2024 04 23 - Paper
2024 04 23 - Paper
2024 04 23 - Paper
Abstract
Today, there is growing interest in the potential epigenetic risk related to assisted reproductive technologies (ART).
Much evidence in the literature supports the hypothesis that adverse pregnancy outcomes linked to ART are
associated with abnormal trophoblastic invasion. The aim of this review is to investigate the relationship between
epigenetic dysregulation caused by ART and subsequent placental response. The dialogue between the endometrium
and the embryo is a crucial step to achieve successful trophoblastic invasion, thus ensuring a non-complicated
pregnancy and healthy offspring. However, as described in this review, ART could impair both actors involved in this
dialogue. First, ART may induce epigenetic defects in the conceptus by modifying the embryo environment. Second, as
a result of hormone treatments, ART may impair endometrial receptivity. In some cases, it results in embryonic growth
arrest but, when the development of the embryo continues, the placenta could bring adaptive responses throughout
pregnancy. Amongst the different mechanisms, epigenetics, especially thanks to a finely tuned network of imprinted
genes stimulated by foetal signals, may modify nutrient transfer, placental growth and vascularization. If these coping
mechanisms are overwhelmed, improper maternal-foetal exchanges occur, potentially leading to adverse pregnancy
outcomes such as abortion, preeclampsia or intra-uterine growth restriction. But in most cases, successful placental
adaptation enables normal progress of the pregnancy. Nevertheless, the risks induced by these modifications during
pregnancy are not fully understood. Metabolic diseases later in life could be exacerbated through the memory of
epigenetic adaptation mechanisms established during pregnancy. Thus, more research is still needed to better
understand abnormal interactions between the embryo and the milieu in artificial conditions. As trophectoderm cells
are in direct contact with the environment, they deserve to be studied in more detail. The ultimate goal of these
studies will be to render ART protocols safer. Optimization of the environment will be the key to improving the
dialogue between the endometrium and embryo, so as to ensure that placentation after ART is similar to that
following natural conception.
Keywords: Assisted Reproductive Technologies, Epigenetic, Imprinted gene, Placenta, Pregnancy
© 2015 Choux et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
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Choux et al. Clinical Epigenetics (2015) 7:87 Page 2 of 20
procedures [10, 11, 4, 12–18]. Even if co-existing mater- expression of non-coding RNAs (such as microRNA,
nal risk factors (such as BMI, maternal age and infertility Piwi, and Miwi).
status) may affect placental development, the artificial For instance, imprinted genes, which are
manipulation of gametes and/or embryos could also play epigenetically regulated, are abundantly expressed in
a role. foetal and placental tissues and are apparently absent
The aim of this review was to investigate the pheno- in non-placental organisms [24, 25]. It is postulated
typic and epigenetic mechanisms by which ART could that genomic imprinting coevolved with placentation
interfere with placental formation and function, resulting or drove the evolution of the placenta [26], sometimes
in placenta-related adverse pregnancy outcomes. The through modifications of retrotransposons [27].
first paragraph will insist on the key role of epigenetics Imprinted genes are expressed in a parent-of-origin
in placental function. Then, the ART-induced placental manner thanks to epigenetic modifications silencing
variations occurring throughout pregnancy will be re- either the paternal or the maternal allele. These
ported. To finish, the potential long-term effects of these epigenetic modifications (DNA methylation being
placental modifications and the future research perspec- the most described) are established in a sex-specific
tives will be addressed. manner during gametogenesis on regulatory sequences
referred to as imprinting control regions (ICRs). After
Proper epigenetic regulation is essential for a functional fertilization, these ICRs act in cis to achieve monoallelic
placenta expression of most imprinted genes. Up to now,
approximately 150 imprinted genes have been
1. Epigenetics in placental function identified in mice and humans. In mice, these are
In mammals, the placenta is a pregnancy-specific under the control of 23 identified ICRs [28–30]
temporary organ that creates intimate contact (http://www.geneimprint.com/site/genes-by-species).
between mother and foetus ensuring the maintenance Interestingly, they are generally not imprinted in
of gestation and foetal well-being by the exchange of all tissues, and the imprinted pattern can be limited to
gases, nutrients and waste products [19]. It originates a precise developmental stage. In addition, the
from the peripheral multipotent cells of the blastocyst conservation of imprinted status or even the sense of
(trophectoderm). In humans, placental the imprinting (maternal or paternal allele expressed)
syncytiotrophoblasts formed by the fusion of may vary between mammalian species [28]. Imprinted
cytotrophoblasts constitute the site of exchange genes, which represent a very small percentage of
between the maternal and foetal circulation. It has genes, appear to play essential roles in embryonic
specific endocrine functions, such as the production growth and placental development by regulating
of placental hormones, but it also functions as a the transport capacity of the placenta thereby
barrier, ensuring a stable environment to a foetus controlling the supply of nutrients [31, 32]. During
deprived of efficient defence mechanisms against preimplantation development, genomic imprinting is
various stresses (oxidative, xenobiotic, chemical) [20]. jeopardized by global DNA demethylation, and some
A finely tuned temporal and spatial regulation of actors such as the complex Zfp57/TRIM28/KAP1 are
trophoblastic invasion is essential for proper future required to protect epigenetic imprinting marks [33].
function of the placenta and foetal development [21]. Moreover, imprinted genes are functionally haploid by
This involves molecular crosstalk between the definition and thus potentially more susceptible to
endometrium and trophoblast [21]. mutations and epimutations [34]. Their dysregulation
Notably, epigenetic regulation is a significant factor may therefore have major consequences on the
in placental development and adaptive function to placental phenotype with long-term consequences for
environmental stress [22]. the developmental programming of adult health and
Epigenetics may be defined as a set of cell-based disease [35].
molecular mechanisms able to modify gene 2. Epigenetic modifications in the placenta and adverse
expression. These mechanisms are heritable through pregnancy outcomes
mitosis or even sometimes meiosis and not sustained To function adequately, the developing placenta
by DNA sequence variation [23]. Epigenetic regulation needs the proper epigenetic regulation of imprinted
controls transcription at two levels: directly on the and non-imprinted genes. Indeed, experimental
DNA (through DNA methylation/hydroxymethylation studies conducted in both humans and animals have
mechanisms) and on the proteins around which the clearly shown the importance of epigenetics in the
DNA is wrapped to constitute the nucleosomes regulation of placental development. For example,
(histone modifications). Epigenetic regulation also drug-induced disruption of DNA methylation was
controls translation or mRNA stability by the able to inhibit human trophoblastic invasion in vitro
Choux et al. Clinical Epigenetics (2015) 7:87 Page 3 of 20
by disturbing the expression of epigenetically In an induced IUGR rat model, Reamon-Buettner and
regulated genes such as E-cadherin [36] as well as colleagues reported decreased expression and aberrant
the proliferation of trophoblast cells in rat placenta DNA methylation patterns of the promoter region of
[37]. The deletion of placental-specific Igf2 in mice the Wnt2 gene, which is known to be implicated in
consistently led to reduced placental growth and placental vascularization [51]. In humans, the same
subsequent foetal growth restriction [38]. pattern was found with lower WNT2 expression and
In addition, numerous findings proved that higher DNA methylation in growth-restricted neonates
disturbed placental epigenetic regulation may cause than in controls [52].
abnormal trophoblastic invasion, which may Interestingly, epigenetic changes were also found on
contribute to the pathophysiology of some repeated sequences. For example, Michels and
spontaneous miscarriages, IUGR and preeclampsia. colleagues found an increased LINE-1 methylation level
Indeed, in humans, DNMT1 expression (DNA in placental tissues from low birth weight infants [53].
methyltransferase 1 involved in DNA methylation Other evidences about preeclampsia reinforce the idea
maintenance) and global DNA methylation were that epigenetic disorders may be involved in abnormal
significantly lower in chorionic villi from early foetus trophoblastic invasion. Actually, mice with induced loss
losses than in those harvested following selective of expression of the imprinted Cdkn1c gene developed
pregnancy termination [39]. a preeclampsia-like syndrome, with hypertension and
Moreover, in humans and animals, a great number proteinuria [54]. Besides, widespread DNA methylation
of associations have been found between IUGR and changes were found in placentas of a cohort of patients
epigenetic variations of imprinted or non-imprinted suffering from early onset preeclampsia but not in
genes in placentas. Notably, by analyzing more than gestational age-matched controls [55]. Some of these
200 human term placentas, Banister and colleagues methylation modifications correlated negatively with
found that the DNA methylation pattern of 22 loci expressional changes, especially for genes implicated in
was highly predictive of IUGR [40]. In mice, induced angiogenesis (such as EPAS 1 and FLT I). Moreover,
loss of imprinting and the subsequent BHLHE40, a gene coding for a protein that can prevent
overexpression of the imprinted Phlda2 gene were trophoblast differentiation exhibited significantly
able to trigger placental and foetal growth decreased DNA methylation and increased expression
retardation in the offspring whereas its deletion in preeclampsia placentas [55]. In addition, the
caused overgrowth [41]. Similarly, in humans, some expression of maspin (SERPINB5), a serine protease
authors demonstrated that PHLDA2 was up-regulated inhibitor and an inhibitor of cell migration [56],
in the placenta in cases of IUGR [42–44] and that its which may modify trophoblast cell invasion in the first
expression level correlated negatively with birth trimester [57], could also be modified in preeclampsia.
weight [45]. As it is considered a negative growth In the same family of genes, SERPIN A3 is a specific
regulator, the authors suggested that this imprinted inhibitor of elastase, which plays a crucial role during
gene potentially plays a direct role in the the implantation process. SERPIN A3 displayed
pathophysiology of IUGR. decreased methylation and increased gene expression
Other imprinted genes were also up-regulated in placentas from pregnancies complicated by
(CDKN1C) or down-regulated (MEG3, GATM, ZAC1, preeclampsia compared with controls [58], through a
GNAS, MEST, IGF2) in IUGR placentas [42, 46, 47, 44]. complex epigenetic regulation [59]. As for IUGR,
Some of these differential expressions were associated several studies highlighted the increased methylation
with decreased placental methylation, as was the case [50] and reduced expression of syncytin-1, as well as
for H19/IGF2 ICR1 [48], or loss of imprinting, as was the down-regulation of WNT2 in preeclamptic
the case for ZAC1 (=PLAGL1) and H19 differentially placentas. These modifications were possibly
methylated regions (DMRs) [42]. responsible for impaired placental function [60].
In addition, other examples of non-imprinted genes Interestingly, epigenetic modifications could also
highlight the possibility that foetal growth potential correlate with the severity of the disease. For instance,
could be negatively impacted by epigenetic hypertension tended to be more severe in preeclamptic
dysregulation in the placenta. Ruebner and colleagues women with biallelic expression of H19, than in
pointed out that expression of Syncytin-1, a protein that women with normally imprinted expression of this
promotes cellular fusion in the syncytiotrophoblast, was gene [61]. Recently, Anton and colleagues
lower in human IUGR placentas than in controls [49]. demonstrated a correlation between disease severity
The same team recently linked decreased expression of and alterations in DNA methylation (hypermethylation
this protein to epigenetic hypermethylation of its of CDH11, COL5A1, TNF, hypomethylation of
promoter [50]. NCAM1) in preeclamptic placentas [62].
Choux et al. Clinical Epigenetics (2015) 7:87 Page 4 of 20
Fig. 1 ART can impair the dialogue between the endometrium and embryo and lead to suboptimal trophoblast invasion. Infertility per se could
be responsible for suboptimal gametes, and several ART steps (such as superovulation and embryo culture) may also be responsible for
suboptimal embryo development, both potentially leading to embryo development arrest. In addition, superovulation may impair endometrium
receptivity. Later, the placentation may be suboptimal and cause miscarriage or placenta-related adverse outcomes. However, a smart dialogue
between the foetus and placenta could bring adaptive responses through regulated epigenetic mechanisms leading to increased weight, cell
proliferation, increased vessel density and increased transport capacity. At birth, epigenetic variations present in cord blood or placentas could
either reflect persisting variations/defects or ongoing compensation at the time of birth
a lower global DNA methylation level, which was 2. ART and endometrial receptivity
associated with growth retardation [39]. These Apart from modifying the epigenetic status of the
results strengthen earlier experimental studies in conceptus, another way in which ART could alter
mice that highlighted the fundamental contribution of trophoblastic invasion could be its effect on the
DNA methylation enzymes to embryonic endometrium.
development [83]. Much evidence has linked poor endometrium quality
In summary, these data support the hypothesis that to abnormal early placentation. Even though some
a suboptimal embryo environment induced by ART genetic causes of endometrial defects leading to
greatly disturbs the epigenetic status of not only the recurrent miscarriages have been described [84–86],
embryo (eventually causing development arrest) but ovarian stimulation, which is required in most ART
also the extra-embryonic tissues. procedures, may also be responsible for poorer
Table 1 Conceptuses and/or placentas in mice: resorption rate, weight, gene expression and/or DNA methylation of imprinted genes
Page 6 of 20
Low levels of misexpression: ↓ Igf2
Snrpn, Mkrn3
Table 1 Conceptuses and/or placentas in mice: resorption rate, weight, gene expression and/or DNA methylation of imprinted genes (Continued)
Page 7 of 20
Choux et al. Clinical Epigenetics (2015) 7:87 Page 8 of 20
endometrium quality. Since the ovary and the uterus selects embryos with normal epigenetic profiles, by
share several signalling pathways, and since hormones unknown putative mechanisms. However, recently,
secreted by the ovary have a direct effect on uterus two different teams highlighted that the risk of large
function, ovarian stimulation probably modifies the for gestational age and preeclampsia could be increased
uterine environment. This is assessed by studies that in frozen embryo cycles compared with fresh cycles or
demonstrated differential expression of genes in the natural conception [99, 100]. Therefore, further studies
endometrium between stimulated and natural cycles, are needed to determine the impact of the different
with a dose-response effect [87, 88]. protocols used in frozen embryo transfer (hormonal
In mice, the implantation rate was lower and treatments used, duration of culture, cryoprotectants,
post-implantation foetal mortality was higher in culture media, etc.).
superovulated recipients than in non-stimulated Other data are in keeping with the hypothesis that
controls [89]. Similar observations were also superovulation and hormone treatment may impair
reported in humans, with a dose-dependent effect: placentation. For example, a recent study examining
the risk of spontaneous abortion was significantly near-term placentas in superovulated mouse recipients
higher in women stimulated with high levels of found altered trophoblast differentiation causing a
hormones than in those stimulated with lower reduced maternal-foetal exchange area [94]. Besides, in
levels [3]. Besides, high serum estradiol levels at humans, pregnancy-associated plasma protein A
ovulation triggering after controlled ovarian (PAPP-A) levels in maternal serum were decreased in
stimulation are associated with placenta-related first-trimester ART pregnancies [101–105]. PAPP-A is
adverse pregnancy outcomes such as growth known to play a critical role in trophoblastic invasion
restriction or preeclampsia [90, 91]. [106] by contributing to maternal tolerance towards the
Other evidences highlight the impact of a suboptimal foetus [107]. Giorgetti et al. confirmed these low levels
endometrium induced by ovarian stimulation on after ART and further added that maternal serum
placental and foetal growth. Notably, hormones are PAPP-A levels correlated strongly and inversely
known to modify birth weight. Indeed, singletons born with estradiol levels at ovulation triggering [108].
after IVF have on average a lower birth weight than Accordingly, PAPP-A values were lower after the
singletons born after natural cycles with mild transfer of fresh embryos (when ovarian stimulation
stimulation [92]. Moreover, an inverse correlation was used) than after the transfer of frozen embryos
between birth weight and estradiol levels achieved in or after unstimulated cycles [101, 109].
case of IVF [93] was found. In mice, the mean weight All these findings highlight a tight relationship
of foetuses was also lower in stimulated than in between high hormone levels and impaired
non-stimulated recipients [89, 94]. trophoblastic invasion presumably through decreased
Surprisingly, birth weight was higher in ART-offspring endometrium receptivity. Exposing the endometrium
after the transfer of cryopreserved/thawed embryos to high levels of estradiol and progesterone produced
than with fresh embryos [95, 96]. While it could be by multiple corpora lutea could possibly render it less
hypothesized that this was caused by a direct effect on efficient for embryo implantation than it is during
the embryo, differences in hormonal treatment natural cycles [110]. Thus, ART processes, and
between the two groups could have an important effect especially hormone treatments, may increase the rate
as well. In the first case (cryopreserved embryos), of adverse pregnancy outcomes by inducing more
women are not treated with follicle-stimulating trophoblastic invasion defects.
hormone (FSH) to induce multifollicular growth, while In addition to hormone treatments, infertility per se
they are treated in the second case. In natural could involve an altered uterine environment. For
conception, when two children from the same example, some authors recently suggested that
mother are compared, the second one is usually endometriosis may be accompanied by epigenetic
heavier [97]. However, when the first is born following modifications implicated in diminished endometrial
transfer of a frozen embryo and the second after IVF, receptivity and altered gene expression. Epigenetic
the situation is reversed [95]. On average, birth weight modifications on the promoter of a mediator of
following frozen embryo transfer is the same as that endometrial receptivity, HOXA10, may be one of the
following natural conception [98]. The fact that frozen mechanisms involved, as reported in women [111–113]
embryos are transferred without controlled and in several animal models [114, 115].
ovarian hyperstimulation suggests that the To summarize, ART, through its negative effect on the
endometrium-embryo dialogue is in this situation endometrium-embryo dialogue, could participate in
closer to the “natural” dialogue and enables preventing successful trophoblastic invasion. This could
normal placentation. It is also possible that freezing potentially explain the occurrence of adverse pregnancy
Choux et al. Clinical Epigenetics (2015) 7:87 Page 9 of 20
outcomes after ART. Depending on the severity of the phenomenon was seen in singletons from ART.
defects, ART could gradually lead to developmental Placentas from ART pregnancies were overrepresented
arrest, miscarriages, preeclampsia or IUGR (Fig. 1). But in the highest quartile of weight, and the placental
in most cases, pregnancies obtained after ART are able weight/birth weight ratio was commonly higher, while
to continue without obvious immediate adverse the mean birth weight was lower, even after adjusting
outcomes. This sustains the hypothesis that initial for potential confounding factors [121]. This
defective trophoblastic invasion could trigger placental increased placental weight after IVF could be the
adaptive responses during pregnancy. result of compensatory responses.
2. Mechanisms involved in placental responses
ART and the possible induction of placental adaptive Metabolic pathways
responses According to Coan and co-workers, the placental
Nuclear transplantation in animals is known to pro- phenotype is responsive to nutritional conditions.
duce placental phenotypic modifications (such as pla- When foetal nutrient availability is compromised, it
centomegaly), to modify placental metabolism and to adapts to maximize the nutrient transfer capacity
disturb imprinted gene expression [116, 117]. Given [122]. These compensatory mechanisms may start
these placental modifications after somatic cell nuclear from the blastocyst stage, within extra-embryonic
transfer, we wondered whether ART could trigger pla- lineages. Actually, using a mouse maternal protein
cental responses. restriction model, some authors demonstrated
increased endocytosis, cell proliferation and
1. Phenotypic placental responses invasiveness in the trophectoderm, which may
In the literature, several studies in animals showed reveal enhanced nutrient capture [123, 124].
that a suboptimal placenta is created by in vitro The up-regulated expression of nutrient supply
conditions but that counterbalancing mechanisms genes such as glucose and system A amino acid
also occurred. First, a smaller quantity of TE cells transporters was shown in small murine placentas
developed in mouse blastocysts from in vitro culture during late gestation, thus reflecting a response to
than in naturally conceived blastocysts [82]. At later foetal demand signals [122]. The foetus itself plays a
stages (12.5 dpc), IVF embryos and placentas were role in its own development and growth by sending
smaller than those in the control group [4] signals to the placenta, which will respond by
(Table 1). However, the placenta was slightly larger regulating genes involved in growth control, specific
(+9 %) at 15.5 dpc and to an even greater extent transport systems and vascularization [125].
(+25 %) at 18.5 dpc, while foetus weight was 16 % These metabolic responses are well-illustrated in
lower at 15.5 dpc but only 9 % lower at 18.5 dpc in IVF studies on animal species [126, 15, 127]. Indeed,
the IVF group than in controls [10] (Table 1). At at early gestation, bovine conceptuses after IVF and
this later stage, cell proliferation was greater in IVF culture displayed placentas with decreased blood
placentas than in controls, in both the labyrinth and vessel density, while at late gestation, placentas had
spongiotrophoblast layers. By birth, IVF foetuses had greater blood vessel density [15, 127]. This impaired
reached the same weight as the controls [10]. In the placental vasculogenesis early in gestation was also
in vitro context, placentas were found to be lighter reported for sheep embryos developed in vitro
than control placentas at early gestation and heavier [128]. This compensatory process could implicate
at late gestation. While a larger placenta is not the angiogenic pathway and particularly an
necessarily synonymous of a higher efficiency in angiogenic transcription factor, peroxisome
nutrient and oxygen transfer, it can in this case proliferator-activated receptor gamma (PPARƳ)
probably contribute to a compensatory growth of protein, which could modulate the density of
the foetus, despite initial functional limitations. maternal blood vessels throughout pregnancy
Similar results were observed in the sheep model: [15]. In addition to the gain in vascularization,
foetuses from in vitro cultured embryos were 60 % increasing cell fusion could improve foeto-maternal
smaller than naturally conceived foetuses at day 24 exchanges. Indeed, two proteins involved in
of gestation, whereas no difference was found at membrane fusion, annexin A3 and α-SNAP, were
later stages [16]. found to be up-regulated in human term placentas
Likewise, in humans, the enlargement of placentas has obtained after ART [19].
been observed in complicated pregnancies associated Besides, in human placentas after ART,
with low birth weight, such as pregnancies with genome-wide mRNA expression revealed the
late-onset preeclampsia, foetal death or advanced overexpression of genes involved in metabolism,
maternal age [118–120]. Interestingly, the same immune response, transmembrane signalling
Choux et al. Clinical Epigenetics (2015) 7:87 Page 10 of 20
and cell cycle control [129, 130]. Similarly, found conflicting data. Indeed, the first published
transcriptomic data in mouse placental tissues study described quantitative differences in global
show that IVF techniques trigger the induction DNA methylation (briefly with a higher and a
of genes involved in cellular proliferation and lower degree of DNA methylation in post-IVF
cell cycle pathways [75]. cord blood and placental samples, respectively)
In summary, the kinetics of placental and foetal and for several imprinted genes [77] (Table 2). In
growth altered by ART may be linked to contrast, two recent studies reported either opposite
modifications in various biological pathways, cord blood findings [139] or none variability in DNA
probably triggering the placental compensation methylation at 25 imprinted DMRs [134] (Table 2).
phenomenon. While the complete picture of the However, the three studies are not comparable
systems that regulate this compensation is still regarding the sample size (10 individuals versus 73),
blurred, epigenetic changes certainly play a part the mode of reproductive treatment (IVF versus
in the adaptive mechanisms. unspecified ART) and the method used.
Imprinted gene network Moreover, other studies focusing on the DNA
Concerning the regulation of this placental methylation of specific imprinted genes also
response, one interesting hypothesis is that generated contradictory results. Indeed, although
potential primary dysfunctions of the placenta some authors reported no epigenetic changes
could be corrected by the imprinted gene network after ART [140, 141], several authors reported
of placental mammals (IGN). The modulation of variations in methylation levels in both cord
this network of coordinated imprinted genes (and blood and/or placentas for a number of imprinted
probably non-imprinted genes), which are involved genes such as MEST [142, 143], H19
in growth control and specific placental transport [144, 142, 145], KCNQ1OT1 [146] or SNRPN
systems, could contribute to the tight regulation of [142]. However, none of them agreed on the
foetal growth during post-implantation changes in DNA methylation and these variations
development. This was described in mice for Igf2, were mild (from 0.6 to 4.5 % differential
Zac1 and H19 [131, 132] and recently in the methylation levels) (Table 2). Once again, these
human placenta for ZAC1 [133]. studies are difficult to compare given the
To support this hypothesis, in mouse placentas after limitations similar to those mentioned above.
ART, most genes of the IGN were up-regulated in a However, most studies focused on normal
coordinated fashion, when compared with the pregnancy, thus excluding placenta-related adverse
control group [5]. The fact that these genes pregnancy outcomes (such as preeclampsia,
with placental reciprocal functions were hypertension, some IUGR) and therefore possibly
up-regulated after ART despite phenotypically ignoring major differences.
and morphologically normal embryos suggests Concerning the expression analysis of imprinted
that placental IGN may participate in the control of genes, conflicting results were also reported.
normal foetal growth in ART pregnancies. However, Dysregulation mainly took place in the placenta and
the methylation status of their DMRs after ART was only for three imprinted genes (H19, IGF2, MEST)
either similar to that in controls or only slightly [77, 144, 145] (Table 2).
modified during gestation [5, 69]. In the same way, Finally, these minimal expressional changes at
the methylation of repeated sequences (ALUYb8, term compared with more significant changes
α-satellites and LINE-1) were reported to be during pregnancy in animals could reflect the
unchanged after ART [134]. Other epigenetic remains of defects that were partially
mechanisms, such as histone modifications, compensated during prenatal life or even
could therefore be involved. In fact, according to methylation allelic polymorphisms (placental
Lewis et al., an ancestral imprinting mechanism, epipolymorphism [147]).
restricted to the placenta, is based on histone Thus, epigenetic “defects” in animals’ placentas
modifications [135], which may confer the after in vitro manipulations are found in most
short-term and flexible response implicated in studies. Most authors consider this variety of
development [136–138]. placental phenotypes triggered by ART to
Regrettably, no evidence is available in animals at originate from epigenetic errors at imprinted
birth concerning the occurrence of epigenetic genes [74], but should we really consider these
modifications in the placenta. In humans, nothing epigenetic modifications as “errors” or should we
is certain (Table 2). Three studies that carried out regard them as smart adaptation mechanisms
large DNA methylation analyses using arrays developed by the placenta? From the results
Table 2 Effects of ART on imprinted genes and retrotransposable element expression and methylation in chorionic villous samples from abortion, peripheral blood, cord blood
Page 11 of 20
GNAS NESPas, GNAS XL-alpha-s,
GNAS Exon1A
Table 2 Effects of ART on imprinted genes and retrotransposable element expression and methylation in chorionic villous samples from abortion, peripheral blood, cord blood
Page 12 of 20
Table 2 Effects of ART on imprinted genes and retrotransposable element expression and methylation in chorionic villous samples from abortion, peripheral blood, cord blood
Page 13 of 20
Choux et al. Clinical Epigenetics (2015) 7:87 Page 14 of 20
above, we can postulate that these “defects” are be written through epigenetic modifications of the concep-
not all harmful for the embryo and that some tus. These plastic responses to the early environment could
could be considered compensatory mechanisms. be kept in memory throughout life, due to epigenetic
Indeed, they reflect the balance between changes such as DNA methylation and histone modifica-
members of the IGN in the placenta. Biallelic tions [165]. We can postulate that ART could trigger
expression as well as the loss of imprinting of similar processes.
parts of the IGN in the placenta could constitute Nevertheless, in humans, there is no evidence of epigen-
a major compensatory mechanism to allow the etic changes persisting into childhood. Indeed, in children
developing foetus to cope with a changing or conceived after IVF, reassuring data have been reported
adverse environment. In response to certain for DNA methylation for four imprinted genes and even
stress factors that modify the early environment on a global scale [166, 167]. Only one recent study ob-
of the embryo, the placenta could amplify these served that some epigenetic errors can still be observed
compensatory mechanisms up to a certain point. during childhood, though this concerned only the
In most cases, efficient compensation ensures imprinted gene SNRPN [168] whose DNA methylation
normal foetal growth up to term. When the levels were not found to be modified at birth after ART
compensation is unbalanced, compensation fails in either cord blood or in the placenta. However, the het-
and pathological features such as miscarriages, erogeneity of biological samples (blood or buccal cells),
low birth weight or preeclampsia could occur. age range, type of reproductive technique and the analysis
However, what remains to be determined is of methylation could hide potential underlying differences.
whether this compensation step per se could be Another hypothesis might reside in tissue-specific epi-
a risk factor for certain diseases later in life. genetic modifications. This could explain the absence of
DNA methylation variations in blood and buccal cells.
Potential long-term effects of ART-related compensation Therefore, studying other tissues may reveal defects linked
during pregnancy to specific metabolic conditions. Notably, Scherrer’s team
These modified maternal-foetal interactions, here after ART, found increased DNA methylation on the promoter of the
might have consequences for outcomes in infancy and even gene encoding eNOS (NO synthase) in vascular tissues in
in adulthood, especially by inducing metabolic and cardio- mice obtained after ART. This resulted in reduced plasma
vascular conditions [148–152]. For instance, in humans, NO concentrations, increased blood pressure and a
new-borns that are either too small or too big may be shorter lifespan [169].
vulnerable to heart disease, hypertension, type II diabetes It is also interesting to consider that tissue-specific epi-
and obesity [153–155]. In addition, the size and shape of mutations for H19, Snrpn and Peg3 genes were described
the placenta have been related to life expectancy in men in individual mice generated by ART (ICSI or superovula-
[156] and their risk for coronary heart disease [157]. Simi- tion) [170].
larly, a high placenta/foetus weight ratio, considered a
marker of intra-uterine stress, has been associated with Ways for medical improvement and future research
hypertension later in life [158]. Ways to improve actual ART protocols Finally, as pla-
As mentioned above, these phenotype modifications of cental defects seem to originate from an altered
the foetus and placenta are found in ART pregnancies. endometrium-embryo dialogue, optimization of the envir-
Thus, the modified intra-uterine environment after ART onment during ART is a cornerstone and may improve
may be one cause of late-onset diseases [159]. Indeed, al- early placentation. Hence, several simple and practical im-
though the majority of ART children are healthy, the provements can be proposed. First, it is possible to optimize
available data about long-term follow-up of ART chil- the quality of oocytes and the endometrial milieu by using
dren revealed cardiovascular and metabolic risk factors lower doses of hormones. Second, the culture media must
[159]. Notably, children born after ART may exhibit in- be optimized to limit trophectoderm cell stress. Even
creases in peripheral adipose tissue mass, in systolic and though the parameters of this optimization are far from
diastolic blood pressure, in fasting glucose levels and being mastered, it has been clearly shown that specific cul-
IGF-I and IGF-II levels as well as changes in the lipid ture media generate a lower degree of stress for the em-
profile [160–164]. In addition, transcriptomic data at bryo [5, 70]. Third, the embryo and endometrium should
birth revealed activation of metabolic pathways impli- be better synchronized either by transferring blastocyst-
cated in chronic disorders such as obesity and type II stage embryos (even if extended embryo culture may have
diabetes [77]. However, further large longitudinal studies per se an impact the epigenetic regulation) and/or by
are needed to confirm these poor outcomes. developing molecular diagnostic tests (for example tran-
Portha and colleagues proposed that the link between the scriptomic, lipidomic and proteomic profiles) to assess the
prenatal environment and adverse long-term effects could quality of the endometrium in order to target the best
Choux et al. Clinical Epigenetics (2015) 7:87 Page 15 of 20
timing of endometrial receptivity [171]. Fourth, another risk of placenta-related adverse pregnancy outcomes [7].
practice recently developed by some teams, could be to These differences may stem from overwhelmed compen-
freeze all embryos and transfer them during subsequent sation mechanisms, which, in certain cases, are not fully
cycles with an optimally prepared endometrium [172]. successful. Several potential reasons may explain this lim-
However, the endometrial tests and the fourth solution ited correction in humans as compared with mice. First,
need to add an embryo cryopreservation step. Recent data although placentation is haemochorial in both humans
reported poorer obstetrical outcomes after frozen embryo and mice [175, 176], their placentas are not organized in
cycles (reported above) and a potential negative impact of the same way (labyrinth and spongiotrophoblast in mice
the cryopreservation itself on the regulation of DNA meth- versus villous trophoblast in humans) and differ in their
yltransferases in preimplantation frozen/thawed embryos morphogenesis and exchange functions [175, 177]. Sec-
[173]. Thus, further studies are required before these strat- ond, in human ART, the cumulative effects are possibly at
egies can be applied safely. their utmost point because the standard method is to
Another way to improve the chances of success could be transfer fresh embryos from a superovulated cycle, which
post-natal correction. Since imprinted genes in the placenta is not performed in mice because pseudopregnant females
appear to be major operators in regulating foetal growth, are used. The effects observed in animal models are there-
further research is needed to better understand the link they fore possibly exacerbated in humans. Third, contrary to
may have with future disease. All in all, imprinted genes animal models, parental infertility is the major reason why
could eventually be used as sensors to predict and better ART is used in humans, and this infertility may be partly
prevent diseases later in life. Interestingly, some studies sug- responsible for the epigenetic disorders and abnormal pla-
gest that customized interventions might be implemented to centation leading to maternal pathologies, such as abrup-
correct effects on phenotypic changes [153]. One example is tio placentae and preeclampsia [178–180]. Therefore, any
the post-natal administration of leptin in rats, which was extrapolation of animal studies to humans should be done
able to reverse the adverse effects of mother-undernutrition: with caution.
the offspring phenotype as well as the expression and Moreover, concerning the methodology, most epigen-
methylation of several hepatic genes were corrected etic studies have addressed the effects of ART stressors
[174]. One other example is the post-natal administra- on DNA methylation at the individual gene level and
tion of butyrate (histone deacetylase inhibitor) in the often analyze one or few CpG. Thus, genome-wide as
mouse model, which normalized both DNA methyla- well as gene-specific approaches that can target regula-
tion of the promoter of the eNOS gene and vascular tory regions (promoters, enhancers, gene body, or else-
function [169]. Further studies in animals are needed to where) and assess functional significance is now needed.
better understand tissue-specific epigenetic regulation High-throughput tools, which are becoming available,
in ART. Thus, screening for epigenetic markers during may be applied more widely to study the epigenomic
early life could be used to identify more vulnerable pa- changes associated with ART. Otherwise, in most stud-
tients and to define an appropriate treatment to poten- ies, only overall expression and methylation levels are
tially correct various epigenetic defects. examined (Tables 1 and 2). Although it could be valu-
able, monoallelic expression of imprinted genes is hard
Future research to assess the impact of ART on health to perform, given the need for informative SNPs in par-
More research is needed to better understand the dis- ents to perform this analysis.
turbed interactions between the embryo and the milieu, From a global DNA methylation point of view, pla-
especially in artificial conditions. New insights about the centa tissue has been shown to display a very low DNA
regulation of actors involved in the protection/mainten- methylation profile compared with other somatic tissues
ance of DNA methylation at imprinted genes in a context [181]. More recently, human studies on placenta samples
of ART are now necessary [33]. Moreover, to our know- using high-throughput tools (methylome) revealed that
ledge, epigenetic defects have not been studied separately placenta presents large partially methylated domains
in TE and ICM cells so far. Nonetheless, knowing whether (PMD) which are stable during pregnancy [182]. This
epigenetic dysregulation occurs in all blastocyst cells or unique property of the placenta might contribute to the
only in TE cells could lead to better understanding of the regulation of the expression of key genes important for
mechanisms implicated in placental defects caused by foetal development. Besides, in placenta samples, the
ART. Knowledge of such mechanisms would be important genes enriched in the highly methylated regions (HMD)
to evaluate possible consequences for the developing indi- are involved in defence responses. The review that we
vidual soon after birth or even later in life. present here focuses on imprinted genes, but research aim-
Furthermore, although placental compensation enables ing to delineate the variations that exist at such loci, be-
mice to reach a normal birth weight [10], evidence in tween placenta from ART and spontaneous pregnancies,
humans shows that ART pregnancies still carry a higher would help us to understand how this alternative epigenetic
Choux et al. Clinical Epigenetics (2015) 7:87 Page 16 of 20
mechanism may contribute to placental remodelling and these adaptation mechanisms per se could have adverse
pregnancy outcomes. effects later in life. More research is thus needed to assess
In addition, to date, no study has focused on histone the real impact of ART on future health. The better un-
modifications in ART placentas, although higher concen- derstanding of the placental mechanisms triggered by
trations of H3K4 trimethylation have been found in mouse ART will aim in fine to render the ART protocols safer.
blastocysts cultured in vivo than in vitro [183]. Recently,
Court and colleagues suggested that placental-specific Competing interests
The authors declare that they have no competing interests.
imprinted loci could be imprinted by an epigenetic
mechanism, such as histone modification, independent Authors’ contributions
of germline methylation [30]. Furthermore, other inter- CC and PF played a role in the conception of the review. CC, VC and PF took
part in the collection and assembly of data. CC, VC and PF carried out data
esting data about miRNAs indicate that they also de- analysis and interpreted the findings. CC, VC, CB, DV, PS and PF drafted the
serve to be studied in more detail [76, 184]. Studies on manuscript. CC, DV and PF wrote the critical discussion. All authors read and
combinations of epigenetic factors would also bring approved the final manuscript.
additional knowledge about the respective roles of the
Acknowledgements
different epigenetic alterations after ART. The authors thank Charlotte, 12WG. The authors also thank Philip Bastable
Besides, since gene expression and DNA methylation for the improvement of the English language and Hélène Jammes for
are sexually dimorphic in male and female placentas it is comments on the manuscript.
also important for future epigenetic placental studies to
Funding
take into account the sex of the foetuses [185]. For ex- The research fellowships of Cécile Choux and Virginie Carmignac were
ample, a study that investigated the epigenetic variations supported by the Burgundy University of Medicine.
of ZAC1 in cases of IUGR revealed down-regulated ex-
Author details
pression in placentas from girls but not boys [133]. 1
Hôpital de Dijon, Université de Bourgogne, Service de
Moreover, the link between placental growth and epi- Gynécologie-Obstétrique, Médecine Fœtale et Stérilité Conjugale, 21079
genetics was not investigated. It would be interesting to Dijon, France. 2Equipe GAD, Génétique des Anomalies du Développement,
EA 4271, Université de Bourgogne, Dijon, France. 3Hôpital de Dijon,
carry out studies comparing placental development during Université de Bourgogne, Laboratoire de Biologie de la Reproduction, 21079
the early steps of foetal life with placental epigenetic re- Dijon, France. 4Institut Cochin, Team "Epigénétique et Physiopathologie de la
sults at birth to unravel the sequence of epigenetic events Reproduction", U1016 Inserm/UMR8104 CNRS/UMR-S8104, 24, rue du
Faubourg St Jacques, 75014 Paris, France.
and distinguish between causal changes and the resulting
epigenetic landscape. Received: 27 May 2015 Accepted: 2 August 2015
Conclusions
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