The adverse effects on offspring of diabetic and/or obese mothers can be passed to the next generation. However, the mechanisms behind this are still unclear. Epigenetics may play a key role during this process.

What are the effects of high-glucose concentrations on DNA methylation of human oocytes? High-glucose concentrations altered DNA methylation levels of Peg3 and Adiponectin in human in vitro maturation oocytes. Maternal diabetes has a detrimental influence on oocyte quality including epigenetic modifications, as shown in non-human mammalian species. Immature metaphase I (MI) stage oocytes of good quality were retrieved from patients who had normal ovarian potential and who underwent ICSI in the Reproductive Medicine Center of People’s Hospital of Zhengzhou University. MI oocytes were cultured in medium with different glucose concentrations (control, 10 mM and 15 mM) in vitro and 48 h later, oocytes with first polar body extrusion were collected to check the DNA methylation levels. MI oocytes underwent in vitro maturation (IVM) at 37°C with 5% mixed gas for 48 h. Then the mature oocytes were treated with bisulfite buffer. Target sequences were amplified using nested or half-nested PCR and the DNA methylation status was tested using combined bisulfite restriction analysis (COBRA) and bisulfite sequencing (BS). High-glucose concentrations significantly decreased the first polar body extrusion rate. Compared to controls, the DNA methylation levels of Peg3 in human IVM oocytes were significantly higher in 10 mM (P < 0.001) and 15 mM (P < 0.001) concentrations of glucose. But the DNA methylation level of H19 was not affected by high-glucose concentrations in human IVM oocytes. We also found that there was a decrease in DNA methylation levels in the promoter of adiponectin in human IVM oocytes between controls and oocytes exposed to 10 mM glucose (P = 0.028). N/A It is not clear whether the alterations are beneficial or not for the embryo development and offspring health. The effects of high-glucose concentrations on the whole process of oocyte maturation are still not elucidated. Another issue is that the number of oocytes used in this study was limited. This is the first time that the effects of high-glucose concentration on DNA methylation of human oocytes have been elucidated. Our result indicates that in humans, the high risk of chronic diseases in offspring from diabetic mothers may originate from abnormal DNA modifications in oocytes. This work was supported by the fund of National Natural Science Foundation of China (81401198) and Doctor Foundation of Qingdao Agricultural University (1116008).The authors declare that there are no potential conflicts of interest relevant to this article.

For humans, ARTs (assisted reproductive technologies) have become the most effective method to treat subfertility/infertility in clinic. To obtain enough oocytes during ART, ovarian stimulation is performed by exogenous hormones, and some patients undergo several ovarian stimulation cycles. Although some adverse effects of ARTs on women and offspring are reported, few studies are focused on the effects of multiple superovulation on ovarian reserve. In the present study, we found that repeated superovulation significantly reduced primordial follicle number and the serum AMH. Compared to the decreased antral follicle number, the expression of genes related to primordial follicle activation, such as Foxo3, Akt, and Rptor, and the atretic follicle number in ovaries were increased by superovulation times. We further found that repeated superovulation reduced the plasma level of FSH, LH, and estradiol, and increased the expression of genes related to apoptosis (Bax, Casp3 (caspase-3), Casp8, and Casp9) in granulosa cells, providing evidence that repeated superovulation disrupted the balance between survival and death in granulosa cells. In summary, our results suggest that repeated superovulation has adverse effects on folliculogenesis.

It is demonstrated that repeated superovulation has deleterious effects on mouse ovaries and cumulus cells. However, little is known about the effects of repeated superovulation on early embryos. Epigenetic reprogramming is an important event in early embryonic development and could be easily disrupted by the environment. Thus, we speculated that multiple superovulations may have adverse effects on histone modifications in the early embryos. Female CD1 mice were randomly divided into four groups: (a) spontaneous estrus cycle (R0); (b) with once superovulation (R1); (c) with three times superovulation at a 7-day interval (R3) and (d) with five times superovulation at a 7-day interval (R5). We found that repeated superovulation remarkably decreased the fertilization rate. With the increase of superovulation times, the rate of early embryo development was decreased. The expression of Oct4, Sox2 and Nanog was also affected by superovulation in blastocysts. The immunofluorescence results showed that the acetylation level of histone 4 at lysine 12 (H4K12ac) was significantly reduced by repeated superovulation in mouse early embryos (P < 0.01). Acetylation level of histone 4 at lysine 16 (H4K16ac) was also significantly reduced in pronuclei and blastocyst along with the increase of superovulation times (P < 0.01). H3K9me2 and H3K27me3 were significantly increased in four-cell embryos and blastocysts. We further found that repeated superovulation treatment increased the mRNA level of histone deacetylases Hdac1, Hdac2 and histone methyltransferase G9a, but decreased the expression level of histone demethylase-encoding genes Kdm6a and Kdm6b in early embryos. In a word, multiple superovulations alter histone modifications in early embryos.

Controlled ovarian stimulation by exogenous gonadotrophins is a key procedure during the in vitro fertilization cycle to obtain a sufficient number of oocytes in humans. Previous studies demonstrated that repeated superovulation had deleterious effects on the ovaries. However, whether repeated superovulation adversely affects the mitochondrial functions of cumulus cells remains unclear. In this study, mice were divided into three groups: superovulation once (R1); superovulation three times (R3), and superovulation five times (R5). We evaluated the effects of repeated superovulation on mitochondrial DNA copies (mtDNA) and observed decreased mtDNA copies per cell with increasing number of superovulation cycles. Further, we investigated the DNA methylation status in exon 2 and the mRNA expression level of nuclear-encoded DNA polymerase gamma A (PolgA). The results showed that the DNA methylation levels of PolgA in R1 and R5 were slightly lower than in R3. Additionally, the altered DNA methylation in PolgA coincided with the changes in PolgA expression in cumulus cells. We also found that the mRNA expression of COX1, CYTB, ND2, and ND4 was altered by repeated superovulation in cumulus cells. Thus, repeated superovulation had adverse effects on mitochondrial function.

Gestational diabetes mellitus (GDM), a common pregnancy disease, has long-term negative effects on offspring health. Epigenetic changes may have important contributions to that, but the underlying mechanisms are not well understood. Here, we report the influence of GDM on DNA methylation of offspring (GDF1) oocytes and the possible mechanisms. Our results show that GDM induces genomic hyper-methylation of offspring oocytes, and at least a part of the altered methylation is inherited by F2 oocytes, which may be a reason for the inheritance of metabolic disorders. We further find that GDM exposure increases the expression of Ezh2 in oocytes. Ezh2 regulates DNA methylation via DNMT1, and Ezh2 knockdown reduces the genomic methylation level of GDF1 oocytes. These results suggest that GDM may induce oocyte genomic hyper-methylation of offspring via enhancing the Ezh2 expression recruiting more DNMT1 into nucleus.