AOGS COM M ENT A R Y

Epigenetics and assisted reproductive technologies

ANJA PINBORG1, ANNE LOFT2, LIV B. ROMUNDSTAD3,4, ULLA-BRITT WENNERHOLM5,
6

7
, CHRISTINA BERGH5 & KRISTIINA AITTOMAKI
VIVECA SODERSTR
OM-ANTTILA
1

Fertility Clinic, Department of Obstetrics/Gynecology, Hvidovre Hospital, University of Copenhagen, Hvidovre, 2Fertility
Clinic, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark, 3Department of Public Health and General
Practice, Norwegian University of Science and Technology (NTNU), Trondheim, 4Spiren Fertility Clinic, Trondheim, Norway,
5
Department of Obstetrics and Gynecology, Institute for Clinical Sciences, Sahlgrenska Academy at the University of
Gothenburg, Gothenburg, Sweden, 6Fertility Clinic, The Family Federation of Finland, Helsinki, and 7Department of
Medical Genetics, Helsinki University Central Hospital (HUCH), Helsinki, Finland

Key words
Infertility, assisted reproduction, delivery,
morbidity, physiology of reproduction
Correspondence
Anja Pinborg, Fertility Clinic, Department of
Obstetrics/Gynaecology, Hvidovre Hospital,
University of Copenhagen, Kettegaard All
e
30, 2650 Hvidovre, Denmark.
E-mail: anja.bisgaard.pinborg.01@regionh.dk
Conflict of interest
The authors have stated explicitly that there
are no conflicts of interest in connection with
this article.
Please cite this article as: Pinborg A, Loft A,
Romundstad LB, Wennerholm U-B,
derstro
m-Anttila V, Bergh C, et al.
So
Epigenetics and assisted reproductive
technologies. Acta Obstet Gynecol Scand
2016; 95:1015.

Abstract
Epigenetic modification controls gene activity without changes in the DNA
sequence. The genome undergoes several phases of epigenetic programming
during gametogenesis and early embryo development, coinciding with assisted
reproductive technologies (ART) treatments. Imprinting disorders have been
associated with ART techniques, but disentangling the influence of the ART
procedures per se from the effect of the reproductive disease of the parents is a
challenge. Epidemiological human studies have shown altered birthweight profiles in ART compared with spontaneously conceived singletons. Conception
with cryopreserved/thawed embryos results in a higher risk of large-for-gestational-age babies, which may be due to epigenetic modification. Further animal
studies have shown altered gene expression profiles in offspring conceived by
ART related to altered glucose metabolism. It is controversial whether human
adolescents conceived by ART have altered lipid and glucose profiles and
thereby a higher long-term risk of cardiovascular disease and diabetes. This
commentary describes the basic concepts of epigenetics and gives a short
overview of the existing literature on the association between imprinting
disorders, epigenetic modification and ART.
ART, assisted reproductive technologies; ICSI, intracytoplasmic
sperm injection; IVF, in vitro fertilization; SNRPN, small nuclear
ribonucleoprotein polypeptide N.

Abbreviations:
Received: 24 April 2015
Accepted: 31 August 2015
DOI: 10.1111/aogs.12799

Introduction
Worldwide, more than 5 million children have been born
after assisted reproductive technologies (ART) and represent 0.55% of the national birth cohorts in Europe (1).
ART includes both standard in vitro fertilization (IVF)
and intracytoplasmic sperm injection (ICSI). After the
implementation of elective single embryo transfer, twin
rates after ART have decreased considerably in the Nordic countries concomitantly with improved perinatal outcomes for children conceived by ART (1,2). Although

10

declining twin rates have improved outcomes for children conceived by ART in general, singletons conceived
by ART still carry a slightly increased risk of preterm
birth and of being small-for-gestational-age, which is
partly explained by the reproductive characteristics of the

Key Message
Basic concepts of epigenetics in relation to assisted
reproductive technologies.

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A. Pinborg et al.

Epigenetics and ART

parents (3). The remaining risk may be related to the

ovarian hormone stimulation or the in vitro techniques
including laboratory parameters and culture conditions,
i.e. culture media and duration of culture. Another finding is that singletons born after frozen embryo transfer
are at increased risk of being large-for-gestational-age
(4). In the early days of IVF, animal studies showed that
IVF can cause intrauterine overgrowth, a phenomenon
known as the large offspring syndrome in cattle and
sheep (5,6). Imprinting disorders may be more common
in children conceived by ART and further epigenetic
modification in gametes or embryos may be caused by
ART, resulting in health consequences in adulthood (6).
There are two ways of considering imprinting problems
associated with ART: (i) the imprinted disorders are
associated with fertility problems of the parents, which
involves at least four genes (H19, LIT1, SNRP, UBE3A)
(7,8) and (ii) the differences in the methylation indices is
a consequence of the adaptation to the early environment, which differs in pregnancies conceived by ART.
The involved genes differ and can be measured as the
level of methylation in many DNA regions (9). This
commentary describes the basic concepts of epigenetics
and gives a short overview of the existing literature on
the association between imprinting disorders, epigenetic
modification and ART.

Material and methods

We searched in PubMed and Cochrane databases for
published articles on this topic during 19802014 and we
included only English language articles. The following
search words were used: imprinting disorder, imprinting
disease, epigenetic, assisted reproductive technology,
in vitro fertilization and intracytoplasmic sperm injection.
We based our commentary on the existing reviews and
meta-analyses and original articles reporting most recent
findings.

Basic concepts
The epigenome modifies the genome without altering the
DNA sequence (Figure 1). The epigenome is a network of
chemical compounds which tags on to the DNA; its role
is to determine which genes are active in a particular cell.
Hence, epigenetic modification is an important form of
controlling gene activity without changes in the DNA
sequence. Several mechanisms of epigenetic control exist,
including DNA methylation, histone modification, noncoding RNA, remodeling of nucleosomes and organization of chromatin structure (10). DNA methylation is the
best studied of these epigenetic mechanisms. The genome
undergoes several phases of epigenetic programming dur-

Figure 1. Basic concepts of epigenetics.

ing gametogenesis and early embryo development coinciding with IVF and ICSI treatments (11). When the
primordial germ cells have migrated to the genital ridge,
their genome undergoes erasure of epigenetic marks. This
enables subsequent epigenetic reprogramming and parent-of-origin controlled activity of specific genes. This
reprogramming is gender-specific, as a proportion of
genes are only active when inherited from the mother
and are inactivated when inherited from the father and
vice versa, a phenomenon called genetic imprinting.
While methylation is completed in the mature spermatozoa, the same phenomenon in oocytes is nearly complete
at the time of ovulation. The first phase of epigenetic
reprogramming takes places during gametogenesis, as
explained above. After fertilization, the second wave of
epigenetic reprogramming occurs with demethylation of
the paternal and maternal genome, after which another
phase of DNA methylation takes place (12). While the
demethylationmethylation occurs in the entire genome
of the early embryo, the imprinted genetic regions are
protected and maintain their gender-specific methylation
patterns created during gametogenesis. The remethylation
is completed by implantation. Aberrancies in epigenetic

2015 Nordic Federation of Societies of Obstetrics and Gynecology, Acta Obstetricia et Gynecologica Scandinavica 95 (2016) 1015

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A. Pinborg et al.

programming, such as hypermethylation or hypomethylation of DNA within a specific genetic region, can result
in disturbances of gene activity and imprinting disorders.
The epimutations causing diseases are maintained through
cell division and can be inherited (13).

Imprinting disorders in children conceived by

ART
The prevalence of the four best known imprinting disorders is reported in Table 1 (14). In the beginning of this
century the first studies suggested an increased incidence
of imprinting disorders associated with children conceived by ART (15,16). Since then diverging results have
been published. The incidence of imprinting disorders

such as BeckwithWiedemann, Angelman, PraderWilli

or SilverRussell syndromes is low in the general population, about 1:10 000 to 1:30 000, with various qualities of
diagnosis and registration of these rare diseases. Therefore, it is a challenge to conduct studies powered to either
confirm or reject an association between imprinting disorders and ART.
The original paper on retinoblastomas in children conceived by ART included only five children and reported
no data on tumor methylation characteristics (16). Ten
years later, Dommering et al. (17) examined the characteristics of the tumors from the five children included in
the original paper and added data on tumor characteristics on two more children conceived by ART. They found
that none of the tumors in either of the children showed

Table 1. Characterization of four human imprinting disorders.

Disorder

Genetic errors

Prevalence

Clinical features

Beckwith
Wiedemann

Chromosome 11p15.5

1:15 000

Fetal macrosomia, macroglossia, renal anomalies, midline

abdominal wall defects, earlobe creases or ear pits, facial
nevus flammeus, neonatal hypoglycemia, and increased
risk of childhood cancer

1: 392 0001:3000

Intrauterine or postnatal growth retardation, learning

disabilities, relative macrocephaly and small triangular
face, limb length asymmetry and a variety of minor
malformations

1: 20 0001:12 000

Severe mental retardation, speech impairment, abnormal

EEG and seizures, often microcephaly, subtle dysmorphic
facial features, jerky movements and hand flapping and
happy disposition

1:30 0001:10 000

Neurodevelopmental disorder with cognitive disabilities,

characteristic facial appearance (narrow temples,
elongated face, thin upper lip, prominent nose), low
muscle tone, short stature and hyperphagia

Paternal UPD (2025%)

Methylation defects (5060%)
Maternal mutations (5%)
Translocation/inversion, duplication and
micro-deletions (each <1%)
Unknown (1020%)

SilverRussell

Genetically heterogeneous condition,

represents a phenotype rather than a
specific disorder
Chromosome 7-related and 11p15.5

Maternal UPD (chromosome 7; 510%)

Maternal UPD (chromosome 11; <1%)
Methylation defect (chromosome 11;
3050%)

Duplication (chromosome 7 or 11;

rare/unknown)

Angelman

Chromosome 15q11-13

PraderWilli

Paternal UPD (27%)

Maternal deletion (7075%)
Methylation defect (25%)
Point mutations (10%)
Translocation, inversion (<1%)
10% unknown

Chromosome 15q11-13

Maternal UPD (2535%)

Paternal de novo deletion (6575%)
Methylation defect (15%)
Micro-deletions and translocations
(rare/unknown)

UPD, uniparental disomy (e.g. two copies of a chromosome, or of part of a chromosome, from one parent and no copy from the other parent).

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A. Pinborg et al.

hypermethylation of the RB1 gene promoter. Only 13%

of cases of retinoblastomas are due to an epigenetic
change, hypermethylation of the RB1 gene promoter. As
none of the children conceived by ART expressed this
hypermethylation in the retinoblastomas, it is not likely
that any of the tumors were associated with ART conception (17).
Studies have reported significant methylation alterations in sperm from men with different degrees of male
factor infertility and in oocytes from women undergoing
ovarian stimulation (18). In 2014, Whitelaw et al. (8)
studied three imprinted genes/regions with known implications for reproduction. They explored aberrant imprinting in the small nuclear ribonucleoprotein polypeptide N
(SNRPN) region, which is known to be associated with
PraderWilli syndrome and Angelman syndrome. The
methylation level of SNRPN was associated with the duration of infertility and was significantly higher in children
conceived by ICSI than by standard IVF and spontaneous
conception. However, the observed differences in methylation levels in children conceived by ART compared with
spontaneously conceived children were relatively small.
In a comprehensive review from 2013, it was estimated
that BeckwithWiedemann syndrome was significantly
associated with ART with a pooled relative risk of 5.2
[95% confidence interval (CI) 1.6, 7.4]) (19). There were
too few data on SilverRussell syndrome to draw a final
conclusion, but the authors stated that a positive association with ART was likely, whereas this was not the case
for Angelman syndrome, PraderWilli syndrome and
retinoblastoma. However, the authors concluded that
Angelman syndrome and PraderWilli syndrome may be
associated with couples having fertility problems (19).
The conflicting results are most likely due to the small
populations in the reported studies.
A recent systematic review with a meta-analysis
demonstrated an association between children conceived
by ART and imprinting disorders when compared with
spontaneously conceived children (18). The combined
odds ratio of any imprinting disorder was 3.67 (95% CI
1.39, 9.74), but weighted mean differences of selected
imprinted genes showed no differences in methylation
levels between children conceived by ART and spontaneously conceived children (18). Lazaraviciute et al. concluded that imprinting disorders are more common after
ART, but the amount of data is both heterogeneous and
limited, thus more controlled studies with adjustment for
the reproductive disease of the couples are needed. Most
importantly, if such changes in the imprinting epigenetic
status in children conceived by ART hold true, the effect
is small (18).
In summary, imprinting disorders have been associated
with ART techniques, but disentangling the effect of the

Epigenetics and ART

ART procedures per se from the effect of the reproductive

disease of the parents is a challenge.

Fetal environmental conditions and epigenetics

While the research on epigenetic programming is mainly
based on animal studies, data from the Dutch and Chinese famines have shown that fetal environmental conditions can cause epigenetic changes in humans that persist
throughout life (20). The epidemiological studies indicated that children conceived during the Dutch Hunger
Winter of 194445, experienced persistent detrimental
health effects such as a higher rate of heart disease later
in life. Children exposed to the famine during the first
10 weeks in utero had lower mean birthweight and
hypomethylation of the imprinted insulin-like growth factor 2 (IGF2) gene compared with their unexposed samesex siblings. By contrast, children exposed to the famine
at the end of pregnancy showed no difference in methylation compared with their unexposed siblings. Similarly,
studies on Chinese children born during the Great Chinese Famine of 195861 have linked prenatal exposure to
famine to an increased risk of diabetes, obesity and
schizophrenia later in life (21). The findings from these
inter-generational studies highlight the fact that environmental exposure during early fetal life may cause longterm health effects even across generations.

Epigenetic modification and ART

Animal studies have shown that gametogenesis, fertilization and early embryo development are stages vulnerable
to epigenetic dysregulation (22,23). The epigenome is
known to be sensitive to environmental changes and to
have the potential to sustainably alter gene expression,
notably during embryonic development (24).
Several animal studies have confirmed that the type of
conditions encountered by the embryo during the preimplantation stage may affect the phenotype of the adult,
and epigenetic modification has been suggested as the
possible mechanism (24). Grace and Sinclair showed that
the various steps of the ART procedure in animal studies
affected the epigenome, as superovulation and in vitro
culture of oocytes caused epigenetic changes in the
embryos and offspring (6). Further in vitro embryo culture conditions have an established epigenetic effect on
cultured embryos in animal models (25) and Horsthemke
et al. (7) showed a difference in the methylation patterns
of various genes associated with the ART process and/or
the etiology of infertility.
Human studies have shown that different ART methods such as ovarian stimulation and supraphysiological
levels of sex steroid hormones, culture media and embryo

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A. Pinborg et al.

cryopreservation may be associated with intrauterine

growth resulting in altered birthweight profiles, which
may be caused by epigenetic modification (4,26,27).
A recent study demonstrated altered gene expression profiles associated with glucose intolerance in children conceived by ART compared with spontaneously conceived
children (28).
Further, some reports indicate that children conceived
by ART have an altered lipid profile, fasting glucose, and
body fat distribution and cardiovascular function (29,30).
Hence, concern has been raised that children conceived
by ART may have a higher risk of developing cardiovascular disease later in life, which may be related to epigenetic changes (30).
In contrast to singletons conceived after fresh embryo
transfer, human singletons conceived after frozen embryo
transfer have a higher risk of being large-for-gestationalage or have a birthweight of more than 4500 g at term
(4,27). This phenomenon is also observed in sibling pairs
where the same mother has given birth to both a singleton conceived after fresh embryo transfer and a singleton
conceived after frozen embryo transfer (4).
This indicates that embryo manipulations performed
during freezing and thawing may cause changes in the
development of the feto-placental unit through epigenetic modification. Whether such early epigenetic alterations and changes in intrauterine growth can cause
long-term diseases, such as diabetes and cardiovascular
diseases later in life, in children conceived by ART is
still unknown.
Whether ART causes changes in the epigenetic programming can be detected by the measuring DNA methylation signatures on the whole genome, as presented by
Tobi et al. (9). As is the case for (mal) nutrition, comprehensive genome-scale studies of differential methylation
following conception by ART are lacking in humans.

Conclusions
The association between imprinting disorders and ART is
still debated; however, if such an association exists, it is
weak and at least partly related to the reproductive disease of the parents. Thus, future research should focus on
epigenetic modifications in children conceived by ART
and the long-term morbidity related to these epigenetic
changes and should be able to adjust for the severity of
the reproductive disease of the parents. For the safety and
quality surveillance of ART techniques, it is of utmost
importance that the Nordic ART registries include cyclespecific parameters, i.e. culture media, duration of culture, freezing techniques, and additionally demographic
data of the couples including cause and duration of
infertility.

14

Recently, several new ART techniques have been introduced, such as prolonged culture time from 2 to 5 days
(blastocyst culture) and ultra-rapid and effective freezing/
thawing methods (vitrification).
To be able to study the long-term effects of the new
techniques, all those working in the field of reproduction
should aim for national ART registers with high quality
registration with linkage between ART treatment and
child outcome.
The Committee of Nordic ART and Safety (CoNARTaS)
has collected information on all Nordic children conceived
by ART in one common database including more than
90 000 children born from 1982 to 2007 (2). The aim of
CoNARTaS is to conduct continuous surveillance of children conceived by ART in all aspects related to morbidity
and development. This database is highly dependent on
data from the Nordic national registers, emphasizing the
importance of including cycle-specific information and
new techniques, which possibly remain the best long-term
quality and safety controls of ART methods.

Funding
The Nordic Expert group research work was unconditionally supported by MSD in Finland, Norway and
Denmark.
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