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

in Reproduction

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

Abstract

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

Introduction

Since the first assisted conception in 1978, assisted reproductive technologies (ARTs) have enabled the birth of ∼4 million children from couples with infertility/subfertility (Zegers-Hochschild et al. 2009). ARTs encompass any treatment modality that is used to improve fertility and establish a pregnancy, including ovarian stimulation, IVF, ICSI, and embryo culture as well as the experimental procedures in vitro oocyte maturation (IVM), and oocyte and ovarian tissue cryopreservation. ARTs can lead to adverse prenatal and postnatal outcomes, including increased risk of intrauterine growth restriction, premature birth, low birth weight, congenital anomalies, and genomic imprinting syndromes (Savage et al. 2011). To reduce these risks, it is paramount to determine which aspects of treatment lead to adverse effects so they may be modified for improved safety. Given that impaired fertility and ARTs alter the gamete and embryo environment, epigenetic instability may be the primary determinant of these suboptimal outcomes.

Genomic imprinting: a model for epigenetic stability

Epigenetics refers to chromatin modifications that regulate gene activity that are not due to DNA sequence changes (Saitou et al. 2012). DNA methylation and histone modifications are two epigenetic mechanisms that alter the functional state of chromatin, activating or repressing gene expression. Genomic imprinting is a specialized epigenetic mechanism that employs repressive modifications to silence one parental allele, while activating modifications on the other parental allele enable expression (Hirasawa & Feil 2010). Disruptions in these asymmetric parental states can have severe consequences for growth and development, including Beckwith–Wiedemann syndrome (BWS) and Angelman syndrome (AS) (Hirasawa & Feil 2010; Box 1).

Box 1 Beckwith–Wiedemann syndrome (BWS) is an overgrowth disorder caused by genetic and epigenetic errors at the KCNQ1OT1 and H19 imprinted domains. ART-conceived BWS children commonly experience maternal KCNQ1OT1 LOM and maternal H19 GOM.

Angelman syndrome (AS) is a neurological disorder that is caused by genetic and epigenetic disturbances at the SNRPN imprinted domain. ART-conceived AS patients often possess maternal SNRPN LOM.

Gamete and early embryo development are important stages when genome-scale epigenetic transitions are orchestrated (Fig. 1). During oogenesis and spermatogenesis, previous somatic epigenetic modifications are erased, and new sex-specific epigenetic marks are acquired. Paternal DNA methylation acquisition occurs during prenatal stages of spermatogenesis and is completed by birth (Saitou et al. 2012; Fig. 1). During spermiogenesis, protamines replace the majority of histones (Carrell 2012). The resulting effect is tight compaction of chromatin into toroids that are punctuated by histone solenoids (Fig. 2). Histone-containing chromatin, which is situated at spermatogenic, developmental, paternally expressed imprinted gene and microRNA promoters, harbors active histone modifications (H3Ac, H4Ac, and H3K4me2/3) or bivalency marks (H3K4me3 and H3K27me3) and is hypomethylated, while that at maternally expressed imprinted genes possesses repressive modifications (H3K9me2/3 and DNA methylation) (Hammoud et al. 2009, Brykczynska et al. 2010, Carrell 2012, Nakamura et al. 2012; Box 2). During oogenesis, acquisition of maternal DNA methylation begins comparatively later (puberty) in primary to antral stage follicles and is mostly complete in MII-ovulated oocytes (Saitou et al. 2012; Fig. 1). The MII oocyte genome also possesses repressive histone modifications (H3K9me2/3 and H4K20me3) (Lepikhov et al. 2010, Hales et al. 2011).

Figure 1
Figure 1

DNA methylation asymmetry during gametogenesis and preimplantation development. Paternal (blue line) and maternal (red line) DNA methylation is erased in primordial germ cells. De novo DNA methylation acquisition occurs earlier in male compared to female germ cell development. Following fertilization, the paternal genome is rapidly and actively demethylated (light blue line) while the maternal genome (light red line) is passively demethylated at each replication cycle. Differential DNA methylation at imprinted genes is protected from demethylation. Infertility/subfertility and various ARTs may cause epigenetic instability at the erasure, acquisition, and maintenance stages. Furthermore, combination of infertility and/or multiple ARTs may impose greater risk for inducing epigenetic errors.

Box 2 Histone modifications are posttranslational covalent modifications to histone tails, which have either activating or repressive functions. Histone 3 lysine 9 (H3K9), histone 3 lysine 27 (H3K27), and histone 4 lysine 20 (H4K20) trimethylation (me3) are repressive modifications while histone 4 acetylation (H4Ac) and histone 3 lysine 4 trimethylation (H3K4me3) are activating modifications.

DNA methylation is the covalent modification of methyl groups to cytosines within CpG dinucleotides. DNA methylation is typically associated with gene silencing.

Citation: REPRODUCTION 144, 4; 10.1530/REP-12-0237

Figure 2
Figure 2

Epigenetic landscape in gametes. In mature sperm, protamines tightly compact chromatin into toroids (90–99% chromatin) that are punctuated by histone solenoids (1–10% chromatin). Sperm DNA is hypermethylated (red; DNA me) except at regions bearing active and bivalent histone modifications. By comparison, histones compact chromatin in the mature oocyte. Chromatin is further condensed into loops that are bound to spindle fibers. Oocyte chromatin is hypermethylated and carries repressive histone modifications. Data from Patrushev & Minkevich (2008) and Carrell (2012).

Citation: REPRODUCTION 144, 4; 10.1530/REP-12-0237

Following fertilization, genome-scale epigenetic reprogramming occurs again with a switch from a gamete-specific to embryonic state. The pronuclear paternal genome is rapidly and actively demethylated (Fig. 1), undergoes protamine to histone replacement, and further acquires active histone modifications (H4Ac, H3Ac, and H3K4me2/3) (Lepikhov et al. 2010, Hales et al. 2011). By comparison, the maternal pronuclear genome contains active (H4Ac, H3Ac, and H3K4me2/3) and repressive modifications (H3K9me2/3, H3K27me2/3, and H4K20me3) and becomes passively demethylated during preimplantation development (Fig. 1). By the four-cell stage, the paternal genome acquires repressive histone modifications (H3K9me2, H3K27me2, and H3K27me3) and is globally no longer distinguishable from the maternal genome (Hales et al. 2011). Thus, before and after fertilization, the paternal and maternal genomes possess asymmetric epigenetic modifications.

Differential imprinted DNA methylation is maintained during the preimplantation remodeling period. Its apparent lack of remodeling has lead to the argument that epigenetic disruption by ARTs is restricted to imprinted genes. However, other genes have now been identified with differential gametic DNA methylation that is retained through early preimplantation development (Smallwood et al. 2011, Kobayashi et al. 2012). Furthermore, while genome-wide methylation analyses are limited, altered DNA methylation has been detected at both imprinted and non-imprinted genes in ART-conceived children (Katari et al. 2009), in vitro cultured mouse blastocysts (Wright et al. 2011), and sperm from infertile men (Houshdaran et al. 2007, Pacheco et al. 2011, Aston et al. 2012), as well as globally in two-cell mouse embryos from superovulated mothers and sperm from men with infertility (Shi & Haaf 2002, Benchaib et al. 2003). Thus, aberrant imprinted methylation may be an indicator of more global epigenetic instability arising from ARTs or underlying infertility. Here, we review the current state of knowledge regarding genomic imprinting following various ARTs and infertility as a model for epigenetic instability. While the literature search was restricted to mouse and human, it is important to note that work in other animal models, such as sheep and cattle, support the studies described herein (for example, see Young et al. (2001), Suzuki et al. (2009), Hori et al. (2010), Barboni et al. (2011) and Heinzmann et al. (2011)). Paramount to assisted reproduction is that timing of ARTs coincides with crucial epigenetic events during gametogenesis and early embryogenesis (Fig. 1). Understanding how ARTs cause epigenetic disruption is crucial for maximizing their efficacy and safety.

Manipulations during female germ cell development

Ovarian stimulation

To produce increased oocyte numbers for assisted reproduction, protocols incorporate large gonadotropin doses. Ovarian stimulation has been linked to BWS and AS in ART-conceived children (Chang et al. 2005, Ludwig et al. 2005, Sutcliffe et al. 2006; Table 1). This has lead to investigations of superovulation as a potential imprinting disruptor. Individual mouse 16-cell embryos recovered from superovulated (7.5 IU eCG/hCG) females had paternal H19 loss of methylation (LOM, Box 3) in 2/10 embryos, maternal Snrpn LOM in 2/10 embryos, and maternal H19 gain of methylation (GOM) in 1/10 embryos. This frequency of imprinting errors was not statistically different from controls (El Hajj et al. 2011). However, as 12–24% of DNA strands per gene per embryo were recovered, additional perturbations may have been missed. Alternatively, methylation perturbations may be initiated at or after the 16-cell stage, as imprinted methylation errors were present in blastocysts after superovulation. Following low (6.25 IU eCG/hCG) and high (10 IU eCG/hCG) hormone regimes, we reported imprinted DNA methylation perturbations in individual mouse blastocysts at maternal alleles of Snrpn (LOM, 4/10 low, 9/10 high blastocysts), Peg3 (LOM, 4/9 low, 5/9 high blastocysts), Kcnq1ot1 (LOM, 2/6 low, 5/9 high blastocysts), and H19 (GOM, 1/10 low, 4/10 high blastocysts) (Market-Velker et al. 2010b). As paternal H19 LOM was also seen (3/10 low, 7/10 high blastocysts), we concluded that superovulation impaired both imprint acquisition in oocytes and imprint maintenance in early embryos in a dose-dependent manner. Moreover, multilocus imprinted methylation perturbations were greater in the high hormone group (10/10 blastocysts) compared with the low hormone (4/10 blastocysts) and control groups (1/10 blastocysts). At midgestation, mouse conceptuses produced via low hormone (5 IU eCG/hCG) treatment showed altered allelic expression of Snrpn, H19, and Igf2, but not Kcnq1ot1 in placentas but not in embryos (Fortier et al. 2008). Additionally, 3/8 superovulation-derived mice (5 IU eCG/hCG) showed LOM at H19 and Peg3, but not Snrpn, in brain and liver tissues (de Waal et al. 2012). These studies indicate that superovulation can lead to imprinting maintenance errors.

Table 1

Studies on ovarian stimulation and genomic imprinting.

ReferenceSpeciesTreatmentMethodSamplesGeneObservations
Chang et al. (2005)HumanOvarian stimulation; IVF/ICSI, embryo culture; Infertility/subfertilityCase series19/341 BWS ART children2/12 stim only
Ludwig et al. (2005)HumanOvarian stimulation; Infertility/subfertilitySurvey; COBRA16/79 subfertile +/− ART childrenSNRPNLOM 4/16 AS children; 1/4 stim only
Blood sample or buccal smear
Sutcliffe et al. (2006)HumanOvarian stimulation; IVF/ICSI; Infertility/subfertilitySurvey11/79 BWS ART children; KCNQ1OT1LOM 8/11 children; 5 stim only
El Hajj et al. (2011)Mouse7.5 IU eCG/hCGLD-BMS10 individual 16-cell embryosH19 patLOM 2/10 embryos
H19 matGOM 1/10 embryos
SnrpnLOM 2/10 embryos
Market-Velker et al. (2010b)Mouse6.25 IU (low) or 10 IU (high) eCG/hCGBMS10 individual blastocystsSnrpnLOM 4/10 low, 9/10 high blastocysts
Peg3LOM 4/9 low, 5/9 high blastocysts
Kcnq1ot1LOM 2/6 low, 5/9 high blastocysts
H19 matLOM 3/10 low, 7/10 high blastocysts
H19 patGOM 1/10 low, 4/10 high blastocysts
Fortier et al. (2008)Mouse5 IU eCG/hCG; Blastocysts transferBMS, RT-PCR36 individual E9.5 embryos and placentasSnrpnLOIE stim 3/36 emb, 10/36 plac
LOIE stim+transfer 5/32 emb, 5/32 plac
LOM 0/4 emb, 0/4 plac
H19LOIE stim 5/36 emb, 22/36 plac
LOIE stim+transfer 13/33 emb, 18/33 plac
LOM 0/4 emb, 0/4 plac
Kcnq1ot1LOIE stim 0/36 emb, 3/36 plac
Igf2LOIE stim 2/36 plac
de Waal et al. (2012)Mouse5 IU eCG/hCG; Culture two-cell to blastocyst; Embryo transferBMS8 individual juvenile liver and brain tissueH19SnrpnPeg3LOM 1/8 livers LOM 0/8 brain LOM 2/8 brain
Anckaert et al. (2009a)Mouse5 IU eCG/hCGBMS3 pools, 100–150 MII oocytesSnrpnLOM 0/14 strands
Peg3LOM 0/13 strands
Igf2rLOM 0/17 strands
H19GOM 0/18 strands
Denomme et al. (2011)Mouse6.25 IU (low) or 10 IU (high) eCG/hCGBMS125 individual MII oocytesSnrpnLOM 0/15 low, 0/15 high oocytes
Peg3LOM 0/15 low, 0/17 high oocytes
Kcnq1ot1LOM 0/15 low, 1/16 high oocytes
H19GOM 0/15 low, 0/17 high oocytes
Sato et al. (2007)Mouse7.5 IU eCG, 3×5 IU hCGCOBRA2 pools, 30–50 MII oocytesPeg1No LOM (94.3 and 94.0%)
2 different strains of miceKcnq1ot1No LOM (96.2 and 93.4%)
Plagl1No LOM (96.7 and 93.4%)
H19GOM (37 and 26%)
Sato et al. (2007)HumanOvarian stimulation; Infertility/subfertilityBMS10 individual MI oocytesPEG1H19LOM 2/7 MI oocytes GOM 2/3 MI oocytes
Khoueiry et al. (2008)HumanOvarian stimulation; Infertility/subfertilityBMS11 pools, 1–3 MII oocytesKCNQ1OT1LOM 2/19 strands

BMS, bisulfite mutagenesis and sequencing; LD-BMS, limited dilution BMS; COBRA, combined bisulfite-PCR restriction analysis; stim, stimulation; LOM, loss of methylation; GOM, gain of methylation; LOIE, loss of imprinted expression; emb, embryo; plac, placenta.

Box 3 LOM and GOM. In our literature evaluation, we defined loss and gain of methylation as having a 50% or greater change in DNA methylation; sporadic methylation loss or gain was not included.

As ovarian stimulation is administered during oogenesis, it may also disrupt imprint acquisition. Five studies have examined the effects of ovarian stimulation on imprint acquisition in oocytes. Following low hormone treatment (5 IU eCG/hCG), methylation of Snrpn, Peg3, Igf2r, and H19 was unaffected in mouse MII oocyte pools (14–18 DNA strands analyzed per gene) (Anckaert et al. 2009a). Likewise, we found no effect of superovulation on imprinted DNA methylation acquisition in 15–17 individual mouse MII oocytes using low (6.25 IU eCG/hCG) and high (10 IU eCG/hCG) hormone dosages at Snrpn, Kcnq1ot1 (LOM 1/16 high oocytes), Peg3, and H19 (Denomme et al. 2011). These results contrast with mouse oocytes collected after sequential hormone treatment (3 days 7.5 IU eCG/1 day 5 IU hCG) where H19 exhibited GOM (26–37%) in pooled MII oocytes, although normal methylation acquisition was present at Peg1 (Mest), Kcnq1ot1, and Plagl1 (Zac1) (Sato et al. 2007). As zona pellucidae were not removed from pooled oocytes, H19 GOM may be the result of cumulus cell contamination. Following ovarian stimulation in humans, individual MI oocytes showed PEG1 LOM (2/7 oocytes) and H19 GOM (2/3 oocytes) (Sato et al. 2007), and in the last study, pooled MII oocytes exhibited maternal KCNQ1OT1 LOM (2/19 strands) (Khoueiry et al. 2008). With respect to the human MI oocyte study, PEG1 may still be in its acquisition phase. Alternatively, human oocytes may be more prone to epigenetic errors and/or encounter more stressors, such as multiple hormone administration, advanced maternal age, and inherent infertility. Considering the frequency of epigenetic perturbations in blastocysts compared with oocytes, ovarian stimulation may have greater adverse impact on maternal factors required for imprint maintenance than on imprint acquisition. Future studies should be directed toward larger numbers of human oocytes as well as identification of maternal effect genes.

In vitro oocyte maturation

Follicle culture and IVM is an alternative, experimental procedure to obtain large oocyte numbers. Long-term IVM is initiated with preantral follicles while short-term IVM begins with GV/MI oocytes, which are then cultured to MII oocytes. Disparate results have been reported for the effects of IVM on genomic imprinting in the mouse. Using long-term IVM and supraphysiological recombinant FSH (rFSH), imprinted methylation perturbations were observed in pools of GV oocytes with H19 GOM (1/7 pools), Peg1 LOM (1/7 pools), and Igf2r LOM (6/7 pools) (Kerjean et al. 2003; Table 2). By comparison, normal H19 (0/39 strands) and Igf2r methylation (0/15 strands) was found in pooled GV oocytes following long-term IVM with physiological rFSH, although LOM was observed at Snrpn (2/47 DNA strands) (Trapphoff et al. 2010). Additionally, independent studies with long-term IVM using physiological or supraphysiological rFSH reported normal Snrpn, Igf2r, Peg3, and H19 methylation in MII oocyte pools (Anckaert et al. 2009a, 2009b, 2010). Furthermore, toxic ammonium levels (through addition of ammonium acetate or mineral oil overlay) and low methyl donor levels in IVM medium had no effect on Snrpn, Igf2r, and H19 methylation (Anckaert et al. 2009b, 2010). In humans, using the short-term protocol on oocytes from stimulated women, H19 GOM was found in 2/10 MII oocyte pools (Borghol et al. 2006) and at 3/34 DNA strands in MII oocyte pools (Al-Khtib et al. 2011). KCNQ1OT1 LOM was also observed in 5/23 and 2/37 DNA strands from IVM MII oocyte pools (Khoueiry et al. 2008, Al-Khtib et al. 2011). This contrasts with short-term IVM MII oocytes that had normal SNRPN (three single oocytes), KCNQ1OT1 (four single oocytes), and MEG3 (GTL2) (three pools of two oocytes) methylation (Geuns et al. 2003, 2007a, 2007b). In the above studies, where zona pellucidae were not removed, there is the possibility of cumulus cell contamination. Overall, the long-term IVM does not appear to pose high risk for imprinted acquisition errors. Further investigations will be required to delineate the incidence and consequent effects of both long-term and short-term IVM.

Table 2

Studies on in vitro oocyte maturation and genomic imprinting.

ReferenceSpeciesTreatmentMethodSamplesGeneObservations
Kerjean et al. (2003)MouseLong protocol; 100 IU/l rFSHBMS7 pools, 5–8 GV oocytesH19GOM 1/7 pools
Peg1LOM 1/7 pools
Igf2rLOM 6/7 pools
Trapphoff et al. (2010)MouseLong protocol; 10 IU/l rFSHBMS7–11 pools, 10 GV oocytesSnrpnLOM 2/47 strands
H19GOM 0/39 strands
Igf2rLOM 0/15 strands
Anckaert et al. (2009a)MouseLong protocol; 10 IU/l rFSH or 100 IU/l rFSHBMS4–8 pools, 100–150 MII oocytesSnrpnLOM 0/22 strands 10 IU/l, 0/20 strands 100 IU/l
Igf2rLOM 0/10 strands 10 IU/l
Peg3LOM 0/15 strands 10 IU/l, 0/21 strands 100 IU/l
H19GOM 0/28 strands 10 IU/l, 0/25 strands 100 IU/l
Anckaert et al. (2009b)MouseLong protocol; 10 IU/l rFSH; 500 μM ammonium acetate and mineral oil overlayBMS2–4 pools, 100 MII oocytesSnrpnLOM 0/26 strands
Igf2rLOM 0/50 strands
H19GOM 0/45 strands
Anckaert et al. (2010)MouseLong protocol; 10 IU/l rFSH; Low methyl donor levelsBMS3 pools, 100 MII oocytesSnrpnLOM 0/36 strands
Igf2rLOM 0/29 strands
H19GOM 0/36 strands
Borghol et al. (2006)HumanShort protocol; Ovarian stimulation; Infertility/subfertilityBMS10 pools, 1–8 MII oocytesH19GOM 2/10 pools
Al-Khtib et al. (2011)HumanShort protocol; Ovarian stimulation; Infertility/subfertilityBMS16 pools, 1–3 MII oocytesH19GOM 3/34 strands
20 individual MII oocytesKCNQ1OT1LOM 2/37 strands
Khoueiry et al. (2008)HumanShort protocol; Ovarian stimulation; Infertility/subfertilityBMS12 pools, 1–6 MII oocytesKCNQ1OT1LOM 5/23 strands
Geuns et al. (2003)HumanShort protocol; Ovarian stimulation; Infertility/subfertilityBMS3 individual MII oocytesSNRPNLOM 0/3 oocytes
Geuns et al. (2007a)HumanShort protocol; Ovarian stimulation; Infertility/subfertilityBMS3 pools, 2 MII oocytesMEG3GOM 0/3 pools
Geuns et al. (2007b)HumanShort protocol; Ovarian stimulation; Infertility/subfertilityBMS4 individual MII oocytesKCNQ1OT1LOM 1/4 oocytes

BMS, bisulfite mutagenesis and sequencing; LOM, loss of methylation; GOM, gain of methylation.

Cryopreservation and vitrification

Given the complications of multi-fetal pregnancies, current clinical practice is to transfer fewer embryos and cryopreserve remaining embryos. Technologies are also advancing for oocyte and ovarian tissue cryogenics. Four studies have investigated cryogenic effects on imprinted methylation (Table 3). Ultra-rapid vitrification of mouse preantral follicles followed by long-term IVM to GV oocytes led to Snrpn LOM (1/50 strands) but not Igf2r LOM (0/15 strands) or H19 GOM (0/58 strands) in pooled vitrified oocytes (Trapphoff et al. 2010). In the second study, mice produced following whole ovary cryopreservation maintained normal H19 and Kcnq1ot1 methylation ratios (Sauvat et al. 2008). Unfortunately, averaging methylation levels from three tissues from 5 to 36 mice may have obscured imprinting defects in individual mice. In humans, vitrification of GV oocytes followed by short-term IVM resulted in H19 GOM in MII oocyte pools (5/29 strands), although this was not significantly different from IVM-only MII oocytes (3/34 strands). Finally, analysis of 17 human vitrified-IVM MII oocytes showed KCNQ1OT1 LOM (1/28 strands), which again was not significantly different from 20 IVM-only MII oocytes (2/37 strands) (Al-Khtib et al. 2011). Overall, these studies suggest that freezing does not impart greater risk than IVM alone. However, more studies are required to understand the effects of cryogenic technologies on genomic imprinting.

Table 3

Studies on cryopreservation and vitrification and genomic imprinting.

ReferenceSpeciesTreatmentMethodSamplesGeneObservations
Trapphoff et al. (2010)MouseVitrification of pre-antral follicles; Long protocol IVM; 10 IU/l rFSHBMS6–12 pools, 10 GV oocytesSnrpnLOM 1/50 strands
H19GOM 0/58 strands
Igf2rLOM 0/15 strands
Sauvat et al. (2008)MouseWhole ovary; cryopreservationSouthernKidney, muscle, and tongue tissues from 5 to 36 miceH19No GOM in mean methylation levels
Kcnq1ot1No LOM in mean methylation levels
Al-Khtib et al. (2011)HumanVitrification of GV oocytes; Short protocol IVM; Infertility/subfertilityBMS12 pools, 1–3 MII oocytesH19GOM 5/29 strands
17 individual MII oocytesKCNQ1OT1LOM 1/28 strands

BMS, bisulfite mutagenesis and sequencing; LOM, loss of methylation; GOM, gain of methylation.

Manipulations to mature oocytes and sperm

IVF and ICSI

Once retrieved, oocytes are fertilized via IVF (oocyte insemination in culture) or ICSI (sperm injection into oocyte). Many studies have reported an increased prevalence of IVF and ICSI (3- to 14-fold) among children with BWS compared with those in the general population (DeBaun et al. 2003, Gicquel et al. 2003, Maher et al. 2003, Halliday et al. 2004, Chang et al. 2005, Rossignol et al. 2006, Sutcliffe et al. 2006, Lim et al. 2009; Table 4). Many of these BWS children display maternal KCNQ1OT1 LOM. Similarly, AS children born after both IVF and ICSI have a greater prevalence of SNRPN imprinting defects than AS children in the general population (Cox et al. 2002, Orstavik et al. 2003, Ludwig et al. 2005). Moreover, in IVF and ICSI BWS children, KCNQ1OT1 LOM is coincident with SNRPN, PEG1, PLAGL1, and IGF2R LOM, indicating that multiple imprinting defects may be present in these children (Rossignol et al. 2006, Lim et al. 2009).

Table 4

Studies on IVF and ICSI and genomic imprinting.

ReferenceSpeciesTreatmentMethodSamplesGeneObservations
DeBaun et al. (2003)HumanIVF/ICSI; Ovarian stimulation; Infertility/subfertilityCase series; Southern7 BWS ART children; 2 IVF, 5 ICSI KCNQ1OT1LOM 5/6 children
Peripheral blood lymphocytes/biopsyH19GOM 1/6 children
Gicquel et al. (2003)HumanIVF/ICSI; Ovarian stimulation; Infertility/subfertilityCase series; Southern6/149 BWS ART children; 4 IVF, 2 ICSIKCNQ1OT1LOM 6/6 children
Blood
Maher et al. (2003)HumanIVF/ICSI; Ovarian stimulation; Infertility/subfertilityCase series6/149 BWS ART children; 3 IVF, 3 ICSIKCNQ1OT1LOM 2/2 children
Halliday et al. (2004)HumanIVF/ICSI; Ovarian stimulation; Infertility/subfertilityCase control4/37 BWS ART children; 3 IVF, 1 ICSI KCNQ1OT1LOM 3/3 children
Chang et al. (2005)HumanIVF/ICSI; Ovarian stimulation; Infertility/subfertilityCase series19/341 BWS ART children; 5/12 IVF, 5/12 ICSI
Rossignol et al. (2006)HumanIVF/ICSI; Ovarian stimulation; Infertility/subfertilitySouthern11/40 BWS ART children; 8 IVF, 3 ICSI KCNQ1OT1LOM 11/11 children
IGF2RLOM 2/11 children
PEG1LOM 0/11 children
SNRPNLOM 1/11 children
Sutcliffe et al. (2006)HumanIVF/ICSI; Ovarian stimulation; Infertility/subfertilitySurvey11/79 BWS ART children; 1 IVF, 5 ICSIKCNQ1OT1LOM 8/11 children
Lim et al. (2009)HumanIVF/ICSI; Ovarian stimulation; Infertility/subfertilityCOBRA, BMS,25 BWS ART children; 12 IVF, 13 ICSI KCNQ1OT1LOM 24/25 children
MS-PCRPeripheral blood lymphocytesPEG1LOM 2/25 children
SNRPNLOM 1/25 children
PLAGL1LOM 1/25 children
Cox et al. (2002)HumanICSI; Ovarian stimulation; Infertility/subfertilityCase series; Southern, MS-PCR2 ICSI AS childrenSNRPNLOM 2/2 children
Blood lymphocytes and cultured skin fibroblasts
Orstavik et al. (2003)HumanICSI; Ovarian stimulation; Infertility/subfertilityCase series; Southern, MS-PCR1 ICSI AS childSNRPNLOM 1/1 child
Ludwig et al. (2005)HumanICSI; Ovarian stimulation; Infertility/subfertilitySurvey; COBRA16/79 subfertile +/− ART AS childrenSNRPNLOM 4/16 children; 1/4 ICSI
Blood sample or buccal smear
Lidegaard et al. (2005)HumanIVF/ICSI; Ovarian stimulation; Infertility/subfertilityCohort study1680 IVF, 4372 ICSI childrenNo imprinting disorders
Bowdin et al. (2007)HumanIVF/ICSI; Ovarian stimulation; Infertility/subfertilitySurvey1524 IVF and ICSI BWS and AS children1 BWS child
174 imprinting disorder phenotype3 BWS-like children (no KCNQ1OT1 LOM)
47 children clinically assessed1 AS-like child (no SNRPN LOM)
Gomes et al. (2009)HumanIVF/ICSI; Ovarian stimulation; Infertility/subfertilityMS-PCR, 18 ART childrenKCNQ1OT1LOM 3/18 children; 2 IVF, 1 ICSI
qMSRD-PCRPeripheral blood, UCB, placenta
Feng et al. (2011)HumanICSI; Ovarian stimulation; Infertility/subfertilityBMS18 IVF/ICSI childrenL3MBTLLOM 1/18 children
RT-PCRUmbilical cord bloodLOIE 1/18 children
Shi et al. (2011)HumanICSI; Ovarian stimulation; Infertility/subfertilityCOBRA, BMS61 ICSI childrenH19LOM 3/61 children
Umbilical cord blood
Turan et al. (2010)HumanIVF; Ovarian stimulation; Infertility/subfertilityqMSRD-PCR, BMS, RT-PCR45 ART, 56 non-ART childrenH19GOM
UCB, cord, placentaGreater intra- and interindividual IVF
Tierling et al. (2010)HumanIVF/ICSI; Ovarian stimulation; Infertility/subfertilityMS-SNUPE35 IVF, 77 ICSI normal childrenPEG1GOM in IVF compared to ICSI
UCB, amnion/chorion tissue
Oliver et al. (2012)HumanIVF/ICSI; Ovarian stimulation; Infertility/subfertilityqMSRD-PCR, BMS34 IVF, 32 ICSI childrenH19No LOM
Peripheral bloodKCNQ1OT1No LOM
SNRPNNo LOM
Manning et al. (2000)HumanICSI; Ovarian stimulation; Infertility/subfertilityMS-PCR92 ICSI childrenSNRPNNo LOM
Blood
Wong et al. (2011)HumanIVF/ICSI; Ovarian stimulation; Infertility/subfertilityMS-SNUPE32 IVF, 45 ICSI childrenSNRPNNo LOM
Placentas
de Waal et al. (2012)MouseICSI; 5 IU eCG/hCG; Culture two-cell to blastocyst; Embryo transferBMS6 individual juvenile H19LOM 1/6 brains, muscles, livers
Brain, muscle and liver tissuePeg3LOM 1/6 brains
SnrpnLOM 1/6 brains
Fauque et al. (2007)MouseIVF; 8 IU eCG, 5 IU hCG; Culture in M16 or G1/G2BMS36 individual blastocystsH19LOM 16/36 blastocysts
Chen et al. (2010)HumanIVF/ICSI; Ovarian stimulation; Culture; Infertility/subfertilityCOBRA26 IVF, 6 ICSI individual preimplantation embryosH19LOM 5/26 IVF, 1/6 ICSI embryos
Ibala-Romdhane et al. (2011)HumanICSI; Ovarian stimulation; Infertility/subfertilityBMS21 ICSI delayed/compromised morulae or blastocystsH19LOM 8/21 embryos
GOM 1/21 embryos

BMS, bisulfite mutagenesis and sequencing; COBRA, combined bisulfite-PCR restriction analysis; qMSRD-PCR, quantitative methylation sensitive restriction digestion and PCR; MS-SNuPe, methylation-sensitive single nucleotide primer extension; LOM, loss of methylation; GOM, gain of methylation; LOIE, loss of imprinted expression; UCB, umbilical cord blood.

Conversely, examination of ART populations for the prevalence of imprinting syndromes or imprinting defects has produced conflicting results. Of 1680 IVF and 4372 ICSI children, none had an imprinting disorder (Lidegaard et al. 2005). In a second study of 1524 IVF and ICSI children, 174 had phenotypic features of an imprinting disorder and of the 47 children clinically assessed, one had BWS, three had BWS-like symptoms (no KCNQ1OT1 LOM), and one had AS-like symptoms (no SNRPN LOM) (Bowdin et al. 2007). With respect to imprinting defects, one ICSI- and two IVF-conceived children out of 18 possessed KCNQ1OT1 LOM (Gomes et al. 2009); one ICSI child out of 18 had L3MBTL LOM (Feng et al. 2011); and three ICSI children out of 61 had H19 LOM (Shi et al. 2011). In addition, greater intra- and interindividual H19 GOM was present in 45 in vitro-conceived compared to 56 in vivo-conceived children (Turan et al. 2010). In another study, higher PEG1 methylation levels were found in 35 IVF-conceived compared to 77 ICSI-conceived and 73 spontaneously conceived children, although no differences in mean methylation levels were identified at nine other imprinted genes (Tierling et al. 2010). By comparison, normal imprinted methylation was detected at H19, KCNQ1OT1, and SNRPN in 34 IVF- and 32 ICSI-conceived children (Oliver et al. 2012); at SNRPN in 92 ICSI children (Manning et al. 2000); and at SNRPN in 32 IVF and 45 ICSI placentas from newborns (Wong et al. 2011). While these later studies argue that the absolute risk of imprinting disorders in ARTs-conceived children is small, it should be noted that many of these studies were performed on buccal and peripheral blood samples by methods that detect 1 or 2 CpGs per gene. In fact, tissue-specific effects of ICSI have been reported with 1/6 ICSI-conceived mice displaying H19 LOM in brain, muscle, and liver compared with 1/6 mice each having Peg3 LOM and Snrpn LOM in brain only (de Waal et al. 2012).

At earlier developmental stages, IVF and ICSI have been implicated in ART-induced epigenetic instability. IVF resulted in significantly more individual mouse blastocysts with aberrant H19 methylation (16/36) compared with embryo production without IVF (0/15) or superovulation alone (0/26) (Fauque et al. 2007). In humans, H19 LOM was identified in five IVF and one ICSI blastocysts out of 32 (Chen et al. 2010). Finally, compared to high-grade control blastocysts, 8/21 ICSI developmentally delayed/morphologically compromised embryos harbored paternal H19 LOM and 1/21 ICSI delayed/compromised embryos possessed maternal H19 GOM (Ibala-Romdhane et al. 2011). Further studies are required to determine the incidence of early developmental and tissue-specific imprinting perturbations induced by IVF and ICSI as well as how these technologies lead to imprinting defects in resulting embryos and offspring.

Manipulations during preimplantation development

In vitro embryo culture

Following IVF or ICSI, embryos are cultured to the eight-cell or blastocyst stage before transfer into the mother. A query of in vitro culture media used in ART-conceived BWS children revealed that multiple media systems were employed, including human tubal fluid (HTF) medium, Cook's sequential medium, preimplantation 1 (P1) medium, and growth 1 and 2 (G1/G2) medium, suggesting that media type per se is not the root factor in generating ART-induced perturbations (Chang et al. 2005; Table 5). Instead, culture environment may be suboptimal, compromising imprint maintenance. The first evidence of this came in 1995 when cultured mouse embryos displayed biallelic H19 expression in postimplantation extraembryonic tissues (Sasaki et al. 1995). Other studies have since shown aberrant imprinting in cultured embryos. In pooled blastocysts, Whitten's but not potassium simplex optimized medium plus amino acids (KSOMaa) cultured embryos exhibited biallelic H19 expression (Doherty et al. 2000). At the individual blastocyst level, 63% (24/38) of Whitten's and 14% (3/21) of KSOMaa-cultured embryos displayed biallelic H19 expression, which correlated with H19 LOM (Whitten's 59% and KSOMaa 77% paternal methylation) (Mann et al. 2004). Snrpn LOM was also greater in embryos cultured in Whitten's (40% maternal methylation) than in KSOMaa (82% maternal methylation) medium. We further compared five commercial media (KSOMaa, Global, HTF, P1/Multiblast, and G1.5/G2.5) with Whitten's medium. While all culture media were suboptimal at maintaining imprinted methylation, some media better maintained H19 (KSOMaa 75%, Global 75%, and other media 52–65% mean paternal methylation), Snrpn (KSOMaa 73%, Global 72%, and other media 54–65% mean maternal methylation), and Peg3 methylation (KSOMaa 93%, HTF 85%, Global 77%, P1/Multiblast 75%, and other media 50–54% mean maternal methylation) (Market-Velker et al. 2010a). Similarly, culture in M16 medium (8/19 blastocysts) caused greater H19 LOM than G1.2/G2.2 (6/17 blastocysts) (Fauque et al. 2007). HTF culture produced 8/23 mouse blastocysts with aberrant H19 expression (Li et al. 2005). In humans, H19 LOM occurred in ∼19% of human embryos cultured in cleavage medium (Chen et al. 2010). Postimplantation, aberrant H19, Ascl2, Snrpn, and Peg3 expression and H19 and Snrpn LOM occurred in placentas but rarely in fetuses subjected to Whitten's or KSOMaa preimplantation culture (Mann et al. 2004).

Table 5

Studies on in vitro embryo culture and genomic imprinting.

ReferenceSpeciesTreatmentMethodSamplesGeneObservations
Chang et al. (2005)HumanOvarian stimulation; IVF/ICSI; Embryo culture; Infertility/subfertilityCase series19/341 BWS ART children10/12 culture; 1 P1, 1 P1+SSS, 1 P1/HTS+SSS,
12 with medical records1 HTF/P1+SSS/BM, 2 HTF+SSS, 2 G1/G2, 2 Cook's
Sasaki et al. (1995)MouseIVF; Culture two-cell to blastocystRT-PCR5 pools, E6.5–E8.5 embryonic and extraembryonic tissueH19LOIE extraembryonic and trophoblast tissues no LOIE in emb
Doherty et al. (2000)Mouse5 IU eCG/hCG; Culture two-cell to blastocyst Whitten's or KSOMaaRT-PCR SSCP3–7 pools, 28 blastocystsH19LOIE Whitten's, no LOIE KSOMaa LOM Whitten's, no LOM KSOMaa
Mann et al. (2004)MouseCulture two-cell to blastocystBMSPool of 25–30 blastocystsH19LOM 11/22 Whitten's, 3/13 strands KSOMaa
Whitten's or KSOMaaRT-PCRSnrpnLOM 11/16 Whitten's, 2/11 strands KSOMaa
17–38 individual blastocystsH19LOIE 24/38 Whitten's, 4/21 KSOMaa
8–15 E9.5 emb and placH19LOM 0/3 emb, 3/3 plac Whitten's,
0/1 emb, 0/1 plac KSOMaa
LOIE 2/15 emb, 11/12 plac Whitten's,
0/8 emb, 6/8 plac KSOMaa
SnrpnLOM 0/3 emb, 1/3 plac Whitten's,
0/1 emb, 1/1 plac KSOMaa
LOIE 1/11 emb, 5/11 plac Whitten's,
0/8 emb, 1/8 plac KSOMaa
Peg3LOIE 2/12 emb, 8/11 plac Whitten's,
0/8 emb, 5/8 plac KSOMaa
Ascl2LOIE 10/11 plac Whitten's, 7/8 plac KSOMaa
Market-Velker et al. (2010a)MouseCulture two-cell to blastocystBMS3 pools, 5 blastocystsH19LOM 61% (mean) Whitten's, 75% KSOMaa, 75%
Whitten's, KSOMaa, Global, HTF,Global, 52% HTF, 65% P1/MB, 55% G1.5/G2.5
P1/MB, G1.5/G2.5SnrpnLOM 58% (mean) Whitten's, 73% KSOMaa, 72%
RT-PCR19–29 individual blastocystsGlobal, 54% HTF, 65% P1/MB, 64% G1.5/G2.5
Peg3LOM 54% (mean) Whitten's, 93% KSOMaa, 77%
Global, 85% HTF, 75% P1/MB, 50% G1.5/G2.5
Culture or stim (6.25 IU)+cultureH19LOIE 40% Whitten's, 81% 6.25 IU+Whitten's
LOIE 60% KSOMaa, 81% 6.25 IU+KSOMaa
LOIE 50% Global, 71% 6.25 IU+Global
LOIE 47% HTF, 76% 6.25 IU+HTF
LOIE 53% P1/MB, 70% 6.25 IU +P1/MB
LOIE 41% G1.5/G2.5, 67% 6.25 IU +G1.5/G2.5
Fauque et al. (2007)Mouse8 IU eCG/5 IU hCG; Culture one-cell to blastocysts M16 or G1/G2BMS15 individual blastocystsH19LOM 0/8 M16 blastocysts LOM 0/7 G1/G2 blastocysts
Li et al. (2005)Mouse10 IU eCG/hCG; IVF; Culture two-cell to morula/blastocyst HTFRT-PCR23 individual IVF embryosH19LOIE 8/23 embryos
Igf2LOIE 2/23 embryos
Cdkn1cLOIE 2/23 embryos
Slc221lLOIE 0/23 embryos
Chen et al. (2010)HumanOvarian stimulation; IVF/ICSI; Culture to day 3 embryos Quinn's, cleavage or HTF; Infertility/subfertilityCOBRA32 individual preimplantation embryosH19LOM 6/32 embryos
Market Velker et al. (2012)MouseCulture two-cell to blastocystBMS, RT-PCR21 individual blastocystsSnrpnLOM 8/11 fast, 4/10 slow blastocysts
Whitten'sH19LOM 5/10 fast, 3/11 slow blastocysts
LOIE 10/19 fast, 0/7 slow blastocysts
Rivera et al. (2008)Mouse0.5 IU eCG/hCG; Blastocyst transfer or culture two-cell blastocyst+transfer KSOMaaBMS, RT-PCRE9.5 emb, ys and placKcnq1ot1LOM 0/4 transfer plac, 3/4 culture+transfer plac
H19LOM 0/4 transfer plac, 2/4 culture+transfer plac
Kcnq1ot1LOIE transfer 0/33 emb, 1/33 ys, 3/30 plac,
LOIE culture+transfer 4/28 emb, 8/28 ys, 9/27 plac
H19LOIE transfer 1/33 emb, 7/33 ys, 14/31 plac,
LOIE culture+transfer 4/29 emb, 20/29 ys, 19/28 plac
Cdkn1cLOIE transfer 0/33 emb, 1/33 ys, 1/31 plac,
LOIE culture+transfer 0/29 emb, 1/29 ys, 1/28 plac
Kcnq1LOIE transfer 0/33 emb, 2/33 ys, 4/31 plac,
LOIE culture+transfer 0/29 emb, 3/29 ys, 6/28 plac
SnrpnLOIE transfer 0/33 emb, 1/33 ys, 4/30 plac,
LOIE culture+transfer 3/29 emb, 5/29 ys, 8/27 plac
Peg3LOIE transfer 0/33 emb, 2/33 ys, 3/31 plac,
LOIE culture+transfer 1/29 emb, 7/29 ys, 8/28 plac

BMS, bisulfite mutagenesis and sequencing; SSCP, single-stranded conformation polymorphism gel electrophoresis; COBRA, combined bisulfite-PCR restriction analysis; stim, stimulation; LOM, loss of methylation; GOM, gain of methylation; LOIE, loss of imprinted expression; emb, embryo; plac, placenta; ys, yolk sac; P1, preimplantation medium; HTF, human tubal fluid medium; SSS, serum substitute supplement; BM, blastocyst medium.

Like other ARTs, imprinting errors occur stochastically in response to suboptimal culture in that not every embryo and not every imprinted locus possesses imprinting defects. To address a possible origin for these stochastic events, we examined imprinted methylation in blastocysts with different developmental rates in culture. We found that a greater number of embryos with fast rates of development had Snrpn and H19 LOM (8/11 and 5/10 respectively) compared with those that developed slower in culture (4/10 and 3/11 respectively) (Market Velker et al. 2012). Future studies are required to determine when epigenetic instability is arising during in vitro development and through which mechanism(s), as well as why some embryos are more sensitive to the adverse effects of culture.

Inherent infertility

Impaired fertility and imprinting defects

The increased incidence of imprinting disorders in the ART population has led to the question of whether infertility predisposes embryos to imprinting errors. In an examination of 16 AS children born to subfertile couples, four were caused by sporadic imprinting defects, including two from parents who conceived without assistance (Ludwig et al. 2005). In addition, a case of two BWS children in the same family, one born via ARTs and the other born naturally, suggests that impaired fertility may be associated with mechanisms leading to imprinting disorders (Strawn et al. 2010).

Female infertility/subfertility

The most common variable influencing natural conception in modern society is reproductive age. Advanced maternal age (>35 years) is directly related to a decline in fertility with a reduced oocyte reserve and poor oocyte quality (Liu & Case 2011). In a Dutch study evaluating ARTs and parental infertility, 6.3% of AS, 8.5% of BWS, and 2.1% of total children were conceived via ARTs, while 6.3% of AS, 8.5% of BWS, and 3.5% of total children were born to subfertile families without ARTs (Doornbos et al. 2007; Table 6). The same relative risk of AS and BWS in subfertile couples with and without ARTs indicates that the increased prevalence of imprinting disorders can be explained by compromised fertility. Significantly, advanced maternal age was increased in AS and BWS mothers compared with the general population, suggesting that advanced maternal age may decrease fertility and increase the risk of imprinting disorders. In mouse, advanced maternal age compromised postimplantation development, although no age-related change in imprinted DNA methylation was detected at Snrpn, U2af1rs1, Kcnq1ot1, Igf2r, Peg1, and H19 in blastocysts and midgestation conceptuses (Lopes et al. 2009). As a means of simulating infertility, our group used connexin37 deletion mice to determine whether compromised gap junctional communication between the oocyte and the cumulus cells would disrupt de novo methylation acquisition in growing oocytes (Denomme et al. 2012). Connexin37 deficiency resulted in loss or delayed methylation acquisition at the late-acquiring gene Peg1, but not at Snrpn and Peg3, suggesting that stored methyl donors or other metabolites normally transported from granulosa cells to the oocyte may have been exhausted during oocyte growth. To understand the etiology of epigenetic instability in infertility, further studies are required to determine when imprinting errors occur (oocyte or embryo development; young or aged oocytes), as well as the type of female factor infertility and the molecular mechanisms leading to imprinting perturbations. Furthermore, to delineate the risk of epigenetic errors resulting from ARTs vs infertility, investigations are required on embryos and children from couples seeking ARTs in the absence of infertility such as for preimplantation genetic diagnosis and same-sex couples.

Table 6

Studies on female infertility/subfertility and genomic imprinting.

ReferenceSpeciesConditionMethodSamplesGeneObservations
Doornbos et al. (2007)HumanImprinting disordersSurvey220 children with imprinting disordersSubfertile+ARTs6.3% AS, 8.5% BWS, 2.1% total population
Subfertile6.3% AS, 8.5% BWS, 3.5% total population
Maternal age30.64 AS, 31.17 BWS, 29.68 total population
Lopes et al. (2009)MouseAged femalesqMSRD-PCR, RT-PCR3 young, 10 aged E10.5 embryosSnrpnNo LOM in mean methylation levels of aged emb or plac
6 young, 12 aged E10.5 placentas No LOIE in blastocyst or emb or plac
U2af1-rs1No LOM in mean methylation levels of aged emb or plac
Kcnq1ot1No LOM in mean methylation levels of aged emb or plac
Igf2rNo LOM in mean methylation levels of aged emb or plac
Peg1No LOM in mean methylation levels of aged emb or plac
H19No LOM in mean methylation levels of aged emb or plac
No LOIE in blastocyst or emb, LOIE 2/12 plac
Denomme et al. (2012)MouseFemale infertilityBMS81 individual connexin37-null GV oocytesSnrpnLOM 0/30 oocytes
Peg3LOM 0/20 oocytes
Peg1Delayed/LOM 3–7/31 oocytes

qMSRD-PCR, quantitative methylation sensitive restriction digestion and PCR; BMS, bisulfite mutagenesis and sequencing; LOIE, loss of imprinted expression; LOM, loss of methylation; emb, embryo; plac, placenta.

Male infertility/subfertility

Male infertility can result from irregular sperm morphology (teratozoospermia), poor motility (asthenozoospermia), low sperm count (oligozoospermia), or absence of sperm (azoospermia) (Krausz 2011). Numerous studies have examined imprinted methylation in sperm from infertile men, with most focusing on oligozoospermia. Analysis of sperm from 79 men with normozoospermia, eight men with moderate oligozoospermia, and ten men with severe oligozoospermia showed greater imprinted methylation perturbations in oligozoospermia compared with normozoospermia at H19 (LOM, 0/79, 1/8, and 3/10), MEG3 (LOM, 5/79, 2/8, and 4/10), PEG1 (GOM, 7/79, 2/8, and 3/10), KCNQ1OT1 (GOM, 3/79, 0/8, and 1/10), PEG3 (GOM, 4/79, 1/8, and 0/10), and SNRPN (GOM, 1/79, 2/8, and 1/10) (Kobayashi et al. 2007; Table 7). Furthermore, multilocus perturbations were greater in moderate (2/8; 25%) and severe oligozoospermic sperm (5/10; 50%) compared with normozoospermic sperm (5/79; 6.3%). Similarly, imprinting perturbations were present in sperm from 0/5 men with normozoospermia, 0/5 men with mild, 2/5 men with moderate (one H19, one H19 plus PEG1), 4/5 men with severe (two H19, two PEG1), and 2/5 men with very severe oligozoospermia (one PEG1, one H19 plus PEG1) (Marques et al. 2008). In a third study, methylation perturbations were again greater in oligozoospermic men compared with normozoospermic men at H19 (LOM, 1/204 normal, 1/61 moderate, and 8/57 severe), MEG3 (LOM, 2/201 normal, 6/56 moderate, and 15/55 severe), ZDBF2 (LOM, 0/119 normal, 1/60 moderate, and 2/47 severe), PEG1 (GOM, 7/129 normal, 4/29 moderate, and 5/23 severe), PEG3 (GOM, 2/127 normal, 1/25 moderate, and 1/21 severe), ZACN (GOM, 0/120 normal, 1/56 moderate, and 4/61 severe), SNRPN (GOM, 0/124 normal, 4/60 moderate, and 2/61 severe), and KCNQ1OT1 (GOM, 1/95 normal, 0/7 moderate, and 1/9 severe) (Sato et al. 2011). Multilocus methylation perturbations were greater in men with severe (10/22; 45%) and moderate (2/11; 18%) oligozoospermia than in normozoospermic men (3/14; 21%). These studies contrast with a lack of SNRPN methylation errors in sperm from 17 normozoospermic, 17 moderate oligozoospermic, and 16 severe oligozoospermic men (Manning et al. 2001). Finally, the prevalence of H19 LOM was greater in sperm from oligozoospermic men (3/3 moderate, 6/6 severe, and 5/6 very severe) compared with men with teratozoospermia (1/9) and astheno-teratozoospemia (0/7) (Boissonnas et al. 2010). Altogether, these results indicate that moderate-to-severe oligozoospermia is associated with imprinting errors.

Table 7

Studies on male infertility/subfertility and genomic imprinting.

ReferenceSpeciesMethodSamplesGeneObservations
Kobayashi et al. (2007)HumanCOBRA, BMSEjaculated spermH19LOM 0/79 norm, 1/8 mod, 3/10 severe
MEG3LOM 5/79 norm, 2/8 mod, 4/10 severe
PEG1GOM 7/79 norm, 2/8 mod, 3/10 severe
KCNQ1OT1GOM 3/79 norm, 0/8 mod, 1/10 severe
PLAGL1GOM 0/79 norm, 0/8 mod, 3/10 severe
PEG3GOM 4/79 norm, 1/8 mod, 0/10 severe
SNRPNGOM 1/79 norm, 2/8 mod, 1/10 severe
Marques et al. (2008)HumanBMSEjaculated spermH19LOM 0/5 norm, 0/5 mild, 2/5 mod, 2/5 severe, 1/5 very severe
PEG1GOM 0/5 norm, 0/5 mild, 1/5 mod, 2/5 severe, 2/5 very severe
Sato et al. (2011)HumanBPLEjaculated spermH19LOM 1/204 norm, 1/61 mod, 8/57 severe
MEG3LOM 2/201 norm, 6/56 mod, 15/55 severe
ZDBF2LOM 0/119 norm, 1/60 mod, 2/47 severe
PEG1GOM 7/129 norm, 4/29 mod, 5/23 severe
PEG3GOM 2/127 norm, 1/25 mod, 1/21 severe
PLAGL1GOM 0/120 norm, 1/56 mod, 4/61 severe
SNRPNGOM 0/124 norm, 4/60 mod, 2/61 severe
KCNQ1OT1GOM 1/95 norm, 0/7 mod, 1/9 severe
Manning et al. (2001)HumanBMSEjaculated spermSNRPNLOM 0/17 norm, 0/17 mod, 0/16 severe
Boissonnas et al. (2010)HumanBMSEjaculated spermH19LOM 3/3 mod, 6/6 severe, 5/6 very severe, 1/9 terato, 0/7 astheno
Minor et al. (2011)HumanBMSTesticular spermH19LOM 5/10 obs, 1/5 nonobs, 5/17 vas
PEG1GOM 0/10 obs, 0/5 nonobs, 2/17 vas
MEG3LOM 0/10 obs, 0/5 nonobs, 0/17 vas
Kobayashi et al. (2009)HumanCOBRA, BMS78 6–9 week abortusesH19LOM 6/78 samples
Trophoblastic villiMEG3LOM 2/78 samples
11 paired abortus – ejaculated sperm PEG1GOM 1/78 samples
KCNQ1OT1GOM 4/78 samples
PLAGL1GOM 1/78 samples
PEG3GOM 1/78 samples
SNRPNGOM 0/78 samples
XISTGOM 5/78 samples
Hammoud et al. (2010)HumanBMSFrozen ejaculated spermKCNQ1OT1GOM 0/7 norm, 3/8 oligo, 2/9 protamine
PEG1GOM 0/5 norm, 3/10 oligo, 1/10 protamine

BMS, bisulfite mutagenesis and sequencing; COBRA, combined bisulfite-PCR restriction analysis; BPL, bisulfite polymerase chain reaction Luminex; norm, normozoospermia; mild, mild oligozoospermia; mod, moderate oligozoospermia; severe, severe oligozoospermia; very severe, very severe oligozoospermia; oligo, oligozoospermia; terato, teratozoospermia; astheno, astheno-teratozoospermia; obs, obstructive azoospermia; nonobs, nonobstructive azoospermia; vas, vasectomy reversal; protamine, abnormal histone–protamine incorporation; LOM, loss of methylation; GOM, gain of methylation.

Testicular sperm from men with azoospermia has also been examined for imprinting errors. H19 LOM occurred in 5/10 men with obstructive azoospermia compared with 1/5 men with nonobstructive azoospermia (Minor et al. 2011). Interestingly, testicular sperm from previously fertile men undergoing vasectomy reversal also showed imprinting errors with 5/17 men possessing PEG1 GOM and/or H19 LOM. Methylation perturbations in these previously fertile men may relate to advanced paternal age. Alternatively, obstruction may generate an aberrant testicular environment, accounting for greater perturbations in vasectomy reversal and obstructive azoospermic men.

The important question is whether sperm-imprinting errors are transmitted to offspring. Analysis of abortuses from 78 males with normozoospermia and moderate-to-severe oligozoospermia identified 17 samples (22%) with abnormal DNA methylation (six H19 LOM, two MEG3 LOM, one PEG1 GOM, four KCNQ1OT1 GOM, one PLAGL1 GOM, one PEG3 GOM, 0 SNRPN GOM, and five XIST GOM), of which six were from normozoospermic and 11 from oligozoospermic males (Kobayashi et al. 2009). Of the 11 paired samples, seven (41%) showed methylation errors in both sperm and abortus (five H19 LOM, one MEG3 LOM, and one H19 plus MEG3 LOM), indicating that imprinting errors can be transmitted to fetuses. However, of these pairs, half the abortuses had a complete LOM and half were mosaic for methylation perturbations. As imprinting errors from sperm would expect to be transmitted to all cells in abortuses, it is also unclear how mosaic methylation patterns arise in offspring following fertilization. Additionally, it is unclear which epigenetic mechanisms would lead to both a LOM at paternally methylated genes and a GOM at normally unmethylated, paternally expressed genes in the same sperm sample.

Finally, imprinting errors have been investigated in sperm with abnormal histone to protamine transition. Imprinting errors occurred almost equally in sperm from men with abnormal protamine incorporation (2/9 KCNQ1OT1 GOM and 1/10 PEG1 GOM) and oligozoospermic men (3/8 KCNQ1OT1 GOM and 3/10 PEG1 GOM) compared with fertile men (0/7 KCNQ1OT1 GOM and 0/5 PEG1 GOM) (Hammoud et al. 2010). As abnormal histone to protamine transition can result in a loss of H3K4me3 at paternally expressed genes and a gain of H3K4me3 at paternally silent genes (Carrell 2012), this may be indicative of chromatin structural changes that produce corresponding GOM and LOM in sperm, and possibly in fetuses and offspring. Further investigations are required to determine whether imprinting errors in sperm originate from incomplete erasure of DNA methylation marks, perturbations in imprinted methylation acquisition, DNA methylation maintenance errors, and/or aberrant chromatin packaging/protamine–histone incorporation. As well, studies are required to identify potential mechanisms for inheritance of imprinting errors in fetuses and offspring from infertile males.

Discussion

Complexity of multiple arts and infertility

The question of gamete and embryo predisposition to ART-induced epigenetic defects is of critical importance. As multiple ARTs are employed, it is difficult to discern the origin of imprinting anomalies in human embryos. In the mouse, superovulation in combination with embryo culture increased the number of blastocysts with biallelic H19 expression above embryo culture alone (Market-Velker et al. 2010a), as well superovulation, embryo culture, and blastocyst transfer generated a greater proportion of conceptuses with aberrant imprinted expression compared with superovulation and embryo transfer without culture (Rivera et al. 2008). These and other studies indicate that combined ART treatments can produce greater numbers of embryos with imprinting perturbations. With this caveat in mind, the literature suggests that ovarian stimulation, IVM, and cryopreservation do not greatly impact DNA methylation acquisition but instead give rise to imprinting maintenance perturbations. Furthermore, as IVF, ICSI, and embryo culture disrupt imprinting maintenance, we propose that ARTs converge on a common imprinting regulatory pathway.

Human embryos produced via ARTs are also the product of underlying infertility/subfertility. This has lead to questions regarding the origin of epigenetic instability, i.e. whether underlying infertility/subfertility compromises epigenetic integrity in gametes/embryos, whether gamete/embryo manipulations cause epigenetic instability, or whether a combination of subfertility and ARTs leads to epigenetic disruption. The relationship between impaired fertility, ARTs, and epigenetic stability is unquestionably complex. However, the possibility exists that ARTs and infertility may disrupt the same biological pathways that lead to epigenetic instability. If this is the case, perturbations induced by infertility/subfertility may be exacerbated by gamete or embryo manipulation, similar to combined ART treatments.

Finally, while the caveats of multiple ARTs and infertility are recognized, it should be noted that further complexity is compounded by a number of methodological and biological issues. These include varied methylation assays (in some cases limited to 1–2 CpGs), nonallelic analyses, lack of controls, small sample sizes, cumulus cell contamination, oocyte, embryo, and tissue pooling (mask or inflate rare epigenetic errors), the use of discarded/failed human reproductive samples (non-fertilized oocytes and fragmented embryos), age (preimplantation, midgestation, and childhood), and type of tissue (embryo, placenta, and peripheral blood cells), type of infertility, as well as social determinants (parental age, smoking, and obesity). To gain further insight into the causes of epigenetic instability arising from ARTs and infertility, future studies need to take these important issues into consideration.

Pathways leading to epigenetic instability

Multiple avenues of investigation are required to delineate the effects of infertility/subfertility and ARTs on epigenetic gene regulation. Foremost, studies are required to determine the molecular and cellular mechanisms giving rise to epigenetic errors. This includes the identification of maternal factors needed for epigenetic regulation during embryo development. Acceleration of oocyte maturation or recovery of atretic oocytes by superovulation, adaptation to suboptimal IVM culture, or stress induced by cryopreservation can affect the oocyte's ability to synthesize and store sufficient amounts of maternal factors (Li et al. 2010). Four maternal effect proteins have been identified that protect imprinted genes from demethylation during preimplantation development. The oocyte-specific DNA methyltransferase 1o maintains imprinted methylation in eight-cell embryos (Cirio et al. 2008). Zinc finger protein 57 together with KAP1 protects imprinted genes from (possibly passive) demethylation (Li et al. 2008, Messerschmidt et al. 2012). Developmental pluripotency-associated 3 (STELLA/PGC7) protects imprinted genes from demethylation by binding to H3K9me2, inhibiting the active conversion of 5-methylcytosine to 5-hydroxymmethylcytosine (Nakamura et al. 2012). Investigation of these and other maternal effect products is required to determine whether their synthesis and storage are affected by oocyte manipulation and/or infertility and whether their ART-induced misregulation leads to epigenetic instability in resulting embryos. We are also intrigued by the possibility that perturbations in bivalency marks in sperm histones of infertile men may lead to epigenetic errors in developing embryos. Further studies are required to investigate perturbations in histone modifications following infertility and ARTs in gametes and embryos. Finally, genome-scale studies are needed to determine the scope of epigenetic instability at nonimprinted genes in gametes and embryos as a result of infertility/subfertility and ARTs.

Assisted reproduction will continue to be a critical medical intervention for infertile couples. To maximize the safety of these ARTs, it is imperative to understand how mechanisms involved in epigenetic regulation are affected by impaired fertility and ART treatments. This will lead to the development of screening procedures to identify at-risk embryos as well as preventative measures that will reduce the occurrence of epigenetic perturbations.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

M M Denomme was supported by a CIHR Training Program in Reproduction, Early Development and the Impact on Health (REDIH) Graduate Scholarship.

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    DNA methylation asymmetry during gametogenesis and preimplantation development. Paternal (blue line) and maternal (red line) DNA methylation is erased in primordial germ cells. De novo DNA methylation acquisition occurs earlier in male compared to female germ cell development. Following fertilization, the paternal genome is rapidly and actively demethylated (light blue line) while the maternal genome (light red line) is passively demethylated at each replication cycle. Differential DNA methylation at imprinted genes is protected from demethylation. Infertility/subfertility and various ARTs may cause epigenetic instability at the erasure, acquisition, and maintenance stages. Furthermore, combination of infertility and/or multiple ARTs may impose greater risk for inducing epigenetic errors.

    Box 2 Histone modifications are posttranslational covalent modifications to histone tails, which have either activating or repressive functions. Histone 3 lysine 9 (H3K9), histone 3 lysine 27 (H3K27), and histone 4 lysine 20 (H4K20) trimethylation (me3) are repressive modifications while histone 4 acetylation (H4Ac) and histone 3 lysine 4 trimethylation (H3K4me3) are activating modifications.

    DNA methylation is the covalent modification of methyl groups to cytosines within CpG dinucleotides. DNA methylation is typically associated with gene silencing.

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    Epigenetic landscape in gametes. In mature sperm, protamines tightly compact chromatin into toroids (90–99% chromatin) that are punctuated by histone solenoids (1–10% chromatin). Sperm DNA is hypermethylated (red; DNA me) except at regions bearing active and bivalent histone modifications. By comparison, histones compact chromatin in the mature oocyte. Chromatin is further condensed into loops that are bound to spindle fibers. Oocyte chromatin is hypermethylated and carries repressive histone modifications. Data from Patrushev & Minkevich (2008) and Carrell (2012).

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