1 Centre for Medical Genetics, and 2 Centre for Reproductive Medicine, University Hospital of the Dutch-speaking Brussels Free University, Laarbeeklaan 101, 1090 Brussels, Belgium
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Abstract |
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Key words: assisted reproductive techniques/epigenetic risks/imprinting/in-vitro culture/methylation
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Introduction |
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Spectrum of assisted reproductive technologies |
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Assisted reproduction together with embryo biopsy procedures and molecular genetic techniques at the single-cell level have led to the development of PGD for couples at risk of transmitting a genetic disease (Handyside et al., 1990). PGD is an early form of prenatal diagnosis, aimed at selecting embryos not affected by the genetic disease under investigation, before their transfer to the uterus. There are two major techniques used in PGD: (i) the presence of chromosomal aberrations is analysed using fluorescence in-situ hybridization (FISH); (ii) single-gene defects are detected using polymerase chain reaction (PCR). Both techniques are becoming increasingly established as reported by the International Working Group on PGD. Up to 2001,
500 healthy babies have been born (International Working Group on Preimplantation Genetics, 2001
).
The procedure of ooplasmic transfer was developed for patients with advanced maternal age and/or with poor embryo development and recurrent implantation failure. About 30 babies have been born using a modified ICSI protocol for the injection of a sperm together with 515% good-quality donor ooplasm (Barritt et al., 2001). Although the presence of donor mitochondrial (mt) DNA was observed in some of the babies, it is not clear whether this had a therapeutic effect (Brenner et al., 2000
). Ooplasmic transfer remains an experimental technique since the underlying factors and mechanisms leading to the observed increased embryo implantation rate are not yet known.
Other techniques, such as metaphase II (MII) spindle transfer, are currently under study in animal models (Wang et al., 2001). Abnormal spindle formation as seen in oocytes from women of advanced age might be due to aberrant control mechanisms in the ooplasm (Battaglia et al., 1996
). Transferring the spindle and bringing it under control of a recipient MII ooplasm might be beneficial in patients with poor oocyte and embryo quality.
Finally, reproductive cloning by nuclear transfer has been achieved in different animal species, but the success rate is low: only one embryo out of 100 will develop into an adult cloned animal (Solter, 2000). The possibility of reproductive human cloning raises major legal, ethical and social issues, and so far it has been banned in many countries.
The widespread, almost immediate clinical application of IVF and ICSI in assisted reproduction, without any adequate experimental phase, as well as the invasive nature of some of the techniques, have led to a debate concerning the safety of these techniques. Retrospective and prospective follow-up studies have shown that the major and minor malformation rates for IVF and ICSI babies were similar to those in the general population. Some studies showed an increase in de-novo chromosomal aberrations in ICSI babies (Tarlatzis and Bili, 1998; Bonduelle et al., 1999
). Neonatal data further indicate that the rates of low and very low birthweight observed in the IVF and ICSI singletons are higher than in the general population (Doyle et al., 1992
; Buitendijk, 1999
; Sutcliffe et al., 2001
; Bonduelle et al., 2002
).
With time, IVF and ICSI technology were complemented with cryopreservation and blastocyst culture/transfer/freezing techniques as a means to increase pregnancy rates and to avoid multiple pregnancies. The cryopreservation process appeared to have no negative influence on neonatal outcome and child development as compared with fresh embryo transfer (Wennerholm, 2000). Blastocyst culture is used in some centres for selected patients as well as in PGD cycles. No differences have been observed in birthweight when comparing blastocyst transfers with early cleavage-stage embryo transfers, but birthweights were lower than in control populations (Ménézo et al., 1999
; Kausche et al., 2001
).
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Epigenetic reprogramming |
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The genetic information of a DNA sequence is complemented by epigenetic modifications. Epigenetics covers a broad range of effects: DNA methylation, imprinting, RNA silencing, co-valent modifications of histones and remodelling by other chromatin-associated complexes. All these effects involve mechanisms in the regulation of gene expression. Epigenetic patterns are imposed on the genome during differentiation through predetermined programmes (genetic factors). Additional epigenetic changes enable cells to respond to environmental factors by altering the expression level of the gene without having to change the DNA code itself. There are at least two critical periods in which epigenetic reprogramming occurs, one during gametogenesis and another during the preimplantation embryonic stage (Reik et al., 2001). Reprogramming during gametogenesis is essential for the imprinting mechanism. Imprinting regulates the differential expression of paternally and maternally derived genes. Imprints are established differentially in sperm and oocyte and are maintained in the zygote and further through all somatic cell divisions. They are reset in the germline in a sex-specific way. Epigenetic germline reprogramming starts when primordial germ cells (PGC) enter the gonads. Demethylation processes occur for non-imprinted genes as well as for imprinted genes, after which cell division is arrested at mitosis for male gametes and at meiosis for female gametes. Erasure of epigenetic modifications in the PGC ensures genetic totipotency which is necessary for the development of a new organism. At cell cycle resumption, methylation and chromatin remodelling take place, providing the genome in the gametes with molecular programmes for oocyte activation and embryonic development. Mature oocytes and sperm have a quite different epigenetic organization. The sperm genome is more methylated than the oocyte genome, chromatin has been compacted with protamines in sperm cells and with histones in oocytes, the oocyte chromatin structure is more repressive than that of sperm cells and, finally, differentially methylated regions have been established at imprinted loci. Both genomes are in a silent state when they come together at fertilization. After fertilization, paternal chromosomes decondense and are remodelled, protamines are exchanged for maternal histones and active, rapid demethylation takes place (Mayer et al., 2000
). The maternal genome is demethylated more gradually by a passive mechanism. At the time of implantation, a genome-wide methylation takes place. Epigenetic reprogramming during the preimplantation period is important for accurate development, as it controls expression of early embryonic genes, cell cleavage and cell determination. In adult tissues, the early embryonic genes are repressed and tissue-specific genes are activated. Imprinted genes maintain their methylation marks from the gamete stage and they escape these general (de)methylation processes of the preimplantation stage.
Disturbance of epigenetic reprogramming may influence gene expression and phenotype characteristics. Moreover, epigenetic changes that occur shortly after fertilization, before specification of the germ line, will involve both somatic cells and germ-line cells and may lead to the inheritance of an epigenetic trait resulting in transgenerational phenotypes.
Possible epigenetic risks linked to assisted reproduction may result from the use of immature sperm cells with incomplete reprogramming or from the use of in-vitro embryo procedures during a time window when epigenetic reprogramming occurs.
Methylation
Very few of the factors involved in epigenetic reprogramming have been identified. The best studied epigenetic modification until now has been DNA methylation of CpG motifs (Jones and Takai, 2001). The establishment and maintenance of methylation patterns depend on at least three different DNA methyltransferases, one maintenance methyltransferase, DNMT1, which prefers hemi-methylated DNA as a substrate, and two de-novo methyltransferases, DNMT3A and DNMT3B, which act preferentially on non-methylated DNA. Loss of Dnmt3b and Dnmt1 genes causes embryonic lethality in mice at mid-gestation. Dnmt3a knockout mice are live-born but they fail to thrive and die shortly after birth (Okano et al., 1999
).
DNA (hypo)methylation is linked to chromatin structure and transcriptional regulation. Transcriptional repression is established through the interaction of methyl-CpG binding proteins with chromatin remodelling complexes. Methyl-CpG binding proteins, such as MECP2, bind selectively to methylated regions, recruit histone deacetylase and direct transcriptional repression by deacetylating histones and assisting the formation of a stable, repressive chromatin structure in promoter regions. Transcriptional activation of a gene has been linked with unmethylated CpG motifs in the promoter region. A transcriptional activator that binds specifically to unmethylated CpG dinucleotides has been identified: CGBP (Voo et al., 2000). Unsurprisingly, mouse embryos lacking the Cgbp gene are viable only up to the blastocyst stage, showing that loss of methylation control in the early embryo will lead to aberrant development and even cell death (Carlone and Skalnik, 2001
). In humans, several genetic diseases have been associated with methylation defects (Robertson and Wolffe, 2000
). ICF, for Immunodeficiency, Centromeric instability and Facial anomalies, involves pericentromeric hypomethylation due to mutations in the methyltransferase DNMT3B gene. Rett syndrome (RTT) is characterized by initial normal development, followed by progressive degeneration of acquired motor skills, loss of speech and mental retardation (Amir and Zoghbi, 2000
). RTT is an X-linked dominant disorder limited to girls, as the few males born alive die before the age of 2 years. Mutations in the methylCpG binding gene MECP2 underlie the disease. Mutations in the same gene, but distinct from those involved in RTT, have been identified as the cause of non-specific X-linked mental retardation (Couvert et al., 2001
). Fragile X mental retardation is associated with aberrant de-novo methylation and histone acetylation of an expanded polymorphic CGG repeat (>200 repeats) in the 5' untranslated region of the FMRI gene (Oberlé et al., 1991
; Verkerk et al., 1991
; Yu et al., 1991
). This methylation spreads to the CpG island in the promoter region and silences the gene. FMR1 plays a role in protein synthesis in neurons. ATR-X (X-linked
-thalassaemia/mental retardation) is characterized by mental retardation, facial dysmorphism,
-thalassaemia and urogenital abnormalities. The ATRX protein is a transcriptional regulator acting through chromatin remodelling. Mutations in ATRX induce changes in the methylation patterns of highly repeated sequences (Gibbons and Higgs, 2000
; Gibbons et al., 2000
). A common aspect to the above diseases is mental retardation. It seems that DNA promoter methylation is an important mechanism of transcriptional regulation in neural cells. Identification of the target genes will help to understand how mutations in MECP2 and DNMT3B relate to defective brain development.
Methylation defects may not only arise by genetic mutations in the methylation machinery, but also by epigenetic alterations at their target genes. Moreover the methyltransferases themselves may be deregulated epigenetically by time variations. DNMT1, 3A and B and chromatin remodelling complexes are cell-cycle regulated. Changes in embryo developmental timing, such as can be caused by embryo culture, may interfere with their activity and finally with methylation patterns and expression levels. Few studies have been performed on the possible effects of assisted reproduction on the expression of non-imprinted genes. Using RTPCR for mRNA phenotyping in bovine preimplantation embryos, it has been shown that the expression patterns of several genes important for development differ between embryos cultured in-vitro and embryos obtained in-vivo. Furthermore, the mRNA levels of in-vitro-produced embryos are altered by the presence or absence of serum in the medium (Niemann and Wrenzycki, 2000). In the follow-up studies of IVF and ICSI children, little attention has been given to methylation defective disorders in the form of, for example, neurodevelopmental problems. Often, the study groups are relatively small and most surveys stop when the children reach the age of 2 years, which means that problems which become manifest only later in development will not be detected. An Australian study showed that there was an increased proportion of 1 year old ICSI children with a mildly delayed mental development, compared with IVF or naturally conceived children (Bowen et al., 1998
). Re-examination of the group of ICSI children at the age of 5 years did not show a difference in full-scale IQ (Leslie et al., 2001
). The overall results of a similar study looking at ICSI and IVF children of 2 years old showed no mental developmental difference between these children and the general population (Bonduelle et al., 1998
). A more recent UK casecontrol study again found no difference in mean neurodevelopmental score between ICSI children and a control group (Sutcliffe et al., 2001
).
Imprinting
Another critical step, apart from the initiation of general (de)methylation patterns at the preimplantation stage, is the resetting and the maintenance of differential methylation marks at imprinted loci in the germline and zygote, followed by their differential expression (Reik and Walter, 2001). Imprinted genes play key roles in embryonic growth and behavioural development and they are also involved in carcinogenesis. Several human syndromes are known to be associated with imprinted genes including SilverRussell, Albright, PraderWilli, Angelman, BeckwithWiedemann and Wilms tumour (Falls et al., 1999
). Aberrant imprinting is also suspected to play a role in other neurobehavioural disorders such as autism, bipolar affective disorder and schizophrenia (Isles and Wilkinson 2000
; Skuse, 2000
). Imprinted genes occasionally function as oncogenes or tumour suppressor genes (IGF2, WT1, M6P/IGF2R, p73). The functional monoallelic loci are particularly vulnerable to proto-oncogene activation and tumour suppressor inactivation (Jirtle et al., 1999
).
The effect of aberrant imprinting during the preimplantation developmental window may have several consequences. There may be consequences in the long term such as cancer susceptibility. Otherwise, early deregulation of imprinted genes may lead to biallelic expression or absence of expression. Taking into account the essential function of imprinted genes in embryonic growth, this may lead to embryo developmental arrest and death, fetal growth retardation or overgrowth, or it may cause one of the clinical syndromes already mentioned. Again, as with the other epigenetic phenomena, the relative timing of imprint processes with respect to the developmental stage is critical. Small differences between the embryonic clock and the timing of the imprinting mechanism may cause major disturbances. This is illustrated by the explicit activity of the oocyte-specific isoform of Dnmt1 during the 8-cell stage of the mouse embryo. This maintenance methyltransferase protein is localized in the cytoplasm of oocytes and blastomeres until postimplantation stage, but at the 8-cell stage it enters the nucleus to ensure maintenance of imprinted methylation marks. Mice deficient in the Dnmt1 isoform die at different times pre- and postnatally (Dean and Ferguson-Smith, 2001; Howell et al., 2001
).
Aberrant imprinting may arise not only at the time of preimplantation, but defects may be generated at an earlier step, at the time of imprint resetting in the gametes. The issue of incomplete imprint resetting during gametogenesis is discussed further in the section about ICSI with immature sperm cells.
There are few reports on imprinting analysis in the context of possible effects of assisted reproduction. In one molecular study focusing on the imprint control region involved in PraderWilli and Angelman syndrome, no imprinting defects were detected in a group of nearly 100 children of which the majority were conceived after ICSI with ejaculated sperm (Manning et al., 2000). In another study, the expression of several imprinted genes was analysed in human oocytes and early embryos (Salpekar et al., 2001
). The expression was variable from embryo to embryo. A follow-up study on 73 infants reported a boy with BeckwithWiedemann syndrome (Olivennes et al., 2001
). Unfortunately this patient was lost to further follow-up and it could not be determined whether the syndrome was due to loss of imprinting.
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Interference of assisted reproduction with epigenetic reprogramming |
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The exact mechanism(s) by which culture media induce abnormal epigenetic modifications are not known. Media components could remove or interact with methyl groups on DNA or on histone tails. Another explanation may be that embryonic developmental timing is disturbed by the synthetic media and that this interferes with epigenetic reprogramming and gene expression.
The use of prolonged culture systems may deregulate epigenetic mechanisms to a further extent. In-vitro culture up to the blastocyst stage is used in some centres for selected patients and in PGD cycles. Follow-up studies comparing blastocyst transfers versus early cleavage-stage embryo transfers showed no differences in birthweight (Ménézo et al., 1999; Kausche et al., 2001
).
Immature sperm cells
Immature sperm cells are epigenetically quite different from mature sperm cells. A first issue of concern is the imprinting status. It is unclear whether imprint establishment has been completed in immature gametes. Most studies on imprint resetting have been performed in mice and almost no studies have addressed the problem in humans. In mice it was demonstrated that imprint resetting for the paternally expressed Snrpn, Igf-2, Peg1 and the maternally expressed Mash2, Igf-2r and H19 has been largely or entirely completed by the time the spermatid stage is reached (Shamanski et al., 1999). In humans, it has been shown that the methylation patterns of H19 and MEST/PEG1 are established during spermatogonial differentiation (Kerjean et al., 2000
). From the limited data available and assuming that the resetting mechanism is similar in mice and humans, it appears that imprint establishment is completed by the time the spermatid stage is reached.
Another difference in epigenetic organization between immature and mature sperm cells is that spermatid chromatin has not yet been so densely packed. The genome may therefore be less protected against events of delayed oocyte activation, such as the delayed inactivation of the metaphase-promoting factor that can cause aneuploidy in the embryo (Tesarik, 1998). Another point of concern relates to the finding that spermatogenesis-specific genes undergo late epigenetic reprogramming at the level of the epididymis (Ariel et al., 1994
). Genome-wide methylation patterns have been initiated at the prospermatogonia stage in the testes. However, genes involved in spermatogenesis are specifically demethylated for transcription and they become remethylated again in the epididymis. This implies that methylation patterns of spermatids and sperm derived from testes and epididymis will differ from the hypermethylated patterns found in ejaculated sperm. Follow-up studies of children born after ICSI with epididymal and testicular sperm have shown no additional risks as compared with children born after ICSI with ejaculated sperm (Bonduelle et al., 2002
). However, there is a case report of two major malformations out of four pregnancies obtained after ICSI with elongated spermatids (Zech et al., 2000
). Another study on a larger series did not detect an increased incidence of malformations (Sousa et al., 2000
).
Furthermore, the mature sperm genome is in a silent state, but the spermatid genome is transcriptionally active and the introduction of spermatid transcripts into the oocyte may interfere with epigenetic reprogramming during the preimplantation stage. A comparative expression study performed on preimplantation mouse embryos derived from oocytes injected with round spermatids or sperm showed that activation of the embryonic genome is much less efficient with spermatids. A higher rate of developmental arrest has indeed been found in embryos derived from round spermatid injection compared with embryos obtained after standard ICSI (Vicdan et al., 2001). The expression patterns of several genes were disturbed in early embryos (4-cell stage) derived from spermatids. The results also suggested that regulatory mechanisms were activated in the oocyte to repress inappropriate spermatid transcription (Ziyyat and Lefèvre, 2001
).
Ooplasmic transfer and cloning
Some small series of clinical cases of ooplasmic transfer have been reported (Barritt et al., 2001). Very little is known about the factors and organelles that are possibly transferred. The presence of donor mtDNA was observed in some of the babies, but it is not clear whether this had a therapeutic effect (Brenner et al., 2000
). The function of mitochondria is controlled by interactions between both nuclear and mitochondrial genes. The dual nature of this control sets up potential conflicts between the different genome parts. Ooplasmic transfer into human oocytes may induce conflicts between the multiple genome parts (nuclear DNA, recipient mtDNA, donor mtDNA) and lead to unpredictable outcomes (Cummins, 2001
).
Cloning involves nuclear transfer (NT) into an enucleated oocyte. The nucleus may come from an undifferentiated cell such as an embryonic stem cell (ES) or from a differentiated somatic cell. So far, the efficiency of the technique is low and most clones die before birth. The few clones that develop to term have often developmental abnormalities (LOS). Successful cloning requires that the epigenetic state of the donor nucleus is reprogrammed to that of the zygote (Rideout et al., 2001). The donor nuclei are not silent and their chromatin organization is likely to be different from that of the gametes. Moreover, there is a time limit to this reprogramming as it must be completed by the time that the embryonic genome is activated. The available data clearly demonstrate that reprogramming is incomplete in most NT embryos. A comparison of global methylation patterns in cloned and normal bovine preimplantation embryos showed disturbances in methylation dynamics in the cloned embryos (Bourchis et al., 2001
).
The epigenetic state of undifferentiated ES cells may resemble that of the early embryo better and require less reprogramming. On this basis, a higher cloning efficiency is expected for ES cell nuclear transfer. The percentage of ES cell NT blastocysts that develop to term is 1020-fold higher than with somatic nucleus transfer. However, the percentage of initial clones that reach blastocyst stage is much lower with ES cell NT. This is probably related to the cell stage as well as to the epigenetic instability of the ES cells. Donor nuclei should preferentially be in the G0/G1 or G2 phase, and many ES cells are in the S phase. Examination of imprinted gene expression in cloned mice and in donor ES cell lines from which they were derived showed that the expression of the imprinted H19 and Peg1 genes varied between ES cell subclones and even among mice derived from cells of the same ES cell subclone. These results indicate an unstable epigenetic state of ES cells (Humpherys et al., 2001).
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Epigenetic inheritance |
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Conclusions |
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It has been suggested that the accumulation of epigenetic defects during embryo culture may lead to aberrant phenotypes. Several lines of evidence support this hypothesis. The accumulation of severe epigenetic disturbances above a certain threshold may lead to early mortality. Embryos that develop to term may still have epigenetic defects and these may result in obvious aberrant phenotypes or in subtle changes in gene expression that can be easily overlooked.
Epigenetic disturbances induced by in-vitro culture may underlie the phenotype of lower birthweight. It is difficult on the basis of the available data to deduce whether there are other aberrations with an epigenetic origin in the population of children as a result of assisted reproduction. This is because the evaluation of epigenetic risks in the follow-up studies has been inadequate so far. Data on imprinting and methylation defects have often been collected in relatively small study groups. Given that the incidence of PraderWilli or Angelman syndrome is 1/15000, a large study population is needed to detect increased risks with sufficient power. Data on the incidence of imprinting disorders in IVF and ICSI children should therefore be collected preferentially from multiple centres. Another reason why epigenetic risks have been studied insufficiently is that the phenotypes associated with epigenetic defects may be difficult to recognize in short-term studies. Phenotypes due to imprinting defects may be obvious and show up early in life while clinical findings of neurodevelopmental delays and mental retardation may point in the direction of phenotypes caused by methylation defects. Other phenotypes may be subtler, such as the phenotypes of cancer predisposition that can be measured only later in life.
In conclusion, continuous follow-up studies are necessary in larger series of children in order to be able to assess long term risks linked to epigenetic disturbances after assisted reproduction. A complete safety evaluation may even require studies from a two-generation perspective.
Meanwhile, additional information on epigenetic reprogramming may be derived from molecular studies on preimplantation embryos. Preferentially and where possible, studies should be done on human embryos, as results from animal studies cannot always be extrapolated to humans.
Finally, the further elucidation of the mechanisms and factors involved in epigenetic regulation in fundamental research programmes will yield valuable information and identify elements that can be used to evaluate the safety of assisted reproductive technologies.
As the impact of epigenetic disturbances on later life of humans born as a result of assisted reproduction is not known, a definite answer about the safety of assisted reproduction cannot be given. Long-term clinical follow-up studies of the children born as a result of assisted reproduction as well as further molecular research are recommended.
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Acknowledgements |
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Notes |
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References |
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