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. 2012 May 15;27(7):2208–2216. doi: 10.1093/humrep/des151

Methylation levels at imprinting control regions are not altered with ovulation induction or in vitro fertilization in a birth cohort

RC Rancourt 1,2, HR Harris 1, KB Michels 1,2,*
PMCID: PMC3376159  PMID: 22587996

Abstract

STUDY QUESTION

Do fertility treatments, including ovulation induction (OI), alter epigenetic mechanisms such as DNA methylation at imprinted loci?

SUMMARY ANSWER

We observed small but statistically significant differences in certain imprinting control regions (ICRs) based on the method of conception, however, these small changes in methylation did not correlate to the overall transcriptional levels of the genes adjacent to the ICRs (such as KCNQ1 and SNRPN).

WHAT IS KNOWN AND WHAT THIS PAPER ADDS

Assisted reproductive technology (ART) has been associated with an increase in the risk of rare childhood disorders caused by loss of imprinting (LOI). This study provides novel epigenetic analyses on infants conceived by OI and examines how methylation levels correlate with gene expression.

DESIGN

Data and biospecimens used in this study were from 147 participants of the Epigenetic Birth Cohort comprising 1941 mother–child dyads recruited between June 2007 and June 2009 at the Department of Obstetrics, Gynecology and Reproductive Biology at Brigham and Women's Hospital (BWH) in Boston, MA, USA. Wilcoxon rank-sum tests were used to examine the differences in median percent methylation at each differentially methylated region (DMR) between the spontaneous conception control group and the fertility treatment groups (OI and IVF).

PARTICIPANTS AND SETTING

For each woman who reported IVF we selected a woman who conceived spontaneously matched on age (±2 years). To increase efficiency, we matched the same controls from the spontaneously conceived group to participants who reported OI. If an appropriate control was not identified that had been previously matched to an IVF participant, a new control was selected. The final analytic sample consisted of 61 spontaneous, 59 IVF and 27 OI conceptions.

MAIN RESULTS AND THE ROLE OF CHANCE

No functionally relevant differences in methylation levels were observed across five (out of six) imprinted DMRs in either the placenta or cord blood of infants conceived with OI or IVF compared with infants conceived spontaneously. While KCNQ1, SNRPN and H19 DMRs demonstrated small but statistically significant differences in methylation based on the method of conception, expression levels of the genes related to these control regions only correlated with the methylation levels of H19.

BIAS, CONFOUNDING AND OTHER REASONS FOR CAUTION

Limitations of our study include the limited sample size, lack of information on OI medication used and culture medium for the IVF procedures and underlying reasons for infertility among OI and IVF patients. We did not perform allele-specific expression analyses and therefore cannot make any inferences about LOI.

GENERALIZABILITY TO OTHER POPULATIONS

These results are likely to be generalizable to non-Hispanic white individuals in populations with similar ART and fertility treatments.

STUDY FUNDING/COMPETING INTEREST(S)

This project was supported by the Milton Fund, Harvard University (P.I.: K.B.M) and by Public Health Research Grant 5R21CA128382 from the National Cancer Institute, National Institutes of Health (P.I.: K.B.M.). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. No conflict of interests to declare.

Trial registration number

N/A.

Keywords: ovulation induction, IVF, DNA methylation, genomic imprinting, fertility treatment

Introduction

Assisted reproductive technology (ART) has been one of the most significant advances in human developmental biology over the past three decades. The use of intricate techniques and treatments has enabled many couples to overcome fertility challenges to successfully conceive. In the USA alone, more than 1% of all infants born annually are conceived using ART (Centers for Disease Control and Prevention, 2009). However, little is currently known regarding possible health outcomes of the offspring conceived with these technologies. Studies conducted with animal models and with human cultured embryos have highlighted potential risks for abnormal development that may arise from hormone stimulation, and the manipulation and culturing of oocytes/fertilized eggs (Mann et al., 2004; Li et al., 2005; Santos et al., 2010; reviewed in van Montfoort et al., 2012). Retrospective case–control studies and a large patient survey study have reported associations between ART and rare imprinting disorders [reviewed in (Manipalviratn et al., 2009; Owen and Segars, 2009; Banks and Segars, 2012)] and (Sutcliffe et al., 2006).

Genomic imprinting is the process by which a gene is expressed from one of the two parentally inherited chromosomes and repressed on the other in a parent-of-origin fashion. Imprinted genes are frequently found in clusters that contain differentially methylated regions (DMRs). For germline DMRs, DNA methylation is established during oogenesis or spermatogenesis and maintained upon fertilization and throughout development (Reik et al., 2001; Morgan et al., 2005). Germline DMRs usually act as imprinting control regions (ICRs), which can function over a long-range distance to control the parental origin-specific gene expression/repression of imprinted genes in the cluster, as well as secondary somatic DMRs, which tend to be tissue-specific. Disruption of the ICR causes loss of imprinting (LOI) at the surrounding genes resulting in biallelic expression and can lead to major phenotypic changes as seen in imprinting disorders (Butler, 2009).

The human chromosome 11 contains a large cluster of imprinted genes with two distinct ICRs: the maternally methylated KCNQ1 ICR in the KCNQ1 domain and the paternally methylated H19 ICR in the IGF2/H19 domain. Disruption of these ICRs can result in the Beckwith–Wiedemann syndrome (BWS) phenotype (Smilinich et al., 1999; Catchpoole et al., 2000; Maher and Reik, 2000; Weksberg et al., 2005). LOI in the SNPRN cluster, located on human chromosome 15q11, can lead to two distinct neurological conditions. The loss of maternal imprinting is seen in patients with Angelman syndrome (AS) while the loss of paternal imprinting is characteristic for Prader–Willi syndrome (PWS) (Knoll et al., 1989; Nicholls et al., 1989; Buiting, 2010). There is no consensus as to which an imprinted region has direct involvement in the congenital developmental disorder known as Silver–Russell syndrome (SRS). However, several candidate imprinted loci have been implicated in various SRS patient cases, including GRB10, MEST and H19/IGF2 (Preece et al., 1999; Monk et al., 2000; Kobayashi et al., 2001; Kagami et al., 2007). Case–control studies have suggested that patients with BWS (Rossignol et al., 2006; Gomes et al., 2007), AS (Cox et al., 2002; Orstavik et al., 2003) and SRS (Kagami et al., 2007; Douzgou et al., 2008) were more likely conceived with ART than healthy individuals suggesting that reproductive technologies contribute to the disruption of imprint methylation marks (e.g. loss of methylation at DMRs at the normally methylated allele) but these studies were compromised by limited sample sizes.

Which aspect of ART (if any) may affect DNA methylation at ICRs and allele-specific expression in humans has remained unresolved. While the culture medium has been implicated based on findings from animal studies, ovulation stimulation is a likely candidate since ovulation stimulation coincides with the timing of the establishment of the imprinted marks in the female gametes and external disruption may disturb re-methylation at the ICRs. Problems with ovulation are a common cause of female fertility problems and hormones (e.g. clomiphene citrate and human chorionic gonadotrophin) are often given to stimulate ovulation. Ovulation induction (OI) is also used to optimize the timing and collection of multiple oocytes for downstream ART procedures. It is possible that the use of such ovulation inducing hormones may result in improper establishment of key epigenetic marks (e.g. DNA methylation) in the germline during oogenesis, potentially resulting in immature oocytes and/or incomplete methylation patterns (Santos et al., 2010; Obata et al., 2011). The possible effect of OI on imprinting patterns has not been studied in humans.

We examined whether different methods of conception (e.g. IVF, OI and spontaneous) were associated with changes in the establishment of methylation marks at imprinted DMRs, which are known to be critical in embryonic and extra-embryonic development. In order to examine the effect of fertility treatments on the establishment of methylation marks, analyses of five ICRs were performed by bisulfite pyrosequencing in both the cord blood (embryonic) and the placenta (extra-embryonic). We analyzed the extent of methylation at the ICRs of the maternally methylated MEST, GRB10, KCNQ1, SNRPN, the paternally methylated H19 ICR and the maternally methylated IGF2DMR0, which is established somatically during embryonic development.

Materials and Methods

Study population

The data and biospecimen used in this study were collected between June 2007 and June 2009 at the Department of Obstetrics, Gynecology and Reproductive Biology at Brigham and Women's Hospital (BWH) in Boston, MA, USA, as a part of the Epigenetic Birth Cohort (EBC), which comprises 1941 mother–child dyads (Michels et al., 2011). Details of the study protocol have been described previously by Michels et al. (2011). Briefly, pregnant women completed a 2-page questionnaire and gave permission to have information abstracted from their pregnancy charts and to donate samples from umbilical cord and placenta for research purposes after detachment. The study protocol for the sample collection and for the data analysis was approved by the BWH Institutional Review Board (IRB).

We restricted our study population to non-Hispanic white individuals, since methylation has been reported to vary by race (Zhang et al., 2011) and to singleton births resulting from one implanted placenta. The participants self-reported their method of conception (spontaneous planned or unplanned, use of OI medication or in-vitro fertilization) on the questionnaire; if this information was not available from the questionnaire, the method of conception was abstracted from the medical charts. We selected 27 patients who reported having used OI and 59 patients who reported having conceived with IVF and for each participant who reported IVF we selected a spontaneous participant matched on age (±2 years). To increase efficiency, we matched these same spontaneous controls to participants who reported OI whenever possible but if an appropriate control was not available that had been previously matched to an IVF participant, a new control was selected.

Sample preparation

Genomic DNA was isolated from the buffy coat layer of the cord blood using the QIAamp DNA Blood Mini Kit (Qiagen, Valencia, CA, USA). Total RNA was isolated from whole cord blood with the PAXgene blood RNA kit (Qiagen) according to the manufacturer's protocol. For placenta tissue collection, a 2 cm incision was made in the amnion to access the placenta tissue. The samples (<1cm3) were taken at the placenta perimeter from the upper layer (nearest to the umbilical cord). Genomic DNA and RNA were extracted from fresh frozen placenta tissue with the QIAamp DNA Mini Kit and RNeasy Mini Kit (respectively) according to manufacturer's protocol (Qiagen). Genomic DNA was bisulfite treated using the EZ DNA Methylation Gold kit (Zymo Research, Irvine, CA, USA) according to the manufacturer's protocol (alternative protocol 2). The bisulfite treatments were performed in duplicate on every sample.

DNA methylation assays

Pyrosequencing was performed on bisulfite-converted DNA. Percent methylation was analyzed across CpG sites located within the following DMRs: GRB10, MEST, SNRPN, KCNQ1, H19 and IGF2DMR0. Methylation assays were designed with the PyroMark Assay Design Software 2.0 (www.qiagen.com) using sequences encompassing the CpG-rich islands, which have been previously described in the literature as the DMR and confirmed with UCSC Genome Browser website CpG island characterization. Bisulfite-converted DNA was mixed with 0.2 μM of each primer and amplified using the HotstarTaq plus Master Mix (Qiagen). Primers sequence and chromosomal locations for pyroassays can be found in Supplementary data, Table SI. The primers for IGF2DMR0 (Ito et al., 2008) and H19 (Guo et al., 2008) have been previously described. Each assay included a bisulfite conversion check to verify full conversion of the DNA. Methylation levels for all CpG sites were assessed using the Pyromark Q24 pyrosequencer (Qiagen). Values for each methylation assay were calculated by taking the average across CpG sites: 6 CpGs for GRB10, 9 CpGs for MEST, 7 CpGs for SNRPN, 9 CpGs for KCNQ1, 8 CpG sites for H19 and 6 CpGs for IGF2DMR0. In the case of SNRPN the fourth CpG site was excluded from analyses as this site exhibited variability in methylation values (this exclusion resulted in a total of 7 CpG sites being used for analysis). Each assay was validated with a methylation scale (0–100%), which was created from whole genome amplified DNA (representing hypomethylation), and DNA treated with CpG methyltransferase M.SssI (New England Biolabs, Ipswich, MA, USA; representing the expected hypermethylation). The methylation scales were tested in duplicate for each assay (an example scale pyrorun is shown in Supplementary data, Fig. S1). For each sample, PCRs were performed on both bisulfite treatments and was rerun if the difference between the two replicates exceeded two standard deviations based on the entire study population.

Overall expression analysis

cDNA was synthesized from the cord blood and placenta on a subset of samples from each conception group. cDNA was synthesized in duplicate, with negative controls (RT−) according to the manufacturer's protocol of the iScript™ cDNA Synthesis Kit (BioRad, Hercules, CA, USA). Quantitative PCR was used to measure the relative fold expression levels for the genes KCNQ1, SNRPN and H19, which were normalized to control housekeeping gene GAPDH and ALPHA-TUBULIN. Primer information is available in Supplementary data, Table SII. Reactions were performed using the FastStart SYBR Green master mix (Roche, Indianapolis, IN, USA) according to the manufacturer's protocol with gene-specific exon-spanning primers and were run on an Eppendorf Mastercycler® ep Realplex Thermal Cycler (Eppendorf, Hauppauge, NY, USA). Expression assays were performed in duplicate and the relative fold expression was calculated using ΔΔCT corrected for the amplification efficiency calculated from the standard curve of the each primer set [outlined in references (Pfaffl, 2001; Stahlberg et al., 2004)].

Statistical analyses

Differences in median percent methylation at each DMR between the spontaneous conception control group and the fertility treatment groups (OI or IVF) were not normally distributed and therefore compared using the Wilcoxon rank-sum test. The standard error was calculated from the mean of replicates. Spearman correlation coefficients between overall expression and methylation levels were calculated.

A 5% significance level was used. All statistical analyses were performed using SAS version 9.2 (SAS Institute Inc, Cary, NC, USA).

Results

The final study population included 61 spontaneous conception, 27 OI and 59 IVF mother–child dyads. Maternal and infant characteristics of the study population are provided in Table I. The mean maternal age (range) for each subgroup was 35.5 years (23–44) for spontaneous conception, 34.5 (25–43) for OI and 36.5 (24–44) for IVF (Table I). The mean infant birth weights across the three subgroups were 3469g (spontaneous), 3556g (OI) and 3354g (IVF), respectively.

Table I.

Maternal and infant characteristics of the study population by type of conception.

Spontaneous (n = 61) OI (n = 27) IVF (n = 59)
Maternal characteristics
 Age (years) 35.5 (4.7) 34.5 (4.6) 36.5 (4.5)
 Pre-pregnancy BMI (kg/m2) 24.9 (4.3) 26.4 (5.8) 24.0 (4.4)
Infant characteristics
 Female (%) 47.5 40.7 52.5
 Preterm (<37 weeks) (%) 1.6 0.0 6.8
 Birthweight (g) 3469 (462) 3556 (610) 3354 (625)
 Race/Ethnicity Non-Hispanic White
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Data represent means (standard deviation) unless otherwise indicated.

The anticipated values of DNA methylation for an imprinted DMR are 50% with one parental chromosome being fully methylated and the other not being methylated at all. The methylation assays performed in this study exhibited values that were close to the expected percent methylation for an imprinted DMR with a range between 32 and 59%. For each DMR, a similar methylation pattern across the CpG sites tested was seen in both the cord blood and placenta samples. The ranges of percent methylation in the placenta and cord blood are listed in Supplementary data, Table SIII. None of the DMRs examined in this study, displayed methylation levels above 60%, consistent with maintenance of imprinting.

Small but statistically significant differences in median methylation levels were observed comparing OI to spontaneous conceptions: in the placenta for H19 (40.2 versus 44.6% in OI versus spontaneous, P < 0.0001), in the cord blood for KCNQ1 (43.6 versus 42.3% in OI versus spontaneous P = 0.003) and in both the cord blood and the placenta for SNRPN (42.5 versus 40.4%, P = 0.047 and 43.2 versus 41.1%, P = 0.005, in OI versus spontaneous in the cord blood and placenta, respectively; Fig. 1 and Supplementary data, Table SIII). No statistically significant differences were observed in methylation levels for GRB10, MEST or IGF2DMR0 in the cord blood or placenta, or for H19 in the cord blood and KCNQ1 in the placenta between the OI and spontaneous conception subgroups.

Figure 1.

Figure 1

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Percent methylation across six imprinted DMRs in the placenta (A) and the cord blood (B) reveals no large difference according to conception modes. The mean methylation values were calculated across the CpG sites of GRB10 ICR, MEST ICR, H19 ICR, IGF2DMR0, SNRPN ICR and KCNQ1 ICR. Samples were analyzed with the OI group (shown in blue), and IVF group (shown in red) compared with the spontaneous (Spon) group (shown in green). Individual values are depicted by x, with the median of each group shown with a horizontal black line. Statistically significant differences between each values are denoted with an asterisk (*) and represent P-values ≤ 0.05. Expected levels of methylation are indicated with dotted line.

To investigate whether IVF treatment altered methylation patterns, the same analysis was performed across the six DMRs comparing IVF to spontaneous conception in both the cord blood and the placenta. Similarly to the OI group, small but statistically significant differences in median methylation levels were detected for H19 (43.4 versus 44.7% in IVF versus spontaneous, P = 0.01) and SNRPN (42.1 versus 40.4% in IVF versus spontaneous, P = 0.008) in the placenta and KCNQ1 (42.9 versus 42.3% in IVF versus spontaneous, P = 0.02) in the cord blood. Additionally, in the IVF group, MEST had lower methylation levels in the placenta (48 versus 51.4% in IVF versus spontaneous, P < 0.0001). No further significant differences were identified in the additional DMRs tested (Fig. 1 and Supplementary data, Table SIII).

When stratifying by maternal pregnancy BMI, we observe that methylation levels of the DMRs (MEST, SNRPN and KCNQ1) in the cord blood and placenta were statistically significantly different between OI and spontaneous (or IVF and spontaneous) among women with the BMI <25 group, while no significant differences were observed in the groups with BMI 25 to <30 and BMI 30+. Statistically significant differences for H19 were observed in the BMI <25 and BMI 25 to <30 groups. However, most of our participants had a BMI <25 and our numbers are quite low for the two higher BMI categories resulting in less power to detect a significant difference in these strata (Supplementary data, Table SV). Additional analyses of the methylation levels stratified by the sex of the infant did not reveal any sex-specific pattern (Supplementary data, Table SVI).

To assess whether the small but statistically significant differences in methylation levels of H19, KCNQ1 and SNRPN may affect the transcription levels of the genes corresponding to these control elements, overall expression was analyzed. Quantitative PCR on H19, KCNQ1 and SNRPN was performed using the cord blood and placenta on a subset of individuals with high and low methylation levels in each conception group (H19, n = 19 placenta, KCNQ1, n = 24 cord blood, SNRPN, n = 32 placenta and n = 33 cord blood). Expression levels were normalized to the housekeeping gene GAPDH and ALPHA-TUBULIN (data not shown) relative fold expression was calculated according to ΔΔCT corrected for primer set amplification efficiencies (Pfaffl, 2001; Stahlberg et al., 2004). Differences in methylation levels did not translate into differences in overall gene expression (Fig. 2). The methylation level ranges were 36–53% for H19 placenta samples, 32–55 and 36–47% for SNRPN (placenta and cord blood, respectively) and 37–50% for KCNQ1 cord blood samples. The relative fold expression levels for each subgroup ranged from 0.98–7.36 for H19, 0.68–8.99 and 2.32–20.4 for SNRPN (placenta and cord blood, respectively) and 2.12–7.06-for KCNQ1. The expression levels assessed for H19 were significantly correlated with their corresponding methylation levels while those for SNRPN, and KCNQ1 were not (Supplementary data, Table SIV). Allele-specific expression was not examined in this study.

Figure 2.

Figure 2

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Small changes in methylation levels are not linked to overall gene expression. Expression graphs (A1–D1) show quantitative PCR analysis of H19 in the placenta (A1), SNRPN in the placenta (B1) and the cord blood (D1) and KCNQ1 in the cord blood (C1) values normalized to GAPDH. ICR methylation levels of the corresponding samples from spontaneous (SPON, shown in green), OI (shown in blue) and IVF (shown in red) groups are shown in graphs A2–D2. Expression experiments were performed in duplicate with error bars showing the calculated standard error of the mean for the replicate values [s.e.(m)].

Discussion

We examined methylation at six imprinted DMRs which are known to be important in embryonic and extra-embryonic development. Disruption in the methylation pattern of these ICRs has previously been described in patients with imprinting disorders. Overall, we found that the methylation levels across the ICRs of GRB10, MEST, H19, SNRPN and KCNQ1 as well as IGF2DMR0 were not disrupted by fertility treatments (IVF and OI) in the cord blood and placenta in our birth cohort. We observed statistically significant differences in certain ICRs, but these small differences in methylation did not relate to the overall transcriptional levels of the genes adjacent to the ICRs for KCNQ1 and SNRPN. Only for H19 were the marginally lower mean methylation levels in placentas from infants conceived with OI and IVF correlated with expression levels. Whether these differences have any functional implications needs to be examined in future studies with larger samples size permitting the assessment of allele-specific expression and thus differences in imprinting patterns. At large our analyses suggests that both small changes in methylation (0–3%) across our conception subgroups as well as considerable variation in methylation (over 20%) within groups do not correlate with the gene expression activity of the genes we examined. We were interested in identifying a methylation level threshold that must be maintained for proper gene expression and function; however, we were not able to determine such level.

Our results are in agreement with large prospective epidemiologic studies, which did not observe an increase in the frequency of imprinting disorders in ART conceptions (Lidegaard et al., 2005; Bowdin et al., 2007; Doornbos et al., 2007). In a Danish cohort consisting of 442 349 spontaneous conception group and 6052 IVF children, Lidegaard et al. (2005) examined the prevalence of various diseases and no reported cases of PWS, BWS or SRS were observed within the IVF group, while three cases of PWS and two cases of SRS were found in the non-IVF group. In another cohort consisting of 1524 children conceived with ART (in Republic of Ireland and Central England), Bowdin et al. (2007) reported one case of BWS and no cases of AS. Both studies concluded that the risk of imprinting disorders among ART conceptions is small and highlighted the need for large observational studies to address such questions.

Previous case–control studies have not reported important differences in methylation at imprinted loci among ART conceptions (such as IVF and ICSI) compared with spontaneous conceptions (Katari et al., 2009; Tierling et al., 2010). Tierling et al. (2010) investigated methylation levels at 10 imprinted DMRs (such as MEST, H19, SNRPN, KCNQ1 and GRB10) in a cohort consisting of 77 ICSI, 35 IVF and 73 spontaneous conceptions and also observed no disruption of methylation patterns. The sole statistically significant difference in methylation levels they found was at MEST among the IVF group in the cord blood and aminoic chorion tissue. We also found statistically significant differences at MEST in the IVF conception group, yet we only observed this in placenta and not in the cord blood. In a methylation microarray performed by Katari et al. on the cord blood and placenta of 10 ART and 13 spontaneous conceptions, small quantitative differences in methylation associated with ART were observed for both imprinted regions and non-imprinted regions (Katari et al., 2009). These authors concluded that higher mean methylation at CpG sites in the cord blood and lower methylation in the placenta among IVF compared with spontaneous conceptions were seen, however, a statistically significant disruption of methylation was not described. While microarrays provide large CpG coverage spanning the genome, relying on one CpG site may not provide the best representation for the methylation levels at a regulatory element (e.g. promoter or ICR).

Case–control studies have linked imprinting disorders (e.g. BWS and AS) to ART conceptions, however, these studies have generally relied on limited sample sizes. Moreover, these studies could not address any effect of the underlying fertility problem itself on these childhood disorders (Fig. 3). The occurrence of epigenetic alterations in the germline in either parent may result in sub/infertility. Unfortunately, it will be difficult if not impossible to separate any effects of primary infertility on methylation pattern of the child from that of ART since pregnancy is only accomplished with the help of OI or IVF.

Figure 3.

Figure 3

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Three possible paths that may induce imprinting disorders in children conceived with fertility treatments. (A) Imprinting errors during OI induced by hormone stimulation during critical oocyte developmental time points. (B) With IVF treatment possible alterations may be introduced by culture conditions and the manipulation of gametes. (C) Outlines the possibility of an underlying epigenetic errors originating from one or both of the parental germlines present in the gametes, which may be linked to the infertility itself.

The establishment of germline methylation marks in the oocyte, which for ICRs is the final methylation state thought to be maintained, is known to be critical for embryogenesis (Monk and Salpekar, 2001; Obata and Kono, 2002). In previous studies with animal models and human oocytes, hormone stimulation caused disruption of methylation marks of imprinted genes (e.g. H19 and MEST; Sato et al., 2007; Market-Velker et al., 2010), which altered expression and induced LOI (e.g. H19 and Snrpn; Fortier et al., 2008). Perhaps hormone stimulation brings forth the presence of oocytes which are epigenetically immature, carrying improper marks, resulting in LOI (Obata et al., 2011). However, we did not observe a disrupted methylation pattern at the imprinted loci studied (GRB10, MEST, H19/IGF2, SNRPN and KCNQ1) and it is possible that our OI group represents examples of successful conception due to mature oocytes with the proper epigenetic marks which may be preferentially fertilized. Downstream ART procedures for collection and culturing of oocytes following hormone stimulation have long been scrutinized for potentially introducing imprinting errors. Studies of human oocytes cultured at different stages have observed the occurrence of incorrect methylation patterns at imprinted loci (e.g. H19 and KCNQ1; Borghol et al., 2006; Geuns et al., 2007). In mouse studies examining the effects different culture techniques (e.g. differences in culture medium) may have on the development of culture oocytes, embryos and extra-embryonic tissues at different gestational time points have suggested differences in DNA methylation at imprinted loci (e.g. H19, Snrpn and Peg3) depending on the culture conditions (Doherty et al., 2000; Mann et al., 2004). While in vitro or animal models may not accurately reflect human biology, these studies make important contributions to understanding human developmental biology.

Limitations of our study include the limited sample size, lack of information on exact OI medication and culture medium for the IVF procedures, and underlying reasons for infertility among OI and IVF patients. To our knowledge this is the first methylation analysis of imprinted DMRs of the offspring of women treated with OI. The OI conception group provided the unique opportunity to separate the possible effects of manipulating the ovum at critical points in the development of epigenetic marks from other effects of the ART procedures. Allele-specific expression studies on a larger cohort would provide interesting additional insight into LOI, which may not necessarily be linked to any changes in methylation at imprinted DMRs. Larger fertility treatment subgroups will be necessary to ensure a sufficient number of informative individuals [heterozygous for Single-nucleotide polymorphism (SNP)] for proper LOI analyses. Interestingly, several case studies have reported solely methylation data while no allele-specific gene expression was examined which would be necessary to define LOI (Orstavik et al., 2003; Rossignol et al., 2006; Gomes et al., 2007; Kagami et al., 2007; Douzgou et al., 2008).

Even though disruption in imprinted methylation patterns were not identified, we cannot rule out LOI as allele-specific expression was not investigated in this study as an insufficient number of participants had informative SNP for the genes studied nor can we exclude the possibility that other epigenetic mechanisms may be affected by OI and/or IVF, such as histone modifications, chromatin structure, transcriptional regulation and the nucleosome composition. As growing research using birth cohorts reveals limited adverse effects of reproductive technology on human development, the research focus should shift to understand how ART may influence long-term health outcomes as these children age.

Supplementary data

Supplementary data are available at http://humrep.oxfordjournals.org/.

Authors' roles

R.C.R., H.R.H., K.B.M.: conceived and designed the experiments. R.C.R.: performed the experiments. H.R.H., R.C.R.: analyzed the data. K.B.M.: contributed reagents/materials/analysis tools. R.C.R., H.R.H., K.B.M.: manuscript drafting.

Funding

This project was supported by the Milton Fund, Harvard University (P.I.: K.B.M) and by Public Health Research Grant 5R21CA128382 from the National Cancer Institute, National Institutes of Health (P.I.: K.B.M.). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Conflict of interest

No conflict of interests to declare.

Supplementary Material

Supplementary Data
supp_27_7_2208__index.html (1KB, html)

Acknowledgements

We gratefully acknowledge the EBC participants. We thank Joelle Perkins, Eliza Gardiner, Michelle Peters and Seema Sannesy for help with data and specimen collection. We would also like to thank Sonia Hernández-Diaz and especially the members of the Michels' lab for helpful discussions of the project: Ludovic Barault, Amy Non, Alexandra Binder and Timothy Barrow.

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