DNA methylation and gene expression
differences in children conceived
in vitro vs. in vivo
Carmen Sapienza
Fels Institute for Cancer Research and Molecular Biology
Temple University School of Medicine
Philadelphia, PA 19140
ART vs. in vivo conception:
•
•
•
•
Neural tube defects - 3X (Ericson and Kallen, 2001)
“Major” birth defects - 2X (Hansen et al., 2002)
Respiratory disease - 3.5X (Koivurova et al., 2003)
Low birth weight - 2.6X (Schieve et al., 2002)
ART and imprinting defects:
• Beckwith-Wiedemann syndrome - 5.4 - 8.1X
• Angelman syndrome - ?X (3/3 imp.def.)
• Animal models demonstrate “LOI”, “LOS”
Genes
Effectors of
cellular stress
responses
Environment
Changes in Epigenotype
Long-Term, Somatically Heritable
Changes in Gene Expression
“Genes + Environment” Phenotype
Do Children Conceived in vitro
Differ from Children Conceived
in vivo in “Epigenotype” ?
Experimental Designs for Small Effects
• Compare many individuals at few “likely suspect”
loci
• Compare few individuals at many loci
– Placenta and cord blood DNA - 10 ART children versus
12 Control children
– Methylation at 1,536 CpGs in promoters of more than
700 genes (highly biased selection)
• 205 CpGs in DMRs of all genes known or suspected to be
imprinted/expressed from only one allele
– Use DNA methylation differences to select candidate
genes for analysis of mRNA levels in a larger number
of individuals
“GoldenGate” assay (Illumina, Inc.): fraction of methyl C at a CpG site
hybridization/synthesis/ligation/PCR in bisulfite treated/untreated DNA
Data Filtering for Candidate Genes:
Reduction of false positives:
require 2 CpGs differ in the same gene
Reduction of false negatives:
reduce stringency CpGs differ at P<0.08
Result: 78 genes “different” in cord blood
40 genes “different” in placenta
Genes
Effectors of
cellular stress
responses
Environment
Changes in Epigenotype
Long-Term, Somatically Heritable
Changes in Gene Expression
“Genes + Environment” Phenotype
Absolute range of difference small…
Absolute range of difference moderate…
Absolute range of difference large…
PYY in Placenta
0.6
ART
Control
cg22028686
0.5
0.4
0.3
0.2
0.1
0.0
0.0
0.2
0.4
0.6
cg03384000
0.8
1.0
Genes
Effectors of
cellular stress
responses
Environment
Changes in Epigenotype
Long-Term, Somatically Heritable
Changes in Gene Expression
“Genes + Environment” Phenotype
Assume cis-acting epigenetic differences
linked to steady-state mRNA levels
Do Children Conceived in vitro
Differ in “Epigenotype” from
Children Conceived in vivo?
As a group, yes. But most
individuals are within the
“normal” range.
Associated Phenotype?
CEBPA, MEST, NNAT and SERPINF1 all
involved in adipocyte
differentiation/development or insulin
signaling
Link between low birthweight – later
obesity/hypertension type 2 diabetes?
Conclusions
• in vitro conception is associated with
significant DNA methylation differences in 610% of genes assayed
• in vitro conception-associated methylation
differences are associated with significant
differences in steady state mRNA levels of 2040% of affected (highly selected) genes
• the level of expression of individual genes in
some in vitro children is more than two
standard deviations from the in vivo mean
Many Questions…
• Infertility, per se, ART or both?
• If infertility - genetic, environmental, male
factor, female factor?
• If ART – gonadotropins, ICSI, IVF, culture?
• Are differences between groups (preexisting
or alleged “changes”) random or are some
genes more susceptible than others? (useful
in predicting phenotype/treatment?)
Acknowledgments
Fels Institute for Cancer Research,
Temple University School of Medicine
Nahid Turan, Ph.D. (Temple)
Sunita Katari, M.D. (Temple)
Oluwatoyin Erinle, B.A. (Temple)
Halleh Asadpour, B.S. (Temple)
Margaret Brannigan, B.A. (Temple)
Noa Holzman (Cornell)
Leigh Gerson, B.S. (Temple)
Sidd Kosaraju (Duke)
Division of Reproductive Endocrinology and Infertility,
University of Pennsylvania
Christos Coutifaris, M.D., Ph.D. (Penn)
Raffi Chalian, M.D. (Penn)
Michael Foster, B.A. (Penn)
Research supported by NIH R01 HD048730