Skip Navigation

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (58)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Lucifero, D.
Right arrow Articles by Trasler, J. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lucifero, D.
Right arrow Articles by Trasler, J. M.
Social Bookmarking
 Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Human Reproduction Update, Vol.10, No.1 pp.3-18, 2004
© European Society of Human Reproduction and Embryology 2004; all rights reserved

Potential significance of genomic imprinting defects for reproduction and assisted reproductive technology

Diana Lucifero1, J.Richard Chaillet2 and Jacquetta M. Trasler1,3

1 McGill University–Montreal Children’s Hospital Research Institute and Departments of Paediatrics, Human Genetics, and Pharmacology & Therapeutics, McGill University, Montreal, Quebec, H3H 1P3, Canada and 2 University of Pittsburgh, Department of Molecular Genetics and Biochemistry, Pittsburgh, PA 15213, USA 3 To whom correspondence should be addressed at: McGill University-Montreal Children’s Hospital Research Institute, 2300 Tupper Street, Montreal, Quebec. Canada H3H 1P3. e-mail: jacquetta.trasler{at}mcgill.ca


    Abstract
 TOP
 Abstract
 Introduction
 Genomic imprinting
 Imprint dynamics and timing...
 Mechanisms of genomic imprinting
 Roles of imprinted genes...
 Imprinting defects in...
 Evidence of imprinting defects...
 Imprinting mechanisms and...
 Perspectives and outlook
 Acknowledgements
 References
 
Recent studies suggest a possible link between human assisted reproductive technology and genomic imprinting disorders. Assisted reproductive technology includes the isolation, handling and culture of gametes and early embryos at times when imprinted genes are likely to be particularly vulnerable to external influences. Evidence of sex-specific differences in imprint acquisition suggests that male and female germ cells may be susceptible to perturbations in imprinted genes at specific prenatal and postnatal stages. Imprints acquired first during gametogenesis must be maintained during preimplantation development when reprogramming of the overall genome occurs. In this review, we will discuss both new developments in our understanding of genomic imprinting including the mechanisms and timing of imprint erasure, acquisition and maintenance during germ cell development and early embryogenesis as well as the implications of this research for future epigenetic studies in reproduction and assisted reproductive technology.

Key words: Angelman syndrome/assisted reproductive technology/Beckwith–Wiedemann syndrome/genomic imprinting/reproduction


    Introduction
 TOP
 Abstract
 Introduction
 Genomic imprinting
 Imprint dynamics and timing...
 Mechanisms of genomic imprinting
 Roles of imprinted genes...
 Imprinting defects in...
 Evidence of imprinting defects...
 Imprinting mechanisms and...
 Perspectives and outlook
 Acknowledgements
 References
 
The functional asymmetry of mammalian parental genomes was discovered in the mid-1980s through elegant nuclear transplantation experiments, which demonstrated the non-viability of uniparental embryo development (McGrath and Solter, 1984Go; Barton et al., 1984Go; Surani et al., 1984Go). A subset of our genes, ~60 to date, is known to be subject to genomic imprinting, the allele-specific expression of a gene where the allele that is expressed depends on whether it is maternal or paternal in origin. The monoallelic expression of imprinted genes results from the two parental alleles maintaining different epigenetic profiles. ‘Epigenetics’ refers to a process that regulates gene activity without affecting the genetic (DNA) code and is heritable through cell division. Germ cell development and early embryogenesis are crucial windows in the erasure, acquisition and maintenance of genomic imprints. Moreover, a number of genes regulated by imprinting have been shown to be essential to fetal growth and placental function. Increasing attention has recently focused on potential epigenetic disturbances resulting from embryo culture, somatic cell nuclear cloning and assisted reproductive technology (De Rycke et al., 2002Go; Gosden et al., 2003Go), indicating that a better understanding of genomic imprinting or parent-of-origin effects on gene expression is highly significant to the current study of reproduction and development.

In particular, publications over the last year have seeded concern about the possibility of an increased incidence of rare genomic imprinting diseases in children born of assisted reproductive technology. Cases of Angelman syndrome (AS) (Cox et al., 2002Go; Orstavik et al., 2003Go) and Beckwith–Wiedemann syndrome (BWS) (De Baun et al., 2003Go; Maher et al., 2003Go; Gicquel et al., 2003Go) with methylation imprint defects have been documented in children conceived via IVF and/or ICSI. It is unclear at present, but worth considering in the light of the BWS and AS cases, whether less rare imprinting abnormalities, such as those associated with placental dysfunction or postnatal cancer, are associated with assisted reproductive technology.

This overview will discuss the relevance of genomic imprinting to reproduction and highlight how aberrant imprinting can have deleterious consequences for the developing embryo. Specific points that will be addressed include the importance of proper establishment and maintenance of genomic imprints to the development of the embryo as well as to the function of extraembryonic tissues. We will also evaluate the existing evidence suggesting that imprinted genes may be particularly vulnerable to gamete and embryo manipulations in assisted reproductive technology and suggest directions for future research in this area.


    Genomic imprinting
 TOP
 Abstract
 Introduction
 Genomic imprinting
 Imprint dynamics and timing...
 Mechanisms of genomic imprinting
 Roles of imprinted genes...
 Imprinting defects in...
 Evidence of imprinting defects...
 Imprinting mechanisms and...
 Perspectives and outlook
 Acknowledgements
 References
 
The functional and sex-specific non-equivalence of imprinted alleles explains the developmental failure of uniparental embryos and confirms the requirement of both parental genomes for normal development (Barton et al., 1984Go; McGrath and Solter, 1984Go; Surani et al., 1984Go). Mouse androgenotes created from the activation of a zygote with two male pronuclei devoid of any maternal genome exhibit proliferation of extraembryonic tissues and poor embryonic development. In contrast, parthenogenetically activated oocytes give rise to relatively normal embryos that survive to an early somite stage with incomplete formation of extraembryonic structures. These observations suggest that genes expressed by the paternal genome are directed towards the development of extraembryonic tissues essential to support the growth of the embryo, while the maternal genome appears to be geared towards expressing genes that contribute to proper embryo development. The opposing tendencies of the male and female genomes as well as the elucidation from mouse studies that Igf2 and Igf2r are imprinted genes with conflicting functions led to the development of the most widely recognized theory of imprinting, the ‘parental conflict’ hypothesis (Haig and Graham, 1991Go; Moore and Haig, 1991Go). This theory proposes that the paternal genome has evolved to express genes that favour the extensive use of maternal resources and lead to optimal fetal development and growth, thus ensuring transmission of the father’s genes to the next generation. On the other hand, genes expressed by the maternal genome serve to counteract the effort made by paternally expressed genes, and limit investments in embryo development and growth in favour of salvaging resources for future pregnancies. The ‘conflict theory’ is relevant, not only to mothers and their embryos/fetuses during growth, but has been extended to postnatal effects as well, including effects on maternal behaviour (e.g. Peg3, Peg1). Mothers homozygous for targeted mutations in the paternally expressed Peg3 and Peg1 genes have defects in nurturing behaviour (Lefebvre et al., 1998Go; Li et al., 1999Go). Alternate and additional theories have also been proposed (e.g. Varmuza and Mann, 1994Go; McGowan and Martin, 1997Go; Pardo-Manuel de Villena et al., 2000Go; Beaudet and Jiang, 2002Go; Bestor, 2003Go; Kaneko-Ishino et al., 2003Go; reviewed in Wilkins and Haig, 2003Go).

To date, genomic imprinting is thought to be restricted to eutherian mammals although studies have shown that imprinting is conserved in some marsupials (the South American opossum, the tammar wallaby) (Toder et al., 1996Go; Killian et al., 2000Go; O’Neill et al., 2000Go; Ding et al., 2003Go). In flowering plants (angiosperms), parental imprinting governs development of the endosperm (non-embryonic, placenta-like tissue); increasing dosage of paternally derived genomes promotes growth of the endosperm, while increasing maternal dosage has the opposite effect (Spielman et al., 2001Go). Amphibians, insects and unicellular fungi show no evidence of imprinted genes in their genomes. However, similar epigenetically regulated phenomena of restricted gene expression have been described in species within these groups. For example, DNA methylation is used by Neurospora crassa to silence transposons and for X chromosome inactivation, and chromatin remodelling proteins are found in both Drosophila melanogaster and C. elegans. These observations suggest that gene control mechanisms used in lower organisms gained additional functions in higher organisms, including the control of genomic imprinting.

Imprinting is an epigenetically controlled phenomenon because something other than DNA sequence must distinguish the parental alleles and determine sex-specific gene expression. The role of DNA methylation in genomic imprinting has been extensively investigated. Although evidence suggests that it is not the only epigenetic mark used to distinguish the parental alleles of imprinted genes (Davis et al., 2000Go), numerous studies have confirmed its crucial role in the process. Mice completely deficient in the DNA methyltransferase (DNMT), Dnmt1, and subsequently in DNA methylation exhibit biallelic expression of several imprinted genes (Li et al., 1993Go). Mice lacking two additional DNMT, Dnmt1o and Dnmt3L, also display aberrant expression of a subset of imprinted genes (Bourc’his et al., 2001aGo; Howell et al., 2001Go; Hata et al., 2002Go). Secondly, many characterized imprinted genes have been shown to have sequences that are differentially methylated in the gametes (Tycko and Morison, 2002Go; Spahn and Barlow, 2003Go). In general, the two parental alleles have different levels of DNA methylation, and in many cases the methylation is concentrated in a single area, called a differentially methylated domain (DMD), within or near the imprinted gene. DNA methylation is a heritable yet reversible epigenetic mark that can be stably propagated after DNA replication and influence gene expression, properties that make it a particularly attractive imprinting mechanism.

In addition to allele-specific methylation, imprinting is associated with histone modifications, antisense transcripts and non-coding RNA including microRNA (Spahn and Barlow, 2003Go). For example, several imprinted loci, including Igf2r (Hu et al., 2000Go), the Igf2-H19 domain (Grandjean et al., 2001Go), Snrpn (Gregory et al., 2001Go) and U2af1-rs1 (Gregory et al., 2001Go) display acetylation of particular histone amino acid residues exclusively on the expressed allele. These observations, as well as the fact that DNA methyltransferase complexes associate with histone deacetylases (Fuks et al., 2000Go), suggest co-operation between DNA methylation, histone modifications, and overall chromatin state in the regulation of imprinted gene allele-specific expression.

As more detailed information about imprinted genes was unveiled, it became obvious that this subset of genes shares common characteristic features such as genomic clustering, regulation by antisense transcripts, and the presence of repeat elements near or within their DMD. Two imprinting clusters have been extensively described: a cluster on human chromosome 11p15 is linked to the pathogenesis of BWS, and a cluster on 15q11–13 is linked to the AS/Prader–Willi syndromes (AS/PWS) (Figure 1A and B). The mouse loci for these regions are located on chromosome 7.



View larger version (31K):
[in this window]
[in a new window]
 
Figure 1. Organization of human imprinting clusters at (A) 15q11-13 and (B) 11p15. Angelman and Beckwith–Wiedemann syndromes are associated with defects within these imprinting clusters respectively. Genes shown in blue represent paternally expressed imprinted genes, while maternally expressed imprinted genes are shown in red. White boxes represent genes that are either non-imprinted or whose imprinting status has not yet been confirmed. DMD within the imprinting clusters described are illustrated by ovals. Red-striped ovals represent a DMD that is normally methylated on the maternal allele; blue-striped ovals represent a DMD that inherits methylation from the paternal genome. The direction of the arrow indicates the transcriptional orientation of the gene. (C) BWS methylation defects are classified into two groups: (i) BWSIC1 and (ii) BWSIC2 imprinting defects; the brackets indicate the area affected in each case. The maternal alleles of patients with BWSIC1 defects acquire aberrant methylation at the H19 DMD, while the DMD within KCNQ1OT1 remains normally methylated resulting in the silencing of the H19 and the biallelic expression of IGF2. A BWSIC2 defect is characterized by loss of methylation within the KCNQ1OT1 DMD, again on the maternal allele, while the H19 DMD remains unmethylated as normal (as shown by absence of a striped oval). Consequently, KCNQ1OT1 expression is biallelic while CDKN1C and KCNQ1 are silenced on both alleles in these patients. Drawings are not to scale.

 
The clustering of several imprinted genes in distinct areas of the genome is postulated to allow coordinated regulation of genes in a given chromosomal region. Supporting this concept, imprinting centres (IC) or imprint control elements have been found in some clusters, and microdeletions within the IC can affect the imprinting status of genes kilobases away (Spahn and Barlow, 2003Go). Genes within both the BWS and the AS/PWS clusters also have antisense transcripts which are often non-coding and may serve to regulate the imprinted expression of their sense genes (e.g. KCNQ1/KCNQ10T1, UBE3A/UBE3A-AS). It has also been shown that the short tandem repeat elements within the DMD of several imprinted genes are important for the maintenance of their imprinting status (Reinhart et al., 2002Go). This is the case for the Igf2r DMD2 and is also suspected in the other genes such as Snrpn because of the repeat-rich nature of their DMD.


    Imprint dynamics and timing during gametogenesis and early embryogenesis
 TOP
 Abstract
 Introduction
 Genomic imprinting
 Imprint dynamics and timing...
 Mechanisms of genomic imprinting
 Roles of imprinted genes...
 Imprinting defects in...
 Evidence of imprinting defects...
 Imprinting mechanisms and...
 Perspectives and outlook
 Acknowledgements
 References
 
Mouse studies

Experimental evidence in mice suggests that genomic imprints in the parental gametes are erased upon every reproductive cycle, re-established in the immature germ cells of the developing embryo according to their fate as either male or female gametes, and maintained through both the preimplantation period (when the rest of the genome is demethylated) as well as postimplantation development (Figure 2). As such, imprints are dynamically changing during both germ cell and embryo development. Gametogenesis and the zygotic stage of embryogenesis are the only times when the maternal and paternal genomes are separate. As imprinted alleles are differentially marked to allow for their sex-specific expression, this period during which they are uniquely separate must be when the marking event occurs. Prior to the establishment of these sex-specific marks in the germ line, imprints are erased. In the mouse, there is evidence that this erasure occurs around the time that primordial germ cells enter the gonad, at approximately days 10.5–12.5 of gestation (Szabo and Mann, 1995bGo; Kato et al., 1999Go; Hajkova et al., 2002Go; Lee et al., 2002Go; Szabo et al., 2002Go).



View larger version (59K):
[in this window]
[in a new window]
 
Figure 2. Methylation dynamics of imprinted genes during germ cell and preimplantation embryo development. (A) The top panel illustrates the methylation dynamics of maternally and paternally methylated imprinted genes, shown by the red and blue lines respectively. During gametogenesis the pattern of non-imprinted gene methylation closely resembles that of imprinted genes. However, after fertilization the methylation status of the maternal and paternal genomes does not follow that of imprinted genes, as shown by the paler red and blue lines. (B) The progression of methylation imprint acquisition during female and male germ cell development is illustrated in the bottom panel and shown by the red and blue shading above and below the female and male germ cells respectively. The maintenance of maternal and paternal methylation imprints in the preimplantation embryo is shown by the purple shading. Arrows represent the stage of germ cell development at birth. Adapted and reprinted with permission from Elsevier (Gosden et al., 2003). PGC = primordial germ cell; Oog = oogonia; Sp = spermatogonia; PL = preleptotene; L = leptotene; Z = zygotene; P = pachytene; D = diplotene; NG = non-growing oocyte; G = growing oocyte; RS = round spermatid; MII = metaphase II oocyte; IVM = in vitro maturation; ROSI = round spermatid injection; PGD = preimplantation genetic diagnosis.

 
Subsequent to this erasure, the timing of acquisition of genomic imprints is significantly different between the two germ lines. Studies investigating the erasure and acquisition of genomic imprints have generally used evidence of DNA methylation status, as well as evidence of mono- or biallelic expression of the genes of interest, as an indication of imprint acquisition. Other approaches have been nuclear transplantation and cloning experiments using germ cell nuclei at different stages (Obata et al., 1998Go; Bao et al., 2000Go; Lee et al., 2002Go; Obata and Kono, 2002Go; Yamazaki et al., 2003Go). In these nuclear transplantation experiments, evidence of normal imprint acquisition (in the nuclear donor cells from gametes at different stages of their development) is tested by assessing postimplantation embryos for monoallelic expression of imprinted genes.

The paternal methylation imprint on H19, a maternally expressed gene, has been extensively characterized (Bartolomei et al., 1991Go, 1993; Ferguson-Smith et al., 1993Go; Tremblay et al., 1995Go; Davis et al., 1999Go; Ueda et al., 2000Go). A DMD was shown to span a 2 kb region roughly 2–4 kb from the transcription start site of the gene. This region is methylated in mature sperm and unmethylated in oocytes (Olek and Walter, 1997Go; Lucifero et al., 2002Go). The methylation imprint at this locus was shown to be acquired in prospermatogonia and complete by the pachytene stage of meiosis (Davis et al., 1999, 2000; Ueda et al., 2000Go). Additional experiments on H19 showed that paternally inherited alleles in male germ cells may have some mechanism for ‘remembering’ their origin and thus acquire their methylation imprint before maternally inherited alleles (Davis et al., 1999, 2000). A paternally inherited methylation imprint has also been described for two additional imprinted genes in the mouse, Gtl2 (Takada et al., 2002Go) and Rasgrf1 (Yoon et al., 2002Go).

Further proof that paternal imprints are complete by the haploid phase of spermatogenesis comes from round spermatid microinjection experiments. Shamanski et al. (1999Go) examined the imprinting status of three maternally expressed and three paternally expressed genes in embryos generated by injecting Mus castaneus spermatids into Mus musculus oocytes. Expression of imprinted genes was similar in embryos derived from round spermatids as compared to control embryos (from normal matings or derived by mature sperm microinjection).

In the female germ line, a number of studies have suggested that imprint acquisition occurs in the postnatal growth phase while oocytes are arrested at the diplotene stage of prophase I (Chaillet et al., 1991Go; Ueda et al., 1992Go; Brandeis et al., 1993Go; Stoger et al., 1993Go; Kono et al., 1996Go; Obata et al., 1998Go, 2002; Bao et al., 2000Go; Lucifero et al., 2002Go; Obata and Kono, 2002Go). We have previously shown that the methylation imprint on the paternally expressed gene Snrpn is acquired during this stage and that the establishment of the maternal methylation imprint on Igf2r, Peg1 and Peg3 is complete in metaphase II oocytes (Lucifero et al., 2002Go). Nuclear transplantation experiments using postnatal oocytes at various stages of maturation point to this same window of oocyte development as the time when functional imprints are acquired (Bao et al., 2000Go; Obata and Kono, 2002Go). Interestingly, and of potential relevance for the practice of human in vitro oocyte culture, one of the imprinted genes examined by Obata and Kono (2002Go), Impact, was only imprinted late in follicular development in the antral follicle stage.

Imprints established in the gametes must be faithfully maintained during preimplantation development while the methylation status of non-imprinted genes undergoes dynamic changes (Figure 2). In the mouse, within 4 h of fertilization the male pronucleus undergoes rapid demethylation by a presumably active mechanism (Mayer et al., 2000Go; Oswald et al., 2000Go; Santos et al., 2002Go). The maternal genome remains protected from this process and is therefore likely to undergo passive demethylation (Monk et al., 1987Go; Rougier et al., 1998Go). The overall methylation status of non-imprinted genes reaches a minimum at the blastocyst stage of development after which de novo methylation begins. During this wave of genome-wide methylation loss in the preimplantation embryo, imprinted genes maintain the marks inherited from the gametes, which finally translate into monoallelic sex-specific gene expression (Szabo and Mann, 1995aGo). The dynamics of methylation imprints at H19 (Olek and Walter, 1997Go), Igf2r (Stoger et al., 1993Go), U2afbp-rs (Shibata et al., 1997Go) and Ndn (Hanel and Wevrick, 2001Go) have been described during various stages of preimplantation development.

Human studies

In humans, limited information is available on the methylation status of imprinted genes during gametogenesis and embryogenesis, although the data available suggest some conservation of the methylation imprint acquisition and maintenance dynamics described in the mouse. The paternally inherited methylation imprint on H19 has been shown to be conserved in human sperm (Kerjean et al., 2000Go; Hamatani et al., 2001Go). In fetal gonocytes H19 is unmethylated, while in adult spermatogonia and subsequent stages of male germ cell development the H19 methylation imprint is acquired and maintained (Kerjean et al., 2000Go). Jinno et al. (1996Go) have shown that the paternal imprint on H19 is conserved in the preimplantation embryo.

An initial study on the methylation status of the SNURF-SNRPN locus within 15q11–13 in a small number of human oocytes suggested that the methylation imprint in fully grown human oocytes is absent and that this imprint must be acquired during or following fertilization in humans (El Maarri et al., 2001Go). However, more in keeping with the mouse data, a recent study on a larger number of oocytes at different developmental stages indicated that SNRPN is methylated in human oocytes (Geuns et al., 2003Go). From the latter study, human methylation imprints on SNRPN were present by the germinal vesicle stage and maintained in metaphase I and metaphase II oocytes. Certainly, from other data, a stable distinguishing feature must be established before the 4-cell stage as monoallelic (parental allele-specific) expression of SNRPN is initiated in 4-cell human embryos (Huntriss et al., 1998Go).


    Mechanisms of genomic imprinting
 TOP
 Abstract
 Introduction
 Genomic imprinting
 Imprint dynamics and timing...
 Mechanisms of genomic imprinting
 Roles of imprinted genes...
 Imprinting defects in...
 Evidence of imprinting defects...
 Imprinting mechanisms and...
 Perspectives and outlook
 Acknowledgements
 References
 
Although a number of different enzymes have been postulated to be involved in either erasing imprints or ‘marking’ imprinted genes for parental allele-specific expression including DNA demethylases, histone modification enzymes and DNA methyltransferases (DNMT), with the exception of the DNMT, little is known. DNMT are the family of de novo and maintenance enzymes responsible for the addition of a methyl group to the 5-position of cytosine within CpG dinucleotides. Investigations into the dynamics of DNMT expression during germ cell and preimplantation development as well as studies of mice and humans with mutations in these enzymes have provided insight into the mechanisms responsible for imprint acquisition and propagation. DNMT1 is the predominant mammalian DNA methylating enzyme; methylation of the genome is only ~5% of normal levels in mice homozygous for a targeted deletion in Dnmt1 (Lei et al., 1996Go). As DNMT1 has a preference for hemimethylated DNA, its role is postulated to be most important for the maintenance and propagation of methylation patterns. More recently, DNMT3A, DNMT3B and DNMT3L have been characterized (Okano et al., 1998Go; Aapola et al., 2000Go). DNMT3A and DNMT3B are thought to perform an important function as de novo methyltransferases, whereas DNMT3L plays a role in DNA methylation but does not appear to have DNA methyltransferase activity (Okano et al., 1998Go).

Erasure of imprints

Recent evidence from mouse studies indicates that erasure may take place over a very short time, in as little as 24 h, at about the time when the germ cells initially enter the gonad. This observation suggests an active erasure process, although the identity of the enzymes or the molecular complex that is responsible for this demethylation is unknown (Hajkova et al., 2002Go).

Acquisition of imprints

The DNMT involved in the acquisition of methylation imprints in the male germ line are currently unknown; however, DNMT3A and DNMT3L are postulated to be involved since male mice with targeted knockouts of the genes encoding these enzymes have abnormalities in spermatogenesis (Bourc’his et al., 2001aGo; Hata et al., 2002Go). The fact that methylation imprints are normal in mouse oocytes deficient in the only form of DNMT1, i.e. DNMT1o, that is present in growing oocytes, indicates that DNMT1 is not required for the acquisition of imprints in female germ cells (Howell et al., 2001Go). In contrast, mouse knockout studies of Dnmt3L suggest a critical role for this protein in the acquisition of maternal methylation imprints in the oocyte (Bourc’his et al., 2001aGo; Hata et al., 2002Go). Interestingly, DNMT3L does not share any of the conserved DNMT catalytic motifs responsible for enzymatic activity. The involvement of DNMT3A and DNMT3B in the acquisition of imprints is suggested by experiments done by Hata et al. (2002Go). The authors transplanted the ovaries from Dnmt3a–/–, Dnmt3b+/– mice (such mice do not normally survive to adulthood) into normal recipient mice, derived embryos from the oocytes from these ovaries and showed that Igf2r, Peg1, Peg3 and Snrpn have completely unmethylated DMD in the embryos (Hata et al., 2002Go). Thus, DNMT3L is postulated to be a regulator of maternal imprint establishment, which may interact with known (e.g. DNMT3A or DNMT3B) (Hata et al., 2002Go) or yet-to-be-discovered DNA methyltransferases.

Maintenance of imprints

DNMT1o is produced in mouse oocytes during their postnatal growth phase and is present throughout preimplantation development when genome epigenetic reprogramming occurs. Gene-targeting experiments have shown that DNMT1o is essential for the maintenance of methylation on imprinted genes at the 8-cell stage, the only time-point when this normally cytoplasmic isoform translocates to the nucleus; all embryos developing in the absence of DNMT1o during the preimplantation period implant successfully but then die, prior to birth (Dean and Ferguson-Smith, 2001Go; Howell et al., 2001Go). That this DNMT1 variant is present to faithfully propagate imprints at this single stage of preimplantation development is remarkable, and suggests that maintaining imprints is a crucial and highly regulated process. Although other DNMT are postulated to maintain gametic methylation imprints at stages of preimplantation development other than the 8-cell stage, the identity of such enzymes is currently unknown. Gene-targeting studies indicate that DNMT1 is required for the maintenance of DNA methylation patterns on imprinted and non-imprinted genes in the postimplantation period (Li et al., 1992Go, 1993); it is possible that other DNMT are also involved.

Errors in erasure, acquisition or maintenance of imprints

Genomic imprinting defects may occur at any of the described stages of imprint erasure, acquisition or maintenance. A complete defect of imprint erasure would result in 50% of the gametes maintaining an inappropriate imprint and carrying the opposite sex’s epigenotype at certain imprinted loci. Establishment defects could result in absence of an imprint at a specific locus and again lead to the gametes harbouring the opposite sex’s imprint epigenotype. Defects in imprint maintenance could occur at any stage of pre- or postimplantation embryo development. Maintenance defects in post-zygotic embryos could lead to mosaicism with a subset of cells affected by the maintenance defect and the remainder of cells unaffected (i.e. with normal imprints).

Defects at any of these stages may arise because of problems with the machinery (enzymes) responsible for erasing, setting down, or maintaining imprints. Alternatively, epigenetic insults may cause changes in the methylation status or chromatin conformation within imprinted genes, leading to abnormal (i.e. other than monoallelic) expression patterns.

Few studies have directly investigated imprinting defects in normal gametes and embryos. Croteau et al. (2001Go) have shown that spontaneous imprinting defects resulting in biallelic H19 and Igf2 expression occur in 1.6 and 0.5% of day 7.5 or 8.5 mouse post-implantation embryos respectively. The incidence of biallelic expression of either gene was dependent on the genetic background of the embryo, and thus presumably on trans acting factors. These observations suggest that imprinting defects may occur sporadically in normal embryos and that the processes of imprint erasure, establishment and maintenance are vulnerable to errors.

There appears to be little experimental evidence that defects in genomic imprinting can be repaired. Due to the parental allele-specific nature of imprints, it is difficult to envisage a mechanism that would allow damaged imprints to be repaired post-zygotically in the embryo.


    Roles of imprinted genes in fetal development, placental function and human disease
 TOP
 Abstract
 Introduction
 Genomic imprinting
 Imprint dynamics and timing...
 Mechanisms of genomic imprinting
 Roles of imprinted genes...
 Imprinting defects in...
 Evidence of imprinting defects...
 Imprinting mechanisms and...
 Perspectives and outlook
 Acknowledgements
 References
 
Mouse gene targeting experiments and studies of mutations in imprinted genes associated with human imprinting disorders have revealed a number of important physiological functions for imprinted genes. Since these findings have been the subject of a number of recent reviews (e.g. Reik and Walter, 2001Goa; Tycko and Morison, 2002Go), only a few examples of the functions of imprinted genes are illustrated here in order to underline the variety of phenotypes that would be expected following perturbations in imprinting in the germ line or embryo.

Fetal development

It is estimated that the total number of imprinted genes in the mouse and human genomes may range between 100 and 200. Of those that have been identified to date, a significant number appear to have important roles in fetal development (Table I). IGF2 is among these genes. Knockout mice deficient in Igf2 were found to have an absence of phenotype when the targeted allele was inherited through the female germ line, yet when transmitted through the male germ line, the offspring were shown to be severely growth-retarded (De Chiara et al., 1991Go). It was subsequently concluded that Igf2 was both an imprinted gene and a potent fetal-specific growth factor whose signalling was required for normal embryo development. Other imprinted genes that have since been shown also to be important for fetal growth include H19, IGF2R, PEG3 as well as others listed in Table I. Their role in fetal development was revealed as a result of gene targeting strategies similar to that described for Igf2. These genes belong to various families of signalling molecules or transcription factors. In humans, IGF2R is not imprinted in most individuals, although cases of polymorphic imprinting have been reported (Tycko and Morison, 2002Go).


View this table:
[in this window]
[in a new window]
 
Table I. Functions, chromosomal locations and roles of several imprinted genes
 
Placental function

Many of the imprinted genes that were first identified in the mouse were shown to be expressed in the placenta. It has been argued that imprinted genes play essential roles in controlling the placental supply of maternal nutrients to the fetus, by regulating the growth of the placenta and/or the activity of transplacental transport systems (Reik et al., 2003Go). ASCL2 is a maternally expressed placental-specific transcription factor and is expressed in the spongiotrophoblast and labyrinthine layers. Ascl2 knockout mice die within 19 days of embryonic development because of placental failure (Guillemot et al., 1994Go). Human studies have mapped ASCL2 to the same chromosomal imprinting cluster where IGF2 and H19 reside. In human extraembryonic tissue, ASCL2 expression is confined to extravillous trophoblast cells. Recent studies suggest that ASCL2 is either not imprinted or escapes imprinting in humans (Miyamoto et al., 2002Go).

Tssc3 is a maternally expressed gene whose expression is limited to the labyrinth. Targeted deletion of Tssc3 results in global hyperplasia of placental tissues with expansion of spongiotrophoblast (Frank et al., 2002Go). Two other imprinted genes that are implicated in placental function are Peg1 and Peg3. Peg1 has been shown to be expressed in murine extraembryonic mesoderm derivatives and in human trophoblast. Peg3 has recently been shown to be expressed in the extraembryonic lineages of the mouse and in human placental tissue (Hiby et al., 2001Go).

Igf2, in addition to being an important regulator of fetal growth, also has profound effects on placental development. Recently a targeted deletion of the labyrinthine trophoblast-specific isoform of Igf2, Igf2P0, resulted in impaired placental growth and reduced placental transfer functions with subsequent fetal growth retardation (Constancia et al., 2002Go). It was proposed from this study that imprinted genes expressed in extraembryonic tissues may serve to regulate the placental supply of maternal resources and nutrients to the developing fetus (Constancia et al., 2002Go). A number of additional imprinted genes are specific transporters or channels involved in nutrient exchange (Reik et al., 2003Go). For example, a knockout of the maternally expressed extraneuronal monoamine transporter, Slc22a3, affects placental transfer functions (Zwart et al., 2001Go).

The fact that many imprinted genes are essential for placental function in the mouse and that they have human counterparts, which also appear to be imprinted and expressed in similar extra-embryonic tissues, make these an important factor that may explain cases of placental dysfunction in humans. There is increasing evidence that imprinted genes play important roles in the placenta to control the balance between supply and demand for nutrients, suggesting that defects in imprinted genes expressed in the placenta may be associated with clinical syndromes such as intrauterine growth retardation (IUGR).

Human disease

Loss of function of a number of imprinted genes has been found to be linked to human genetic diseases, the progression of certain cancers, and has also been implicated in a number of neurological disorders. Two different syndromes that were initially described in the 1950s and 1960s, namely AS and PWS, were found over 25 years later both to be associated with a deletion at the same chromosomal location, 15q11-q13. This locus carries a cluster of imprinted genes, which includes SNRPN, UBE3A, ZNF127, IPW and NDN, and is syntenic to the group of imprinted genes found centrally on mouse chromosome 7 (Figure 1A).

Angelman syndrome is caused by a loss of function of the maternal allele or duplication of the paternal allele within a region that spans UBE3A. Ube3a/UBE3A shows tissue-specific imprinting, being biallelically expressed in most tissues with the paternal allele silenced selectively in hippocampal and cerebellar neurons (Tycko and Morison, 2002Go). AS is characterized by ataxia, hypotonia, severe mental and motor retardation, epilepsy and absence of speech. The methylation defect associated with AS involves loss of methylation within the SNRPN imprinting centre (IC) which subsequently assumes a paternal (undermethylated) profile. Only a small percentage (~5%) of AS patients have disrupted SNRPN IC methylation.

Prader–Willi syndrome is associated with a loss of function of the paternal allele or maternal duplication at the SNRPN locus, which is harboured within the same 15q11-q13 region. Patients with PWS are generally obese, mentally retarded, of short stature, suffer from muscular hypotonia, hypogonadotropic hypogonadism and have characteristic reduced fetal activity in the womb. In a subset of PWS patients, a methylation defect within the SNRPN IC has been described. This defect results in a gain of methylation at the IC resulting in the paternal allele assuming a maternal (methylated) profile and loss of function of the paternally expressed genes within this region (ZNF127, NDN, MAGEL2, SNRPN and IPW).

Another disease that exhibits parent-of-origin effects in its inheritance is BWS. This disorder is linked to a loss of function of the maternal allele at 11p15 where the imprinting cluster that includes H19, IGF2, CDKN1C, KCNQ1 and KCNQ1OT1 resides (Figure 1B). BWS is an overgrowth disorder and its main features are exomphalos, macroglossia, visceromegaly, neonatal hypoglycaemia, umbilical and abdominal wall abnormalities, as well as characteristic indentations of the ear. Children with BWS are predisposed to developing embryonic and childhood cancers.

Two methylation defects have been described in BWS patients. A BWS imprinting centre 1 (BWSIC1) defect results in a gain of methylation within the H19 DMD on the maternal allele such that it assumes a paternal (methylated) profile. As a result the normally expressed H19 maternal allele is silenced and IGF2 is biallelicly expressed (Figure 1Ci). The second methylation defect occurs at BWSIC2 and involves loss of maternal methylation at KCNQ1OT1, the KCNQ1 antisense transcript. Expression of KCNQ1OT1 becomes biallelic while both CDKN1C and KCNQ1 are silenced (Figure 1Cii). Wilms’ tumours are not seen in patients with BWSIC2 effects. Five to 10% of sporadic BWS cases have a BWSIC1 defect, while 40% manifest a BWSIC2 defect (Maher and Reik, 2000Go).

Monozygotic twinning occurs more frequently among BWS patients than it does in the general population (Weksberg et al., 2002Go). Many of these twins are discordant for BWS, with the affected twin showing a loss of methylation and subsequent biallelic expression at KCNQ1OT1 (Weksberg et al., 2002Go). The authors propose that this discordance in KCNQ1OT1 imprinting may arise via two possible mechanisms; (i) during twinning an unequal splitting of the inner cell mass may result in mosaicism where only one twin carries the defect resulting in BWS; or (ii) an imprinting defect during preimplantation development could lead to a problem in the imprint maintenance at KCNQ1OT1, suggesting that this gene may be particularly susceptible to imprinting insults.

Silver–Russell syndrome is a disorder characterized by low birthweight, dwarfism and lateral asymmetry and has been linked to the loss of function of a gene(s) within a less well described imprinted cluster (Preece, 2002Go). About 7–10% of patients with Silver–Russell syndrome show maternal uniparental disomy for a region on chromosome 7, while patients with paternal uniparental disomy of the same region are unaffected; these findings implicate an imprinted gene(s) in the aetiology of the disease in a subset of patients. Although PEG1 was proposed to be a likely candidate, the gene affected in this disease remains unclear (Nishita et al., 1996Go; Riesewijk et al., 1998Go).

In addition to the diseases described above, loss of imprinting is also involved in the progression of certain cancers (Tycko and Morison, 2002Go). Tumours that show imprinting effects include Wilms’ tumour where, in a subset of cases, loss of imprinting occurs at chromosomal region 11p15, which is in close proximity to the region involved in the pathogenesis of BWS. Loss of function of the maternal allele leading to the suppression of H19 and biallelic expression of IGF2 appears to be involved. Evidence suggests that other cancers show loss of imprinting, including hepatoblastoma, neuroblastoma, sporadic osteosarcoma, rhabdomyosarcoma and choriocarcinoma. Some imprinted genes act as tumour suppressor genes, the best characterized being IGF2R and WT1. Compared to non-imprinted tumour suppressor genes where loss of function requires mutations on both alleles, loss of function of both IGF2R (IGF2R imprinting is a polymorphic trait in humans) and WT1 require inactivation of only one allele.

Much like the observation that a number of imprinted genes are localized in extraembryonic tissues, it has also been noted that many are also expressed in the central nervous system. Studies of Peg1, Peg3, Ube3a, Grf1 and Gabrb3 knockout mice, as well as mice carrying a uniparental disomy at chromosome 2, suggest a functional role of imprinted genes in cognition and behaviour. Mouse knockouts of some imprinted genes show significant neurological defects ranging from abnormal maternal behaviour (Peg3, Peg1) and impaired memory (Grf1, Gabrb3) to motor dysfunction with seizures (Ube3a). In addition, several of the human imprinting diseases described have a neurodevelopmental impairment component to them. Other neurological disorders also appear to be inherited in a parent-of-origin-dependent manner. Some examples include bipolar affective disorder, autism, epilepsy, schizophrenia, Tourette syndrome, Turner’s syndrome and late onset Alzheimer’s disease (reviewed in Isles and Wilkinson, 2000Go; Nicholls, 2000Go).


    Imprinting defects in uniparental and molar pregnancies
 TOP
 Abstract
 Introduction
 Genomic imprinting
 Imprint dynamics and timing...
 Mechanisms of genomic imprinting
 Roles of imprinted genes...
 Imprinting defects in...
 Evidence of imprinting defects...
 Imprinting mechanisms and...
 Perspectives and outlook
 Acknowledgements
 References
 
Spontaneous uniparental development has been well documented in humans and occurs at a significant frequency (Mutter, 1997Go). Ovarian teratomas are the product of gynogenetic development derived from the parthenogenetic activation of an unfertilized oocyte within the ovary. These benign tumours are characterized by the presence of several types of differentiated but disorganized tissues including teeth, hair and adipose tissue, with no evidence of extraembryonically derived tissues. Teratomas constitute roughly 20% of all ovarian tumours.

Consistent with observations made in mice, human androgenetic conceptuses exhibit hyperplasia of extraembryonic trophoblastic tissues with a lack of embryo development. These hydatidiform moles, as they are referred to, originate from the fertilization of an enucleated egg or develop after the loss of maternal chromosomes in the zygote. Hydatidiform moles account for ~1 in 1000 pregnancies and lead to gestational loss (Grimes, 1984Go). The incidence of molar pregnancies varies in different regions, with Japan, Eastern Asia, Indonesia and Iran having a 5–15-fold higher rate of occurrence (Lindor et al., 1992Go). The initial development of hydatidiform moles also results in a subsequent increased risk for future incidence of trophoblastic tumours.

Hydatidiform moles belong to a group of gestational trophoblastic diseases which also include choriocarcinoma and placental site trophoblastic tumour (reviewed in Li et al., 2002Go). These molar pregnancies can be characterized as partial or complete moles depending on whether they are triploid (partial hydatidiform moles or PHM with two paternal contributions and one maternal contribution) or androgenetic (complete hydatidiform moles or CHM with only two paternal contributions) in addition to histopathological features. However, it is recognized that a number of reported CHM are biparental in origin (Helwani et al., 1999Go; Moglabey et al., 1999Go; Fisher et al., 2000Go; Judson et al., 2002Go). These biparental moles are proposed to result from normal fertilization (i.e. maternal and paternal contributions) where the maternal alleles carry paternal imprints (or the paternal epigenotype). Biparental CHM appear to be recurrent with some being familial in origin in contrast to PHM, which are exclusively sporadic. Paternal contribution to the development of these biparental CHM is unlikely as one patient was reported to have had three biparental CHM by two different partners (Fisher et al., 2000Go).

Judson et al. postulated that familial and repetitive biparental CHM may in fact result from the global disruption of maternal imprinting (likely disrupted during oocyte development when imprints are acquired in the female) thus explaining their uniparental phenotype. They examined the methylation status of a number of genes (KCNQ1OT1, SNRPN, PEG1, PEG3) which are normally maternally methylated and confirmed that in biparental moles, these genes assumed a paternal epigenotype (Judson et al., 2002Go). In contrast, H19, which is methylated in the paternal germ line, remained unaffected. These results suggest that biparental CHM are caused by a recessive maternal effect mutation which leads to the failure of establishment of maternal imprints and leads the maternal genome to assume a paternal epigenetic appearance. It was proposed by Judson et al. that a mutation in DNMT3L, the enzyme shown to be essential to maternal methylation imprint establishment, may explain this phenotype. Although they looked for a mutation in this enzyme, none was identified (Judson et al., 2002Go). In addition, the same group did not find evidence of mutations in DNMT1, DNMT1o, DNMT2, DNMT3A or DNMT3B, leading them to suggest that factors other than the known DNMT are involved in imprint establishment in the female germ line (Hayward et al., 2003Go). Another group, Moglabey et al. (1999Go), mapped a maternal locus responsible for biparental CHM to 19q13.4. In members of the same family, El Maarri et al. (2003Go) went on to show that NESP55, SNRPN and H19 were abnormally methylated on the maternal alleles in the biparental CHM and suggested that abnormal methylation in biparental CHM as a result of a defective 19q13.4 locus is due to an error in establishment of maternal methylation during oogenesis or its maintenance during preimplantation development.

A case of confined placental mosaicism for complete hydatidiform mole with a normal female infant in a singleton pregnancy was recently reported (Makrydimas et al., 2002Go). This child was a chromosomally and phenotypically normal female infant. The mechanism resulting in this confined placental mosaicism must have taken place post-zygotically and resulted in selective loss of the maternal genome in cells that would give rise to extraembryonic tissues. Alternatively, a global loss of maternal imprints could have affected a subset of cells (destined to contribute to extraembryonic tissue) in the early embryo.

BWS is sometimes misdiagnosed in utero as partial hydatidiform mole. The characteristics of a BWS placenta include gross umbilical edema, scattered and enlarged villi, placentomegaly, and placental chorangioma (Genest, 2001Go). Placental abnormalities in BWS patients may be linked to the disregulation of ASCL2 or IGF2, both located within the 11p15 imprinting cluster.


    Evidence of imprinting defects associated with assisted reproductive technology procedures
 TOP
 Abstract
 Introduction
 Genomic imprinting
 Imprint dynamics and timing...
 Mechanisms of genomic imprinting
 Roles of imprinted genes...
 Imprinting defects in...
 Evidence of imprinting defects...
 Imprinting mechanisms and...
 Perspectives and outlook
 Acknowledgements
 References
 
Much debate has recently surrounded the issue of possible epigenetic alterations brought about by embryo culture and manipulation in assisted reproductive technology (De Rycke et al., 2002Go; Thompson et al., 2002Go; Gosden et al., 2003Go). While it is generally accepted that somatic cell nuclear transfer technology bypasses essential reprogramming steps in gametogenesis and early embryogenesis and that an epigenetic component is disregulated (reviewed in Wilmut et al., 2002Go), controversy surrounds the idea that assisted reproductive technologies may be vulnerable to similar yet admittedly less pronounced effects. The expression of imprinted genes as well as overall methylation status has been shown to be abnormal in a subset of cloned animals (Bourc’his et al., 2001bGo; Dean et al., 2001Go; Humpherys et al., 2001Go; Kang et al., 2001Go; Ohgane et al., 2001Go; Humpherys et al., 2002Go; Kang et al., 2002Go; Mann et al., 2003Go).

Animal studies

The observation that in vitro culture may affect embryo outcome was initially made in ruminants. A connection to imprinting was proposed, as some of the lambs and calves born after embryo culture exhibited overgrowth abnormalities, which are now collectively referred to as ‘large offspring syndrome’. This proposed link was confirmed when it was found that sheep with ‘large offspring syndrome’ showed both lack of expression and aberrant methylation of Igf2r (Young et al., 2001Go).

Studies using mouse models have also established a relationship between embryo culture and the disrupted expression and methylation of imprinted genes. Khosla et al. have shown that the addition of fetal calf serum to M16 medium can disrupt imprinted gene function. They allowed the preimplantation development of 1- and 2-cell mouse embryos to proceed in a chemically defined M16 culture medium in the presence or absence of 5 and 10% fetal calf serum. Cultured blastocysts were then transferred to recipients to determine effects on postimplantation development. Analysis of the fetuses was carried out at day 14 of gestation and it was observed that M16 + FCS fetuses were lower in weight than control fetuses and exhibited decreased H19 and Igf2 expression. The decreased expression of H19 was correlated with an increase in methylation of its DMD. Grb7 and Grb10, two additional imprinted genes, also showed altered expression. In contrast, Peg1 did not show an altered expression profile, suggesting that some genes may be more resistant to epigenetic insults (Khosla et al., 2001Go).

Another study investigated the effect of different culture media on the expression of H19 and Snrpn in blastocysts that had been allowed to develop in vitro from the 2-cell stage (Doherty et al., 2000Go). Whitten’s medium was compared to KSOM supplemented with amino acids. Results indicated that the culture of preimplantation embryos in Whitten’s medium leads to aberrant expression of the normally silent paternal allele of H19. This abnormal expression correlated with a lack of methylation on the paternal allele. In contrast, embryos cultured in KSOM with amino acids showed no sign of abnormal H19 expression or methylation. Much like Peg1 in the above study (Khosla et al., 2001Go), blastocyst Snrpn expression was shown to be unaffected by either culture medium.

Human studies

In light of the concern caused by these animal studies, investigations into the occurrence of genomic imprinting diseases among children conceived with assisted reproductive technology have been conducted. The first one published was carried out to determine whether ICSI manipulations induced abnormal methylation patterns at 15q11-13, the locus linked to the pathogenesis of both AS and PWS (Manning et al., 2000Go). No evidence of abnormal methylation was found in samples from the 92 children examined, none of whom showed any clinical symptoms of either syndrome.

More recently, five studies have presented cases of ICSI- and IVF-conceived children afflicted with imprinting diseases (reviewed in Gosden et al., 2003Go). Cox et al. (2002Go) describe two children and Orstavik et al. (2003Go) a third child conceived using ICSI who were born with AS. Analysis within the 15q11-13 locus revealed an abnormal methylation pattern within SNRPN in samples from all three children. In each case, a loss of methylation on the maternal allele was found. Parents of the children were shown to have normal methylation profiles, and it was also confirmed that the childrens’ phenotype was not caused by a chromosomal deletion at this locus. AS normally occurs in 1 in 15 000 children, and when looking at the basis for the disorder, abnormal imprinting accounts for <5% of cases, deletion being the main aberration observed in patients. The authors therefore suggest that a causal relationship between ICSI and increased incidence of imprinting disorders is likely (Cox et al., 2002Go; Orstavik et al., 2003Go).

Three studies point to an association between IVF and ICSI and the development of another imprinting disease, BWS. De Baun et al. (2003Go) initiated a BWS registry in the USA in an effort to follow-up the incidence of cancer in these high-risk patients. In analysing information that was collected from these BWS children, they observed that several among them had been conceived via assisted reproductive technology. They proceeded to conduct a prospective study of BWS assisted reproductive technology-born children and identified a total of seven IVF- and/or ICSI-conceived children afflicted with BWS (four from their initial registry and three from their prospective study). ICSI was implicated in the conception of five of these children (four with ejaculated sperm, one with testicular sperm), one child was conceived via IVF, and it was unknown whether another child was conceived via IVF and/or ICSI. To determine the genetic defect responsible for the disease they performed methylation analysis on samples from these patients. Hypomethylation of KCNQ1OT1, corresponding to a BWSIC2 defect, was observed in five of the six children for whom samples were available. The methylation profile of H19 was also found to be abnormal in one of these children (BWSIC1 defect). One BWS-afflicted child showed normal methylation patterns of both H19 and KCNQ1OT1 (De Baun et al., 2003Go). At about the same time as the results from the USA were published, concern for an association between BWS and assisted reproductive technologies was raised independently in separate studies in two other countries. In a British BWS registry, six of 149 cases were in children conceived with assisted reproductive technologies (Maher et al., 2003Go) and in a French registry the same numbers, six of 149, were reported (Gicquel et al., 2003Go); eight out of the 12 patients (two from the British study and all six from the French study) had epigenetic BWSIC2 defects. In a previous report, we evaluated whether there was a significant increase in the risk of BWS associated with assisted reproductive technologies (Gosden et al., 2003Go). Based on the studies by De Baun (2003Go), Maher (2003Go) and Gicquel (2003Go), if we assume that assisted reproductive technologies make up 1% of all births, there is a significant 4.2-fold increase in the risk of BWS for children born using assisted reproductive technologies; however, if the frequency of assisted reproductive technology births is >1.9%, then the increase in BWS is no longer significant. Together, the results suggest the need for further studies to monitor children conceived with assisted reproductive technologies for epigenetic disorders such as BWS.

In response to these observations, we were interested in examining whether previous studies looking into the outcome of children conceived via assisted reproductive technology noted any evidence of children born with imprinting diseases. We performed independent full-text literature or abstract searches of studies looking at the outcome of assisted reproductive technology manipulations using the words Beckwith, Angelman, Prader–Willi and Wilms’ tumour. Our findings, in addition to the studies described above, are summarized in Table II. We were surprised to find a number of studies where reports of major congenital malformations within the IVF or ICSI group included children afflicted with BWS (Sutcliffe et al., 1995aGo; Koudstaal et al., 2000Go; Olivennes et al., 2001Go; Bonduelle et al., 2002Go; Boerrigter et al., 2002Go). However, as we have argued previously, these studies were small and it is likely that internationally coordinated, much larger, studies in the range of 100 000 births, will be needed to detect a doubling in the risk of BWS associated with assisted reproductive technologies (Gosden et al., 2003Go). Nevertheless, the appearance of BWS among assisted reproductive technology-conceived children may be due to a unique vulnerability of the imprinting locus on chromosome 11p15. Observations made in the study describing discordant KCNQ1OT1 methylation and expression in monozygotic twins discordant for BWS may support this suggestion (Weksberg et al., 2002Go). Also, in the mouse studies looking at the outcome of imprinted gene expression and methylation in embryos after culture, H19 has been shown to be particularly vulnerable (Doherty et al., 2000Go). The fact that H19 resides in this cluster and is linked to the pathogenesis of BWS may also predispose assisted reproductive technology children to increased risk of BWS particularly among imprinting diseases.


View this table:
[in this window]
[in a new window]
 
Table II. Reported cases of assisted reproductive technology-born children with genomic imprinting diseases
 
In addition, we noted a case of Silver–Russell syndrome among the IVF children identified in the study of the MRC Working Party on Children Conceived by IVF in Great Britain from 1978 to 1987 (MRC Working Party, 1990Go). Among the malformations listed by Hansen et al. (2002, Appendix 2), a case of PWS is included. However, because of confidentiality reasons cited by the authors, children with malformations could not be classified according to whether they were in the IVF, ICSI or naturally conceived group.

Based on the observed roles that imprinted genes play in the development of cancer, as well as in cognition and behaviour, one should examine whether assisted reproductive technology children may also be at increased risk for certain cancers and neurodevelopmental problems. Studies have investigated both these endpoints in assisted reproductive technology children and not found any evidence of an overall increased risk (Sutcliffe et al., 1995bGo, 2001; Bergh et al., 1999Go; Klip et al., 2001Go), although one study suggests that ICSI babies at 1 year of age are significantly developmentally delayed (Bowen et al., 1998Go). More detailed analysis into the subset of cancers that show loss of imprinting and the neurodevelopmental problems shown to be associated with imprinted gene disruption may reveal an association. In light of the data presented here, these analyses are warranted. It is also feasible that assisted reproductive technology pregnancies affected by an imprinting defect are more likely to result in placental abnormalities, which may be reflected in the overall health of fetus. Schieve et al. (2002Go) have shown that IVF- and ICSI-born children are at increased risk for low and very low birthweight. Disregulation of imprinted genes important for both embryonic and placental development may provide a rationale for this observation. Two studies investigating the outcome of singleton pregnancies after assisted reproductive technology noted a significant increase in the placenta-to-birthweight ratio among IVF infants (Daniel et al., 1999Go; Koudstaal et al., 2000Go).


    Imprinting mechanisms and timing: an explanation for the association of human imprinting disorders and assisted reproductive technology?
 TOP
 Abstract
 Introduction
 Genomic imprinting
 Imprint dynamics and timing...
 Mechanisms of genomic imprinting
 Roles of imprinted genes...
 Imprinting defects in...
 Evidence of imprinting defects...
 Imprinting mechanisms and...
 Perspectives and outlook
 Acknowledgements
 References
 
BWS and AS, the imprinting diseases that have been associated in a number of studies with IVF or ICSI technologies (Cox et al., 2002Go; Orstavik et al., 2003Go; De Baun et al., 2003Go; Gicquel et al., 2003Go; Maher et al., 2003Go), are related in that they are imprinting disorders characterized by a defective maternal allele. The main defect found in both the AS and BWS cases associated with assisted reproductive technology was a loss of methylation on the maternal alleles. BWS and AS both involve duplication of the paternal allele or a loss of function of the maternal allele at different imprinted loci. This is also the case when looking at the defect in IGF2R imprinting that causes ‘large offspring syndrome’ and suggests that the maternal genome is perhaps more vulnerable than the paternal genome to epigenetic and imprinting insults. An explanation for this increased sensitivity may lie in the fact that the majority of imprinted genes are regulated by a maternally derived methylation imprint. To date, only a few known imprinted genes harbour a methylation imprint that is inherited from the male germ line (H19, Rasgrf1, Gtl2). This apparent difference between the imprint methylation status of the maternal and paternal genomes may have evolved in response to the active demethylation process which targets the paternal genome within hours after fertilization (Reik and Walter, 2001Gob). As a result, imprinting defects in humans potentially brought about by embryo culture and other manipulations may be more likely to perturb imprinted genes regulated by maternal methylation. Ideally, however, to address underlying mechanisms, one would like to know whether specific techniques used in human assisted reproductive technology predispose embryos to epigenetic defects. To date, the numbers of cases of assisted reproductive technology-conceived children with imprinting defects are too small to allow such an analysis. In this section we will discuss how current gamete and embryo manipulations used in human assisted reproductive technology (reviewed in De Rycke et al. 2002Go; Winston and Hardy, 2002Go) might interfere with genomic imprinting, particularly by altering the erasure, acquisition and maintenance of imprints during germ cell formation or during early embryogenesis. A schematic representation relating the timing of imprinting events and procedures used in assisted reproductive technology is shown in Figure 2.

Possible effects of assisted reproductive technology on male germ cells

It is unlikely that assisted reproductive technology involving male gametes (e.g. the use of surgically obtained elongated spermatids or immature sperm) interferes with either the erasure or acquisition of imprints, as both processes appear to be complete by the spermatid phase of spermatogenesis. It is theoretically possible, although not supported by animal or human data, that freezing of mature sperm might perturb established imprints if either the freezing process or the cryoprotectants were to alter chromatin structure and/or DNA methylation. Perturbations associated with ICSI could include disruption of the oocyte cytoskeleton, the introduction of exogenous material into the early embryo or the leakage of cytoplasm, events that could lead to loss or inability of enzymes, i.e. DNMT, to maintain imprints during preimplantation development. Although studies by Shamanski et al. (1999Go) suggest that imprinting is not perturbed by ICSI in mouse, to our knowledge, similar experiments have not been performed in other species or in primates.

Possible effects of assisted reproductive technology on female germ cells

The two important processes associated with imprinting that occur during oocyte growth and maturation are the acquisition of maternal methylation imprints (e.g. on genes such as Snrpn) and the protection of imprinted genes that are normally unmethylated in the female germ line (e.g. H19) from becoming methylated. Either of these two processes could be affected by assisted reproductive technology procedures involving female gametes such as superovulation or in vitro maturation (IVM). It is unclear from experiments published to date whether or not the clinical use of high dose gonadotrophins, to mature many oocytes simultaneously, can alter imprint acquisition in oocytes. Gonadotropins could cause the premature release of oocytes that had not completed the imprinting process; genes that acquire their imprints late in oocyte development would be predicted to be the most sensitive to hormone-induced perturbations. Although superovulation in the mouse leads to delayed embryo development, an increased number of abnormal blastocysts, an increased number of resorptions and fetal growth retardation, it is not known whether any of these are associated with imprinting defects (Van der Auwera and D’Hooghe, 2001Go). Nevertheless, studies are warranted, in particular in light of evidence that immunofluorescence staining with an antibody against 5-methylcytosine revealed a higher incidence of abnormal methylation patterns in 2-cell embryos from superovulated females as compared to non-superovulated females (Shi and Haaf, 2002Go).

The IVM of oocytes is used clinically and involves culturing of oocytes from the germinal vesicle to the metaphase II stage. Preliminary studies in the mouse suggest that a 12 day culture period can lead to loss of methylation at Igf2r and Peg1 and gain of methylation at H19 (Kerjean et al., 2003Go). It is unknown whether the shorter time required for IVM might also affect imprints, in particular for genes that are imprinted late in oocyte development.

Possible effects of assisted reproductive technology on early embryogenesis

Preimplantation embryo development coincides with the time when gametic methylation imprints must be maintained, while most of the remainder of the genome is being stripped of its methylation (Figure 2). Potential problems that could arise as a result of embryo manipulation, cryopreservation or culture include the lack of maintenance of imprints that were acquired during gametogenesis, a perturbation of existing imprints, and a lack of protection of the normally unmethylated allele leading to methylation on both alleles. The fact that embryo culture conditions can alter imprinting in mice and sheep (Doherty et al., 2000Go; Khosla et al., 2001Go; Young et al., 2001Go) suggests that effects of the different culture media and prolonged culture (to the blastocyst stage) used clinically should be studied in more detail in both animal models and human. Although it has not been examined experimentally, embryo cryopreservation could potentially affect the cytoskeleton and the availability of enzymes associated with methylation and demethylation of the genome during preimplantation development.

Studies required

There is clearly a need for more basic research on animal gametes and embryos to model procedures (e.g. ICSI, cryopreservation, superovulation, embryo culture) used in human assisted reproductive technology and test for effects on imprinted gene expression and methylation. The mouse is an excellent model but other models where early embryo development may be more similar to human, such as bovine or non-human primate, should also be examined. Techniques such as bisulphite genomic sequencing and PCR-based expression assays (e.g. Mann et al., 2003Go; Schoenherr et al., 2003Go) now permit imprinting abnormalities (deviation from monoallelic expression or alterations in methylation) to be assessed in single blastocysts. These advances may allow critical human studies to be performed using single embryos.


    Perspectives and outlook
 TOP
 Abstract
 Introduction
 Genomic imprinting
 Imprint dynamics and timing...
 Mechanisms of genomic imprinting
 Roles of imprinted genes...
 Imprinting defects in...
 Evidence of imprinting defects...
 Imprinting mechanisms and...
 Perspectives and outlook
 Acknowledgements
 References
 
Key events in the timing and mechanisms of imprint erasure, establishment and maintenance have been uncovered in recent years. These crucial events of genomic imprinting occur in gametes and embryos and their timing coincides with the use of assisted reproductive technology. A growing amount of scientific evidence suggests a role for genomic imprinting defects in both uni- and bi-parental molar pregnancies, and of more concern, in assisted reproductive technology-born children who may be at risk for inheriting imprinting diseases. In light of these results, there should be more rigorous and thorough animal testing of existing techniques used in assisted reproductive technology for effects on imprinting and such testing should also precede the introduction of new embryo manipulations and technologies into the clinic. With respect to studies following the development of assisted reproductive technology-born children, a greater effort to include more subjects from multiple registries is needed. Prospective studies looking at the incidence of imprinting disorders in assisted reproductive technology-conceived children should be initiated. These studies should enrol a large number of patients and include registries from multiple countries, as a large number of cases will be needed to attain enough power to detect a difference in an unlikely outcome (Gosden et al., 2003Go). Detecting imprinting defects in children conceived by assisted reproductive technology is likely to require long-term follow-up since an increased cancer risk or neurodevelopmental problems may only manifest themselves in later years.

Efforts should also be continued to obtain blood or tissue samples from children conceived via ICSI or IVF that are afflicted with imprinting diseases. A more detailed analysis of the expression and methylation profiles of the affected imprinted loci in these patients may allow us to reach a better understanding of the epigenetic insult resulting in these imprinting defects. Animal models of the imprinting diseases described in this review are available (e.g. Sun et al., 1997Go; Gabriel et al., 1999Go) and would provide an interesting avenue for research into the susceptibilities of these conditions to assisted reproductive technology. Finally, more detailed disclosure of the specific procedures used in the assisted reproductive technology cycles (e.g. the use of ovarian stimulation, prolonged embryo culture, type of culture media or cryopreservation) is needed to determine whether an individual procedure leads to imprinting defects.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Genomic imprinting
 Imprint dynamics and timing...
 Mechanisms of genomic imprinting
 Roles of imprinted genes...
 Imprinting defects in...
 Evidence of imprinting defects...
 Imprinting mechanisms and...
 Perspectives and outlook
 Acknowledgements
 References
 
This work was supported by grants from the Canadian Institutes of Health Research (to J.M.T.) and the National Institutes of Health (to J.R.C.). J.M.T. is a William Dawson Scholar of McGill University and a Scholar of the Fonds de la recherche en santé du Québec. D.L. is a recipient of studentships from the Canadian Institutes of Health Research and the Fonds de la recherche en santé du Québec.


    References
 TOP
 Abstract
 Introduction
 Genomic imprinting
 Imprint dynamics and timing...
 Mechanisms of genomic imprinting
 Roles of imprinted genes...
 Imprinting defects in...
 Evidence of imprinting defects...
 Imprinting mechanisms and...
 Perspectives and outlook
 Acknowledgements
 References
 

    Aapola U, Kawasaki K, Scott HS, Ollila J, Vihinen M, Heino M, Shintani A, Kawasaki K, Minoshima S, Krohn K et al (2000) Isolation and initial characterization of a novel zinc finger gene, DNMT3L, on 21q22.3, related to the cytosine-5-methyltransferase 3 gene family. Genomics 65,293–298.[CrossRef][Web of Science][Medline]

    Bao S, Obata Y, Carroll J, Domeki I and Kono T (2000) Epigenetic modifications necessary for normal development are established during oocyte growth in mice. Biol Reprod 62,616–621.[Abstract/Free Full Text]

    Bartolomei MS, Zemel S and Tilghman SM (1991) Parental imprinting of the mouse H19 gene. Nature 351,153–155.[CrossRef][Medline]

    Bartolomei MS, Webber AL, Brunkow ME and Tilghman SM (1993) Epigenetic mechanisms underlying the imprinting of the mouse H19 gene. Genes Dev 7,1663–1673.[Abstract/Free Full Text]

    Barton SC, Surani MA and Norris ML (1984) Role of paternal and maternal genomes in mouse development. Nature 311,374–376.[CrossRef][Medline]

    Beaudet AL and Jiang YH (2002) A rheostat model for a rapid and reversible form of imprinting-dependent evolution. Am J Hum Genet 70,1389–1397.[CrossRef][Web of Science][Medline]

    Bergh T, Ericson A, Hillensjo T, Nygren KG and Wennerholm UB (1999) Deliveries and children born after in-vitro fertilisation in Sweden 1982–95: a retrospective cohort study. Lancet 354,1579–1585.[CrossRef][Web of Science][Medline]

    Bestor TH (2003) Cytosine methylation mediates sexual conflict. Trends Genet 19,185–190.[CrossRef][Web of Science][Medline]

    Boerrigter PJ, de Bie JJ, Mannaerts BM, van Leeuwen BP and Passier-Timmermans DP (2002) Obstetrical and neonatal outcome after controlled ovarian stimulation for IVF using the GnRH antagonist ganirelix. Hum Reprod 17,2027–2034.[Abstract/Free Full Text]

    Bonduelle M, Liebaers I, Deketelaere V, Derde MP, Camus M, Devroey P and Van Steirteghem A (2002) Neonatal data on a cohort of 2889 infants born after ICSI (1991–1999) and of 2995 infants born after IVF (1983–1999). Hum Reprod 17,671–694.[Abstract/Free Full Text]

    Bourc’his D, Xu GL, Lin CS, Bollman B and Bestor TH (2001a) Dnmt3L and the establishment of maternal genomic imprints. Science 294,2536–2539.[Abstract/Free Full Text]

    Bourc’his D, Le Bourhis D, Patin D, Niveleau A, Comizzoli P, Renard JP and Viegas-Pequignot E (2001b) Delayed and incomplete reprogramming of chromosome methylation patterns in bovine cloned embryos. Curr Biol 11,1542–1546.[CrossRef][Web of Science][Medline]

    Bowen JR, Gibson FL, Leslie GI and Saunders DM (1998) Medical and developmental outcome at 1 year for children conceived by intracytoplasmic sperm injection. Lancet 351,1529–1534.[CrossRef][Web of Science][Medline]

    Brandeis M, Kafri T, Ariel M, Chaillet JR, McCarrey J, Razin A and Cedar H (1993) The ontogeny of allele-specific methylation associated with imprinted genes in the mouse. EMBO J 12,3669–3677.[Web of Science][Medline]

    Chaillet JR, Vogt TF, Beier DR and Leder P (1991) Parental-specific methylation of an imprinted transgene is established during gametogenesis and progressively changes during embryogenesis. Cell 66,77–83.[CrossRef][Web of Science][Medline]

    Constancia M, Hemberger M, Hughes J, Dean W, Ferguson-Smith A, Fundele R, Stewart F, Kelsey G, Fowden A, Sibley C et al (2002) Placental-specific IGF-II is a major modulator of placental and fetal growth. Nature 417,945–948.[CrossRef][Medline]

    Cox GF, Burger J, Lip V, Mau UA, Sperling K, Wu BL and Horsthemke B (2002) Intracytoplasmic sperm injection may increase the risk of imprinting defects. Am J Hum Genet 71,162–164.[CrossRef][Web of Science][Medline]

    Croteau S, Polychronakos C and Naumova AK (2001) Imprinting defects in mouse embryos: stochastic errors or polymorphic phenotype? Genesis 31,11–16.[CrossRef][Web of Science][Medline]

    Daniel Y, Schreiber L, Geva E, Amit A, Pausner D, Kupferminc MJ and Lessing JB (1999) Do placentae of term singleton pregnancies obtained by assisted reproductive technologies differ from those of spontaneously conceived pregnancies? Hum Reprod 14,1107–1110.[Abstract/Free Full Text]

    Davis TL, Trasler JM, Moss SB, Yang GJ and Bartolomei MS (1999) Acquisition of the H19 methylation imprint occurs differentially on the parental alleles during spermatogenesis. Genomics 58,18–28.[CrossRef][Web of Science][Medline]

    Davis TL, Yang GJ, McCarrey JR and Bartolomei MS (2000) The H19 methylation imprint is erased and re-established differentially on the parental alleles during male germ cell development. Hum Mol Genet 9,2885–2894.[Abstract/Free Full Text]

    DeRycke M, Liebaers I and Van Steirteghem A (2002) Epigenetic risks related to assisted reproductive technologies: risk analysis and epigenetic inheritance. Hum Reprod 17,2487–2494.[Abstract/Free Full Text]

    Dean W and Ferguson-Smith A (2001) Genomic imprinting: mother maintains methylation marks. Curr Biol 11,R527–R530.[CrossRef][Web of Science][Medline]

    Dean W, Santos F, Stojkovic M, Zakhartchenko V, Walter J, Wolf E and Reik W (2001) Conservation of methylation reprogramming in mammalian development: aberrant reprogramming in cloned embryos. Proc Natl Acad Sci USA 98,13734–13738.[Abstract/Free Full Text]

    DeBaun MR, Niemitz EL and Feinberg AP (2003) Association of in vitro fertilization with Beckwith-Wiedemann syndrome and epigenetic alterations of LIT1 and H19. Am J Hum Genet 72,156–160.[CrossRef][Web of Science][Medline]

    DeChiara TM, Robertson EJ and Efstratiadis A (1991) Parental imprinting of the mouse insulin-like growth factor II gene. Cell 64,849–859.[CrossRef][Web of Science][Medline]

    Ding F, Patel C, Ratnam S, McCarrey JR and Chaillet JR (2003) Conservation of Dnmt1o cytosine methyltransferase in the marsupial Monodelphis domestica. Genesis 36,209–213.[CrossRef][Web of Science][Medline]

    Doherty AS, Mann MR, Tremblay KD, Bartolomei MS and Schultz RM (2000) Differential effects of culture on imprinted H19 expression in the preimplantation mouse embryo. Biol Reprod 62,1526–1535.[Abstract/Free Full Text]

    ElMaarri O, Buiting K, Peery EG, Kroisel PM, Balaban B, Wagner K, Urman B, Heyd J, Lich C, Brannan CI et al (2001) Maternal methylation imprints on human chromosome 15 are established during or after fertilization. Nature Genet 27,341–344.[CrossRef][Web of Science][Medline]

    ElMaarri O, Seoud M, Coullin P, Herbiniaux U, Oldenburg J, Rouleau G and Slim R (2003) Maternal alleles acquiring paternal methylation patterns in biparental complete hydatidiform moles. Hum Mol Genet 12,1405–1413.[Abstract/Free Full Text]

    Ferguson-Smith AC, Sasaki H, Cattanach BM and Surani MA (1993) Parental-origin-specific epigenetic modification of the mouse H19 gene. Nature 362,751–755.[CrossRef][Medline]

    Fisher RA, Khatoon R, Paradinas FJ, Roberts AP and Newlands ES (2000) Repetitive complete hydatidiform mole can be biparental in origin and either male or female. Hum Reprod 15,594–598.[Abstract/Free Full Text]

    Frank D, Fortino W, Clark L, Musalo R, Wang W, Saxena A, Li CM, Reik W, Ludwig T and Tycko B (2002) Placental overgrowth in mice lacking the imprinted gene Ipl. Proc Natl Acad Sci USA 99,7490–7495.[Abstract/Free Full Text]

    Fuks F, Burgers WA, Brehm A, Hughes-Davies L and Kouzarides T (2000) DNA methyltransferase Dnmt1 associates with histone deacetylase activity. Nature Genet 24,88–91.[CrossRef][Web of Science][Medline]

    Gabriel JM, Merchant M, Ohta T, Ji Y, Caldwell RG, Ramsey MJ, Tucker JD, Longnecker R and Nicholls RD (1999) A transgene insertion creating a heritable chromosome deletion mouse model of Prader–Willi and Angelman syndromes. Proc Natl Acad Sci USA 96,9258–9263.[Abstract/Free Full Text]

    Genest DR (2001) Partial hydatidiform mole: clinicopathological features, differential diagnosis, ploidy and molecular studies, and gold standards for diagnosis. Int J Gynecol Pathol 20,315–322.[CrossRef][Web of Science][Medline]

    Geuns E, De Rycke M, Van Steirteghem A and Liebaers I (2003) Methylation imprints of the imprint control region of the SNRPN-gene in human gametes and preimplantation embryos. Hum Mol Genet. 12,2873–2879.[Abstract/Free Full Text]

    Gicquel C, Gaston V, Mandelbaum J, Siffroi JP, Flahault A, and Le Bouc Y (2003) In vitro fertilization may increase the risk of Beckwith–Wiedemann syndrome related to the abnormal imprinting of the KCN1OT gene. Am J Hum Genet 72,1338–1341.[CrossRef][Web of Science][Medline]

    Gosden R, Trasler J, Lucifero D and Faddy M (2003) Rare congenital disorders, imprinted genes, and assisted reproductive technology. Lancet 361,1975–1977.[CrossRef][Web of Science][Medline]

    Grandjean V, O’Neill L, Sado T, Turner B and Ferguson-Smith A (2001) Relationship between DNA methylation, histone H4 acetylation and gene expression in the mouse imprinted Igf2-H19 domain. FEBS Lett 488,165–169.[CrossRef][Web of Science][Medline]

    Gregory RI, Randall TE, Johnson CA, Khosla S, Hatada I, O’Neill LP, Turner BM and Feil R (2001) DNA methylation is linked to deacetylation of histone H3, but not H4, on the imprinted genes Snrpn and U2af1-rs1. Mol Cell Biol 21,5426–5436.[Abstract/Free Full Text]

    Grimes DA (1984) Epidemiology of gestational trophoblastic disease. Am J Obstet Gynecol 150,309–318.[Web of Science][Medline]

    Guillemot F, Nagy A, Auerbach A, Rossant J and Joyner AL (1994) Essential role of Mash-2 in extraembryonic development. Nature 371,333–336.[CrossRef][Medline]

    Haig D and Graham C (1991) Genomic imprinting and the strange case of the insulin-like growth factor II receptor. Cell 64,1045–1046.[CrossRef][Web of Science][Medline]

    Hajkova P, Erhardt S, Lane N, Haaf T, El Maarri O, Reik W, Walter J and Surani MA (2002) Epigenetic reprogramming in mouse primordial germ cells. Mech Dev 117,15–23.[CrossRef][Web of Science][Medline]

    Hamatani T, Sasaki H, Ishihara K, Hida N, Maruyama T, Yoshimura Y, Hata J and Umezawa A (2001) Epigenetic mark sequence of the H19 gene in human sperm. Biochim Biophys Acta 1518,137–144.[Medline]

    Hanel ML and Wevrick R (2001) Establishment and maintenance of DNA methylation patterns in mouse Ndn: implications for maintenance of imprinting in target genes of the imprinting center. Mol Cell Biol 21,2384–2392.[Abstract/Free Full Text]

    Hansen M, Kurinczuk JJ, Bower C and Webb S (2002) The risk of major birth defects after intracytoplasmic sperm injection and in vitro fertilization. N Engl J Med 346,725–730.[Abstract/Free Full Text]

    Hata K, Okano M, Lei H and Li E (2002) Dnmt3L cooperates with the Dnmt3 family of de novo DNA methyltransferases to establish maternal imprints in mice. Development 129,1983–1993.[Web of Science][Medline]

    Hayward BE, De Vos M, Judson H, Hodge D, Huntriss J, Picton HM, Sheridan E and Bonthron DT (2003) Lack of involvement of known DNA methyltransferases in familial hydatidiform mole implies the involvement of other factors in establishment of imprinting in the human female germline. BMC Genet 4,2.[CrossRef][Medline]

    Helwani MN, Seoud M, Zahed L, Zaatari G, Khalil A and Slim R (1999) A familial case of recurrent hydatidiform molar pregnancies with biparental genomic contribution. Hum Genet 105,112–115.[CrossRef][Web of Science][Medline]

    Hiby SE, Lough M, Keverne EB, Surani MA, Loke YW and King A (2001) Paternal monoallelic expression of PEG3 in the human placenta. Hum Mol Genet 10,1093–1100.[Abstract/Free Full Text]

    Howell CY, Bestor TH, Ding F, Latham KE, Mertineit C, Trasler JM and Chaillet JR (2001) Genomic imprinting disrupted by a maternal effect mutation in the Dnmt1 gene. Cell 104,829–838.[CrossRef][Web of Science][Medline]

    Hu JF, Pham J, Dey I, Li T, Vu TH and Hoffman AR (2000) Allele-specific histone acetylation accompanies genomic imprinting of the insulin-like growth factor II receptor gene. Endocrinology 141,4428–4435.[Abstract/Free Full Text]

    Humpherys D, Eggan K, Akutsu H, Hochedlinger K, Rideout WM III, Biniszkiewicz D, Yanagimachi R and Jaenisch R (2001) Epigenetic instability in ES cells and cloned mice. Science 293,95–97.[Abstract/Free Full Text]

    Humpherys D, Eggan K, Akutsu H, Friedman A, Hochedlinger K, Yanagimachi R, Lander ES, Golub TR and Jaenisch R (2002) Abnormal gene expression in cloned mice derived from embryonic stem cell and cumulus cell nuclei Proc Natl Acad Sci USA 99,12889–12894.[Abstract/Free Full Text]

    Huntriss J, Daniels R, Bolton V and Monk M (1998) Imprinted expression of SNRPN in human preimplantation embryos. Am J Hum Genet 63,1009–1014.[CrossRef][Web of Science][Medline]

    Isles AR and Wilkinson LS (2000) Imprinted genes, cognition and behaviour. Trends Cogn Sci 4,309–318.[CrossRef][Web of Science][Medline]

    Jinno Y, Sengoku K, Nakao M, Tamate K, Miyamoto T, Matsuzaka T, Sutcliffe JS, Anan T, Takuma N, Nishiwaki K et al (1996) Mouse/human sequence divergence in a region with a paternal-specific methylation imprint at the human H19 locus. Hum Mol Genet 5,1155–1161.[Abstract/Free Full Text]

    Judson H, Hayward BE, Sheridan E and Bonthron DT (2002) A global disorder of imprinting in the human female germ line. Nature 416,539–542.[CrossRef][Medline]

    Kaneko-Ishino T, Kohda T and Ishino F (2003) The regulation and biological significance of genomic imprinting in mammals. J Biochem (Tokyo) 133,699–711.[Abstract/Free Full Text]

    Kang YK, Koo DB, Park JS, Choi YH, Chung AS, Lee KK and Han YM (2001) Aberrant methylation of donor genome in cloned bovine embryos. Nature Genet 28,173–177.[CrossRef][Web of Science][Medline]

    Kang YK, Park JS, Koo DB, Choi YH, Kim SU, Lee KK and Han YM (2002) Limited demethylation leaves mosaic-type methylation states in cloned bovine pre-implantation embryos. EMBO J 21,1092–1100.[CrossRef][Web of Science][Medline]

    Kato Y, Rideout WM III, Hilton K, Barton SC, Tsunoda Y and Surani MA (1999) Developmental potential of mouse primordial germ cells. Development 126,1823–1832.[Abstract]

    Kerjean A, Dupont JM, Vasseur C, Le Tessier D, Cuisset L, Paldi A, Jouannet P and Jeanpierre M (2000) Establishment of the paternal methylation imprint of the human H19 and MEST/PEG1 genes during spermatogenesis. Hum Mol Genet 9,2183–2187.[Abstract/Free Full Text]

    Kerjean A, Couvert P, Heams T, Chalas C, Poirier K, Chelly J, Jouannet P, Paldi A, and Poirot C (2003) In vitro follicular growth affects oocyte imprinting establishment in mice. Eur J Hum Genet 11,493–496.[CrossRef][Web of Science][Medline]

    Khosla S, Dean W, Brown D, Reik W and Feil R (2001) Culture of preimplantation mouse embryos affects fetal development and the expression of imprinted genes. Biol Reprod 64,918–926.[Abstract/Free Full Text]

    Killian JK, Byrd JC, Jirtle JV, Munday BL, Stoskopf MK, MacDonald RG and Jirtle RL (2000) M6P/IGF2R imprinting evolution in mammals. Mol Cell 5,707–716.[CrossRef][Web of Science][Medline]

    Klip H, Burger CW, de Kraker J and van Leeuwen FE (2001) Risk of cancer in the offspring of women who underwent ovarian stimulation for IVF. Hum Reprod 16,2451–2458.[Abstract/Free Full Text]

    Kono T, Obata Y, Yoshimzu T, Nakahara T and Carroll J (1996) Epigenetic modifications during oocyte growth correlates with extended parthenogenetic development in the mouse. Nature Genet 13,91–94.[CrossRef][Web of Science][Medline]

    Koudstaal J, Braat DD, Bruinse HW, Naaktgeboren N, Vermeiden JP and Visser GH (2000) Obstetric outcome of singleton pregnancies after IVF: a matched control study in four Dutch university hospitals. Hum Reprod 15,1819–1825.[Abstract/Free Full Text]

    Lee J, Inoue K, Ono R, Ogonuki N, Kohda T, Kaneko-Ishino T, Ogura A and Ishino F (2002) Erasing genomic imprinting memory in mouse clone embryos produced from day 11.5 primordial germ cells. Development 129,1807–1817.[Web of Science][Medline]

    Lefebvre L, Viville S, Barton SC, Ishino F, Keverne EB and Surani MA (1998) Abnormal maternal behaviour and growth retardation associated with loss of the imprinted gene Mest. Nature Genet 20,163–169.[CrossRef][Web of Science][Medline]

    Lei H, Oh SP, Okano M, Juttermann R, Goss KA, Jaenisch R and Li E (1996) De novo DNA cytosine methyltransferase activities in mouse embryonic stem cells. Development 122,3195–3205.[Abstract]

    Li E, Bestor TH and Jaenisch R (1992) Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell 69,915–926.[CrossRef][Web of Science][Medline]

    Li E, Beard C and Jaenisch R (1993) Role for DNA methylation in genomic imprinting. Nature 366,362–365.[CrossRef][Medline]

    Li HW, Tsao SW and Cheung AN (2002) Current understandings of the molecular genetics of gestational trophoblastic diseases. Placenta 23,20–31.[CrossRef][Web of Science][Medline]

    Li L, Keverne EB, Aparicio SA, Ishino F, Barton SC and Surani MA (1999) Regulation of maternal behavior and offspring growth by paternally expressed Peg3. Science 284,330–333.[Abstract/Free Full Text]

    Lindor NM, Ney JA, Gaffey TA, Jenkins RB, Thibodeau SN and Dewald GW (1992) A genetic review of complete and partial hydatidiform moles and nonmolar triploidy. Mayo Clin Proc 67,791–799.[Web of Science][Medline]

    Lucifero D, Mertineit C, Clarke HJ, Bestor TH and Trasler JM (2002) Methylation dynamics of imprinted genes in mouse germ cells. Genomics 79,530–538.[CrossRef][Web of Science][Medline]

    Maher ER and Reik W (2000) Beckwith–Wiedemann syndrome: imprinting in clusters revisited. J Clin Invest 105,247–252.[Web of Science][Medline]

    Maher ER, Brueton LA, Bowdin SC, Luharia A, Cooper W, Cole TR, Macdonald F, Sampson JR, Barratt CL, Reik W et al (2003) Beckwith–Wiedemann syndrome and assisted reproduction technology (ART). J Med Genet 40,62–64.[Free Full Text]

    Makrydimas G, Sebire NJ, Thornton SE, Zagorianakou N, Lolis D and Fisher RA (2002) Complete hydatidiform mole and normal live birth: a novel case of confined placental mosaicism: case report. Hum Reprod 17,2459–2463.[Abstract/Free Full Text]

    Mann MR, Chung YG, Nolen LD, Verona RI, Latham KE and Bartolomei MS (2003) Disruption of imprinted gene methylation and expression in cloned preimplantation stage mouse embryos. Biol Reprod 69,902–914.[Abstract/Free Full Text]

    Manning M, Lissens W, Bonduelle M, Camus M, De Rijcke M, Liebaers I and Van Steirteghem A (2000) Study of DNA-methylation patterns at chromosome 15q11-q13 in children born after ICSI reveals no imprinting defects. Mol Hum Reprod 6,1049–1053.[Abstract/Free Full Text]

    Mayer W, Niveleau A, Walter J, Fundele R and Haaf T (2000) Demethylation of the zygotic paternal genome. Nature 403,501–502.[Medline]

    McGowan RA and Martin CC (1997) DNA methylation and genome imprinting in the zebrafish, Danio rerio: some evolutionary ramifications. Biochem Cell Biol 75,499–506.[CrossRef][Web of Science][Medline]

    McGrath J and Solter D (1984) Completion of mouse embryogenesis requires both the maternal and paternal genomes. Cell 37,179–183.[CrossRef][Web of Science][Medline]

    Miyamoto T, Hasuike S, Jinno Y, Soejima H, Yun K, Miura K, Ishikawa M and Niikawa N (2002) The human ASCL2 gene escaping genomic imprinting and its expression pattern. J Assist Reprod Genet 19,240–244.[CrossRef][Web of Science][Medline]

    Moglabey YB, Kircheisen R, Seoud M, El Mogharbel N, Van den Veyrer and Slim R (1999) Genetic mapping of a maternal locus responsible for familial hydatidiform moles. Hum Mol Genet 8,667–671.[Abstract/Free Full Text]

    Monk M, Boubelik M and Lehnert S (1987) Temporal and regional changes in DNA methylation in the embryonic, extraembryonic and germ cell lineages during mouse embryo development. Development 99,371–382.[Abstract]

    Moore T and Haig D (1991) Genomic imprinting in mammalian development: a parental tug-of-war. Trends Genet 7,45–49.[Web of Science][Medline]

  1. MRC Working Party on Children Conceived by In Vitro Fertilisation (1990) Births in Great Britain resulting from assisted conception, 1978–87. Br Med J 300,1229–1233.[Abstract/Free Full Text]

    Mutter GL (1997) Role of imprinting in abnormal human development. Mutat Res 396,141–147.[Web of Science][Medline]

    Nicholls RD (2000) The impact of genomic imprinting for neurobehavioral and developmental disorders. J Clin Invest 105,413–418.[Web of Science][Medline]

    Nishita Y, Yoshida I, Sado T and Takagi N (1996) Genomic imprinting and chromosomal localization of the human MEST gene. Genomics 36,539–542.[CrossRef][Web of Science][Medline]

    Obata Y and Kono T (2002) Maternal primary imprinting is established at a specific time for each gene throughout oocyte growth. J Biol Chem 277,5285–5289.[Abstract/Free Full Text]

    Obata Y, Kaneko-Ishino T, Koide T, Takai Y, Ueda T, Domeki I, Shiroishi T, Ishino F and Kono T (1998) Disruption of primary imprinting during oocyte growth leads to the modified expression of imprinted genes during embryogenesis. Development 125,1553–1560.[Abstract]

    Obata Y, Kono T and Hatada I (2002) Gene silencing: maturation of mouse fetal germ cells in vitro. Nature 418,497.[CrossRef][Medline]

    Ohgane J, Wakayama T, Kogo Y, Senda S, Hattori N, Tanaka S, Yanagimachi R and Shiota K (2001) DNA methylation variation in cloned mice. Genesis 30,45–50.[CrossRef][Web of Science][Medline]

    Okano M, Xie S and Li E (1998) Cloning and characterization of a family of novel mammalian DNA (cytosine-5) methyltransferases. Nature Genet 19,219–220.[CrossRef][Web of Science][Medline]

    Olek A and Walter J (1997) The pre-implantation ontogeny of the H19 methylation imprint. Nature Genet 17,275–276.[CrossRef][Web of Science][Medline]

    Olivennes F, Mannaerts B, Struijs M, Bonduelle M and Devroey P (2001) Perinatal outcome of pregnancy after GnRH antagonist (ganirelix) treatment during ovarian stimulation for conventional IVF or ICSI: a preliminary report. Hum Reprod 16,1588–1591.[Abstract/Free Full Text]

    O’Neill MJ, Ingram RS, Vrana PB and Tilghman SM (2000) Allelic expression of IGF2 in marsupials and birds. Dev Genes Evol 210,18–20.[CrossRef][Web of Science][Medline]

    Orstavik KH, Eiklid K, van der Hagen CB, Spetalen S, Kierulf K, Skjeldal O and Buiting K (2003) Another case of imprinting defect in a girl with Angelman syndrome who was conceived by intracytoplasmic semen injection. Am J Hum Genet 72,218–219.[CrossRef][Web of Science][Medline]

    Oswald J, Engemann S, Lane N, Mayer W, Olek A, Fundele R, Dean W, Reik W and Walter J (2000) Active demethylation of the paternal genome in the mouse zygote. Curr Biol 10,475–478.[CrossRef][Web of Science][Medline]

    Pardo-Manuel de Villena F, Casa-Esperon E and Sapienza C (2000) Natural selection and the function of genome imprinting: beyond the silenced minority. Trends Genet 16,573–579.[CrossRef][Web of Science][Medline]

    Preece MA (2002) The genetics of the Silver–Russell syndrome. Rev Endocr Metab Disord 3,369–379.[CrossRef][Medline]

    Reik W and Walter J (2001a) Genomic imprinting: parental influence on the genome. Nat Rev Genet 2,21–32.[Web of Science][Medline]

    Reik W and Walter J (2001b) Evolution of imprinting mechanisms: the battle of the sexes begins in the zygote. Nature Genet 27,255–256.[CrossRef][Web of Science][Medline]

    Reik W, Constancia M, Fowden A, Anderson N, Dean W, Ferguson-Smith A, Tycko B and Sibley C (2003) Regulation of supply and demand for maternal nutrients in mammals by imprinted genes. J Physiol 547,35–44.[Abstract/Free Full Text]

    Reinhart B, Eljanne M and Chaillet JR (2002) Shared role for differentially methylated domains of imprinted genes. Mol Cell Biol 22,2089–2098.[Abstract/Free Full Text]

    Riesewijk AM, Blagitko N, Schinzel AA, Hu L, Schulz U, Hamel BC, Ropers HH and Kalscheuer VM (1998) Evidence against a major role of PEG1/MEST in Silver–Russell syndrome. Eur J Hum Genet 6,114–120.[CrossRef][Web of Science][Medline]

    Rougier N, Bourc’his D, Gomes DM, Niveleau A, Plachot M, Paldi A and Viegas-Pequignot E (1998) Chromosome methylation patterns during mammalian preimplantation development. Genes Dev 12,2108–2113.[Abstract/Free Full Text]

    Santos F, Hendrich B, Reik W and Dean W (2002) Dynamic reprogramming of DNA methylation in the early mouse embryo. Dev Biol 241,172–182.[CrossRef][Web of Science][Medline]

    Schieve LA, Meikle SF, Ferre C, Peterson HB, Jeng G and Wilcox LS (2002) Low and very low birth weight in infants conceived with use of assisted reproductive technology. N Engl J Med 346,731–737.[Abstract/Free Full Text]

    Schoenherr CJ, Levorse JM and Tilghman SM (2003) CTCF maintains differential methylation at the Igf2/H19 locus. Nature Genet 33,66–69.[CrossRef][Web of Science][Medline]

    Shamanski FL, Kimura Y, Lavoir MC, Pedersen RA and Yanagimachi R (1999) Status of genomic imprinting in mouse spermatids. Hum Reprod 14,1050–1056.[Abstract/Free Full Text]

    Shi W and Haaf T (2002) Aberrant methylation patterns at the two-cell stage as an indicator of early developmental failure. Mol Reprod Dev 63,329–334.[CrossRef][Web of Science][Medline]

    Shibata H, Ueda T, Kamiya M, Yoshiki A, Kusakabe M, Plass C, Held WA, Sunahara S Katsuki M, Muramatsu M et al (1997) An oocyte-specific methylation imprint center in the mouse U2afbp- rs/U2af1-rs1 gene marks the establishment of allele-specific methylation during preimplantation development. Genomics 44,171–178.[CrossRef][Web of Science][Medline]

    Spahn L and Barlow DP (2003) An ICE pattern crystallizes. Nature Genet 35,11–12.[CrossRef][Web of Science][Medline]

    Spielman M, Vinkenoog R, Dickinson HG and Scott RJ (2001) The epigenetic basis of gender in flowering plants and mammals. Trends Genet 17,705–711.[CrossRef][Web of Science][Medline]

    Stoger R, Kubicka P, Liu CG, Kafri T, Razin A, Cedar H and Barlow DP (1993) Maternal-specific methylation of the imprinted mouse Igf2r locus identifies the expressed locus as carrying the imprinting signal. Cell 73,61–71.[CrossRef][Web of Science][Medline]

    Sun FL, Dean WL, Kelsey G, Allen ND and Reik W (1997) Transactivation of Igf2 in a mouse model of Beckwith–Wiedemann syndrome. Nature 389,809–815.[CrossRef][Medline]

    Surani MA, Barton SC and Norris ML (1984) Development of reconstituted mouse eggs suggests imprinting of the genome during gametogenesis. Nature 308,548–550.[CrossRef][Medline]

    Sutcliffe AG, D’Souza SW, Cadman J, Richards B, McKinlay IA and Lieberman B (1995a) Minor congenital anomalies, major congenital malformations and development in children conceived from cryopreserved embryos. Hum Reprod 10,3332–3337.[Abstract/Free Full Text]

    Sutcliffe AG, D’Souza SW, Cadman J, Richards B, McKinlay IA and Lieberman B (1995b) Outcome in children from cryopreserved embryos. Arch Dis Child 72,290–293.[Abstract/Free Full Text]

    Sutcliffe AG, Taylor B, Saunders K, Thornton S, Lieberman BA and Grudzinskas JG (2001) Outcome in the second year of life after in-vitro fertilisation by intracytoplasmic sperm injection: a UK case–control study. Lancet 357,2080–2084.[CrossRef][Web of Science][Medline]

    Szabo PE and Mann JR (1995a) Allele-specific expression and total expression levels of imprinted genes during early mouse development: implications for imprinting mechanisms. Genes Dev 9,3097–3108.[Abstract/Free Full Text]

    Szabo PE and Mann JR (1995b) Biallelic expression of imprinted genes in the mouse germ line: implications for erasure, establishment, and mechanisms of genomic imprinting. Genes Dev 9,1857–1868.[Abstract/Free Full Text]

    Szabo PE, Hubner K, Scholer H and Mann JR (2002) Allele-specific expression of imprinted genes in mouse migratory primordial germ cells. Mech Dev 115,157–160.[CrossRef][Web of Science][Medline]

    Takada S, Paulsen M, Tevendale M, Tsai CE, Kelsey G, Cattanach BM and Ferguson-Smith AC (2002) Epigenetic analysis of the Dlk1-Gtl2 imprinted domain on mouse chromosome 12: implications for imprinting control from comparison with Igf2-H19. Hum Mol Genet 11,77–86.[Abstract/Free Full Text]

    Thompson JG, Kind KL, Roberts CT, Robertson SA and Robinson JS (2002) Epigenetic risks related to assisted reproductive technologies: Short- and long-term consequences for the health of children conceived through assisted reproduction technology: more reason for caution? Hum Reprod 17,2783–2786.[Abstract/Free Full Text]

    Toder R, Wilcox SA, Smithwick M and Graves JA (1996) The human/mouse imprinted genes IGF2, H19, SNRPN and ZNF127 map to two conserved autosomal clusters in a marsupial. Chromosome Res 4,295–300.[CrossRef][Web of Science][Medline]

    Tremblay KD, Saam JR, Ingram RS, Tilghman SM and Bartolomei MS (1995) A paternal-specific methylation imprint marks the alleles of the mouse H19 gene. Nature Genet 9,407–413.[CrossRef][Web of Science][Medline]

    Tycko B and Morison IM (2002) Physiological functions of imprinted genes. J Cell Physiol 192,245–258.[CrossRef][Web of Science][Medline]

    Ueda T, Yamazaki K, Suzuki R, Fujimoto H, Sasaki H, Sakaki Y and Higashinakagawa T (1992) Parental methylation patterns of a transgenic locus in adult somatic tissues are imprinted during gametogenesis. Development 116,831–839.[Abstract]

    Ueda T, Abe K, Miura A, Yuzuriha M, Zubair M, Noguchi M, Niwa K, Kawase Y, Kono T, Matsuda Y et al (2000) The paternal methylation imprint of the mouse H19 locus is acquired in the gonocyte stage during foetal testis development. Genes Cells 5,649–659.[Abstract]

    Van der Auwera I and D’Hooghe T (2001) Superovulation of female mice delays embryonic and fetal development. Hum Reprod 16,1237–1243.[Abstract/Free Full Text]

    Varmuza S and Mann M (1994) Genomic imprinting–defusing the ovarian time bomb. Trends Genet 10,118–123.[CrossRef][Web of Science][Medline]

    Weksberg R, Shuman C, Caluseriu O, Smith AC, Fei YL, Nishikawa J, Stockley TL, Best L, Chitayat D, Olney A et al (2002) Discordant KCNQ1OT1 imprinting in sets of monozygotic twins discordant for Beckwith–Wiedemann syndrome. Hum Mol Genet 11,1317–1325.[Abstract/Free Full Text]

    Wilkins JF and Haig D (2003) What good is genomic imprinting: the function of parent-specific gene expression. Nat Rev Genet 4,359–368.[CrossRef][Web of Science][Medline]

    Wilmut I, Beaujean N, de Sousa PA, Dinnyes A, King TJ, Paterson LA, Wells DN and Young LE (2002) Somatic cell nuclear transfer. Nature 419,583–586.[CrossRef][Medline]

    Winston RM and Hardy K (2002) Are we ignoring potential dangers of in vitro fertilization and related treatments? Nat Cell Biol 4(Suppl),s14–s18.[CrossRef][Medline]

    Yamazaki Y, Mann MR, Lee SS, Marh J, McCarrey JR, Yanagimachi R and Bartolomei MS (2003) Reprogramming of primordial germ cells begins before migration into the genital ridge, making these cells inadequate donors for reproductive cloning. Proc Natl Acad Sci USA 100,12207–12212.[Abstract/Free Full Text]

    Yoon BJ, Herman H, Sikora A, Smith LT, Plass C and Soloway PD (2002) Regulation of DNA methylation of Rasgrf1. Nature Genet 30,92–96.[CrossRef][Web of Science][Medline]

    Young LE, Fernandes K, McEvoy TG, Butterwith SC, Gutierrez CG, Carolan C, Broadbent PJ, Robinson JJ, Wilmut I and Sinclair KD (2001) Epigenetic change in IGF2R is associated with fetal overgrowth after sheep embryo culture. Nature Genet 27,153–154.[CrossRef][Web of Science][Medline]

    Zwart R, Verhaagh S, Buitelaar M, Popp-Snijders C and Barlow DP (2001) Impaired activity of the extraneuronal monoamine transporter system known as uptake-2 in Orct3/Slc22a3-deficient mice. Mol Cell Biol 21,4188–4196.[Abstract/Free Full Text]


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
EndocrinologyHome page
A. M. Zama and M. Uzumcu
Fetal and Neonatal Exposure to the Endocrine Disruptor Methoxychlor Causes Epigenetic Alterations in Adult Ovarian Genes
Endocrinology, October 1, 2009; 150(10): 4681 - 4691.
[Abstract] [Full Text] [PDF]


Home page
Arch. Dis. Child. Fetal Neonatal Ed.Home page
I R Makhoul, A Tamir, D Bader, A Rotschild, Z Weintraub, S Yurman, D Reich, Y Bental, J Jammalieh, T Smolkin, et al.
In vitro fertilisation and use of ovulation enhancers may both influence childhood height in very low birthweight infants
Arch. Dis. Child. Fetal Neonatal Ed., September 1, 2009; 94(5): F355 - F359.
[Abstract] [Full Text] [PDF]


Home page
Mol Hum ReprodHome page
M.V. Gomes, J. Huber, R.A. Ferriani, A.M. Amaral Neto, and E.S. Ramos
Abnormal methylation at the KvDMR1 imprinting control region in clinically normal children conceived by assisted reproductive technologies
Mol. Hum. Reprod., August 1, 2009; 15(8): 471 - 477.
[Abstract] [Full Text] [PDF]


Home page
Hum Mol GenetHome page
A. L. Fortier, F. L. Lopes, N. Darricarrere, J. Martel, and J. M. Trasler
Superovulation alters the expression of imprinted genes in the midgestation mouse placenta
Hum. Mol. Genet., June 1, 2008; 17(11): 1653 - 1665.
[Abstract] [Full Text] [PDF]


Home page
Hum ReprodHome page
M. Toppings, C. Castro, P. H. Mills, B. Reinhart, G. Schatten, E. T. Ahrens, J. R. Chaillet, and J. M. Trasler
Profound phenotypic variation among mice deficient in the maintenance of genomic imprints
Hum. Reprod., April 1, 2008; 23(4): 807 - 818.
[Abstract] [Full Text] [PDF]


Home page
Am. J. Clin. Nutr.Home page
A D. Smith, Y.-I. Kim, and H. Refsum
Is folic acid good for everyone?
Am. J. Clinical Nutrition, March 1, 2008; 87(3): 517 - 533.
[Abstract] [Full Text] [PDF]


Home page
Biol. Reprod.Home page
B Mahsoudi, A Li, and C O'Neill
Assessment of the Long-Term and Transgenerational Consequences of Perturbing Preimplantation Embryo Development in Mice
Biol Reprod, November 1, 2007; 77(5): 889 - 896.
[Abstract] [Full Text] [PDF]


Home page
J AndrolHome page
K. Biermann and K. Steger
Epigenetics in Male Germ Cells
J Androl, July 1, 2007; 28(4): 466 - 480.
[Full Text] [PDF]


Home page
Proc. Natl. Acad. Sci. USAHome page
Z. Rosenwaks and K. Bendikson
Further evidence of the safety of assisted reproductive technologies
PNAS, April 3, 2007; 104(14): 5709 - 5710.
[Full Text] [PDF]


Home page
Toxicol SciHome page
T. M. Price, S. K. Murphy, and E. V. Younglai
Perspectives: The Possible Influence of Assisted Reproductive Technologies on Transgenerational Reproductive Effects of Environmental Endocrine Disruptors
Toxicol. Sci., April 1, 2007; 96(2): 218 - 226.
[Abstract] [Full Text] [PDF]


Home page
Hum Reprod UpdateHome page
C. Allegrucci and L.E. Young
Differences between human embryonic stem cell lines
Hum. Reprod. Update, March 1, 2007; 13(2): 103 - 120.
[Abstract] [Full Text] [PDF]


Home page
Hum ReprodHome page
A. Sato, E. Otsu, H. Negishi, T. Utsunomiya, and T. Arima
Aberrant DNA methylation of imprinted loci in superovulated oocytes
Hum. Reprod., January 1, 2007; 22(1): 26 - 35.
[Abstract] [Full Text] [PDF]


Home page
EndocrinologyHome page
H.-S. Chang, M. D. Anway, S. S. Rekow, and M. K. Skinner
Transgenerational Epigenetic Imprinting of the Male Germline by Endocrine Disruptor Exposure during Gonadal Sex Determination
Endocrinology, December 1, 2006; 147(12): 5524 - 5541.
[Abstract] [Full Text] [PDF]


Home page
Biol. Reprod.Home page
D. Lucifero, J. Suzuki, V. Bordignon, J. Martel, C. Vigneault, J. Therrien, F. Filion, L. C. Smith, and J. M. Trasler
Bovine SNRPN Methylation Imprint in Oocytes and Day 17 In Vitro-Produced and Somatic Cell Nuclear Transfer Embryos
Biol Reprod, October 1, 2006; 75(4): 531 - 538.
[Abstract] [Full Text] [PDF]


Home page
Hum ReprodHome page
G. Ptak, K. Matsukawa, C. Palmieri, L. D. Salda, P. A. Scapolo, and P. Loi
Developmental and functional evidence of nuclear immaturity in prepubertal oocytes
Hum. Reprod., September 1, 2006; 21(9): 2228 - 2237.
[Abstract] [Full Text] [PDF]


Home page
Hum Reprod UpdateHome page
B. Horsthemke and M. Ludwig
Assisted reproduction: the epigenetic perspective
Hum. Reprod. Update, September 1, 2005; 11(5): 473 - 482.
[Abstract] [Full Text] [PDF]


Home page
Hum Reprod UpdateHome page
E. Seli and D. Sakkas
Spermatozoal nuclear determinants of reproductive outcome: implications for ART
Hum. Reprod. Update, July 1, 2005; 11(4): 337 - 349.
[Abstract] [Full Text] [PDF]


Home page
Hum Reprod UpdateHome page
ESHRE Capri Workshop Group
Fertility and ageing
Hum. Reprod. Update, May 1, 2005; 11(3): 261 - 276.
[Abstract] [Full Text] [PDF]


Home page
Hum ReprodHome page
C. E. Boklage
The epigenetic environment: secondary sex ratio depends on differential survival in embryogenesis
Hum. Reprod., March 1, 2005; 20(3): 583 - 587.
[Abstract] [Full Text] [PDF]


Home page
ReproductionHome page
H. Fulka, M. Mrazek, O. Tepla, and J. Fulka Jr
DNA methylation pattern in human zygotes and developing embryos
Reproduction, December 1, 2004; 128(6): 703 - 708.
[Abstract] [Full Text] [PDF]


Home page
Biol. Reprod.Home page
T. P. Fleming, W. Y. Kwong, R. Porter, E. Ursell, I. Fesenko, A. Wilkins, D. J. Miller, A. J. Watkins, and J. J. Eckert
The Embryo and Its Future
Biol Reprod, October 1, 2004; 71(4): 1046 - 1054.
[Abstract] [Full Text] [PDF]


Home page
Hum Mol GenetHome page
D. Lucifero, M. R.W. Mann, M. S. Bartolomei, and J. M. Trasler
Gene-specific timing and epigenetic memory in oocyte imprinting
Hum. Mol. Genet., April 15, 2004; 13(8): 839 - 849.
[Abstract] [Full Text] [PDF]


This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (58)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Lucifero, D.
Right arrow Articles by Trasler, J. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lucifero, D.
Right arrow Articles by Trasler, J. M.
Social Bookmarking
 Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?