Hum. Reprod. Advance Access originally published online on August 21, 2006
Human Reproduction 2007 22(1):26-35; doi:10.1093/humrep/del316
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Aberrant DNA methylation of imprinted loci in superovulated oocytes
1 St Luke Clinic, Tsumori, Oita, Japan and 2 Department of Molecular Genetics, Division of Molecular and Cell Therapeutics, Medical Institute of Bioregulation, Kyusyu University, Beppu, Oita, Japan
3 To whom correspondence should be addressed at: Department of Molecular Genetics, Division of Molecular and Cell Therapeutics, Medical Institute of Bioregulation, Kyusyu University, 4546, Tsurumihara, Beppu, Oita 874-0838, Japan. E-mail: tarima{at}tsurumi.beppu.kyushu-u.ac.jp
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Abstract |
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BACKGROUND: There is an increased incidence of rare imprinting disorders associated with assisted reproduction technologies (ARTs). The sex-specific epigenetic modifications that are imposed during gametogenesis act as a primary imprint to distinguish maternal and paternal alleles. The most likely candidate for the gametic mark is DNA methylation. However, the timing of DNA methylation acquisition in adult oocytogenesis and the effects of superovulation are unknown. METHODS: We examined the maternal methylation of PEG1(MEST), LIT1(KCNQ1OT1) and ZAC(PLAGL1) and the paternal methylation of H19 in adult growing oocytes of humans and mice and compared them with the methylation status of mouse neonatal growing oocytes by using bisulphite sequencing. Furthermore, we examined the effects of superovulation in the human and mouse. RESULTS: Maternal methylation of these genes has already been initiated to some extent in adult human and mouse non-growing oocytes but not in mouse neonates. In addition, the methylation dynamics during adult human and mouse oocyte development changed more gradually than those during neonatal oocyte development. Furthermore, we found the demethylation of PEG1 in growing oocytes from some ART-treated infertile women and a gain in the methylation of H19. We also detected methylation changes in superovulated mice. CONCLUSION: Our studies in the human and mouse suggest that superovulation can lead to the production of oocytes without their correct primary imprint and highlight the need for more research into ARTs.
Key words: ART/DNA methylation/genomic imprinting/human adult oocyte/superovulation
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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Genomic imprinting, which describes the allele-specific expression of certain genes, accounts for the requirement of both maternal and paternal genomes in normal development (Ohlsson et al., 1998
; Reik and Walter, 1998; Surani, 1998; Tilghman, 1999). Many imprinted genes play important roles in regulating embryonic growth, placental function and neurobehavioural processes (McGrath and Solter, 1984; Surani et al., 1984), and the aberrant expression of several imprinted genes has been linked to a number of diseases, developmental abnormalities and malignant tumours in humans (Paulsen and Ferguson-Smith, 2001).
In the mouse, the sex-specific epigenetic modifications are imposed during gametogenesis and act as markers to distinguish the maternal and paternal alleles (Surani, 1998). Imprints are erased in primordial germ cells (Hajkova et al., 2002; Lee et al., 2002) and re-established during gametogenesis in a sex-specific manner to establish the correct parental expression pattern for the next generation. Allele-specific DNA methylation has been observed in the vicinity of most imprinted genes in somatic cells and, in some cases, in germ cells (Reik and Walter, 1998; Paulsen and Ferguson-Smith, 2001). As DNA methylation is both a heritable and a reversible epigenetic modification that is stably propagated after DNA replication, it is a strong candidate for the primary epigenetic mark in the germ line (Li, 2002). The timing of the acquisition of the DNA methylation at imprinted loci differs between the two germ lines. In the male germ line, H19, Rasgrf1 and Gtl2 methylation imprints are initiated prenatally during embryonic germ cell development and are complete by the pachytene phase of post-natal spermatogenesis in mice (Davis et al., 1999, 2000; Ueda et al., 2000; Li et al., 2004). In contrast, in the female germ line, Igf2r, Snrpn, Peg1 and Peg3 methylation imprints are acquired asynchronously in a gene-specific manner, whereas oocytes are arrested at prophase I and during the transition from primordial to antral follicles in the post-natal growth phase (post-pachytene) (Lucifero et al., 2004a). Nuclear transplantation using post-natal oocytes at various stages of maturation points to this same window of oocyte development as the time when functional imprints are acquired (Bao et al., 2000; Obata and Kono, 2002).
In humans, limited information is available on the methylation status of imprinted genes during gametogenesis and embryogenesis, but available data suggest some conservation of the timing of DNA methylation acquisition and maintenance dynamics described in mice. The paternally inherited methylation imprint on H19 is present in mature human sperm but absent in fetal spermatogonia (Kerjean et al., 2000). The only available study on maternal imprinting in human oocytes suggested that the methylation imprint on SNRPN was present by the germinal vesicle (GV) stage and maintained during subsequent stages (Geuns et al., 2003).
Several studies published over the last few years have suggested that there is an increased incidence of rare imprinting disorders associated with human assisted reproduction technologies (ARTs) (Cox et al., 2002; Hansen et al., 2002; Schieve et al., 2002; Weksberg et al., 2002; DeBaun et al., 2003; Gicquel et al., 2003; Maher et al., 2003; Orstavik et al., 2003; Halliday et al., 2004). ARTs are important treatments for infertile people of reproductive age in which the oocytes and/or sperm are manipulated in the laboratory. These include IVF, ICSI and in vitro maturation (IVM) of oocytes (Wright et al., 2003). ART involves the isolation, handling and culture of gametes and early embryos, generally after hormone stimulation protocols, at times when the epigenetic marks at imprinted loci are potentially vulnerable to external influences. ART (IVF and ICSI) is associated with an increased risk of imprinting disorders, including cases of Beckwith–Wiedemann syndrome (BWS) (NIM130650) and Angelman’s syndrome (AS) (NIM105830), with the loss of maternal DNA methylation at differentially methylated regions (DMRs) (DeBaun et al., 2003; Gicquel et al., 2003; Maher et al., 2003; Orstavik et al., 2003). Much debate has recently surrounded the issues of possible epigenetic alterations brought about by human ART (Lucifero et al., 2004b). One of the important issues is the artificial induction of ovulation with high doses of gonadotrophin. In ART procedures, a large amount of gonadotrophin is used to obtain the mature oocytes. Although it is uncertain whether exogenous gonadotrophins alter the maturation process of oocytes or the physiological environment of the uterus, it has been shown that implantation may be more likely when large amounts of gonadotrophin are avoided (Winston and Hardy, 2002). Furthermore, studies in mice and hamsters have suggested that superovulation decreases the viability of embryos (McKiernan and Bavister, 1998; Van der Auwera and D’Hooghe, 2001).
In this study, we have determined the DNA methylation status at the DMRs of four imprinted genes (PEG1, LIT1, ZAC and H19) in humans and mice using the bisulphite multiplex PCR sequencing technique. Mutations in these human genes have been implicated in Silver–Russell syndrome (OMIM 180860
[OMIM]
), BWS and transient neonatal diabetes (OMIM 601410
[OMIM]
), and this technique is a highly sensitive approach to analyse every potential methylation site in a target sequence. We have used the mouse model to examine DNA methylation status of the Lit1 and Zac DMRs during neonatal female germ cell development to compare our findings with those for Peg1, which has been characterized as one of the latest known imprinted genes to acquire a DNA methylation imprint (Obata and Kono, 2002; Lucifero et al., 2004b). We have also examined these marks in oocytes obtained from naturally cycling mouse and human adult ovaries and in material obtained after superovulation. Our data showed the occurrence of methylation errors on imprinted genes in fully grown oocytes because of superovulation, which, as far as we know, is the first evidence that any manipulation of oocyte maturation or folliculogenesis can result in altered timing of the imprinting of genes and perhaps lead to congenital malformations in humans.
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Materials and methods |
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Oocyte collection
BDF and ICR mice were used for all experiments. Oocytes were collected from neonate ovaries at 1, 10, 15 and 30 days post-partum (dpp) at the primordial, primary, pre-antral and antral follicle stage (GV fully grown oocytes). Oocytes of the same stages were also removed from the adult ovaries (10–15 weeks). The ovaries were immersed in 3.5 ml of M2 medium (Fulton and Whittingham, 1978
) containing 1.5 mg/ml of collagenase (Wako, Tokyo, Japan). Oocyte–granulosa cell complexes from the various follicle stages were transferred to M2 containing 1.5 mg/ml of trypsin (Sigma, Tokyo, Japan) and 1.5 mg/ml of collagenase. After 15 min, the complexes were washed, and granulosa cells were removed by mouth pipetting. The granulosa cell-free oocytes were placed in M2 medium to remove the zona pellucida and attached cumulus cells. Oocytes were washed repeatedly and placed in phosphate-buffered saline as reported previously (Bao et al., 2000; Obata and Kono, 2002). The oocytes were classified regarding whether they were in the primordial follicle stage (30–60 µm, a small oocyte surrounded by a single layer of pregranulosa cells), primary follicle stage (60–120 µm, a single layer of enlarged granulosa cells), pre-antral–antral follicle stage (120–200 µm, two- to three-layer follicle) or GV stage (200 µm, perivitelline space present in the follicle). The GV, metaphase I (MI) and metaphase II (MII) oocytes were collected from 10-week-old females that ovulated naturally or were superovulated by the injection of 7.5 IU of pregnant mare’s serum gonadotrophin (PMSG) (Teikoku Zouki, Tokyo, Japan) for 3 days followed 24 h later by injection of 5.0 IU of hCG (Teikoku Zouki). These oocytes were removed 24 h after the hCG injection, and the oocyte–granulosa cell complex was picked up from the oviduct and then the oocytes were collected as mentioned above. In the case of adult human oocytes, we harvested immature oocytes from biopsy samples obtained from laparoscopic examinations and superovulated GV and MI oocytes from IVF procedures after receiving the patients’ agreement and with the approval of the Institutional Ethics Committee and Japan’s Society of Obstetrics and Gynecology. We were able to obtain material from normal ovaries of women of reproductive age (28–40 years old) at laparoscopic biopsy. On average, 20 usable oocytes at the appropriate developing follicle stages (primary, pre-antral and antral follicle stages) could be obtained from each small (0.3–0.5 mm3) biopsy. Human oocytes during the growing phase were hand-picked using the same procedure as for the mouse oocyte collection. As a control, normal leukocyte DNA of humans and mice was used.
In the case of superovulated oocytes, we focused on the mouse Peg1 and human PEG1 genes. In both humans and mice, the DMR spans the promoter, the first exon and part of the first intron and is unmethylated on the active paternal allele (Kobayashi et al., 1997; Lefebvre et al., 1997). Paternal transmission of a methylated Peg1 gene results in growth-retarded embryos and increased post-natal death. Abnormal adult maternal behaviour has been noted in Peg1-deficient females (Lefebvre et al., 1998; Li et al., 1999). The acquisition of the methylation imprint of the Peg1 is the latest of the known methylation imprints (Obata and Kono, 2002; Lucifero et al., 2004b). Seventy-two GV and MI oocytes were collected by needle biopsy during ultrasound examination from infertile patients aged 23–41 years, who had received hyperstimulation therapy (hMG 150–300 IU/day, for 7–14 days) to generate mature oocytes and initiate IVF therapy. The patients were healthy women with no habitual drug use and no particular past or familial disease history.
Bisulphite treatment PCR
The methylation assay was performed at the DMRs of four imprinted genes (PEG1, LIT1, ZAC and H19) in humans and mice using the bisulphite genomic sequencing method (Olek et al., 1996). DNA of 30–50 oocytes at several stages and of single human GV and MI oocytes was prepared, and bisulphite treatment was carried out in low-melting-point agarose (Sigma), and the products were amplified by PCR as follows. A multiplex PCR reaction mix containing 1 pM of each of the four following primer sets—200 mM dNTPs, 1x PCR buffer, 2 mM MgCl2, 1.25 U LA Taq DNA polymerase (Takara, Tokyo, Japan)—in a total volume of 25 µl was used. The following PCR programme was used for the first round: 5 min of denaturation at 96°C followed by 25 cycles of 30 s at 96°C, 30 s at 55°C and 30 s at 72°C and a final extension for 10 min at 72°C in a Perkin-Elmer 9600 thermal cycler. Three microlitres of the first-round multiplex PCR product was used as DNA input for amplification in the second-round nested PCR with the following programme: 5 min of denaturation at 96°C followed by 30 cycles of 30 s at 96°C, 30 s at 50–57°C and 30 s at 72°C and a final extension for 10 min at 72°C. Primers specific to bisulphite-converted DNA for human PEG1, ZAC and H19 were previously described (Kamiya et al., 2000; Kerjean et al., 2000). For LIT1, the first-round primers were 5'-GTGTTAYGGYGGTGGAGATTTTGT-3' and 5'-AACCAAAAACARAACCAATTCTCTA-3' and the second-round PCR primers were 5'-GTGTTAYGGYGGTGGAGATTTTGT-3' and 5'-AACCAATTCTCTACRTAATATATTCA-3'. The region analysed for each of these genes was within a CpG island. We examined 23 CpG sites in a 223-bp fragment of PEG1 (MEST) (AC144863
[GenBank]
, 17954–18263), 18 CpG sites in a 190-bp fragment of LIT1 (KCNQ1OT1) (AC021424
[GenBank]
, 128558–128807), 12 CpG sites in a 116-bp fragment of ZAC (AL109755
[GenBank]
, 52885–52733) and 15 CpG sites in a 230-bp fragment of H19 (AF087017
[GenBank]
, 6099–6329). The mouse primers for Peg1, Lit1, Zac and H19 were described previously (Hata et al., 2002; Arima et al., 2005). We examined 21 CpG sites in a 462-bp fragment of Peg1 (AC146699
[GenBank]
, 7961–7499), 17 CpG sites in a 336-bp fragment of Lit1 (AC012540
[GenBank]
, 190291–189956), 21 CpG sites in a 329-bp fragment of Zac (AF314094
[GenBank]
, 1227–1556) and 11 CpG sites in a 419-bp fragment of H19 (AF049091
[GenBank]
, 2594–3016). We summarized the primer sequences in Table I.
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All PCR products were purified using Gene Pure (Nippon Gene, Japan), and half of the purified PCR products were digested with appropriate restriction enzymes (BsiEI, RsaI, Sau3AI and HhaI) and electrophoresed on 2.5% agarose gels. The remaining portion of the PCR-amplified products was directly sequenced and/or cloned into the TOPO TA vector (Invitrogen), and individual clones were sequenced using T7 and/or M13 reverse primers and an automated ABI 3100 sequencer. Twenty clones for each individual were sequenced on average. At least two separate sodium modification treatments were carried out for each DNA sample, and at least three independent amplification experiments were performed for each individual examined.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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Ontogeny of primary imprint methylation during adult mouse oocyte development
The ontogeny of the methylation mark in the adult germ line is still not known. Therefore, we collected mouse oocytes at different stages from unstimulated neonatal and adult ovaries and compared them to examine the dynamics of the methylation changes that occur (Figure 1, Table II). Oocytes at the primordial, primary, pre-antral and antral follicle stages were collected and subjected to the bisulphite multiplex PCR methylation method. We examined the timing of methylation acquisition of the DMRs of three maternally imprinted genes from different chromosomal regions: Peg1, Lit1 and Zac. We also examined the methylation of the paternally imprinted gene H19. The DMR for this gene should remain unmethylated in the female germ line.
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In mouse neonatal developing oocytes, the methylation acquisition patterns of the Lit1 and Zac DMRs were quite similar to the previously reported methylation dynamics of Snrpn, Igf2r and Peg3 (Obata and Kono, 2002
; Lucifero et al., 2004b)—i.e. only a few of the potential methylation sites were found to be methylated in the primordial follicle-stage oocytes. The number of methylated strands increased from the primary to pre-antral follicle-stage oocytes. By the antral follicle-stage oocytes, all of the sites were found to be fully methylated. The DMR of Peg1 first began to show de novo methylation in pre-antral follicle-stage oocytes and was fully established in fully grown GV. The H19 DMR was unmethylated in all samples. This firstly demonstrates that no DNA methylation of this DMR occurs in naturally obtained mouse oocytes, but this also serves as a control against contamination because the presence of material from somatic cells would result in the detection of some fully methylated sequences. In adult mouse oocytes, all of the maternally methylated regions we examined had, to some extent, already acquired some DNA methylation at the primordial and primary follicle stage and were completely methylated in antral follicle-stage oocytes (Figure 1A and C). The methylation dynamics during adult oocyte development changed more gradually than during neonatal oocyte development.
To ensure that the sequencing results from a limited number of templates accurately reflected the overall methylation pattern for these DMRs in the isolated germ cell populations and to further confirm that the cloning was not biased towards either treated or untreated temples, we carried out restriction analysis on germ cell and somatic cell DNA, cutting the DNA with enzymes that could cleave only the methylated templates of the same bisulphite-treated PCR samples that were used for cloning and sequencing shown in Figure 1B. PCR of each of the DMRs was followed by digestion with the enzyme RsaI for Peg1 and Zac, with BsaAI for Lit1 and with HhaI for H19, so that the undigested and digested products indicated unmethylated and methylated templates. About half methylated and half unmethylated templates, representing paternal and maternal alleles, were obtained after the treatment of DNA from normal somatic leukocytes, indicating a lack of bias in the PCR.
Mouse superovulated oocytes
Infertile women are treated with various stimulation regimens. As these patients have a low fertility rate and an increased reproductive loss rate and are generally of advanced age (Shiota and Yamada, 2005), it is difficult to isolate the effect of the hormonal stimulation from other factors to evaluate the risk in ART procedures. We therefore examined the effect of hormonal stimulation of ovulation in mice. We used large amounts of gonadotrophin to induce maturation of oocytes in two strains of mice (ICR and BDF). Mouse GV, MI and MII oocytes were collected. Using the DNA extracted from the mouse GV, MI and MII oocytes, the bisulphite PCR sequencing and restriction assays were performed. All of the maternally imprinted genes were over 90% methylated, and no significant differences were found between normal and superovulated GV oocytes (Figure 2, Table III). In contrast, we found that H19 DMR, which is normally only methylated in the paternal germ line, showed DNA methylation in some samples similar to that seen in the human samples.
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Analysis of the methylation imprint in human adult oocytes
We next extended our analysis to human adult oocytes. Three hundred oocytes were collected from biopsy samples obtained from laparoscopic examinations. On average, 20 usable oocytes at the appropriate developing follicle stages (primary, pre-antral and antral follicle stages) could be obtained from each small (0.3–0.5 mm3) biopsy. These were subjected to methylation analysis. The human PEG1 DMR methylation profile during adult oogenesis was found to be similar to the mouse profile. Approximately 50% methylation was already detected in the primary follicle stage, and full methylation was shown in the oocytes with fully grown GVs at the antral follicle stage (Figure 3). To confirm the results, we digested the PCR products with the appropriate restriction enzymes and electrophoresed the products on 3% agarose gels. We found that the sequencing results showed no bias in the choice of the PCR clones.
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Analysis of the methylation imprint of human superovulated oocytes by single-cell bisulphite PCR
We examined the methylation states of human superovulated oocytes. As human material has limited availability and may show some differences among individual oocytes, we adapted the bisulphite PCR sequencing technique to the single-cell level and focused on the mouse Peg1 and human PEG1 genes. Table IV summarizes the clinical characterization of patients: age, ART indication, method of ovarian stimulation and number of ART procedures. The overall efficiency of amplification at the single-cell level was 22.2% (16/72: contamination rate 9.7%). The results of single-cell bisulphite PCR sequencing showed the methylated pattern in only 10 of the 16 cases (10/16) (Figure 4A). All single-cell PEG1 PCR was followed by digestion with the enzyme Sau3AI. The lack of a mixture of both the methylated and unmethylated patterns within a single sample indicates that there was no somatic cell contamination (Figure 4B). We next examined the methylation status of a paternally imprinted gene H19 by single-cell bisulphite PCR sequencing. The success rate of PCR amplification was 18.7% (6/32: contamination rate 6.2%). Two MI oocytes were methylated, whereas four were unmethylated (Figure 4C). We confirmed by the restriction assay method that there was no somatic cell contamination (Figure 4D). This result suggests that DNA methylation can be lost or gained by the artificial induction of ovulation in humans.
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Discussion |
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Methylation dynamics of imprinted genes in the adult oocytes
In the neonatal female germ line, the establishment of the imprint signal occurs in a gene-specific manner at a specific time during oocyte growth for all genes throughout the primary to antral follicle stage (Lucifero et al., 2002
, 2004b; Obata et al., 1998; Obata and Kono, 2002). To better understand the process of genomic imprinting and assess the risks linked to ART, we determined the DNA methylation profiles of the DMRs for four imprinted genes, the maternally methylated Lit1, Zac and Peg1 genes and the paternally methylated H19 gene in normal adult mouse oocytes. Consistent with the previous reports, both maternally and paternally methylated DMRs were unmethylated in the neonate mouse oocytes (Lucifero et al., 2002, 2004b; Obata and Kono, 2002). The three maternally methylated DMRs acquired DNA methylation of 
50% in the adult oocytes at the early follicle stage. The paternally methylated H19 was unmethylated at all stages examined as predicted. Our analysis of the available human material suggests a similar pattern for the methylation dynamics of the human loci.
The methylation error because of the excess ovarian stimulations
A complex developmental programme occurs during oogenesis and relies on timed and highly orchestrated interactions between the oocyte, the follicular compartment and the neuroregulatory axis and paracrine and autocrine hormonal stimuli (Eppig et al., 2002). Meiosis is initiated during fetal life and arrested after pachytene in the diplotene stage (termed dictyate stage) for long periods. The resumption of meiosis takes place in adult females under hormonal control (Crisp, 1992), and only fully grown oocytes can respond to the maturational stimulus after they acquired maturational competence during preceding stages of oocyte growth and follicular development. The different stages of oogenesis and folliculogenesis each may exhibit specific sensitivities to environmental chemicals (Hoyer and Sipes, 1996; Hirshfield, 1997). In addition, natural changes in gene expression levels occur during ageing such as changes in the expression of DNA methyltransferase (Dnmt) 3 proteins required in the establishment of the germ line imprint (Okano et al., 1998, 1999; Bourc’his et al., 2001; Hata et al., 2002; Hamatani et al., 2004; Kaneda et al., 2004) which may, in turn, be because of changes in the endocrine environment. We have found that superovulated GV and MI oocytes from infertile women show a gain of H19 methylation and a loss of PEG1 methylation in our single-cell methylation assay. We cannot distinguish whether these changes in DNA methylation are because of the process of superovulation or the age of the patients or are inherent to the infertility problems suffered by some of these individuals. However, we have also demonstrated DNA methylation at the normally unmethylated H19 DMR in murine superovulated material which suggests that the changes we see at the human H19 locus are due, at least in part, to the superovulation procedure. The difference in the loss of maternal methylation at Peg1 between the mouse and human may be a consequence of the number of treatments because the human patients were subjected to numerous superovulations over a long time period. Alternatively, the H19 DMR might be more susceptible to changes in mice than the PEG1 DMR. The exposure of mouse embryos to different culture conditions can alter H19 imprinting (Doherty et al., 2000; Khosla et al., 2001; Lonergan et al., 2003; Mann et al., 2004). In addition, Croteau et al. (2001) have shown that spontaneous imprinting defects resulting in biallelic H19 and Igf2 expression occur in 1.6 and 0.5% of day 7.5 and 8.5 mouse post-implantation embryos, respectively. The incidence of biallelic expression of either gene was dependent on the genetic background of the embryo, and thus presumably on trans-acting factors. These observations suggest that imprinting defects may occur sporadically in normal embryos and that the processes of imprint erasure, establishment and maintenance are vulnerable to errors. Others genes such as LIT1, ZAC and PEG1 may be more resistant.
Immature oocytes are also collected during controlled ovarian stimulations. These oocytes are usually discarded because of the possibility of abnormal embryonic development or an increased rate of abortion (Smith et al., 2000). However, in cases of poor responders and in patients with an unsynchronized cohort of follicles, where the presence of immature oocytes is frequent after stimulation (Smith et al., 2000), the use of immature oocytes for IVF is important to increase the number of embryos obtained in each cycle. However, our data would suggest that immature GV and MI oocytes might not have completed their full imprinting programme. Pregnancies obtained after in vitro maturation of these oocytes might therefore be at a significant risk for an imprinting disorder.
Our data, and those of others, strongly support the need for further research, particularly in animals, before translating new techniques into practice in humans. In addition, a retrospective examination of children born after each ART method might reveal the safest and most ethical approach to use.
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Acknowledgements |
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We thank Miss M. Nasu for technical assistance and all the members of our laboratory for their support and valuable suggestions. In particular, we thank Dr R. John for comments on the manuscript. This work was supported by a grant from the Ministry of Health and Welfare of Japan (15591758, 16045212) (T.A.).
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Footnotes |
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* These authors contributed equally to this work.
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Submitted on February 6, 2006; resubmitted on May 12, 2006; resubmitted on June 26, 2006; accepted on July 11, 2006.
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