O-192 DNA methylation at H19/IGF2 ICR1 in the placenta of pregnancies conceived by IVF and ICSI

vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI), has recent- ly caused concerns about its effect on the epigenome and ultimately the risk to the fetus. In particular, studies on the effects of in vitro manipulation of mouse oocytes and embryos have shown that DNA methylation at imprinted genes can be altered and can result in reduced fetal growth. Furthermore, studies of sperm from infertile men have also revealed an association between oligozoospermia and aberrant DNA methylation. In the majority of ART-born children diagnosed with imprinting disorders, namely Beckwith Wiedemann Syndrome and Angel- man Syndrome, aberrant DNA methylation was shown to be the cause. These studies suggest that ART or infertility may introduce epigenetic modifications that affect proper fetal growth and imprinting abnormalities. Therefore, in this study we investigated the whether IVF and ICSI pregnancies lead to aberrant DNA methylation at the imprinting control region 1 (ICR1) of H19 and IGF2, in the placenta. We also investigated whether placentas from small for gestational age (SGA) pregnancies were particularly associated with reduced methylation. Materials and Methods: Women that have conceived by IVF (n = 32) or ICSI (n = 45) were included in this study. Women that have conceived naturally (NC; n = 12) were also recruited for this study. Each type of pregnancy was further classified as appropriate for gestational age (AGA) or small for gestational age (SGA). These were determined using a Canadian perinatal surveillance report. The placenta from each pregnancy was sampled for whole villi at two sites. DNA methylation in the placenta was then ascertained using the methylation- sensitive single nucleotide primer extension (MS-SNuPE) assay for two CpG sites in the ICR1(C10 and C12). Results: The mean methylation in IVF-AGA (n = 25), IVF-SGA (n = 7), ICSI-AGA (n = 32), and ICSI-SGA (n = 13) placentas were 45.52 ± 4.86%, 47.25 ± 5.77%, 45.64 ± 6.06%, and 42.73 ± 4.39%, respectively. The mean methylation in the controls, NC-AGA (n = 7) and NC SGA (n = 5), were 44.68 ± 4.18% and 44.63 ± 3.60%, respectively. No significant differences in DNA methylation at the H19 ICR was observed between all sample and con- trol groups (ANOVA, p = 0.49). Hypomethylation, which we defined as below 31.5% based on our results, was observed in eight cases in at least one CpG site. Hypomethylation at an individual CpG was not restricted to the IVF and ICSI cases (IVF-AGA, n = 1; IVF-SGA, n = 1; ICSI-AGA, n = 1; ICSI-SGA, n = 3), as it was also observed in natural conception cases (NC-AGA, n = 1; NC-SGA, n = 1). Furthermore, methylation in umbilical cord blood was also obtained from seven ART cases, showing no significant correlation between methylation in the cord blood and the placenta. Conclusions: Studies in both human and mice have shown that methods in- volved in ART may lead to aberrant DNA methylation at imprinted genes. Con- trary to these findings, our results suggest that ARTs may not alter imprinting of H19 and IGF2 in most cases. We also did not detect any differences in methyla- tion between SGA and AGA pregnancies, which suggest that in our cases fetal growth restriction is not due to epigenetic modifications at the H19 ICR1. It is unknown whether the cases showing hypomethylation altered the expression of H19 and IGF2. However, in a separate study we have shown that expression of these genes was not altered in ART placentas compared to those of natural con- ception. Thus, further study is required, and those that relate epigenetic status of imprinted genes with transcriptional activity will be important in delineating the consequences of aberrant DNA methylation.