doi: 10.1111/j.1365-2796.2011.02445.x
Epigenetics and assisted reproductive technology
A. N. Iliadou1, P. C. J. Janson2 & S. Cnattingius3
Fromthe1DepartmentofMedicalEpidemiologyandBiostatistics; 2ClinicalAllergyResearchUnit,DepartmentofMedicine;and3Clinical
EpidemiologyUnit,DepartmentofMedicine;KarolinskaInstitutet,Stockholm,Sweden
Abstract. Iliadou AN, Janson PCJ, Cnattingius S (Karo-
linska Institutet, Stockholm; Sweden). Epigenetics
and assisted reproductive technology (Review). J In-
ternMed2011;270: 414–420.
During gametogenesis, the female and male germ
cells undergo a process whereby imprinting marks
are erased from the genome. During the later stages
of germ-cell development, the methylation marks of
the female and male germ lines are re-established. A
second phase of demethylation of the genome occurs
at the time of fertilization, and during development of
the early embryo. Assisted reproductive technology
involves several steps that subject the gametes and
early developing embryos to environmental stress,
and this is the primary reason for an increased inter-
est in the putative link between these techniques and
imprinting disorders. Although animal studies sup-
port a link between assisted reproductive techniques
(ARTs) and imprintingdisorders, viaalteredmethyla-
tion patterns, data in humans are inconsistent. Here
we provide an overview of the field of epigenetics in
relationtoARTs.
Keywords: assisted reproductive techniques, DNA
methylation,epigenetics,genomic imprinting.
Introduction
People throughout the world can appreciate and re-
late to the findings of Professor Robert Edwards, the
2010Nobel Prizewinner inphysiologyormedicine for
the development of in vitro fertilization (IVF) tech-
niques, which have led to the births of over 4 million
babies. Although initially controversial, with both
financial and ethical opposition, assisted reproduc-
tive techniques (ARTs) are now generally well ac-
cepted. Currently, ARTs account for between 1% and
4%of births annually inmanywestern countries, and
theglobaluseof these techniques isgrowing [1,2].
Recent studies suggest a possible link between hu-
man ARTs and genomic imprinting disorders [3–6].
Concern for epigenetic effects of theseassistedproce-
dures in humans has arisen from the observation of
an increased incidence of rare genomic imprinting
diseases, such as Beckwith–Wiedemann syndrome
(BWS)andAngelmansyndrome (AS), inchildrenborn
after the use of ART [7–9]. One of the symptoms of
BWS is overgrowth in children and the disease has
been associated with altered imprinting of genes,
including the IGF2R gene [7, 10]. AS is a neurocogni-
tive disorder associated with loss of methylation in
imprinted gene clusters. It is interesting that most of
the imprinted genes identified to date have a role in
regulatingpre-and ⁄orpostnatalgrowth [11,12].
In this short review,wewill start bypresenting the re-
ported risks of pregnancy by ARTs and then discuss
the current understanding of the link between ARTs
and epigenetic disorders in both animals and
humans.
Outcome of pregnancies by ARTs
Scientific activity to investigate the possible conse-
quences and adverse effects of ARTs has increased
exponentially during the last decade. In recent years,
several studies have reported both short- and long-
term adverse outcomes of ARTs. The safety of the
procedures and the health of children conceived by
ARTs have been questioned [13]. However, little is
known about whether these adverse effects are the
result of ARTs per se or the consequence of parental
subfertility.
Assisted reproductive techniques have been associ-
ated with pregnancy complications (pre-eclampsia
and placental complications), adverse perinatal out-
comes(lowbirthweight,pretermbirth,perinatalmor-
tality and congenital malformations) and epigenetic
alternations. However, studies of adverse outcomes
after theuse ofARTshavebeenquestioned, primarily
because multiple births are over-represented in
assisted pregnancies. Hence, it has been difficult to
determine whether pregnancy complications and
414 ª 2011 The Association for the Publication of the Journal of Internal Medicine
Review |

adverse outcomes are a result of ARTs or owing to
multiple births and their associated problems. Of
interest, in a Danish national cohort study, nomajor
differences in physical health were found between
twins conceived through ARTs (ART twins) and those
conceivednaturally (non-ARTtwins) [14]. It shouldbe
noted,however, thatARTtwinsarealmostexclusively
dizygotic (DZ) whereas one-third of non-ART twins
are monozygotic (MZ), and hence comparisons be-
tween ART and non-ART twins are not equivalent.
Thus, to investigate differences in outcomes between
ART and non-ART twin pregnancies, comparisons
should be made after taking zygosity into account. A
study of umbilical cords of DZ ART twins showed
more pathological characteristics compared to cords
fromDZnon-ARTtwins [15]. Furthermore,whenART
twinswerecomparedtosingletonsconceivedthrough
ARTs (ART singletons), twins were more often admit-
ted to neonatal intensive care units and had poorer
speechdevelopment, evenafterstratification forbirth
weight [16, 17]. A systematic review andmeta-analy-
sis showed that, compared to non-ART twins, ART
twinsweremoreoftendeliveredbycaesareansection,
bornpretermandadmitted toneonatal intensive care
units [18]. It was also noted that mothers who had
undergone intracytoplasmic sperm injection (ICSI),
which involves direct injection of sperm into the oo-
cyte, took more sick leave and were more frequently
hospitalized during pregnancy [19]. No other results
were significantly different between mothers with
ART twin pregnancies and those with spontaneously
conceivedtwinpregnancies.
To reduce the risks of pregnancy and perinatal com-
plications, a single embryo transfer was introduced
as standard practice in some countries in the early
2000s. Results from the many independent pub-
lished reviews and meta-analyses of perinatal out-
comes in ART singletons show even stronger differ-
ences between ART and non-ART singletons
compared to ART and non-ART twins [20–24]. ART
singletons have on average a 2-fold increased risk of
perinatal mortality and preterm birth (<37 weeks), a
50% increased risk of low birth weight (<2500 g) and
an even greater risk of very low birth weight
(<1500 g).ARTsingletonsarealsoat increasedriskof
caesarean delivery, being small for gestational age,
admission to a neonatal intensive care unit and birth
defects. The aetiology and biological mechanisms
underlying these risks in singleton ART pregnancies
remain essentially unresolved. However, recently a
Norwegianstudy, comparingnaturally andART-con-
ceived siblings, showed no significant differences in
small for gestational age foetuses andpretermbirths,
suggesting that reported differences may be owing to
parents’ infertility rather than the ART procedures
[25].
Register studies in Sweden investigating pregnancy
outcomes and maternal and childhood morbidity
have also found increased risks of low birth weight,
small forgestationalage foetuses (5.1%inARTsingle-
tons vs. 2.8% in the general population of singletons)
and preterm births (9.6% in ART singletons vs. 5.3%
in the general population of singletons) [26–33].
Adjusting for years of involuntary childlessness re-
duced the risks,whichnevertheless remainedsignifi-
cantly increased for low birth weight and preterm
birth [34]. A recent study showed that these risks
were greater in infants conceived after embryo trans-
fer at the blastocyst stage than after two-stage cleav-
age, suggesting that the duration of the embryo in
culture medium could be one possible explanation
for the increased risks [35]. Hence, there is evidence
to suggest that ART singleton foetuses may have an
increased risk of low birth weight and ⁄or preterm
birth. There is a plethora of epidemiological evidence
to support the foetal programminghypothesis,which
states that restricted intrauterine growth has impor-
tant lifelonghealth implications, therefore it is essen-
tial to investigate theunderlyingmechanisms [36]. In
the light of evidence that adverse environmental con-
ditions early in human gestation can be recorded as
persistentchanges inepigenetic information [37], it is
of even greater importance to study and understand
potential epigeneticchangesrelated toARTs.
Epigenetics and genomic imprinting
It iswell known that the phenotype of an individual is
not exclusively determined by the genotype. In 1942,
Waddington [38] introduced the term epigenetics,
defined as ‘heritable changes in gene expression that
occurwithoutanychanges ingenesequence’.
Epigenetic modifications are heritable in the sense
that the ‘epigenetic status’ of the chromatin is pre-
served during cell mitosis. There are many different
types of epigenetic modifications that are known to
affect expression, including changes in nucleosome
positioning and conformation, and histonemodifica-
tions such as acetylation, phosphorylation,methyla-
tionandubiquitinylation [39].
Themost thoroughly studied epigeneticmodification
toDNA ismethylation.DNAmethylation inmammals
is almost exclusively restricted to CpGdinucleotides.
Clusters of CpG dinucleotides, i.e. CpG islands, are
A. N. Iliadou et al. | Review: Epigenetics and assisted reproductive technology
ª 2011 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine 270; 414–420 415