High-glucose concentrations change DNA methylation levels in human IVM oocytes

High-glucose concentrations change DNA methylation levels in human IVM oocytes Abstract STUDY QUESTION What are the effects of high-glucose concentrations on DNA methylation of human oocytes? SUMMARY ANSWER High-glucose concentrations altered DNA methylation levels of Peg3 and Adiponectin in human in vitro maturation oocytes. WHAT IS KNOWN ALREADY Maternal diabetes has a detrimental influence on oocyte quality including epigenetic modifications, as shown in non-human mammalian species. STUDY DESIGN, SIZE, DURATION 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. PARTICIPANTS/MATERIALS, SETTING, METHODS 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). MAIN RESULTS AND THE ROLE OF CHANCE 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). LARGE SCALE DATA N/A LIMITATIONS REASONS FOR CAUTION 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. WIDER IMPLICATIONS OF THE FINDINGS 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. STUDY FUNDING/COMPETING INTEREST(S) 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. human oocyte, DNA methylation, high glucose concentrations, in vitro maturation, diabetes, epigenetics Introduction Diabetes is defined as ‘fasting blood glucose equal to or higher than 7 mmol/l, or on medication for raised blood glucose, or with a history of diagnosis diabetes’ by the World Health Organization (WHO). If the distribution of fasting plasma glucose in a population is higher than the theoretical distribution, it is termed as high-blood glucose. The WHO has estimated that, globally, 422 million adults who are over 18 years would live with diabetes in 2014. The global incidence of diabetes has increased from 4.7% in 1980 to 8.5% in 2014, while the prevalence of diabetes in the WTO Mediterranean Region is as high as 13.7% (http://www.who.int/mediacentre/factsheets/fs312/en/). Not only are there complication of diabetes in patients themselves but also there are deleterious effects on their offspring (Vrachnis et al., 2012a,b,c). The long-term consequences for offspring of diabetic parents include a higher risks of insulin resistance, abnormal glucose metabolism and chronic diseases (Vrachnis et al., 2012a,b,c). The complications of diabetes are mainly caused by hyperglycemia which can induce a delay in embryo development, a higher incidence of degenerate and fragmented embryos, growth retardation and failure of anterior neuropore closure (Jungheim and Moley, 2008). High-blood glucose also changes the micro-environment of follicle development which is important for oocyte maturation, oocyte quality and embryonic development. At physiological levels, the oocyte is exposed to glucose levels of 31–82% of follicle fluid concentrations (Stokes et al., 2008). During oocyte maturation in vitro, proper glucose concentrations in the medium can increase the oocyte maturation rate and quality (Sutton-McDowall et al., 2010). But in vivo and in vitro experiments have demonstrated that high-glucose levels lead to poor developmental potential of oocytes and embryos (Sutton-McDowall et al., 2005, 2010) because of poor oocyte quality (Colton et al., 2002; Wang et al., 2009; Wang and Moley, 2010). A high glucose environment increases the glucose levels within the follicle and causes glucose accumulation in oocytes (Moley et al., 1998; Wang et al., 2012). Although oocytes have a poor capacity to utilize glucose (Biggers et al., 1967), the up-take glucose and glucose metabolism has been detected in denuded oocytes of different species (Downs, 1995; Wang et al., 2012). Furthermore, glucose is essential for the nuclear maturation of oocytes (Krisher and Bavister, 1998; Rose-Hellekant et al., 1998). Establishing proper DNA methylation is a crucial event during oocyte nuclear maturation (Stewart et al., 2016). However, the DNA methylation status is prone to be altered by changes in the internal and external environment, especially during gametogenesis and embryonic development (Feil and Fraga, 2012; Tammen et al., 2013). Previous studies have demonstrated that maternal hyperglycemia changes DNA methylation in mouse oocytes (Ge et al., 2013). But the effects of hyperglycemia on DNA methylation in human oocytes are not well elucidated. We know that it is difficult to obtain oocytes from diabetic women and the detrimental effects of diabetes on patients are mainly caused by the high-blood glucose level. Therefore, we used an in vitro maturation (IVM) model to check the influence of high glucose levels on the DNA methylation status in human oocytes. Generally, the glucose concentration of human follicle fluid is about 3.3 mM (Leese and Lenton, 1990). It is detrimental to oocytes if the glucose concentration is over 10 mM in medium (Sutton-McDowall et al., 2010). Thus, in the present study, we investigated the effects of various glucose concentrations (control (5.6 mM), 10 mM and 15 mM glucose in IVM medium) on DNA methylation in human IVM oocytes. Another study reported that the DNA methylation level in the differentially methylated region (DMR) of imprinted gene Peg3 was changed in oocytes of diabetic mouse (Ge et al., 2013). Therefore, we checked the methylation patterns of the maternal imprinted gene Peg3 and the paternal imprinted gene H19 which was used as control. Meanwhile, we also tested the DNA methylation level at the promoter region of Adiponectin because it plays a key role in onset of metabolic diseases (Lee and Shao, 2014) and the offspring of diabetic women are prone to obesity and other chronic diseases (Vrachnis et al., 2012a,b,c). Materials and Methods All of the procedures in the present study were reviewed and supported by the Ethic Committee of Life Science of Zhengzhou University. We received agreement, permission and signed consent from all patients included in the present study. All reagents were purchased from Sigma-Aldrich, unless indicated specifically. Criteria for choosing patients Women who were infertile due to non-ovarian reasons and who underwent IVF using ICSI intracytoplasmic sperm injection in the Reproductive Center of People’s Hospital of Zhengzhou University were included in this study. The women had no any other diseases which may affect ovarian function, such as polycystic ovarian syndrome (PCOS), diabetes, and other endocrine and metabolic diseases. All patients were younger than 35 years old. Oocyte collection and in vitro maturation Oocytes surrounded with cumulus cells (COCs) of patients were retrieved from fully grown follicles by ultrasound-guided at 36–37 h after hCG injection. COCs were cultured in vitro at 37°C with 5% mixed gas (O2, CO2 and N2) for 5 h. Then cumulus cells were removed using 80 IU/ml hyaluronidase in IVF medium. If oocytes were mature, they were used for ICSI. If oocytes were immature (at MI stage) with normal morphology and cytoplasm (Fig. 1A), they were collected and used in the present study. Figure 1 View largeDownload slide High-glucose concentrations reduced the first PB extrusion rate. (A) Representative pictures of human MI oocytes used for IVM and MII oocytes after IVM. 20×, magnification; (B) The first PB extrusion rate was calculated in each group and expressed as percentage. *P < 0.05; **P < 0.01; first PB, the first polar body. Figure 1 View largeDownload slide High-glucose concentrations reduced the first PB extrusion rate. (A) Representative pictures of human MI oocytes used for IVM and MII oocytes after IVM. 20×, magnification; (B) The first PB extrusion rate was calculated in each group and expressed as percentage. *P < 0.05; **P < 0.01; first PB, the first polar body. IVM medium was made using Tissue Culture Medium 199 (TCM199) supplemented with 5 mg/ml human transferrin, 5 ng/ml sodium selenite,10 ng/ml human recombinant insulin, 10 mIU/ml FSH (Merk, Germany), 1 mM L-glutamine, 0.3 mM sodium pyruvate, 0.8%HSA, 100 IU/ml penicillin G, 100 mg/ml streptomycin sulfate, 10 ng/ml epidermal growth factor, 1 mg/ml estradiol and 0.5 IU/ml hCG (Lizhu, China). Oocytes were divided into three groups: the control group in which oocytes were cultured in IVM medium; the 10 mM group in which oocytes were cultured in IVM medium with 10 mM glucose and the 15 mM group in which oocytes were cultured in IVM medium with 15 mM glucose. The oocyte maturation was at 37°C with 5% mixed gas. After 48 h, oocytes developed to MII stage (with the first polar body (first PB) extruded) were collected and stored at −80°C for the following experiments. Bisulfite treatment and PCR amplification DNA bisulfite transformation was carried out as in a previous study (Ge et al., 2014a,,b). Briefly, oocytes were lysed for 37 min at 37°C and then added to 15 μl low melting agarose in a previous tube. The agarose with DNA was transferred into a 2.0 ml Eppendorf tube with 200 μl pre-cooled mineral oil. Then we carefully transferred the agarose bead into a new 2.0 ml Eppendorf tube and added 500 μl pre-prepared bisulfite buffer. These tubes with the agarose bead and bisulfite buffer were cooled on ice for 15 min and then incubated in water bath for 4 h at 50°C. The incubated beads were washed for 15 min three times with TE (Tris-HCl and EDTA), for 15 min two times with 0.3 M NaOH, and for 15 min three times with distilled water. After washing, beads were stored at −20°C until used. Targeted DNA fragments were amplified using nested or half-nested PCR. For the first round PCR, beads were melted at 70°C in 200 μl tubes and then an amplification mix, including buffer, dNTP, hot-start DNA polymerase, water and the first round PCR primers of Peg3, H19 and Adiponectin, was added into tubes. A1 μl sample of the product of the first round PCR was used as template for the secondary round of PCR for Peg3, H19 and adiponectin, respectively. Primers used in this study are shown in Table I. Table I Oligonucleotides used for nested and half-nested PCR. Primer name  Primer sequence  H19-outer forward  5′-AGGTGTTTTAGTTTTATGGATGATGG-3′  H19-inner forward  5′-TGTATAGTATATATATGGGTATTTTTGGAGGTTT-3′  H19-reverse  5′-TCCTATAAATATCCTATTCCCAAATAACC-3′  Peg3-forward  5′-GGTTGTTGATTGGTTAGTATAG-3′  Peg3-outer reverse  5′-CACTCACCTCACCTCAATAC-3′  Peg3-inner reverse  5′-ACCTCACCTCAATACTAC-3′  Adiponectin-outer forward  5′-GGGTAGGTAGATATTTGTTT-3′  Adiponectin-inner forward  5′-GGGGTAGGTAGATATTTGTTTTGTTT-3′  Adiponectin-outer reverse  5′-TCAACACCTTAAACTTTCTTAACA-3′  Adiponectin-inner reverse  5′-AAACTACCACCCACTTAA-3′  Primer name  Primer sequence  H19-outer forward  5′-AGGTGTTTTAGTTTTATGGATGATGG-3′  H19-inner forward  5′-TGTATAGTATATATATGGGTATTTTTGGAGGTTT-3′  H19-reverse  5′-TCCTATAAATATCCTATTCCCAAATAACC-3′  Peg3-forward  5′-GGTTGTTGATTGGTTAGTATAG-3′  Peg3-outer reverse  5′-CACTCACCTCACCTCAATAC-3′  Peg3-inner reverse  5′-ACCTCACCTCAATACTAC-3′  Adiponectin-outer forward  5′-GGGTAGGTAGATATTTGTTT-3′  Adiponectin-inner forward  5′-GGGGTAGGTAGATATTTGTTTTGTTT-3′  Adiponectin-outer reverse  5′-TCAACACCTTAAACTTTCTTAACA-3′  Adiponectin-inner reverse  5′-AAACTACCACCCACTTAA-3′  View Large Combined bisulfite restriction analysis and bisulfite sequencing To test the DNA methylation status of H19 and Peg3, combined bisulfite restriction analysis (COBRA) and and bisulfite sequencing (BS) were used in the present study as previous described (Ge et al., 2013). Briefly, the PCR products were digested by endogenous restriction enzymes (TaqαI, RsaI and/or BstUI, NEB, China) for which CpG sites are included in the recognition sites. For BS, the PCR products were pooled together for each group, cloned into a T vector and sequenced (GENEWIZ, China). The sequencing result was analyzed using CpGViewer. Statistical analysis Average data were presented as mean ± standard deviation and the significance between groups was tested using one-way analysis of variance. The methylation level was presented as a percentage and the significance between groups was checked using Chi-square test. If the P-value was <0.05, it was considered as showing a statistical difference between groups. Results High-glucose levels decreased the first PB extrusion rate In total, 109 good quality MI oocytes (Fig. 1A) were retrieved from 68 patients. These oocytes were randomly divided into the three groups: control (n = 34), 10 mM (n = 39) and 15 mM (n = 36). We analyzed the average age, average body mass index (BMI), average duration of infertility, antral follicle count (AFC), total dosages of gonadotropin and basic hormone levels including FSH, LH, estradiol (E2) and anti-Mullerian hormone (AMH). The results showed that all of these parameters were similar between groups (Table II). Table II Characteristics of patients. Group  Control (n = 23)  10 mM (n = 25)  15 mM (n = 20)  P-value  Age (years)  30.18 ± 4.76  30.38 ± 4.73  29.86 ± 5.03  0.933  Duration of infertility (years)  4.07 ± 2.84  4.19 ± 3.72  2.35 ± 1.50  0.064  BMI  23.22 ± 3.54  22.32 ± 3.13  21.55 ± 2.43  0.205  bFSH (IU/L)  5.92 ± 1.64  6.47 ± 1.22  6.10 ± 1.34  0.447  bLH (IU/L)  4.67 ± 3.00  5.97 ± 5.01  4.53 ± 1.84  0.392  bE2 (pg/ml)  39.46 ± 17.91  40.00 ± 14.47  31.59 ± 15.25  0.273  AMH  8.39 ± 11.60  7.93 ± 3.08  6.81 ± 3.73  0.78  AFC  11.82 ± 3.71  10.17 ± 5.58  11.14 ± 5.92  0.1  Total dose of Gn (IU)  2584.24 ± 1501.91  2037.98 ± 929.84  2285.12 ± 796.92  0.237  Group  Control (n = 23)  10 mM (n = 25)  15 mM (n = 20)  P-value  Age (years)  30.18 ± 4.76  30.38 ± 4.73  29.86 ± 5.03  0.933  Duration of infertility (years)  4.07 ± 2.84  4.19 ± 3.72  2.35 ± 1.50  0.064  BMI  23.22 ± 3.54  22.32 ± 3.13  21.55 ± 2.43  0.205  bFSH (IU/L)  5.92 ± 1.64  6.47 ± 1.22  6.10 ± 1.34  0.447  bLH (IU/L)  4.67 ± 3.00  5.97 ± 5.01  4.53 ± 1.84  0.392  bE2 (pg/ml)  39.46 ± 17.91  40.00 ± 14.47  31.59 ± 15.25  0.273  AMH  8.39 ± 11.60  7.93 ± 3.08  6.81 ± 3.73  0.78  AFC  11.82 ± 3.71  10.17 ± 5.58  11.14 ± 5.92  0.1  Total dose of Gn (IU)  2584.24 ± 1501.91  2037.98 ± 929.84  2285.12 ± 796.92  0.237  View Large Then we analyzed the first PB extrusion rate between the groups, which was 70.58% (control, 24/34), 56.41% (10 mM, 22/39) and 30.56% (15 mM, 11/36), respectively. With the increase in glucose level in IVM medium, the first PB extrusion rate significantly decreased (Fig. 1B, P = 0.003). High-glucose concentrations changed DNA methylation levels in the DMR of Peg3 The COBRA result showed that three samples of H19 were not completely digested by enzyme TaqαI and RsaI in the control group (Fig. 2A, red box). The same three samples of Peg3 in control group were also not completely digested by TaqαI and BstUI in control group (Fig. 3A, red box). So, we concluded that these three samples may be contaminated by somatic cells. Thus these three samples were excluded from the following experiments. All the other samples of H19 were completely cut by both enzymes TaqαI and RsaI in the control, 10 mM and 15 mM groups. This indicated that the DNA methylation level of H19 in oocytes may be not affected by different glucose concentrations in IVM medium and that the samples were not contaminated by somatic cells. We further confirmed these results using BS. As shown in Fig. 2B, the DNA methylation levels in the DMR of H19 were similar between the groups (99.5, 98.4 and 99.4%, P > 0.05). These results indicated that the DNA methylation level in the DMR of H19 in human IVM oocytes was not affected by increased glucose concentrations in IVM medium. Figure 2 View largeDownload slide DNA methylation patterns of H19 in human IVM oocytes. (A) COBRA result of H19. The size of the target fragment is 218 bp. The red box means these samples may be contaminated by somatic cells and these were therefore excluded from the following experiments. M, DNA ladder; the number with red arrow is the size of the DNA ladder bands; (B) Bisulfite sequencing result. The percentage number is the DNA demethylation level of H19 in each group. Black circles, methylated CpG sites; white circles, unmethylated CpG sites; gray circles, lost CpG sites. Figure 2 View largeDownload slide DNA methylation patterns of H19 in human IVM oocytes. (A) COBRA result of H19. The size of the target fragment is 218 bp. The red box means these samples may be contaminated by somatic cells and these were therefore excluded from the following experiments. M, DNA ladder; the number with red arrow is the size of the DNA ladder bands; (B) Bisulfite sequencing result. The percentage number is the DNA demethylation level of H19 in each group. Black circles, methylated CpG sites; white circles, unmethylated CpG sites; gray circles, lost CpG sites. Figure 3 View largeDownload slide DNA methylation patterns of Peg3 in human IVM oocytes. (A) COBRA result of Peg3. The size of the target fragment is 224 bp. The red box means these samples may be contaminated by somatic cells and these were therefore excluded from the following experiments. The blue arrows represent the samples which were not completely digested by one or both enzymes. M, DNA ladder; the number with red arrow is the size of the DNA ladder bands; (B) Bisulfite sequencing result. The percentage number is the DNA methylation level of Peg3 in each group. Black circles, methylated CpG sites; white circles, unmethylated CpG sites; gray circles, the lost CpG sites. Figure 3 View largeDownload slide DNA methylation patterns of Peg3 in human IVM oocytes. (A) COBRA result of Peg3. The size of the target fragment is 224 bp. The red box means these samples may be contaminated by somatic cells and these were therefore excluded from the following experiments. The blue arrows represent the samples which were not completely digested by one or both enzymes. M, DNA ladder; the number with red arrow is the size of the DNA ladder bands; (B) Bisulfite sequencing result. The percentage number is the DNA methylation level of Peg3 in each group. Black circles, methylated CpG sites; white circles, unmethylated CpG sites; gray circles, the lost CpG sites. However, we found that some samples (blue arrows) were not completely digested by TaqαI and BstUI in the three groups (Fig 3A, blue arrows) and the number of undigested samples showed a decreased trend with an increased of glucose concentration in IVM medium (Fig. 3A). This indicated that DNA methylation levels in the DMR of Peg3 may be affected by the increased glucose concentration in IVM medium. So we further tested the DNA methylation level in DMR of Peg3 using BS. The result showed that with the increase in glucose concentration in IVM medium, the DNA methylation level in DMR of Peg3 showed a significant increase from 66.8% (control) to 97.8% (15 mM, Fig. 3B, P < 0.0001). DNA methylation in the promoter of adiponectin was affected by glucose concentrations In the present study, we also analyzed the DNA methylation level in the promoter of adiponectin in oocytes using BS. As shown in Fig. 4A, the methylation levels in the promotor of adiponectin in the control, 10 mM and 15 mM groups were 24.0, 9.1 and 15.2%, respectively. Statistical results showed that the DNA methylation level in control was significantly higher than that in 10 mM group (P = 0.028). The DNA methylation level in promoter of adiponectin was slightly lower in the 10 mM than in 15 mM (P = 0.451). In 15 mM, the DNA methylation level of adiponectin was lower than in control, but there was no statistical difference between them (P = 0.135). Figure 4 View largeDownload slide DNA methylation status in promoter of adiponectin in human IVM oocytes. (A) The bisulfite sequencing result. The percentage number is the DNA methylation level in each group. Black circles, methylated CpG sites; white circles, unmethylated CpG sites; yellow circles, lost CpG sites; (B) CpG sites in the target fragment. The red bold bases are the CpG sites in the target sequence. The sites were marked as Site 1, Site 2 and Site 3, respectively. (C) Methylation levels at each CpG site in groups. The DNA methylation level is represented as percentage. * means P < 0.05. Figure 4 View largeDownload slide DNA methylation status in promoter of adiponectin in human IVM oocytes. (A) The bisulfite sequencing result. The percentage number is the DNA methylation level in each group. Black circles, methylated CpG sites; white circles, unmethylated CpG sites; yellow circles, lost CpG sites; (B) CpG sites in the target fragment. The red bold bases are the CpG sites in the target sequence. The sites were marked as Site 1, Site 2 and Site 3, respectively. (C) Methylation levels at each CpG site in groups. The DNA methylation level is represented as percentage. * means P < 0.05. We further analyzed the DNA methylation level at each CpG site (red bases) which was marked as Site 1, Site 2 and Site 3 (Fig. 4B). The DNA methylation level was 33.3, 0 and 27.3% at Site 1, 14.3, 0 and 0% at Site 2, and 42.9, 27.3 and 18.2% at Site 3, respectively (Fig. 4C). Although the DNA methylation level at Sites 2 (P = 0.189) and 3 (P = 0.397) showed no significant differences between groups, it showed a decreased trend with the increase in glucose concentration in IVM medium. But at Site 1, the methylation level was higher in control than in 10 mM group (P = 0.035).There were no significant differences between the control and 15 mM groups (P = 0.191), and the 10 mM and 15 mM groups (P = 0.062) at Site 1 (Fig. 4C). These results showed that there is a decreasing trend in DNA methylation level in the promoter of Adiponectin in human IVM oocytes with the increase in glucose concentration in IVM medium. Discussion Oocyte maturation includes cytoplasmic maturation and nuclear maturation (Coticchio et al., 2015), both of which are important for fertilization and embryo development. One of the most crucial events during nuclear maturation is establishing proper DNA methylation (Geiman and Robertson, 2002; Seisenberger et al., 2013). We know that DNA methylation can be affected by internal and external environment. Maternal diabetes mellitus is a crucial internal factor, causing a decrease in oocyte quality and abnormalities in embryo development (Wang and Moley, 2010), which are mainly caused by the high-blood glucose level. In the present study, we found the first PB extrusion rate of human oocytes was significantly decreased by a higher glucose concentration in IVM medium. In streptozotocin-induced mouse model, DNA methylation levels in the DMR of imprinted gene Peg3 in oocytes were altered (Ge et al., 2013). But the effects of high glucose level on the DNA methylation level in human oocytes were not clear. The effects of environmental changes on different oocyte stages are different (Ge et al., 2013). In the present study, the MI stage oocytes were chosen to test the effects of glucose levels on DNA methylation patterns in human IVM oocytes. We found that the DNA methylation level in the DMR of the maternal imprinted gene Peg3 showed a significant increase in human oocytes with an increase in glucose concentrations in IVM medium. Peg3 is a paternally monoallelic expressed gene which is controlled by the DNA methylation status in the DMR established during gametogenesis and is involved in embryo development, cell growth, muscle development, autophagy and control of the nutritional supply between pups and mothers (Relaix et al., 1996; Dowdy et al., 2005; Feng et al., 2008; Broad and Keverne, 2011; Buraschi et al., 2013; Flisikowski et al., 2010). But alterations of maternal internal environment and environmental pollutants can induce changes inDNA methylation patterns of Peg3 in oocytes and embryos (Liang et al., 2011; Chao et al., 2012). For example, diabetes alters DNA methylation of Peg3 in mouse oocytes (Ge et al., 2013). But in human, there were few studies about the effects of hyperglycemia on DNA methylation of oocytes, because it is difficult to obtain materials from diabetic women. Thus, we used an in vitro maturation model to test the effects of high glucose on DNA methylation of human oocytes for the first time. We found that the methylation level in DMR of H19 was not influenced by the increased glucose concentration in human IVM oocytes. But the DNA methylation level in the DMR of maternal imprinted gene Peg3 was increased with the increasing glucose concentrations. To avoid somatic cell contamination, the paternal imprinted gene H19 was used as control. Thus, we concluded that the high glucose concentration in IVM medium induced the alteration of the DNA methylation level of Peg3. Studies showed that oocytes could directly take up glucose and the high environmental glucose concentration can induce glucose accumulation in mouse oocytes (Wang et al., 2012; Frank et al., 2013). Oocytes may utilize glucose through the pentose phosphate pathway (PPP) which is important for nuclear maturation and the progression of all stages of oocyte maturation, such as meiotic resumption, MI–MII transition and the resumption of meiosis after fertilization (Sutton-McDowall et al., 2005, 2010; Herrick et al., 2006). We know that glucose concentrations which are too low (<2.3 mM) or too high (>10 mM) in vitro are detrimental to oocyte maturation (Sutton-McDowall et al., 2005, 2010). However, we found the DNA methylation level in DMR of Peg3 in 15 mM group oocytes was more close to 100% compared to the other groups. According to the research results in non-human mammalian oocytes (Bromfield et al., 2008; Hales et al., 2011; Ge et al., 2013), Peg3 should have a high DNA methylation level in human oocytes. This contradiction may be caused by few studies about the natural dynamics of DNA methylation status of the imprinted gene Peg3. Although a study reported that increased imprinting errors in human oocyte were not associated with IVM, this study only tested the DNA methylation status at three CpG sites of Peg3 and oocytes were from PCOS patients (Kuhtz et al., 2014). So it cannot represent the DNA methylation status of Peg3 in the natural situation. In macaques, the DNA methylation level in the DMR of Peg3 is low in oocytes but it is high in tissues, and the higher DNA methylation level in the DMR of Peg3 was established during embryo development (Cheong et al., 2015). Another study showed the expression of Peg3 in human ovary was obviously higher than in other tissues (Kim et al., 1997). These studies indicate that the DNA methylation level in DMR of Peg3 may be low in human oocytes in the natural condition. Therefore, the high DNA methylation level of Peg3 in oocytes in the 15 mM group would have been induced by high-glucose concentrations. Adiponectin expression is regulated by DNA methylation modification in the promoter (Houde et al., 2014) and it maintains systematic energy homeostasis (Kishida et al., 2014; Lee and Shao, 2014). Abnormal adiponectin level in blood is associated with many chronic diseases, such as diabetes, obesity, hypertension and vascular disease (Kishida et al., 2014). A previous study showed that offspring of diabetic mothers had a high risk of obesity, diabetes, hypertension, and cardiovascular diseases (Battista et al., 2011). The risk of these diseases may originate from the altered epigenetic modifications in oocytes (Ge et al., 2014a,b). We found that the methylation of Adiponectin was 24% in the control oocytes, which was similar to the methylation level in placenta (about 30%) (Bouchard et al., 2012). But there was a decreasing trend in the DNA methylation level of Adiponectin in human IVM oocytes with an increase in glucose levels. We further analyzed the methylation level at each CpG site in human IVM oocytes and found that there was a significant decrease in DNA methylation at CpG site 1 with the increase in glucose level in IVM medium. In the placenta in gestational diabetes mellitus, the DNA methylation level at CpG sites of Adiponectin is also lower after the second trimester and that is significantly associated with the higher maternal serum adiponectin levels (Bouchard et al., 2012). If obese children have insulin resistance, the DNA methylation level of adiponectin is significantly decreased and is significantly associated with serum adiponectin levels (Garcia-Cardona et al., 2014). Adiponectin is also crucial for fetal fat deposition and embryo development (Cikos et al., 2010; Reverchon et al., 2014). Thus, the decreased DNA methylation of adiponectin in human oocytes caused by high-glucose concentrations may be adverse to fetal development and offspring health. Although we found that high-glucose concentrations altered the DNA methylation status of Peg3 and adiponectin in human IVM oocytes, it is not clear whether the alteration is good or bad for embryo development and offspring health. The effects of high-glucose concentration on the whole process of oocyte maturation are still not elucidated. Another issue is that the oocyte number used in this study was limited. Therefore, more work is required to elucidate the influence of high-glucose levels on DNA methylation status in human oocytes. However as a start, we have found for the first time that DNA methylation status in human IVM oocytes is altered by high glucose concentrations. Authors’ roles W.Q. and T.S.-B. designed the study, analyzed the data and interpreted the data; S.X.-B. D-T.-F. and Z.T.-T. interpreted the data; Y.S., L.S.-M., S.W. and Z-C.-L. participated in conception of the study design and revised the manuscript; G.Z-J. conceived the study design, participated in the data analysis and interpretation and wrote the manuscript. Funding This work was supported by the fund of National Natural Science Foundation of China (81401198) and Doctor Foundation of Qingdao Agricultural University (1116008). Conflict of interest The authors declare that there are no potential conflicts of interest relevant to this article. Acknowledgements We acknowledge Juan-Ke Xie, Ya-Nan Zhang, Qi Liu, Duo Wei, Lin Hu and other staff working in the Reproductive Medicine Center of People’s Hospital of Zhengzhou University for their contribution to this work. References Battista MC, Hivert MF, Duval K, Baillargeon JP. 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High-glucose concentrations change DNA methylation levels in human IVM oocytes

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© The Author(s) 2018. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com
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Abstract

Abstract STUDY QUESTION What are the effects of high-glucose concentrations on DNA methylation of human oocytes? SUMMARY ANSWER High-glucose concentrations altered DNA methylation levels of Peg3 and Adiponectin in human in vitro maturation oocytes. WHAT IS KNOWN ALREADY Maternal diabetes has a detrimental influence on oocyte quality including epigenetic modifications, as shown in non-human mammalian species. STUDY DESIGN, SIZE, DURATION 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. PARTICIPANTS/MATERIALS, SETTING, METHODS 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). MAIN RESULTS AND THE ROLE OF CHANCE 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). LARGE SCALE DATA N/A LIMITATIONS REASONS FOR CAUTION 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. WIDER IMPLICATIONS OF THE FINDINGS 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. STUDY FUNDING/COMPETING INTEREST(S) 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. human oocyte, DNA methylation, high glucose concentrations, in vitro maturation, diabetes, epigenetics Introduction Diabetes is defined as ‘fasting blood glucose equal to or higher than 7 mmol/l, or on medication for raised blood glucose, or with a history of diagnosis diabetes’ by the World Health Organization (WHO). If the distribution of fasting plasma glucose in a population is higher than the theoretical distribution, it is termed as high-blood glucose. The WHO has estimated that, globally, 422 million adults who are over 18 years would live with diabetes in 2014. The global incidence of diabetes has increased from 4.7% in 1980 to 8.5% in 2014, while the prevalence of diabetes in the WTO Mediterranean Region is as high as 13.7% (http://www.who.int/mediacentre/factsheets/fs312/en/). Not only are there complication of diabetes in patients themselves but also there are deleterious effects on their offspring (Vrachnis et al., 2012a,b,c). The long-term consequences for offspring of diabetic parents include a higher risks of insulin resistance, abnormal glucose metabolism and chronic diseases (Vrachnis et al., 2012a,b,c). The complications of diabetes are mainly caused by hyperglycemia which can induce a delay in embryo development, a higher incidence of degenerate and fragmented embryos, growth retardation and failure of anterior neuropore closure (Jungheim and Moley, 2008). High-blood glucose also changes the micro-environment of follicle development which is important for oocyte maturation, oocyte quality and embryonic development. At physiological levels, the oocyte is exposed to glucose levels of 31–82% of follicle fluid concentrations (Stokes et al., 2008). During oocyte maturation in vitro, proper glucose concentrations in the medium can increase the oocyte maturation rate and quality (Sutton-McDowall et al., 2010). But in vivo and in vitro experiments have demonstrated that high-glucose levels lead to poor developmental potential of oocytes and embryos (Sutton-McDowall et al., 2005, 2010) because of poor oocyte quality (Colton et al., 2002; Wang et al., 2009; Wang and Moley, 2010). A high glucose environment increases the glucose levels within the follicle and causes glucose accumulation in oocytes (Moley et al., 1998; Wang et al., 2012). Although oocytes have a poor capacity to utilize glucose (Biggers et al., 1967), the up-take glucose and glucose metabolism has been detected in denuded oocytes of different species (Downs, 1995; Wang et al., 2012). Furthermore, glucose is essential for the nuclear maturation of oocytes (Krisher and Bavister, 1998; Rose-Hellekant et al., 1998). Establishing proper DNA methylation is a crucial event during oocyte nuclear maturation (Stewart et al., 2016). However, the DNA methylation status is prone to be altered by changes in the internal and external environment, especially during gametogenesis and embryonic development (Feil and Fraga, 2012; Tammen et al., 2013). Previous studies have demonstrated that maternal hyperglycemia changes DNA methylation in mouse oocytes (Ge et al., 2013). But the effects of hyperglycemia on DNA methylation in human oocytes are not well elucidated. We know that it is difficult to obtain oocytes from diabetic women and the detrimental effects of diabetes on patients are mainly caused by the high-blood glucose level. Therefore, we used an in vitro maturation (IVM) model to check the influence of high glucose levels on the DNA methylation status in human oocytes. Generally, the glucose concentration of human follicle fluid is about 3.3 mM (Leese and Lenton, 1990). It is detrimental to oocytes if the glucose concentration is over 10 mM in medium (Sutton-McDowall et al., 2010). Thus, in the present study, we investigated the effects of various glucose concentrations (control (5.6 mM), 10 mM and 15 mM glucose in IVM medium) on DNA methylation in human IVM oocytes. Another study reported that the DNA methylation level in the differentially methylated region (DMR) of imprinted gene Peg3 was changed in oocytes of diabetic mouse (Ge et al., 2013). Therefore, we checked the methylation patterns of the maternal imprinted gene Peg3 and the paternal imprinted gene H19 which was used as control. Meanwhile, we also tested the DNA methylation level at the promoter region of Adiponectin because it plays a key role in onset of metabolic diseases (Lee and Shao, 2014) and the offspring of diabetic women are prone to obesity and other chronic diseases (Vrachnis et al., 2012a,b,c). Materials and Methods All of the procedures in the present study were reviewed and supported by the Ethic Committee of Life Science of Zhengzhou University. We received agreement, permission and signed consent from all patients included in the present study. All reagents were purchased from Sigma-Aldrich, unless indicated specifically. Criteria for choosing patients Women who were infertile due to non-ovarian reasons and who underwent IVF using ICSI intracytoplasmic sperm injection in the Reproductive Center of People’s Hospital of Zhengzhou University were included in this study. The women had no any other diseases which may affect ovarian function, such as polycystic ovarian syndrome (PCOS), diabetes, and other endocrine and metabolic diseases. All patients were younger than 35 years old. Oocyte collection and in vitro maturation Oocytes surrounded with cumulus cells (COCs) of patients were retrieved from fully grown follicles by ultrasound-guided at 36–37 h after hCG injection. COCs were cultured in vitro at 37°C with 5% mixed gas (O2, CO2 and N2) for 5 h. Then cumulus cells were removed using 80 IU/ml hyaluronidase in IVF medium. If oocytes were mature, they were used for ICSI. If oocytes were immature (at MI stage) with normal morphology and cytoplasm (Fig. 1A), they were collected and used in the present study. Figure 1 View largeDownload slide High-glucose concentrations reduced the first PB extrusion rate. (A) Representative pictures of human MI oocytes used for IVM and MII oocytes after IVM. 20×, magnification; (B) The first PB extrusion rate was calculated in each group and expressed as percentage. *P < 0.05; **P < 0.01; first PB, the first polar body. Figure 1 View largeDownload slide High-glucose concentrations reduced the first PB extrusion rate. (A) Representative pictures of human MI oocytes used for IVM and MII oocytes after IVM. 20×, magnification; (B) The first PB extrusion rate was calculated in each group and expressed as percentage. *P < 0.05; **P < 0.01; first PB, the first polar body. IVM medium was made using Tissue Culture Medium 199 (TCM199) supplemented with 5 mg/ml human transferrin, 5 ng/ml sodium selenite,10 ng/ml human recombinant insulin, 10 mIU/ml FSH (Merk, Germany), 1 mM L-glutamine, 0.3 mM sodium pyruvate, 0.8%HSA, 100 IU/ml penicillin G, 100 mg/ml streptomycin sulfate, 10 ng/ml epidermal growth factor, 1 mg/ml estradiol and 0.5 IU/ml hCG (Lizhu, China). Oocytes were divided into three groups: the control group in which oocytes were cultured in IVM medium; the 10 mM group in which oocytes were cultured in IVM medium with 10 mM glucose and the 15 mM group in which oocytes were cultured in IVM medium with 15 mM glucose. The oocyte maturation was at 37°C with 5% mixed gas. After 48 h, oocytes developed to MII stage (with the first polar body (first PB) extruded) were collected and stored at −80°C for the following experiments. Bisulfite treatment and PCR amplification DNA bisulfite transformation was carried out as in a previous study (Ge et al., 2014a,,b). Briefly, oocytes were lysed for 37 min at 37°C and then added to 15 μl low melting agarose in a previous tube. The agarose with DNA was transferred into a 2.0 ml Eppendorf tube with 200 μl pre-cooled mineral oil. Then we carefully transferred the agarose bead into a new 2.0 ml Eppendorf tube and added 500 μl pre-prepared bisulfite buffer. These tubes with the agarose bead and bisulfite buffer were cooled on ice for 15 min and then incubated in water bath for 4 h at 50°C. The incubated beads were washed for 15 min three times with TE (Tris-HCl and EDTA), for 15 min two times with 0.3 M NaOH, and for 15 min three times with distilled water. After washing, beads were stored at −20°C until used. Targeted DNA fragments were amplified using nested or half-nested PCR. For the first round PCR, beads were melted at 70°C in 200 μl tubes and then an amplification mix, including buffer, dNTP, hot-start DNA polymerase, water and the first round PCR primers of Peg3, H19 and Adiponectin, was added into tubes. A1 μl sample of the product of the first round PCR was used as template for the secondary round of PCR for Peg3, H19 and adiponectin, respectively. Primers used in this study are shown in Table I. Table I Oligonucleotides used for nested and half-nested PCR. Primer name  Primer sequence  H19-outer forward  5′-AGGTGTTTTAGTTTTATGGATGATGG-3′  H19-inner forward  5′-TGTATAGTATATATATGGGTATTTTTGGAGGTTT-3′  H19-reverse  5′-TCCTATAAATATCCTATTCCCAAATAACC-3′  Peg3-forward  5′-GGTTGTTGATTGGTTAGTATAG-3′  Peg3-outer reverse  5′-CACTCACCTCACCTCAATAC-3′  Peg3-inner reverse  5′-ACCTCACCTCAATACTAC-3′  Adiponectin-outer forward  5′-GGGTAGGTAGATATTTGTTT-3′  Adiponectin-inner forward  5′-GGGGTAGGTAGATATTTGTTTTGTTT-3′  Adiponectin-outer reverse  5′-TCAACACCTTAAACTTTCTTAACA-3′  Adiponectin-inner reverse  5′-AAACTACCACCCACTTAA-3′  Primer name  Primer sequence  H19-outer forward  5′-AGGTGTTTTAGTTTTATGGATGATGG-3′  H19-inner forward  5′-TGTATAGTATATATATGGGTATTTTTGGAGGTTT-3′  H19-reverse  5′-TCCTATAAATATCCTATTCCCAAATAACC-3′  Peg3-forward  5′-GGTTGTTGATTGGTTAGTATAG-3′  Peg3-outer reverse  5′-CACTCACCTCACCTCAATAC-3′  Peg3-inner reverse  5′-ACCTCACCTCAATACTAC-3′  Adiponectin-outer forward  5′-GGGTAGGTAGATATTTGTTT-3′  Adiponectin-inner forward  5′-GGGGTAGGTAGATATTTGTTTTGTTT-3′  Adiponectin-outer reverse  5′-TCAACACCTTAAACTTTCTTAACA-3′  Adiponectin-inner reverse  5′-AAACTACCACCCACTTAA-3′  View Large Combined bisulfite restriction analysis and bisulfite sequencing To test the DNA methylation status of H19 and Peg3, combined bisulfite restriction analysis (COBRA) and and bisulfite sequencing (BS) were used in the present study as previous described (Ge et al., 2013). Briefly, the PCR products were digested by endogenous restriction enzymes (TaqαI, RsaI and/or BstUI, NEB, China) for which CpG sites are included in the recognition sites. For BS, the PCR products were pooled together for each group, cloned into a T vector and sequenced (GENEWIZ, China). The sequencing result was analyzed using CpGViewer. Statistical analysis Average data were presented as mean ± standard deviation and the significance between groups was tested using one-way analysis of variance. The methylation level was presented as a percentage and the significance between groups was checked using Chi-square test. If the P-value was <0.05, it was considered as showing a statistical difference between groups. Results High-glucose levels decreased the first PB extrusion rate In total, 109 good quality MI oocytes (Fig. 1A) were retrieved from 68 patients. These oocytes were randomly divided into the three groups: control (n = 34), 10 mM (n = 39) and 15 mM (n = 36). We analyzed the average age, average body mass index (BMI), average duration of infertility, antral follicle count (AFC), total dosages of gonadotropin and basic hormone levels including FSH, LH, estradiol (E2) and anti-Mullerian hormone (AMH). The results showed that all of these parameters were similar between groups (Table II). Table II Characteristics of patients. Group  Control (n = 23)  10 mM (n = 25)  15 mM (n = 20)  P-value  Age (years)  30.18 ± 4.76  30.38 ± 4.73  29.86 ± 5.03  0.933  Duration of infertility (years)  4.07 ± 2.84  4.19 ± 3.72  2.35 ± 1.50  0.064  BMI  23.22 ± 3.54  22.32 ± 3.13  21.55 ± 2.43  0.205  bFSH (IU/L)  5.92 ± 1.64  6.47 ± 1.22  6.10 ± 1.34  0.447  bLH (IU/L)  4.67 ± 3.00  5.97 ± 5.01  4.53 ± 1.84  0.392  bE2 (pg/ml)  39.46 ± 17.91  40.00 ± 14.47  31.59 ± 15.25  0.273  AMH  8.39 ± 11.60  7.93 ± 3.08  6.81 ± 3.73  0.78  AFC  11.82 ± 3.71  10.17 ± 5.58  11.14 ± 5.92  0.1  Total dose of Gn (IU)  2584.24 ± 1501.91  2037.98 ± 929.84  2285.12 ± 796.92  0.237  Group  Control (n = 23)  10 mM (n = 25)  15 mM (n = 20)  P-value  Age (years)  30.18 ± 4.76  30.38 ± 4.73  29.86 ± 5.03  0.933  Duration of infertility (years)  4.07 ± 2.84  4.19 ± 3.72  2.35 ± 1.50  0.064  BMI  23.22 ± 3.54  22.32 ± 3.13  21.55 ± 2.43  0.205  bFSH (IU/L)  5.92 ± 1.64  6.47 ± 1.22  6.10 ± 1.34  0.447  bLH (IU/L)  4.67 ± 3.00  5.97 ± 5.01  4.53 ± 1.84  0.392  bE2 (pg/ml)  39.46 ± 17.91  40.00 ± 14.47  31.59 ± 15.25  0.273  AMH  8.39 ± 11.60  7.93 ± 3.08  6.81 ± 3.73  0.78  AFC  11.82 ± 3.71  10.17 ± 5.58  11.14 ± 5.92  0.1  Total dose of Gn (IU)  2584.24 ± 1501.91  2037.98 ± 929.84  2285.12 ± 796.92  0.237  View Large Then we analyzed the first PB extrusion rate between the groups, which was 70.58% (control, 24/34), 56.41% (10 mM, 22/39) and 30.56% (15 mM, 11/36), respectively. With the increase in glucose level in IVM medium, the first PB extrusion rate significantly decreased (Fig. 1B, P = 0.003). High-glucose concentrations changed DNA methylation levels in the DMR of Peg3 The COBRA result showed that three samples of H19 were not completely digested by enzyme TaqαI and RsaI in the control group (Fig. 2A, red box). The same three samples of Peg3 in control group were also not completely digested by TaqαI and BstUI in control group (Fig. 3A, red box). So, we concluded that these three samples may be contaminated by somatic cells. Thus these three samples were excluded from the following experiments. All the other samples of H19 were completely cut by both enzymes TaqαI and RsaI in the control, 10 mM and 15 mM groups. This indicated that the DNA methylation level of H19 in oocytes may be not affected by different glucose concentrations in IVM medium and that the samples were not contaminated by somatic cells. We further confirmed these results using BS. As shown in Fig. 2B, the DNA methylation levels in the DMR of H19 were similar between the groups (99.5, 98.4 and 99.4%, P > 0.05). These results indicated that the DNA methylation level in the DMR of H19 in human IVM oocytes was not affected by increased glucose concentrations in IVM medium. Figure 2 View largeDownload slide DNA methylation patterns of H19 in human IVM oocytes. (A) COBRA result of H19. The size of the target fragment is 218 bp. The red box means these samples may be contaminated by somatic cells and these were therefore excluded from the following experiments. M, DNA ladder; the number with red arrow is the size of the DNA ladder bands; (B) Bisulfite sequencing result. The percentage number is the DNA demethylation level of H19 in each group. Black circles, methylated CpG sites; white circles, unmethylated CpG sites; gray circles, lost CpG sites. Figure 2 View largeDownload slide DNA methylation patterns of H19 in human IVM oocytes. (A) COBRA result of H19. The size of the target fragment is 218 bp. The red box means these samples may be contaminated by somatic cells and these were therefore excluded from the following experiments. M, DNA ladder; the number with red arrow is the size of the DNA ladder bands; (B) Bisulfite sequencing result. The percentage number is the DNA demethylation level of H19 in each group. Black circles, methylated CpG sites; white circles, unmethylated CpG sites; gray circles, lost CpG sites. Figure 3 View largeDownload slide DNA methylation patterns of Peg3 in human IVM oocytes. (A) COBRA result of Peg3. The size of the target fragment is 224 bp. The red box means these samples may be contaminated by somatic cells and these were therefore excluded from the following experiments. The blue arrows represent the samples which were not completely digested by one or both enzymes. M, DNA ladder; the number with red arrow is the size of the DNA ladder bands; (B) Bisulfite sequencing result. The percentage number is the DNA methylation level of Peg3 in each group. Black circles, methylated CpG sites; white circles, unmethylated CpG sites; gray circles, the lost CpG sites. Figure 3 View largeDownload slide DNA methylation patterns of Peg3 in human IVM oocytes. (A) COBRA result of Peg3. The size of the target fragment is 224 bp. The red box means these samples may be contaminated by somatic cells and these were therefore excluded from the following experiments. The blue arrows represent the samples which were not completely digested by one or both enzymes. M, DNA ladder; the number with red arrow is the size of the DNA ladder bands; (B) Bisulfite sequencing result. The percentage number is the DNA methylation level of Peg3 in each group. Black circles, methylated CpG sites; white circles, unmethylated CpG sites; gray circles, the lost CpG sites. However, we found that some samples (blue arrows) were not completely digested by TaqαI and BstUI in the three groups (Fig 3A, blue arrows) and the number of undigested samples showed a decreased trend with an increased of glucose concentration in IVM medium (Fig. 3A). This indicated that DNA methylation levels in the DMR of Peg3 may be affected by the increased glucose concentration in IVM medium. So we further tested the DNA methylation level in DMR of Peg3 using BS. The result showed that with the increase in glucose concentration in IVM medium, the DNA methylation level in DMR of Peg3 showed a significant increase from 66.8% (control) to 97.8% (15 mM, Fig. 3B, P < 0.0001). DNA methylation in the promoter of adiponectin was affected by glucose concentrations In the present study, we also analyzed the DNA methylation level in the promoter of adiponectin in oocytes using BS. As shown in Fig. 4A, the methylation levels in the promotor of adiponectin in the control, 10 mM and 15 mM groups were 24.0, 9.1 and 15.2%, respectively. Statistical results showed that the DNA methylation level in control was significantly higher than that in 10 mM group (P = 0.028). The DNA methylation level in promoter of adiponectin was slightly lower in the 10 mM than in 15 mM (P = 0.451). In 15 mM, the DNA methylation level of adiponectin was lower than in control, but there was no statistical difference between them (P = 0.135). Figure 4 View largeDownload slide DNA methylation status in promoter of adiponectin in human IVM oocytes. (A) The bisulfite sequencing result. The percentage number is the DNA methylation level in each group. Black circles, methylated CpG sites; white circles, unmethylated CpG sites; yellow circles, lost CpG sites; (B) CpG sites in the target fragment. The red bold bases are the CpG sites in the target sequence. The sites were marked as Site 1, Site 2 and Site 3, respectively. (C) Methylation levels at each CpG site in groups. The DNA methylation level is represented as percentage. * means P < 0.05. Figure 4 View largeDownload slide DNA methylation status in promoter of adiponectin in human IVM oocytes. (A) The bisulfite sequencing result. The percentage number is the DNA methylation level in each group. Black circles, methylated CpG sites; white circles, unmethylated CpG sites; yellow circles, lost CpG sites; (B) CpG sites in the target fragment. The red bold bases are the CpG sites in the target sequence. The sites were marked as Site 1, Site 2 and Site 3, respectively. (C) Methylation levels at each CpG site in groups. The DNA methylation level is represented as percentage. * means P < 0.05. We further analyzed the DNA methylation level at each CpG site (red bases) which was marked as Site 1, Site 2 and Site 3 (Fig. 4B). The DNA methylation level was 33.3, 0 and 27.3% at Site 1, 14.3, 0 and 0% at Site 2, and 42.9, 27.3 and 18.2% at Site 3, respectively (Fig. 4C). Although the DNA methylation level at Sites 2 (P = 0.189) and 3 (P = 0.397) showed no significant differences between groups, it showed a decreased trend with the increase in glucose concentration in IVM medium. But at Site 1, the methylation level was higher in control than in 10 mM group (P = 0.035).There were no significant differences between the control and 15 mM groups (P = 0.191), and the 10 mM and 15 mM groups (P = 0.062) at Site 1 (Fig. 4C). These results showed that there is a decreasing trend in DNA methylation level in the promoter of Adiponectin in human IVM oocytes with the increase in glucose concentration in IVM medium. Discussion Oocyte maturation includes cytoplasmic maturation and nuclear maturation (Coticchio et al., 2015), both of which are important for fertilization and embryo development. One of the most crucial events during nuclear maturation is establishing proper DNA methylation (Geiman and Robertson, 2002; Seisenberger et al., 2013). We know that DNA methylation can be affected by internal and external environment. Maternal diabetes mellitus is a crucial internal factor, causing a decrease in oocyte quality and abnormalities in embryo development (Wang and Moley, 2010), which are mainly caused by the high-blood glucose level. In the present study, we found the first PB extrusion rate of human oocytes was significantly decreased by a higher glucose concentration in IVM medium. In streptozotocin-induced mouse model, DNA methylation levels in the DMR of imprinted gene Peg3 in oocytes were altered (Ge et al., 2013). But the effects of high glucose level on the DNA methylation level in human oocytes were not clear. The effects of environmental changes on different oocyte stages are different (Ge et al., 2013). In the present study, the MI stage oocytes were chosen to test the effects of glucose levels on DNA methylation patterns in human IVM oocytes. We found that the DNA methylation level in the DMR of the maternal imprinted gene Peg3 showed a significant increase in human oocytes with an increase in glucose concentrations in IVM medium. Peg3 is a paternally monoallelic expressed gene which is controlled by the DNA methylation status in the DMR established during gametogenesis and is involved in embryo development, cell growth, muscle development, autophagy and control of the nutritional supply between pups and mothers (Relaix et al., 1996; Dowdy et al., 2005; Feng et al., 2008; Broad and Keverne, 2011; Buraschi et al., 2013; Flisikowski et al., 2010). But alterations of maternal internal environment and environmental pollutants can induce changes inDNA methylation patterns of Peg3 in oocytes and embryos (Liang et al., 2011; Chao et al., 2012). For example, diabetes alters DNA methylation of Peg3 in mouse oocytes (Ge et al., 2013). But in human, there were few studies about the effects of hyperglycemia on DNA methylation of oocytes, because it is difficult to obtain materials from diabetic women. Thus, we used an in vitro maturation model to test the effects of high glucose on DNA methylation of human oocytes for the first time. We found that the methylation level in DMR of H19 was not influenced by the increased glucose concentration in human IVM oocytes. But the DNA methylation level in the DMR of maternal imprinted gene Peg3 was increased with the increasing glucose concentrations. To avoid somatic cell contamination, the paternal imprinted gene H19 was used as control. Thus, we concluded that the high glucose concentration in IVM medium induced the alteration of the DNA methylation level of Peg3. Studies showed that oocytes could directly take up glucose and the high environmental glucose concentration can induce glucose accumulation in mouse oocytes (Wang et al., 2012; Frank et al., 2013). Oocytes may utilize glucose through the pentose phosphate pathway (PPP) which is important for nuclear maturation and the progression of all stages of oocyte maturation, such as meiotic resumption, MI–MII transition and the resumption of meiosis after fertilization (Sutton-McDowall et al., 2005, 2010; Herrick et al., 2006). We know that glucose concentrations which are too low (<2.3 mM) or too high (>10 mM) in vitro are detrimental to oocyte maturation (Sutton-McDowall et al., 2005, 2010). However, we found the DNA methylation level in DMR of Peg3 in 15 mM group oocytes was more close to 100% compared to the other groups. According to the research results in non-human mammalian oocytes (Bromfield et al., 2008; Hales et al., 2011; Ge et al., 2013), Peg3 should have a high DNA methylation level in human oocytes. This contradiction may be caused by few studies about the natural dynamics of DNA methylation status of the imprinted gene Peg3. Although a study reported that increased imprinting errors in human oocyte were not associated with IVM, this study only tested the DNA methylation status at three CpG sites of Peg3 and oocytes were from PCOS patients (Kuhtz et al., 2014). So it cannot represent the DNA methylation status of Peg3 in the natural situation. In macaques, the DNA methylation level in the DMR of Peg3 is low in oocytes but it is high in tissues, and the higher DNA methylation level in the DMR of Peg3 was established during embryo development (Cheong et al., 2015). Another study showed the expression of Peg3 in human ovary was obviously higher than in other tissues (Kim et al., 1997). These studies indicate that the DNA methylation level in DMR of Peg3 may be low in human oocytes in the natural condition. Therefore, the high DNA methylation level of Peg3 in oocytes in the 15 mM group would have been induced by high-glucose concentrations. Adiponectin expression is regulated by DNA methylation modification in the promoter (Houde et al., 2014) and it maintains systematic energy homeostasis (Kishida et al., 2014; Lee and Shao, 2014). Abnormal adiponectin level in blood is associated with many chronic diseases, such as diabetes, obesity, hypertension and vascular disease (Kishida et al., 2014). A previous study showed that offspring of diabetic mothers had a high risk of obesity, diabetes, hypertension, and cardiovascular diseases (Battista et al., 2011). The risk of these diseases may originate from the altered epigenetic modifications in oocytes (Ge et al., 2014a,b). We found that the methylation of Adiponectin was 24% in the control oocytes, which was similar to the methylation level in placenta (about 30%) (Bouchard et al., 2012). But there was a decreasing trend in the DNA methylation level of Adiponectin in human IVM oocytes with an increase in glucose levels. We further analyzed the methylation level at each CpG site in human IVM oocytes and found that there was a significant decrease in DNA methylation at CpG site 1 with the increase in glucose level in IVM medium. In the placenta in gestational diabetes mellitus, the DNA methylation level at CpG sites of Adiponectin is also lower after the second trimester and that is significantly associated with the higher maternal serum adiponectin levels (Bouchard et al., 2012). If obese children have insulin resistance, the DNA methylation level of adiponectin is significantly decreased and is significantly associated with serum adiponectin levels (Garcia-Cardona et al., 2014). Adiponectin is also crucial for fetal fat deposition and embryo development (Cikos et al., 2010; Reverchon et al., 2014). Thus, the decreased DNA methylation of adiponectin in human oocytes caused by high-glucose concentrations may be adverse to fetal development and offspring health. Although we found that high-glucose concentrations altered the DNA methylation status of Peg3 and adiponectin in human IVM oocytes, it is not clear whether the alteration is good or bad for embryo development and offspring health. The effects of high-glucose concentration on the whole process of oocyte maturation are still not elucidated. Another issue is that the oocyte number used in this study was limited. Therefore, more work is required to elucidate the influence of high-glucose levels on DNA methylation status in human oocytes. However as a start, we have found for the first time that DNA methylation status in human IVM oocytes is altered by high glucose concentrations. Authors’ roles W.Q. and T.S.-B. designed the study, analyzed the data and interpreted the data; S.X.-B. D-T.-F. and Z.T.-T. interpreted the data; Y.S., L.S.-M., S.W. and Z-C.-L. participated in conception of the study design and revised the manuscript; G.Z-J. conceived the study design, participated in the data analysis and interpretation and wrote the manuscript. Funding This work was supported by the fund of National Natural Science Foundation of China (81401198) and Doctor Foundation of Qingdao Agricultural University (1116008). Conflict of interest The authors declare that there are no potential conflicts of interest relevant to this article. Acknowledgements We acknowledge Juan-Ke Xie, Ya-Nan Zhang, Qi Liu, Duo Wei, Lin Hu and other staff working in the Reproductive Medicine Center of People’s Hospital of Zhengzhou University for their contribution to this work. References Battista MC, Hivert MF, Duval K, Baillargeon JP. 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Human ReproductionOxford University Press

Published: Mar 1, 2018

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