AOGS COM M ENT A R Y

Epigenetics and assisted reproductive technologies

ANJA PINBORG1, ANNE LOFT2, LIV B. ROMUNDSTAD3,4, ULLA-BRITT WENNERHOLM5,
€
VIVECA SODERSTR €
OM-ANTTILA 6 € 7
, CHRISTINA BERGH5 & KRISTIINA AITTOMAKI
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 Abstract

Infertility, assisted reproduction, delivery,
morbidity, physiology of reproduction Epigenetic modification controls gene activity without changes in the DNA
sequence. The genome undergoes several phases of epigenetic programming
Correspondence during gametogenesis and early embryo development, coinciding with assisted
Anja Pinborg, Fertility Clinic, Department of reproductive technologies (ART) treatments. Imprinting disorders have been
Obstetrics/Gynaecology, Hvidovre Hospital,
associated with ART techniques, but disentangling the influence of the ART
University of Copenhagen, Kettegaard All e
procedures per se from the effect of the reproductive disease of the parents is a
30, 2650 Hvidovre, Denmark.
E-mail: anja.bisgaard.pinborg.01@regionh.dk challenge. Epidemiological human studies have shown altered birthweight pro-
files in ART compared with spontaneously conceived singletons. Conception
Conflict of interest with cryopreserved/thawed embryos results in a higher risk of large-for-gesta-
The authors have stated explicitly that there tional-age babies, which may be due to epigenetic modification. Further animal
are no conflicts of interest in connection with studies have shown altered gene expression profiles in offspring conceived by
this article.
ART related to altered glucose metabolism. It is controversial whether human
adolescents conceived by ART have altered lipid and glucose profiles and
Please cite this article as: Pinborg A, Loft A,
Romundstad LB, Wennerholm U-B, thereby a higher long-term risk of cardiovascular disease and diabetes. This
€derstro
So €m-Anttila V, Bergh C, et al. commentary describes the basic concepts of epigenetics and gives a short
Epigenetics and assisted reproductive overview of the existing literature on the association between imprinting
technologies. Acta Obstet Gynecol Scand disorders, epigenetic modification and ART.
2016; 95:10–15.
Abbreviations:ART, assisted reproductive technologies; ICSI, intracytoplasmic
Received: 24 April 2015 sperm injection; IVF, in vitro fertilization; SNRPN, small nuclear
Accepted: 31 August 2015 ribonucleoprotein polypeptide N.

DOI: 10.1111/aogs.12799

declining twin rates have improved outcomes for chil-

dren conceived by ART in general, singletons conceived
Introduction by ART still carry a slightly increased risk of preterm
birth and of being small-for-gestational-age, which is
Worldwide, more than 5 million children have been born
partly explained by the reproductive characteristics of the
after assisted reproductive technologies (ART) and repre-
sent 0.5–5% 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 Key Message
rates after ART have decreased considerably in the Nor- Basic concepts of epigenetics in relation to assisted
dic countries concomitantly with improved perinatal out- reproductive technologies.
comes for children conceived by ART (1,2). Although

10 ª 2015 Nordic Federation of Societies of Obstetrics and Gynecology, Acta Obstetricia et Gynecologica Scandinavica 95 (2016) 10–15
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 find-
ing 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 environ-
ment, 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

Figure 1. Basic concepts of epigenetics.
We searched in PubMed and Cochrane databases for
published articles on this topic during 1980–2014 and we ing gametogenesis and early embryo development coin-
included only English language articles. The following ciding with IVF and ICSI treatments (11). When the
search words were used: imprinting disorder, imprinting primordial germ cells have migrated to the genital ridge,
disease, epigenetic, assisted reproductive technology, their genome undergoes erasure of epigenetic marks. This
in vitro fertilization and intracytoplasmic sperm injection. enables subsequent epigenetic reprogramming and par-
We based our commentary on the existing reviews and ent-of-origin controlled activity of specific genes. This
meta-analyses and original articles reporting most recent reprogramming is gender-specific, as a proportion of
findings. 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.
Basic concepts
While methylation is completed in the mature spermato-
The epigenome modifies the genome without altering the zoa, the same phenomenon in oocytes is nearly complete
DNA sequence (Figure 1). The epigenome is a network of at the time of ovulation. The first phase of epigenetic
chemical compounds which tags on to the DNA; its role reprogramming takes places during gametogenesis, as
is to determine which genes are active in a particular cell. explained above. After fertilization, the second wave of
Hence, epigenetic modification is an important form of epigenetic reprogramming occurs with demethylation of
controlling gene activity without changes in the DNA the paternal and maternal genome, after which another
sequence. Several mechanisms of epigenetic control exist, phase of DNA methylation takes place (12). While the
including DNA methylation, histone modification, non- demethylation–methylation occurs in the entire genome
coding RNA, remodeling of nucleosomes and organiza- of the early embryo, the imprinted genetic regions are
tion of chromatin structure (10). DNA methylation is the protected and maintain their gender-specific methylation
best studied of these epigenetic mechanisms. The genome patterns created during gametogenesis. The remethylation
undergoes several phases of epigenetic programming dur- is completed by implantation. Aberrancies in epigenetic

ª 2015 Nordic Federation of Societies of Obstetrics and Gynecology, Acta Obstetricia et Gynecologica Scandinavica 95 (2016) 10–15 11
Epigenetics and ART A. Pinborg et al.

programming, such as hypermethylation or hypomethyla- such as Beckwith–Wiedemann, Angelman, Prader–Willi

tion of DNA within a specific genetic region, can result or Silver–Russell syndromes is low in the general popula-
in disturbances of gene activity and imprinting disorders. tion, about 1:10 000 to 1:30 000, with various qualities of
The epimutations causing diseases are maintained through diagnosis and registration of these rare diseases. There-
cell division and can be inherited (13). fore, it is a challenge to conduct studies powered to either
confirm or reject an association between imprinting dis-
orders and ART.
Imprinting disorders in children conceived by
The original paper on retinoblastomas in children con-
ART
ceived by ART included only five children and reported
The prevalence of the four best known imprinting disor- no data on tumor methylation characteristics (16). Ten
ders is reported in Table 1 (14). In the beginning of this years later, Dommering et al. (17) examined the charac-
century the first studies suggested an increased incidence teristics of the tumors from the five children included in
of imprinting disorders associated with children con- the original paper and added data on tumor characteris-
ceived by ART (15,16). Since then diverging results have tics on two more children conceived by ART. They found
been published. The incidence of imprinting disorders 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– Chromosome 11p15.5 1:15 000 Fetal macrosomia, macroglossia, renal anomalies, midline
Wiedemann abdominal wall defects, earlobe creases or ear pits, facial
• Paternal UPD (20–25%) nevus flammeus, neonatal hypoglycemia, and increased
• Methylation defects (50–60%) risk of childhood cancer
• Maternal mutations (5%)
• Translocation/inversion, duplication and
micro-deletions (each <1%)
• Unknown (10–20%)

Silver–Russell Genetically heterogeneous condition, 1: 392 000–1:3000 Intrauterine or postnatal growth retardation, learning
represents a phenotype rather than a disabilities, relative macrocephaly and small triangular
specific disorder face, limb length asymmetry and a variety of minor
Chromosome 7-related and 11p15.5 malformations

• Maternal UPD (chromosome 7; 5–10%)

• Maternal UPD (chromosome 11; <1%)
• Methylation defect (chromosome 11;
30–50%)
• Duplication (chromosome 7 or 11;
rare/unknown)

Angelman Chromosome 15q11-13 1: 20 000–1:12 000 Severe mental retardation, speech impairment, abnormal
EEG and seizures, often microcephaly, subtle dysmorphic
• Paternal UPD (2–7%) facial features, jerky movements and hand flapping and
• Maternal deletion (70–75%) happy disposition
• Methylation defect (2–5%)
• Point mutations (10%)
• Translocation, inversion (<1%)
• 10% unknown

Prader–Willi Chromosome 15q11-13 1:30 000–1:10 000 Neurodevelopmental disorder with cognitive disabilities,
characteristic facial appearance (narrow temples,
• Maternal UPD (25–35%) elongated face, thin upper lip, prominent nose), low
• Paternal de novo deletion (65–75%) muscle tone, short stature and hyperphagia
• Methylation defect (1–5%)
• 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. Epigenetics and ART

hypermethylation of the RB1 gene promoter. Only 13% ART procedures per se from the effect of the reproductive
of cases of retinoblastomas are due to an epigenetic disease of the parents is a challenge.
change, hypermethylation of the RB1 gene promoter. As
none of the children conceived by ART expressed this
Fetal environmental conditions and epigenetics
hypermethylation in the retinoblastomas, it is not likely
that any of the tumors were associated with ART concep- While the research on epigenetic programming is mainly
tion (17). based on animal studies, data from the Dutch and Chi-
Studies have reported significant methylation alter- nese famines have shown that fetal environmental condi-
ations in sperm from men with different degrees of male tions can cause epigenetic changes in humans that persist
factor infertility and in oocytes from women undergoing throughout life (20). The epidemiological studies indi-
ovarian stimulation (18). In 2014, Whitelaw et al. (8) cated that children conceived during the Dutch Hunger
studied three imprinted genes/regions with known impli- Winter of 1944–45, experienced persistent detrimental
cations for reproduction. They explored aberrant imprint- health effects such as a higher rate of heart disease later
ing in the small nuclear ribonucleoprotein polypeptide N in life. Children exposed to the famine during the first
(SNRPN) region, which is known to be associated with 10 weeks in utero had lower mean birthweight and
Prader–Willi syndrome and Angelman syndrome. The hypomethylation of the imprinted insulin-like growth fac-
methylation level of SNRPN was associated with the dura- tor 2 (IGF2) gene compared with their unexposed same-
tion of infertility and was significantly higher in children sex siblings. By contrast, children exposed to the famine
conceived by ICSI than by standard IVF and spontaneous at the end of pregnancy showed no difference in methyla-
conception. However, the observed differences in methy- tion compared with their unexposed siblings. Similarly,
lation levels in children conceived by ART compared with studies on Chinese children born during the Great Chi-
spontaneously conceived children were relatively small. nese Famine of 1958–61 have linked prenatal exposure to
In a comprehensive review from 2013, it was estimated famine to an increased risk of diabetes, obesity and
that Beckwith–Wiedemann syndrome was significantly schizophrenia later in life (21). The findings from these
associated with ART with a pooled relative risk of 5.2 inter-generational studies highlight the fact that environ-
[95% confidence interval (CI) 1.6, 7.4]) (19). There were mental exposure during early fetal life may cause long-
too few data on Silver–Russell syndrome to draw a final term health effects even across generations.
conclusion, but the authors stated that a positive associa-
tion with ART was likely, whereas this was not the case
Epigenetic modification and ART
for Angelman syndrome, Prader–Willi syndrome and
retinoblastoma. However, the authors concluded that Animal studies have shown that gametogenesis, fertiliza-
Angelman syndrome and Prader–Willi syndrome may be tion and early embryo development are stages vulnerable
associated with couples having fertility problems (19). to epigenetic dysregulation (22,23). The epigenome is
The conflicting results are most likely due to the small known to be sensitive to environmental changes and to
populations in the reported studies. have the potential to sustainably alter gene expression,
A recent systematic review with a meta-analysis notably during embryonic development (24).
demonstrated an association between children conceived Several animal studies have confirmed that the type of
by ART and imprinting disorders when compared with conditions encountered by the embryo during the pre-
spontaneously conceived children (18). The combined implantation stage may affect the phenotype of the adult,
odds ratio of any imprinting disorder was 3.67 (95% CI and epigenetic modification has been suggested as the
1.39, 9.74), but weighted mean differences of selected possible mechanism (24). Grace and Sinclair showed that
imprinted genes showed no differences in methylation the various steps of the ART procedure in animal studies
levels between children conceived by ART and sponta- affected the epigenome, as superovulation and in vitro
neously conceived children (18). Lazaraviciute et al. con- culture of oocytes caused epigenetic changes in the
cluded that imprinting disorders are more common after embryos and offspring (6). Further in vitro embryo cul-
ART, but the amount of data is both heterogeneous and ture conditions have an established epigenetic effect on
limited, thus more controlled studies with adjustment for cultured embryos in animal models (25) and Horsthemke
the reproductive disease of the couples are needed. Most et al. (7) showed a difference in the methylation patterns
importantly, if such changes in the imprinting epigenetic of various genes associated with the ART process and/or
status in children conceived by ART hold true, the effect the etiology of infertility.
is small (18). Human studies have shown that different ART meth-
In summary, imprinting disorders have been associated ods such as ovarian stimulation and supraphysiological
with ART techniques, but disentangling the effect of the levels of sex steroid hormones, culture media and embryo

ª 2015 Nordic Federation of Societies of Obstetrics and Gynecology, Acta Obstetricia et Gynecologica Scandinavica 95 (2016) 10–15 13
Epigenetics and ART A. Pinborg et al.

cryopreservation may be associated with intrauterine Recently, several new ART techniques have been intro-
growth resulting in altered birthweight profiles, which duced, such as prolonged culture time from 2 to 5 days
may be caused by epigenetic modification (4,26,27). (blastocyst culture) and ultra-rapid and effective freezing/
A recent study demonstrated altered gene expression pro- thawing methods (vitrification).
files associated with glucose intolerance in children con- To be able to study the long-term effects of the new
ceived by ART compared with spontaneously conceived techniques, all those working in the field of reproduction
children (28). should aim for national ART registers with high quality
Further, some reports indicate that children conceived registration with linkage between ART treatment and
by ART have an altered lipid profile, fasting glucose, and child outcome.
body fat distribution and cardiovascular function (29,30). The Committee of Nordic ART and Safety (CoNARTaS)
Hence, concern has been raised that children conceived has collected information on all Nordic children conceived
by ART may have a higher risk of developing cardiovas- by ART in one common database including more than
cular disease later in life, which may be related to epige- 90 000 children born from 1982 to 2007 (2). The aim of
netic changes (30). CoNARTaS is to conduct continuous surveillance of chil-
In contrast to singletons conceived after fresh embryo dren conceived by ART in all aspects related to morbidity
transfer, human singletons conceived after frozen embryo and development. This database is highly dependent on
transfer have a higher risk of being large-for-gestational- data from the Nordic national registers, emphasizing the
age or have a birthweight of more than 4500 g at term importance of including cycle-specific information and
(4,27). This phenomenon is also observed in sibling pairs new techniques, which possibly remain the best long-term
where the same mother has given birth to both a single- quality and safety controls of ART methods.
ton conceived after fresh embryo transfer and a singleton
conceived after frozen embryo transfer (4).
Funding
This indicates that embryo manipulations performed
during freezing and thawing may cause changes in the The Nordic Expert group research work was uncondition-
development of the feto-placental unit through epige- ally supported by MSD in Finland, Norway and
netic modification. Whether such early epigenetic alter- Denmark.
ations and changes in intrauterine growth can cause
long-term diseases, such as diabetes and cardiovascular References
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