- Email: [email protected]
Aberrant DNA methylation of imprinted H19 gene in human preimplantation embryos
Aberrant DNA methylation of imprinted H19 gene in human preimplantation embryos DNA methylation patterns of the H19 differentially methylated region were successfully determined in 32 of 50 human day 3 embryos produced and discarded after assisted reproductive technology procedures. We found methylation patterns similar to those of somatic cells in 81.3% of embryos and demethylation or hypomethylation patterns in 18.7% of embryos. (Fertil Steril 2010;94:2356–8. 2010 by American Society for Reproductive Medicine.) Key Words: Assisted reproductive technologies, DNA methylation, H19, human embryos
Recent studies suggest that assisted reproductive technology (ART) may be associated with an increased incidence of Angelman syndrome (AS) and Beckwith-Wiedemann syndrome (BWS) (1, 2). More than 90% of ART-conceived children with BWS showed imprinting defects, compared with 40%–50% of naturally conceived children with BWS; the majority of ART-conceived children with AS also demonstrated imprinting defects (3). Concerns about imprinting disorders linked to ART are therefore increasing. DNA methylation is a major mechanism for genetic imprinting. Aberrant DNA methylation at the H19 differentially methylated region (DMR) has been described in about 2%–7% of BWS cases (4). The H19 DMR is located upstream of the H19 gene transcription start site in chromosome 11p15.5 and is a paternally imprinted gene. Furthermore, H19 is a good epigenetic marker for susceptibility to environmental injuries because the methylation status of H19 DMR seems to be vulnerable to culture conditions (5).
Some steps of manipulation in the ART procedure, such as in vitro maturation (IVM) and superovulation with high-dose hormone, may cause abnormal methylation profiles in oocytes (6, 7). Men with severe oligozoospermia or azoospermia exhibit abnormal methylation profiles in sperm DNA, suggesting that imprinting errors could also be related to infertility itself (8–10). Thus, aberrant DNA methylation in embryos could either from result in vitro culture (11) or from imprinting errors in parental gametes (12, 13). However, thus far the data have been primarily obtained from animal studies; therefore, human studies are needed.
Shi-Ling Chen, M.D., M.Sc. Xiao-Yun Shi, M.D., M.Sc. Hai-Yan Zheng, M.D. Fang-Rong Wu, M.D. Chen Luo, M.Sc. Center for reproductive Medicine, Department of Obstetrics and Gynecology, Nanfang Hospital, Southern Medical University, Guangzhou, People’s Republic of China
The study protocol was approved by the Institutional Ethical Committee and Institutional Review Board. Informed consent was obtained from all patients before embryos were donated for research. In our ART procedure, Quinn’s Advantage HEPES Medium, HTF Medium, and Cleavage Medium (Sage, Pasadena, CA) are used to manipulate gametes and embryos. The culture conditions were 37 C, 5% CO2, 5% O2, and 90% N2. We collected 50 day 3 embryos (from 48 patients) that were normally fertilized and confirmed by the presence of two pronuclei but were unsuitable for transfer or freezing due to poor quality (summarized in Table 1; online-only appendix). After removal of the zona pellucida using acidic Tyrode’s solution (Sage), the single embryo was washed repeatedly and then transferred to a 0.5-mL polymerase chain reaction (PCR) tube containing 2 mL lysis buffer (10 mM Tris-HCl, 1 mM EDTA, 10% SDS, 1% proteinase K, pH 8.0). The samples were incubated for 1 hour at 37 C and then stored at 80 C until use. An aliquot from the last washing droplet served as a blank. Adult leukocytes, human oocytes (metaphase I stage) and human sperm were used as controls.
Received October 26, 2009; revised January 13, 2010; accepted January 29, 2010; published online March 19, 2010. Supported by National Key Basic Research Development Plan of China (973 Program) (2007CB948104). S.-L.C. has nothing to disclose. X.-Y.S. has nothing to disclose. H.-Y.Z. has nothing to disclose. F.-R.W. has nothing to disclose. C.L. has nothing to disclose. The first two authors contributed equally to this work. The preliminary results of this trial have been presented at the 65th American Society of Reproductive Medicine meeting, which was held in Atlanta, Georgia, in October 2009. Reprint requests: Shi-Ling Chen, M.D., M.Sc., Center for Reproductive Medicine, Department of Obstetrics and Gynecology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, People’s Republic of China (FAX: 0086-20-87280183; E-mail: [email protected]).
2356
Related studies on human embryos are limited owing to ethical and legal restrictions. In this pilot study, we collected 50 human preimplantation embryos produced during ART procedures. We determined their H19 DMR methylation status at the singleembryo level using a combined bisulfite restriction analysis (COBRA) and sequencing technique to evaluate epigenetic risks linked to ART.
DNA of leukocytes and sperm were bisulfite converted with the EpiTect Bisulfite Kit (QIAGEN, Hilden, Germany). For embryos
Fertility and Sterility Vol. 94, No. 6, November 2010 Copyright ª2010 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/$36.00 doi:10.1016/j.fertnstert.2010.01.120
FIGURE 1 DNA methylation patterns of H19 DMR were determined by COBRA using the Taq I restriction enzyme. The digested PCR products were separated on 4% agarose gels; gel images were saved as TIFF files. The 230-bp bands represented unmethylated maternal alleles; 100-bp and 130-bp bands represented methylated paternal alleles. The percentage of methylation was calculated as the density ratio of the two methylated bands to the total three bands. (A) Leukocyte controls showed the differential methylation pattern (about half methylated and half unmethylated), whereas oocytes and sperm exhibited the unmethylated pattern and methylated pattern, respectively. (B) Methylation patterns in most embryos were very similar to those of somatic leukocytes, except that embryo no. 7 (another four embryos not shown here) showed a demethylation pattern and embryo no. 22 exhibited a hypomethylation pattern (14% methylation). (C) To determine the source of altered H19 DMR methylation in six embryos, we examined methylation of the six sperm samples that were used to fertilize the corresponding embryos (no. 7, 16, 17, 22, 31, and 32). Normal hypermethylation patterns were confirmed in all six sperm samples.
Chen. Correspondence. Fertil Steril 2010.
and oocytes, the bisulfite-conversion protocol using low melting point (LMP) agarose was modified based on the description of Geuns et al. (14). Briefly, the sodium bisulfite solution was prepared using the CpGenome DNA Modification Kit (Chemicon International, Temecula, CA), according to the manufacturer’s instructions. After retrieval from 80 C, each sample was embedded by adding 6 mL melted 2% LMP agarose (80 C). Then 50 mL cold mineral oil was added to each sample, and DNA was denatured by incubating samples for 15 minutes at 80 C. The tube was subsequently kept on ice for 2 minutes, and then 500 mL prepared sodium bisulfite solution was added. The agarose bead was naturally formed. The sample was incubated at 50 C for 12 hours. Mineral oil and bisulfite were then removed, and the bead was equilibrated with 1 mL TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0; 4 10 minutes). This was followed by desulfonation in 0.5 mL of 20 mM NaOH (2 10 minutes). Finally, the bead was washed in 1 mL TE (1 10 minutes) and 1 mL H2O (2 10 minutes) for direct use in a PCR reaction. The H19 DMR was amplified by hemi-nested single-embryo PCR of the bisulfite-converted DNA; this 230-bp sequence encompasses 18 CpG sites (GenBank AF125183, nucleotide positions 7870–8100). The primers were (outer forward primer F1) 50 -AGG
Fertility and Sterility
TGT TTT AGT TTT ATG GAT GAT GG-30 , (inner forward primer F2) 50 -TGT ATA GTA TAT ATA TGG GTA TTT TTG GAG GTT T-30 , and (reverse primer R) 5 0 -TCC TAT AAA TAT CCT ATT CCC AAATAA CC-30 (6). The 25-mL PCR reaction mix contained 0.2 mM of each primer, 2 PCR HotStart premix buffer (TaKaRa Bio, Tokyo, Japan); reactions were carried out with a GeneAmp PCR system 9700. First-round PCR used primers F1 and R with the following conditions: 5-minute denaturation at 95 C, followed by 35 cycles of 94 C for 1 minute, 58 C for 1 minute, and 72 C for 1 minute, with a final extension at 72 C for 5 minutes. Three microliters of the first-round PCR product were used as DNA input for amplification in the second-round nested PCR (using primers F2 and R) with the following conditions: 5-minute denaturation at 95 C, followed by 40 cycles of 94 C for 1 minute, 60 C for 30 seconds, and 72 C for 30 seconds, with a final extension at 72 C for 5 minutes. To determine the overall methylation pattern, COBRA was performed with the Taq I restriction enzyme, which recognizes a single site in the bisulfite-converted H19 DMR sequence and cuts only methylated DNA. The purified PCR product was digested with 5 U Taq I restriction enzyme (Promega, Madison, WI) at 65 C for 2 hours in a total volume of 20 mL and separated by 4% agarose
2357
electrophoresis. Gel images were saved as TIFF files. Band densities were determined using Labworks image acquisition and analysis software (Ultra-violet Products, Ltd., Cambridge, UK). Percent methylation was calculated as the density ratio of the two methylated bands to the three total bands (15). To confirm COBRA results, PCR products for samples of interest were cloned into the PGEM-T vector (Promega), and approximately 20 clones of each individual sample were sequenced. As expected, methylation patterns for H19 DMR showed the differential methylation pattern (about half methylated and half unmethylated) for leukocytes, the methylated pattern for sperm and the unmethylated pattern for oocytes, indicating a lack of bias in bisulfite PCR (Fig. 1A). Of the 32 embryos that were successfully analyzed (amplification efficiency, 64%), 26 embryos (81.3%) showed the differential methylation pattern (49.4% 9.7% methylation), similar to that of somatic leukocytes. Abnormal methylation was considered when results were 2 SD of mean methylation (12). Six embryos (18.7%) exhibited aberrant methylation patterns: five showed the demethylation pattern, and one showed a hypomethylation pattern (14% methylation; Fig. 1B). Subsequent sequencing confirmed these results (Table 1, online-only appendix). A total of 50 negative PCR controls were tested, and none produced amplification products, indicating no PCR contamination. Because demethylation or hypomethylation patterns of H19 DMR in embryos may be due to imprinting errors in spermatozoa, we next examined
DNA methylation patterns in the sperm samples that were used to fertilize the six embryos. The COBRA assays confirmed normal hypermethylation patterns in all six sperm samples (Fig. 1C). In summary, we found methylation patterns similar to those of somatic cells in 81.3% of the ART embryos, indicating that differential methylation of H19 DMR was established in these embryos. However, 18.7% of embryos exhibited a demethylation or hypomethylation pattern. After confirming no imprinting errors of H19 DMR in the sperm samples, we hypothesized that ART manipulation, especially in vitro embryo culture, was a possible cause. Previous animal studies have reported that culture medium may produce methylation defects of H19 DMR. The present study was restricted to one imprinted gene and poor-quality embryos produced by ART. Without comparing these embryos with normal human embryos conceived naturally, we cannot conclude that ART specifically caused the altered methylation we observed. Ethical considerations prevented us from using normal human embryo controls in the present study. Therefore, our results should be confirmed by studies involving normal human embryos and multiple imprinted genes. Acknowledgments: The authors thank all members of the Research Center of Clinical Medicine of Nanfang Hospital for their technical supports and valuable suggestions. In particular, we thank Mr. Zuo-Lin Qiu, Mr. Weiqing Zhang, and Mr. Jie Yang for their daily work in the IVF laboratory of Nanfang Hospital.
REFERENCES 1. Ludwig M, Katalinic A, Gross S, Sutcliffe A, Varon R, Horsthemke B. Increased prevalence of imprinting defects in patients with Angelman syndrome born to subfertile couples. J Med Genet 2005;42:289–91. 2. Maher ER, Brueton LA, Bowdin SC, Luharia A, Cooper W, Cole TR, et al. Beckwith-Wiedemann syndrome and assisted reproduction technology (ART). J Med Genet 2003;40:62–4. 3. Manipalviratn S, DeCherney A, Segars J. Imprinting disorders and assisted reproductive technology. Fertil Steril 2009;91:305–15. 4. Weksberg R, Shuman C, Beckwith JB. BeckwithWiedemann syndrome. Eur J Hum Genet 2010; 18:8–14. 5. Khosla S, Dean W, Brown D, Reik W, Feil R. Culture of preimplantation mouse embryos affects fetal development and the expression of imprinted genes. Biol Reprod 2001;64:918–26. 6. Sato A, Otsu E, Negishi H, Utsunomiya T, Arima T. Aberrant DNA methylation of imprinted loci in superovulated oocytes. Hum Reprod 2007;22:26–35.
2358
Chen et al.
Correspondence
7. Borghol N, Lornage J, Blachere T, Sophie GA, Lefevre A. Epigenetic status of the H19 locus in human oocytes following in vitro maturation. Genomics 2006;87:417–26. 8. Marques CJ, Costa P, Vaz B, Carvalho F, Fernandes S, Barros A, et al. Abnormal methylation of imprinted genes in human sperm is associated with oligozoospermia. Mol Hum Reprod 2008;14: 67–74. 9. Kobayashi H, Sato A, Otsu E, Hiura H, Tomatsu C, Utsunomiya T, et al. Aberrant DNA methylation of imprinted loci in sperm from oligospermic patients. Hum Mol Genet 2007;16:2542–51. 10. Marques CJ, Francisco T, Sousa S, Carvalho F, Barros A, Sousa M. Methylation defects of imprinted genes in human testicular spermatozoa. Fertil Steril 2010;94:585–94. 11. Fauque P, Jouannet P, Lesaffre C, Ripoche MA, Dandolo L, Vaiman D, et al. Assisted reproductive technology affects developmental kinetics, H19 imprinting control region methylation and H19 gene
12.
13.
14.
15.
expression in individual mouse embryos. BMC Dev Biol 2007;7:116. Kobayashi H, Hiura H, John RM, Sato A, Otsu E, Kobayashi N, et al. DNA methylation errors at imprinted loci after assisted conception originate in the parental sperm. Eur J Hum Genet 2009;17: 1582–91. Shi W, Haaf T. Aberrant methylation patterns at the two-cell stage as an indicator of early developmental failure. Mol Reprod Dev 2002;63:329–34. 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;12:2873–9. Leakey TI, Zielinski J, Siegfried RN, Siegel ER, Fan CY, Cooney CA. A simple algorithm for quantifying DNA methylation levels on multiple independent CpG sites in bisulfite genomic sequencing electropherograms. Nucl Acids Res 2008;36:e64.
Vol. 94, No. 6, November 2010
TABLE 1 Results of methylation analysis of H19 DMR in human preimplantation embryos. Day 2 quality Embryo no. ART 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
IVF IVF IVF ICSI IVF ICSI IVF IVF IVF IVF IVF IVF IVF IVF ICSI IVF ICSI IVF ICSI IVF IVF IVF IVF IVF IVF IVF IVF ICSI IVF IVF IVF IVF
Cells 2 Uncleaved 4 2 Uncleaved 4 2 2 4 3 4 2 2 2 2 Uncleaved Uncleaved 3 5 2 5 5 3 4 3 2 4 2 4 3 5 4
Day 3 quality
Grade Cells 1 — 3 1 — 3 1 1 2 2 1 1 3 2 1 — — 1 3 1 2 3 2 2 3 1 1 1 2 2 3 2
4 2 12 3 6 5 2 4 4 5 5 2 3 4 5 4 3 4 5 3 6 8 3 4 5 2 5 5 4 4 5 4
Grade
Methylationa (%)
2 1 3 3 3 3 2 1 2 4 1 1 3 3 3 3 3 3 4 3 3 4 2 2 4 1 3 3 2 4 3 2
46.9 51.1 42.0 52.8 51.6 52.2 (49.0) 0 (0.3)b 41.6 44.9 39.2 47.5 56.4 45.9 56.9 57.7 0 (1.7)b 0 (0.3)b 52.7 55.9 43.5 41.2 14.0 (10.5)b 53.5 47.8 61.7 57.9 54.4 44.0 65.5 55.8 0 (0)b 0 (0)b
Note: ICSI ¼ intracytoplasmic sperm injection. a Each value is the percent methylation determined by COBRA. The results of sequencing analysis are given in the parentheses. b the abnormal values. Chen. Correspondence. Fertil Steril 2010.
Fertility and Sterility
2358.e1
Recent studies suggest that assisted reproductive technology (ART) may be associated with an increased incidence of Angelman syndrome (AS) and Beckwith-Wiedemann syndrome (BWS) (1, 2). More than 90% of ART-conceived children with BWS showed imprinting defects, compared with 40%–50% of naturally conceived children with BWS; the majority of ART-conceived children with AS also demonstrated imprinting defects (3). Concerns about imprinting disorders linked to ART are therefore increasing. DNA methylation is a major mechanism for genetic imprinting. Aberrant DNA methylation at the H19 differentially methylated region (DMR) has been described in about 2%–7% of BWS cases (4). The H19 DMR is located upstream of the H19 gene transcription start site in chromosome 11p15.5 and is a paternally imprinted gene. Furthermore, H19 is a good epigenetic marker for susceptibility to environmental injuries because the methylation status of H19 DMR seems to be vulnerable to culture conditions (5).
Some steps of manipulation in the ART procedure, such as in vitro maturation (IVM) and superovulation with high-dose hormone, may cause abnormal methylation profiles in oocytes (6, 7). Men with severe oligozoospermia or azoospermia exhibit abnormal methylation profiles in sperm DNA, suggesting that imprinting errors could also be related to infertility itself (8–10). Thus, aberrant DNA methylation in embryos could either from result in vitro culture (11) or from imprinting errors in parental gametes (12, 13). However, thus far the data have been primarily obtained from animal studies; therefore, human studies are needed.
Shi-Ling Chen, M.D., M.Sc. Xiao-Yun Shi, M.D., M.Sc. Hai-Yan Zheng, M.D. Fang-Rong Wu, M.D. Chen Luo, M.Sc. Center for reproductive Medicine, Department of Obstetrics and Gynecology, Nanfang Hospital, Southern Medical University, Guangzhou, People’s Republic of China
The study protocol was approved by the Institutional Ethical Committee and Institutional Review Board. Informed consent was obtained from all patients before embryos were donated for research. In our ART procedure, Quinn’s Advantage HEPES Medium, HTF Medium, and Cleavage Medium (Sage, Pasadena, CA) are used to manipulate gametes and embryos. The culture conditions were 37 C, 5% CO2, 5% O2, and 90% N2. We collected 50 day 3 embryos (from 48 patients) that were normally fertilized and confirmed by the presence of two pronuclei but were unsuitable for transfer or freezing due to poor quality (summarized in Table 1; online-only appendix). After removal of the zona pellucida using acidic Tyrode’s solution (Sage), the single embryo was washed repeatedly and then transferred to a 0.5-mL polymerase chain reaction (PCR) tube containing 2 mL lysis buffer (10 mM Tris-HCl, 1 mM EDTA, 10% SDS, 1% proteinase K, pH 8.0). The samples were incubated for 1 hour at 37 C and then stored at 80 C until use. An aliquot from the last washing droplet served as a blank. Adult leukocytes, human oocytes (metaphase I stage) and human sperm were used as controls.
Received October 26, 2009; revised January 13, 2010; accepted January 29, 2010; published online March 19, 2010. Supported by National Key Basic Research Development Plan of China (973 Program) (2007CB948104). S.-L.C. has nothing to disclose. X.-Y.S. has nothing to disclose. H.-Y.Z. has nothing to disclose. F.-R.W. has nothing to disclose. C.L. has nothing to disclose. The first two authors contributed equally to this work. The preliminary results of this trial have been presented at the 65th American Society of Reproductive Medicine meeting, which was held in Atlanta, Georgia, in October 2009. Reprint requests: Shi-Ling Chen, M.D., M.Sc., Center for Reproductive Medicine, Department of Obstetrics and Gynecology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, People’s Republic of China (FAX: 0086-20-87280183; E-mail: [email protected]).
2356
Related studies on human embryos are limited owing to ethical and legal restrictions. In this pilot study, we collected 50 human preimplantation embryos produced during ART procedures. We determined their H19 DMR methylation status at the singleembryo level using a combined bisulfite restriction analysis (COBRA) and sequencing technique to evaluate epigenetic risks linked to ART.
DNA of leukocytes and sperm were bisulfite converted with the EpiTect Bisulfite Kit (QIAGEN, Hilden, Germany). For embryos
Fertility and Sterility Vol. 94, No. 6, November 2010 Copyright ª2010 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/$36.00 doi:10.1016/j.fertnstert.2010.01.120
FIGURE 1 DNA methylation patterns of H19 DMR were determined by COBRA using the Taq I restriction enzyme. The digested PCR products were separated on 4% agarose gels; gel images were saved as TIFF files. The 230-bp bands represented unmethylated maternal alleles; 100-bp and 130-bp bands represented methylated paternal alleles. The percentage of methylation was calculated as the density ratio of the two methylated bands to the total three bands. (A) Leukocyte controls showed the differential methylation pattern (about half methylated and half unmethylated), whereas oocytes and sperm exhibited the unmethylated pattern and methylated pattern, respectively. (B) Methylation patterns in most embryos were very similar to those of somatic leukocytes, except that embryo no. 7 (another four embryos not shown here) showed a demethylation pattern and embryo no. 22 exhibited a hypomethylation pattern (14% methylation). (C) To determine the source of altered H19 DMR methylation in six embryos, we examined methylation of the six sperm samples that were used to fertilize the corresponding embryos (no. 7, 16, 17, 22, 31, and 32). Normal hypermethylation patterns were confirmed in all six sperm samples.
Chen. Correspondence. Fertil Steril 2010.
and oocytes, the bisulfite-conversion protocol using low melting point (LMP) agarose was modified based on the description of Geuns et al. (14). Briefly, the sodium bisulfite solution was prepared using the CpGenome DNA Modification Kit (Chemicon International, Temecula, CA), according to the manufacturer’s instructions. After retrieval from 80 C, each sample was embedded by adding 6 mL melted 2% LMP agarose (80 C). Then 50 mL cold mineral oil was added to each sample, and DNA was denatured by incubating samples for 15 minutes at 80 C. The tube was subsequently kept on ice for 2 minutes, and then 500 mL prepared sodium bisulfite solution was added. The agarose bead was naturally formed. The sample was incubated at 50 C for 12 hours. Mineral oil and bisulfite were then removed, and the bead was equilibrated with 1 mL TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0; 4 10 minutes). This was followed by desulfonation in 0.5 mL of 20 mM NaOH (2 10 minutes). Finally, the bead was washed in 1 mL TE (1 10 minutes) and 1 mL H2O (2 10 minutes) for direct use in a PCR reaction. The H19 DMR was amplified by hemi-nested single-embryo PCR of the bisulfite-converted DNA; this 230-bp sequence encompasses 18 CpG sites (GenBank AF125183, nucleotide positions 7870–8100). The primers were (outer forward primer F1) 50 -AGG
Fertility and Sterility
TGT TTT AGT TTT ATG GAT GAT GG-30 , (inner forward primer F2) 50 -TGT ATA GTA TAT ATA TGG GTA TTT TTG GAG GTT T-30 , and (reverse primer R) 5 0 -TCC TAT AAA TAT CCT ATT CCC AAATAA CC-30 (6). The 25-mL PCR reaction mix contained 0.2 mM of each primer, 2 PCR HotStart premix buffer (TaKaRa Bio, Tokyo, Japan); reactions were carried out with a GeneAmp PCR system 9700. First-round PCR used primers F1 and R with the following conditions: 5-minute denaturation at 95 C, followed by 35 cycles of 94 C for 1 minute, 58 C for 1 minute, and 72 C for 1 minute, with a final extension at 72 C for 5 minutes. Three microliters of the first-round PCR product were used as DNA input for amplification in the second-round nested PCR (using primers F2 and R) with the following conditions: 5-minute denaturation at 95 C, followed by 40 cycles of 94 C for 1 minute, 60 C for 30 seconds, and 72 C for 30 seconds, with a final extension at 72 C for 5 minutes. To determine the overall methylation pattern, COBRA was performed with the Taq I restriction enzyme, which recognizes a single site in the bisulfite-converted H19 DMR sequence and cuts only methylated DNA. The purified PCR product was digested with 5 U Taq I restriction enzyme (Promega, Madison, WI) at 65 C for 2 hours in a total volume of 20 mL and separated by 4% agarose
2357
electrophoresis. Gel images were saved as TIFF files. Band densities were determined using Labworks image acquisition and analysis software (Ultra-violet Products, Ltd., Cambridge, UK). Percent methylation was calculated as the density ratio of the two methylated bands to the three total bands (15). To confirm COBRA results, PCR products for samples of interest were cloned into the PGEM-T vector (Promega), and approximately 20 clones of each individual sample were sequenced. As expected, methylation patterns for H19 DMR showed the differential methylation pattern (about half methylated and half unmethylated) for leukocytes, the methylated pattern for sperm and the unmethylated pattern for oocytes, indicating a lack of bias in bisulfite PCR (Fig. 1A). Of the 32 embryos that were successfully analyzed (amplification efficiency, 64%), 26 embryos (81.3%) showed the differential methylation pattern (49.4% 9.7% methylation), similar to that of somatic leukocytes. Abnormal methylation was considered when results were 2 SD of mean methylation (12). Six embryos (18.7%) exhibited aberrant methylation patterns: five showed the demethylation pattern, and one showed a hypomethylation pattern (14% methylation; Fig. 1B). Subsequent sequencing confirmed these results (Table 1, online-only appendix). A total of 50 negative PCR controls were tested, and none produced amplification products, indicating no PCR contamination. Because demethylation or hypomethylation patterns of H19 DMR in embryos may be due to imprinting errors in spermatozoa, we next examined
DNA methylation patterns in the sperm samples that were used to fertilize the six embryos. The COBRA assays confirmed normal hypermethylation patterns in all six sperm samples (Fig. 1C). In summary, we found methylation patterns similar to those of somatic cells in 81.3% of the ART embryos, indicating that differential methylation of H19 DMR was established in these embryos. However, 18.7% of embryos exhibited a demethylation or hypomethylation pattern. After confirming no imprinting errors of H19 DMR in the sperm samples, we hypothesized that ART manipulation, especially in vitro embryo culture, was a possible cause. Previous animal studies have reported that culture medium may produce methylation defects of H19 DMR. The present study was restricted to one imprinted gene and poor-quality embryos produced by ART. Without comparing these embryos with normal human embryos conceived naturally, we cannot conclude that ART specifically caused the altered methylation we observed. Ethical considerations prevented us from using normal human embryo controls in the present study. Therefore, our results should be confirmed by studies involving normal human embryos and multiple imprinted genes. Acknowledgments: The authors thank all members of the Research Center of Clinical Medicine of Nanfang Hospital for their technical supports and valuable suggestions. In particular, we thank Mr. Zuo-Lin Qiu, Mr. Weiqing Zhang, and Mr. Jie Yang for their daily work in the IVF laboratory of Nanfang Hospital.
REFERENCES 1. Ludwig M, Katalinic A, Gross S, Sutcliffe A, Varon R, Horsthemke B. Increased prevalence of imprinting defects in patients with Angelman syndrome born to subfertile couples. J Med Genet 2005;42:289–91. 2. Maher ER, Brueton LA, Bowdin SC, Luharia A, Cooper W, Cole TR, et al. Beckwith-Wiedemann syndrome and assisted reproduction technology (ART). J Med Genet 2003;40:62–4. 3. Manipalviratn S, DeCherney A, Segars J. Imprinting disorders and assisted reproductive technology. Fertil Steril 2009;91:305–15. 4. Weksberg R, Shuman C, Beckwith JB. BeckwithWiedemann syndrome. Eur J Hum Genet 2010; 18:8–14. 5. Khosla S, Dean W, Brown D, Reik W, Feil R. Culture of preimplantation mouse embryos affects fetal development and the expression of imprinted genes. Biol Reprod 2001;64:918–26. 6. Sato A, Otsu E, Negishi H, Utsunomiya T, Arima T. Aberrant DNA methylation of imprinted loci in superovulated oocytes. Hum Reprod 2007;22:26–35.
2358
Chen et al.
Correspondence
7. Borghol N, Lornage J, Blachere T, Sophie GA, Lefevre A. Epigenetic status of the H19 locus in human oocytes following in vitro maturation. Genomics 2006;87:417–26. 8. Marques CJ, Costa P, Vaz B, Carvalho F, Fernandes S, Barros A, et al. Abnormal methylation of imprinted genes in human sperm is associated with oligozoospermia. Mol Hum Reprod 2008;14: 67–74. 9. Kobayashi H, Sato A, Otsu E, Hiura H, Tomatsu C, Utsunomiya T, et al. Aberrant DNA methylation of imprinted loci in sperm from oligospermic patients. Hum Mol Genet 2007;16:2542–51. 10. Marques CJ, Francisco T, Sousa S, Carvalho F, Barros A, Sousa M. Methylation defects of imprinted genes in human testicular spermatozoa. Fertil Steril 2010;94:585–94. 11. Fauque P, Jouannet P, Lesaffre C, Ripoche MA, Dandolo L, Vaiman D, et al. Assisted reproductive technology affects developmental kinetics, H19 imprinting control region methylation and H19 gene
12.
13.
14.
15.
expression in individual mouse embryos. BMC Dev Biol 2007;7:116. Kobayashi H, Hiura H, John RM, Sato A, Otsu E, Kobayashi N, et al. DNA methylation errors at imprinted loci after assisted conception originate in the parental sperm. Eur J Hum Genet 2009;17: 1582–91. Shi W, Haaf T. Aberrant methylation patterns at the two-cell stage as an indicator of early developmental failure. Mol Reprod Dev 2002;63:329–34. 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;12:2873–9. Leakey TI, Zielinski J, Siegfried RN, Siegel ER, Fan CY, Cooney CA. A simple algorithm for quantifying DNA methylation levels on multiple independent CpG sites in bisulfite genomic sequencing electropherograms. Nucl Acids Res 2008;36:e64.
Vol. 94, No. 6, November 2010
TABLE 1 Results of methylation analysis of H19 DMR in human preimplantation embryos. Day 2 quality Embryo no. ART 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
IVF IVF IVF ICSI IVF ICSI IVF IVF IVF IVF IVF IVF IVF IVF ICSI IVF ICSI IVF ICSI IVF IVF IVF IVF IVF IVF IVF IVF ICSI IVF IVF IVF IVF
Cells 2 Uncleaved 4 2 Uncleaved 4 2 2 4 3 4 2 2 2 2 Uncleaved Uncleaved 3 5 2 5 5 3 4 3 2 4 2 4 3 5 4
Day 3 quality
Grade Cells 1 — 3 1 — 3 1 1 2 2 1 1 3 2 1 — — 1 3 1 2 3 2 2 3 1 1 1 2 2 3 2
4 2 12 3 6 5 2 4 4 5 5 2 3 4 5 4 3 4 5 3 6 8 3 4 5 2 5 5 4 4 5 4
Grade
Methylationa (%)
2 1 3 3 3 3 2 1 2 4 1 1 3 3 3 3 3 3 4 3 3 4 2 2 4 1 3 3 2 4 3 2
46.9 51.1 42.0 52.8 51.6 52.2 (49.0) 0 (0.3)b 41.6 44.9 39.2 47.5 56.4 45.9 56.9 57.7 0 (1.7)b 0 (0.3)b 52.7 55.9 43.5 41.2 14.0 (10.5)b 53.5 47.8 61.7 57.9 54.4 44.0 65.5 55.8 0 (0)b 0 (0)b
Note: ICSI ¼ intracytoplasmic sperm injection. a Each value is the percent methylation determined by COBRA. The results of sequencing analysis are given in the parentheses. b the abnormal values. Chen. Correspondence. Fertil Steril 2010.
Fertility and Sterility
2358.e1