Jayant G. Mehta1
Sub-fertility Laboratory and Quality Control, Queen’s Hospital, Barking, Havering and Redbridge University Hospitals NHS Trust, Rom Valley Way, Romford, Essex, RM7 0AG, UK
Jayant G. Mehta
In recent years, a number of reports have linked epigenetics and ovarian stimulation to human-assisted reproductive techniques (ART). These reports have alluded to the fact that the pathological causes of diseases such as Beckwith-Wiedemann syndrome (BWS-OMM #130650) and Angelman syndrome (AS-OMIM #105830) may be associated with epigenetic disruption of chromosomal regions, or epimutations, as a consequence of defective DNA methylation status of imprinted genes. The acquisition of a unique epigenetic profile in a small subset of genes in the male and female germlines is time specific during the development of gametes. It involves a well-orchestrated expression of enzymes. Three important mechanisms that are involved in the imprinting process include DNA methylation, posttranslational modification of histone proteins, and remodeling of chromatin and RNA-based mechanisms. Genomic imprinting once established in the germline must remain unaltered following fertilization of these gametes and throughout the life of the offspring. This observation raises possibility for ART-induced epigenetic disturbance during the maintenance of these imprints in early embryonic life. How genomic imprinting may be influenced by ovarian stimulation is explored here. These differential epigenetic marks in the gametes result in a parent-of-origin-specific expression of these imprinted genes in the offspring. Based on the mouse model and limited ART human observations, the consequences of dose-dependent hormonal superovulation and how it may affect the genomic imprinting are discussed. The mechanisms involved in epigenetic deregulation and reprogramming of gametes as well as early embryos are also considered.
EpigeneticsOvulation inductionDNA methylationCpGGenomic imprintingOvarian stimulation
The birth of Louise Brown, in 1978, heralded an era of assisted reproduction. The number of children born with the help of assisted reproductive technologies (ART) has been steadily growing, approaching almost 2–3 % of the total births in developed countries [1, 2]. Although the use of ART continues to grow exponentially, animal studies have suggested the possibilities of superovulation and embryo culture media affecting non-genomic inheritance, lending biological plausibility to the concern that ART may increase the risk of imprinting disorders [3–5]. In the early 2000s, a number of reports were published that have linked human-ART by alluding to the fact that the pathological causes of diseases such as Beckwith-Wiedemann syndrome (BWS-OMM #130650) [6–10] and Angelman syndrome (AS-OMIM #105830) [9, 11, 12] are a direct result of epigenetically based defective DNA methylation status of imprinted genes. Realization and understanding of the causes and the mechanisms involved in epimutations have emerged as an urgent task for all specialists in genetics and reproductive medicine and have triggered efforts aimed at ensuring safety of ART. The aim of this chapter is to review the evidence associated with abnormal epigenetic modification of the genome, primarily defective DNA methylation, within ART processes and possible contribution of ovarian stimulation to these epigenetic modifications.
35.2 Epigenetics, DNA Methylation, and Genomic Imprinting
Epigenetics (epi- Greek: epί- over, outside of, around, above -genetics), a term coined by Conrad Waddington (1942), describes changes in the gene expression, gene activity, and phenotype caused by mechanisms, not involving base pair changes in the underlying DNA sequence, during the development of an individual. However, the classical definition describes epigenetics as a science-investigating mechanism by means of which a genotype produces the phenotype [13, 14]. Jean-Baptiste Lamarck (1744–1829) formulated the theory that the environment shapes genes, and these changes are passed on to the offspring. It was Charles Darwin (1809–1882) who postulated that genes are not changed by the environment but formulated through natural selection. Epigenetic phenomena, therefore, manifest upon any changes in the realization of hereditary information from DNA transcription to RNA translation and processing of the protein molecule. It is understood that epigenetic modifications are inherited over cell generations and express long-term position effect in the genome. These modifications are reversible [15, 16]. Epigenetics and biological processes are known to influence reprogramming in early development, X-chromosome inactivation, cancer, obesity, and biobehavioral reproduction. It is therefore not surprising that ART interventions may have the potential to affect epigenetic processes at multiple stages of gametogenesis and embryo development. The mechanisms and control of the epigenetic alternations of DNA are still being unraveled. However, the three mechanisms that have been identified so far include:
35.2.2 DNA Methylation and ART Procedures
One of the most intensely studied and first identified epigenetic modification in mammals is DNA methylation. In normal cells, it ensures the proper regulation of gene expression and stable gene silencing. During DNA methylation, a methyl group (−CH3) is transferred from S-adenosylmethionine (SAM) and covalently added to the fifth carbon of a cytosine base on a cytosine guanine (CG) dinucleotide . This process is further associated with histone modifications (acetylation/deacetylation and methylation). Functionally, significant DNA methylation in the mammalian genome occurs mainly at cytosine bases included in CpG dinucleotides (p indicates the phosphate group linking the two bases).
The interplay of these epigenetic modifications is crucial in regulating the functioning of the genome by changing chromatin architecture, and it is sometimes associated with antisense RNAs [18, 19]. Generally speaking, methylation silences gene transcription through structural blocking of transcriptional factor which binds to DNA by the presence of a methyl group . While DNA methylation is responsible for silencing of imprinted genes and inactivation of X-chromosome, few genes, such as IGF2 and IGF2R, are activated by methylation . DNA methyltransferase (DNMT) catalyzes the methylation process and acts in conjunction with methyl-CpG-binding proteins, interacting corepressors, and transcription factors. Moreover, DNMT1 acts as a sequence-independent methyltransferase to conserve the patterns of methylation status in the process of DNA replication and cell division in somatic cells . During the process of gametogenesis and early life, de novo methylation is regulated by two “constituent” isoforms, DNMT3a and DNMT3b. DNMT1 is a “maintaining” methylase, is specific to hemi-methylated sequences, and provides reproduction of the DNA methylation pattern on the daughter strand after replication. Inheritance of the methylation status is provided by this enzyme which ensures gene expression in cell generations.
The most critical role of DNA methylation in the gene expression is “genomic imprinting.” This activity differentially marks maternal and paternal gene in the individual genome through epigenetic process. Furthermore, this marking further ensures that monoallelic expression of imprinted genes depends on their maternal or paternal origin. The monoallelic expression of imprinted genes is provided by supramolecular chromatin modifications, which are differentially responsible for marking parental alleles. The main regulator of this process is allele-specific DNA methylation established upon germline cell maturation .
35.2.3 Differentially Methylated Regions
Differentially methylated regions (DMRs) have been identified in all imprinted genes studied. These are CpG sequences, whose methylation status considerably differs from that of the parental homologs. It has to be appreciated that not all the CpG dinucleotides are methylated. An area of the genome (usually at least 200 base pairs (BP) long) with high incidence of CpGs (exceeding 50 %) is normally referred to as “CpG Island.” Approximately half of the CpG islands are located near the transcription start site of genes, particularly for housekeeping genes; these CpGs are generally not methylated nor have low levels of methylation. The remaining 50 % of CpG islands are intragenic or intergenic and are believed to represent the transcription start site of noncoding RNAs ; these CpG islands are usually methylated .
The methylation pattern established during germline cell development for these regions is strictly specific. It has been observed that in some imprinted loci, methylation occurs exclusively in oogenesis but not during spermatogenesis. In contrast, in some other loci, DMRs are methylated during spermatogenesis rather than in oogenesis. After fertilization, differential methylation is preserved in somatic cells under the influence of DNA methyltransferase activity. Although some DMRs regulate the activity of a particular imprinted gene (micro-imprinted domains), others are known to coordinate the expression of a whole gene cluster. An important feature of imprinted genes in the mammals is the presence of organized clusters in the genome. The presence of DMRs within a cluster of imprinted genes permits the establishment and maintenance of monoallelic expression of a gene group. In the human genome, three large clusters of imprinted genes with their own imprinting centers have been identified and are located in distinct regions: two in region 11p15.5 and one in region 15q11–q13 .
Moreover, the allele-specific methylation of primary DMRs provides a heritable “memory” that is maintained throughout fertilization and embryo development . However, abnormal expression of imprinted genes can also result from genetic disorders (deletion or duplication, mutation or uniparental disomy) and epimutations (methylation anomalies). While gene activation is driven by gradients of signaling molecules and transcription factors, gene silencing is supported by DNA methylation and chromatin modifications carried out by specialized enzymatic activities. It is therefore highly probable that mutations in many components of the epigenetic gene silencing machinery could lead to a variety of human disorders as observed after ART treatment.
22.214.171.124 Posttranslational Modification of Histone Tails
Posttranslational modifications of histone tails involve addition of an acetyl, methyl, and phosphate group or, more rarely, ubiquitination, sumoylation, ADP-ribosylation, deamination, and non-covalent proline isomerization. It has been observed that histone modifications work often in conjunction or independently of DNA methylation . During the histone tail modifications, the affinity of the basic histone proteins to the acidic DNA is changed. Positive charge of lysine is neutralized by acetylation, facilitating the disassociation of the histone protein from the negatively charged DNA, thus allowing chromatin conformation to open up more.
It has also been observed that DNA is least transcriptionally active when it is methylated and when bound with unacetylated histones. The reality is that the “histone code” is extremely complex and is still being deciphered. As an example, arginine residues can be mono- or dimethylated, while lysine residues can accept one, two, or three methyl residues [27–29]. Additionally, the epigenetic control at the chromatin level is regulated by two chromatin remodeling complexes. The Brahma/SWI/SNF complex changes the position of nucleosomes along the DNA, whereas the SNF2H/ISWI complex mobilizes nucleosomes.
126.96.36.199 Noncoding RNAs (ncRNA)
Mattick and colleagues  have reviewed the third model of epigenetic control which involves noncoding RNAs (ncRNA). These ncRNAs play a central role in genetic and epigenetic processes and are generated in abundance by vast nonprotein-coding segments of DNA. In fact, transcription factors and proteins involved in epigenetic modification (e.g., DNMTs and methyl DNA-binding domain proteins) are known to bind RNA, which in turn contributes to chromatin structural organization .
One of the prominent features of the epigenome is its plasticity. Although epigenetic marks are maintained through cellular mitosis and are considered to be stable, they have been known to be modified reversibly by the environment. Moreover, important epigenetic changes are associated with aging and involve global DNA hypomethylation and isolated hypermethylation of specific loci . Furthermore, it has also been established that epigenetic programming can change during specific windows of sensitivity .
35.2.4 Genomic Imprinting
Genomic imprinting is an epigenetic process that is responsible for regulating gene transcription and is known to lead to the expression of only one allele of a particular gene in a parental-specific manner, in other words, parental inheritance. Mouse embryos created with two female pronuclei or two male pronuclei failed to survive in utero [32–34]. These experiments elegantly demonstrated the concept of genomic imprinting. In these studies, embryos derived from female pronuclei showed near-normal embryos, with abnormal extra embryonic tissue. However, embryos derived from male pronuclei demonstrated poorly developed embryonic tissue [32, 34–36]. These studies imply that for normal embryogenesis to occur, both maternal and paternal genomes were required.
The two rare disorders of imprinting associated with ART raised questions about the effect of ART on early development [37, 38]. Because humans contain two sets of autosomal genes, a copy is inherited from each parental gamete. During classical Mendelian inheritance, independent of parental origin, the genes from each parent are expressed in the offspring in equal measures. Therefore, the disease phenotype is highly dependent on uniparental expression as observed in a small handful of known genetic disorders.
Currently, more than 150 imprinted genes have been identified in mice and humans [39–41]. They play crucial roles in embryonic growth and development, placental function , postnatal metabolic pathways, and behavior associated with the control resources .
Recently, few studies in humans have reported widespread methylation patterns in children born after ART and have suggested the notion of biologic plausibility (reviewed in Ref. ).
35.3 ART and Epigenetics
As discussed earlier, gene imprinting studies using mouse nuclear transplants have demonstrated that for normal embryogenesis to occur, both maternal and paternal genomes are required [32–34]. As epigenetic reprogramming of DNA methylation occurs during germ cell and preimplantation development, it is highly probable that ART manipulations of conception, which include ovarian stimulation, in vitro maturation of oocytes, the use of ICSI, the use of immature sperm, in vitro culture of embryos, and cryopreservation of both embryos and gametes, could theoretically disturb the normal conception process leading to epigenetic errors [22, 44, 45]. It is therefore not surprising that ART interventions have the potential to influence epigenetic processes at various stages of gametogenesis and embryo development.
Although it has not been established which ART procedures are involved in epigenetic anomalies, the “timing” of the manipulation relative to the erasure and establishment of imprinting marks is a key contributory factor. Furthermore, because oocytes are vulnerable to reestablishment of methylation marks, which occurs just before ovulation, the artificial induction of ovulation in the course of an ART cycle could conceivably affect oocyte imprinting [9, 46, 47].
Furthermore, one can argue in favor of hormonal influence on epigenetic processes disrupting imprinting marks laid down asynchronously in both gametogenesis. In the male haplogenome, imprinting marks undergo erasure actively and are initiated during prenatal stages of spermatogenesis and are completed at postnatal stage. While, in the female haplogenome, the erasure is passive and begins after puberty in growing oocytes from primordial to antral follicles . During this phase, the entire PGC genome, including imprinted genes, is demethylated and then remethylated in a sex-specific manner. This ensures that the egg and sperm have the appropriate respective imprinted marks. The time course for remethylation of gametes is different for the two sexes. While the imprinting process progresses over time with increasing oocyte diameter, different imprinted genes complete the process at different times and, for several imprinted genes, the process is not completed until ovulation [48, 49]. Therefore, possible disruption at different points during this process may influence and result in varying degrees of epigenetic aberrations. In addition, incomplete erasure of the imprints can result in epigenetic inheritance to the next generation. More specifically, any epigenetic modification may result in transmission to the offspring even if one epigenetic mark is not erased in the parental germ cells.
As discussed in earlier chapters, females with low ovarian reserves or advanced maternal age have very poor prognosis of a successful outcome. It is therefore likely that use of high doses of exogenous hormones during this period may disrupt the acquisition of imprints in oocyte maturation. This forced oocyte maturation may also lead to the loss of maternal-specific expression and the development of imprinting disorders in some or all of the oocytes that are normally non-ovulated.
It is also necessary to consider that some cells in the embryo appear to be more susceptible to alteration in imprint marks during in vitro manipulation. It has been reported that in mouse model in vitro culture, the placenta exhibited a greater loss of imprinting than did the embryo [50, 51]. Although the placenta epigenetic marks might seem irrelevant since the placenta is discarded at birth, this observation is noteworthy when considering the transgenerational effects of altered epigenetic marks in the placenta in animals [51, 52] and their relevance to human diseases [42, 53, 54] and how altered placental function theoretically could affect future generations .
35.4 Epigenetic Reprogramming (EP) of Oocytes
As patient is exposed to different procedures during ART treatment, it is difficult to determine which individual techniques might perturb epigenetic events in oocytes and embryos. The availability of large numbers of oocytes and preimplantation embryos from a known genetic strain of mouse, at precise stages of development, has allowed mechanisms responsible for aberrant genomic imprinting to be investigated .
35.4.1 Epigenetic Programming in Mice Oocytes
Seki et al.  observed in mice that around embryonic day 7.25 (E7.25), EP starts with germ cell development from epiblast cells, continues after the primordial germ cells (PGCs) have reached the genital ridge at E10.5, and lasts until E13.5 (42–44 days in humans). They further observed a marked genome-wide DNA demethylation once the PGCs had migrated into the developing gonads, as high as 73.2–85 % in embryonic stem cells compared with less than 10 % in female PGCs at E13.5. At this stage, both imprinted and nonimprinted genomic loci are demethylated. This erasure is necessary to maintain the totipotency of germline. Moreover, it has been suggested that demethylation provides erasure of the accumulated aberrant epigenetic modifications (epimutations). Although the chromatin rearrangements are very transient (from E11.5 to E12.5) , it is worth noting that the erasure of differential DNA demethylation of imprinted genes persists until new imprints are imposed later in the embryo in a sex-specific manner. Realistically, demethylated chromatin state is maintained during arrest of meiosis in female germline cells. In spermatogonia, methylation is initiated on resumption of mitotic divisions and is completely terminated at meiosis I pachytene. By contrast, in oocytes, methylation is established only during their maturation and terminates at metaphase II [23, 58]. It has been confirmed that the de novo methylase DNMT3 in collaboration with DNMT3L is responsible for establishing a new DNA methylation state at repeated sequences and developmental genes [59, 60] and for resetting the sex-specific germline DMR imprint [61, 62]. As a consequence, sex-dependent methylation of imprinted genes is observed in germline cells during their maturation. Mature oocytes and spermatozoa significantly differ in their epigenetic organization: the sperm genome is more methylated and its chromatin is more condensed as compared to that in oocytes, owing to replacement of histones by protamines. Interestingly, most regulatory imprinted domains in the human genome are methylated in oocytes. So far, only three imprinted domains (H19, MEG3, and GNAS) are known whose methylation is established in the male germline. This marked difference of methylation of imprinted genes in different parental chromosomes may have appeared during evolution as an additional protection against active demethylation of the paternal genome, which is switched immediately after fertilization .
Because the maternal imprint appears to be established at the same time for all the analyzed imprinted genes, methylation dynamics seem to be more progressive during adult mouse follicle growth, as compared with those in neonatal period .
35.4.2 Epigenetic Programming in Human Oocytes
Sato and colleagues , studying the human oocyte development competence by cumulus cell morphology and circulating hormone profile, observed that the timing of maternal imprinting appears to be identical to the mouse model. They further observed that in late antral follicle stage oocytes, DNA methylation on maternally methylated DMRS was completed. This was in contrast to only 50 % DNA methylation in early follicle stages (primordial and primary follicle stages). Interestingly, the paternally methylated DMRs remained unmethylated at all the stage of development.
Exploring this further, it was observed that the de novo methylation of KCNQ1OT1 DMR (KvDR1) occurred very slowly with the meiosis II progression , and only about two thirds of alleles were observed to be methylated on this DMR in fully grown germinal vesicle (GV) oocytes. Contrary to this observation, Geuns and colleagues , looking at a different region within KvDR1, reported an overall methylation pattern for this imprinted gene as early as GV stage. It is likely that the discrepancy observed by the two groups can be attributed to methylation acquisition dissociated between the two different regions of this DMR.
It should be noted that, of the 20 identified gDMRs/ICRs, 17 maternal ICRs are methylated in the oocyte, whereas only three are methylated in the sperm (paternal gDMRs). Because of the high number of maternal ICRs being methylated in the oocytes, it is highly feasible that the frequency of imprinting errors during maternal epigenetic reprogramming could be statistically higher than in the sperm. Moreover, maternal ICRs are CpG island promoters, whereas paternal ICRs are relatively CpG poor and intergenic. It begs to ask the questions whether these sexual discrepancies observed is linked to the different developmental kinetics of male and female gametogenesis and does it suggest evolutionary reasons for this observation [66, 67]. Studies have also highlighted the crucial role of maternal reprogramming. Studies demonstrated that maternal ICRs play a dominant role in early development regulating the biologic pathways related to the establishment of the fetomaternal interface .
35.5 Epigenetic Effects of Hormonal Superovulation
It is a common practice in in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) treatment cycles to stimulate ovaries using exogenous gonadotropins. Depending on the age and ovarian reserves of the patient, different stimulation protocols have been employed (discussed in earlier chapters). Due to lack of sufficient clinical material, most of the observed epigenetic changes come from animal studies. A comparative study of two cell mouse embryos from superovulated mice revealed a higher incidence of methylation abnormalities when compared with nonsuperovulated mice . Furthermore, the loss of methylation observed on the maternal allele at SNRPN, PEG3, and KCNQ1OT1 was dose dependent and statistically significant at the higher dosage of hormone stimulation. The authors concluded that hormonal stimulation of the ovaries may affect gene imprinting .
To understand the EP and consequences of superovulation, Sato’s group  studied the methylation of three maternally methylated genes (Peg1, Zac, and Kcnq1ot1) and one paternally methylated gene (H19) in pooled superovulated MII oocytes from two different strains of mice (ICR and BDF). The group observed that all of the maternally methylated genes had normal methylation after superovulation; however, H19 had gained methylation. These results suggested that the acquisition of maternal methylation imprints is unaffected but that oocyte quality was affected such that abnormal methylation occurred on H19.
In a separate study, Fauque and colleagues  reported decreased levels of expression of H19 in blastocysts after superovulation. The possible explanation for these results could be an alteration in oocyte quality affecting H19 EP marking or a delay in embryo development. This is feasible as H19 is normally expressed first at the blastocyst stage .
The effects of superovulation were further examined at different times in the development. Using a low-dose superovulation protocol, Fortier et al.  demonstrated alteration in the expression of maternally and paternally methylated imprinted genes in the midgestation mouse placenta, suggesting that trophectoderm-derived tissues may be more susceptible to disruption of imprinted genes than the embryo proper.
Based on these studies, the authors concluded that superovulation could possibly have two distinct effects, one to disrupt the acquisition of methylation imprints during oocyte growth and the second to impair the proper maintenance of imprints during preimplantation development. These observations further confirmed that imprinting errors occur in a dose-dependent manner, with more frequent disturbances at the higher dosage of hormone stimulation than low hormone doses. Furthermore, both the maternal and paternal H19 alleles were perturbed by superovulation suggesting that maintenance of imprinting after fertilization may be affected. Consistent with the above observation, in a human study, the authors confirmed that hormonal stimulation of the ovary affects gene imprinting, leading to a loss of methylation at PEG1 and a gain of methylation at H19 in superovulated immature human oocytes .
Based on both the animal and limited human studies, it is highly probable that superovulation may be responsible for modifications in maternal-effected gene products that are later required for imprinting maintenance in developing embryos .
35.6 Use of Immature Gametes: In Vitro Growth (IVG) and In Vitro Maturation (IVM)
As clinics attempt to use immature gametes by culturing them in media with different concentrations of gonadotropins, growth factors, and other proteins, there has been a concern that the use of immature gametes may be associated with imprinting defects in the offspring. The processes of IVG and IVM of oocytes are fairly complex and with different efficiency in different species (see for review) . Although live offspring have been obtained following IVG/IVM of mice primordial follicles , only isolated preantral follicles have been grown in nonrodent species .
In contrast, the process of IVM in humans involves the maturation of germinal vesicle (GV) oocytes to metaphase II (MII), with primary clinical indications for women with PCOS or fertility preservation in patients preparing to undergo cytotoxic cancer therapy .
Although a mouse study has reported significant histone acetylation changes in MII oocytes and early cleavage embryos, after IVM , in human, no increase was detected in congenital malformation in children conceived by IVM .
However, Khoueiry and colleagues  observed that the KCNQ10T1 DMR (KvDMR) is more methylated in the GV and metaphase I (MI) oocytes of natural cycles than those from stimulated cycles (62.55 vs. 67.8 % for GV and 70.3 % vs. 63.6 % for the MI, respectively). This observations implied that gonadotropin stimulation is likely to modify the dynamics of de novo methylation during oocyte maturation or/and may be responsible for recruiting too young follicle. It is therefore probable that these imprinting disruptions observed may be due to the developmental delay in the oocytes preventing imprint establishment at the right time or by ovarian stimulation or in vitro culture interfering with the imprint acquisition in the oocytes.
Moreover, evidence exists that additional manipulations may also be associated with altered methylation of imprinted genes. IVG of mouse oocytes has been reported to result in hypomethylation of IGF2 and PEG1/MEST and hypermethylation of H19 . In contrast, Anckaert and colleagues , using a well-established follicle culture system with controlled titrating exposure to follicle stimulating hormone, detected no abnormalities in the methylation of a number of imprinted genes.
Because IVG and IVM are either being evaluated for or are clinically used, it is necessary that more controlled studies should be undertaken, allowing better understanding of how EP influences the clinical use of immature oocytes. It will be important for future studies to establish optimal culture techniques in order to pursue the physiological significance of any defects detected in oocytes by closely examining the offspring and their placentae at later times in gestation.
From a genetic perspective, it is reassuring that in a transgenic mouse model, various ART techniques including IVF, ICSI, round spermatid injection (ROSI), and cell culture have not been associated with an increase in the frequency or spectrum of point mutations .
Diseases such as Beckwith-Wiedemann syndrome (BWS-OMM #130650) and Angelman syndrome (AS-OMIM #105830) have been reported to be associated with epigenetic disruption of chromosomal regions, or epimutations, as a consequence of defective DNA methylation status of imprinted genes. Three important mechanisms that are involved in the imprinting process include DNA methylation, posttranslational modification of histone proteins, and remodeling of chromatin and RNA-based mechanisms. The acquisition of a unique epigenetic profile in the male and female germlines is time specific during the development of gametes. It involves a well-orchestrated expression of enzymes. Furthermore, genomic imprinting once established in the germline must remain unaltered following fertilization of these gametes and throughout the life of the offspring.
It is highly probable that epimutations found in babies conceived using ART may be a consequence of the ART procedures rather than an inherent condition in the infertile population. No studies to date have identified at which stage(s) during the ART procedures the epigenetic alterations could arise. However, tangible evidence from animal and limited human studies points to the fact that superovulation is responsible for an increased incidence of epimutations. Because acquisition of maternal imprints extends over a relatively long period of time, there is a danger of these imprints being exposed to disturbances. Furthermore, it has been demonstrated that the exogenous gonadotropins influence the kinetics of oocyte maturation by inducing accelerated follicular growth in some cases . This ovarian stimulation has profound effects on the gene imprinting resulting in poor development of the embryos, placentation, and possibly leading to failed implantation(s). As discussed previously, the acquisition of methylation in the oocyte is a complex phenomenon requiring proper oocyte growth. Human oocytes are more prone to epigenetic errors as they encounter more stressors—such as multiple hormone administration, advanced maternal age, environmental factors, or inherent infertility . In contrast, animal studies excluding infertility effects have highlighted the negative impact of ovulation induction per se in this critical period.
One major disadvantage of using exogenous hormones in ART is the release of some MII oocytes with incomplete or labile imprints. Considering several studies in humans and mice, the administration of exogenous gonadotropins has been shown to induce molecular changes in the oocyte with a negative impact on the maintenance of genomic imprints during subsequent embryogenesis. As suggested, ovarian stimulation may have a greater adverse impact on the maternal factors required for imprint maintenance than on imprint acquisition . It is therefore likely that exogenous hormone treatments may be responsible for the epigenetic reprogramming of the gametes as well as the early embryos. The scope of this chapter has restricted in-depth discussion on the EP contributions to implantation, endometrial receptivity, and placentation. Collectively, it is likely that deleterious effects of EP may also alter the physiologic environment of the uterus.
Due to limited human data being available, there is a need for well-designed and controlled study to better understand the mechanisms involved in epimutations. Finally, because superovulation may have deleterious effects on imprinting maintenance, research in humans needs to be performed not only on oocytes but also on embryos. Better insight will improve risk assessment among ART practitioners of the impact of ovarian induction protocols on epigenetic control of the genome.
Zegers-Hochschild F, Adamson GD, de Mouzon J, Ishihara O, Mansour R, Nygren K, et al; International Committee for Monitoring Assisted ReproductiveTechnology (ICMART) and the World Health Organization (WHO). International Committee for Monitoring Assisted ReproductiveTechnology (ICMART) and the World Health Organization (WHO) revised glossary of ART terminology, 2009. Fertil Steril. 2009;92(5):1520–4.
Maher ER. Imprinting and assisted reproductive technology. Hum Mol Genet. 2005;14(1):R133–8.CrossRefPubMed
Doherty AS, Mann MR, Tremblay KD, Bartolomei MS, Schultz RM. Differential effects of culture on imprinted H19 expression in the preimplantation mouse embryo. Biol Reprod. 2000;62(6):1526–35.CrossRefPubMed
Khosla S, Dean W, Brown D, Reik W, Feil R. Culture of preimplantation mouse embryos affects fetal development and the expression of imprinted genes. Biol Reprod. 2001;64(3):918–26.CrossRefPubMed
Zaitseva I, Zaitsev S, Alenina N, Bader M, Krivokharchenko A. Dynamics of DNA demethylation in early mouse and rat embryos developed in vivo and in vitro. Mol Reprod Dev. 2007;74(10):1255–61.CrossRefPubMed
DeBaun MR, Niemitz EL, Feinberg AP. Association of in vitro fertilization with Beckwith-Wiedemann syndrome and epigenetic alterations of LIT1 and H19. Am J Hum Genet. 2003;72(1):156–60.PubMedCentralCrossRefPubMed
Gicquel C, Gaston V, Mandelbaum J, Siffroi JP, Flahault A, Le Bouc Y. In vitro fertilization may increase the risk of Beckwith-Wiedemann syndrome related to the Abnormal imprinting of the KCN1OT gene. Am J Hum Genet. 2003;72(5):1338–41.PubMedCentralCrossRefPubMed
Halliday J, Oke K, Breheny S, Algar E, Amor DJ. Beckwith-Wiedemann syndrome and IVF: a case-control study. Am J Hum Genet. 2004;75(3):526–8.PubMedCentralCrossRefPubMed
Ludwig H. Archives of gynecology and obstetrics: 135 years. Arch Gynecol Obstet. 2005;271(1):1–5.CrossRefPubMed
Maher ER, Brueton LA, Bowdin SC, Luharia A, Cooper W, Cole TR, et al. Beckwith-Wiedemann syndrome and assisted reproduction technology (ART). J Med Genet. 2003;40(1):62–4.PubMedCentralCrossRefPubMed
Cox GF, Burger J, Lip V, Mau UA, Sperling K, Wu BL, Horsthemke B. Intracytoplasmic sperm injection may increase the risk of imprinting defects. Am J Hum Genet. 2002;71(1):162–4.PubMedCentralCrossRefPubMed
Orstavik KH, Eiklid K, van der Hagen CB, Spetalen S, Kierulf K, Skjeldal O. Another case of imprinting defect in a girl with Angelman syndrome who was conceived by intracytoplasmic semen injection. Am J Hum Genet. 2003;72(1):218–9.PubMedCentralCrossRefPubMed
Waddington CH. The epigenotype. 1942. Endeavour. 1942;1:18–20.
Bird A. DNA methylation patterns and epigenetic memory. Genes Dev. 2002;16(1):6–21.CrossRefPubMed
Swales AK, Spears N. Genomic imprinting and reproduction. Reproduction. 2005;130(4):389–99.CrossRefPubMed
Robertson KD. DNA methylation and human disease. Nat Rev Genet. 2005;6(8):597–610. Review.CrossRefPubMed
Jaenisch R, Bird A. Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nat Genet. 2003;33(Suppl):245–54.CrossRefPubMed
Holmes R, Soloway PD. Regulation of imprinted DNA methylation. Cytogenet Genome Res. 2006;113(1–4):122–9.CrossRefPubMed
Lewis A, Reik W. How imprinting centres work. Cytogenet Genome Res. 2006;113(1–4):81–9.CrossRefPubMed
Bird A, Wolffe A. Methylation-induced repression belts, braces, and chromatin. Cell. 1999;99(5):451–4.CrossRefPubMed
Li E, Beard C, Jaenisch R. Role for DNA methylation in genomic imprinting. Nature. 1993;366(6453):362–5.CrossRefPubMed
Reik W, Dean W, Walter J. Epigenetic reprogramming in mammalian development. Science. 2001;293(5532):1089–93.CrossRefPubMed
Arnaud PH, Feil R. Epigenetic deregulation of genomic imprinting in human disorders and following assisted reproduction. Birth Defects Res C Embryo Today. 2005;75(2):81–97.CrossRefPubMed
Illingworth R, Kerr A, Desousa D, Jørgensen H, Ellis P, Stalker J, et al. A novel CpG island set identifies tissue-specific methylation at developmental gene loci. PLoS Biol. 2008;6(1), e22.PubMedCentralCrossRefPubMed
Gibney ER, Nolan CM. Epigenetics and gene expression. Heredity. 2010;105(1):4–13.CrossRefPubMed
Constancia M, Kelsey G, Reik W. Resourceful imprinting. Nature. 2004;432(7013):53–7.CrossRefPubMed
Delcuve GP, Rastegar M, Davie JR. Epigenetic control. J Cell Physiol. 2009;219(2):243–50.CrossRefPubMed
Jenuwein T, Allis CD. Translating the histone code. Science. 2001;293(5532):1074–80.CrossRefPubMed
Peterson CL, Laniel MA. Histones and histone modifications. Curr Biol. 2004;14(14):R546–51.CrossRefPubMed
Mattick JS, Amaral PP, Dinger ME, Mercer TR, Mehler MF. RNA regulation of epigenetic processes. Bioessays. 2009;31(1):51–9.CrossRefPubMed
Calvanese V, Lara E, Kahn A, Fraga MF. The role of epigenetics in aging and age-related diseases. Ageing Res Rev. 2009;8(4):268–76.CrossRefPubMed
Barton SC, Adams CA, Norris ML, Surani MA. Development of gynogenetic and parthenogenetic inner cell mass and trophectoderm tissues in reconstituted blastocysts in the mouse. J Embryol Exp Morphol. 1985;90:267–85.PubMed
McGrath J, Solter D. Completion of mouse embryogenesis requires both the maternal and paternal genomes. Cell. 1984;37(1):179–83.CrossRefPubMed
Surani MA, Barton SC, Norris ML. Development of reconstituted mouse eggs suggests imprinting of the genome during gametogenesis. Nature. 1984;308(5959):548–50.CrossRefPubMed
Surani MA, Barton SC. Development of gynogenetic eggs in the mouse: implications for parthenogenetic embryos. Science. 1983;222(4627):1034–6.CrossRefPubMed
Barton SC, Surani MA, Norris ML. Role of paternal and maternal genomes in mouse development. Nature. 1984;311(5984):374–6.CrossRefPubMed
Manipalviratn S, DeCherney A, Segars J. Imprinting disorders and assisted reproductive technology. Fertil Steril. 2009;91(2):305–15.PubMedCentralCrossRefPubMed
Amor DJ, Halliday J. A review of known imprinting syndromes and their association with assisted reproduction technologies. Hum Reprod. 2008;23(12):2826–34.CrossRefPubMed
Reik W, Walter J. Genomic imprinting: parental influence on the genome. Nat Rev Genet. 2001;2(1):21–32.CrossRefPubMed
Morison IM, Ramsay JP, Spencer HG. A census of mammalian imprinting. Trends Genet. 2005;21(8):457–65.CrossRefPubMed
Morison I. Catalogue of Parent of origin effects: Imprinted genes and related effects - parental origin of de novo mutation. Available from: http://www.igc.ac.nz/home.html
Wadhwa PD, Buss C, Entringer S, Swanson JM. Developmental origins of health and disease: a brief history of the approach and current focus on epigenetic mechanisms. Semin Reprod Med. 2009;27(5):358–68.PubMedCentralCrossRefPubMed
Batcheller A, Maguire M, DeCherney AH, Segars JH. Are there subtle, genome-wide epigenetic alterations in normal offspring conceived from assisted reproductive technologies? Fertil Steril. 2011;96(6):1306–11.PubMedCentralCrossRefPubMed
Wilkins-Haug L. Assisted reproductive technology, congenital malformations, and epigenetic disease. Clin Obstet Gynecol. 2008;51(1):96–105.CrossRefPubMed
Owen C, Segars J. Imprinting disorders and assisted reproductive technology. Semin Reprod Med. 2009;27(5):417–28.PubMedCentralCrossRefPubMed
Sutcliffe AG, Peters CJ, Bowdin S, Temple K, Reardon W, Wilson L, et al. Assisted reproductive therapies and imprinting disorders—a preliminary British survey. Hum Reprod. 2006;21(4):1009–11.CrossRefPubMed
Chang AS, Moley KH, Wangler M, Feinberg AP, Debaun MR. Association between Beckwith-Wiedemann syndrome and assisted reproductive technology: a case series of 19 patients. Fertil Steril. 2005;83(2):349–54.CrossRefPubMed
Market-Velker BA, Zhang L, Magri LS, Bonvissuto AC, Mann MR. Dual effects of superovulation: loss of maternal and paternal imprinted methylation in a dose-dependent manner. Hum Mol Genet. 2009;19(1):36–51.CrossRef
Lucifero D, Mann MR, Bartolomei MS, Trasler JM. Gene-specific timing and epigenetic memory in oocyte imprinting. Hum Mol Genet. 2004;13(8):839–49.CrossRefPubMed
Mann M, Lee S, Doherty A, Verona R, Nolen L, Schultz R, Bartolomei MS. Selective loss of imprinting in the placenta following preimplantation development in culture. Development. 2004;131(15):3727–35.CrossRefPubMed
Rivera RM, Stein P, Weaver J, Mager J, Schultz R, Bartolomei M. Manipulations of mouse embryos prior to implantation result in aberrant expression of imprinted genes on day 9.5 of development. Hum Mol Genet. 2008;17(1):1–14.CrossRefPubMed
Bocock PN, Aagaard-Tillery K. Animal models of epigenetic inheritance. Semin Reprod Med. 2009;27(5):369–79.CrossRefPubMed
Coan PM, Burton GJ, Ferguson-Smith AC. Imprinted genes in the placenta—a review. Placenta. 2005;26(Suppl A):S10–20.CrossRefPubMed
Barker D. The origins of the developmental origins theory. J Intern Med. 2007;261(5):412–7.CrossRefPubMed
Huntriss J, Picton HM. Epigenetic consequences of assisted reproduction and infertility on the human preimplantation embryo. Hum Fertil. 2008;11(2):85–94.CrossRef
Seki Y, Hayashi K, Itoh K, Mizugaki M, Saitou M, Matsui Y. Extensive and orderly reprogramming of genome-wide chromatin modifications associated with specification and early development of germ cells in mice. Dev Biol. 2005;278(2):440–58.CrossRefPubMed
Hajkova P, Ancelin K, Waldmann T, Lacoste N, Lange UC, Cesari F, et al. Chromatin dynamics during epigenetic reprogramming in the mouse germ line. Nature. 2008;452(7189):877–81.CrossRefPubMed
Trasler JM. Gamete imprinting: setting epigenetic patterns for the next generation. Reprod Fertil Dev. 2006;18(1–2):63–9.CrossRefPubMed
Oda M, Yamagiwa A, Yamamoto S, Nakayama T, Tsumura A, Sasaki H, et al. DNA methylation regulates long-range gene silencing of an X-linked homeobox gene cluster in a lineage-specific manner. Genes Dev. 2006;20(24):3382–94.PubMedCentralCrossRefPubMed
Okano M, Bell DW, Haber DA, Li E. DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell. 1999;99(3):247–57.CrossRefPubMed
Kaneda M, Hirasawa R, Chiba H, Okano M, Li E, Sasaki H. Genetic evidence for Dnmt3a-dependent imprinting during oocyte growth obtained by conditional knockout with Zp3-Cre and complete exclusion of Dnmt3b by chimera formation. Genes Cells. 2010;15(3):169–79.CrossRefPubMed
Shovlin TC, Bourc’his D, La Salle S, O’Doherty A, Trasler JM, Bestor TH, Walsh CP. Sex-specific promoters regulate Dnmt3L expression in mouse germ cells. Hum Reprod. 2007;22(2):457–67.CrossRefPubMed
Sato C, Shimada M, Mori T, Kumasako Y, Otsu E, Watanabe H, Utsunomiya T. Assessment of human oocyte developmental competence by cumulus cell morphology and circulating hormone profile. Reprod Biomed Online. 2007;14(1):49–56.CrossRefPubMed
Khoueiry R, Ibala-Rhomdane S, Mery L, Blachere T, Guerin JF, Lornage J, Lefevre A. Dynamic CpG methylation of the KCNQ1OT1 gene during maturation of human oocytes. J Med Genet. 2008;45(9):583–8.CrossRefPubMed
Geuns E, Hilven P, Van Steirteghem A, Liebaers I, De Rycke M. Methylation analysis of KvDMR1 in human oocytes. J Med Genet. 2007;44(2):144–7.PubMedCentralCrossRefPubMed
Bourc’his D, Bestor TH. Origins of extreme sexual dimorphism in genomic imprinting. Cytogenet Genome Res. 2006;113(1–4):36–40.PubMed
Schulz R, Proudhon C, Bestor TH, Woodfine K, Lin CS, Lin SP, et al. The parental non-equivalence of imprinting control regions during mammalian development and evolution. PLoS Genet. 2010;6(11), e1001214.PubMedCentralCrossRefPubMed
Shi W, Haaf T. Aberrant methylation patterns at the two-cell stage as an indicator of early developmental failure. Mol Reprod Dev. 2002;63(3):329–34.CrossRefPubMed
Sato A, Otsu E, Negishi H, Utsunomiya T, Arima T. Aberrant DNA methylation of imprinted loci in superovulated oocytes. Hum Reprod. 2007;22(1):26–35.CrossRefPubMed
Fauque P, Jouannet P, Lesaffre C, Ripoche MA, Dandolo L, Vaiman D, Jammes H. Assisted reproductive technology affects developmental kinetics, H19 imprinting control region methylation and H19 gene expression in individual mouse embryos. BMC Dev Biol. 2007;7:116.PubMedCentralCrossRefPubMed
Market-Velker BA, Zhang L, Magri LS, Bonvissuto AC, Mann MRW. Dual effects of superovulation: loss of maternal and paternal imprinted methylation in a dose-dependent manner. Hum Mol Genet. 2010;19(1):36–51.CrossRefPubMed
Fortier AL, Lopes FL, Darricarrere N, Martel J, Trasler JM. Superovulation alters the expression of imprinted genes in the midgestation mouse placenta. Hum Mol Genet. 2008;17(11):1653–65.CrossRefPubMed
Linke M, May A, Reifenberg K, Haaf T, Zechner U. The impact of ovarian stimulation on the expression of candidate reprogramming genes in mouse preimplantation embryos. Cytogenet Genome Res. 2013;139(2):71–9.CrossRefPubMed
Picton HM, Harris SE, Muruvi W, Chambers EL. The in vitro growth and maturation of follicles. Reproduction. 2008;136(6):703–15.CrossRefPubMed
Eppig JJ, O’Brien MJ. Development in vitro of mouse oocytes from primordial follicles. Biol Reprod. 1996;54(1):197–207.CrossRefPubMed
Siristatidis CS, Maheshwari A, Bhattacharya S. In vitro maturation in sub fertile women with polycystic ovarian syndrome undergoing assisted reproduction. Cochrane Database Syst Rev. 2009;(1):CD006606.
Wang N, Le F, Zhan QT, Li L, Dong MY, Ding GL, et al. Effects of in vitro maturation on histone acetylation in metaphase II oocytes and early cleavage embryos. Obstet Gynecol Int. 2010;2010:989278.PubMedCentralPubMed
Buckett WM, Chian RC, Holzer H, Dean N, Usher R, Tan SL. Obstetric outcomes and congenital abnormalities after in vitro maturation, in vitro fertilization, and intracytoplasmic sperm injection. Obstet Gynecol. 2007;110(4):885–91.CrossRefPubMed
Kerjean A, Couvert P, Hearns T, Chalas C, Poirier K, Chelly J, Jouannet P, Andras Paldi A, Poirot C. In vitro follicular growth affects oocyte imprinting establishment in mice. Eur J Hum Genet. 2003;11(7):493–6.CrossRefPubMed
Anckaert E, Adriaenssens T, Romero S, Dremier S, Smitz J. Unaltered imprinting establishment of key imprinted genes in mouse oocytes after in vitro follicle under variable follicle stimulating hormone exposure. Int J Dev Biol. 2009;53(4):541–8.CrossRefPubMed
Caperton L, Murphey P, Yamazaki Y, McMahan CA, Walter CA, Yanagimachi R, McCarrey JR. Assisted reproductive technologies do not alter mutation frequency or spectrum. Proc Natl Acad Sci U S A. 2007;104(12):5085–90.PubMedCentralCrossRefPubMed
Baerwald AR, Walker RA, Pierson RA. Growth rates of ovarian follicles during natural menstrual cycles, oral contraception cycles, and ovarian stimulation cycles. Fertil Steril. 2009;91(2):440–9.CrossRefPubMed
Minocherhomji S, Athalye AS, Madon PF, Kulkarni D, Uttamchandani SA, Parikh FR. A case–control study identifying chromosomal polymorphic variations as forms of epigenetic alterations associated with the infertility phenotype. Fertil Steril. 2009;92(1):88–95.CrossRefPubMed
Denomme MM, Mann MR. Genomic imprints as a model for the analysis of epigenetic stability during assisted reproductive technologies. Reproduction. 2012;144(4):393–409.CrossRefPubMed