DNA Methylation in Preimplantation Embryos

Mellissa Mann, PhDMagee-Womens Research Institute

Department of Obstetrics & Gynecology, and Reproductive SciencesUniversity of Pittsburgh School of Medicine

Passive loss

passive loss H3K9me3

~70 million couples involuntarily infertile

1 in 6 people of reproductive age

1-4% children born by ART

Infertility and Assisted Reproductive Technologies

AngelmanSyndrome

Beckwith-WeidemannSyndrome

ART-Associated Imprinting DisordersSilver-Russell

Syndrome

General population: <5% Imprinting DNA methylation errorsGeneral population: 55% General population: <5%

ART population: 45%ART population: 90% ART population: 92%

BWS Normal BWS BWS BWS BWS/Normal

BWS Normal

Error arise between the2-cell and blastocyst stages

Imprinting Disorders among Twins

McNamara, AJOG 2016

Genomic ImprintingPredominant expression of a gene from the maternal or the paternal copy

Imprinted or marked with their gametic-origin

Paternal allele Maternal alleleMaternal allelePaternal allele

Methylated CpGUnmethylated CpG

DNA Methylation Programming and ARTs

PrimordialGerm Cell

Paternal specific imprint Maternal specific imprintPaternal genome Maternal genome

MaintenanceAcquisitionErasure

In Vitro Culture

Ovarian Freezing

In Vitro Oocyte Growth/MaturationOvarian Stimulation Embryo Freezing

DN

A M

ethy

latio

n

Embryos are predisposed to DNA methylation errors because ARTs disrupt crucial DNA methylation programming in early embryos.

Hypothesis

B6(CAST7) Female

Imprinted Regions

CASTChromosome 7

X

Snrpn

H19

Kcnq1ot1

Silver-Russell Syndrome

Beckwith-Wiedemann Syndrome

Angelman Syndrome

B6

B6 Male

B6(CAST7) Female

CAST B6

Allelic Analyses with B6(CAST7) X B6 Mice

F1

CASTChromosome 7

X

Bisulfite Mutagenesis

Genomic DNA

Sequencing98%DNA strand

Methylated CpGUnmethylated CpGB6

B6 Male

96% 11%Paternal Maternal

Superovulation

Determine whether superovulation result in changes in imprinted DNA methylation in oocytes.

Single MII Oocytes

Determine whether superovulation results in loss of imprinted methylation in MII oocytes

1. Spontaneous Ovulation2. Superovulation 6.25 IU 3. Superovulation 10 IU

B6(CAST7) X B6 Female

SnrpnKcnq1ot1

Peg3H19

imprinted methylation analysis

eCG hCG

Superovulation

0h 44-48h

SpontaneousOvulation

Imprinted Methylation Acquisition Not Affected by Superovulation

Snr

pnK

cnq1

ot1

Peg

3H

19

MaintenanceM

ethy

latio

n

Fert

iliza

tion

Superovulation Does Not Disrupts Imprinted Methylation Acquisition

PrimordialGerm Cell

Acquisition

Superovulation

Superovulation and Embryo Culture

Determine whether superovulation, embryo culture or both result in changes in imprinted DNA methylation in blastocysts.

1. Spontaneous Ovulation2. Superovulation3. Spontaneous Ovulation plus Embryo Culture4. Superovulation plus Embryo Culture

In Vivo Development

SingleBlastocysts

Superovulation and/or Embryo Culture Paradigm

eCG hCG

Superovulation (10 IU)

0h 44-48h

B6(CAST7) Female

Nature mating with B6 males

X

SnrpnKcnq1ot1

H19imprinted DNA

methylation analysisWhitten’s

Embryo Culture

SpontaneousOvulation

% M

ethy

latio

n

ab

b,c

c

c ab

b,cc

c a

bb

bb

Snrpn Kcnq1ot1 H19

Superovulation and/or Embryo Culture and DNA Methylation Programming

Met

hyla

tion

PrimordialGerm Cell

Fert

iliza

tion

PrimordialGerm Cell

In Vitro CultureSuperovulation

Maintenance

Superovulation and/or embryo culture resulted imprinted DNA methylation errors in blastocysts

20-35 36-50 50-60 70-80 >8010-19Birth-10 years

EducationJob/career, PartnerMatureFinancially stableLife experience

Child Bearing

Optimal fertility

Last 3 Decades

Maternal Age

Virgin Young Middle Advanced0

5

10

15

20

25

30

35

40

45

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Hum

an A

ge (y

ears

)

Mouse Age (months)

Human and Mouse Maternal Age Comparison

Dutta and Sengupta, Life Sciences 152: 244-248, 2016.Fernando et al., Nature Neuroscience 19:1197-1202, 2016.

Blastocysts

Natural mating with B6 males

SnrpnKcnq1ot1

H19 imprinted methylation

analysis

B6(CAST7) Female

Retired Breeders

2 monthsVirgin

4 months

8 months

12 months

Maternal Age Paradigm

% S

nrpn

met

hyla

tion

Virgin Young Middle Advanced

Mouse Age (months)

Snrpn Imprinted Methylation Not Disrupted in Blastocysts by Maternal Age

% K

cnq1

ot1

met

hyla

tion

Virgin Young Middle Advanced

Kcnq1ot1 Imprinted Methylation Not Disrupted in Blastocysts by Maternal Age

Mouse Age (months)

% H

19 m

ethy

latio

n

Virgin Young Middle AdvancedMouse Age (months)

H19 Imprinted Methylation Not Disrupted in Blastocysts by Maternal Age

Met

hyla

tion

Fert

iliza

tion

Imprinted DNA Methylation Not Disrupted by Advanced Maternal Age

PrimordialGerm Cell

Maintenance

ADVANCED MATERNAL AGE

Advanced Maternal Age and ARTs

Determine whether advanced maternal age in combination with ARTs contributes to imprinted methylation loss in blastocysts.

1. Spontaneous Ovulation <2 months2. Spontaneous Ovulation >10 months3. Superovulation plus Embryo Culture <2 months4. Superovulation plus Embryo Culture >10 months

In Vivo Development

SingleBlastocysts

ARTs and Maternal Age Paradigm

eCG hCG

Superovulation (10 IU)

0h 44-48h

B6(CAST7) Female

Nature mating with B6 males

X

SnrpnKcnq1ot1

H19imprinted methylation

analysisWhitten’s

Embryo Culture

SpontaneousOvulation

1.5-2 monthsVirgin

10-12 monthsRetired Breeder

Advanced maternal age did not increase imprinted methylation errors with ARTs

Snrpn Kcnq1ot1 H19

% M

ethy

latio

n

aa

b bb

a a

bb

aa

bb

MaintenanceM

ethy

latio

n

Fert

iliza

tion

Imprinted DNA Methylation Errors by ARTs not by Advanced Maternal Age

PrimordialGerm Cell

Superovulation In Vitro Culture

Advanced Maternal Age

PrimordialGerm Cell

Fert

iliza

tionPrimordial

Germ Cell

Met

hyla

tion

DNMT1TRIM28

ZFP57DPPA3

2. When?

4. How?

3. What developmental parameter?

TET3

ARTs Disrupt Imprinted DNA Methylation

In Vitro CultureSuperovulation

Maintenance

1. MEF?

Mann LabAudrey KindsfatherBrenna MarketMichelle DenommeCarlee WhiteCat PressimoreWill MacDonaldLiyue Zhang

Acknowledgements

UndergraduatesMegan CzekalskMargaret ErismanTiffany RicketsSadie SignorellaAlisha BesterJosef IanniMorgan McWilliamsRachelle Pettapiece-PhillipsMichelle GabrielleLiana KaufmanNicole Perrier

Ben ShafferFelicia WranitzAshley FoulkrodSarah LaloneLauren MagriSaqib SachaniMichael GoldingAnne Pin

High SchoolMalaika Miles-RossouwFatima Ba’abbadYunZhi ChenDorothy Kessler

Community CollegeTaylor Smith