Perturbations in imprinted methylation from assisted reproductive technologies but not advanced maternal age in mouse preimplantation embryos

Abstract

Background: Over the last several decades, the average age of first-time mothers has risen steadily. With increasing maternal age comes a decrease in fertility, which in turn has led to an increase in the use of assisted reproductive technologies by these women. Assisted reproductive technologies (ARTs), including superovulation and embryo culture, have been shown separately to alter imprinted DNA methylation maintenance in blastocysts. However, there has been little investigation on the effects of advanced maternal age, with or without ARTs, on genomic imprinting. We hypothesized that ARTs and advanced maternal age, separately and together, alter imprinted methylation in mouse preimplantation embryos. For this study, we examined imprinted methylation at three genes, Snrpn, Kcnq1ot1, and H19, which in humans are linked to ART-associated methylation errors that lead to imprinting disorders.

Results: Our data showed that imprinted methylation acquisition in oocytes was unaffected by increasing maternal age. Furthermore, imprinted methylation was normally acquired when advanced maternal age was combined with superovulation. Analysis of blastocyst-stage embryos revealed that imprinted methylation maintenance was also not affected by increasing maternal age. In a comparison of ARTs, we observed that the frequency of blastocysts with imprinted methylation loss was similar between the superovulation only and the embryo culture only groups, while the combination of superovulation and embryo culture resulted in a higher frequency of mouse blastocysts with maternal imprinted methylation perturbations than superovulation alone. Finally, the combination of increasing maternal age with ARTs had no additional effect on the frequency of imprinted methylation errors.

Conclusion: Collectively, increasing maternal age with or without superovulation had no effect of imprinted methylation acquisition at Snrpn, Kcnq1ot1, and H19 in oocytes. Furthermore, during preimplantation development, while ARTs generated perturbations in imprinted methylation maintenance in blastocysts, advanced maternal age did not increase the burden of imprinted methylation errors at Snrpn, Kcnq1ot1, and H19 when combined with ARTs. These results provide cautious optimism that advanced maternal age is not a contributing factor to imprinted methylation errors in embryos produced in the clinic. Furthermore, our data on the effects of ARTs strengthen the need to advance clinical methods to reduce imprinted methylation errors in in vitro-produced embryos.

Keywords: Assisted reproductive technologies; DNA methylation; Fertility; Genomic imprinting; Maternal age; Mouse.

Fig. 1

Imprinted methylation acquisition at the (a) Snrpn, (b) Kcnq1ot1, and (c) H19 ICRs in germinal vesicle oocytes was unaltered by increasing maternal age. Each box shows oocytes from spontaneously ovulating females with maternal age in months (mths) (n = 3–4 females per age group). Each line of circles represents the CpGs in the Snrpn, Kcnq1ot1, or H19 ICRs in an individual oocyte (n = 13–19 oocytes per age group). Oocytes were excluded if sequenced DNA clones (n = 5–8/denuded oocyte) exhibited more than one methylation pattern, suggestive of cumulus cell contamination. Black circles, methylated CpGs; white circles, unmethylated CpGs; %5mC, percentage of 5-methyl CpGs over the total number of CpGs for a specific ICR. Same letter (a), no statistically significant difference between groups

Fig. 2

Imprinted methylation acquisition at the (a) Snrpn, (b) Kcnq1ot1, and (c) H19 ICRs in MII oocytes was unchanged by increasing maternal age combined with superovulation. Each box shows oocytes from superovulated females with maternal age in months (mths) (n = 3–4 females per age group; n = 10–13 oocytes per age group). See Fig. 1 for details. Same letter (a), no statistically significant difference between groups

Fig. 3

Advanced maternal age did not perturb imprinted methylation maintenance of the (a) Snrpn, (b) Kcnq1ot1, and (c) H19 ICRs in blastocysts. Embryos were derived from spontaneously ovulating females. Diamonds represent the mean methylation levels of maternal (red) or paternal (blue) alleles for the Snrpn, Kcnq1ot1, and H19 ICRs in individual blastocysts (n = 9–22 embryos, n = 3–7 females per age group). Same letter (a), no statistically significant difference between groups

Fig. 4

ARTs generated a loss of imprinted methylation maintenance at the (a) Snrpn, (b) Kcnq1ot1, and (c) H19 ICRs in blastocysts from young mothers. Diamonds represent the mean methylation levels of maternal (red) or paternal (blue) alleles for Snrpn, Kcnq1ot1, and H19 ICRs in individual embryos (n = 9–17 blastocysts; n = 3–4 females per treatment group). SO, superovulation; EC, embryo culture. Letters (a, b, c), a statistically significant difference between groups (Additional file 1: Table S1)

Fig. 5

Increasing maternal age plus ARTs did not further increase imprinted methylation errors at the (a) Snrpn, (b) Kcnq1ot1, and (c) H19 ICRs in blastocysts. Diamonds represent the mean methylation levels of maternal (red) or paternal (blue) alleles for Snrpn, Kcnq1ot1, and H19 ICRs in individual embryos (n = 10–17 blastocysts; n = 2–5 females per treatment group). Letters (a, b, c), a statistically significant difference between groups (Additional file 1: Table S2)