227 Ovarian stimulation in aged mice results in placental genomic imprinting dysregulation

It has been shown in mice that ovarian stimulation with exogenous gonadotropin alone can lead to aberrant genomic imprinting, an epigenetic phenomenon that restricts gene expression based on parental origin. This effect from ovarian stimulation has been reported in various offspring tissues including embryos and placenta. However, there has been little investigation on the effects of advanced maternal age, with or without ovarian stimulation, on genomic imprinting regulation in offspring placenta. As a reproductively aging society and with an increase in demand for assisted reproductive technology, it is key to understand the impact aging has on pregnancy and offspring health. Young (3–4 months) CD1 females and naturally aged (15 months) CD1 females were either ovarian stimulated (YS n = 6; AS n = 13) or unstimulated (YU n = 6; AU n = 15) before mating. Stimulated females received IP injections of low dose 5 IU eCG followed by 5 IU hCG 46 h later. Pregnant females were sacrificed at E16 of fetal development and F1 placentas were collected for DNA and RNA isolation (Qiagen). Targeted bisulfite pyrosequencing (PyroMark Q24 Advanced System, Qiagen) examined average placental DNA methylation levels at imprinted regions (n = 6–15 per group) analysed as a ratio of C:T peaks from each CpG. Statistical analysis was calculated using Student’s t-test, with significance at P < 0.05. Relative gene expression was performed on placentas from aged females by qPCR (Applied Biosystems) for genes associated with the same imprinted regions (n = 3 per group). Gene expression was normalized to a stable housekeeping gene (Rpl19) and statistical analysis was performed by REST 2009 software (Qiagen) with significance at P < 0.05. Imprinted methylation in placentas derived from young females was comparable between ovarian stimulated and unstimulated groups at all imprinted regions (Kcnq1ot1, P = 0.28; Snrpn, P = 0.12; Peg3, P = 0.29). Similarly, placental imprinted methylation was unaffected in unstimulated females, irrespective of age (Kcnq1ot1, P = 0.97; Snrpn, P = 0.26; Peg, P = 0.31). However, placentas derived from ovarian stimulated aged females resulted in a significant loss of methylation at maternally methylated imprinting control regions when compared with unstimulated aged females (Kcnq1ot1 AS: 43.3%, AU: 44.6%, P = 0.007; Snrpn AS: 34.9%, AU: 37.1%, P = 0.046; Peg3 AS: 43.6%, AU: 47.0%, P = 0.008). Hypomethylation resulted in increased gene expression from the normally silenced maternal allele (Kcnq1ot1 fold change = 1.96, P < 0.05; Snrpn fold change = 3.03, P < 0.05; Peg3 fold change = 2.28, P < 0.05) following ovarian stimulation in the same aged females relative to unstimulated aged females. In this study, placental imprinted methylation was unchanged in young females following ovarian stimulation, as well as in advanced maternal age without ovarian stimulation. However, despite a low hormone dosage, ovarian stimulated aged females resulted in embryonic placentas with hypomethylated imprinted regions and compromised imprinted gene regulation compared with their unstimulated aged counterparts. Overall, the inclusion of ovarian stimulation for aged females resulted in genomic imprinting dysregulation in mouse offspring placentas, which can lead to severe placental abnormalities and subsequent fetal growth defects.