- Email: [email protected]
Igf2 differentially methylated domain and the expression of H19 and Igf2
Embryo vitrification affects the methylation of the H19/ Igf2 differentially methylated domain and the expression of H19 and Igf2 Zengyan Wang, M.D.,a,b Ling Xu, M.D.,a and Fangfang He, M.D.a a
Department of Obstetrics and Gynecology, Peking Union Medical College Hospital, Peking Union Medical College, Beijing; and b Reproductive Medicine Center, First Affiliated Hospital, Sun Yat-sen University, Guangzhou, People’s Republic of China
Objective: To investigate the effects of embryo vitrification on the methylation status of the H19/Igf2 differentially methylated domain (DMD) and the expression of those genes. Design: Comparative and controlled study. Setting: A university-affiliated hospital. Animals: ICR female and male mice. Intervention(s): Day 14 fetuses that arose from copulation of male and female mice were taken as the control group. Embryos resulting from superovulation, IVF, in vitro culture, and embryo transfer were taken as the IVF group. Embryos that were vitrified were taken as the vitrified group. Main Outcome Measure(s): The methylation status of H19/Igf2 DMD was analyzed by cloning and sequencing following DNA bisulfite treatment. The expression of H19 and Igf2 was quantified by real-time reverse-transcription polymerase chain reaction. Result(s): Loss of methylation was found in the H19/Igf2 DMD of the fetuses from the IVF group, but it was more severe in the vitrified group. H19 expression was significantly increased in the IVF and vitrified groups, as compared with the control group; however, the quantity in the vitrified group was less than that detected in the IVF group. Igf2 expression in the IVF and vitrified groups was found to have decreased significantly, whereas the Igf2 expression in the vitrified group was greater than in the IVF group. Conclusion: Embryo vitrification aggravated the loss of methylation in the H19/Igf2 DMD, and compensated for the perturbed expression of H19 by IVF procedures. (Fertil Steril 2010;93:2729–33. 2010 by American Society for Reproductive Medicine.) Key Words: Embryo, vitrification, mouse, imprinting
Although imprinting disorders are relatively rare, the effects on those children born from assisted reproductive technologies (ARTs) should not be neglected. Many imprinted genes are involved in regulating proper embryo development and cell proliferation; therefore, imprinting disorders can be associated with intrauterine growth retardation (IUGR), low birth weight (LBW), and tumor genesis. Genomic imprinting is an epigenetic mechanism wherein one allele is silenced in a parental specific manner. The genes that undergo such a process are termed imprinted genes. Most of the imprinted genes are located in clusters throughout the genome and are regulated through epigenetic modifications in their corresponding imprinted control regions (ICRs), also known as the differentially methylated domain (DMD). The epigenetic modifications that affect these regions include differential methylation of DNA, methylation/ acetylation of histones, and production of antisense RNA (1, 2). The differential methylation of DNA in ICRs is the most studied model of epigenetic modification. During embryo development, imprinting Received August 27, 2009; revised March 5, 2010; accepted March 8, 2010; published online April 18, 2010. Z.W. has nothing to disclose. L.X. has nothing to disclose. F.H. has nothing to disclose. Supported by grant no. 2008BAI57B02 from the Chinese Ministry of Science and Technology. Reprint requests: Fangfang He, M.D., Department of Obstetrics and Gynecology, Peking Union Medical College Hospital, Peking Union Medical College, 1 Shuaifuyuan Alley, Dongcheng District, Beijing, People’s Republic of China (FAX: 86-010-65296223; E-mail: hefangfangmd@ yahoo.com.cn).
0015-0282/$36.00 doi:10.1016/j.fertnstert.2010.03.025
is first erased in primary germ cells (PGCs), re-established during gametogenesis, and then maintained after fertilization and throughout subsequent development. The genome-wide demethylation and remethylation process occurs in the embryos during the preimplantation period, while the DMDs of the imprinted genes are maintained (3). During the methylation changing period, some environmental conditions—such as ovarian stimulation, IVF, embryo culture, embryo transfer (ET) and embryo cryopreservation—may cause modifications to genomic imprinting regions and affect the development of the fetus. Evidence obtained from animal studies has shown that various ART manipulations can affect imprinted genes (4–6), including ovarian stimulation, IVF, embryo culture, and ET. However, relatively few studies (7–9) have considered the safety of embryo cryopreservation on the DNA level, and most of them focused on the apoptosis and gene expression; none were involved in imprinted genes. The aim of this study was to explore the effects of embryo cryopreservation on the fetus and placenta in a murine animal model.
MATERIALS AND METHODS This study was approved by the Institutional Review Board of the Peking Union Medical College.
Animals All ICR mice used in this study were of the specific pathogen free (SPF) class and fed in SPF animal rooms that were maintained by the Beijing Vital River
Fertility and Sterility Vol. 93, No. 8, May 15, 2010 Copyright ª2010 American Society for Reproductive Medicine, Published by Elsevier Inc.
2729
FIGURE 1 Illustration of experiment design. Ovulating ICR females were selected to mate with ICR males and checked for a vaginal plug the morning following mating. The day that the vaginal plug was first seen was designated as day 0.5. On day 14, females were euthanized and the fetuses and placentas were immediately removed. To avoid contamination of trophoblastic tissue with the fetuses, we completely split the membrane outside of the conceptus with a pair of fine forceps. Samples were snap-frozen in liquid nitrogen. In the IVF group and vitrified group, the superovulation, IVF and culture protocols were the same as previously described (10). All the morulae were divided into two subgroups (IVF and vitrified). Those in the IVF group were cultured to blastocysts and transferred into the uteruses of the pseudopregnant ICR females; those in the vitrified group were cryopreserved, warmed when needed, and cultured to blastocysts and transferred. A total of six blastocysts were transferred to each uterine horn of the pseudopregnant ICR female. Fresh embryos were transferred into the left horns and cryopreserved embryos were transferred into the right. The day of transfer occurred on day 4 according to the embryo age. The fetuses and placentas were collected on day 14.
Wang. Embryo vitrification affects imprinting. Fertil Steril 2010.
Experimental Animals Centre. All culture media used in this study were similar to those used in our previous publication concerning oocyte vitrification (10). The protocol used for embryo vitrification and warming was previously described for the IVF cycles in our hospital (11). Figure 1 illustrates the experimental design.
Methylation Analysis of the H19/Igf2 DMD Fetal tissues and placentas were removed at specified time points and finely chopped with a stainless-steel razor blade. Each sample was then divided into two equal portions; one was used for genomic DNA extraction, the other for total RNA extraction via commercially supplied kit according to the manufacturer’s instructions (Qiagen, Crawley, West Sussex, U.K.). Bisulfite treatment of DNA was performed according to a modified protocol by Mann et al. (12). DNA (2–5 mg) was dissolved in 18 mL sterile water and denatured at 95 C for 20 min; 2 mL of 3 M NaOH was added to the solution, and DNA was allowed to incubate at 42 C for 20 min; 380 mL of bisulfite and hydroquinone solution (5 M bisulfite and 125 mM hydroquinone, freshly prepared) was then added. The mixtures were overlaid with
2730
Wang et al.
Embryo vitrification affects imprinting
mineral oil and incubated at 50 C for 12–16 h in the dark. The converted DNA was purified by a kit (Promega Wizard Cleanup DNA system, Madison, WI). A total of 11 mL of 3 M NaOH was added, and the solution was placed in a 37 C water bath for 15 min to terminate the bisulfite conversion. Next, 166 mL of 5 M ammonium acetate and 750 mL of 2.5 volume alcohol was added. The solution remained at –70 C for 30 minutes to allow for efficient precipitation of DNA. DNA was then washed twice using 200 mL of 70% alcohol, dissolved in 10 mM TE (Tris-HCl, EDTA, pH 5.0), and stored at –20 C until use. Bisulfite converted DNA was amplified by nested PCR. The primers were designed using Methyl Primer Express V1.0 software (Foster City, CA); the first and second round PCR programs were identical (Table 1). The presence of amplified products was analyzed by electrophoresis on 1.5% agarose gel. The PCR products were ligated into the pEasy-T3 vector (Transgene, Beijing, China), and transformed into Top10 cells (Transgene, Beijing, China). The pEasy-T3 vector includes an LacZ gene. Therefore, we put X-gal (Sigma-Aldrich, St. Louis, MO) and IPTG (Sigma-Aldrich) on the surface of the agar plates and performed white-blue plaque selection to pick 15–20
Vol. 93, No. 8, May 15, 2010
TABLE 1 Primers used for DNA methylation and gene expression analysis.
Gene
Primers
5’-3’ sequence
Methylation analysis H19/Igf2 DMD Primers 1 Sense gag tat tta gga ggt ata aga att Antisense atc aaa aac taa cat aaa ccc ct Primers 2-Nested Sense gta agg aga tta tgt tta ttt ttg g Antisense cta acc tca taa aac cca taa cta t Expression analysis H19 Sense tgc act ggt ttg gag tcc cg Antisense gct tcc aga cta ggc gag gg Igf2 Sense atc tgt gac ctc ttg agc agg Antisense ggg ttg ttt aga gcc aat caa Gapdh Sense tac ccc caa tgt gtc cgt cgt Antisense ggg tgg tcc agg gtt tct tac
Annealing temperature
Cycle number
PCR product (bp)
Sequence reference
55
35
473
55
35
427
58
40
180
NR_001592.1
58
40
198
NM_001122737.1
58
40
318
XM_001473623.1
U19619
Wang. Embryo vitrification affects imprinting. Fertil Steril 2010.
positive clones for each sample. Next, we used M13 primers to amplify the positive clones by PCR. If the length of the PCR products included the inverted fragment and the sequences in the T vector, the clone was designated as truly positive and subsequently sequenced (ABI PRISM-77, Applied Biosystems, Foster City, CA). We sorted the clones into two categories according to the percentages of methylated CpG Cytosine Guanine dinucleotide: >50% as hypermethylated and <50% as hypomethylated. The percentages of hypermethylated and hypomethylated clones in each fetus and placenta of all three groups were calculated. Figure 2 provides a sample of how the calculation was performed.
Quantification of H19 and Igf2 Expression by Real-Time Reverse Transcription PCR Total RNA were extracted with Trizol (Invitrogen, Carlsbad, CA) and dissolved in 10 mL Tris (10 mM, pH 7.6) and stored at 80 C until use. The total RNA was reverse transcribed to cDNA by the RT enzyme Superscript III (Takara Bio Inc., Otsu, Shiga, Japan). The housekeeping gene glyceraldehyde phosphate dehydrogenase (Gapdh) was analyzed as an internal control. The primers for H19, Igf2, and Gapdh are listed in Table 1. The calculation of gene expression was as follows, using H19 in the fetus (F) of the IVF group as an example: H19F ¼ 2DDCt;DD Ct ¼ [Ct(H19F) Ct(GapdhF)]IVF [Ct(H19F) Ct(GapdhF)]control.
Statistical Analysis Data were analyzed by the SPSS 13.0 software package (Cary, NC, USA). The proportions of hypermethylated clones among different groups were determined to be normally distributed, with homogeneity of variance; they were compared by one way ANOVA. The H19DDCt and Igf2DDCt between different groups were all normally distributed, and H19 fetuses (F)DDCt were without homogeneity of variance; they were compared with Dunnett’s T3 for multiple comparisons, whereas H19 placenta (P)DDCt, Igf2FDDCt, and Igf2PDDCt were with homogeneity of variance and were compared by Student-Newmann-Keuls test for multiple comparisons.
group) and 50 fetuses and placentas from the right horns (vitrified group). The implantation rates for the two experimental subgroups were 88.3% and 83.3%, respectively, with no statistically significant difference between the groups. There were no significant differences in the morphology of embryos between the IVF and vitrified groups, and no differences in the morphology of fetuses and placentas among all three groups.
Percentages of Hypermethylated Clones in the Fetuses and Placentas Approximately 50% of hypermethylated clones were from fetuses in the control group were, with an average of 51.4% 1.13% (mean SD). Compared with the controls, the percentages of hypermethylated clones in the IVF and vitrified groups were significantly lower, with average percentages of 37.79% 0.76% and 20.16% 0.97%, respectively. Multiple comparisons revealed that the differences between groups were statistically significant (P < 0.05; Fig. 3).
FIGURE 2 The sequencing result of a representative fetus from the control group. Seven hypermethylated and seven hypomethylated clones were obtained, accounting for 50% each. Each row represents a unique methylation status of the 14 successfully sequenced clones of the analyzed fetus, with the frequency of that methylation state shown in parentheses to the right. Each circle within the row represents a single CpG site; open and closed circles represent unmethylated and methylated CpGs, respectively.
RESULTS Implantation Rate of Fresh and Cryopreserved Embryos In the control group, five females became pregnant and yielded 51 fetuses and placentas. In the study group, 10 pseudopregnant ICR females underwent successful embryo transfer, from which we obtained 53 fetuses and placentas from the left uterine horns (IVF Fertility and Sterility
Wang. Embryo vitrification affects imprinting. Fertil Steril 2010.
2731
FIGURE 3 (A) Comparisons of the sequencing results from the fetuses and placentas. (B) Comparisons of H19 expression. (C) Comparisons of Igf2 expression. Control population, n ¼ 51; IVF group, n ¼ 53; vitrified group, n ¼ 50. *Statistically significant compared with the control. *Statistically significant compared with the IVF group. F ¼ fetuses; P ¼ placentas.
Wang. Embryo vitrification affects imprinting. Fertil Steril 2010.
The average percentages of hypermethylated clones in the placentas of the above three groups was 53.24% 1.11% (control), 32.49% 1.24% (IVF), 36.87% 0.97% (vitrified). Upon multiple comparisons, the average percentages in the IVF and vitrified groups were found to be significantly lower than in the control group, whereas no significant differences were found between the vitrified group and the IVF group (Fig. 3).
Expression Levels of H19 in the Fetuses and Placentas Compared with the control group, the H19FDDCt in the IVF group and the vitrified group were 3.53 0.43 and 1.77 0.32, respectively. The differences in the three groups, or between any two, were statistically significant (P < 0.05; Fig. 3). The H19PDDCt in the three groups was 0.00 0.37 (control), 2.44 0.56 (IVF) and 3.79 0.81 (vitrified). The differences in the three groups, or between any two, were statistically significant (P < 0.05; Fig. 3).
Expression Levels of Igf2 in the Fetuses and Placentas The Igf2FDDCt in the control, IVF, and vitrified groups was 0.00 0.29, 2.01 0.43, and 1.21 0.24, respectively. The differences among the three groups, or between any two, were statistically significant (P < 0.05; Fig. 3). The Igf2PDDCt in the three groups was 0.00 0.37 (control), 1.48 0.23 (IVF), and 1.69 0.45 (vitrified). The differences among the three groups, between the IVF group and the control, and between the vitrified group and the control, were statistically significant. However, the differences between the IVF group and the vitrified group were not statistically significant (P ¼ 0.05; Fig. 3).
DISCUSSION In the mouse, H19 is located at the distal end of chromosome 7. The regulation mechanism of this gene, however, is not completely clear. The expression of both H19 and Igf2 would then be regulated by the methylation of the H19/Igf2 DMD located upstream of H19, wherein both of them reciprocally compete for the enhancer downstream of H19 (4). In the locus of H19/Igf2, enhancers can activate maternal H19 and paternal Igf2 (13). Differential expression is caused by the epigenetic switch located upstream of H19, a region that contains the DMD. In the maternal allele, this region was unmethylated or hypomethylated and could bind with a kind of zinc finger protein named
2732
Wang et al.
Embryo vitrification affects imprinting
CTCF. The enhancer activated H19 expression and resulted in inhibition of Igf2 expression. In the paternal allele, this region was hypermethylated and could not bind CTCF, thus the enhancer could access the Igf2 promoter and drive the paternal transcription, whereas H19 expression was inhibited by methylation. The epigenetic switch acted as an insulator between the enhancer and Igf2. Engel et al. (14) proved that H19/Igf2 activity was associated with the location of the promoter and the enhancer by tracking the enhancer of H19/Igf2 through three-dimensional analysis. The different expression quantities of H19/Igf2 in our study indicated differential regulation status of H19/Igf2 DMD.
Methylation Status of the H19/Igf2 DMD Studies (5, 6) on animals indicated that H19 was sensitive to manipulations such as ovarian stimulation and in vitro culture. H19 was shown to regulate the development of the fetus and placenta through the expression of Igf2 (3, 15). Children born via ART and who have Beckwith-Wiedemann syndrome also showed an association with H19 disorders (16). Both the fetuses and placentas were obviously affected by IVF and vitrification. In contrast, Rivera et al. (6) showed that fetuses were affected less than placentas. These contradictory findings may be the result of using different mouse lines, different manipulations, or different culture media. The disordered methylation of ART manipulations to mouse H19 ICRs included gain of methylation (17) and loss of methylation (6), which persisted in the postimplantation embryos. The manipulations in our IVF group fetuses decreased the methylation status of the H19/Igf2 DMD; vitrification appeared to aggravate this condition by comparison. Significant loss of methylation was also found in the placentas; however, the differences in the fetuses between IVF group and vitrified group were significant, however in the placentas were not significant. The cells surrounding the morulae developed to trophectoderm and became placenta, while the cells inside developed into the inner cell mass and became the fetus. We postulated that in vitrification, the cells surrounding the morulae are exposed for a longer period of time to cryoprotectants, and that the cooling speed would also be faster than experienced by the cells inside. These differences might explain the different methylation effects in fetuses and placentas. Vol. 93, No. 8, May 15, 2010
Expression of H19 and Igf2 H19ICR is an important regulator of the expression of H19 (18), so we postulated that H19 expression in the IVF and vitrified groups might increase, and that the reciprocally regulated Igf2 might decrease, so we quantified the H19 and Igf2 expression by real-time reverse transcription PCR. Compared with the controls, the expression of H19 in the fetuses of the IVF and vitrified groups were significantly increased, although those in the vitrified group were lower than in the IVF group. The expression in the placentas of the IVF group was significantly decreased, and it was decreased more in the vitrified group. The results obtained for the fetuses coincided with the methylation status of the H19/Igf2 DMD. However, there were several unexpected findings. Although the methylation in the fetuses of the vitrified group decreased more than those of the IVF group, the H19 expression did not increase in a corresponding manner. Fauque et al. (5) analyzed blastocysts and also found that the expression of H19 was not associated with the methylation of ICRs. The imprinting and expression of H19 was not only regulated by the methylation of ICRs, but also by the modification of histones. Vitrification can affect other factors associated with H19 expression. The H19/Igf2 DMD methylation decreased in the placentas of the IVF and vitrified groups, whereas the H19 expression decreased. The fact that H19 expression in the placentas disagreed with the H19/Igf2 DMD methylation status might be attributable to the theory that the imprint of
H19 in the fetus was mainly caused by DNA methylation, whereas in the placenta it was mainly a result of histone modification (6). The Igf2 expression in the fetuses of the IVF and vitrified groups decreased significantly compared with the control group, but increased significantly in placentas. The trend was opposite to that observed for H19 expression, but consistent with the imprint model of H19/Igf2 (19). Compared with the IVF group, the Igf2 expression in the fetuses of the vitrified group increased relatively. In the placentas, no significant changes were found, indicating that vitrification did not aggravate the disordered Igf2 expression in the IVF group and might have resulted in a compensation effect. This result may be attributable to other factors associated with Igf2 expression that were affected by vitrification, or to the fact that vitrification affected other factors in IVF.
CONCLUSIONS Although we could not analyze the methylation status and expression of each parental allele, the result of this research indicates that embryo vitrification could cause abberant methylation to H19 ICRs, with compensation of the disordered H19/Igf2 expression in IVF, but not affecting the H19 or Igf2 expression in placentas. To fully understand the underlying mechanism, further study is required. We are unable to generalize these results directly to humans because of the genetic differences between humans and mice.
REFERENCES 1. Holmes R, Soloway PD. Regulation of imprinted DNA methylation. Cytogenet Genome Res 2006;113:122–9. 2. Lewis A, Reik W. How imprinting centres work. Cytogenet Genome Res 2006;113:81–9. 3. Reik W, Constancia M, Fowden A, Anderson N, Dean W, Ferguson-Smith A, et al. Regulation of supply and demand for maternal nutrients in mammals by imprinted genes. J Physiol 2003;547:35–44. 4. Li T, Vu TH, Ulaner GA, Littman E, Ling JQ, Chen HL, et al. IVF results in de novo DNA methylation and histone methylation at an Igf2-H19 imprinting epigenetic switch. Mol Hum Reprod 2005;11: 631–40. 5. 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 expression in individual mouse embryos. BMC Dev Biol 2007;7:116. 6. Rivera RM, Stein P, Weaver JR, Mager J, Schultz RM, Bartolomei MS. Manipulations of mouse embryos prior to implantation result in aberrant expression of imprinted genes on day 9.5 of development. Hum Mol Genet 2008;17:1–14. 7. Park SY, Kim EY, Cui XS, Tae JC, Lee WD, Kim NH, et al. Increase in DNA fragmentation and apoptosis-
Fertility and Sterility
8.
9.
10.
11.
12.
13.
related gene expression in frozen-thawed bovine blastocysts. Zygote 2006;14:125–31. Mamo S, Bodo S, Kobolak J, Polgar Z, Tolgyesi G, Dinnyes A. Gene expression profiles of vitrified in vivo derived 8-cell stage mouse embryos detected by high density oligonucleotide microarrays. Mol Reprod Dev 2006;73:1380–92. Dhali A, Anchamparuthy VM, Butler SP, Pearson RE, Mullarky IK, Gwazdauskas FC. Gene expression and development of mouse zygotes following droplet vitrification. Theriogenology 2007;68:1292–8. Wang Z, Sun Z, Chen Y, He F. A modified cryoloop vitrification protocol in the cryopreservation of mature mouse oocytes. Zygote 2009;17:217–24. Sun ZY, He FF, Yu Q, Deng CY. The application of vitrification in human blastocyst cryopreservation: Analysis of clinical outcome of 117 cycles. J Reprod Med 2006;43–7. Mann MR, Chung YG, Nolen LD, Verona RI, Latham KE, Bartolomei MS. Disruption of imprinted gene methylation and expression in cloned preimplantation stage mouse embryos. Biol Reprod 2003;69:902–14. Kaffer CR, Grinberg A, Pfeifer K. Regulatory mechanisms at the mouse Igf2/H19 locus. Mol Cell Biol 2001;21:8189–96.
14. Engel N, Raval AK, Thorvaldsen JL, Bartolomei SM. Three-dimensional conformation at the H19/Igf2 locus supports a model of enhancer tracking. Hum Mol Genet 2008;17:3021–9. 15. Eggenschwiler J, Ludwig T, Fisher P, Leighton PA, Tilghman SM, Efstratiadis A. Mouse mutant embryos overexpressing IGF-II exhibit phenotypic features of the Beckwith-Wiedemann and Simpson-GolabiBehmel syndromes. Genes Dev 1997;11:3128–42. 16. Gicquel C, Gaston V, Mandelbaum J, Siffroi JP, Flahault A, Le Bouc Y. In vitro fertilization may increase the risk of Beckwith-Wiedemann syndrome related to the abnormal imprinting of the KCN1OT gene. Am J Hum Genet 2003;72:1338–41. 17. 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. 18. 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: 3727–35. 19. Sasaki H, Ishihara K, Kato R. Mechanisms of Igf2/ H19 imprinting: DNA methylation, chromatin and long-distance gene regulation. J Biochem 2000;127: 711–5.
2733
Department of Obstetrics and Gynecology, Peking Union Medical College Hospital, Peking Union Medical College, Beijing; and b Reproductive Medicine Center, First Affiliated Hospital, Sun Yat-sen University, Guangzhou, People’s Republic of China
Objective: To investigate the effects of embryo vitrification on the methylation status of the H19/Igf2 differentially methylated domain (DMD) and the expression of those genes. Design: Comparative and controlled study. Setting: A university-affiliated hospital. Animals: ICR female and male mice. Intervention(s): Day 14 fetuses that arose from copulation of male and female mice were taken as the control group. Embryos resulting from superovulation, IVF, in vitro culture, and embryo transfer were taken as the IVF group. Embryos that were vitrified were taken as the vitrified group. Main Outcome Measure(s): The methylation status of H19/Igf2 DMD was analyzed by cloning and sequencing following DNA bisulfite treatment. The expression of H19 and Igf2 was quantified by real-time reverse-transcription polymerase chain reaction. Result(s): Loss of methylation was found in the H19/Igf2 DMD of the fetuses from the IVF group, but it was more severe in the vitrified group. H19 expression was significantly increased in the IVF and vitrified groups, as compared with the control group; however, the quantity in the vitrified group was less than that detected in the IVF group. Igf2 expression in the IVF and vitrified groups was found to have decreased significantly, whereas the Igf2 expression in the vitrified group was greater than in the IVF group. Conclusion: Embryo vitrification aggravated the loss of methylation in the H19/Igf2 DMD, and compensated for the perturbed expression of H19 by IVF procedures. (Fertil Steril 2010;93:2729–33. 2010 by American Society for Reproductive Medicine.) Key Words: Embryo, vitrification, mouse, imprinting
Although imprinting disorders are relatively rare, the effects on those children born from assisted reproductive technologies (ARTs) should not be neglected. Many imprinted genes are involved in regulating proper embryo development and cell proliferation; therefore, imprinting disorders can be associated with intrauterine growth retardation (IUGR), low birth weight (LBW), and tumor genesis. Genomic imprinting is an epigenetic mechanism wherein one allele is silenced in a parental specific manner. The genes that undergo such a process are termed imprinted genes. Most of the imprinted genes are located in clusters throughout the genome and are regulated through epigenetic modifications in their corresponding imprinted control regions (ICRs), also known as the differentially methylated domain (DMD). The epigenetic modifications that affect these regions include differential methylation of DNA, methylation/ acetylation of histones, and production of antisense RNA (1, 2). The differential methylation of DNA in ICRs is the most studied model of epigenetic modification. During embryo development, imprinting Received August 27, 2009; revised March 5, 2010; accepted March 8, 2010; published online April 18, 2010. Z.W. has nothing to disclose. L.X. has nothing to disclose. F.H. has nothing to disclose. Supported by grant no. 2008BAI57B02 from the Chinese Ministry of Science and Technology. Reprint requests: Fangfang He, M.D., Department of Obstetrics and Gynecology, Peking Union Medical College Hospital, Peking Union Medical College, 1 Shuaifuyuan Alley, Dongcheng District, Beijing, People’s Republic of China (FAX: 86-010-65296223; E-mail: hefangfangmd@ yahoo.com.cn).
0015-0282/$36.00 doi:10.1016/j.fertnstert.2010.03.025
is first erased in primary germ cells (PGCs), re-established during gametogenesis, and then maintained after fertilization and throughout subsequent development. The genome-wide demethylation and remethylation process occurs in the embryos during the preimplantation period, while the DMDs of the imprinted genes are maintained (3). During the methylation changing period, some environmental conditions—such as ovarian stimulation, IVF, embryo culture, embryo transfer (ET) and embryo cryopreservation—may cause modifications to genomic imprinting regions and affect the development of the fetus. Evidence obtained from animal studies has shown that various ART manipulations can affect imprinted genes (4–6), including ovarian stimulation, IVF, embryo culture, and ET. However, relatively few studies (7–9) have considered the safety of embryo cryopreservation on the DNA level, and most of them focused on the apoptosis and gene expression; none were involved in imprinted genes. The aim of this study was to explore the effects of embryo cryopreservation on the fetus and placenta in a murine animal model.
MATERIALS AND METHODS This study was approved by the Institutional Review Board of the Peking Union Medical College.
Animals All ICR mice used in this study were of the specific pathogen free (SPF) class and fed in SPF animal rooms that were maintained by the Beijing Vital River
Fertility and Sterility Vol. 93, No. 8, May 15, 2010 Copyright ª2010 American Society for Reproductive Medicine, Published by Elsevier Inc.
2729
FIGURE 1 Illustration of experiment design. Ovulating ICR females were selected to mate with ICR males and checked for a vaginal plug the morning following mating. The day that the vaginal plug was first seen was designated as day 0.5. On day 14, females were euthanized and the fetuses and placentas were immediately removed. To avoid contamination of trophoblastic tissue with the fetuses, we completely split the membrane outside of the conceptus with a pair of fine forceps. Samples were snap-frozen in liquid nitrogen. In the IVF group and vitrified group, the superovulation, IVF and culture protocols were the same as previously described (10). All the morulae were divided into two subgroups (IVF and vitrified). Those in the IVF group were cultured to blastocysts and transferred into the uteruses of the pseudopregnant ICR females; those in the vitrified group were cryopreserved, warmed when needed, and cultured to blastocysts and transferred. A total of six blastocysts were transferred to each uterine horn of the pseudopregnant ICR female. Fresh embryos were transferred into the left horns and cryopreserved embryos were transferred into the right. The day of transfer occurred on day 4 according to the embryo age. The fetuses and placentas were collected on day 14.
Wang. Embryo vitrification affects imprinting. Fertil Steril 2010.
Experimental Animals Centre. All culture media used in this study were similar to those used in our previous publication concerning oocyte vitrification (10). The protocol used for embryo vitrification and warming was previously described for the IVF cycles in our hospital (11). Figure 1 illustrates the experimental design.
Methylation Analysis of the H19/Igf2 DMD Fetal tissues and placentas were removed at specified time points and finely chopped with a stainless-steel razor blade. Each sample was then divided into two equal portions; one was used for genomic DNA extraction, the other for total RNA extraction via commercially supplied kit according to the manufacturer’s instructions (Qiagen, Crawley, West Sussex, U.K.). Bisulfite treatment of DNA was performed according to a modified protocol by Mann et al. (12). DNA (2–5 mg) was dissolved in 18 mL sterile water and denatured at 95 C for 20 min; 2 mL of 3 M NaOH was added to the solution, and DNA was allowed to incubate at 42 C for 20 min; 380 mL of bisulfite and hydroquinone solution (5 M bisulfite and 125 mM hydroquinone, freshly prepared) was then added. The mixtures were overlaid with
2730
Wang et al.
Embryo vitrification affects imprinting
mineral oil and incubated at 50 C for 12–16 h in the dark. The converted DNA was purified by a kit (Promega Wizard Cleanup DNA system, Madison, WI). A total of 11 mL of 3 M NaOH was added, and the solution was placed in a 37 C water bath for 15 min to terminate the bisulfite conversion. Next, 166 mL of 5 M ammonium acetate and 750 mL of 2.5 volume alcohol was added. The solution remained at –70 C for 30 minutes to allow for efficient precipitation of DNA. DNA was then washed twice using 200 mL of 70% alcohol, dissolved in 10 mM TE (Tris-HCl, EDTA, pH 5.0), and stored at –20 C until use. Bisulfite converted DNA was amplified by nested PCR. The primers were designed using Methyl Primer Express V1.0 software (Foster City, CA); the first and second round PCR programs were identical (Table 1). The presence of amplified products was analyzed by electrophoresis on 1.5% agarose gel. The PCR products were ligated into the pEasy-T3 vector (Transgene, Beijing, China), and transformed into Top10 cells (Transgene, Beijing, China). The pEasy-T3 vector includes an LacZ gene. Therefore, we put X-gal (Sigma-Aldrich, St. Louis, MO) and IPTG (Sigma-Aldrich) on the surface of the agar plates and performed white-blue plaque selection to pick 15–20
Vol. 93, No. 8, May 15, 2010
TABLE 1 Primers used for DNA methylation and gene expression analysis.
Gene
Primers
5’-3’ sequence
Methylation analysis H19/Igf2 DMD Primers 1 Sense gag tat tta gga ggt ata aga att Antisense atc aaa aac taa cat aaa ccc ct Primers 2-Nested Sense gta agg aga tta tgt tta ttt ttg g Antisense cta acc tca taa aac cca taa cta t Expression analysis H19 Sense tgc act ggt ttg gag tcc cg Antisense gct tcc aga cta ggc gag gg Igf2 Sense atc tgt gac ctc ttg agc agg Antisense ggg ttg ttt aga gcc aat caa Gapdh Sense tac ccc caa tgt gtc cgt cgt Antisense ggg tgg tcc agg gtt tct tac
Annealing temperature
Cycle number
PCR product (bp)
Sequence reference
55
35
473
55
35
427
58
40
180
NR_001592.1
58
40
198
NM_001122737.1
58
40
318
XM_001473623.1
U19619
Wang. Embryo vitrification affects imprinting. Fertil Steril 2010.
positive clones for each sample. Next, we used M13 primers to amplify the positive clones by PCR. If the length of the PCR products included the inverted fragment and the sequences in the T vector, the clone was designated as truly positive and subsequently sequenced (ABI PRISM-77, Applied Biosystems, Foster City, CA). We sorted the clones into two categories according to the percentages of methylated CpG Cytosine Guanine dinucleotide: >50% as hypermethylated and <50% as hypomethylated. The percentages of hypermethylated and hypomethylated clones in each fetus and placenta of all three groups were calculated. Figure 2 provides a sample of how the calculation was performed.
Quantification of H19 and Igf2 Expression by Real-Time Reverse Transcription PCR Total RNA were extracted with Trizol (Invitrogen, Carlsbad, CA) and dissolved in 10 mL Tris (10 mM, pH 7.6) and stored at 80 C until use. The total RNA was reverse transcribed to cDNA by the RT enzyme Superscript III (Takara Bio Inc., Otsu, Shiga, Japan). The housekeeping gene glyceraldehyde phosphate dehydrogenase (Gapdh) was analyzed as an internal control. The primers for H19, Igf2, and Gapdh are listed in Table 1. The calculation of gene expression was as follows, using H19 in the fetus (F) of the IVF group as an example: H19F ¼ 2DDCt;DD Ct ¼ [Ct(H19F) Ct(GapdhF)]IVF [Ct(H19F) Ct(GapdhF)]control.
Statistical Analysis Data were analyzed by the SPSS 13.0 software package (Cary, NC, USA). The proportions of hypermethylated clones among different groups were determined to be normally distributed, with homogeneity of variance; they were compared by one way ANOVA. The H19DDCt and Igf2DDCt between different groups were all normally distributed, and H19 fetuses (F)DDCt were without homogeneity of variance; they were compared with Dunnett’s T3 for multiple comparisons, whereas H19 placenta (P)DDCt, Igf2FDDCt, and Igf2PDDCt were with homogeneity of variance and were compared by Student-Newmann-Keuls test for multiple comparisons.
group) and 50 fetuses and placentas from the right horns (vitrified group). The implantation rates for the two experimental subgroups were 88.3% and 83.3%, respectively, with no statistically significant difference between the groups. There were no significant differences in the morphology of embryos between the IVF and vitrified groups, and no differences in the morphology of fetuses and placentas among all three groups.
Percentages of Hypermethylated Clones in the Fetuses and Placentas Approximately 50% of hypermethylated clones were from fetuses in the control group were, with an average of 51.4% 1.13% (mean SD). Compared with the controls, the percentages of hypermethylated clones in the IVF and vitrified groups were significantly lower, with average percentages of 37.79% 0.76% and 20.16% 0.97%, respectively. Multiple comparisons revealed that the differences between groups were statistically significant (P < 0.05; Fig. 3).
FIGURE 2 The sequencing result of a representative fetus from the control group. Seven hypermethylated and seven hypomethylated clones were obtained, accounting for 50% each. Each row represents a unique methylation status of the 14 successfully sequenced clones of the analyzed fetus, with the frequency of that methylation state shown in parentheses to the right. Each circle within the row represents a single CpG site; open and closed circles represent unmethylated and methylated CpGs, respectively.
RESULTS Implantation Rate of Fresh and Cryopreserved Embryos In the control group, five females became pregnant and yielded 51 fetuses and placentas. In the study group, 10 pseudopregnant ICR females underwent successful embryo transfer, from which we obtained 53 fetuses and placentas from the left uterine horns (IVF Fertility and Sterility
Wang. Embryo vitrification affects imprinting. Fertil Steril 2010.
2731
FIGURE 3 (A) Comparisons of the sequencing results from the fetuses and placentas. (B) Comparisons of H19 expression. (C) Comparisons of Igf2 expression. Control population, n ¼ 51; IVF group, n ¼ 53; vitrified group, n ¼ 50. *Statistically significant compared with the control. *Statistically significant compared with the IVF group. F ¼ fetuses; P ¼ placentas.
Wang. Embryo vitrification affects imprinting. Fertil Steril 2010.
The average percentages of hypermethylated clones in the placentas of the above three groups was 53.24% 1.11% (control), 32.49% 1.24% (IVF), 36.87% 0.97% (vitrified). Upon multiple comparisons, the average percentages in the IVF and vitrified groups were found to be significantly lower than in the control group, whereas no significant differences were found between the vitrified group and the IVF group (Fig. 3).
Expression Levels of H19 in the Fetuses and Placentas Compared with the control group, the H19FDDCt in the IVF group and the vitrified group were 3.53 0.43 and 1.77 0.32, respectively. The differences in the three groups, or between any two, were statistically significant (P < 0.05; Fig. 3). The H19PDDCt in the three groups was 0.00 0.37 (control), 2.44 0.56 (IVF) and 3.79 0.81 (vitrified). The differences in the three groups, or between any two, were statistically significant (P < 0.05; Fig. 3).
Expression Levels of Igf2 in the Fetuses and Placentas The Igf2FDDCt in the control, IVF, and vitrified groups was 0.00 0.29, 2.01 0.43, and 1.21 0.24, respectively. The differences among the three groups, or between any two, were statistically significant (P < 0.05; Fig. 3). The Igf2PDDCt in the three groups was 0.00 0.37 (control), 1.48 0.23 (IVF), and 1.69 0.45 (vitrified). The differences among the three groups, between the IVF group and the control, and between the vitrified group and the control, were statistically significant. However, the differences between the IVF group and the vitrified group were not statistically significant (P ¼ 0.05; Fig. 3).
DISCUSSION In the mouse, H19 is located at the distal end of chromosome 7. The regulation mechanism of this gene, however, is not completely clear. The expression of both H19 and Igf2 would then be regulated by the methylation of the H19/Igf2 DMD located upstream of H19, wherein both of them reciprocally compete for the enhancer downstream of H19 (4). In the locus of H19/Igf2, enhancers can activate maternal H19 and paternal Igf2 (13). Differential expression is caused by the epigenetic switch located upstream of H19, a region that contains the DMD. In the maternal allele, this region was unmethylated or hypomethylated and could bind with a kind of zinc finger protein named
2732
Wang et al.
Embryo vitrification affects imprinting
CTCF. The enhancer activated H19 expression and resulted in inhibition of Igf2 expression. In the paternal allele, this region was hypermethylated and could not bind CTCF, thus the enhancer could access the Igf2 promoter and drive the paternal transcription, whereas H19 expression was inhibited by methylation. The epigenetic switch acted as an insulator between the enhancer and Igf2. Engel et al. (14) proved that H19/Igf2 activity was associated with the location of the promoter and the enhancer by tracking the enhancer of H19/Igf2 through three-dimensional analysis. The different expression quantities of H19/Igf2 in our study indicated differential regulation status of H19/Igf2 DMD.
Methylation Status of the H19/Igf2 DMD Studies (5, 6) on animals indicated that H19 was sensitive to manipulations such as ovarian stimulation and in vitro culture. H19 was shown to regulate the development of the fetus and placenta through the expression of Igf2 (3, 15). Children born via ART and who have Beckwith-Wiedemann syndrome also showed an association with H19 disorders (16). Both the fetuses and placentas were obviously affected by IVF and vitrification. In contrast, Rivera et al. (6) showed that fetuses were affected less than placentas. These contradictory findings may be the result of using different mouse lines, different manipulations, or different culture media. The disordered methylation of ART manipulations to mouse H19 ICRs included gain of methylation (17) and loss of methylation (6), which persisted in the postimplantation embryos. The manipulations in our IVF group fetuses decreased the methylation status of the H19/Igf2 DMD; vitrification appeared to aggravate this condition by comparison. Significant loss of methylation was also found in the placentas; however, the differences in the fetuses between IVF group and vitrified group were significant, however in the placentas were not significant. The cells surrounding the morulae developed to trophectoderm and became placenta, while the cells inside developed into the inner cell mass and became the fetus. We postulated that in vitrification, the cells surrounding the morulae are exposed for a longer period of time to cryoprotectants, and that the cooling speed would also be faster than experienced by the cells inside. These differences might explain the different methylation effects in fetuses and placentas. Vol. 93, No. 8, May 15, 2010
Expression of H19 and Igf2 H19ICR is an important regulator of the expression of H19 (18), so we postulated that H19 expression in the IVF and vitrified groups might increase, and that the reciprocally regulated Igf2 might decrease, so we quantified the H19 and Igf2 expression by real-time reverse transcription PCR. Compared with the controls, the expression of H19 in the fetuses of the IVF and vitrified groups were significantly increased, although those in the vitrified group were lower than in the IVF group. The expression in the placentas of the IVF group was significantly decreased, and it was decreased more in the vitrified group. The results obtained for the fetuses coincided with the methylation status of the H19/Igf2 DMD. However, there were several unexpected findings. Although the methylation in the fetuses of the vitrified group decreased more than those of the IVF group, the H19 expression did not increase in a corresponding manner. Fauque et al. (5) analyzed blastocysts and also found that the expression of H19 was not associated with the methylation of ICRs. The imprinting and expression of H19 was not only regulated by the methylation of ICRs, but also by the modification of histones. Vitrification can affect other factors associated with H19 expression. The H19/Igf2 DMD methylation decreased in the placentas of the IVF and vitrified groups, whereas the H19 expression decreased. The fact that H19 expression in the placentas disagreed with the H19/Igf2 DMD methylation status might be attributable to the theory that the imprint of
H19 in the fetus was mainly caused by DNA methylation, whereas in the placenta it was mainly a result of histone modification (6). The Igf2 expression in the fetuses of the IVF and vitrified groups decreased significantly compared with the control group, but increased significantly in placentas. The trend was opposite to that observed for H19 expression, but consistent with the imprint model of H19/Igf2 (19). Compared with the IVF group, the Igf2 expression in the fetuses of the vitrified group increased relatively. In the placentas, no significant changes were found, indicating that vitrification did not aggravate the disordered Igf2 expression in the IVF group and might have resulted in a compensation effect. This result may be attributable to other factors associated with Igf2 expression that were affected by vitrification, or to the fact that vitrification affected other factors in IVF.
CONCLUSIONS Although we could not analyze the methylation status and expression of each parental allele, the result of this research indicates that embryo vitrification could cause abberant methylation to H19 ICRs, with compensation of the disordered H19/Igf2 expression in IVF, but not affecting the H19 or Igf2 expression in placentas. To fully understand the underlying mechanism, further study is required. We are unable to generalize these results directly to humans because of the genetic differences between humans and mice.
REFERENCES 1. Holmes R, Soloway PD. Regulation of imprinted DNA methylation. Cytogenet Genome Res 2006;113:122–9. 2. Lewis A, Reik W. How imprinting centres work. Cytogenet Genome Res 2006;113:81–9. 3. Reik W, Constancia M, Fowden A, Anderson N, Dean W, Ferguson-Smith A, et al. Regulation of supply and demand for maternal nutrients in mammals by imprinted genes. J Physiol 2003;547:35–44. 4. Li T, Vu TH, Ulaner GA, Littman E, Ling JQ, Chen HL, et al. IVF results in de novo DNA methylation and histone methylation at an Igf2-H19 imprinting epigenetic switch. Mol Hum Reprod 2005;11: 631–40. 5. 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 expression in individual mouse embryos. BMC Dev Biol 2007;7:116. 6. Rivera RM, Stein P, Weaver JR, Mager J, Schultz RM, Bartolomei MS. Manipulations of mouse embryos prior to implantation result in aberrant expression of imprinted genes on day 9.5 of development. Hum Mol Genet 2008;17:1–14. 7. Park SY, Kim EY, Cui XS, Tae JC, Lee WD, Kim NH, et al. Increase in DNA fragmentation and apoptosis-
Fertility and Sterility
8.
9.
10.
11.
12.
13.
related gene expression in frozen-thawed bovine blastocysts. Zygote 2006;14:125–31. Mamo S, Bodo S, Kobolak J, Polgar Z, Tolgyesi G, Dinnyes A. Gene expression profiles of vitrified in vivo derived 8-cell stage mouse embryos detected by high density oligonucleotide microarrays. Mol Reprod Dev 2006;73:1380–92. Dhali A, Anchamparuthy VM, Butler SP, Pearson RE, Mullarky IK, Gwazdauskas FC. Gene expression and development of mouse zygotes following droplet vitrification. Theriogenology 2007;68:1292–8. Wang Z, Sun Z, Chen Y, He F. A modified cryoloop vitrification protocol in the cryopreservation of mature mouse oocytes. Zygote 2009;17:217–24. Sun ZY, He FF, Yu Q, Deng CY. The application of vitrification in human blastocyst cryopreservation: Analysis of clinical outcome of 117 cycles. J Reprod Med 2006;43–7. Mann MR, Chung YG, Nolen LD, Verona RI, Latham KE, Bartolomei MS. Disruption of imprinted gene methylation and expression in cloned preimplantation stage mouse embryos. Biol Reprod 2003;69:902–14. Kaffer CR, Grinberg A, Pfeifer K. Regulatory mechanisms at the mouse Igf2/H19 locus. Mol Cell Biol 2001;21:8189–96.
14. Engel N, Raval AK, Thorvaldsen JL, Bartolomei SM. Three-dimensional conformation at the H19/Igf2 locus supports a model of enhancer tracking. Hum Mol Genet 2008;17:3021–9. 15. Eggenschwiler J, Ludwig T, Fisher P, Leighton PA, Tilghman SM, Efstratiadis A. Mouse mutant embryos overexpressing IGF-II exhibit phenotypic features of the Beckwith-Wiedemann and Simpson-GolabiBehmel syndromes. Genes Dev 1997;11:3128–42. 16. Gicquel C, Gaston V, Mandelbaum J, Siffroi JP, Flahault A, Le Bouc Y. In vitro fertilization may increase the risk of Beckwith-Wiedemann syndrome related to the abnormal imprinting of the KCN1OT gene. Am J Hum Genet 2003;72:1338–41. 17. 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. 18. 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: 3727–35. 19. Sasaki H, Ishihara K, Kato R. Mechanisms of Igf2/ H19 imprinting: DNA methylation, chromatin and long-distance gene regulation. J Biochem 2000;127: 711–5.
2733