1. 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

2. 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”

3. EnvironmentGenes Changes in Epigenotype Long-Term, Somatically Heritable Changes in Gene Expression “Genes + Environment” Phenotype Effectors of cellular stress responses

4. Do Children Conceived in vitro Differ from Children Conceived in vivo in “Epigenotype” ?

5. 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

6. “GoldenGate” assay (Illumina, Inc.): fraction of methyl C at a CpG site hybridization/synthesis/ligation/PCR in bisulfite treated/untreated DNA

7. 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

9. EnvironmentGenes Changes in Epigenotype Long-Term, Somatically Heritable Changes in Gene Expression “Genes + Environment” Phenotype Effectors of cellular stress responses

10. Absolute range of difference small…

12. Absolute range of difference moderate…

13. Absolute range of difference large…

14. EnvironmentGenes Changes in Epigenotype Long-Term, Somatically Heritable Changes in Gene Expression “Genes + Environment” Phenotype Effectors of cellular stress responses

15. Assume cis-acting epigenetic differences linked to steady-state mRNA levels

21. 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.

22. 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?

23. Conclusions in vitro conception is associated with significant DNA methylation differences in 6- 10% of genes assayed in vitro conception-associated methylation differences are associated with significant differences in steady state mRNA levels of 20- 40% 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

24. 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?)

25. 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