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How might IVF (and related technologies) alter epigenetic marks?


I was reading up on KCNQ1, which encodes a voltage-gated potassium channel, and I discovered that it happens to be only maternally expressed. This is regulated by KCNQ1OT1, a non-coding RNA, which is also epigenetically regulated (expressed only paternally). Mutations in KCNQ1OT1 are associated with Beckwith-Wiedemann Syndrome (BWS).

Further reading led to the discovery that there are ART-related (assisted reproductive technology) cases of BWS due to loss of methylation at KCNQ1.

However, this article found epigenetic stability of KCNQ1OT1 methylation in cultured human embryonic stem cells, which leads me to think that KCNQ1 might also have been properly methylated.

So what exactly are assisted reproductive technologies doing that can disrupt inheritance of epigenetic marks (that culturing hESCs doesn't do)? (Is this understood for any gene?)


I was at a talk last year where some geneticists were starting to look at the effect of IVF techniques on genetic issues. Because of the work being done and the inclinations of the clients involved, this has not been studied very much. One presumes that since zygotes are still used, there will not be much epigenetic effects due to methylation patterns - its the same cell stocks being used in vitro and in vivo.

There may be an effect on genetics because the natural selection process. Spermatogenesis and oogenesis is a highly selective process; only a small fraction of the ova generated by the ovaries ever mature and a large percentage of sperm generated are not motile or have other irregularities. During fertilization, sperm competition obviously throws out all but one in 250 million. It also appears that there are processes in the fallopian tubes that can store the sperm for several (up to 5) days before fertilization.

So while its not strongly established one can see how various IVF processes vary from in vivo fertilization. This could have an impact on epigenetic factors, although different sperm vary genetically due to Meiosis and this would be a big source of genetic variation in the outcome.


This year, the Keystone Symposia hosted concurrent meetings on DNA methylation and epigenomics. Multiple sessions were jointly held between the two meetings, and in total the number of participants at both meetings was one of the largest ever at Keystone. A notable aspect of the two meetings was the relatively large number of new and increasingly powerful epigenetic technologies that have been developed recently, ranging from novel single-cell epigenetic profiling to ligation-free Hi-C (Table 1). Many new findings and novel concepts were also discussed at the meeting, particularly around the epigenetics of differentiation and development, as well as disease, pluripotency and stem cells, to name just a few. The meeting opened with a Keynote Address by Adrian Bird (University of Edinburgh, UK), who reported on altered DNA methylation regions in cancer, and brain genomes that coincide with regions of altered base composition. AT-rich DNA regions are relatively less methylated, while CG-rich regions are relatively more methylated, leading to speculation that base composition in the genome impacts the methylome. The proteins bound to DNA are known to influence DNA methylation, and these proteins are in turn influenced by DNA base composition.

Key questions in epigenetics are what drives cell differentiation, and what is the role in this process of the establishment of chromatin marks or DNA methylation? Related to this, how are the patterns of chromatin or DNA methylation initially primed, and how does genetic or epigenetic variation impact development? A number of talks focused on these questions. First, Alexander Meissner (Harvard University, Broad Institute, USA) discussed targeted DNA methylation as embryonic stem cells (ESCs) differentiate into the endoderm. Knockout (KO) of DNA (cytosine-5)-methyltransferase 3A (DNMT3A) in differentiating ESCs leads to a lack of methylation acquired de novo at many sites, including genes such as Nanog and Foxa2. Nonetheless, the cells are still able to form teratomas when they are injected into mice. Knockout of both DNMT3A and DNMT3B causes passage-dependent loss of DNA methylation, albeit at very slow rates. Interestingly, deletion of DNA (cytosine-5)-methyltransferase 1 (DNMT1) causes cell death in ESCs. Ryan Lister (University of Western Australia, Australia) addressed whether de novo methylation occurring in gene promoters is (solely) responsible for gene silencing. He described epigenetic manipulation in cells by inducing the expression of a zinc finger-DNMT3A fusion protein, resulting in high levels of widespread, de novo methylation of gene promoters. Strikingly, gains in promoter DNA methylation simultaneously coexist with active histone marks on many gene promoters, and are insufficient to transcriptionally repress most genes. Removal of zinc finger-DNMT3A overexpression leads to a rapid return to an unmethylated state. Bing Ren (University of California, San Diego, USA) described the creation of a novel, chromosome-spanning haplotype reconstruction strategy (HaploSeq Table 1), which revealed extensive allelic biases and considerable variation in both chromatin state and transcription from identical human tissues in different individuals. Allelic differences of chromatin state involve cis-regulatory elements and are associated with allelic differences in transcription factor binding due to local sequence variations.


Background

Assisted reproductive technologies (ART), mostly intrauterine insemination (IUI) and in vitro fertilization (IVF), have helped many couples to overcome infertility. Worldwide, millions of children have been born via ART, and they now account for > 4% of births in some European countries [1].

Even though most of these children are considered healthy, there is increasing awareness about the potential consequences of ART on a number of complications potentially linked to epigenetic deregulation [2].

Epidemiological studies suggest that singletons born following the use of ART have an increased risk of adverse perinatal outcomes (e.g. low birth weight after fresh embryo transfers and abnormal placentation) [3, 4]. Furthermore, ART-conceived offspring also have an increased risk of rare imprinting disorders, such as Beckwith–Wiedemann (BWS), Russell–Silver (SRS), Angelman (AS) and Prader–Willi (PWS) syndromes [5,6,7,8,9,10]. However, there are considerable differences in the reported relative risks (Additional file 1: Table S1), which can mostly be explained by the substantial methodological heterogeneity (findings mostly obtained from voluntary registries or relatively small populations). The systematic reviews on this field have nevertheless made it possible to assess a substantial number of children. In a review, Vermeiden and Bernardus estimated that the birth of a child with BWS is significantly associated with ART, with a pooled relative risk of 5.2 [95% CI 1.6–7.4] [11]. Similarly, a systematic review demonstrated that the combined odds ratio of any imprinting syndromes in children conceived by ART is 3.67 [95% CI 1.39–9.74] when compared with naturally conceived children [12]. In a recent meta-analysis, a positive association was still found between conception after ART and four imprinting conditions (BWS, SRS, AS, PWS), among which BWS had a summary odds ratio of 5.8 [95% CI 3.1–11.1] [13]. It is difficult to draw any conclusions concerning SRS because it is extremely rare [11], but a positive association with ART treatment is likely [13]. No significant associations were found between the incidence of AS or PWS and IVF treatments [11]. Fertility problems could be involved in these two last syndromes in ART children [11], but the results of any meta-analysis must be interpreted in light of the limitations of the contributing studies.

During the periconceptional period, genome-wide epigenetic reprogramming occurs [14, 15]. This reprogramming includes imprinting, which is crucial for the proper development and future health of offspring. The many manipulations and processes of ART (e.g. hormonal stimulation, embryo manipulation, culture and cryopreservation) are concurrent with epigenetic reprogramming and imprinting (i.e. during female gametogenesis and preimplantation embryo development), leading to concerns that the ART themselves could negatively affect epigenetics and the establishment/maintenance of genomic imprints. Importantly, contrary to the molecular aetiologies of BWS children conceived naturally, almost all ART-conceived BWS typically occurred though loss of epigenetic marks in imprinting control regions (close to 95% of children with BWS born after IVF/ICSI vs 50% in the general population) [11]. However, the infertility/subfertility status of the parents may also play a role in the increased incidence of these disorders, as underlined in a Dutch study performed in families with a child with BWS, PWS, or AS [9]. Interestingly, there is increasing evidence that some female infertility syndromes such as polycystic ovary syndrome (PCOS) [16] or endometriosis [17] are associated with epigenetic alterations.

However, so far, the risk of imprinting-related diseases in relation to specific types of ART or underlying causes of female infertility has not been assessed. Therefore, the first aim of this extensive national cohort study was to compare the prevalence of imprinting-related disorders in singletons born after fresh (fresh-ET) or frozen (FET) embryo transfers, intrauterine insemination (IUI), or following natural conception (NC). Our second aim was to study the role of the three major types of female infertility (i.e. endometriosis, PCOS and primary ovarian insufficiency [POI]) on the prevalence of these imprinting-related diseases.


Links to Disease

Among all the epigenetics research conducted so far, the most extensively studied disease is cancer, and the evidence linking epigenetic processes with cancer is becoming 𠇎xtremely compelling,” says Peter Jones, director of the University of Southern California’s Norris Comprehensive Cancer Center. Halfway around the world, Toshikazu Ushijima is of the same mind. The chief of the Carcinogenesis Division of Japan’s National Cancer Center Research Institute says epigenetic mechanisms are one of the five most important considerations in the cancer field, and they account for one-third to one-half of known genetic alterations.

Many other health issues have drawn attention. Epigenetic immune system effects occur, and can be reversed, according to research published in the November�mber 2005 issue of the Journal of Proteome Research by Nilamadhab Mishra, an assistant professor of rheumatology at the Wake Forest University School of Medicine, and his colleagues. The team says it’s the first to establish a specific link between aberrant histone modification and mechanisms underlying lupus-like symptoms in mice, and they confirmed that a drug in the research stage, trichostatin A, could reverse the modifications. The drug appears to reset the aberrant histone modification by correcting hypoacetylation at two histone sites.

Lupus has also been a focus of Bruce Richardson, chief of the Rheumatology Section at the Ann Arbor Veterans Affairs Medical Center and a professor at the University of Michigan Medical School. In studies published in the May𠄺ugust 2004 issue of International Reviews of Immunology and the October 2003 issue of Clinical Immunology, he noted that pharmaceuticals such as the heart drug pro-cainamide and the antihypertensive agent hydralazine cause lupus in some people, and demonstrated that lupus-like disease in mice exposed to these drugs is linked with DNA methylation alterations and interruption of signaling pathways similar to those in people.


Affiliations

Systems Biology Program, Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain

Marcos Francisco Perez & Ben Lehner

Universitat Pompeu Fabra (UPF), Barcelona, Spain

Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain

You can also search for this author in PubMed Google Scholar

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Contributions

M.F.P. and B.L. wrote the manuscript.

Corresponding author


4. The Reach of Epigenetic Research in the Life Sciences

4.1. Intragenerational and Transgenerational Epigenetics

The first phase of the survey determined the relative frequency of the occurrence of intragenerational vs. transgenerational epigenetic papers. The actual use of these two specific terms as the secondary search term was not productive: only slightly above 1% of all epigenetic papers actually contained either or both of these adjectives. Subsequently, five major categories of epigenetic papers were formed for this survey. Epigenetic papers including the terms “mechanism”, 𠇍isease” and �velopment and ageing” (and their related topics) were considered to be more representative of an intragenerational perspective, while epigenetic studies, including the terms 𠇎volution” and “inheritance”, were considered to be more representative of epigenetic papers with a transgenerational component to them (however little that might be) Of course, there are transgenerational epigenetic papers that discuss the mechanism of inheritance, and these would be represented in both the “mechanism” and 𠇎volution” category. However, as is evident from Figure 1 , the majority of the focus of epigenetic studies was on mechanism and disease states in approximately equal measure. Indeed, χ% of papers referencing epigenetics also mentioned either evolution and/or inheritance. Noteworthy is that while transgenerational epigenetics studies have revealed many instances of epigenetic inheritance of disease/pathologies (e.g., [3,27,28]), the epigenetic inheritance of mal-adaptive modified phenotypes receives little attention compared to the “here and now” of diseases that develop in an individual’s life span. These findings are not surprising, as even a quick examination of a sample of papers comprising the epigenetic literature reveals intensive discussion of mechanisms of epigenetic phenomena, especially as they relate to human health and disease. Additionally, to no one’s surprise, funding follows disease and its prevention and cure, which has greatly enabled the growth of epigenetic studies.

Radar diagram showing the relative distribution of publications drawn from the PubMed database (http://www.ncbi.nlm.nih.gov/pubmed/) that contain the search terms 𠇎pigenetic(s)” and one of five focus areas. The graphic to the right indicates a gradient between intragenerational and transgenerational epigenetics based on the percentage of epigenetic papers emerging from each area of study indicated in the radar diagram. Thus, epigenetic papers with the terms �velopment and ageing” or 𠇍isease” are assumed to be more likely to be addressing intragenerational issues, such as evolution, while epigenetic papers mentioning 𠇎volution” or “inheritance” are viewed as more likely to be focusing on transgenerational epigenetic events. See the text for an additional discussion.

4.2. Epigenetics and Taxon

The survey next explored the taxonomic distribution of epigenetic papers using the secondary search terms (and their adjectives) of 𠇊nimals”, “plants”, 𠇏ungi”, “protists”, �teria”, 𠇊rchaebacteria” and “viruses” [29]. Approximately 60% of epigenetic papers contained the search term 𠇊nimal(s)”

10% contained “plant(s)” and near negligible numbers of epigenetic papers specifically mentioned any of the other major taxa ( Figure 2 A).

Radar diagram showing the relative distribution of publications on epigenetics drawn from the PubMed database (http://www.ncbi.nlm.nih.gov/pubmed/). (A) distribution of publications that contain the search terms 𠇎pigenetic(s)” and one of seven biological taxa (B) distribution of publications that contain the search terms 𠇎pigenetic(s)” and one of 12 biological fields.

4.3. Epigenetics and the Biological Field

The survey next considered epigenetic papers that included one of 12 major biological fields ( Figure 2 B). The vast majority (

95%) of epigenetic papers that even mentioned, if not actually discussed, a particular biological field was clustered in just six areas: chemistry/biochemistry, molecular biology, genetics, physiology, cellular biology or anatomy/morphology. Occurring at a very low frequency in the epigenetic literature were the fields of behavior (

2% of papers), taxonomy/systematics (1%𠄲%), evolution (a little above 1%) and, all being less than 0.5% of the epigenetic papers, development, ecology and evo-devo.

Combing the survey on biological fields and taxa reveals how some areas of epigenetics are almost completely unexplored. For example, combining 𠇎pigenetics” + “plant” + 𠇎vo-devo” yielded only two papers among the

50,000 epigenetics papers warehoused in PubMed. Similarly, 𠇎pigenetics” + “virus” + �ology” yielded just three papers. Yet, as we will now turn to, the role of epigenetics in the biology of all of these taxa may be profound.


Conclusion

Oocyte maturation is a complex process involving multiple steps and is regulated by many molecules and signaling pathways. In recent years, due to the rapid development and popularization of technologies like the genetic modification of animal models, molecular biology, and biochemistry, researchers have gained a better understanding of oocyte GV arrest and meiosis I resumption. The major cellular and molecular affairs, especially the epigenetic modification events related to oocyte maturation in response to hormone induction, and the major advances in this field, are highlighted in this review.

Since the development of an oocyte depends not only on the oocyte itself, but on mutual communication and physical contact with follicular granulosa cells, it is important to focus more on epigenetic changes within oocytes, ovarian granulosa cells in response to hormones, and other extracellular molecules induction. Besides, applying microscopes with high resolution and a high-throughput analysis technique, such as mono-cellular based sequencing and omics techniques, should be emphasized to present clearer 3D or even time-dependent 4D representations of critical affairs that happen during oogenesis. Finding more specific oocyte-expressed proteins, such as RBPs and oocyte-derived paracrine molecules, may contribute to uncover the mysterious mechanisms of oocyte meiosis as well. Further, integration of analysis of sequencing data, comparing the data collected from different breeds, and verifying the function of each individual molecule in vitro and in vivo simultaneously based on multiple animal models are also plausible.


Epigenetics in Translational and Personalized Medicine

For the past several decades genetics has been at the forefront in terms of understanding human disease. A recent addition to genetics has been epigenetics, which includes the role of the environment, both social and natural, including day-to-day habits, lifestyle and personal experiences on human health. Epigenetics establishes a scientific basis for how external factors and the environment can shape an individual both physically and mentally.

The knowledge that environment and lifestyle can alter health brings with it awareness that habits, social environment, diet and other factors shape health beyond our acquired genetic traits. Moreover, despite the risk presented by inherited genes and mutations, epigenetic factors play a decisive role in the actual development of disease. Research into epigenetics could lead to insights into how factors like diet and exercise can be customized to an individual in concordance with their naturally inherited genome in order to minimize the risk of developing a disease to which he/she is naturally predisposed.

Advances in epigenetic profiling technology such as genome-scale DNA methylation analysis points to the future possibilities of how epigenetic profiling can help in determining the risk of an individual with a particular type of genetic makeup developing a specific disease. Also the same epigenetic profile, along with knowledge of the genomic sequence, can help to determine which medications or alternative medicine approaches would be effective in preventing or curing a particular disease. Such approaches toward improving health and lifestyle and reducing the risk of developing an otherwise inevitable disease might be possible in the near future. It will require extensive knowledge of the biochemical and physiological mechanisms of epigenetics, and we are still on this challenging path, but we have good reasons to be optimistic.


This study was supported by grants from the National Key Research & Developmental Program of China (2018YFC1003701 and 2018YFC1003801) the National Basic Research Program of China (2013CB945501) the Institution of Higher Education Projects of Building First-Class Discipline Construction in Ningxia Region (Biology) (NXYLXK2017B05) the National Natural Science Foundation of China (31872792, 32071132, and 32070839) and the Project of the State Key Laboratory of Agrobiotechnology (2015SKLAB4-1).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Future efforts in human environmental epigenetic research

One area of increasing interest in epigenetic research is the concept of transgenerational inheritance. Initially, it was believed that because of both the reprogramming of epigenetic patterns in gametogenesis and the erasure of DNA methylation and other marks during the early phases of embryogenesis, epigenetic regulation would not be passed on to successive generations. Yet, it is well known that genomic imprinting, the patterning of parent-of-origin allele-specific expression of a subset of mammalian genes is established in the germline and is maintained in all somatic lineages (Jaenisch, 1997). Thus, mechanisms must exist to maintain some level of epigenetic patterning across successive generations.

It is important to differentiate transgenerational from intergenerational inheritance. The latter would represent effects on offspring of the parents' environment, including the effects of nutrition or environment exposures during pregnancy on the developing fetus and its germline, and thus would encompass effects in the F1 and F2 generations. True transgenerational effects, in contrast, would be observable in the F3 generation and beyond (Daxinger and Whitelaw, 2012 Ferguson-Smith and Patti, 2011 Lim and Brunet, 2013). This strict consideration will make it difficult if not impossible to conclusively demonstrate the existence of transgenerational inheritance in humans, where control or at least adequate assessment of the environments experienced over three generations is not feasible. Studies in plants and in model organisms where experimental design can allow for exquisite control across multiple generations are truly the only way in which the issue of transgenerational inheritance can be appropriately assessed (Heard and Martienssen, 2014). Studies in model systems will eventually shed light on the key mechanisms involved in this type of inheritance and then the presence and prevalence of those mechanisms can be assessed in human populations in more tractable time frames to provide evidence, but not definitive proof, of the existence of transgenerational inheritance.

There is a growing interest in the study of environmental exposures and epigenetic alterations, including exposures from the natural and man-made environment, as well as the environment more broadly, including the impact of psychosocial stressors and community context. As this research moves forward, it would be of use to consider these environments even more broadly. Given that epigenetic variation may denote a mode of short-term adaptation to the environment, considering significant environmental issues such as climate change on the epigenome would be of great interest. Although challenging for human studies, more focused work on transgenerational effects will also help to elucidate the potential role of epigenetic mechanisms in short-term adaptation.

In summary, human population and epidemiological studies can play an important role in understanding the impact of the environment on the epigenome. Through cross-disciplinary research, careful study design and novel application of new molecular technologies, it will be possible to begin to better understand these impacts and to put the role of epigenetic variation in humans in a wider context.