Epigenetics and ART

Epigenetics

• ‘Epigenetics’ refers to a process that regulates gene activity without affecting the genetic (DNA) code and is heritable through cell division.

• DNA-Gene- Genetic code- aminoacid-protein(gene expression)

• Functional asymmetry of mammalian parental genomes.

• Non viability of uniparental embryo ‐development.

• 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.

• Germ cell development and early embryogenesis are crucial windows in the erasure, acquisition and maintenance of genomic imprints.

• 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 IVF/ICSI and embryo culture.

• Angelman syndrome (AS) • Beckwith–Wiedemann syndrome (BWS) have

been documented in children conceived via IVF and/or ICSI

Genomic Imprinting

• 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 .

• 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 led to the development of the most widely recognized theory of imprinting, the ‘parental conflict’ hypothesis.

• 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.

• Genomic imprinting is thought to be restricted to mammals.

• 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.

• In general, the two parental alleles have different levels of DNA methylation.

• DNA methylation is a heritable yet reversible epigenetic mark that can be stably propagated after DNA replication and influence gene expression

• Evidence suggests suggest co operation ‐between DNA methylation, histone modifications, and overall chromatin state in the regulation of imprinted gene allele‐specific expression.

• Imprinted genes share common characteristic features such as genomic clustering.

• 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)

Imprint dynamics and timing during gametogenesis and early embryogenesis

• Paternal imprints are complete by the haploid phase of spermatogenesis.

• In the female germ line, imprint acquisition occurs in the postnatal growth phase while oocytes are arrested at the diplotene stage of prophase I

• 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.‐

Mechanisms of genomic imprinting

• Enzyme involved in gene imprinting- DNA methyl transferase.

• Erasure of imprints• 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.

• Acquisition of imprints• DNMT in male• Other mech in female• Maintenance of imprints• . 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

Errors in erasure, acquisition or maintenance of 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.

• Evidence suggests that imprinting defects may occur sporadically in normal embryos and that the processes of imprint erasure, establishment and maintenance are vulnerable to errors.

• 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

• Fetal development• IGF2• Placental function• Imprinted genes play essential roles in

controlling the placental supply of maternal nutrients to the fetus, by regulating the growth of the placenta. For eg. Tssc3

• 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• Angelman syndrome• Prader willi Syndrome Chromosome 15 long

arm.• Beckwith Weidman syndrome(overgrowth

disorder+ childhood cancer)• Numner of imprinted genes are expressed in

extra embryonic tissues and the nervous system.

Imprinting defects in uniparental and molar pregnancies

• Spontaneous uniparental development has been well documented in humans

• Ovarian teratomas are the product of gynogenetic development derived from the parthenogenetic activation of an unfertilized oocyte within the ovary.

• No evidence of extraembryonically derived tissues.

• Human androgenetic conceptuses exhibit hyperplasia of extraembryonic trophoblastic tissues with a lack of embryo development- Complete mole

Evidence of imprinting defects associated with assisted reproductive technology procedures

• Animal studies• lambs and calves , overgrowth abnormalities,

‘large offspring syndrome’.• Human studies• AS, BWS- IVF/ICSI children.

• 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.

• To address underlying mechanisms, one would like to know whether specific techniques used in human ART 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.

• 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

• Freezing of mature sperm- Chromatin damage.

• 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

• Possible effects of assisted reproductive technology on female germ cells

• The two important processes associated with imprinting that occur during oocyte growth are the acquisition of maternal methylation imprints and the protection of imprinted genes that are normally unmethylated in the female germ line (e.g. H19) from becoming methylated

• 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

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.

• Possible adverse effects 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

• 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 ‐now permit imprinting abnormalities to be assessed in single blastocysts.

• These advances may allow critical human studies to be performed using single embryos.

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