Human assisted reproductive techniques (ART)

In recent decades, human assisted reproductive techniques (ART) have been widely applied in treatment of infertility. ART involves follicular stimulation, oocyte isolation, gamete manipulation, in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI) and other related procedures. Laboratory manipulation of gametes and embryos coincides with a period when genes at various imprinted loci are extensively reprogrammed1 and it has been suggested that this reprogramming can be disrupted by ART procedures resulting in babies born with rare genomic imprinting disorders including Beckwith—Wiedemann syndrome (BWS), Angelman syndrome (AS) and Silver—Russell syndrome (SRS), but not Prader—Willi syndrome (PWS)2. Infact, BWS is nine times more common in ART than natural conception3, Figure11.

Genomic imprinting is a process controlled by epigenetic regulatory mechanisms whereby certain maternal and paternal gene alleles are silenced in a specific manner leading to monoallelic expression of one parental allele. It has a crucial role in normal embryonic development and placental functions4. Imprinted genes are organized in clusters and regulated by DNA methylation (the main epigenetic modification) at differentially methylated regions (DMR) of imprinted loci creating unique patterns related to parental origin. This pattern of imprinting must be retained when genome-wide epigenetic reprogramming takes place during embryogenesis. Deviation from the normal epigenetic process would result in a disruption of embryonic development 5. Table (1) highlights potential areas of impacts on ART6.

ART procedures are associated with epigenetic disruption in mammalian preimplantation embryos


To date it is unclear whether epigenetic anomalies are exacerbated by ART procedures. The main concerns about epigenetic modification in oocyte global methylation involve ovulation induction7. In humans, it is very difficult to determine whether it is ART procedures or the underlying cause of the infertility that affects the genomic imprinting8. In males, global demethylation takes place in primordial germ cells during fetal life and remethylation occurs in early gametogenesis. In females, the process is gradual and completes during oocyte maturation. It is possible that oocyte epimutation is induced by superovulation where forced maturation may affect imprinting maintenance machinery or lead to lost expression of the specific genome imprinting pattern. This notion was supported in a study involving mouse blastocysts generated after induced ovulation. There was a loss of maternal methylation in Snrpn, Kcnq1ot1 and peg3 as compared with a control group (spontaneously ovulated females)9. Another study identified a gain in maternal methylation for allele H19 10. Using methylcytosine immunofluorescence techniques, abnormal methylation patterns have been demonstrated in embryos from superovulated versus non superovlated mice11. In contract, other studies showed normal methylation patterns in imprinted genes (Snrpn, Peg3, and H19) of mature oocytes from superovulated mice12. Denomme et al (2011) observed methylated and unmethylated patterns of Peg1/Mest genes in superovulated mature oocytes suggesting that, during superovulation oocyte maturation was not fully established. The data variation may be explained by the fact that ovulation induction is intended to recruit oocytes likely destined for atresia13. The impact on imprinting patterns has not been considered and the forced maturation may not leave the oocytes enough time to complete its epigenetic programming14.

Artificial oocyte growth and maturation

In vitro growth and in vitro maturation of mammalian oocytes represent great approaches to treat infertility patients, however these techniques may result in epigenetic perturbation15. In vitro folliculogenesis studies in mice showed that methylation was disrupted in oocytes at imprinted loci of genes (H19, Mest/Peg1, and Igf2r)16. In contract, normal methylation patterns were seen at (H19, Igf2r, and Snrpn) in another study, suggesting that maternal imprinting is more vulnerable to the external environment12.

Culture media

In vitro embryo culture represents another factor which may exacerbate epigenetic aberration. Numerous studies have indicated a link between perturbation in DNA methylation patterns and culture media17,18,19,20. Large offspring syndrome has been observed in cattle conceived by ART as a result of aberrant methylation in imprinted loci. This aberration leads to loss of maternal allelic methylation and overexpression of IGF2R. It has been hypothesized that imprinted loci are protected from global demethylation by certain mechanisms and this protection is compromised by ART procedures21.

Amino acids are important constituents of embryo culture media (specially glutamine). Embryonic development rate is significantly increased when embryos are cultured in media supplemented with glutamine22,23. On the other hand, ammonium is a major product of amino acid consumption and breakdown. Ammonium has cytotoxic effect disrupting the development of the embryo24. When cultured in elevated levels of NH+4, mouse embryos exhibited a reduction inner cell mass (ICM) and trophectoderm cell numbers in blastocysts as well as a high rate of apoptosis. Increased levels of ammonium correlated with an aberration in expression of the imprinted H19 gene25. Pyruvate is an important substrate during the cleavage stage of embryonic development and elevated ammonium levels in media have been shown to decrease pyruvate oxidation and increase the glycolytic activity leading to a reduction in intracellular pH. The pH shift can cause epigenetic perturbation although this effect is less at the post-compaction stage. These data suggested that sensitivity of epigenetic mechanisms may vary according to the embryonic stage24.

O2 concentration was also seen as an important factor in epigenetic expression. Some studies have revealed that fluctuating O2 concentrations can alter the production of mitochondrial reactive oxygen species leading to increase random genomic expression errors which can affect embryonic metabolism and ultimately compromise development26. Collectively, these data suggest that metabolic stress on embryos can have consequences which affect health later in life.


Vitrification known an important technique in ART allowing preservation of excess embryos/oocytes. Bakhtari et al (2014)27 conducted a study in mice which demonstrated a decrease in global DNA methylation of ICM in vitrified blastocysts. This was supported by other studies which also observed reduced DNA methylation 28,29,30. Contradicted significant increases in gene methylation of oocytes/ blastocysts/ fetuses after vitrification were noted in some studies 31,32. Birth weight of singleton derived from vitrified blastocysts were remarkably higher than those of singletons born from fresh transfer30. These variations could also be due to differences in stimulation protocols, culture conditions, developmental stage as well as the length of investigated genomic regions used in each study33.

Possible effects caused by micromanipulation procedures

ICSI and IVF are novel techniques widely applied in human fertility treatment. Extensive studies have been investigated whether these techniques have consequences on the developing embryo and/or postnatal life. Microarray analysis using DNA obtained from tissues (brain, liver and kidney) derived from mice produced using IVF or ICSI indicated significant differences in gene expression for the ICSI but not the IVF group. These differences can be observed as early as the blastocyst stage. No phenotypic differences in these tissues were observed between two groups34. This was supported by another study which found perturbations in DNA methylation of CpG islands of DMR in mice conceived by ICSI34. Sperm chromatin decondensation has also been shown to be delayed in embryos derived from ICSI. This delay can have an epigenetic effect on the zygote; affecting global methylation in male pronucleus35.

In 2014, Whitelaw et al reported that the DNA methylation status for SNRPN was higher in children conceived in ICSI versus IVF. However a high correlation between epigenetic perturbation and the duration of infertility has been identified regardless of the conception method36. In 2012, Oliver et al reported a high incidence of hyper DNA methylation in SNPRN of children born by IVF as compare to spontaneous conception37. It has also been found hyper DNA methylation in placenta but not in cord blood for infants conceived by IVF compared to natural conception38. A large study involved 308,974 women, observed high incident of defects in births conceived by ICSI but not IVF. This is may be due to the type of the subsubfertility itself. ICSI bypasses many natural barriers and has been a critical tool for treating male factor infertility. In fact, oligozoospermia, teratozoospermia and asthenozoospermia are correlated with significant deviations in methylation patterns of sperm DNA and these perturbations can have consequences on later embryo/fetal development39. Differences in epigenetic profile could contribute to the slight increase in blood pressure of ART children versus spontaneous conception as well as lower birth weight and shorter gestation duration40,41,42,43. In contrast, another study found no difference in blood pressure between the two cohorts44. It was further suggested that the low birth weight may be related to other maternal and paternal factors45.


ART procedures coincide with the timing of epigenetic processes in where global changes in DNA methylation profiles occur. This overlap may influence early epigenome development5. Imprinting disorders may be expected in children born after ART because genomic imprinting development and maintenance are vulnerable to environmental factors. Some loci may be more susceptible to external influences and this could explain why BWS, SRS and AS but not PWS are more common in ART1. PWS results from a disjunction error at meiosis I associated with advanced maternal age46,47. On the other hand, it is still unclear whether epimutation results from ART procedures or that it is a consequence of underling infertility causes. Many fertility clinics favour culturing embryos to blastocyst stage to allow embryos to select themselves before transferring the best quality embryo. However several studies have suggested that, while short-term culture is safe, extended culture may affect genomic imprints and specially in placenta tissues48. While many protocols continue to involve aggressive stimulation for advanced maternal age and low ovarian reserve, there is increasing concern that this will have epigenetic consequences by birth and later in life. Comprehensive studies are required to investigate the influence of ART procedures on imprint methylation disorders and to determine if there are precautions that can be taken in order to avoid development of these disorders.

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