The loss of imprinted DNA methylation in mouse blastocysts is inflicted to a similar extent by in vitro follicle culture and ovulation induction.

STUDY HYPOTHESIS: Does in vitro follicle culture (IFC) have an effect on maintenance of imprinted DNA methylation in preimplantation mouse embryos? STUDY FINDING: We report similar alterations in the methylation pattern of H19 imprinted maternally expressed transcript (H19), small nuclear ribonucleoprotein polypeptide N (Snrpn) and mesoderm specific transcript (Mest) imprinted genes in mouse blastocysts obtained after ovulation induction and IFC. Furthermore, we observed no differences in the gene expression of maternal effect proteins related with imprinting maintenance between superovulated in vivo grown or IFC oocytes. WHAT IS KNOWN ALREADY: Assisted reproductive technology is associated with adverse post-natal outcomes such as increased risk of premature birth, altered birthweight, congenital anomalies and genomic imprinting syndromes in human and in animal models. Previous studies have shown that ovulation induction allowed normal imprinting establishment in mouse oocytes, but interfered with imprinting maintenance during preimplantation . Normal imprinting establishment was also observed in mouse oocytes derived from a standardized IFC from the early pre-antral follicle stage. STUDY DESIGN, SAMPLES/MATERIALS,
METHODS: The methylation profiles of differentially methylated regions (DMRs) of three key imprinted genes (H19, Snrpn and Mest) were compared at hatched blastocyst stage between embryos obtained from IFC or superovulated oocytes, each subjected to IVF and preimplantation in vitro culture (IVC); in non-manipulated in vivo produced late blastocyst (control) and in in vivo produced 2-cell embryos that were in vitro cultured until the hatched blastocyst stage (to assess the effect of IVC). Two different mice strains (Mus musculus C57BL/6J X CBA/Ca and Mus musculus B6 (CAST7)) were used to discriminate between maternal and paternal alleles of imprinted genes. Additionally, a limiting-dilution bisulfite-sequencing technique was carried out on individual embryos in order to avoid amplification bias. To assess whether IFC and ovulation induction differentially affect the mRNA expression of imprinting maintenance genes in the oocyte, a comparison of DNA methyltransferase 1 (Dnmt1o), methyl-CpG binding domain protein 3 (MBD3) and developmental pluripotency-associated 3 (Dppa3) was performed by qPCR between in vivo and in vitro grown oocytes at the germinal vesicle and metaphase II (MII) stage. MAIN RESULTS AND THE ROLE OF CHANCE: Results showed a loss of global imprinted DNA methylation in all in vitro manipulated embryos, due to an increase in the amount of abnormal alleles (<50% methylated). Importantly, there were no differences in blastocysts obtained from IFC and ovulation induction. Moreover, similar mRNA expression levels for Dnmt1o, MBD3 and Dppa3 genes were observed in IFC and stimulated oocytes. LIMITATIONS, REASONS FOR CAUTION: The methylation analysis was restricted to a number of well-selected imprinted genes. Future studies need to determine whether ovulation induction and IFC affect maternal effect factors at the protein level. WIDER IMPLICATIONS OF THE
FINDINGS: In vitro maturation of oocytes (IVM) is a patient-friendly alternative to conventional ovarian stimulation in PCOS patients. IFC is an emerging technology in human oncofertility. The results of this study show for the first time that in vitro oocyte culture induces no additional epigenetic alterations compared with conventional ovulation induction, at least for imprinted genes at the hatched blastocyst stage. The mouse IFC system can be used to test the sensitivity of the oocyte during its growth and maturation to several nutritional, metabolic and hormonal conditions possibly linked to epigenetic alterations. LARGE SCALE DATA: N/A. STUDY FUNDING AND COMPETING INTERESTS: This study received funding by Strategic Research Programs-Groeiers (OZR/2014/97), IWT/TBM/110680 and by UZ Brussel Fonds Willy Gepts (WFWG 2013). There is no conflict of interest.