Dual effects of superovulation: loss of maternal and paternal imprinted methylation in a dose-dependent manner.

Superovulation or ovarian stimulation is currently an indispensable assisted reproductive technology (ART) for human subfertility/infertility treatment. Recently, increased frequencies of imprinting disorders have been correlated with ARTs. Significantly, for Angelman and Beckwith-Wiedemann Syndromes, patients have been identified where ovarian stimulation was the only procedure used by the couple undergoing ART. In many cases, increased risk of genomic imprinting disorders has been attributed to superovulation in combination with inherent subfertility. To distinguish between these contributing factors, carefully controlled experiments are required on spontaneously ovulated, in vivo-fertilized oocytes and their induced-ovulated counterparts, thereby minimizing effects of in vitro manipulations. To this end, effects of superovulation on genomic imprinting were evaluated in a mouse model, where subfertility is not a confounding issue. This work represents the first comprehensive examination of the overall effects of superovulation on imprinted DNA methylation for four imprinted genes in individual blastocyst stage embryos. We demonstrate that superovulation perturbed genomic imprinting of both maternally and paternally expressed genes; loss of Snrpn, Peg3 and Kcnq1ot1 and gain of H19 imprinted methylation were observed. This perturbation was dose-dependent, with aberrant imprinted methylation more frequent at the high hormone dosage. Superovulation is thought to primarily affect oocyte development; thus, effects were expected to be limited to maternal alleles. Our study revealed that maternal as well as paternal H19 methylation was perturbed by superovulation. We postulate that superovulation has dual effects during oogenesis, disrupting acquisition of imprints in growing oocytes, as well as maternal-effect gene products subsequently required for imprint maintenance during pre-implantation development.