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Does ART Increase Imprinting Disorders?
Henry Malter and Jacques Cohen
Tyho-Galileo Research Laboratories and Reprogenetics Inc., West Orange, NJ 07052,
Several recent reports have suggested a relationship between assisted reproduction
technology (ART) and the incidence of rare medical syndromes associated with abnormal
imprinting (Cox et al., 2002; DeBraun et al., 2003; Maher et al., 2003; Orstavik et al.,
2003; Giquel et al., 2003). While it is entirely possible that such a connection exists (and
data from experimental animals supports this premise to some extent), the substance of
these reports does little to define this connection and merely suggests that ART as a form
of conception is “over-represented” in the population of affected individuals. Obviously,
suggesting that technological aspects of ART themselves are causative factorsbased on
such analyses is confounded by a variety of issues, not the least of which is the
improbable concept that the relevant characteristics of ART patients are identical (in the
absence of ART treatment) with “control” populations used for comparison. In the only
study with an extensive discussion of ART patient history, the mother of a sporadic
Angelman’s syndrome child (and the maternal grandmother as well) exhibited an
established history of substantial reproductive dysfunction and multiple prior
spontaneous abortions both in ART and natural conception (Orstavik et al., 2003). It
There is little question that many processes involved with the creation of a
developmentally viable genome take place during the pre-implantation period. Epigenetic
modification of the genome is a critical component of these processes (El-Maarri et al.,
2001). In its most basic sense, imprinting provides a method for identifying and choosing
a gene for expression based on whether the gene was inherited from a male or female
lineage. It is thought that imprinting is in fact an effect of the “battle of the sexes” i.e the
selection process for each gender’s evolutionary “interest” during the gestational process
(Reik and Walter, 2001a; 2001b). This was manifested through controlling the growth of
the fetus for conditions optimal to either the male or female parent’s interest. The
outcome of this process is that some genes are only expressed on chromosomes that were
derived from a maternal line and others are only expressed from paternal chromosomes.
This may seem to be a self-evident “fact of life” but it is only the existence of imprinted
genes that makes it an absolute requirement for an organism’s genome to be derived from
both a mother’s and father’s chromosomes. In fact, this was demonstrated in early
experiments where mouse embryos were produced through nuclear transfer with 2 sets of
either all maternal or all paternal chromosomes (McGrath and Solter, 1984; Surani et al.,
1984). Such embryos do not survive and exhibit uniquely abnormal development due to
the skewed gene expression such all-maternal or all-paternal complements manifest. In
other words, if a gene has evolved to be expressed only on the maternal chromosome, this
gene will be completely silenced in all-paternal embryos and they will lack its function –
and vice-versa. Also, since imprinted genes are normally only present in one functional
copy, (since the other chromosome is effectively turned-off); the presence of 2 identically
imprinted active chromosomes in maternal or paternally-derived embryos can result in
too much expression.
Obviously, experiments creating such grossly abnormal embryos would seem to have
little bearing on normal clinical ART techniques. However, limited imprinting
abnormalities have also been described through more subtle manipulations during the
reproductive process. This is apparently the case due to the fact that important events of
the imprinting process can occur during early development – when typical “in-vitro”
manipulations take place. The most extreme form of such abnormalities has been
described following nuclear transfer “cloning”. Embryos produced through nuclear
transfer can exhibit abnormal imprinting and subsequent expression at several genes. The
resulting incorrect pattern of gene expression has been associated with often highly
abnormal development.
One of the most common developmental problems associated with cloning is the “large
offspring” syndrome (LOS) in which fetal growth and gestation length control is lost
leading to the development of abnormally large fetuses manifesting a variety of health
problems (Young et al., 1998; Young et al., 2001). As mentioned, imprinted genes such
as those for some growth factors, are theoretically involved in a gender “tug-of war”
controlling the gestational process. In nuclear transfer cloning, the chromosomes from a
late embryonic or adult somatic cell nucleus are used to replace the early embryonic
genome and direct development. No doubt the processes through which the existing
imprints of these chromosomes are recognized and processed is imperfect during cloning
scenarios (Kang et al., 2001). However, it is now realized that more subtle manipulations
to the early embryo can also result in similar disturbance to the imprinting process
(Doherty et al., 2000; Khosla et al., 2001; Stojanov and O’Neil, 2001). For instance, the
addition of abnormal components such as serum to culture media is known to result in
some of the same imprinting abnormalities including LOS in some animal species. It is
based on such results (from mouse and large animal experiments) that some have
speculated that the normal manipulations associated with human ART could result in
similar deficits or disturbances to the imprinting process.
While gross imprinting related abnormalities, such as in LOS, have yet to be
characterized in the human, several rare but well characterized syndromes are caused by
imprinting errors. Angelman’s, and Prader-Willi syndromes are serious conditions that
involve multiple developmental abnormalities and Beckwith-Wiedemann syndrome
mimics to some extent a fetal overgrowth phenotype. While the complete genetic basis of
these diseases is as yet unclear, they all apparently derive from perturbations at distinct
loci in the human genome that are normally expressed only from one parental
chromosome. When this normal pattern of expression is lost, either through actual
chromosomal lesion or through failure of the normal imprinting process, developmental
abnormalities result. For instance, human chromosome 15 contains a region that is
subject to imprinting - with some genes expressed only on maternal chromosomes and
others expressed only paternally. When normal (maternal only) expression of the
imprinted Ube3A gene is perturbed, a variety of developmental neurological
abnormalities occur leading to Angelman’s Syndrome. Conversely, expression
abnormalities of paternal-only genes in this region apparently lead to the completely
different phenotype associated with Prader-Willi syndrome. As mentioned, such
expression anomalies can ultimately result from a variety of causes including
chromosomal deletions in the critical regions. However, sporadic loss of the correct
parental imprint i.e. during the transmission of the maternal or paternal chromosome to
the affected child has been clearly described during natural conception. It is this type of
sporadic error in the imprinting process that could be associated with the application of
ART techniques in infertile couples (although it is definitely a naturally occurring
phenomenon as well).
Critical components of the imprinting process occur in the early embryo during the time
that in vitro culture and manipulation must occur in ART treatment. As discussed above,
some animal models suggest that imprinting can apparently be disturbed by “artificial”
conditions during this in vitro period. However, the connection between the specifics of
these studies and the reality of human ART has yet to be established. In fact, the majority
of what is definitively known suggests that this connection is questionable.
For one thing, clear differences are known to exist in the specifics of the imprinting
process and imprinted genes between the rodent and ruminant animals used for
imprinting research and the human. This is most obvious in the situation regarding the
insulin-like growth factor 2 receptor gene (IGF2R). Until recently, it had been speculated
that potential ART-induced perturbations to imprinting of this gene might arise from in
vitro manipulation of human embryos. There is no doubt that IGF2R is imprinted in the
mouse, sheep and bovine and that loss of imprinting control of this gene during cloning
or serum-augmented culture has been associated with LOS and severe developmental
problems. Also, the structure of IGF2R is strikingly similar between these species and the
human suggesting that similar imprinting control regions might be present. However, a
recent study on IGF2R in the human and other primates has definitively shown that this
gene is not, in fact, subject to imprinting in the primate lineage and therefore could not
play a role in ART-associated perturbations (Killian et al, 2001). The human IMPACT
gene is also known to avoid imprinting, despite imprinted status in the mouse (Okamura
et al., 2000). Most recently, the GRB10 gene, which exhibits complex imprinting
phenomenon in the mouse has been shown to be expressed from both maternal and
paternal alleles in most human tissues despite highly conserved gene structure and overt
patterns of molecular imprinting markers between the two species (Arnaud et al., 2003).
These studies do not, of course, suggest that human genes are not subject to imprinting.
However, they do strongly suggest that real differences exist in the specifics of the
imprinting process between experimental animals and the human. Therefore, suggesting
that phenomena observed in rodents and ruminants such as the apparent culture-induced
imprinting disturbance, will necessarily be a problem in the human are not well founded.
Furthermore, the only direct studies examining imprinted human genes following ART
treatment, although limited in scope, have shown no evidence of perturbations to date.
Analysis of the imprinting control region of the human SNRPN gene in ICSI offspring
and demonstrated no evidence of abnormalities (Manning et al., 2000). This elegant study
but was however limited and will require more extensive follow-up. A recent more
detailed study in human oocytes and early embryos has revealed that maternal imprints at
the SNRPN locus are, in fact, established and stabilized prior to the preimplantation
period (Geuns et al. 2003). This definitive result obtained by a highly sensitive molecular
sequencing technique, contradicts a previous study that suggested that this imprint was
altered at or following fertilization. This result suggests that imprints at this locus would
not be perturbed during ART treatment, since they seem to be stably established prior to
the preimplantation period. This is a particularly important result, since expression
abnormalities at the SNRPN locus are associated with human Angelman and Prader-Willi
syndromes. Whether this could potentially have an effect on procedures involving in vitro
maturation needs to be determined.
As for the recent patient cohort studies suspecting imprinting disorders after ART, the
authors themselves are quick to point out that they are as yet inconclusive. One criticism
is that it is unclear how each of these unique cohorts of affected individuals reflect the
overall population of affected individuals worldwide and how these groups can be
correctly related to the population of ART individuals. For instance, in one study an ART
derived cohort of affected individuals apparently collected over a 2-year period is simply
compared with the proportion of ART births for a preceding year. This forms the basis
for suggesting that ART is responsible for a six-fold increase in disease incidence
(DeBraun et al., 2003). The absolute numbers of affected individuals identified following
ART are still very small and compared with the total number of ART offspring still fall
well short of the baseline incidence of these diseases in the “normal” human population.
While the authors suggest that the ascertainment of these cases would be expected to
underestimate the true rate of affected ART individuals, this is as yet unproven.
Obviously, a much more substantial direct analysis will be required before even the basic
incidence of imprinting-related disorders following ART is clear. Such an analysis will
still fail to delineate the underlying cause of any observed change in this incidence
between the application of assisted reproduction techniques and the intrinsic
characteristics of the ART patient population.
However, taken together, the research data and population studies suggesting that
epigenetic changes with deleterious consequences could result from the application of
ART techniques in the human must be taken seriously. Though we must be careful
considering technological and etiological causes separately (Ludwig and Diedrich, 2002).
This situation calls for an immediate response by ART practitioners in several areas.
First, ART patients should be informed and counseled concerning the current state of
knowledge about imprinting disorders and ART. While the relationship between ART
and these disorders is as yet unclear, the potential for a relationship and other as yet
unidentified epigenetic consequences is a serious issue that patients have a right to
understand and consider in making their assisted reproductive choices. Second, a careful
and rigorous assessment of the true incidence of imprinting-related conditions in the ART
population needs to be undertaken, with emphasis on specific population controlled
causes and potential effects of technologically induced changes. For the latter, two
separate technologies must be regarded; one that controls oocyte recrutement by ovarian
hyper-stimulation and the other that uses culture and manipulation conditions. This would
seem to be a perfect subject for support by governmental and professional societies
worldwide. Also, current clinical record keeping and follow-up procedures need to take
the requirements of such assessment into account. Third, much further research is
indicated in the direct assessment of epigenetic status in ART embryos. Standard
molecular techniques already provide for such analysis and require only further
refinement and application (Salpekar et al., 2001). Such research would be vital not only
to an understanding of the true nature of epigenetic processing during the preimplantation period in the human; it would also be critical to diagnostic strategies for
identification of embryos harboring imprinting errors. This could possibly lead to
alteration of follicular stimulation protocols and refinement of culture conditions.
An unfortunate recent news item about the ART/imprinting studies ended with yet
another questionable pun postulating that: “ART does not imitate life”. Assisted
reproductive medicine has never claimed or attempted to “imitate life”. It is simply an
evolving “best attempt” at circumventing the plethora of complex infertility problems
plaguing the human population. On a daily basis, ART addresses such infertility
problems (many of which were previously intractable) with great success. It is well past
time to accept that such intervention is, like all medicine, an imperfect process. However,
the “bottom line” on ART treatment, as revealed through clinical data reporting and
follow-up studies, does not suggest any substantial negative consequences and the vast
majority of ART children are healthy. Despite this, criticism of ART based on vague
pessimistic assertions and selective analysis is rampant. In the week following these latest
publications on imprinting disorders, just as many infertile couples will present
themselves at ART clinics seeking assistance with their medical issues. The possibility of
a connection between ART techniques and imprinting-related disorders simply presents a
further challenge in counseling and assisting such patients with the birth of a healthy
child. This challenge is certainly not beyond our capabilities. Furthermore, as suggested,
the supposedly ART-related increase in the incidence of such “spontaneous” imprinting
disorder births may in fact be related to intrinsic factors in the infertile population being
treated. As has already been the case for a variety of other developmental genetic issues,
ART combined with diagnostic strategies may emerge as a powerful method of avoiding
such births - rather than their “cause”.
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