General imprinting status is stable in assisted reproductionconceived offspring

Chun Feng, M.D.,a Shen Tian, Ph.D.,a Yu Zhang, M.D.,a Jing He, M.D.,b Xiao-Ming Zhu, Ph.D.,a Dan Zhang, Ph.D.,a Jian-Zhong Sheng, Ph.D.,c and He-Feng Huang, M.D.a,d
a Department of Reproductive Endocrinology and b Department of Obstetrics, Womens Hospital, and c Department of Pathophysiology, School of Medicine, Zhejiang University, and d Key Laboratory of Reproductive Genetic, Ministry of Education, Hangzhou, Zhejiang, Peoples Republic of China

Objective: To evaluate whether the genomic imprinting status of assistant reproductive technology (ART) conceived offspring is stable. Design: Prospective clinical observational study. Setting: In vitro fertilization (IVF) center, university-afliated teaching hospital. Patient(s): Sixty ART-conceived babies (30 IVF and 30 intracytoplasmic sperm injection [ICSI]) and 60 naturally conceived babies. Intervention(s): Collection of umbilical cord blood and peripheral blood samples. Main Outcome Measure(s): Expression prole was examined by microarray and real-time reverse-transcription polymerase chain reaction (PCR), allele-specic expression was studied by direct sequencing after PCR, and DNA methylation status was investigated by sodium bisulte sequencing. Result(s): Hierarchic clustering demonstrated no obvious clustering between the ART- and naturally conceived offspring, suggesting similar genomic imprinting expression between the two groups. Three differentially expressed genes were identied in ART-conceived offspring, with PEG10 and L3MBTL up-regulated and PHLDA2 down-regulated. Allele-specic expression of the differentially expressed imprinted genes was maintained in the majority of the ART- and naturally conceived offspring. However, in one ICSI case, monoallelic expression of L3MBTL was disrupted and all CpGs were completely unmethylated. These were not inherited from the parents. Conclusion(s): The global prole of imprinting is stable in children conceived through ART. However, imprinting of a few specic imprinted genes may be vulnerable in a fraction of ART-conceived children. (Fertil Steril 2011;96:141723. 2011 by American Society for Reproductive Medicine.) Key Words: Assisted reproductive technology (ART), epigenetic, genomic imprinting, methylation, offspring

Since the application of in vitro fertilization (IVF) about 30 years ago, >4,000,000 babies have been conceived through assisted reproductive technology (ART) worldwide (1); >200,000 ART babies are born worldwide each year (2). ART-conceived offspring are a substantial part of the general population, and this proportion continues to grow rapidly. The health of the ART-conceived offspring has become increasingly important, because guaranteeing the health of these children offers potential benets to the world. There are mounting concerns about the health of ART-conceived offspring. Increasing risks associated with ART have been reported,
Received May 27, 2011; revised and accepted September 19, 2011; published online October 8, 2011. C.F. has nothing to disclose. S.T. has nothing to disclose. Y.Z. has nothing to disclose. J.H. has nothing to disclose. X.-M.Z. has nothing to disclose. D.Z. has nothing to disclose. J.-Z.S. has nothing to disclose. H.-F.H. has nothing to disclose. C.F. and S.T. contributed equally to this work. Supported by grants from the National Basic Research Program of China (973 program; no. 2012CB944901), the Specialized Research Fund for the Doctoral Program of Higher Education of China (no. 20100101120155), and the National Natural Science Foundation of China (nos. 81170310, 81070541, 81170620, 81170587, and 30901604). Reprint requests: He-Feng Huang, M.D., Department of Reproductive Endocrinology, Womens Hospital, School of Medicine, Zhejiang University, 2 Xueshi Road, Hangzhou, Zhejiang 310006, Peoples Republic of China (E-mail: huanghefg@hotmail.com).

including spontaneous abortion, low or very low birth weight, small for gestational age, major malformations, cerebral palsy, and so on (3). Imprinting disorders, including Beckwith-Wiedemann syndrome and Angelman syndrome, have recently attracted considerable attention regarding the safety of ART (4). Different case series and case-control studies have consistently suggested the increased risks of imprinting disorders in pregnancies after ART (46). Genomic imprinting is an epigenetic phenomenon of the nonequivalent expression of maternally and paternally derived alleles in an individual. Genomic imprinting affects several dozen mammalian genes, and one allele is usually suppressed during development. Imprinted genes tend to affect the growth of the embryo in the womb and the behavior of the offspring after birth. Aberrant imprinting disturbs development and causes various disease syndromes (7). DNA methylation is the key molecular mechanism in imprinting establishment and maintenance. The accurate regulation of imprinted gene expression necessitates precise epigenetic regulation through DNA methylation at differentially methylated regions (DMRs) (8, 9). ART procedures, including hormone-induced superovulation, in vitro fertilization (IVF), and embryo culture, involve the manipulation of gametes and embryos during gametogenesis, fertilization, and preimplantation stages, during which the methylation reprogramming of imprinted genes takes place (10, 11). Therefore, ART is likely to induce the inappropriate methylation of imprinted genes and disrupt imprinting. Furthermore, epigenetic

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Fertility and Sterility Vol. 96, No. 6, December 2011 Copyright 2011 American Society for Reproductive Medicine, Published by Elsevier Inc.

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alterations that occur during gametogenesis are not corrected during postimplantation development and are associated with aberrant imprinted gene expression and phenotypic abnormalities (12). Because disruption of the allele-specic expression of imprinted genes is associated with various human genetic diseases, certain types of cancers, and a number of neurologic disorders (1315), the assessment of the imprinting status of ART-conceived children is an urgent requirement. Experimental studies performed in oocyte, peripheral blood, placenta, and umbilical cord blood samples indicate that superovulation, manipulation, and in vitro embryo culture may lead to disrupted patterns of DNA methylation and gene expression (1618). In contrast, several other studies on humans found no detectable alterations in imprinted gene expression, indicating that the imprints are stably maintained during the ART procedure (1921). Owing to the discrepancies between studies, the correlation between ART procedures and the expression of imprinted genes in offspring remains unclear. To elucidate the imprinting status of offspring conceived through ART, two main questions need to be addressed: 1) Is there any evidence of imprinting variation in ART-conceived offspring? and 2) To what extent does the epigenetic disruption of imprinted genes occur in ART-conceived offspring? To answer these questions, we carried out a general mRNA expression survey of imprinted genes in both ART- and naturally conceived offspring using Affymetrix oligonucleotide microarray as an initial step in estimating the risks of imprinting alterations after ART. The imprinted genes differentially expressed between the two groups were then selected as candidates for real-time reverse-transcription polymerase chain reaction (RT-PCR) validation. The monoallelic expression of the candidate genes were examined after the validation of the expression variations, and the correlation between DNA methylation and imprinting instability was investigated.

rally. In the control group, RNA samples from every two children were pooled for one chip assay. Total RNA was isolated as described previously and further puried with RNeasy Mini kit (Qiagen). The labeled cRNA was subsequently hybridized to human U133 Plus 2.0 Genechip (Affymetrix) according to standard protocols provided by the manufacturer (Supplemental Table 2, available online at www.fertstert.org). Expression levels were normalized using robust multichip average (RMA). Unsupervised hierarchic clustering was performed with Cluster 3.0 by centroid linkage and visualized with Treeview. We performed signicance analysis of microarrays (SAM, Stanford University) (22) and t test using RMA data to search the imprinted genes differently expressed between ART- and naturally conceived offspring.

Real-Time Quantitative RT-PCR

Real-time RT-PCR was performed for 80 samples, with 40 samples from each group. The 16 samples participated in the above-mentioned microarray analysis were also included. Total RNA was extracted as described previously. Then a standard real-time RT-PCR assay was carried out using Sybr PrimeScript RT-PCR Kit (Takara) on an ABI 7500 system (Applied Biosystems; the primers and the cycling conditions are provided in Supplemental Table 3, available online at www.fertstert.org). The relative gene expression levels were then calculated using the 2DDCt method (23).

Identication of Genomic Polymorphisms

Sixty ART-conceived and 60 naturally conceived children were involved in the genomic polymorphism analysis, including the 80 samples included in the previous RT-PCR analysis. Genomic DNA was extracted as described previously. PCR was carried out on a Peltier thermal cycler PTC200 (MJ Research; the primers and the cycling conditions are provided in Supplemental Table 4, available online at www.fertstert.org). All PCR products were subjected to direct sequencing using a BigDye terminator cycle sequencing kit (ABI).

RT-PCR for Analyzing Allele-Specic Expression

The heterozygous cases were picked up for allele-specic expression analysis. First-strand cDNA was synthesized from 500 ng total RNA with oligo dT and random hexamers using PrimeScript RT reagent Kit (Takara, Dalian, China). PCR was carried out as described above (the primers and the cycling conditions are provided in Supplemental Table 5, available online at www.fertstert.org). All RT-PCR products were directly sequenced using a BigDye terminator cycle sequencing kit (ABI).

MATERIALS AND METHODS Patients

A total of 120 babies were recruited in this study, including 60 babies conceived through ART (30 IVF and 30 intracytoplasmic sperm injection [ICSI]) as the study group and 60 babies conceived naturally as the control group. The inclusion criteria and the main characteristics of the pregnancies are summarized in Supplemental Table 1 (available online at www.fertstert.org). All specimens and their corresponding clinical information used in this study were procured according to the Institutional Review Board protocols. Umbilical cord blood samples (10 mL) were collected during cesarean section, anticoagulated with heparin, put immediately on ice, and transfered to the laboratory within 30 minutes for blood processing. Lymphocytes were isolated by gradient centrifugation with lymphocyte separation medium (Hengxin) and stored at 80 C.

Bisulphite Genomic Sequencing

One microgram of genomic DNA was denatured by treatment with NaOH and modied with sodium bisulte using the CpGenome DNA modication kit (Chemicon International) according to the manufacturers instructions. The methylation patterns of all sequences were determined for one strand (the primers and the cycling conditions are provided in Supplemental Table 6, available online at www.fertstert.org). The PCR products were subsequently cloned into the pMD19-T vector (Takara). Randomly picked clones were sequenced using the universal sequencing primer M13.

Preparation of RNA and DNA

Total RNA was extracted from umbilical cord blood lymphocytes with the use of Trizol Reagent (Invitrogen) followed by DNase I treatment (Takara) according to the manufacturers instructions. Purity and RNA integrity were evaluated by absorbance at 260 nm and 280 nm and by electrophoresis through agarose gels with the characteristic 2:1 ethidium bromide staining ratio of 28S to 18S RNA. The qualied samples were stored at 80 C. Genomic DNA was extracted from umbilical cord lymphocytes with the use of blood genome DNA extraction kit (Takara) according to the manufacturers instructions. The DNA was precipitated in 70% ethanol, resuspended in hydration solution, and stored at 80 C.

RESULTS Expression Proling of the Imprinted Genes of the ARTand Naturally Conceived Offspring by Microarray
Approximately 70 imprinted genes have been identied in the human genome so far (http://igc.otago.ac.nz/home.html). We determined the gene expression prole in 16 blood samples, eight from each group, with the use of the Affymetrix U133 Plus 2.0 Genechip, which covers 65 unique imprinted genes (Supplemental Table 2), corresponding to 179 probe sets. Given the hypothesis that imprinting was prone to disruption after ART procedures, the blood samples of the ART- and naturally conceived children could be distinguished by the expression of the imprinted genes. We therefore performed an Vol. 96, No. 6, December 2011

Microarray Analysis
The study group consisted of four children conceived through IVF and four through ICSI. The control group consisted of eight children conceived natu-

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Imprinting is stable in ART offspring

FIGURE 1
Expression prole of imprinted genes in umbilical cord blood specimens of ART- and naturally conceived offspring by microarray and realtime RT-PCR. (A) Unsupervised hierarchic clustering of imprinted genes. Rows represent genes, and columns represent samples. Colored pixels capture the magnitude of the response for any gene, where red, green, and black represent induction, repression, and no change, respectively. There was no obvious clustering of the two groups. N naturally conceived offspring; IVF IVF-conceived offspring; ICSI ICSI-conceived offspring. (B) Real-time RT-PCR for imprinted genes differentially expressed. Each column and bar represents the mean and standard error, respectively. *P< .05. (C) Microarray and real-time RT-PCR for the three differentially expressed imprinted genes. The y-axis and x-axis represent the RT-PCR and the microarray results, respectively. For the real-time RT-PCR results, only the data of 16 samples involved in the microarray assay (four IVF-, four ICSI-, and eight naturally conceived children) are shown.

Feng. Imprinting is stable in ART offspring. Fertil Steril 2011.

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TABLE 1
Genomic polymorphisms and imprinting of PEG10, L3MBTL, and PHLDA2 genes in umbilical cord blood of ART- and naturally conceived offspring. Genomic polymorphisms Gene PEG10 A/C A/A C/C L3MBTL C/T C/C T/T PHLDA2 C/T C/C T/T ART Naturally Loss of imprinting ART Naturally

FIGURE 2
Imprinting of PEG10, L3MBTL, and PHLDA2 genes of ART- and naturally conceived offspring. The left and right columns represent the sequencing result of gDNA and cDNA, respectively. (A, C, E) Results of children conceived naturally. (B, D, F) Results of children conceived through ART.

22 (36.7%) 3 (5%) 35 (58.3%) 18 (30%) 40 (66.7%) 2 (3.3%) 8 (13.3%) 50 (83.3%) 2 (3.3%)

15 (25%) 1 (1.7%) 44 (73.3%) 15 (25%) 45 (75%) 0 8 (13.3%) 52 (86.7%) 0

0/22 1/18

0/15 0/15

Note: The genomic polymorphisms of PEG10, L3MBTL, and PHLDA2 were investigated in 60 ART-conceived offspring and 60 naturally conceived offspring. A/C, C/T genomic umbilical blood DNA is heterozygous for the A/C and C/T alleles, respectively; A/A, C/C, T/T genomic umbilical blood DNA is homozygous for the A, C, and T alleles, respectively; ART assisted reproduction technologyconceived offspring; Naturally naturally conceived offspring. Feng. Imprinting is stable in ART offspring. Fertil Steril 2011.

unsupervised analysis using all 179 probe sets. However, clustering analysis showed that there was no obvious clustering between the two groups (Fig. 1A), indicating that the expression of the imprinted genes was not signicantly affected by ART. To further assess the accuracy of the microarray and to identify the differentially expressed imprinted genes between the ART and the control groups, we used SAM and t test to search imprinted genes with expression variation between ART- and naturally conceived offspring. Because only a small number of individuals were involved in the microarray assay, we assumed that there could be many false-positive and false-negative results at individual genes. To limit false-positive differences, the differentially expressed genes were conrmed with real-time RT-PCR. To decrease false-negative differences, we used the criteria of q-value < 43.07% (SAM) and P<.1 (t test), rather than 5% and .05, respectively. The top nine discriminatory imprinted genes were selected as initial candidates to be evaluated by real-time RT-PCR (Supplemental Table 7, available online at www.fertstert.org).

Feng. Imprinting is stable in ART offspring. Fertil Steril 2011.

Identication of Differentially Expressed Imprinted Genes Between ART- and Naturally Conceived Offspring by RealTime RT-PCR
The initially identied candidate genes were assayed by real-time RT-PCR on a sample set of 40 ART-conceived and 40 control offsprng. The relative gene expression levels were calculated using the 2DDCt method (23), and a t test was performed subsequently. The expression levels of PEG10 (P.018) and L3MBTL (P.000) in ART-conceived offspring were signicantly higher than those in naturally conceived ones, whereas the mRNA level of PHLDA2 (P.000) was lower in ART-conceived offspring compared with the control offspring. There were no signicant Feng et al. Imprinting is stable in ART offspring

differences in the mRNA levels of the other six imprinted genes between ART- and naturally conceived offspring specimens (Fig. 1B). The 16 samples involved in the microarray assay were also included in the real-time RT-PCR validation. The microarray and real-time RT-PCR results for PEG10, L3MBTL, and PHLDA2 of the 16 samples were shown in Figure 1C. We found that the microarray and RT-PCR results were generally in accordance with each other. Aas sample size increased to 40 for each group, statistically signicant differences were observed in the expression of these three genes by real-time RT-PCR.

Imprinting of PEG10, L3MBTL, and PHLDA2 Genes

Because of the signicantly increased PEG10 and L3MBTL expression and decreased PHLDA2 expression found in ARTconceived offspring, we then asked if these changes might result from a loss of imprinting (LOI) and therefore further investigated the imprinting status of PEG10, L3MBTL, and PHLDA2. Because of the rare incidence of LOI, another 20 ART-conceived and 20 naturally conceived children were included in this imprinting assay, in Vol. 96, No. 6, December 2011

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FIGURE 3
Methylation prole of L3MBTL differentially methylated region. Methylated and unmethylated CpGs are shown as solid and open circles, respectively. (A) All of the CpGs were moderately methylated in one naturally conceived child. (C) The 15th CpG showed hypomethylation in one IVF case. (E) All of the CpGs were completely unmethylated in one ICSI case. (B, D, F) Summary of bisulte-sequencing PCR (BSP) results of naturally, IVF-, and ICSI-conceived children. (G) BSP results of the parents of the child with LOI.

Feng. Imprinting is stable in ART offspring. Fertil Steril 2011.

addition to the 80 samples involved in the real-time RT-PCR. Genomic DNA specimens from ART- and naturally conceived offspring were rst analyzed for heterozygosity using singlenucleotide polymorphisms (SNPs) in the exons of PEG10, L3MBTL, and PHLDA2 genes. Among the 60 blood samples from the naturally conceived offspring, we observed 15 heterozygous cases for the SNP of PEG10, 15 heterozygous cases for the SNP of L3MBTL, and eight heterozygous cases for the SNP of PHLDA2. The SNPs of the 60 blood samples derived from the ART-conceived offspring revealed 22 heterozygous cases of PEG10, 18 cases of L3MBTL, and eight cases of PHLDA2 (Table 1). Illustrations of PEG10, L3MBTL, and PHLDA2 polymorphisms are provided in Figure 2. Fertility and Sterility

The PEG10 sequencing of the corresponding umbilical cord blood cDNA revealed entirely monoallelic expression in 37 heterozygous cases, and the imprinting of PEG10 was not disrupted in both naturally and ART-conceived offspring. The L3MBTL sequence analysis of the corresponding umbilical cord blood cDNA revealed that in the majority (17/18) of the ART-conceived children monoallelic expression of L3MBTL was maintained, and in one (1/18) of the heterozygous samples a biallelic expression was observed. In the naturally conceived offspring, however, no biallelic expression was detected. Figure 2 shows the monoallelically expressed L3MBTL in the naturally conceived offspring blood and the LOI of L3MBTL in the ART-conceived offspring. Sixteen heterozygous samples of PHLDA2 were screened for their imprinting status in

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total. The sequencing of the corresponding umbilical cord blood cDNA revealed entirely biallelic expression in both ART- and naturally conceived offspring (Table 1; Fig. 2).

DNA Methylation Status of L3MBTL

To determine whether the LOI of L3MBTL is due to CpG methylation variation, we examined the DNA methylation status of L3MBTL in six naturally conceived, six IVF-conceived, and six ICSI-conceived children, including the child with LOI of L3MBTL. Human L3MBTL gene contains a CpG island with a length of 112 bp, an overall G C content of 68%, and CpGobs/ CpGexp of 0.62. We investigated a 310-bp-long fragment carrying this CpG island with 20 CpG nucleotides in the reported DMR (24) by sodium bisulte sequencing. Figure 3B, 3D, and 3F show that the mean methylation fractions of L3MBTL in the three groups were approximately 50%, 50% and 40%, respectively. As a whole, the methylation fractions were quite similar among the three groups, and all of the CpGs were moderately methylated. However, minor methylation variation did exist individually. In the naturally conceived offspring, all of the CpGs were moderately methylated (Fig. 3A). However, in the ICSI case which underwent LOI, all of the CpGs were completely unmethylated and monoallelic expression was disrupted (Fig. 3E). In one of the IVF cases, the 15th CpG showed hypomethylation, although monoallelic expression was maintained (Fig. 3C). To clarify whether the loss of methylation originated from the parents or arose de novo, the peripheral blood of the parents was collected for bisulte-sequencing PCR (BSP) of L3MBTL. No hypomethylation of L3MBTL was observed, and all of the CpGs were moderately methylated in the peripheral blood samples of both the mother and the father (Fig. 3G).

DISCUSSION
In this study, the mRNA expression level, allelic-specic expression, and DNA methylation of imprinted genes in umbilical cord blood samples were investigated to elucidate the general imprinting status in offspring conceived through ART. The main conclusions are as follows: 1) The global imprinted gene expression prole in the ART-conceived offspring is similar to that of naturally conceived ones, except for the PEG10, L3MBTL, and PHLDA2 genes; 2) the imprinting of a small part of the imprinted genes is prone to disruption in a fraction of the ART-conceived children, indicating the individual and gene variability of imprinting vulnerability; and 3) the loss of imprinting in ART-conceived children can be partially attributed to dysregulated DNA methylation. These ndings addressed the global prole of imprinting safety in children conceived through ART and may facilitate further research through the identication of the most vulnerable imprinted genes and the potential mechanisms. Using microarray analysis and real-time RT-PCR, we investigated the expression proles of most of the known human imprinted genes in the umbilical cord blood samples of children conceived through ART and naturally. No obvious clustering between the two groups was observed, an indication of the similarity of the expression prole of the imprinted genes of the ART- and naturally conceived offspring. To decrease false-negative differences, the top nine discriminatory imprinted genes were selected as initial candidates to be evaluated by real-time RT-PCR. Only three imprinted genes, namely, PEG10, L3MBTL and PHLDA2, revealed altered expressions in ART-conceived children. The expression is stable for the majority of the imprinted genes (For the importance and Feng et al. Imprinting is stable in ART offspring

function of PEG10, L3MBTL, and PHLDA2, refer to the Supplemental Discussion, available online at www.fertstert.org). To investigate the possibility that the expression variation of PEG10, L3MBTL, and PHLDA2 might result from LOI, we assessed the allele-specic expression of the three imprinted genes in ART- and naturally conceived offspring. The PEG10 sequencing revealed entirely monoallelic expression in both naturally and ART-conceived children. The L3MBTL sequencing revealed that in the majority of the naturally and ART-conceived children, monoallelic expression of L3MBTL was maintained, whereas a biallelic expression was observed in one ICSI-conceived child. This is consistent with the imprinting stability previously observed in human embryonic stem cells (25). The imprinting status of two maternally methylated regions (KvDMR1 and PEG1) and one paternally methylated region (H19/IGF2 DMR) was analyzed using BSP in our laboratory, and no signicant imprint variability at these DMRs was detected (26). These ndings suggest that the global expression prole of the imprinted genes in ART-conceived offspring is similar to that of the naturally conceived ones. However, such interpretations should be treated with caution, because the size of the samples analyzed in the present study is too small to exclude rare imprinting disorders. The single ICSI-conceived child with the biallelic expression of L3MBTL indicates that the monoallelic expression of a small part of the imprinted genes can be vulnerable to disruption in a fraction of the ART-conceived children. A series of reports on epigenetic instability in sheep embryos (27), mouse embryos (28), mouse embryonic stem cells (29), and human embryonic stem cells (30) have been published. In the present study, one ART-conceived child exhibited LOI at the L3MBTL gene, whereas most of the imprinted genes maintained monoallelic expression in most of the ARTconceived children. This result suggests gene-specic differences and individual variation in the stability of imprinted loci. An allele-specic expression study in human embryonic stem cells demonstrated generally monoallelic gene expression, but during prolonged passage one cell line became biallelic in H19 (31), supporting the assumption on the gene and individual variability of susceptibility to imprinting perturbation. PHLDA2 exhibited biallelic expression in all of the umbilical blood samples, indicating that PHLDA2 is not imprinted in umbilical blood. The imprinting status of PHLDA2 varies among different human organs. It is reported to be imprinted in placenta, liver, intestine, limb, and adrenal gland, whereas biallelic expression is observed in brain, adult blood, kidney, heart, and testis (3234). The results of this study serve as a supplement to the earlier reports. Because one ART child exhibited LOI at L3MBTL, and DNA methylation is a crucial mechanism of imprinting regulation (11), we examined the CpG methylation status of L3MBTL. The mean methylation fractions were quite similar among the three groups when six cases in each group were taken as a whole, and all of the CpGs were moderately methylated, suggesting that there is no obvious increase of methylation variation in ART-conceived offspring. This result is in accordance with those of the earlier studies which demonstrated no signicant methylation differences in a single DMR in 92 healthy ICSI cases (19) and in ten DMRs in 105 ART-conceived children (21). When the cases were considered individually, however, minor methylation variation did exist. In the ICSI-conceived child with biallelic expression, all of the CpG islands were completely unmethylated. In one IVF-conceived child who maintained monoallelic expression, one CpG site was hypomethylated. In all of the naturally conceived cases and the other ART-conceived cases, the CpG islands of L3MBTL were moderately methylated. It appears that the LOI of L3MBTL may be partly attributed to aberrant hypomethylation, but Vol. 96, No. 6, December 2011

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hypomethylation in a single CpG site could not disturb natural imprinting. The explanation for the loss of methylation is outlined in the Supplemental Discussion. In conclusion, our data support the assumption that the general prole of imprinting is stable in children conceived through ART.

However, the imprinting of a few specic imprinted genes may be vulnerable in a fraction of the ART-conceived children. More data are needed to examine the imprinting status in ART-conceived children further. The limitations of this study are discussed in the supplemental material.

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Study of DNA-methylation patterns at chromosome 15q11-q13 in children born after ICSI reveals no imprinting defects. Mol Hum Reprod 2000;6:104953. 20. Geuns E, De Rycke M, Van Steirteghem A, Liebaers I. Methylation imprints of the imprint control region of the SNRPN-gene in human gametes and preimplantation embryos. Hum Mol Genet 2003;12:28739. 21. Tierling S, Souren NY, Gries J, Loporto C, Groth M, Lutsik P, et al. Assisted reproductive technologies do not enhance the variability of DNA methylation imprints in human. J Med Genet 2010;47:3716. 22. Tusher VG, Tibshirani R, Chu G. Signicance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci U S A 2001;98: 511621. 23. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(Delta Delta C(t)) method. Methods 2001;25:4028. 24. Bench AJ, Li J, Huntly BJ, Delabesse E, Fourouclas N, Hunt AR, et al. Characterization of the imprinted polycomb gene L3MBTL, a candidate 20q tumour suppressor gene, in patients with myeloid malignancies. Br J Haematol 2004;127:50918. Monk M, Boubelik M, Lehnert S. Temporal and regional changes in DNA methylation in the embryonic, extraembryonic and germ cell lineages during mouse embryo development. Development 1987;99:37182. Li L, Wang L, Le F, Liu X, Yu P, Sheng J, et al. Evaluation of DNA methylation status at differentially methylated regions in IVF-conceived newborn twins. Fertil Steril 2011;95:19759. Young LE, Fernandes K, McEvoy TG, Butterwith SC, Gutierrez CG, Carolan C, et al. Epigenetic change in IGF2R is associated with fetal overgrowth after sheep embryo culture. Nat Genet 2001;27:1534. Mann MR, Lee SS, Doherty AS, Verona RI, Nolen LD, Schultz RM, et al. Selective loss of imprinting in the placenta following preimplantation development in culture. Development 2004;131:372735. Humpherys D, Eggan K, Akutsu H, Hochedlinger K, third Rideout WM, Biniszkiewicz D, et al. Epigenetic instability in ES cells and cloned mice. Science 2001;293:957. Kim KP, Thurston A, Mummery C, Ward-van Oostwaard D, Priddle H, Allegrucci C, et al. Genespecic vulnerability to imprinting variability in human embryonic stem cell lines. Genome Res 2007; 17:173142. Rugg-Gunn PJ, Ferguson-Smith AC, Pedersen RA. Epigenetic status of human embryonic stem cells. Nat Genet 2005;37:5857. Lee MP, Feinberg AP. Genomic imprinting of a human apoptosis gene homologue, TSSC3. Cancer Res 1998;58:10526. Qian N, Frank D, OKeefe D, Dao D, Zhao L, Yuan L, et al. The IPL gene on chromosome 11p15.5 is imprinted in humans and mice and is similar to TDAG51, implicated in Fas expression and apoptosis. Hum Mol Genet 1997;6:20219. Muller S, van den Boom D, Zirkel D, Koster H, Berthold F, Schwab M, et al. Retention of imprinting of the human apoptosis-related gene TSSC3 in human brain tumors. Hum Mol Genet 2000;9:75763.

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SUPPLEMENTAL DISCUSSION
L3MBTL is a paternally expressed imprinted gene. It is reported that overexpression of L3MBTL results in improper nuclear segregation and cytokinesis, indicating that L3MBTL plays a role in proper progression of cell division (1). The paternally expressed gene PEG10 plays an essential role in embryonic development (2), liver regeneration, and hepatocarcinogenesis (3). The imprinting of PEG10 is disrupted in human embryonic stem cells (4) and aborted cloned bovine fetuses (5). Lack of the expression of the maternal allele expressed gene PHLDA2 in hydatidiform moles (6) and Wilms tumor (WT) (7) suggests that it is pathogenetically signicant for tumor formation. The expression level of PHLDA2 is negatively correlated with birth weight, and it may play an important role by constraining fetal growth (8). The increased expression of PEG10 and L3MBTL and the decreased expression of PHLDA2 in ART-conceived offspring may be involved in the increased risk of low birth weight and potential risks of tumorigenesis in later life. Long-term follow-up study is required to make clear the phenotype of these alterations. There seem to be two likely explanations for the loss of methylation of L3MBTL. First, it could be passed down from the parents. Second, some aspects of the ART procedures could result in the epigenetic differences. The methylation of imprinted genes is erased in primordial germ cells, established during gametogenesis, and maintained during preimplantation development (9, 10). ART procedures, including hormone-induced superovulation, in vitro fertilization, and embryo culture, manipulate gametes and embryos during these crucial stages of methylation reprogramming of imprinted genes. Therefore, ART is likely to induce inappropriate methylation of imprinted

genes and thus to disrupt imprinting. To further investigate the origin of methylation variation, the peripheral blood of the parents was collected for bisulte-sequencing PCR of L3MBTL. No hypomethylation of L3MBTL was observed in the peripheral blood of both the mother and the father, and all of the CpGs were moderately methylated, which suggested that ART procedures probably play a role in the loss of methylation. Its reported that spermatozoa from oligozoospermic patients carry a raised risk of transmitting imprinting errors (11, 12). The indications for ART in this child were tubal factor and asthenospermia. It should be taken into consideration that infertile couples may be more likely to have gametes with abnormal methylation although the methylation in their peripheral blood remains accurate. In human studies, it is difcult to distinguish the different effects of ART procedures and infertility background. Therefore further animal experiments should be conducted to conrm the inuence of various ART procedures and parental infertility. Our study covered the majority of the imprinted genes identied in the human genome, adopted microarray to search for genes prone to aberrant imprinting status, and found one gene with disrupted imprinting in one child. Considering the fact that the incidence of imprinting disorders in the general population is very low (1 in 15,000 newborns), the result that one ICSI child showed an LOI for one gene in 60 ART offspring should be taken seriously, and more experimental evidence is required to draw a conclusion. Nevertheless, this study at least showed that the disruption of imprinting in ARTconceived children is not as severe as supposed. One limitation of the present study is that there is still a fraction of imprinted genes that were not included. The conclusion would be more convincing if all of the imprinted genes were covered.

SUPPLEMENTAL REFERENCES
1. Koga H, Matsui S, Hirota T, Takebayashi S, Okumura K, Saya H. A human homolog of Drosophila lethal(3)malignant brain tumor (l(3)mbt) protein associates with condensed mitotic chromosomes. Oncogene 1999;18:3799809. 2. Ono R, Nakamura K, Inoue K, Naruse M, Usami T, Wakisaka-Saito N, et al. Deletion of Peg10, an imprinted gene acquired from a retrotransposon, causes early embryonic lethality. Nat Genet 2006;38:1016. 3. Jie X, Lang C, Jian Q, Chaoqun L, Dehua Y, Yi S, et al. Androgen activates PEG10 to promote carcinogenesis in hepatic cancer cells. Oncogene 2007;26: 574151. 4. Kim KP, Thurston A, Mummery C, Ward-van Oostwaard D, Priddle H, Allegrucci C, et al. Genespecic vulnerability to imprinting variability in human embryonic stem cell lines. Genome Res 2007;17:173142. 5. Liu JH, Yin S, Xiong B, Hou Y, Chen DY, Sun QY. Aberrant DNA methylation imprints in aborted bovine clones. Mol Reprod Dev 2008;75:598607. 6. Saxena A, Frank D, Panichkul P, van den Veyver IB, Tycko B, Thaker H. The product of the imprinted gene IPL marks human villous cytotrophoblast and is lost in complete hydatidiform mole. Placenta 2003;24:83542. 7. Schwienbacher C, Angioni A, Scelfo R, Veronese A, Calin GA, Massazza G, et al. Abnormal RNA expression of 11p15 imprinted genes and kidney developmental genes in Wilms tumor. Cancer Res 2000;60:15215. 8. Lee MP, Feinberg AP. Genomic imprinting of a human apoptosis gene homologue, TSSC3. Cancer Res 1998;58:10526. 9. Razin A, Cedar H. DNA methylation and genomic imprinting. Cell 1994;77:4736. 10. Reik W, Dean W, Walter J. Epigenetic reprogramming in mammalian development. Science 2001; 293:108993. 11. Marques CJ, Carvalho F, Sousa M, Barros A. Genomic imprinting in disruptive spermatogenesis. Lancet 2004;363:17002. 12. Hammoud SS, Purwar J, Pueger C, Cairns BR, Carrell DT. Alterations in sperm DNA methylation patterns at imprinted loci in two classes of infertility. Fertil Steril 2010;94:172833. 13. Saveanu L, Carroll O, Lindo V, Del Val M, Lopez D, Lepelletier Y, et al. Concerted peptide trimming by human ERAP1 and ERAP2 aminopeptidase complexes in the endoplasmic reticulum. Nat Immunol 2005;6:68997. 14. Apostolidou S, Abu-Amero S, ODonoghue K, Frost J, Olafsdottir O, Chavele KM, et al. Elevated placental expression of the imprinted PHLDA2 gene is associated with low birth weight. J Mol Med 2007;85:37987. Lux A, Beil C, Majety M, Barron S, Gallione CJ, Kuhn HM, et al. Human retroviral gag- and gagpol-like proteins interact with the transforming growth factor-beta receptor activin receptor-like kinase 1. J Biol Chem 2005;280:848293. Li J, Bench AJ, Vassiliou GS, Fourouclas N, Ferguson-Smith AC, Green AR. Imprinting of the human L3MBTL gene, a polycomb family member located in a region of chromosome 20 deleted in human myeloid malignancies. Proc Natl Acad Sci U S A 2004;101:73416. Herzing LB, Kim SJ, Cook EH Jr, Ledbetter DH. The human aminophospholipid-transporting ATPase gene ATP10C maps adjacent to UBE3A and exhibits similar imprinted expression. Am J Hum Genet 2001;68:15015. Schule B, Albalwi M, Northrop E, Francis DI, Rowell M, Slater HR, et al. Molecular breakpoint cloning and gene expression studies of a novel translocation t(4;15)(q27;q11.2) associated with Prader-Willi syndrome. BMC Med Genet 2005;6:18.

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SUPPLEMENTAL TABLE 1
Characteristics of the naturally conceived, IVF-conceived, and ICSI-conceived pregnancies. Characteristic n Gestational age (wk) Birth weight (g) Birth length (cm) Maternal age (y) Maternal body height (cm) Maternal body mass (kg) Maternal BMI (kg/m2) Maternal weight gain (kg) Naturally 60 38.5 1.3 3287 383 49.9 1.1 29.7 4.2 160.9 4.4 71.2 9.7 27.5 3.6 16.7 5.4 IVF 30 38.2 1.0 3193 462 49.7 1.0 31.0 3.7 162.1 3.3 75.0 7.8 28.5 2.7 16.5 4.8 ICSI 30 38.1 1.6 3178 470 49.8 1.1 29.1 3.6 161.5 3.8 72.8 9.2 27.9 2.9 16.8 5.8 P value .517 .430 .866 .146 .415 .169 .340 .983

Note: Data are expressed as mean SD. Pregnant women who conceived by ART treatment and delivered in our hospital from the year 2004 to 2006 were enrolled, and those conceived naturally and delivered in our hospital during the same phase were involved as the control group. The inclusion criterion was full-term uncomplicated singleton pregnancy. The exclusion criteria included women >35 years old, premature labor, and twin pregnancy. The indications for ART included tubal disease, severe endometriosis, oligoasthenoteratospermia, and unexplained infertility. There were no differences in gestation age, birth weight, and maternal age between the study and the control groups (P>.05). BMI body mass index. Feng. Imprinting is stable in ART offspring. Fertil Steril 2011.

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SUPPLEMENTAL TABLE 2
Human imprinted genes identied by microarray. Gene symbol DIRAS3 TP73 PRIM2A HYMAI PLAGL1 IGF2R SLC22A2 SLC22A3 GRB10 PEG10 PPP1R9A CALCR PON1 SGCE CPA4 DLX5 MEST COPG2 KLF14 KCNK9 CTNNA3 INPP5F WT1 CDKN1C KCNQ1 KCNQ1DN KCNQ1OT1 OSBPL5 ZNF215 H19 IGF2 IGF2AS INS ASCL2 PHLDA2 SLC22A18 SLC22A18AS SDHD HTR2A DLK1 MEG3 ATP10A PWCR1 SNRPN SNORD108 SNORD64 SNURF GABRG3 Unigene title DIRAS family, GTP-binding RAS-like 3 Tumor protein p73 Primase, DNA, polypeptide 2 Hydatidiform mole associated and imprinted Pleiomorphic adenoma gene-like 1 Insulin-like growth factor 2 receptor Solute carrier family 22 (organic cation transporter), member 2 Solute carrier family 22 (extraneuronal monoamine transporter), member 3 Growth factor receptor-bound protein 10 Paternally expressed 10 Protein phosphatase 1, regulatory (inhibitor) subunit 9A Calcitonin receptor Paraoxonase Sarcoglycan, epsilon Carboxypeptidase A4 Distal-less homeobox 5 Mesoderm specic transcript homolog (mouse) Coatomer protein complex, subunit gamma 2 Kruppel-like factor 14 Potassium channel, subfamily K, member 9 Catenin (cadherin-associated protein), alpha 3 Inositol polyphosphate-5-phosphatase F Wilms tumor 1 Cyclin-dependent kinase inhibitor 1C Potassium voltage-gated channel, KQT-like subfamily, member 1 KCNQ1 downstream neighbor KCNQ1 overlapping transcript 1 Oxysterol binding protein-like 5 Zinc nger protein 215 H19, imprinted maternally expressed transcript Insulin-like growth factor 2 Insulin-like growth factor 2 antisense Insulin Achaete-scute complex homolog 2 (Drosophila) Pleckstrin homology-like domain, family A, member 2 Solute carrier family 22 (organic cation transporter), member 18 Solute carrier family 22 (organic cation transporter), member 18 antisense Succinate dehydrogenase complex, subunit D, integral membrane protein 5-hydroxytryptamine (serotonin) receptor 2A Delta-like 1 homolog (Drosophila) Maternally expressed 3 ATPase, class V, type 10A Prader-Willi syndrome chromosome region 1 Small nuclear ribonucleoprotein polypeptide N Small nucleolar RNA, C/D box 108 Small nucleolar RNA, C/D box 64 SNRPN upstream reading frame Gamma-aminobutyric acid (GABA) A receptor, gamma 3 Gene no. (Gene ID) 9077 7161 5558 57061 5325 3482 6582 6581 2887 23089 55607 799 5444 8910 51200 1749 4232 26958 136259 51305 29119 22876 7490 1028 3784 55539 10984 114879 7762 283120 3481 51214 3630 430 7262 5002 5003 6392 3356 8788 55384 57194 692236 6638 338427 347686 8926 2567 Location 1p31 1p36.3 6p12-p11.1 6q24 6q24-q25 6q26 6q26 6q26-q27 7p12-p11.2 7q21 7q21.3 7q21.3 7q21.3 7q21-q22 7q32 7q22 7q32 7q32 7q32.2 8q24.3 10q22.2 10q26.11 11p13 11p15 11p15.5 11p15 11p15 11p15.4 11p15.4 11p15.5 11p15.5 11p15.5 11p15.5 11p15.5 11p15.5 11p15.5 11p15.5 11q23 13q14-q21 14q32 14q32 15q11.2 15q11.2 15q11.2 15q11.2 15q12 15q12 15q12 Expressed allele Paternal Maternal Maternal Paternal Paternal Polymorphic imprinting Maternal Maternal Isoform dependent Paternal Maternal Maternal Maternal Paternal Maternal Maternal Paternal Paternal Maternal Maternal Maternal Paternal Paternal Maternal Maternal Maternal Paternal Maternal Maternal Maternal Paternal Paternal Paternal Maternal Maternal Maternal Maternal Paternal Maternal Paternal Maternal Maternal Paternal Paternal Paternal Paternal Paternal Paternal

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SUPPLEMENTAL TABLE 2
Continued. Gene symbol GABRB3 GABRA5 NDN MAGEL2 MKRN3 UBE3A C15orf2 ZNF597 TCEB3C PEG3 ZIM2 ZNF264 USP29 NNAT L3MBTL GNAS SANG Unigene title Gamma-aminobutyric acid (GABA) A receptor, beta 3 Gamma-aminobutyric acid (GABA) A receptor, alpha 5 Necdin homolog (mouse) MAGE-like 2 Makorin, ring nger protein, 3 Ubiquitin protein ligase E3A Chromosome 15 open reading frame 2 Zinc nger protein 597 Transcription elongation factor B polypeptide 3C Paternally expressed 3 Zinc nger, imprinted 2 Zinc nger protein 264 Ubiquitin specic peptidase 29 Neuronatin l(3)mbt-like (Drosophila) GNAS complex locus GNAS1 antisense Gene no. (Gene ID) 2562 2558 4692 54551 7681 7337 23742 146434 162699 5178 23619 9422 57663 4826 26013 2778 149775 Location 15q11.2-q12 15q11.2-q12 15q11.2-q12 15q11-q12 15q11-q13 15q11-q13 15q11-q13 16p13.3 18q21.1 19q13.4 19q13.4 19q13.4 19q13.43 20q11.2-q12 20q13.12 20q13.3 20q13.32 Expressed allele Paternal Paternal Paternal Paternal Paternal Maternal Monoallelic Maternal Maternal Paternal Paternal Paternal Paternal Paternal Paternal Maternal Paternal

Note: The study group consisted of four children conceived through IVF (two female and two male; maternal age 3035 years; gestational age 3839 weeks) and four through ICSI (two female and two male; maternal age 3134 years; gestational age 3738 weeks). The control group consisted of eight children conceived naturally (four female and four male; maternal age 2436 years; gestational age 3740 weeks). In the control group, RNA samples from every two children were pooled for one assay. Affymetrix human genome U133 Plus 2.0 Genechip was used, which contains >55,000 probe sets representing >47,000 transcripts derived from $39,500 human genes. Feng. Imprinting is stable in ART offspring. Fertil Steril 2011.

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SUPPLEMENTAL TABLE 3
Primer sequences for real-time RT-PCR. Gene PHLDA2 PWCR1 SNRPN UBE3A L3MBTL MEG3 TP73 GNAS PEG10 ACTB Primer forward 50 -GAGCGCACGGGCAAGTA-30 50 -TCGATGATGAGTCCCCCATAA-30 50 -GGAACCACCATTTGTCTATGATCC-30 50 -CTCTTCTTGCAGTTTACAACG-30 50 -AAGAAGCCTCGCCATCACG-30 50 -CCTTCAGTGTCTGCATGTGG-30 50 -CACCACGTTTGAGCACCTCT-30 50 -TGACTGCCATCATCTTCGTGG-30 50 -CTCAATCAGTGACTGTGTGC-30 50 -CTGGAACGGTGAAGGTGACA-30 Primer reverse 50 -CAGCGGAAGTCGATCTCCTT-30 50 -CATTTTGTTCAGCTTTTCCAAGG-30 50 -CTGCAGGTGGTGACCATGTG-30 50 -CTTGAGTATTCCGGAAGTAAAAGC-30 50 -GACATGAAGAGGGACTGGTGC-30 50 -AGCCCTGTGCTTTGGAACC-30 50 -GCCCACCACCTCATTATTCC-30 50 -CAGCAAGGACTTTCTCAGCGAG-30 50 -TCCATAGCCGTTTCAGAGAG-30 50 -AAGGGACTTCCTGTAACAATGCA-30

Note: GNAS GNAS complex locus; SNRPN small nuclear ribonucleoprotein polypeptide N; PEG10 paternally expressed 10; UBE3A ubiquitin protein ligase E3A; PWCR1 Prader-Willi syndrome chromosome region 1; PHLDA2 pleckstrin homology-like domain, family A, member 2; TP73 tumor protein p73-like; L3MBTL l(3)mbt-like 3 (Drosophila); MEG3 maternally expressed 3. First-strand cDNA was synthesized from 1 mg total RNA with oligo dT and random hexamers. For PCR, the primers were designed using Primer3 online or derived from previously published studies (1318). The cycling condition was: 95 C for 10 seconds; 40 cycles of 95 C for 5 seconds and 62 C for 34 seconds. b-Actin was simultaneously quantied as an internal control. All PCR reactions were done in duplicate for both target genes and internal controls. The respective Ct values of the target genes were rst normalized by subtracting the Ct value of the b-actin control (DCt Ct [target] Ct [housekeeping]). The level of gene-specic mRNA in ART children relative to control children was calculated by subtracting the normalized Ct values of the control from those of ART children (DDCt DCt [ART] average DCt [control]), and the relative level was determined (2DDCt). Feng. Imprinting is stable in ART offspring. Fertil Steril 2011.

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SUPPLEMENTAL TABLE 4
Primer sequences for identication of genomic polymorphisms. Gene PHLDA2 L3MBTL PEG10 Primer forward 50 -CTCTGACCCAGTCCTTTCCC-30 50 -AGAGCCATTGTGAGCATT-30 50 -ATTCACTGTCCGAACCTG-30 Primer reverse 50 -GGTTCCCAGCGCCTTTCA-30 50 -GTTCCCACCATAGCAGTC-30 50 -GATCTTCCTTGTCCGTCT-30 SNP A/G [rs1056819] C/T [rs1062943] A/C [rs3750105]

Note: PCR was carried out in a 50-mL reaction mixture containing 1 PCR buffer, 1.5 mmol/L MgCl2, 0.2 mmol/L dNTP, 200 pmol of each forward and reverse primer, and 1.25 U Taq polymerase (Takara) on a Peltier thermal cycler PTC200 (MJ Research). The cycling condition was: 94 C for 5 minutes; 35 cycles of 94 C for 30 seconds, 55 C for 30 seconds, and 72 C for 30 seconds (PEG10 and L3MBTL) or 35 cycles of 94 C for 60 seconds and 68 C for 60 seconds (PHLDA2); 72 C for 10 minutes. Feng. Imprinting is stable in ART offspring. Fertil Steril 2011.

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SUPPLEMENTAL TABLE 5
Primer sequences for analyzing allele-specic expression. Gene PHLDA2 L3MBTL PEG10 Primer forward 50 -GACTGGATGAGGGTGTCCTG-30 50 -AGAGCCATTGTGAGCATT-30 50 -ATTCACTGTCCGAACCTG-30 Primer reverse 50 -GCCATACGCTGGACGAGT-30 50 -GTTCCCACCATAGCAGTC-30 50 - GATCTTCCTTGTCCGTCT-30 Accession number NM_003311 NM_015478 NM_001040152

Note: The PCR reaction (50 mL) was as described in Supplemental Table 4. The cycling condition was: 94 C for 5 minutes; 35 cycles of 94 C for 30 seconds, 55 C for 30 seconds, and 72 C for 30 seconds (PEG10 and L3MBTL) or 35 cycles of 94 C for 60 seconds, 62 C for 45 seconds, and 72 C for 60 seconds (PHLDA2); 72 C for 10 minutes. Feng. Imprinting is stable in ART offspring. Fertil Steril 2011.

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SUPPLEMENTAL TABLE 6
Primer sequences for bisulphite genomic sequencing. Gene L3MBTL Primer forward 50 -TGTTTTGGATGTGTTTTGTTTTTT-30 Primer reverse 50 -AAACCCATCATCTAAACCAACTTC-30 Accession number NW_927339

Two microliters of bisulphite-treated DNA (50 ng) were subjected to PCR amplication in a 25-mL reaction containing 1 PCR buffer, 1.5 mmol/L MgCl2, 0.2 mmol/L dNTP, 200 pmol of each forward and reverse primer, and 1 U Taq polymerase (Takara) under the following touch down cycle conditions: 94 C for 5 minutes; 94 C for 30 seconds, 65 C55 C (0.5 C/cycle) for 45 seconds, and 72 C for 45 seconds for 20 cycles; 94 C for 30 seconds, 55 C for 45 seconds, and 72 C for 45 seconds for 15 cycles; nal extension 72 C for 10 minutes. Methylation-unspecic primers complementary to the bisulphite-treated DNA were used to amplify the fragments in the DMR region. They were designed using Methprimer online (http://www.urogene.org/methprimer/ index1.html). Feng. Imprinting is stable in ART offspring. Fertil Steril 2011.

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SUPPLEMENTAL TABLE 7
The relative changes found in the microarray for the nine top discriminatory imprinted genes. Gene symbol PWCR1 SNRPN UBE3A TP73 GNAS MEG3 PEG10 L3MBTL PHLDA2 Gene ID 221974_at 1559343_at 211575_s_at 1554379_a_at 228173_at 210795_s_at 212092_at 206822_s_at 209803_s_at ART/natural 0.34 0.61 0.40 1.46 0.28 1.35 1.39 1.49 0.23

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