Human Reproduction Update, Vol.23, No.2 pp.

156–165, 2017
Advanced Access publication on November 14, 2016 doi:10.1093/humupd/dmw041

Potential of human twin embryos

generated by embryo splitting in
assisted reproduction and research
Laila Noli1,2, Caroline Ogilvie3, Yacoub Khalaf1,2, and Dusko Ilic1,2,*

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1
Division of Women’s Health, Guy’s Hospital, London SE1 9RT, UK 2Assisted Conception Unit, Guy’s Hospital, London SE1 9RT, UK
3
Genetics Laboratories, King’s College London, Guy’s Hospital, London SE1 9RT, UK

*Correspondence address. E-mail: [email protected]

Submitted on May 25, 2016; resubmitted on October 15, 2016; editorial decision on October 20, 2016; accepted on November 2, 2016

TABLE OF CONTENTS
...........................................................................................................................
• Introduction
Methods of embryo splitting
Blastomere biopsy/separation
Bisection
• Methods
• Results
Potential benefits of embryo splitting
Embryo splitting in farm animals
Embryo splitting in non-human primates
• Embryo splitting in humans
Ethical considerations
Regulatory framework
Controversies of the initial studies
• Molecular mechanisms
Human development is under strict temporal control
The role of cell–cell interactions in fate specification and Hippo signalling
Lineage commitment and reproductive competence
• Conclusion and prospectives

BACKGROUND: Embryo splitting or twinning has been widely used in veterinary medicine over 20 years to generate monozygotic twins with
desirable genetic characteristics. The first human embryo splitting, reported in 1993, triggered fierce ethical debate on human embryo cloning.
Since Dolly the sheep was born in 1997, the international community has acknowledged the complexity of the moral arguments related to this
research and has expressed concerns about the potential for reproductive cloning in humans. A number of countries have formulated bans either
through laws, decrees or official statements. However, in general, these laws specifically define cloning as an embryo that is generated via nuclear
transfer (NT) and do not mention embryo splitting. Only the UK includes under cloning both embryo splitting and NT in the same legislation.
On the contrary, the Ethics Committee of the American Society for Reproductive Medicine does not have a major ethical objection to transfer-
ring two or more artificially created embryos with the same genome with the aim of producing a single pregnancy, stating that ‘since embryo
splitting has the potential to improve the efficacy of IVF treatments for infertility, research to investigate the technique is ethically acceptable’.
OBJECTIVE AND RATIONALE: Embryo splitting has been introduced successfully to the veterinary medicine several decades ago and
today is a part of standard practice. We present here an overview of embryo splitting experiments in humans and non-human primates
and discuss the potential of this technology in assisted reproduction and research.

© The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
For Permissions, please email: [email protected]
Potential use of embryo splitting 157

SEARCH METHODS: A comprehensive literature search was carried out using PUBMED and Google Scholar databases to identify stud-
ies on embryo splitting in humans and non-human primates. ‘Embryo splitting’ and ‘embryo twinning’ were used as the keywords, alone or
in combination with other search phrases relevant to the topics of biology of preimplantation embryos.
OUTCOMES: A very limited number of studies have been conducted in humans and non-human primates. The published material, espe-
cially the studies with human embryos, is controversial. Some reports suggest that twinning technology will find clinical use in reproductive
medicine in the future, whereas others conclude the opposite that human twin embryos created in vitro are unsuitable not only for clinical,
but also for research, purposes.
WIDER IMPLICATIONS: The blastomere biopsy technique of embryo splitting seems to be unsuitable for either clinical or research
purposes; however, embryo bisection, a preferable method of cloning in veterinary medicine, has not yet been tested on human
embryos.

Key words: embryo splitting / embryo twinning / blastomere biopsy / lineage commitment / developmental clock

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Introduction Methods of embryo splitting
Using a range of different techniques, embryo splitting or in vitro twin-
The in vitro production of genetically identical copies of organisms can
ning has been performed in several animal species. Early in the 20th
be done in two ways: somatic cell nuclear transfer (SCNT) and
century, experiments in fish showed that lowering the incubation tem-
embryo twinning or splitting. Embryo splitting mimics the natural pro-
perature or reducing the oxygen concentration decreased the rate of
cess that creates identical twins, whereas SCNT is completely artifi-
development and thereby increased the incidence of monozygotic
cial. Depending on the developmental stage of an embryo, splitting
(MZ) twins (Stockard, 1921). Similarly, a large number of more recent
can be done using either blastomere biopsy (for cleavage-stage
studies have demonstrated that delayed fertilization in rabbits also led
embryos) or bisection (for morula or blastocysts). Unlike cloning by
to MZ twinning (Hall, 2003; Aston et al., 2008). It has been suggested
SCNT that theoretically can produce multiple, genetically identical,
that, in these cases, twinning may have been induced by disruptions in
copies, the number of clones that can be produced by embryo split-
communication between blastomeres at various stages of development
ting is limited by the degree to which preimplantation embryos can
(Otsuki et al., 2016). The latest improvements in microscopy and
be efficiently subdivided.
micromanipulation technologies have allowed the mechanical induction
Research into embryo splitting dates back to the late 1800s, with
of MZ twinning via blastomere biopsy or blastocyst bisection.
early studies by Hans Dreisch on sea urchin embryos providing
proof-of-concept evidence that individual blastomeres from 2- and 4-
cell embryos could develop into larvae (Driesch, 1894). Subsequent
studies on salamanders by Hans Spemann demonstrated that individ-
Blastomere biopsy/separation
ual blastomeres of 2-cell stage embryos possess the potential to The blostomere biopsy technique involves the removal of one or more
develop into full organisms. Hans Spemann also performed the first blastomeres from a cleavage-stage embryo and their insertion into a
nuclear transfer (NT) experiments in 1914 (Spemann, 1921). previously prepared empty zona pellucida (ZP) for further development
The obvious advantage of using genetically identical animals is in (Fig. 1). To achieve this, the donor embryo is treated first with acidified
research, reducing the number of test animals needed for compara- Tyrode’s solution, which produces an opening in the ZP. Blastomeres
tive studies (Biggers, 1986; Yang and Anderson, 1992). Nowadays, are then removed via an aspirating pipette that is inserted through the
however, embryo splitting has also been extensively used in veterin- ZP hole. The free blastomere is subsequently transferred to a ZP that
ary medicine and breeding to maintain high quality and healthy live- was previously emptied by removing its cellular content. Embryo split-
stock with desirable genetic characteristics (Yang et al., 2007). In ting using the blastomere biopsy/separation technique has been favour-
most cases, the artificially generated twin embryos are transferred able and the pregnancies have been achieved in most of the large
into different recipients to avoid the risks of multiple births (Norman animal species tested, including sheep (Williadsen, 1979), cattle
et al., 2004). The first calves generated by embryo splitting were (Williadsen and Polge, 1981), horses (Allen and Pashen, 1984) and pigs
registered with the Holstein Association USA in 1982 and by 2002, a (Ash et al., 1989). Similar success rates of the procedure in these spe-
total of 2319 such animals were registered. So far, studies have not cies were confirmed in later studies from multiple groups. However, in
detected any differences between products from cloned and non- non-human primates such as Rhesus monkeys, the studies with a similar
cloned animals (Norman et al., 2004; Yang et al., 2007). strategy were not successful (Chan et al., 2000; Mitalipov et al., 2002),
Until 1997, many thought that cloning from somatic cells would be and the molecular basis for this difference has not been addressed.
impossible; however, the turning point came following the first suc-
cessful cloning of a mammal, Dolly the sheep, 83 years after NT was
first described by Spemann (1921). Much of the ethical debate sur- Bisection
rounding embryo research since then has become focused on the This method is used to mechanically divide post-compaction embryos
future potential of cloning adult humans. into two equal halves which, in case of the blastocyst, include an even
158 Noli et al.

1984) and pigs (Nagashima et al., 1989). Numerous studies have fol-
lowed, most of them in cattle.
In general, no difference has been found in the proportion of preg-
nancies or twins born from demi-embryos created by either of these
two methods (Tagawa et al., 2008).

Methods
A comprehensive literature search was carried out using PUBMED and
Google Scholar databases to identify studies on embryo splitting in humans
and non-human primates. ‘Embryo splitting’ and ‘embryo twinning’ were
used as the keywords, alone or in combination with other search phrases
relevant to the topic of biology of preimplantation embryos.

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Results
Potential benefits of embryo splitting
Figure 1 Embryo splitting of 8-cell embryo using blastomere Embryo splitting has potential benefits in both assisted reproduction
biopsy approach. The donor embryo (future Twin A) is treated first
programmes and research.
with acidified Tyrode’s solution or a laser beam to generate an
For patients with low response to hormonal stimulation, the tech-
opening in the zona pellucida (ZP). Blastomeres are then removed
via an aspirating pipette (AP) that is inserted through the ZP hole.
nology, if proven to be safe, may provide additional embryos for
The free blastomeres are subsequently transferred to a ZP that was intrauterine transfer and in such a way as to increase the likelihood of
previously emptied by removing its cellular content (future Twin B). pregnancy (Illmensee et al., 2010).
b1–b8, individual blastomeres of 8-cell cleavage-stage embryo; Em, Currently, there is a severe shortage of human embryos that have
embryo; HP, holding pipette. been donated for research purposes, and there is an associated
potential for future restrictions on human developmental research.
Since the policy to transfer embryos at the blastocyst stage of devel-
opment was introduced, there has been a reduction in the number of
cleavage-stage embryos that are available for research. Therefore,
splitting embryos at the cleavage stage offers the potential to increase
the number of viable embryos, and it may therefore be a suitable
means of addressing the current shortage in the availability of
research embryos. In addition, embryo splitting provides the oppor-
tunity to obtain genetically identical embryos, a feature that is ideal
for comparative research. Having genetically identical embryos in
control (non-treated) and experimental (treated) groups would elim-
inate the bias of genetic background and fewer embryos would be
needed to reach conclusions .

Figure 2 Embryo splitting using blastocyst dissection approach. Embryo splitting in farm animals
Post-compaction embryos are divided mechanically into two equal The most common outcome of producing MZ offspring is twins or
halves which, in the case of the blastocyst, include an even distribu- singletons, but triplets and quadruplets have also been reported in
tion of inner cell mass (ICM) and trophectoderm (TE) between the
cattle following the transfer of quartered embryos (Willadsen and
resultant demi-embryos. SMb, surgical microblade; ZP, zona
Polge, 1981; Johnson et al., 1995). An increase in the production of
pellucida.
cattle from the transfer of split embryos produced using these
approaches has been reported (Leibo and Rall, 1987). In addition to
fresh transfer, the use of frozen-thawed demi-embryos has also been
distribution of inner cell mass (ICM) and trophectoderm (TE) between attempted (Seike et al., 1991). Remarkably, in the case of cattle, the
the resultant demi-embryos (Fig. 2). Using this technique, The MZ twin normal pregnancy rate from a whole embryo transfer is ~70%. The
embryos are then immediately cultured in vitro using a culture medium equivalent rate for a demi-embryo is ~50–55%, and this method,
that encourages further development. Although this procedure has not therefore, provides a 30–40% increase in the chance of conception
been attempted in humans, blastocyst bisection has been effective in a (Seike et al., 1989; Wood and Trounson, 2000). In addition, no
number of large mammalian species, including goat (Udy, 1987), sheep developmental or physiological defects have been reported in the off-
(Széll and Hudson, 1991), cattle (Ozil et al., 1982; Williams et al., spring resulting from these split embryos, which develop into healthy
Potential use of embryo splitting 159

animals. However, a study in horses (Allen and Pashen, 1984) has the pregnancies were singletons (Mitalipov et al., 2002). While blasto-
shown that the size of twins born from embryos of unequal alloca- cyst bisection led to the formation of higher numbers of demi-
tions of cells (one versus two blastomeres) is different and that the embryos, the number of clinical pregnancies per oocyte was higher
disparity persists into adult life. for embryos produced by blastomere separation (Mitalipov et al.,
2002). However, in spite of the fact that pregnancies have been
established using both methods of embryo splitting in rhesus mon-
Embryo splitting in non-human primates keys, they have both resulted in only singleton offspring, whether
Paving the way towards the splitting of human embryos, the technology they were implanted in different or the same recipients (Chan et al.,
was investigated first in rhesus macaques (Macaca mulatta). Rhesus 2000; Mitalipov et al., 2002).
macaques are a non-human primate model that is highly related to There could be multiple reasons why twinning approaches in rhe-
humans in evolutionary, genetic and physiological terms. Therefore, sus monkeys have not met with similar success to that seen in mice
they can be used to gain crucial information for human research (Van or large domestic animals. Normally, rhesus monkeys do not carry
twins; only ~0.25% of naturally occurring pregnancies are twin preg-

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de Berg and Williams-Blangero, 1996). Specifically, the successful devel-
opment of methods for producing MZ twins in monkeys could lead to nancies. Even then, the offspring rarely survive due to various compli-
significant advancements in the scientific understanding of human dis- cations (Hendrickx and Binderd, 1980; Schramm et al., 2002).
ease, MZ twinning and the effects of the maternal environment on the Pregnancy rates following transfer of two embryos generated in vitro
epigenetic profile of a developing embryo. In addition, these studies are in range of 25–40%, with <15% resulting in twin gestations
could also lead to the development of better animal models for vaccine (Lanzendorf et al., 1990; Mitalipov et al., 2002; Schramm et al., 2002).
trials and tissue transplantation studies (Schramm and Paprocki, 2004a, In both seminal primate studies (Chan et al., 2000; Mitalipov et al.,
b). However, current strategies aimed at producing MZ twins in rhesus 2002), more than one twin embryo was transferred into each recipi-
monkeys have met with only limited success (Schramm and Paprocki, ent. Single twin embryo transfers into separate recipients might
2004a,b). Blastomere separation studies performed in rhesus monkeys improve outcomes greatly. In addition, rhesus monkeys cannot be
gave rise to blastocysts with significantly different total cell numbers synchronized and optimal timing for transfer requires cryopreserva-
within a given demi-embryo pair (Mitalipov et al., 2002). This may have tion, which has not been very successful for split embryos regardless
resulted from the asymmetric distribution of cytoplasm between the of species (Weston et al., 1996).
blastomeres during separation or a difference in the polarity of cells
within the embryo. There were 22 pairs of demi-embryos created using
blastomere separation and then transferred, resulting in a pregnancy Embryo splitting in humans
rate of 33% (7 out of 22). Among these pregnancies, two twin pregnan-
The first human embryo splitting procedure was reported by a team
cies (9%) were initiated, but neither of the twin pairs developed to
of researchers including Robert Stillman and Jerry Hall from George
term (Mitalipov et al., 2002). In another study, a total of 368 embryos
Washington University in Washington, DC, in October 1993, at a
were created by splitting 107 rhesus embryos at the 8-cell stage. The
joint meeting of the American Fertility Society and the Canadian
compaction rate was not affected by number of identical clones pro-
Fertility and Andrology Society (Hall et al., 1993). Researchers used
duced from one embryo. However, the blastocyst formation was
polyspermic embryos that would not survive and would have been
reduced by each identical clone produced and no blastocyst was
routinely discarded. They separated blastomeres from 17 2- to 8-cell
formed when splitting beyond sextuplets was attempted. In an attempt
embryos, covered them in an artificial ZP and cultured them for up
to produce sets of identical quadruplets, each originally consisting of
to 32-cell divisions. The researchers claimed that their results pointed
two blastomeres, a pair of the quadruplet embryos was transferred to
a way for enhanced infertility treatment in humans. However, it was
each of two fertile surrogates. However, only one monkey was born: a
later found that the study did not possess the valid Institutional
healthy female named Tetra (Chan et al., 2000).
Review Board approval, and the authors were reprimanded and
Data from Chan et al. showed a reduction in the developmental
instructed to destroy their data (Fackelmann, 1994; Macklin, 1995).
potential of the blastocysts when blastomere separation was per-
The case led to fierce ethical debate on embryo cloning (Cohen and
formed at later cleavage stages between the 8- and 16-cell stages
Tomkin 1994; Cohen, 1994; National Advisory Board on Ethics in
(Chan et al., 2000). Mitalipov demonstrated that blastomere separ-
Reproduction, 1994; Verhey, 1994; Macklin, 1995; Burke 1996). In
ation at the 2- or 4- cell stage can produce demi-embryos that
the wake of protests from the scientific community and media, the
develop into blastocysts comparable to non-manipulated control
American Society for Regenerative Medicine’s (ASRM’s) Ethics
embryos (Mitalipov et al., 2002). The ratio of ICM to TE and the ratio
Committee formulated a statement concerning embryo splitting and
of ICM to total cells in these split blastocysts were similar to the
its use in infertility treatment, which was subsequently accepted by
ratios in non-manipulated control blastocysts. However, the total
the Board of Directors in December 1995 (The Ethics Committee of
number of cells in the split blastocysts was almost 50% lower than
the American Society for Reproductive Medicine, 2004).
the number in the controls, similar to results recorded in other spe-
cies (Willadsen and Polge, 1981; Willadsen et al., 1981; Willadsen,
1981; Willadsen, 1989).
In the case of the demi-embryos that were developed using blasto- Ethical considerations
cyst bisection methods, a pregnancy rate of 33% (4 out of 12) was Despite the fact that spontaneous MZ twinning is a natural form of
achieved. However, no twin pregnancies were established, and all of cloning, twinning of human embryos in vitro continues to be a matter
160 Noli et al.

of ethical debate. The ethical considerations have given rise to a regu- Since Dolly the sheep was born in 1997, the international commu-
latory framework to restrict research and development in human nity acknowledged the complexity of the moral arguments that are
cloning, which in the UK, includes the particular methodology of related to this research and has expressed concern about the poten-
embryo splitting. The process of embryo splitting falls under the gen- tial for reproductive cloning in humans. Numerous countries have
eric heading of human cloning, which is an emotive and controversial formulated bans either through laws, decrees or official statements
topic and the ethical debate regarding embryo splitting is, therefore, (UNESCO, 2004).
more likely to attract public attention and scrutiny.
Biological barriers are likely to prevent human reproductive cloning
by NT in the foreseeable future and, if the cloning ever happens, it is Regulatory framework
more likely to be achieved by embryo splitting. There is an argument In terms of regulation at the international level, the General Conference
that embryos produced from splitting are not artificial clones and that of UNESCO unanimously acclaimed the Universal Declaration on the
they should be available for use in therapy. First, embryo splitting is Human Genome and Human Rights in 1997. This international instru-
distinct from the process of NT, which transfers nuclear content ment was subsequently endorsed by the General Assembly of the

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from somatic cells for the purposes of creation of a child or thera- United Nations in 1998, which declared that human reproductive clon-
peutic application. NT effectively duplicates a fully formed human ing is a practice against human dignity (UNESCO, 2004).
being, whereas embryo splitting replicates the natural process that At the European level, the Additional Protocol to the Convention
forms MZ twins during embryogenesis. NT circumvents normal gam- of the Council of Europe for the Protection of Human Rights and
etogenesis and fertilization and prevents the normal programming of Dignity of the Human Being with regard to the Application of Biology
an embryo’s genome. Second, the split embryos are dichorionic and and Medicine on the Prohibition of Cloning Human Beings was devel-
diamniotic, with a separate placenta and amnion, which reduces the oped in 1998 and took effect in 2001. It states that ‘any intervention
risk of common complications in twin pregnancies, such as cord seeking to create a human being genetically identical to another
entanglement or twin–twin transfusion. Third, since MZ twinning is human being, whether living or dead, is prohibited’ (Council of
also a natural phenomenon, significant information can be obtained Europe, 1997).
from analysing the behaviour of twins. Finally, the embryo splitting In terms of the UK and the US regulations, following controversy
procedure familiarizes parents with the possibility of twin pregnancies over the original research in the USA, the ASRM published a state-
and their risk, which better prepares them for these events. ment concerning embryo splitting in 1995 stating that ‘splitting one
Most mammalian reproductive cloning that is performed using embryo into two or more embryos could serve the needs of infertile
SCNT gives rise to offspring that either die during gestation or suffer couples in several ways’ and that they did not recognize a significant
from large offspring syndrome, which is typified by respiratory and ethical objection to the placement of two or more embryos with the
metabolic abnormalities and an enlarged, dysfunctional placenta same genome in the recipient uterus with the aim of producing a sin-
(Jaenisch, 2004). Clones that do survive usually have a normal pheno- gle pregnancy, as long as the parents undergoing the fertility treat-
type and are physiologically able to produce healthy offspring (French ment were duly apprised of the outcome of this procedure (The
et al., 2006). In addition, no significant behavioural or psychological Ethics Committee of the American Society for Reproductive
problems related to MZ twining have so far ever been reported and Medicine, 2004). A bill was subsequently passed by the House of
the technology therefore seems to be safe. Hence, the ethical debate Representatives in 2003 that banned reproductive and therapeutic
centres on whether human reproductive cloning by embryo splitting, cloning. The bill paved the way for legislation to be passed in different
if possible in the foreseeable future without increasing the risk of states that outlawed either reproductive cloning or both therapeutic
abnormalities in the child, is ethically justifiable. and reproductive cloning. Fifteen states have laws on human cloning.
In order to avoid complications of the twin pregnancies, only one These laws specifically define cloning as an embryo that is achieved
of the split embryos might be transferred, whereas the other one via NT (National Conference of State Legislatures, 2003; 2008) and
could be cryopreserved for future use. However, this would likely do not include embryo splitting. Thus, in the USA, legislation on
give rise to an ethical debate on MZ twins of different age. reproductive cloning relates specifically and exclusively to NT meth-
There are various issues to consider in this ethical debate, including odologies. Furthermore, since the 1995 statement by the ASRM,
the right to life of the embryo and the interests of the child, the soci- legislation has allowed embryo splitting as an infertility treatment. The
etal consequences and teleological perspectives (Strong, 2005). For UK includes cloning both by embryo splitting and NT in the same
example, one controversial and highly discussed aspect of embryo legislation. There is, therefore, a major difference in laws between
splitting is whether artificial twinning violates the right of an unborn these two countries.
child to be unique. However, given that embryo splitting replicates a The Human Fertilisation and Embryology Authority (HFEA) have
natural process, none of these arguments carry sufficient ethical justi- not supported the views of the ASRM. The original HFEA Act 1990
fication to warrant a total ban on human reproductive cloning using (The Human Fertilisation and Embryology Authority, 1990), which
this methodology. It is widely accepted that embryo splitting must regulates the medical and scientific manipulation of embryos, defined
not be used for unethical purposes, such as the generation of histo- an embryo as a ‘live human embryo where fertilization is complete’,
compatible embryos with the intention of organ transplantation. and therefore the Human Reproductive Cloning Act was brought
Therefore, the main consideration, from both a scientific and a clinical into force in 2001 to cover embryos created by reproductive cloning
perspective, is whether this methodology can be used without an techniques. It prohibits reproductive cloning and states in Chapter 23
increased risk of abnormalities. that ‘a person who places in a woman a human embryo which has
Potential use of embryo splitting 161

been created otherwise than by fertilization is guilty of an offence and Split embryos were evaluated in terms of their size, biological behav-
this offence carries up to 10 years and/or an unlimited fine’ (The iour, morphology and expression of an ICM marker, NANOG, using
Human Reproductive Cloning Act, 2001). In 2002, a ruling came into immunocytochemistry (Van de Velde et al., 2008). Blastocysts that
force that allowed for clones produced by nuclear replacement to be were derived from individually cultured blastomeres resulted in
classified as embryos, and reproductive cloning therefore subse- embryos that were one-quarter the size of regular human embryos
quently fell under the HFEA Act (The Human Fertilisation and that were cultured in vitro. It was also shown that in spite of their smal-
Embryology Authority, 1990). Furthermore, the 6th HFEA Code of ler size, the blastocysts underwent compaction on Day 4 and cavitation
Practice (paragraph 8.9 ii) specifies that the embryo splitting proced- on Day 5, similar to the control human embryos. On Day 6, the major-
ure must not be used by fertility clinics to produce embryos for treat- ity of these split embryos were able to form complete blastocysts that
ment purposes (The Human Fertilisation and Embryology Authority, possessed a distinct ICM and TE, even though the yield of cells per
2003). The HFEA now stipulates that a license must be granted for embryo was very low. The presence of NANOG-positive cells sug-
therapeutic cloning research. The first license was awarded by the gested that the ICM cells in the split embryos are pluripotent. In one
HFEA in 2004 to scientists from the University of Newcastle to cre- embryo, all four blastomeres developed into viable blastocysts, each

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ate human embryonic stem cells (hESC) via cell NT (The Human with a cohesive TE and a tightly packed ICM, with some cells expres-
Fertilisation and Embryology Authority, 2004). sing NANOG. Although the sample size was small, the authors suc-
In Australia, under a license issued by the National Health and cessfully demonstrated that the single cells isolated from a 4-cell stage
Medical Research Council (NHMRC) Embryo Research Licensing human embryo could individually develop into mini-blastocysts with
Committee, cloning by SCNT for therapeutic purposes is permitted, delineated ICM and TE cells (Van de Velde et al., 2008).
whereas reproductive cloning is banned (The Prohibition of Human Illmensee et al. (2010) demonstrated that the ideal developmental
Cloning for Reproduction Act, 2002). Other countries are even stage for splitting human embryos is the 6–8 cells stage, in terms of
more restrictive. Hong Kong prohibits the ‘replacing of the nucleus of both splitting and developmental efficiency. The authors claimed that
a cell of an embryo with a nucleus taken from any other cell’ as well the number of blastocyst-stage embryos that formed in the study sig-
as the ‘cloning of any embryo’ (Human Reproductive Technology nificantly exceeded the original number of embryos that were split at
Ordinance, 2000). The scope of the latter, therefore, is arguably the this stage. The split embryos appeared to hatch earlier, however,
widest prohibition, as it rules out all cloning techniques, such as cell possibly because of compromised ZP integrity from the blastomere
nucleus replacement, embryo splitting, parthenogenesis and cloning biopsy. Earlier hatching may enhance the implantation capacity of
using stem cell lines. At the present, there is no country which per- embryos, especially in patients who may have experienced multiple
mits reproductive cloning of humans by legislation or guidelines implantation failures (Primi et al., 2004; Petersen et al., 2005). In a
(National Legislation Concerning Human Reproductive and second study by the same group, the authors showed that the MZ
Therapeutic Cloning. UNESCO. 2004). However, only a few, such as characteristics of triploid embryos were not altered by embryo split-
the UK (The Human Fertilisation and Embryology Authority, 2003), ting, with the resulting twin embryos containing the same allelic short
Australia (The Prohibition of Human Cloning for Reproduction Act, tandem repeats (STR) sequences as expected (Illmensee et al.,
2002), Singapore (Human Cloning and Other Prohibited Practices 2011). Six selected polymorphic STR markers in the HLA locus on
Act, 2004) and India (Ethical Guidelines for Biomedical Research on Chromosome 6 were selected and subjected to nested multiplex
Human Participants, 2006), define embryo splitting. Even when they PCR analysis. Fluorograms from five pairs of twin blastocysts showed
do, the embryo splitting technology always ends up under the that peak positions for the detected STR profiles were identical
umbrella of forbidden activity of human cloning. between twin embryos. This was the first study to demonstrate the
monozyogocity of twinned human embryos at the DNA level
(Illmensee et al., 2011).
Controversies of the initial studies Our group has analysed the largest number of twin embryos
More recent studies from two groups has suggested that the use of (n = 176) reported to date. Twin embryos created by splitting either
these types of embryo splitting techniques may result in the forma- early (2–5 blastomeres, n = 43) or late (6–10 blastomeres, n = 45)
tion of viable and morphologically adequate blastocysts in humans cleavage-stage embryos were compared with IVF embryos that later
(Van de Velde et al., 2008; Illmensee et al., 2010, 2011). However, resulted in pregnancy and live birth upon single blastocyst transfer
neither of these three studies presented comprehensive qualitative (n = 42) (Noli et al., 2015a). The comparative methods used include
analyses of the embryos that were created using splitting techniques. morphokinetics and immunodetection of lineage markers. In addition,
In addition, the results have been somewhat contradictory. For we attempted a derivation of hESC lines following our standard pro-
example, Van de Velde et al. (2008) reported that blastomeres tocols (Ilic et al., 2012; Stephenson et al., 2012). We found that twin
derived from 4-cell embryos possessed sufficient plasticity to form a embryos were smaller and that the size of the twin blastocyst was
proper blastocyst. Illmensee et al (2010) could not reproduce that proportional to the number of blastomeres used for creation of the
result; they reported that the blastomeres from 8-cell embryos, embryo. In addition, the ICM was generally relatively poorly devel-
rather than the blastomeres derived from embryos at earlier stages, oped, if distinguishable at all. Immunostaining revealed that the major-
led to the development of the blastocysts. The most recent study by ity of the cells expressed markers of both ICM and TE, which raises a
Noli et al. (2015a) suggested that human twin embryos created question about their developmental competence. Indeed, none of
in vitro using a blastomere biopsy technique were unsuitable for not the 10 twin embryos with a morphologically distinguishable ICM-like
only for clinical but also research purposes, regardless of the stage of structure gave rise to hESC lines, even though the success rate of the
development of the parental embryos. protocol is 30–50% when using IVF embryos.
162 Noli et al.

Molecular mechanisms control (Noli et al., 2015a). The size of twin embryos was propor-
tionate to the number of cells used for their creation: more cells
Human development is under strict used as a starting material gave bigger blastocysts (Fig. 3). The aver-
age diameter of blastocysts generated from a single blastomere of 2-
temporal control
or 3-cell stage embryos was 86.93 µm, and from four blastomeres of
Embryogenesis follows a precise and specific programme shared by all 8- or 9-cell embryo, it was 102.25 µm, whereas an average diameter
individuals of the same species including human. The programme is of blastocysts formed from intact embryos was 120.87 µm. The esti-
strictly regulated by developmental timers that are set at fertilization mated time of development from the pronuclear (2PN) stage to
and inherited in every daughter cell. Timers are not cell–cell contact expanding blastocyst for participating blastomeres is quite similar
dependent; they are intrinsic to each blastomere. For example, the between control intact embryos (116.22 hours) and the twin
transition from rapid and symmetrical cell divisions to slow and asym- embryos, regardless of the time of splitting (112.70–115.92 hours).
metrical divisions in Xenopus laevis embryos always occurs at the 12th Similar to the ICM in mouse (Morris et al., 2012), sheep (Willadsen,
cleavage after fertilization (Masui and Wang, 1998; Wang et al., 2000). 1981) and rhesus monkey demi-embryos (Schramm et al., 2002), the

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After the 12th cleavage, the cells become contact dependent and cell ICM in human twin embryos had fewer cells in comparison with the
cycle durations become variable. The molecular mechanisms governing ICM in intact controls (Noli et al., 2015a). Whether the number of
such strict schedules are unclear. It could be simply due to the physical pluripotent cells in human twin embryos could be increased by
time needed for successive gene activations and biochemical reactions manipulating fibroblast growth factor (FGF) and WNT signalling or
to take place or to the nuclear/cytoplasmic ratio (Masui and Wang, some other pathways, such as IGF1 (Kimber et al., 2008; Noli et al.,
1998; Wang et al., 2000). Research on the spatial and temporal moni- 2015b) remains to be investigated.
toring of mouse embryonic development has suggested that a develop-
mental clock also exists in mammals (Morris et al., 2012). The
landmark developmental events of cell compaction, lineage commit-
The role of cell–cell interactions in fate
ment and cavitation took place at the same time in the embryos split
at the 2-cell stage as in the intact non-manipulated controls. However,
specification and Hippo signalling
their developmental potential was not the same. Unless the ICM con- Previous research in the murine system has supported the importance
tained a minimum of four NANOG-positive pluripotent stem cells at of continuous cell–cell interaction in the regulation of blastomere fate,
the time of implantation, development did not proceed. Modulation of as biopsied blastomeres tend to re-establish cell–cell interactions sub-
Fgf and Wnt signalling increased the number of pluripotent cells in sequent to their transfer into recipient ZP (Johnson and Ziomek, 1981;
demi-embryos and rescued the developmental failure phenotype. Lorthongpanich et al., 2012). A pre-patterning model (Piotrowska and
Interestingly, the same treatment did not work if the blastomeres were Zernicka-Goetz, 2001; Piotrowska et al., 2001) posits that the ICM
separated at the 4-cell stage indicating that at that stage, at least in and TE lineages undergo predetermination due to the asymmetrical
mouse, all blastomeres do not have equal developmental potential. localization of molecular determinants in the oocyte; conversely, the
Indeed, cell fate in 4-cell mouse embryos is biased through heterogen- inside–out (Tarkowski and Wroblewska, 1967) and cell polarity models
eity in Oct4 and Sox2 targets (Goolam et al., 2016). (Johnson and Ziomek, 1981) hypothesize that a decision-making pro-
Data from our study with human embryos suggested that the cess is dependent on cell position within the embryo.
human preimplantation development is also subject to strict temporal Our results have suggested that lineage determination in human
embryos takes place through the cell position-dependent inside–out
or cell polarity models (Noli et al., 2015a). In the pre-patterning
model, the number of blastomeres used for the creation of twin
embryos is not a governing factor; therefore, the probability of form-
ing twin embryos with better quality ICMs would have been higher
than what we observed.
Recently completed studies in a mouse model have implied that the
Hippo pathway is responsible for the translation of positional informa-
tion to lineage specification, acting primarily through the downstream
mediator proteins YAP1 and TEAD1-4 (reviewed in Lorthongpanich
and Issaragrisil, 2015). The immunostaining studies of YAP1 expression
in twin embryos suggested that this mechanism may also be conserved
in human embryonic development (Noli et al., 2015a).

Lineage commitment and reproductive

competence
Figure 3 The size of twin embryos is proportionate to the num- The first embryonic cell fate commitment, ICM and TE lineage segre-
ber of cells used for their creation: more cells used as the starting gation, begins at the compaction/morula stage, when asymmetric cell
material produce bigger blastocysts. division pushes one cell inwards and the other daughter cell remains
outside (Bruce and Zernicka-Goetz, 2010; Lorthongpanich et al., 2012).
Potential use of embryo splitting 163

This decision, however, is not ultimate. The outer cells from the morula unique miRNA profile in spent media of twin blastocysts might be a
and the early blastocyst tend to retain their plasticity for a short period result of differential lineage commitment in these embryos.
of time. These cells express the pluripotency markers POU5F1
(OCT4), SOX2 and SALL4 and the TE markers HLA-G and KRT18 but
not CDX2 (Cauffman et al., 2009; Chen et al., 2009; Verloes et al., Conclusion and prospectives
2011). NANOG expression has also been reported in the polar TE cells
of the early blastocyst (Cauffman et al., 2009). Upon isolation from fully Data from the most recent studies on split human embryos have sug-
developed human blastocysts and their subsequent reaggregation, TE gested that embryo splitting using the blastomere biopsy or separation
cells were able to develop into blastocysts expressing the pluripotency techniques may be neither a suitable source of genetically identical
marker NANOG (De Paepe et al., 2013). Furthermore, most of the iso- embryos for research purposes, nor a novel-assisted reproduction
lated TE cells did not regain their original position when placed in the treatment (Noli et al., 2015a). Aberrant lineage commitment and a
centre of the embryo; instead, they integrated into the ICM with subse- strict developmental clock demonstrated in human split embryos may
quent expression of NANOG, indicating that the TE cells at that stage also explain the poor results reported from twinning experiments in

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of embryonic development were not yet fully committed (De Paepe non-human primates (Chan et al., 2000; Mitalipov et al., 2002).
et al., 2013). However, data provided by the UK HFEA showed that in 1104 live
We found that Day 5 twin embryos expressed NANOG almost births from cycles involving single embryo transfer, the rate of multiple
universally, with NANOG-positive cells co-localizing with the TE pregnancies was 2.3%, which is higher than the rate of 0.4–0.45%
markers CDX2 and GATA2 (Noli et al., 2015a). Although TE cells reported in in vivo conceptions (Blickstein et al. 2003; Aston et al.,
co-expressing NANOG, CDX2 and GATA2 were reduced in num- 2008). This suggests that IVF may lead to embryo splitting in vivo and
ber by Day 6, they still constituted a significant fraction of the TE. that further exploration of in vitro techniques is warranted.
However, at Day 6, we could find only 1–4 NANOG-positive cells Embryo bisection, a preferable method of cloning in veterinary medi-
that lost the expression of CDX2 and GATA2, indicating the initial cine, has not yet been used on human embryos. If the parental embryo
formation of the ICM. SOX17, a marker of primitive endoderm, was is of high quality, especially with a large ICM, a careful bisection might
also detected in twins with larger ICMs on Day 6. The data indicated yield two developmentally competent embryos. Although such a tech-
that the molecular events responsible for first and second fate com- nical approach might benefit fertility patients, the approach is limited by
mitments of the embryos were taking place in the split embryos; lack of quality control, and there is as yet no country in which regula-
however, they lagged behind the control intact blastocysts obtained tions permit embryo splitting for clinical purposes. The possibilities of
by IVF. Irrespective of these findings, in almost all twins with distin- maintaining human embryos in culture beyond Day 7 are still very lim-
guishable ICM-like structure on Day 6, the ICM was small and of ited and there would be little room for comparative studies. However,
poor quality. This may suggest that, similar to the experimental the most recent advances in extending the culture of human embryos
results with mouse split embryos, the epiblast possessed an insuffi- beyond Day 7 may allow some progress (Deglincerti et al., 2016;
cient number of cells to continue the post-implantation development Shahbazi et al., 2016); this extended culture will allow more detailed
of the conceptus, rendering the twin embryos reproductively incom- research into the early development of the embryo, leading to a better
petent (Balbach et al., 2010; Morris et al., 2012). understanding of the limitations of embryo manipulation.
miRNAs are known regulators (mostly repressors) of target gene As IVF techniques improve, the need to provide additional embryos
expression; they are secreted (Valadi et al., 2007) from embryos for transfer should become less pressing, and this, together with the
in vitro, and attempts have been made to link specific miRNAs ethical problems associated with this type of embryo manipulation,
detected in spent blastocyst medium (SBM) with embryo ploidy sta- may mean that this approach may never reach the clinic.
tus and reproductive competence (McCallie et al., 2010, Kropp et al.,
2014, Rosenbluth et al., 2014, Capalbo et al., 2016). Analyses of
spent culture media collected at different stages of preimplantation Acknowledgements
embryo development showed a marked increase in the number of
We thank science illustrator Nikola Kolundzic ([email protected])
miRNA detected at the blastocyst stage (Capalbo et al., 2016).
for schematic drawings.
These authors compared miRNAs in SBM from 25 implanted euploid
embryos which had later resulted in the birth of a healthy baby fol-
lowing a single embryo transfer, with 28 non-implanted embryos and
identified miR-20a and miR-30c as statistically significantly more abun- Authors’ roles
dant. Analysis of miRNA detected in SBM of twin blastocysts L.N. conducted the review of literature and wrote the first draft of
revealed a unique profile (Noli et al., 2016). Nearly a quarter (11 out the paper. D.I. and Y.K. supervised the work, helped in performing
of 48) of the miRNA found in SBM of twin embryos were not the review of literature and corrected the paper. C.O. critically
detected in SBM or TE samples of normal blastocysts. Furthermore, reviewed the manuscript. All authors approved the final version of
levels of those detected in spent culture media of both twin and nor- the manuscript.
mal blastocysts were consistently different, including miR-30c, which
was significantly lower in media of twin embryos. Although the exact
role of miRNAs secreted from developing embryos is unclear, it likely
reflects development-related specific gene activity. Since the majority
Funding
of cells in twin embryos express both TE and ICM markers, the L.N. is supported by Saudi Arabia government studentship.
164 Noli et al.

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