This document discusses epigenetics and genomic imprinting in the context of assisted reproductive technologies (ART). It provides background on epigenetics and how imprinting marks on genes from sperm and egg are established and maintained. Errors in imprinting establishment or maintenance can cause developmental disorders. The document reviews evidence that ART procedures like in vitro fertilization could potentially disturb imprinting by affecting gametes, early embryo development, or the maintenance of imprints. More research is needed using animal models and new techniques to better understand any risks and their underlying mechanisms.

1. Epigenetics and ART

2. 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- aminoacidprotein(gene expression)

3. • 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.

4. • 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.

5. • 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.

6. • Angelman syndrome (AS)
• Beckwith–Wiedemann syndrome (BWS) have
been documented in children conceived via
IVF and/or ICSI

7. 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 .

8. • 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.

9. • 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.

10. • 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.

11. • 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.

12. • 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.

13. • 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

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

15. • 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)

16. 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

17. • The overall methylation status of
non‐imprinted genes reaches a minimum at
the blastocyst stage of development after
which de novo methylation begins.

18. • 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.

19. 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.

20. •
•
•
•
•
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

21. 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.

22. • 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.

23. Roles of imprinted genes in fetal development,
placental function and human disease
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•
•
•
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

24. • 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).

25. • 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.

26. 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.

27. • Human androgenetic conceptuses exhibit
hyperplasia of extraembryonic trophoblastic
tissues with a lack of embryo developmentComplete mole

28. 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.

29. • 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.

30. • 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.

31. • 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

32. • 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

33. • 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

34. • 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

35. 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.

36. • 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

37. • 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.

38. 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

39. • 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.

40. • 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.

41. • THANK YOU!