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High Frequency of Imprinted Methylation Errors in Human Preimplantation Embryos.

ABSTRACT: Assisted reproductive technologies (ARTs) represent the best chance for infertile couples to conceive, although increased risks for morbidities exist, including imprinting disorders. This increased risk could arise from ARTs disrupting genomic imprints during gametogenesis or preimplantation. The few studies examining ART effects on genomic imprinting primarily assessed poor quality human embryos. Here, we examined day 3 and blastocyst stage, good to high quality, donated human embryos for imprinted SNRPN, KCNQ1OT1 and H19 methylation. Seventy-six percent day 3 embryos and 50% blastocysts exhibited perturbed imprinted methylation, demonstrating that extended culture did not pose greater risk for imprinting errors than short culture. Comparison of embryos with normal and abnormal methylation didn't reveal any confounding factors. Notably, two embryos from male factor infertility patients using donor sperm harboured aberrant methylation, suggesting errors in these embryos cannot be explained by infertility alone. Overall, these results indicate that ART human preimplantation embryos possess a high frequency of imprinted methylation errors.

SUBMITTER: White CR

PROVIDER: S-EPMC4667293 | biostudies-literature | 2015 Dec

REPOSITORIES: biostudies-literature

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High Frequency of Imprinted Methylation Errors in Human Preimplantation Embryos.

White Carlee R CR Denomme Michelle M MM Tekpetey Francis R FR Feyles Valter V Power Stephen G A SG Mann Mellissa R W MR

Scientific reports 20151202

Assisted reproductive technologies (ARTs) represent the best chance for infertile couples to conceive, although increased risks for morbidities exist, including imprinting disorders. This increased risk could arise from ARTs disrupting genomic imprints during gametogenesis or preimplantation. The few studies examining ART effects on genomic imprinting primarily assessed poor quality human embryos. Here, we examined day 3 and blastocyst stage, good to high quality, donated human embryos for impri ...[more]

PMID: 26626153

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Project description: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.

Project description:PURPOSEThe purpose of this paper is to study whether human preimplantation embryos regulate endometrial stromal cell (hESC) migration.METHODSPrimary hESCs were isolated from fertile patients undergoing hysterectomy for benign conditions (uterine scar niche n?=?3, dysmenorrhea n?=?2; no hormonal treatment). Migration and proliferation assays were performed by culturing decidualized or non-decidualized hESCs in the presence of embryo conditioned medium (ECM) from high-quality embryos (fragmentation ??20%) or from low-quality embryos (fragmentation >?20%) or in non-conditioned medium from the same dishes (control). ECM samples from 425 individually cultured human embryos were used in this study.RESULTSECM from high-quality embryos, i.e., with a low percentage of fragmentation, actively stimulated decidualized hESC migration (p?<?0.001). This effect was consistent throughout embryonic development from cleavage stage embryos with 2-7 cells (high quality vs. control; p?=?0.036), 8-18 cells (high quality vs. control; p?<?0.001) to morulae (high quality vs. control; p?=?0.003). Additionally, linear regression analysis showed that hESC migration was influenced by embryo quality (fragmentation, ? -?0.299; p?=?0.025) and not developmental stage (cell number, ? 0.177; p?=?0.176) or maternal age (? -?0.036; p?=?0.78). Opposite to decidualized hESCs, the migration response of non-decidualized hESCs was inhibited by ECM from high-quality embryos (p?=?0.019). ECM from low-quality embryos, i.e., with a high percentage of fragmentation, did not cause an altered migration response in decidualized hESCs (p?=?0.860) or non-decidualized hESCs (p?=?0.986). Furthermore, ECM of both high- and low-quality human embryos did not influence the number of proliferating cells (p?=?0.375) and the cell cycle time (p?=?0.297) of non-decidualized or decidualized hESCs.CONCLUSIONThis study reveals a mechanism by which high-quality human preimplantation embryos actively interact with the endometrium to increase their chances of successful implantation.

Project description:The methylation of cytosine at CG sites in the mammalian genome is dynamically reprogrammed during gametogenesis and preimplantation development. It was previously shown that oocyte-derived DNMT1 (a maintenance methyltransferase) is essential for maintaining and propagating CG methylation at imprinting control regions in preimplantation embryos. In mammalian somatic cells, hemimethylated-CG-binding protein UHRF1 plays a critical role in maintaining CG methylation by recruiting DNMT1 to hemimethylated CG sites. However, the role of UHRF1 in oogenesis and preimplantation development is unknown. In the present study, we show that UHRF1 is mainly, but not exclusively, localized in the cytoplasm of oocytes and preimplantation embryos. However, smaller amounts of UHRF1 existed in the nucleus, consistent with the expected role in DNA methylation. We then generated oocyte-specific Uhrf1 knockout (KO) mice and found that, although oogenesis was itself unaffected, a large proportion of the embryos derived from the KO oocytes died before reaching the blastocyst stage (a maternal effect). Whole genome bisulfite sequencing revealed that blastocysts derived from KO oocytes have a greatly reduced level of CG methylation, suggesting that maternal UHRF1 is essential for maintaining CG methylation, particularly at the imprinting control regions, in preimplantation embryos. Surprisingly, UHRF1 was also found to contribute to de novo CG and non-CG methylation during oocyte growth: in Uhrf1 KO oocytes, transcriptionally-inactive regions gained less methylation, while actively transcribed regions, including the imprinting control regions, were unaffected or only slightly affected. We also found that de novo methylation was defective during the late stage of oocyte growth. To the best of our knowledge, this is the first study to demonstrate the role of UHRF1 in de novo DNA methylation in vivo. Our study reveals multiple functions of UHRF1 during the global epigenetic reprogramming of oocytes and early embryos.

Dataset Information

High Frequency of Imprinted Methylation Errors in Human Preimplantation Embryos.

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High Frequency of Imprinted Methylation Errors in Human Preimplantation Embryos.

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