Transcript Epigenetic effects of ART

Assisted reproduction treatment
and epigenetic inheritance
Part II
Human Reproduction Update, Vol.18, No.2 pp. 171-197
Presented by Hsing Chun Tsai
2012.05.29
Outlines
 Epigenetic inheritance and germline reprogramming
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Mitotic inheritance of epigenetic marks
Reprogramming the Genome towards Totipotency
Transgenerational epigenetic inheritance
Stress, Hormone, and Nutrition Induced Transgenerational
Epigenetic Variation
 Epigenetic effects of ART
– Studies on mice designed to evaluate epigenetic and physiological
aspects of ART
– Epigenetic aspects of ART
 Conclusions
Epigenetically crucial phase
 in order to prepare the cells for pluri- and toti-potency and
down-regulate the inheritance of epigenetic information
between generations
 between generations, the germ line is subjected to two
distinct reprogramming events …
– Primordial germ cells (PGCs)
– Preimplantation embryo ------- ART
The questions ??
1. If the conditions during gametogenesis and
in vitro phases intrinsic to ART could elicit
epigenetic effects ?
2. If the assumed epigenetic effects of ART can
be transmitted to the next generation ?
Outlines
 Epigenetic inheritance and germline reprogramming
–
–
–
–
Mitotic inheritance of epigenetic marks
Reprogramming the Genome towards Totipotency
Transgenerational epigenetic inheritance
Stress, Hormone, and Nutrition Induced Transgenerational
Epigenetic Variation
 Epigenetic effects of ART
– Studies on mice designed to evaluate epigenetic and physiological
aspects of ART
– Epigenetic aspects of ART
 Conclusions
Epigenetic effects on ART
- Studies on mice designed to evaluate epigenetic
and physiological aspects of ART
- Epigenetic aspects of ART
Mice model
 Epigenesis and physiological, behavioral readouts after ART
in the mouse
– The effects of ovulation induction and preimplantation
embryo culture on maintenance of imprinting  up to
mid-gestation
– Imprinting status followed using methylation status of
DMR (differentially methylated region)  paternally
imprinted H19
 All details of regulation by CpG methylation of imprinted
gene expression are not yet available.
 ovulation induction and in vitro embryo culture ↔
maintenance of DMR methylation
 Physiological status of maternal tract after a ovulation
induction procedure adds to deregulation of imprinting
 From experiments reported:
– Oocyte inbred genotype could already confer cell biological stress
that exacerbates after hormonal priming and during in vitro culture
 The placenta is much more vulnerable than the embryo ↔
great significance of imprinting regulation for placental gene
expression
– Might be related to the overall lower level of 5methylCpG
– In the mouse at least, effects on placental imprinted gene expression
of H19 (fine regulator of prenatal growth) translate into deregulation
of imprinted gene network
 Epigenetic effects also influence non-imprinting gene
expression
– Avy and Axinfu genetic system: in vitro culture  hypomethylation of
IAP cryptic promoters
– in the mouse, ART  physiological parameters at adult age, such as
insulin sensitivity and blood pressure; adult behavior
Epigenetic aspects of ART in human
imprinting disorders in children born after ART
Beckwith-Wiedemann syndrome
 irrespective of cause of
3.1~16.1
infertility
 after IVF and ICSI
 ET or FET on Day 2,3,5
 different stimulation
after IUI + COS or COS alone  BWS reported
 Not ART practice but subfertility is at the heart of this increase in BWS
Clinical presentation
 ↑ risk of childhood cnacer
 macroglossia
 macrosomia (birth weight and length > 90th percentile)
 midline abdominal wall defects (omphalocele/exomphalos,
umbilical hernia, diastasis recti)
 ear creases or ear pits
 neonatal hypoglycemia
Link between ART and epigenetic regulation
in BWS
 in general population BWS: DMR CpG methylation error  50-60%
 in ART-BWS:
– almost all cases related to hypomethylation of maternal KCNQ1OT1 DMR
– more often other maternal methylated regions are hypomethylated
Angelman syndrome (AS)
 neuro-genetic disorder: characterized by intellectual and
developmental disability, sleep disturbance, seizures, jerky movements
(especially hand-flapping), frequent laughter or smiling, and usually a
happy demeanor
 Caused by a shortage of maternal UBE3A expression in the
SNRPN imprinting cluster
7 AS cases from ovulation
induction and/or IUI
Silver-Russell Syndrome (SRS)
 Dwarfism
 5 cases published in children born after IVF or ICSI
– 1/5  hypermethylation of paternal MEST DMR
 Mechanism:
– ~ 44% of SRS caused by H19 DMR hypomethylation
– 5-10% by maternal uniparental disomy of Chr 7  So far, no
imprinted candidate gene on Chr 7 could be identified
 # of cases involving ART  too small
Retinoblastoma (RB)
Prader-Willi syndrome (PWS)
 (epi)genetic disorder involving imprinting
 Mechanism:
– most are (point)mutation or a deletion, ex: 3 reported
PWS-ART cases and 2/7 RB-ART cases
– other 5 cases had no gene defect found and methylation
not analyzed
Epidemiological data
 IVF children in Sweden (n = 31,850)  1 BWS, 2 SRS and 4
PWS
 Danish National Cohort study (n = 6,052)  none with a
genomic imprinting disease
 French cohort IVF children (n = 15,162)  6 BWS
– cf. spontaneous BWS incidence  1/13,700
 tendency towards ↑ risk after ART
Effects of ART on epigenetic parameters in
human gametes and embryos
oocytes
 Spontaneous oogenesis
 in human, studies on imprinting directed epigenetic
reprogramming during oogenesis are vary limited for ethical
reasons.
– Only 1 study used immature oocyte from growing follicles in nonstimulated fertile patients after LSC (Sato et al., 2007)
same as in mice
different from mice  the imprint is
removed in E13.5 PGCs
 Ovulation induction **
 The analysis of a possible effect of hormonal priming on imprinting could
be confounded with maternal age and/or general suboptimal oogenesis.
 Maternal DMR methylation level
– the cause of subfertility was male factor or tuba obstruction 
genuine ovulation induction effect
– low incidence of AS and PWS after ART  not be expected
– ~10% MII oocytes can lead to BWS but not in agreement with true
incidence
– PCOS & methylation  no difference
 Paternal DMR demethylation
 In vitro maturation
 IVM of oocytes for IVF  avoiding exogenous gonadotropin,
especially for patients at risk of OHSS and/or PCOS
 Small and medium-sized antral follicles aspirated  culture for
24-48h  fertilization
 at antral follicle stage, most DMR CpG methylation has been
established although not completely so
 IVM could interfere with imprint establishment or
maintenance
– maturation time (28h in vitro when compared with ~36h in vivo)
might be too short to finish methylation process
– H19 DMR is more vulnerable to the environment
• methylation error: GV  MII >> MI  MII
Spermatozoa
 in human male germline: both maternal MEST and paternal
H19 DMRs are completely erased in fetal prospermatogonia
– For maternal MEST DMR  remains unmethylated
during spermatogenesis
– For paternal H19 DMR  the imprint established during
adult spermatogonial stage or at least before the
spermatocytes enter meiosis I and maintained thereafter
(resembling mice reprogramming)
 in mature spermatozoa: paternally imprinted DMR
completely methylated while maternally imprinted
unmethylated
 effect of male subfertility on the epigenetic status of
DMRs in spermatozoa
 ART is unlikely to affect methylation pattern in spermatozoa
including paternal imprints (∵established before manipulation)
 disturbed spermatogenesis is associated with incorrect
imprinting
– in oligozoospermia: hypermethylation of several maternally imprinted
DMRs or hypomethylation of H19 and IG-DMR ↑, esp. in <10x106/ml
– Boissonnas et al. (2010): H19 DMR in teratozoospermic (TZ) and oligoastheno-teratozoospermic (OAT) pts
• TZ: 2/16 CpGs significantly hypomethylated
• OAT: methylation drastically reduced for all CpGs, significant in
subgroup with conc. < 10 x 106 /ml
 Sperm concentration is positively correlated with H19
methylation and negatively correlated with MEST methylation
 Alteration of protamine 1 to protamine 2 ratio (should be ~ 1)
generally denotes affected spermatogenesis (cause or
consequence?)  methylation defects
DMR methylation defects are associated with poor spermatogenesis!
 Azoospermia, sperm motility < 40% or normal morphology <
5%  related to ↑ MEST methylation
 The methylation of non-imprinted genes and repetitive
sequence was also affected.
 effects of spermatozoa methylation defects on IVF
outcome
 To what extent DMR methylation defects in both degree and
prevalence before germline transmission ??
1. Kobayashi et al. (2009): compare the methylation defect found
in trophoblastic villi from ART-miscarriages between 6-9 wks
with imprints in the semen from the father
– 7/17 ART pregnancies with placental H19 methylation
defect, also found in spermatozoa  transfer from father!!
2. Marques et al. (2010): in a patient with hypospermatogenesis
and almost complete hypomethylation of H19 DMR 
embryos obtained after ICSI all showed arrest
3. Salpekar et al. (2001): in the human, H19 is not expressed up
to blastocyst stage  a common paternal factor might be at
stake !!
 no analysis, no formal proof of paternal inheritance of H19
DMR hypomethylation
4. Boisnnas et al. (2010): in OAT with partial hypomethylation
of H19, the fertilization rate after ICSI was reduced.
– Developmental parameters (embryo quality, implantation rate, GA,
BW)  similar to normally methylated controls
5. Kobayashi et al. (2007): spermatozoa with both maternal /
paternal methylation imprinting error  normal pregnancy
with normal methylation
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both parents had normal methylation profile  de novo
methylation  maybe due to oligozoospermia of father
The preimplantation embryo
 Loss of 5methylCpG immunostaining in human embryos
after fertilization resembles in mammalian embryos.
– Paternal active demethylation
– Maternal passive demethylation
 abnormal chromatin organization correlated in arrested IVF
embryos  proper chromatin organization for early
development
 transcripts of several imprinted genes like SNRPN, MEST,
UBE3A and IGF2 already present at the preimplantation
embryonic stages (not H19)
 primary imprints laid down during oogenesis and
spermatogenesis are resistant to active and passive
demethylation during cleavage divisions
Effects of IVF and embryo culture
 Effects of IVF or subsequent development in culture medium
alone are difficult to investigate in the human
– inevitably connected with each other
– no in vivo comparison
 in vitro condition could affect maintenance of imprinting:
– At Day 3, 19% of human surplus embryos of low-quality showed
hypomethylation of H19  paternal transmission unlikely as
corresponding sperm samples were normal
– hypomethylation  growth arrest?? Or growth arrest (induced by in
vitro condition)  loss of methylation??
Mode of IVF
 ICSI or IVF elevates the risk for epigenetic abnormalities 
no convincing evidence
– Nuclear structure and methylation levels seen in arrested
embryos and fully grown blastocysts did not differ
between IVF and ICSI
 ↑ risk of imprinting disorders applies to pregnancies
originating from both IVF and ICSI
Effects of culture medium
 IVF children derived from embryos cultured in two different
media showed a significant difference in birthweight of
almost 250 gm
 Similar to animal study but the effect in human seems less
severe and a causative epigenetic variable not be found!!
Epigenetic effects of IVF on offspring other than
imprinting diseases
 induced epigenetic variants (do not have clear phenotypical
effects) might be transmitted to the offspring
– all part of a dizygotic twin with co-twin showing normal methylation
– methylation level:
• 41.5% in naturally conceived children
• ~14% in these 3 probands without clinical symptoms
• 1% in BWS patients
(significant)t
hypomethylation of H19 (trend)
Zhang et al.
(2010)
IVF
3
n=3
placenta
global gene expression
26 genes differentially expressed,
none was imprinted
 methylation effect in ART children
– methylation of the single investigated CpG within the
analyzed tissue was never completely (100%) methylated
or demethylated (0%)
methylation defects are not transmitted from the oocyte or
sperm cell
– The type of analyses and the presentation of the results do
not allow us to accurately specify mosaicism.
Perinatal, congenital and physiological outcome
of IVF children; an epigenetic response ?
 For perinatal outcome, 3 meta-analyses (n= 5,361 ~ 31,000
singleton)  ↑ risk in IVF group for …
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Very preterm birth (RR = 3.0 ~3.3)
Preterm birth (RR = 1.9~2.0)
Very low birth weight (RR = 2.7~3.8)
Low birth weight (RR = 1.4~1.8)
Small for gestational age (RR = 1.4~1.6)
Cesarean section (RR = 1.5~2.1)
Admittance to NICU (RR = 1.3~1.6)
Mortality (RR = 1.7~2.4)
 For congenital malformations … more controversy, mainly
due to relative small sample sizes!!
 Swedish group analyzed 2 cohorts (n > 15,000 singleton IVF children)
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Esophageal atresia (OR = 5.2)
Urogenital defects (OR = 2.3)
Limb reduction (OR = 1.7~2.0)
Neural tube defects (OR = 2.9~4.2)
Cardiovascular malformation (OR = 1.3~1.7)
Syndromes associated with imprinting defects like PWS (RR = 4.0)
 For cancer …RR = 1.4 (Kallen et al., cohort of 27,000 IVF children)
 For physical development …
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sBP, dBP
Peripheral skinfold thickness
Fasting glucose level
Weight/height gain
DHEAS and LH level in puberty girls
↑ in IVF group (after
adjustment for potential
confounders)
 A younger IVF group of ~6 y/o  IVF children were taller
and had a slightly more favorable lipid profile
Epigenetic adaptive response to the (preimplantation) environment!
Conclusions
 in the mouse:
– Effect of ART: from imprinting maintenance to physiological
homeostasis to behavior found
– Placenta  much more vulnerable to the influence of ART on
imprinting compared with the embryo
– OAT model, effects of ART at advanced maternal age, effect of in
vitro culture without ovulation induction  lacking
 The effect of ovulation induction on maintenance of imprinting:
1.
2.
On maintenance of imprinting at recruitment of an antral follicle
• Not much work in human; none in mouse
• Mice maturation of multiple oocytes in one cycle is natural.
• imprinting erasure timing: human (later, during 1st meiotic prophase)
 mouse (before 1st meiotic prophase)
Maternal early embryonic cellular effect on maintenance of the imprint
after gamete fusion
3. Expressed via maternal tractus
• after transferring the embryo to a non-stimulated uterus (changing
environment to normal)  ovulation induction effect on imprinting ↓
• at mid-gestation, in vitro culture aggravated the impact on ovulation
induction
 Superovulated in vivo matured human MII oocytes
– # of oocytes not methylated at KCNQ1OT1 much higher
than the prevalence of BWS after ART
Great majority of embryos derived from these are not
viable!!
Non-arrested embryos showed normal methylation!!
 Whether mild ovarian stimulation would lead to a reduction
in BWS cases ?
 in human:
– poor spermatogenesis  methylation abnormality 
sperm as a potential vehicle for transmitting paternal
methylation abnormality (small chance)
– type of IVF  no influence
– in vitro culture  methylation defects (ex: in arrested embryo,
H19 hypomethylation reported without corresponding sperm defect) (no in
vivo comparison)
– different methylation in the in vitro group:
• UCB: most CpG hypermethylated
• Placenta: most CpG hypomethylated
 An affected CpG methylation maintenance can be without
any effect but might bring IVF progeny closer to a threshold
– making them more vulnerable to physiological effects at adolescence
or late-onset diseases, like CV diseases, DM, cancer …
– maternal imprinting gene KLF14  DM type II and adipocyte-related
metabolic disease risk
 More evidence for epigenetic effects of ART in the mouse
than in man
– further research on poor spermatogenesis and oocytes from older
women
Take home massage
 ART can induce epigenetic variation that might be
transmitted to the next generation.
 Ovulation induction and in vitro culture
 DMR methylation defects are associated with poor
spermatogenesis !!
 A multigenerational study of systematic ART on
epigenetic parameter is lacking.
Thank You !!
Whether children born after ART might have
subclinical form of BWS?
 Bowdin et al. (2007): 1524 probands for clinical features
linked to BWS
– 4 children had at least one of the signs  one diagnosed as BWS
– none of other 3 children showed loss of methylation at KCNQ1OT1
– No milder forms of BWS