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Susceptibility of Genomic Imprinting to ART
Mellissa MannChildren’s Health Research Institute
Department of Obstetrics & Gynecology, and Biochemistry
University of Western Ontario
35-70 million couples involuntarily infertile
1 in 6 people of reproductive age
ART
1-2% children born by ART
Infertility and Assisted Reproductive Technologies
Children conceived via ARTs are at increased risk
Intrauterine growth retardation Premature birth Low birth weight Possibly genetic disorders
ART-induced perturbations in the mouse
Reduced viability Intrauterine growth retardation Developmental abnormalities Deviation in behaviour
Developmental abnormalities in in vitro produced livestock
Large Offspring Syndrome
• Higher perinatal mortality
• Breathing difficulties• Reluctance to suckle• Skeletal anomalies• Large organs• Cerebellar dysplasia
•Increased prenatal loss•Large placentas•Large fetuses•Polyhydramnios•Parturition problems
Duke University Medical Center
Epigenetics: Heritable alterations in gene activity without a change in DNA sequence
Obese and YellowSkinny and Brown
Epigenetics
EstrogenDisruptors
HerbicidesPesticides
Nutrition DrugsVitamins
Epigenetics: Mediator of Environment, Development, and Disease
Pathology
Cancer
CardiovascularDisorders
GrowthDisorders
ImprintingDisorders
Lupus
NeurologicalDisorders
PediatricDisorders
ReproductiveDisorders
Paternal alleleMaternal allele
Genomic ImprintingThe unequal expression of the maternal and paternal alleles of
a gene
Imprinted or markedwith their gametic-origin
Maternal allele Paternal allele
Mouse/Human Imprinted Domains
Kcnq1
Kcnq1ot1
Cd81
Ascl2Th Ins2H19
Igf2Nap1l4
Slc22a1l
Tssc3 Cdkn1c
Mouse Distal 7/ Human 11p15.5
Beckwith-Wiedemann Syndrome Critical Region 1
IC IC
Prader-Willi Syndrome Angelman Syndrome
Frat3
Magel2NdnMouse Central 7/ Human 15q11-13
Ipw Ube3A Atp10c
Mkrn3
SnurfSnrpn snoRNA Genes
IC
IC Antisense
Paternal MaternalBiallelic Imprinting Center
IC Non-coding RNA
Methylated imprinted allele
Unmethylated imprinted allele
PaternalGenome
MaternalGenome
Methylation changes during mouse preimplantation development
Are genomic imprints maintained in preimplantation embryos after in vitro
culture?
CASTChromosome 7
B6(CAST7)
B6
B6
X CAST B6
B6(CAST7) mice for use in allelic analyses
F1
Peg3
Snrpn
H19
Kcnq1ot1
Trophectoderm
ICM
Blastocyst
Whitten’s
KSOMaa
2-cell
In vitro preimplantation culture regimes
PaternalMaternal
14%p=0.002
6%p=0.006
63%
Loss of H19 imprinted expression occurs in a subset of individual
blastocysts after culture in Whitten’s medium
0
20
40
60
80
100
0
20
40
60
80
100
0
20
40
60
80
100
Vivo
KSOMaa
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Whitten’s
10080
6040200
100
80604020
0
100
8060
4020
0
100806040200
100
8060
4020
0
Blastocyst
Loss of imprinting occurred during preimplantation development in culture, indicating that mechanisms that operate to maintain imprinting were disrupted.
What are the long-term effects of preimplantation development in
culture?
Cultured Blastocysts
Recipient Mothers
Postimplantation embryos recovered at 9.5 days of pregnancy after preimplantation culture in
Whitten’s medium.
8%3% 3% 5%
81%
10%
30%
11% 14%
32%
0%
20%
40%
60%
80%
100%
Empty SeverelyAbnormal
Abnormal Delayed Normal
Controls Whitten's
0
20
40
60
80
100
0
20
40
60
80
100
0
20
40
60
80
100
0
20
40
60
80
100
Embryo Placenta
H19
Ascl2
Snrpn
Peg3
1008060
40
200
100
8060
40
20
0
100
80
6040
200
100
80
60
4020
0
Paternal
Maternal
Whitten’sKSOMaa Whitten’sKSOMaaVivoVivo
Loss of imprinted expression occurs primarily in day 9.5 placentasafter preimplantation culture
Loss of imprinted expression occurs primarily in day 9.5 placentas after preimplantation culture
100 10087
100
7592
825
13
0
20
40
60
80
100
100 100 92 10088
55
12
45
8
0
20
40
60
80
100
Embryo Placenta
H19
Ascl2
Snrpn
Peg3
WK WKVV
Paternal
Maternal
Biallelic
100
7592
825
0
20
40
60
80
100
100 10083
100
6373
372716
0
20
40
60
80
100
Perturbations in imprinting persist long after embryos have been removed from culture.
Loss of imprinting occurs more frequently in extraembryonic than embryonic lineages
Placenta
Embryo
Day 9.5
Trophectoderm
ICM
Blastocyst
Loss of impr
inting
Maintains/restores
imprinting
Preimplantation
Culture Proximity to culture?
ICM vs TE?Less redundancy?
Sporadic imprinting defects may arise during ART procedures
Beckwith-Wiedemann Syndrome
X XOR OR
Biparental origin
ART children diagnosed with imprinting disorders
Loss ofmaternalmethylation
Loss of maternalmethylation
Normal
X
IM
OR OR
Angelman Syndrome
X X
Biparental origin
PGC Oocytematuration
HumansSuperovulation
+/- GnRH treatment
Oocyte retrieval
IVF/ICSI
In vitro culture
Superovulation In vitro culture
Mice
Assisted Reproductive Technologies
PGC Oocytematuration
Superovulation In vitro cultureMice
Future StudiesAssisted Reproductive Technologies
1
1. Do imprinting defects result from superovulation?
2,3
2. When is imprinting lost during preimplantation development in culture?
3. Does loss of imprinting occur in mouse embryos cultured in media used in human assisted reproduction?
4,5
4. Is loss of imprinting tissue-specific?
5. What are the long-term affects of preimplantation culture on genomic imprinting and development?
1. Determine whether superovulation contributes to loss of imprinting
Spontaneous vs Induced Ovulation
Imprint Acquisition
Extraovarian Sensitivity
Preantral Early antral Preovulatory
2. Determine when imprint maintenance is lost during preimplantation development in
culture.
Examine imprinting of H19, Snrpn, Kcnq1ot1, Peg3
In vitro culture
3. Does loss of imprinting occur in mouse embryos cultured in media used in human
assisted reproduction? Trophectoderm
ICM
Blastocyst
Global
G1.2/G2.22-cell
HTF
P-1/Bl +SSS
One Step
Two Step
H19Snrpn Peg3Kncq1ot1
H19SnrpnPeg3Kncq1ot1
Day 5.5 Day 6.5 Day 7.5 Day 8.5
Right horn
4. Determine how disruptions lead to the selective loss of imprinting in the
placenta
In VivoderivedWhitten’s
Left horn Right horn
Examine imprinting of H19, Snrpn, Kcnq1ot1, Peg3
Ultrasound biomicroscopyDay 9.5, Day 13.5, Day 17.5
In VivoderivedWhitten’s
Left horn Right horn
5. Determine the long-term affects of preimplantation culture on genomic
imprinting and development
U
D
E
P
U
D
U
D
EP
UD
NTNT
Ultrasound biomicroscopy at Day 9.5-10.5 of gestation Growth and Viability
Crown-rumplength
Variable embryonic growth Calcium hydroxyapatite deposits
Resorption
Beckwith-Wiedemann Syndrome
Macrosomia
Visceromegaly (liver, heart)
Macroglossia ( tongue)
Abdominal wall defects
Polydactyly (limb)
Cardiac defects (heart)
Adrenal defects
Lens defects (eye)
Vertebral defects
Hemihypertrophy
Polyhydramnios
Placentomegaly/Placental hydrops
Ultrasound biomicroscopy at Day 13.5 of gestation
Limb
Heart Eye
Tongue
Liver
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Michael GoldingAnne PinLiyue ZhangSarah LaloneJulia FosterBrenna MarketLauren Magri
Michelle GabrielMorgan McWilliam
Acknowledgements
University of Pennsylvania
Marisa BartolomeiRichard Schultz