Title: Generalized disturbance of DNA methylation in the uterine decidua in the CBA/J x DBA/2 mouse model of pregnancy failure Running title: Abnormal methylation in murine pregnancy failure Summary Sentence:DNA methylation in the uterine decidua of CBA/J x DBA/2 mouse pregnancies is highly abnormal, suggesting a role for epigenetics in pregnancy failure.

Non-chromosomal pregnancy failure is a common but poorly understood phenomenon. Because recent data have suggested that epigenetic abnormalities such as abnormal placental DNA methylation may play a role in human pregnancy failure, we undertook experiments designed to test whether decidual and/or placental DNA methylation abnormalities are present in a mouse model of pregnancy failure. A large number of studies have shown that crosses between CBA/J female mice and DBA/2 males result in pregnancies with a high rate of failure/resorption, while other crosses with CBA/J females produce normal pregnancies. Although CBA/J x DBA/2 has frequently been used as a model for miscarriage, a detailed explanation for the pregnancy failure phenotype is lacking. We performed timed matings between CBA/J female and DBA/2 male mice as well as the reverse mating, DBA/2 female with CBA/J males. Decidual caps were isolated at E9.5 from both crosses and a microarray-based method was used to comparatively assess genomic methylation at approximately 16,000 loci on mouse chromosome 7. In comparison with decidual caps from DBA/2 x CBA/J pregnancies, CBA/J x DBA/2 decidual caps were characterized by widely and apparently randomly disturbed methylation. In another set of analogous experiments, genomic methylation of placental DNA from E8.5 pregnancies was assessed using the same microarraybased method. This analysis revealed that, in contrast to the decidua, placental DNA methylation from CBA/J x DBA/2 pregnancies was indistinguishable from normal controls. We conclude that abnormal DNA methylation in the uterine decidua is likely to play a role in the CBA/J x DBA/2 model of pregnancy failure. Our studies are the first to demonstrate that epigenetic abnormalities of the decidua are associated with pregnancy failure and set the stage for future efforts aimed at understanding the role of DNA methylation at the maternal-fetal interface.