Effect of folic acid supplementation on adverse morphological and epigenetic outcomes in offspring conceived using assisted reproduction

In Canada, the use of assisted reproductive technologies (ARTs) has risen within the last decade due to the increased prevalence of infertility. Although the majority of ART-conceived children are born healthy, the use of these techniques has been associated with an increased risk of adverse offspring outcomes, such as growth and genomic imprinting disorders. In fact, assisted reproduction has been shown to compromise DNA methylation programming during germ cell and early embryo development. The availability of methyl donors, such as folate from the diet, is critical in the proper establishment and maintenance of DNA methylation patterns throughout development. The goal of this study was to determine whether folic acid supplementation can prevent morphological and epigenetic defects in the offspring associated with ART. Outbred female mice were placed on a folic acid control diet (CD; 2 mg folic acid/kg), a 4-fold folic acid-supplemented diet (4FASD; 8 mg folic acid/kg) or a 10-fold folic acid-supplemented diet (10FASD; 20 mg folic acid/kg) for six weeks prior to assisted reproduction and diets were continued until the collection of midgestation embryos and placentas. Mouse ART consisted of treatments used in human ART: a combination of ovarian stimulation (superovulation), in vitro fertilization, embryo culture to blastocyst and embryo transfer. Comparison between pregnancy outcomes from the CD group and naturally mated mice fed the folic acid control diet (NAT-CD) demonstrated that mouse ART resulted in significantly higher levels of pre- and post-implantation loss, reduced embryo viability, and an increased proportion of delayed embryos. Although both doses of folic acid supplementation did not significantly affect implantation rates, resorption rates or overall embryo viability, the 4FASD was associated with a decrease in embryonic delay. Notably, exposure to the 10FASD resulted in a trend towards an increased incidence of embryonic morphological abnormalities relative to the 4FASD group. In fact, craniofacial malformations were more prevalent in the 10FASD group compared to the CD group. DNA methylation was assessed in embryos and placentas by bisulfite pyrosequencing at four imprinting control regions (ICRs): Snrpn, Kcnq1ot1, Peg1 and H19 ICRs. ART was associated with imprinting defects in placental tissues and to a lesser extent in the embryonic tissues. In the placenta, although both folic acid-supplemented diets did not affect mean methylation at imprinted loci, the 4FASD resulted in significantly reduced variance at the Kcnq1ot1 and H19 ICRs, whereas the 10FASD led to increased variance at the Snrpn ICR. In the embryo, decreased variance was observed in the 4FASD group at the Snrpn, Kcnq1ot1 and H19 ICRs; however, the 10FASD was associated with increased variance at the H19 ICR and lower mean methylation at the Snrpn and H19 ICRs. The 10FASD also resulted in decreased variance at the Kcnq1ot1 ICR, but not to the same extent as the 4FASD. In the CD group, a higher proportion of females exhibited abnormal methylation at the Snrpn, Kcnq1ot1 and H19 ICRs compared to males. Interestingly, after moderate and/or high dose folic acid supplementation, the proportion of females exhibiting abnormal methylation at these ICRs was similar to males. Taken together, the morphological and epigenetic results suggest that a moderate dose of folic acid may be beneficial while high doses may be deleterious in ART pregnancies in a mouse model.

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