Michelle M. Denomme

Genomic imprints as a model for the analysis of epigenetic stability during assisted reproductive technologies

Gamete and early embryo development are important stages when genome-scale epigenetic transitions are orchestrated. The apparent lack of remodeling of differential imprinted DNA methylation during preimplantation development has lead to the argument that epigenetic disruption by assisted reproductive technologies (ARTs) is restricted to imprinted genes. We contend that aberrant imprinted methylation arising from assisted reproduction or infertility may be an indicator of more global epigenetic instability. Here, we review the current literature on the effects of ARTs, including ovarian stimulation, in vitro oocyte maturation, oocyte cryopreservation, IVF, ICSI, embryo culture, and infertility on genomic imprinting as a model for evaluating epigenetic stability. Undoubtedly, the relationship between impaired fertility, ARTs, and epigenetic stability is unquestionably complex. What is clear is that future studies need to be directed at determining the molecular and cellular mechanisms giving rise to epigenetic errors.

Loss of Genomic Imprinting in Mouse Embryos with Fast Rates of Preimplantation Development in Culture

ABSTRACT Currently, the stage of embryo development has been proposed as one of many criteria for identifying healthy embryos in infertility clinics with the fastest embryos being highlighted as the healthiest. However the validity of this as an accurate criterion with respect to genomic imprinting is unknown. Given that embryo development in culture generally requires an extra day compared to in vivo development, we hypothesized that loss of imprinting correlates with slower rates of embryonic development. To evaluate this, embryos were recovered at the 2-cell stage, separated into four groups based on morphological stage at two predetermined time points, and cultured to blastocysts. We examined cell number, embryo volume, embryo sex, imprinted Snrpn and H19 methylation, imprinted Snrpn, H19, and Cdkn1c expression, and expression of genes involved in embryo metabolism—Atp1a1, Slc2a1, and Mapk14—all within the same individual embryo. Contrary to our hypothesis, we observed that faster developing embryos exhibited greater cell numbers and embryo volumes as well as greater perturbations in genomic imprinting and metabolic marker expression. Embryos with slower rates of preimplantation development were most similar to in vivo derived embryos, displaying similar cell numbers, embryo volumes, Snrpn and H19 imprinted methylation, H19 imprinted expression, and Atp1a1 and Slc2a1 expression. We conclude that faster development rates in vitro are correlated with loss of genomic imprinting and aberrant metabolic marker expression. Importantly, we identified a subset of in vitro cultured embryos that, according to the parameters evaluated, are very similar to in vivo derived embryos and thus are likely most suitable for embryo transfer.

Embryonic imprinting perturbations do not originate from superovulation-induced defects in DNA methylation acquisition

OBJECTIVE To investigate whether superovulation disrupts maternal imprint acquisition in oocytes. DESIGN Animal model. SETTING Academic institute. ANIMAL(S) Spontaneously ovulated and superovulated mice. INTERVENTION(S) Low and high hormone dosage treatments were administered to females, and ovulated metaphase II oocytes were collected. MAIN OUTCOME MEASURE(S) Imprinted DNA methylation was analyzed at Snrpn, Kcnq1ot1, Peg3, and H19 in individual oocytes. RESULT(S) Examination of 125 individual oocytes derived from females subjected to low and high hormone treatments revealed normal imprinted methylation patterns that were comparable to oocytes derived from spontaneously ovulated females. CONCLUSION(S) Maternal imprint acquisition was not affected by superovulation. Given its aberrant effects during preimplantation development, superovulation must instead disrupt maternal-effect gene products that are required after fertilization for imprint maintenance. These results eliminate imprint acquisition per se as the initial stage of imprint loss and point to the importance of analyses on early embryos after procedures involving oocyte manipulation.

High Frequency of Imprinted Methylation Errors in Human Preimplantation Embryos.

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.

Maternal control of genomic imprint maintenance

Genomic imprinting is a specialized transcriptional phenomenon that employs epigenetic mechanisms to facilitate parental-specific expression. Perturbations in parental epigenetic asymmetry can lead to the development of imprinting disorders, such as Beckwith-Wiedemann syndrome and Angelman syndrome. DNA methylation is one of the most widely studied epigenetic marks that characterizes imprinted regions. During gametogenesis and early embryogenesis, imprinted methylation undergoes a cycle of erasure, acquisition and maintenance. Gamete and embryo manipulations for the purpose of assisted reproduction are performed during these reprogramming events and may lead to their disruption. Recent studies point to the role of maternal-effect proteins in imprinted gene regulation. Studies are now required to increase understanding of how these factors regulate genomic imprinting as well as how assisted reproduction technologies may alter their function.

Compromised fertility disrupts Peg1 but not Snrpn and Peg3 imprinted methylation acquisition in mouse oocytes

Growth and maturation of healthy oocytes within follicles requires bidirectional signaling and intercellular gap junctional communication. Aberrant endocrine signaling and loss of gap junctional communication between the oocyte and granulosa cells leads to compromised folliculogenesis, oocyte maturation, and oocyte competency, consequently impairing fertility. Given that oocyte-specific DNA methylation establishment at imprinted genes occurs during this growth phase, we determined whether compromised endocrine signaling and gap junctional communication would disrupt de novo methylation acquisition using ERβ and connexin37 genetic models. To compare mutant oocytes to control oocytes, DNA methylation acquisition was first examined in individual, 20–80 μm control oocytes at three imprinted genes, Snrpn, Peg3, and Peg1. We observed that each gene has its own size-dependent acquisition kinetics, similar to previous studies. To determine whether compromised endocrine signaling and gap junctional communication disrupted de novo methylation acquisition,individual oocytes from Esr2- and Gja4-deficient mice were also assessed for DNA methylation establishment. We observed no aberrant or delayed acquisition of DNA methylation at Snrpn, Peg3, or Peg1 in oocytes from Esr2-deficient females, and no perturbation in Snrpn or Peg3 de novo methylation in oocytes from Gja4-null females. However, Gja4 deficiency resulted in a loss or delay in methylation acquisition at Peg1. One explanation for this difference between the three loci analyzed is the late establishment of DNA methylation at the Peg1 gene. These results indicate that compromised fertility though impaired intercellular communication can lead to imprinting acquisition errors. Further studies are required to determine the effects of subfertility/infertility originating from impaired signaling and intercellular communication during oogenesis on imprint maintenance during preimplantation development.

Single oocyte bisulfite mutagenesis.

Epigenetics encompasses all heritable and reversible modifications to chromatin that alter gene accessibility, and thus are the primary mechanisms for regulating gene transcription. DNA methylation is an epigenetic modification that acts predominantly as a repressive mark. Through the covalent addition of a methyl group onto cytosines in CpG dinucleotides, it can recruit additional repressive proteins and histone modifications to initiate processes involved in condensing chromatin and silencing genes. DNA methylation is essential for normal development as it plays a critical role in developmental programming, cell differentiation, repression of retroviral elements, X-chromosome inactivation and genomic imprinting. One of the most powerful methods for DNA methylation analysis is bisulfite mutagenesis. Sodium bisulfite is a DNA mutagen that deaminates cytosines into uracils. Following PCR amplification and sequencing, these conversion events are detected as thymines. Methylated cytosines are protected from deamination and thus remain as cytosines, enabling identification of DNA methylation at the individual nucleotide level. Development of the bisulfite mutagenesis assay has advanced from those originally reported towards ones that are more sensitive and reproducible. One key advancement was embedding smaller amounts of DNA in an agarose bead, thereby protecting DNA from the harsh bisulfite treatment. This enabled methylation analysis to be performed on pools of oocytes and blastocyst-stage embryos. The most sophisticated bisulfite mutagenesis protocol to date is for individual blastocyst-stage embryos. However, since blastocysts have on average 64 cells (containing 120-720 pg of genomic DNA), this method is not efficacious for methylation studies on individual oocytes or cleavage-stage embryos. Taking clues from agarose embedding of minute DNA amounts including oocytes, here we present a method whereby oocytes are directly embedded in an agarose and lysis solution bead immediately following retrieval and removal of the zona pellucida from the oocyte. This enables us to bypass the two main challenges of single oocyte bisulfite mutagenesis: protecting a minute amount of DNA from degradation, and subsequent loss during the numerous protocol steps. Importantly, as data are obtained from single oocytes, the issue of PCR bias within pools is eliminated. Furthermore, inadvertent cumulus cell contamination is detectable by this method since any sample with more than one methylation pattern may be excluded from analysis. This protocol provides an improved method for successful and reproducible analyses of DNA methylation at the single-cell level and is ideally suited for individual oocytes as well as cleavage-stage embryos.

Maintenance of Mest imprinted methylation in blastocyst-stage mouse embryos is less stable than other imprinted loci following superovulation or embryo culture

Abstract Assisted reproductive technologies are fertility treatments used by subfertile couples to conceive their biological child. Although generally considered safe, these pregnancies have been linked to genomic imprinting disorders, including Beckwith–Wiedemann and Silver–Russell Syndromes. Silver–Russell Syndrome is a growth disorder characterized by pre- and post-natal growth retardation. The Mest imprinted domain is one candidate region on chromosome 7 implicated in Silver–Russell Syndrome. We have previously shown that maintenance of imprinted methylation was disrupted by superovulation or embryo culture during pre-implantation mouse development. For superovulation, this disruption did not originate in oogenesis as a methylation acquisition defect. However, in comparison to other genes, Mest exhibits late methylation acquisition kinetics, possibly making Mest more vulnerable to perturbation by environmental insult. In this study, we present a comprehensive evaluation of the effects of superovulation and in vitro culture on genomic imprinting at the Mest gene. Superovulation resulted in disruption of imprinted methylation at the maternal Mest allele in blastocysts with an equal frequency of embryos having methylation errors following low or high hormone treatment. This disruption was not due to a failure of imprinted methylation acquisition at Mest in oocytes. For cultured embryos, both the Fast and Slow culture groups experienced a significant loss of maternal Mest methylation compared to in vivo-derived controls. This loss of methylation was independent of development rates in culture. These results indicate that Mest is more susceptible to imprinted methylation maintenance errors compared to other imprinted genes.