Impact of ART on Genomic Imprinting and Child Health

Introduction

Assisted reproductive techniques (ART) account for approximately 1-2% of all births in developed countries (1). The primary aim of ART is to produce high-quality embryos capable of successful implantation and ensuring a positive perinatal outcome. Despite the widespread acceptance of ART, concerns have arisen regarding the long-term safety of gamete and embryo manipulation and their potential impact on human development (2).

Epigenetic and Genomic Imprinting

The intricate process of normal human development relies heavily on epigenetic modifications, with genetic imprinting playing a pivotal role during early development (3).

Genomic imprinting involves the selective expression of one parental allele of a specific gene while silencing the other allele through chemical modification of nucleotides (4). Two key mechanisms responsible for genetic imprinting are histone modification and DNA methylation (3). Histones undergo covalent modifications that can either activate or suppress gene expression (4). DNA methylation is initiated by DNA Methyltransferase and occurs on cytosine residues of CpG dinucleotides (3). This process can activate certain genes (e.g., IGF2, IGF2R from the paternally inherited allele) while silencing others (e.

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g., H19, exclusively expressed from the maternally inherited allele) (3, 5). Methylation plays a crucial role in development and differentiation, particularly in imprinted genes (6). The initiation of gene imprinting takes place during germ cell proliferation, migration, and development, with further adaptations occurring following fertilization (3). Imprinted genes are vital for controlling placental and fetal growth, somatic differentiation, neurological functions, and postnatal behavioral functions (7). Abnormalities in methylation reprogramming may arise during ovulation induction, in vitro maturation, in vitro oocyte culture, and fertilization, particularly when early embryo development occurs in vitro (7).

Disruptions in established imprinting patterns have been associated with various human genetic diseases (8).

The processes of superovulation, micromanipulation, and embryo culture often coincide with critical epigenetic reprogramming events, potentially leading to interference with genomic imprinting. It is likely that epigenetic marks are susceptible to external environmental stimuli during gametogenesis and early development (6, 9). Medical complications linked to ART may stem from either impairment or permanent alterations in epigenetic gene regulation within the germline and early embryo (7).

Epidemiology

Babies born through ART face a twofold increased risk of major birth defects compared to naturally conceived infants (10, 11), and imprinting disorders are more prevalent among ART-conceived individuals (12, 13).

Animal Studies

While infertility is not a factor in animal models, studies involving animals have implicated ART in epigenetic defects observed in their offspring (2). Lambs and calves born following embryo culture have exhibited a condition known as large offspring syndrome (LOS), which has been linked to imprinting disorders. Abnormal methylation and up-regulation of IGF2R have been associated with LOS in cases involving embryo culture with serum-containing media (14). In mice, the embryo culture environment has been observed to influence epigenetic alterations in genes such as IGF2, H19, Grb7, and Grb10 (15). Fauque et al. (16) found detrimental effects of superovulation, including abnormal methylation, in mice. Kerjean et al. (17) reported aberrant imprinting, with a loss of methylation at the IgF2 locus in oocytes following in vitro growth of mouse follicles. However, it is important to note that findings from animal studies cannot definitively be extrapolated to humans.

ART Processes (Superovulation, Embryo Culture, Intracytoplasmic Sperm Injection, In Vitro Maturation)

Superovulation

Studies by Khouiery et al. (18) have shown that stimulated human cycles result in less methylation of KvDMR1 in germinal vesicle and metaphase oocytes compared to those in natural cycles. Additionally, Sato et al. (19) noted imprinted errors, such as a gain of methylation in H19 and a loss of methylation in PEG1, in human oocytes retrieved after ovulation stimulation. These findings suggest that gonadotropin stimulation may disrupt the methylation process, potentially leading to imprinting disorders.

In Vitro Embryo Culture

Embryo culture media used in vitro can induce specific alterations in gene expression in embryos, ultimately resulting in observable phenotypic changes. Different embryo culture conditions may influence the regulation of cell lineage, blastocyst and fetal developmental rates, and birth weight (20).

Intracytoplasmic Sperm Injection (ICSI)

ICSI bypasses the natural fertilization processes and oocyte activation. This technique may interfere with the establishment of maternal imprints in the oocyte, raising concerns about imprinting defects (21). Abnormal methylation patterns, specifically at the SNRPN differentially methylated region, were observed in two cases reported by Cox et al. (21) in individuals with Angelman syndrome (AS) conceived through ICSI. The methylation imprint of the maternal allele at SNRPN is established at fertilization or shortly thereafter, suggesting a potential link between ICSI and AS (5). Orstvik et al. (22) further supported this association by reporting a case of AS with an imprinting defect following conception via ICSI. AS is rarely caused by sporadic imprinted defects, making ICSI the most likely contributing factor (22). ICSI may also lead to alterations in maternal genome methylation during oocyte or early embryo development (13). Additionally, ICSI has been associated with an increased risk of major birth abnormalities (10).

In Vitro Maturation (IVM)

In IVM, altered methylation patterns have been observed in many M1-arrested oocytes, with 50% of DNA showing methylation at the CTCF-binding site (23). Aberrant hypermethylation at H19 has also been documented in some human M11 oocytes subjected to IVM (23). These changes may result from disruptions in imprint erasure and establishment within oocytes.

Imprinting Syndromes

Concerns have arisen regarding the potential association between ART and imprinting disorders (11). Imprinting disorders such as Beckwith-Wiedemann Syndrome (BWS), Angelman Syndrome (AS), Silver-Russell Syndrome (SRS), and maternal hypomethylation syndrome have been linked to ART (9, 15, 24). The following section explores the evidence of these imprinting disorders and their relationship with ART.

Beckwith-Wiedemann Syndrome (BWS)

BWS affects approximately 1 in 13,700 children, with the majority of cases resulting from epimutation of the maternal allele of one of the two differentially methylated regions (DMRs) at chromosome 11p15 (24). The syndrome presents with variable clinical features, including large pre/post-natal growth, exomphalos, macroglossia, neonatal hypoglycemia, hemihypertrophy, and childhood tumors, particularly Wilms tumor (24). BWS registries have documented cases in children born after ART, and in a significant percentage of these patients, hypomethylation of the maternal alleles of DMR2 has been identified as the underlying molecular mechanism (24). Lim et al. (25) and Gicquel et al. (8) found 96% and 100% of ART-conceived children, respectively, exhibited hypomethylation at KvDMR1. Patients with BWS due to epimutation have a 14-fold higher likelihood of being conceived through ART compared to those without epimutation-related BWS (26). BWS has also been reported in children born after ovulation induction (24). The risk of BWS in ART populations was found to be nine times higher than in the general population by Halliday et al. (27), while Debaun et al. (13) reported a prevalence of 4.6% in ART children compared to the expected background rate. Several studies provide substantial evidence supporting a real association between disordered imprinting causing BWS and ART (5, 8, 9, 13, 24, 28, 29).

Angelman Syndrome (AS)

AS affects 1 in 16,000 children and is characterized by a spectrum of genetic defects (11). Approximately 70% of AS patients have a deletion of 4-6MB in the 15q11.2-15q13 imprinting center (11). Methylation deprivation at the SNRPN imprinting control region has also been observed in AS (5). ART, especially ICSI, has been associated with AS (5, 6, 25). However, Vermeiden et al. (12), in their literature review, found no association between AS and ART.

Silver-Russell Syndrome (SRS)

SRS affects 1 in 100,000 children and is characterized by intrauterine and post-natal growth retardation and learning disabilities (24). The most common defect is hypomethylation of H19/IGF2 at chromosome 11p15.5, but other loci may also be affected, including PEG10, GRB10, MEST, and PEG1 (9, 29). Recent studies indicate that the prevalence of SRS patients in ART is ten times higher than expected.

Maternal Hypomethylation Syndrome (MHS)

This syndrome has been associated with ART conceptions and exhibits features of transient neonatal diabetes mellitus and BWS (9).

Other Imprinted Disorders

Prader-Willi syndrome, MatUPD14 syndrome, PatUPD syndrome, Pseudohypoparathyroidism1b, and Transient neonatal diabetes mellitus have not been associated with ART (15). While retinoblastoma was initially linked to ART, current data does not support such an association (3, 9, 11).

It is important to note that imprinting disorders are rare, and a moderate increase in their occurrence after ART may not be noticeable in a sample of fewer than 10,000 children (28).

Table 2: Relationship between ART and Imprinting Disorders

Relationship between ART and Epigenetic Health

In addition to documented imprinting disorders, it is possible for induced epigenetic variations to be transmitted to offspring without specific phenotypical effects, potentially affecting susceptibility to diseases later in life (5, 9). Hypomethylation of KCNQ10T and H19 has been observed in chorionic villus samples from ART patients with spontaneous abortion and stillbirth (6). Studies have also found an increased prevalence of elevated blood pressure, overweight, obesity, raised fasting blood sugars, and an elevated risk of cardiovascular and metabolic diseases in IVF offspring (11). These health outcomes may be linked to aberrant epigenetic patterns.

Conclusion

In the context of ART, the developing epigenome is exposed to external stimuli that can interfere with the proper establishment and maintenance of genomic imprinting (29). Methylation patterns may differ statistically between ART and non-ART cohorts (6, 7). Specific imprinting disorders, such as BWS, AS, SRS, and MHS, have been associated with ART (9, 24, 31). However, the absolute risk of bearing a child with an imprinting disorder after ART remains low. To minimize the potential negative epigenetic effects, the manipulation of gametes and embryos, as well as the duration of embryo culture, should be minimized (7). Comprehensive evaluation of the impact of ART on the epigenetic process is challenging due to the low prevalence of imprint disorders (30). Therefore, conducting multicenter and international studies to assess ART outcomes is essential.

References

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