Epigenetic status of the H19 locus in human oocytes following in vitro maturation

Abstract

Imprinting is an epigenetic modification that is reprogrammed in the germ line and leads to the monoallelic expression of some genes. Imprinting involves DNA methylation. Maternal imprint is reset during oocyte growth and maturation. In vitro maturation (IVM) of oocytes may, therefore, interfere with imprint acquisition and/or maintenance. To evaluate if maturing human oocytes in vitro would be hazardous at the epigenetic level, we first determined the methylation profile of the H19 differentially methylated region (DMR). The methylation status of the H19 DMR seems particularly vulnerable to in vitro culture conditions. We analyzed oocytes at different stages of maturation following IVM, germinal vesicle (GV), metaphase I (MI), and metaphase II (MII), using the bisulfite mutagenesis technique. Our results indicated that the unmethylated specific maternal profile for the H19 DMR was stably established at the GV stage. The majority of MI-arrested oocytes exhibited an altered pattern of methylation, the CTCF-binding site being methylated in half of the DNA strands analyzed. Of the 20 MII oocytes analyzed, 15 showed the normal unmethylated maternal pattern, while 5 originating from two different patients exhibited a methylated pattern. These findings highlight the need for extended analysis on MII-rescued oocytes to appreciate the epigenetic safety of the IVM procedure, before it becomes a routine and practical assisted reproductive procedure.

Analysis of H19 methylation in control and abnormal human embryos, sperm and oocytes

“…Because of the limited starting material and as bisulfite treatment being deleterious for DNA, identical sequences from separated PCRs are certain to represent distinct chromosomes, but identical sequences obtained from the same PCR product are counted only once, as previously discussed; 15 20 to 30 clones could be scored for control embryos and 12 to 15 clones for embryos that failed to develop (see Supplementary Figures 1 bis and 2 …”

“…15 After treatment with bisulphite and purification, the DNA was immediately used for nested PCR. Five independent nested PCRs were performed per embryo and per sperm sample.…”

“…We previously evidenced such altered pattern of methylation in human oocytes, particularly in oocytes that were immature on the day of retrieval, but also in mature oocytes. 15 Although the maternally originating H19 strands were not methylated in embryo 5.4, they were significantly methylated in embryo 33.3. Both the father and the mother were heterozygous in couple 33.…”

Deleted in Azoospermia-Like Enhances In Vitro Derived Porcine Germ Cell Formation and Meiosis

“…Substantial studies have reported the generation of germ cell-like cells from somatic tissue-derived stem cells such as skin [3][4][5]16,[36][37][38], bone marrow [39][40][41][42], and pancreas [43]. It has been demonstrated that epigenetic imprints are established during gametogenesis [44][45][46], and that the DNA methylation status of imprinted genes is erased and reset during PGC development [47,48]. Using the porcine skin stem cell to the PLC differentiation model utilized in the current study, it was previously revealed that 19% of the 29 individual CpG sites in the differentially methylated region 1 of the 5¢ flanking region of an imprinted gene, H19, were methylated in undifferentiated skin stem cells, while 99% of CpG sites were unmethylated in this region at D25 of differentiation.…”

Study of DNA methylation patterns of imprinted genes in children born after assisted reproductive technologies reveals no imprinting errors: A pilot study

“…During gametogenesis and embryogenesis, existing imprints inherited from previous generations are erased, and new imprints are established. Consequently, many studies have focused on the alteration of DNA methylation patterns in gametes and embryos, which are thought to be linked to various steps in ART (6,28,29). However, the number of human oocytes and embryos, to some extent, were too small to be used to investigate the association between offspring after ART and imprinting defects.…”