Epigenetics in Reproductive Medicine
ARIANE PAOLONI-GIACOBINO
Department of Genetic Medicine and Development, University of Geneva Medical School, 1211 Geneva 4, Switzerland
ABSTRACT: Imprinted genes comprise a small subset of the ge-
nome whose epigenetic reprogramming in the germ line is necessary
for subsequent normal embryonic development. This reprogram-
ming and resetting of the imprints, through an erasure/acquisition/
maintenance cycle, is a subtle and tightly orchestrated phenomenon,
involving specific genomic regions and methylation enzymes. Dys-
regulation of imprinted genes has indeed been shown to lead to
several human disorders as well as to affect placental and fetal
growth. There have been numerous and conflicting studies assessing
the possible association of imprinting disorders with assisted repro-
ductive techniques. This work analyzes all relevant and available
reports with regard to the association between assisted reproductive
techniques and imprinting disorders. It also discusses whether this
possibly increased risk of imprinting disorders may be linked to
specific steps of these reproductive techniques or already present in
the gametes of infertile patients. A better understanding of epigenetic
reprogramming in the germ line is absolutely necessary both to assess
the safety of these methods and of the use of impaired spermatogenesis
gametes for assisted reproduction. (Pediatr Res 61: 51R–57R, 2007)
I
mprinting and epigenetic reprogramming involve, for spe-
cific genes, a sex-specific differential allele DNA methyl-
ation pattern (1), resulting in a parent-of-origin-dependent
pattern of gene expression. Imprinted genes have been dem-
onstrated to play key roles in the regulation of embryonic
growth and placental function at critical stages of develop-
ment as well as in numerous other essential biologic pathways
(1). Disturbed expression of particular imprinted genes has
indeed been linked to fetal growth and development abnor-
malities as well as to various human diseases (2). They may
also play a key role in diseases affecting the placenta, such as
HM, and in overgrowth or intrauterine growth retardation
(IUGR).
Specific imprinting defects have been described in children
conceived by ART. The interpretation of these findings was
either that one or the other of the steps of ART might affect the
process of imprint reprogramming or that the imprinting de-
fect was preexisting. The latter hypothesis implicates that
epimutations in the germinal cells used for ART may be the
cause of imprinting defects in the concerned conceptuses.
Therefore, the exploration of imprinting in defective spermat-
ogenesis is a prerequisite for guaranteeing that the germ cells
used for ART do not carry detrimental epigenetic changes.
Large-scale international follow-up studies of children con-
ceived by ART are also essential to assess the safety of these
techniques.
IMPRINTING AND REPROGRAMMING
The vast majority of genes possess a bi-allelic pattern of
expression. Imprinting corresponds to a specific epigenetic
regulation leading to expression of only one parental allele of
a gene. Some imprinted genes exhibit paternal expression
whether others exhibit maternal expression. The best-
characterized mark of gene imprinting is DNA methylation/
unmethylation (3,4). Usually, methylated DNA sequences are
transcriptionally inactive, whereas unmethylated DNA se-
quences are transcriptionally active (5). There are two mech-
anisms by which DNA methylation inhibits gene transcrip-
tion: the first is interference of the methyl group with the
binding of particular transcription factors to the DNA (6). The
second involves methyl-binding domain proteins mediating
transcriptional repression through binding to the DNA (7).
About 75 imprinted genes have been identified to date in
human, although it is estimated that from 100 to 600 imprinted
genes might exist in the human genome (8,9). Not all im-
printed genes encode proteins. Some of them encode untrans-
lated RNA, antisense RNA, or micro RNA (10) that certainly
play an important role in regulating gene expression. Imprinted
genes are characterized by specific regions up to several
kilobases of length—DMD. At these regions, the levels of
DNA methylation differ between the maternal and paternal
alleles (11). Methylation has been shown to occur at specific
CpG dinucleotide structures within DMD. Within a DMD, one
parental allele is methylated on all/the majority of the CpG
dinucleotides, while the opposite one is methylated on none/a
small percentage of its CpG dinucleotides. Outside the DMD,
similar patterns of methylation are present on both parental
alleles. A constant feature of imprinted genes is that they are
clustered into large chromatin domains, or “imprinted do-
mains,” at specific chromosomal regions. Their clustering may
allow a coordinated regulation of imprinting, imprinted gene
expression, and asynchronous replication timing by imprinting
control centers (12). These are CpG rich and methylated
all/the majority of the CpG dinucleotides on one parental
Received November 1, 2006; accepted November 15, 2006.
Correspondence: Ariane Paoloni-Giacobino, M.D., Department of Genetic Medicine
and Development, Geneva University Medical School, 1, Michel-Servet, 1211 Geneva 4,
Switzerland; e-mail: ariane.giacobino@medecine.unige.ch
DOI: 10.1203/pdr.0b013e318039d978
Abbreviations: ART, assisted reproductive techniques; AS, Angelman syn-
drome; BWS, Beckwith-Wiedemann syndrome; DMD, differentially methyl-
ated domains; Dnmt, DNA methyltransferase; HM, hydatiform mole; ICSI,
intracytoplasmic sperm injection; IVF, in vitro fertilization; LOI, loss of
imprinting; PW, Prader-Willi syndrome; SRS, Silver-Russell syndrome
0031-3998/07/6105-0051R
PEDIATRIC RESEARCH Vol. 61, No. 5, Pt 2, 2007
Copyright © 2007 International Pediatric Research Foundation, Inc. Printed in U.S.A.
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allele only (13). Well-characterized imprinted domains have
been described in human, such as the 11p15.5 and the
15q11-13 regions.
Parental imprints are erased in the immature primordial germ
cells of the developing embryo, subsequently re-established dur-
ing gametogenesis according to a sex-dependent pattern and
maintained through fertilization, pre- and postimplantation em-
bryonic development (14). Imprint re-establishment occurs at late
fetal stages in male germ cells and after birth in growing oocytes
(13). Imprinting reprogramming refers to this erasure/acquisition/
maintenance cycle of DNA methylation, occurring at DMD,
which plays a key role at critical stages of embryonic develop-
ment and fetal growth. Imprinted genes are also thought to play
a role in the control of postnatal growth, brain function and
specific neurobehavioral traits (15).
METHYLATION ENZYMES
Dnmts are responsible for the methylation of DNA; 3 Dnmt
families have been identified so far: Dnmt1, Dnmt2, and
Dnmt3. Dnmt1 is the most abundant DNA methyltransferase
in mammalian cells. Dnmt1 has 3 known isoforms: a somatic
Dnmt1, a splice variant (DNMT1b) and an oocyte-specific
isoform (Dnmt1o). It predominantly methylates hemimethyl-
ated CpG di-nucleotides in the genome and is considered to be
the key maintenance methyltransferase during cell division
(7). The biologic function and the role in the methylation
processes of Dnmt2 is still elusive (16). Dnmt3 is a family of
DNA methyltransferases that could methylate hemimethylated
and previously unmethylated CpG di-nucleotides. Dnmt3a and
Dnmt3b may mediate gene repression through interactions
with transcriptional repressors (17). Also, Dnmt3a adds
methyl groups on imprinting centers (13). Dnmt3L is hypoth-
esized to be required for the establishment of maternal im-
prints in the oocyte. It is expressed during gametogenesis (18).
HUMAN DISEASES INVOLVING IMPRINTED GENES
Abnormal expression of imprinted genes, through genetic
or epigenetic alterations, can lead to a number of diseases.
These diseases are all characterized by a non-mendelian in-
heritance and a parent-of-origin effect. They consist in four
broad categories, including neuron-developmental, metabolic
disorders, and psychiatric/behavioral disorders as well as can-
cer. The first group includes BWS, PWS, and AS (19,20). The
second group includes transient neonatal diabetes mellitus.
The third group includes autism, schizophrenia, and bipolar
disorder. The fourth group includes retinoblastoma (9) (15).
Table 1 provides a selection of human diseases linked to
imprinting defects.
DEFECTIVE IMPRINTING IN ART
Various imprinting disorders have been recently reported
following conception by ART (IVF or ICSI). These tech-
niques (ART) may by themselves have a deleterious effect on
imprinting. New technical steps have been recently added to
the IVF/ICSI procedures, like testicular/ovarian tissue cryo-
preservation and oocyte in vitro maturation (21) as well as
preimplantation genetic diagnosis. It is presently not known
whether these may expose the gametes or early embryos to
risks of imprinting defects.
Recent studies have suggested that a number of specific
imprinting disorders might be more frequent in children con-
ceived by ART than naturally.
BWS
In a prospective study on BWS, DeBaun et al. (22) identi-
fied seven sporadic cases who were conceived by ART. In six
of them, they identified the specific epigenetic alterations
generally associated with BWS, i.e. LOI at KCNQ1OT1 or
H19. Their results showed, in children with BWS, a 6-fold
higher prevalence of ART- versus natural conception (4.6%
versus 0.8%, respectively). Gicquel et al. (23) found in their
BWS patient series a three-time over-representation of ART
compared with the general population (4% versus 1.3). All
their patients presented a KCNQ1OT1 LOI. Maher et al. (24)
studied 149 sporadic BWS cases and looked for a possible
association with ART (24). A conception by ART was re-
corded for 4% of BWS cases to be compared with 1.2% in
their control population. Among the reported cases, 2 had a
KCNQ1OT1 LOI.
Halliday et al. (25), in a large case-control study analyzed
the frequency of BWS in 14=894 babies born after ART
compared with 1=316=500 live births. They detected 37 cases
of BWS, corresponding to an overall risk of BWS 9 times
higher in the ART group, than in their general population.
In a retrospective study, Chang et al. (26) identified 19
BWS children (out of a 341 BWS registry) who were con-
ceived by ART. The latter had similar clinical features as
naturally conceived children. Interestingly, no specific aspect
of the ART procedure, like the use of specific culture media,
or the timing for transfer of embryo could be associated with
BWS.
Table 1. Selected human disorders linked to an imprinting defect,
that have been reported after ART
Disorders
Candidate
chromosomal
location
Reported cases
linked to
ART (Ref)
BWS 11p15 (22–26,36)
AS 15q11-13 (32–34)
PWS 15q11-13 (35,36)
SRS 7 (35)
Isolated hemihyperplasia 11p15 (38)
Autism 15q11-13 NR
Bipolar disorder 18p11.2 NR
Schizophrenia 18p11.2 NR
Late-onset Alzheimer
disease
10 and 12 NR
Transient neonatal diabetes
mellitus
6q24 NR
Albright hereditary
osteodystrophy
20q13.2 NR
Retinoblastoma 13q (39)
Preeclampsia 10q22 NR
Biparental complete HM 19q13.4 NR
NR, not reported.
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