitting, as
documented in our study for the first time, may be advanta-
geous for certain patients enrolled in ART programs (25).
Such novel embryo twinning technology may be applicable
and considered for future clinical use in reproductive medicine.
Indeed, duplication of embryos by microsurgical splitting
should not cause an ethical objection since monozygotic twins
can occasionally occur in natural conception as well as in ART
programs (26,27). Moreover, spontaneous embryo splitting,
although rarely observed, has recently been reported for a pa-
tient who underwent an oocyte donation/ICSI cycle (28). In
general, monozygotic twinning is slightly elevated in ICSI ver-
sus IVF cycles. However, this difference turns out not to be
statistically significant. Currently it is difficult to argue whether
or not invasive ART procedures are causally linked to mono-
zygotic twinning.
There is also some concern that embryo splitting may result
in unequal cell distribution to the twin embryos. Such distribu-
tion however does not seem to interfere with normal develop-
ment. There is evidence for unequal allocation of cells to the
twin embryos, leading to some genetic and phenotypic differ-
ences among healthy monozygotic twins (29). Consequently it
was proposed that epigenetic changes due to DNA methylation
dissimilarity during embryonic and fetal development can con-
tribute to the discordance of monozygotic twins (30). Different
locus-specific 5 methyl cytosin DNA and histone acetylation
can lead to phenotypic variations among adult monozygotic
twins (31). Furthermore, chromosomal mosaicism or point
mutations of nuclear and mitochondrial genes can contribute
to genetic differences between monozygotic twins. These new in-
sights have considerably revised our simplistic view of monozy-
gotic twins being perfect genetic clones (32).
3. Embryo cell cloning
In recent years novel techniques have been developed to culture
in vitro individual blastomeres isolated from cleavage-stage em-
bryos for creating embryos or embryonic stem (ES) cells.
Australian researchers reported on the successful biopsy of
single blastomeres from early mouse embryos and their culture
in vitro, using extracellular matrix components for enhanced
cell proliferation (33). In a study on human embryos, British
researchers successfully isolated cleavage-stage blastomeres
that formed trophoblast colonies during in vitro culture (34).
American researchers demonstrated that individual blasto-
meres biopsied from mouse 8-cell embryos were able to gener-
ate ES cell lines that maintained a diploid karyotype and their
differentiation potential when tested in vitro and in vivo (35).
Such approach does not require the destruction of the embryos
since the biopsied mouse embryos used for ES-cell derivation
were able to develop further into healthy mice.
This new strategy will be most important for future applica-
tions in ART. The previous objection that human embryos
have to be destroyed for creating ES cells can no longer be kept
up as valid ethical argument. Blastomere biopsy as it is rou-
tinely done for PGD or embryonic cell culture will not effect
the developmental potential of biopsied embryos that are being
used worldwide for transvaginal transfer leading to pregnan-
cies and healthy infants.
61
In our recent studies on mouse blastomere cloning, we have
investigated the developmental and morphogenetic potential
of individual blastomeres for blastocyst formation during their
clonal culture in vitro (36). We could document that individual
blastomeres from early cleavage-stage embryos yielded high
rates of blastocysts of good morphological quality. Best clon-
ing results were obtained from 2- to 4- cell embryos, less effi-
ciency was observed from 6- to 8- cell embryos. Outgrowth
and the formation of cellular colonies could be obtained from
these blastocysts originating from single blastomeres (Fig. 4).
A fascinating breakthrough for the extraction of human ES
cells (hESC) was reported very recently by creating hESC
lines from single blastomeres of human 4-cell embryos (37).
From 16 blastomeres cultured in vitro 10 embryos developed
and 2 of them produced cellular outgrowth. Two hESC lines
could be established and showed pluripotency in vivo by
forming various tissues such as teratomas (38). Further
investigations on single blastomeres concerning their capacities
for embryo development and ES cell formation and for their
differentiation potential in vitro and in vivo are of utmost
importance for stem cell research, tissue engineering and
possible therapeutic applications (39,40). As our knowledge
about complex cellular and genetic mechanisms during
mammalian embryogenesis is advancing, embryo bio-
technology will continue to contribute with novel applications
in medicine (41).
4. Perspectives
Embryo splitting has been firmly established in cattle and
other livestock species. Multiplication of healthy offspring de-
rived from embryo splitting has become and will continue to be
an economic factor in animal breeding. Embryo splitting in
nonhuman primates should be further pursued to improve suc-
cess rates for pregnancies and offspring. Genetically identical
rhesus monkeys would be very useful for the study of hu-
man-related twinning and tissue transplantation and may serve
as a model system to investigate epigenetic effects caused by
the maternal environment during pregnancy (42).
From human embryos single blastomeres can be biopsied
for creating ES cells without destroying embryos. These biop-
sied embryos retain their viability and can be used for transfer
in ART programs. Such hESC lines will become an intriguing
source for establishing pluripotent stem cells. Novel strategies
will be designed to unravel and channel their differentiation
capacities for future therapeutic applications. Human embryo
splitting should advance in passing the transition from re-
search to application in ART. Certainly, social and legal issues
concerning embryo splitting have to be in accordance with the
countries’ guidelines for ART (43). Embryo splitting should
become available for patients’ benefits and can be of novel
assistance in increasing the likelihood for pregnancies in repro-
ductive medicine.
Acknowledgements
The authors are grateful to Drs. N. Husami and A. Vidali
(American Fertility Services, New York, USA) and Dr. V.T.
Goudas (Genesis Fertility Center, Patras, Greece) for their
support.
62
References
(1) Willadsen SM. The viability of early cleavage stages containing
half the normal number of blastomeres in the sheep. J Reprod
Fertil 1980;59:57–62.
(2) Johnson WH, Loskutoff NM, Plante Y, Betteridge KJ.
Production of four identical calves by the separation of
blastomeres from an in vitro derived four-cell embryo. Vet
Rec 1995;137:15–6.
(3) Lopes RF, Forell F, Oliveira AT, Rodrigues JL. Splitting and
biopsy for bovine embryo sexing under field conditions. Therio-
genology 2001;56:1383–92.
(4) Seike N, Sakai M, Kanagawa H. Development of frozen-thawed
demiembryos and production of identical twin calves of different
ages. J Vet Med Sci 1991;53:37–42.
(5) Tsunoda Y, Uasui T, Sugie T. Production of monozygotic twins
following transfer of separated half embryos in the goat. Jap J
Zootech Sci 1984;55:643–7.
(6) Oppenheim SM, Moyer AL, Bondurant RH, Rowe JD, Anderson
GB. Successful pregnancy in goats carrying their genetically
identical conceptus. Theriogenology 2000;54:629–39.
(7) Reichelt B, Niemann H. Generation of identical twin piglets
following bisection of embryos at the morula and blastocyst stage.
J Reprod Fertil 1994;100:163–72.
(8) Allen WR, Pashen RL. Production of monozygotic (identical)
horse twins by embryo micromanipulation. J Reprod Fertil
1984;71:607–13.
(9) Tarkowski AK, Wroblewska J. Development of blastomeres of
mouse eggs isolated at the 4- and 8-cell stage. J Embryol Exp
Morphol 1967;18:155–80.
(10) Tsunoda Y, McLaren A. Effect of various procedures on the
viability of mouse embryos containing half the normal number of
blastomeres. J Reprod Fertil 1983;69:315–22.
(11) Papaioannou VE, Mkandawire J, Biggers JD. Development and
phenotypic variability of genetically identical half mouse
embryos. Development 1989;106:817–27.
(12) Illmensee K, Kaskar K, Zavos P. Efficient blastomere biopsy for
mouse embryo splitting for future applications in human assisted
reproduction. Reprod BioMed Online 2005;11:716–25.
(13) Illmensee K, Kaskar K, Zavos P. In-vitro blastocyst development
from serially split mouse embryos and future implications for
human ART. Fertil Steril 2006;86:1112–20.
(14) Mitalipov SM, Yeoman RR, Kuo HC, Wolf DP. Monozygotic
twinning in rhesus monkeys by manipulation of in vitro-derived
embryos. Biol Reprod 2002;66:1449–55.
(15) Chan AW, Dominko T, Luetjens CM, Neuber E, Martinovich C,
Hewitson L, et al.. Clonal propagation of primate offspring by
embryo splitting. Science 2000;287:317–9.
(16) Hall JL, Engel D, Gindoff PR, Mottla GL, Stillman RJ.
Experimental cloning of human polyploid embryos using an
artificial zona pellucida. Fertil Steril 1993:61 [ASRM abstracts
S1].
(17) Jones HW, Edwards RG, Seidel GE. On attempts at cloning in
the human. Fertil Steril 1993;61:423–6.
(18) The Ethics Committee of the American Society of Reproductive
Medicine (ASRM). Embryo splitting for infertility treatment.
Fertil Steril 2004;82:256–7.
(19) Illmensee K, Levanduski M, Vidali A, Husami N, Goudas VT.
Human embryo twinning with applications in reproductive
medicine. Fertil Steril 2009 (Epub ahead of print).
(20) Petersen CG, Mauri AL, Baruffi RL, Oliveira JB, Massaro FC,
Elder K, et al.. Implantation failures: success of assisted hatching
with quarter-laser zona thinning. Reprod BioMed Online
2005;10:224–9.
(21) Primi MP, Senn A, Montag M, Van der Ven H, Mandelbaum J,
Veiga A, et al.. European multicentre prospective randomized
study to assess the use of assisted hatching with a diode laser and
K. Illmensee, M. Levanduski
the benefit of an immunosuppressive/ antibiotic treatment in
different patient populations. Hum Reprod 2004;19:2325–33.
(22) Wood C. Embryo splitting: a role in infertility? Reprod Fertil
Develop 2001;13:91–3.
(23) Hamatani T, MSh Ko, Yamada M, Kuji N, Mizusawa Y, Shoji
M, et al.. Global gene expression profiling of preimplantation
embryos. Hum Cell 2006;19:98–117.
(24) Edwards RG. Genetics, epigenetics and gene silencing in differ-
entiating mammalian embryos. Reprod BioMed Online
2006;13:732–53.
(25) Wood EC, Trounson A. Uses of embryo duplication in humans:
embryology and ethics. Hum Reprod 2000;15:497–501.
(26) Rijnders PM, Van Os HC, Jansen CAM. Increased incidence of
monozygotic twinning following the transfer of blastocysts in
human IVF/ICSI. Fertil Steril 1998;70:S15–6.
(27) Tarlatzis BC, Qublan HS, Sanopoulou T, Zepiridis L, Grimbizis
G, Bontis J. Increase in the monozygotic twinning rate after
intracytoplasmic sperm injection and blastocyst stage embryo
transfer. Fertil Steril 2002;77:196–8.
(28) Robb PA, Williams DB, Robins JC, Thomas MA. Trizygotic
quintuplet pregnancy following simultaneous embryo splitting in
an oocyte donation cycle. J Assist Reprod Genet 2006;23:157–60.
(29) Alikani M, Cekleniak NA, Walters E, Cohen J. Monozygotic
twinning following assisted conception: An analysis of 81
consecutive cases. Hum Reprod 2003;18:1937–43.
(30) Singh SM, Murphy B, O’Reilly R. Epigenetic contributors to
the discordance of monozygotic twins. Clin Genet
2002;62:97–103.
(31) Fraga M, Ballestar E, Paz M, Santiago R, Setien F, Sunner D,
et al.. Epigenetic differences arise during the lifetime of mono-
zygotic twins. Proc Natl Acad Sci USA 2005;102:10604–9.
(32) Shur N. The genetics of twinning: from splitting eggs to breaking
paradigms. Am J Med Genet Part C Semin Med Genet
2009;151C:105–9.
(33) Wilton LJ, Trounson AO. Biopsy of preimplantation mouse
embryos: development of micromanipulated embryos and prolif-
eration of single blastomeres in-vitro. Biol Reprod 1989;40:
145–52.
(34) Geber S, Winston RM, Handyside AH. Proliferation of blasto-
meres from cleavage stage human embryos in-vitro: an alternative
to blastocyst biopsy for preimplantation diagnosis. Hum Reprod
1995;10:1492–6.
(35) Chung Y, Klimanskaya I, Becker S, Marh J, Lu SJ, Johnson J,
et al.. Embryonic and extraembryonic stem cell lines derived from
single mouse blastomeres. Nature 2006;439:216–9.
(36) Illmensee K, Kaskar K, Zavos PM. In-vitro developmental
potential of individual mouse blastomeres cultured with and
without zona pellucida: future implications for human assisted
reproduction. Reprod Biomed Online 2006;13:284–94.
(37) Geens M, Mateizel I, Sermon K, DeRycke M, Spits C, Cauffman
G, et al.. Human embryonic stem cell lines derived from single
blastomeres of two 4-cell stage embryos. Hum Reprod 2009
[Epub ahead of print].
(38) Illmensee K, Stevens LC. Teratomas and chimeras. Sci Am
1979;240:86–98.
(39) Metallo CM, Azarin SM, Moses LE, Ju L, de Pablo J, Palecek S.
Human embryonic stem cell-derived keratinocytes exhibit an
epidermal transcription program and undergo epithelial morpho-
genesis in engineered tissue constructs. Tissue Eng Part A 2009
[Epub ahead of print].
(40) Nasonkin I, Mahairaki V, Xu L, Hatfield G, Cummings BJ,
Eberhart C, et al.. Long-term, stable differentiation of human
embryonic stem cell-derived neural precursors grafted into the
adult mammalian neostriatum. Stem Cells 2009 [Epub ahead of
print].
(41) Illmensee K. Embryo biotechnology in reproductive medicine.
MEFS J 2008;13:1–10.
Embryo splitting 63

(42) Schramm RD, Paprocki AM. Strategies for the production of (43) Jones HJ, Cohen J. Experimentation on the preembryo. Fertil
genetically identical monkeys by embryo splitting. Reprod Biol Steril 2007;87(1 Suppl):52–8.
Endocrinol 2004;16:38–42.