- Email: [email protected]
IGF2 ICR1 in the placenta of pregnancies conceived by in vitro fertilization and intracytoplasmic sperm injection
DNA methylation at H19/IGF2 ICR1 in the placenta of pregnancies conceived by in vitro fertilization and intracytoplasmic sperm injection DNA methylation at H19 ICR1 was investigated in the placenta from pregnancies conceived by in vitro fertilization, intracytoplasmic sperm injection, and from natural conception. Our results showed that there were no significant differences in mean methylation between all pregnancy groups; therefore, assisted reproductive technology may not affect proper imprinting of H19 and IGF2. (Fertil Steril 2011;95:2524–6. 2011 by American Society for Reproductive Medicine.) Key Words: DNA methylation, H19, ICR1, IGF2, placenta, IVF, ICSI, small for gestational age In-vitro fertilization and intracytoplasmic sperm injection (ICSI) are common assisted reproductive technologies (ARTs) in treating infertility. Their associated risks include low birth weight, multiple births, birth defects, chromosome abnormalities, and imprinting disorders (1–7). Recent research has suggested that ART may introduce epigenetic abnormalities that affect proper genomic imprinting, which may contribute to the risks of ART. In animal studies, manipulation of embryo culturing conditions and superovulation can introduce aberrant DNA methylation at imprinted genes, which can affect optimal fetal growth (8, 9). DNA methylation can also be altered during in vitro oocyte maturation (10). In infertile men, an association between oligozoospermia and DNA hypomethylation at the H19 imprinting control region have been reported (11–14). Indeed, other studies have reported differences in DNA methylation and expression of imprinted genes in ART conceptions compared to natural conceptions (15, 16). At the H19/IGF2 imprinting control region 1 (H19 ICR1), aberrant DNA methylation is more common in ART and more variable (17). Edgar Chan Wong, M.Sc.a Chiho Hatakeyama, M.Sc.a Wendy P. Robinson, Ph.D.b Sai Ma, Ph.D.a a Department of Obstetrics and Gynaecology, University of British Columbia, Vancouver, British Columbia, Canada b Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada Received December 23, 2010; revised May 14, 2011; accepted May 16, 2011. E.C.W. has nothing to disclose. C.H. has nothing to disclose. W.P.R. has nothing to disclose. S.M. has nothing to disclose. This study is funded by the Canadian Institutes for Health Research, Ottawa, Ontario, Canada, grant number MOP-4809A, to S.M. E.C.W. and C.H. were recipients of studentships from the Interdisciplinary Women’s Reproductive Health Research Trainee Program. Reprint requests: Sai Ma, Ph.D., D6-4500 Oak Street, Vancouver, BC, Canada V6H-3N1 (E-mail: [email protected]).
2524
In this study, we compared DNA methylation at the H19 ICR1 in placentas conceived by ART and natural conception. We also determined whether aberrant methylation is particularly associated with a small for gestational age (SGA) birth, because H19 and IGF2 are associated with fetal growth and placental development. Women who had conceived by IVF (n ¼ 32) or ICSI (n ¼ 45) were mainly recruited from the University of British Columbia (UBC) Centre of Reproductive Health (Vancouver, British Columbia, Canada), Genesis Fertility Clinic (Vancouver), and Pacific Centre for Reproductive Medicine (Burnaby, British Columbia, Canada). Women who have conceived naturally (NC, n ¼ 12) were recruited from the BC Women’s Hospital and Health Centre in Vancouver. Each case was designated as appropriate for gestational age (IVFAGA, ICSI-AGA, and NC-AGA) or small for gestational age (IVF-SGA, ICSI-SGA, and NC-SGA). For NC-SGA, the patients also displayed intrauterine growth restriction consisting of either: 1. amniotic fluid index of <50 mm; 2. reduced, reversed, or absent diastolic flow in the umbilical artery; or 3. uterine artery notching at 2224þ6 weeks of gestation. SGA is defined as birth weight below the 10th percentile for gestational age. Reference charts from the Canadian Perinatal Surveillance System were used (18). This study was approved by the UBC Research Ethics Boards. The placenta was obtained after delivery. Chorionic villi were sampled at two sites and DNA was extracted. Methylation-sensitive single nucleotide primer extension (MS-SNuPE) was used to determine methylation at two CpG sites (C10 and C12) of the H19 ICR1, as described by Sievers et al. with minor modifications to the polymerase chain reaction protocol (19). Anthropometric data were compared, including maternal age, birth weight, and gestational age (Supplemental Table 1). There were no significant differences in maternal age between each pregnancy group (analysis of variance, P¼.4). However, gestational age and birth weight were significantly different (P<.0001 and
Fertility and Sterility Vol. 95, No. 8, June 30, 2011 Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/$36.00 doi:10.1016/j.fertnstert.2011.05.047
P¼.0006, respectively; analysis of variance). Bonferroni’s post hoc test revealed that for gestational age, NC-SGA was significantly different from NC-AGA, ICSI-AGA, and IVF-AGA (P<.05, for all groups). In most comparisons, birth weight was lower in SGA compared to AGA groups. However, there were no significant differences in birth weight between NC-AGA and IVF-SGA, and between IVFSGA and IVF-AGA. The frequencies of SGA in IVF and ICSI pregnancies were not significantly different (P¼.80, Fisher’s exact test). For each sample, methylation values at C10 and C12 were correlated (r ¼ 0.70, P<.0001). The intra-placental mean methylation values were also correlated (r ¼ 0.72, P<.0001). Given these results, subsequent analyses of DNA methylation comparing each pregnancy group were done using the mean of C10 and C12 at both placental sites. Mean SD methylation values in IVF-AGA (n ¼ 25), IVFSGA (n ¼ 7), ICSI-AGA (n ¼ 32), and ICSI-SGA (n ¼ 13) groups were 45.52% 4.86%, 47.25% 5.77%, 45.64% 6.06%, and 42.73% 4.39%, respectively (Fig. 1). Mean SD methylation values in the control groups, NC-AGA (n ¼ 7) and NC-SGA (n ¼ 5), were 44.68% 4.18% and 44.63% 3.60%, respectively (Fig. 1). No significant difference was detected between mean methylation across all sample groups (P¼.49, analysis of variance). There was also no significant differences in the variance of the mean of each sample group using the Bartlett’s test. In all samples, there was no correlation between birth weight and mean methylation (r ¼ 0.021, P¼.9). There was also no correlation between gestational age and mean methylation (r ¼ 0.0063, P¼.96). Because mean methylation was not significantly different between assisted and natural conceptions, all samples were combined to generate a mean SD of 45.16% 6.83%. Hypomethylation was then defined as a mean value below 31.5% using a cut-off of 2 standard deviations. Hypomethylation was defined as below 31.7% for C10 and 33.2% for C12. One case with mean methylation of less than 31.5% was observed from the ICSI-AGA group (Supplemental Table 2). However, hypomethylation was confined to one placental site, affecting both C10 and C12 (18.63% and 14.32%, respectively). There were eight cases that had at least one CpG hypomethylated, which was observed in IVF and ICSI cases (n ¼ 6) as well as natural conception cases (n ¼ 2) (Supplemental Table 2). The frequency of hypomethylation, occurring in at least one CpG, in IVF and ICSI placentas was not significantly greater than that of natural conception placentas (P¼.24, Fisher’s exact test). Methylation analyses of umbilical cord blood were available from seven ART cases. There was no significant correlation between mean methylation values in the cord blood samples compared to placental tissue (r ¼ 0.54, P¼.21). Perinatal complications were recorded at delivery (Supplemental Table 3). One NC-AGA case had polyhydramnios. One NC-SGA had severe preeclampsia. Velamentous cord insertion was observed in two ICSI-SGA cases, in which one displayed hypomethylation in the placenta. One ICSI-AGA case displayed marginal placenta previa. Further, the newborn of one ICSI-AGA case had mosaic trisomy 21 (46,XY/47,XY þ21; 9/20) and a heart malformation. Aside from one case, hypomethylation was not observed in ART cases that had perinatal complications. Overall, we did not observe any significant differences in H19 ICR1 methylation between ART and natural conception placentas.
Fertility and Sterility
FIGURE 1 Percent methylation at H19 ICR1 in the placenta between pregnancy groups. Each data point represents the mean methylation of C10 and C12 of the H19 ICR1 in two sampled sites of one placenta. The mean methylation for each sample group is represented by the bar in each respective column. No significant differences in mean methylation between groups were observed, P¼ .49.
Chan Wong. H19 ICR1 methylation in ART placentas. Fertil Steril 2011.
This is similar to a recent report in which 10 loci were investigated in different tissues (16). However, significantly higher methylation in MEST was observed in maternal and cord blood in IVF pregnancies (16). In another study, hypomethylation at KvDMR1 was observed in peripheral blood samples in both IVF and ICSI pregnancies (20). Using bead-arrays, differences in DNA methylation at imprinted and nonimprinted loci were detected in IVF placentas and cord blood (15). Significant differences in expression were also detected in cord blood (CEBPA and COPG2) and placenta (SERPINF1 and MEST) (15). Aberrant DNA methylation at the ICR1 was also more frequent in ART conceptions (17). However, significantly lower expression of both H19 and IGF2 were detected in the placenta, which did not correspond to changes in ICR1 methylation (17). We did not observe a difference in methylation in SGA pregnancies conceived by IVF or ICSI, compared with natural conception. In one other study, altered imprinting at six loci including H19/ IGF2 was also not observed in ICSI-SGA pregnancies compared with term-born AGA natural conceptions (21). In natural conceptions, the H19 ICR1 was reported to be significantly less methylated in intrauterine growth restriction placentas compared with normal birth weight placentas (22). Decreased expression of IGF2 and altered expression of other imprinted genes were also observed in placentas from SGA or intrauterine growth restriction natural pregnancies (23). However, in fetal blood, no differences in methylation at IGF2, GNASAS, INSIGF, and LEP were observed between preterm SGA and AGA pregnancies of natural conception (24). In this study, it is likely that hypomethylation at H19 ICR1 did not affect pregnancy or placental complications. Abnormal cord insertion and placenta previa have been associated with ART, but their effects on fetal growth restriction are unclear (25–28).
2525
We also observed that hypomethylation can be restricted to one placental site or one CpG site. It is unknown whether hypomethylation at a single CpG significantly affects expression of the imprinted allele. However, it has been postulated that the placenta is labile to epigenetic changes compared with the fetus (8). This may also be a mechanism of adapting placental physiology to changes in bioavailability of nutrients during pregnancy. We did not observe a correlation in methylation between placenta and cord blood. Katari et al. reported higher mean methylation in cord blood (15). However, methylation in the placenta has been reported to be either higher or lower than maternal or cord blood, depending on which loci (16). At H19, significantly lower methylation in the placenta was reported (16). Nonetheless, differences in methylation between placenta and cord blood have been
suggested to be due to higher plasticity of genomic imprinting in extra-embryonic tissues (16). Overall, we are the first to report on SGA pregnancies derived from IVF and ICSI. However, the effect of ART or infertility on DNA methylation at imprinted genes remains inconclusive or confined to specific genes. It is important for future studies to relate epigenetic status of imprinted genes with transcriptional activity to determine the full clinical effect of epimutations in ART pregnancies. Acknowledgments: The authors thank all the research participants in this study. They further thank the UBC Centre of Reproductive Health, Genesis Fertility Clinic, Pacific Centre for Reproductive Medicine, and other clinics in Canada for their assistance in recruitment. The authors appreciate the technical assistance of WPR’s lab.
REFERENCES 1. Schieve LA, Meikle SF, Ferre C, Peterson HB, Jeng G, Wilcox LS. Low and very low birth weight in infants conceived with use of assisted reproductive technology. N Engl J Med 2002;346: 731–7. 2. Pinborg A. IVF/ICSI twin pregnancies: risks and prevention. Hum Reprod Update 2005;11:575–93. 3. DeBaun MR, Niemitz EL, Feinberg AP. Association of in vitro fertilization with Beckwith-Wiedemann syndrome and epigenetic alterations of LIT1 and H19. Am J Hum Genet 2003;72:156–60. 4. Maher ER, Afnan M, Barratt CL. Epigenetic risks related to assisted reproductive technologies: epigenetics, imprinting, ART and icebergs? Hum Reprod 2003;18:2508–11. 5. Orstavik KH, Eiklid K, van der Hagen CB, Spetalen S, Kierulf K, Skjeldal O, et al. Another case of imprinting defect in a girl with Angelman syndrome who was conceived by intracytoplasmic semen injection. Am J Hum Genet 2003;72:218–9. 6. Halliday J, Oke K, Breheny S, Algar E, J Amor D. Beckwith-Wiedemann syndrome and IVF: a casecontrol study. Am J Hum Genet 2004;75:526–8. 7. Adamson GD, de Mouzon J, Lancaster P, Nygren KG, Sullivan E, Zegers-Hochschild F. International Committee for Monitoring Assisted Reproductive Technology. World collaborative report on in vitro fertilization, 2000. Fertil Steril 2006;85: 1586–622. 8. 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. 9. 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. 10. Borghol N, Lornage J, Blachere T, Sophie Garret A, Lefevre A. Epigenetic status of the H19 locus in human oocytes following in vitro maturation. Genomics 2006;87:417–26. 11. Boissonnas CC, Abdalaoui HE, Haelewyn V, Fauque P, Dupont JM, Gut I, et al. Specific
2526
Chan Wong et al.
12.
13.
14.
15.
16.
17.
18.
19.
epigenetic alterations of IGF2-H19 locus in spermatozoa from infertile men. Eur J Hum Genet 2010;18:73–80. 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. 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. 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. Katari S, Turan N, Bibikova M, Erinle O, Chalian R, Foster M, et al. DNA methylation and gene expression differences in children conceived in vitro or in vivo. Hum Mol Genet 2009;18:3769–78. 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;47:371–6. Turan N, Katari S, Gerson LF, Chalian R, Foster MW, Gaughan JP, et al. Inter- and intraindividual variation in allele-specific DNA methylation and gene expression in children conceived using assisted reproductive technology. PLoS Genet 2010;6:e1001033. Kramer MS, Platt RW, Wen SW, Joseph KS, Allen A, Abrahamowicz M, et al. A new and improved population-based Canadian reference for birth weight for gestational age. Pediatrics 2001;108:E35. Sievers S, Alemazkour K, Zahn S, Perlman EJ, Gillis AJ, Looijenga LH, et al. IGF2/H19 imprinting analysis of human germ cell tumors (GCTs) using the methylation-sensitive singlenucleotide primer extension method reflects the origin of GCTs in different stages of primordial germ cell development. Genes Chromosomes Cancer 2005;44:256–64.
H19 ICR1 methylation in ART placentas
20. Gomes MV, Huber J, Ferriani RA, Amaral Neto AM, Ramos ES. Abnormal methylation at the KvDMR1 imprinting control region in clinically normal children conceived by assisted reproductive technologies. Mol Hum Reprod 2009;15:471–7. 21. Kanber D, Buiting K, Zeschnigk M, Ludwig M, Horsthemke B. Low frequency of imprinting defects in ICSI children born small for gestational age. Eur J Hum Genet 2009;17:22–9. 22. Bourque DK, Avila L, Penaherrera M, von Dadelszen P, Robinson WP. Decreased placental methylation at the H19/IGF2 imprinting control region is associated with normotensive intrauterine growth restriction but not preeclampsia. Placenta 2010;31:197–202. 23. McMinn J, Wei M, Schupf N, Cusmai J, Johnson EB, Smith AC, et al. Unbalanced placental expression of imprinted genes in human intrauterine growth restriction. Placenta 2006;27:540–9. 24. Tobi EW, Heijmans BT, Kremer D, Putter H, Delemarre-van de Waal HA, Finken MJ, et al. DNA methylation of IGF2, GNASAS, INSIGF and LEP and being born small for gestational age. Epigenetics 2011;6:171–6. 25. Daniel Y, Schreiber L, Geva E, Amit A, Pausner D, Kupferminc MJ, et al. Do placentae of term singleton pregnancies obtained by assisted reproductive technologies differ from those of spontaneously conceived pregnancies? Hum Reprod 1999;14:1107–10. 26. Romundstad LB, Romundstad PR, Sunde A, von During V, Skjaerven R, Vatten LJ. Increased risk of placenta previa in pregnancies following IVF/ ICSI; a comparison of ART and non-ART pregnancies in the same mother. Hum Reprod 2006;21:2353–8. 27. Jackson RA, Gibson KA, Wu YW, Croughan MS. Perinatal outcomes in singletons following in vitro fertilization: a meta-analysis. Obstet Gynecol 2004;103:551–63. 28. Ogueh O, Morin L, Usher RH, Benjamin A. Obstetric implications of low-lying placentas diagnosed in the second trimester. Int J Gynaecol Obstet 2003;83:11–7.
Vol. 95, No. 8, June 30, 2011
SUPPLEMENTAL TABLE 1 Mean maternal age, gestational age, and birth weight of each pregnancy group. Pregnancy
n
Maternal age, y (mean ± SD)
Gestational age at birth, wk (mean ± SD)
Birthweight, g (mean ± SD)
IVF-AGA IVF-SGA ICSI-AGA ICSI-SGA NC-AGA NC-SGA
25 7 32 13 7 5
36.4 5.1 37.8 3.2 35.0 4.8 34.5 0.71 33.0 4.9 32.7 5.5
39.1 1.6 37.6 1.7 39.4 1.8 35.5 2.1 38.8 3.3 34.0 5.6
3352.6 581.2 2534.5 315.1 3374.5 542.5 2413.8 448.4 3463 263.7 1866 1036
Note: ICSI ¼ intracytoplasmic sperm injection; NC ¼ natural conception; AGA ¼ appropriate for gestational age; SGA ¼ small for gestational age. Chan Wong. H19 ICR1 methylation in ART placentas. Fertil Steril 2011.
Fertility and Sterility
2526.e1
SUPPLEMENTAL TABLE 2 DNA hypomethylation at individual CpGs (C10 and C12) of the ICR1 at two sites of each placenta. Site 1 Type of conception IVF-AGA (n ¼ 1) Case 17 IVF-SGA (n ¼ 1) Case 2 ICSI-AGA (n ¼ 1) Case 7 ICSI-SGA (n ¼ 3) Case 3 Case 4 Case 7 NC-AGA (n ¼ 1) Case 3 NC-SGA (n ¼ 1) Case 4
Site 2
C10
C12
C10
C12
Mean
31.28a
37.07
40.97
39.6
37.23
37.48
25.19a
60.06
30.89a
39.38
37.91
42.77
18.63a
14.32a
28.41a
31.51a 36.69 29.37a
28.13a 38.1 25.93a
51.03 32.98 46.4
54.63 29.77a 47.77
41.33 34.39 37.37
42.44
38.5
32.68
29.97a
35.9
38.40
39.26
37.14
30.11a
36.23
Note: Hypomethylation at C10 and C12 is defined as a value below 31.7% and 33.2% methylation, respectively. Hypomethylation overall is defined as a value below 31.5% methylation. Hypomethylation was observed in all sample groups, IVF-AGA, IVF-SGA, ICSI-AGA, ICSI-SGA, NC-AGA, and NC-SGA. ICSI ¼ intracytoplasmic sperm injection; NC ¼ natural conception; AGA ¼ appropriate for gestational age; SGA ¼ small for gestational age. a Hypomethylated. Chan Wong. H19 ICR1 methylation in ART placentas. Fertil Steril 2011.
2526.e2
Chan Wong et al.
H19 ICR1 methylation in ART placentas
Vol. 95, No. 8, June 30, 2011
SUPPLEMENTAL TABLE 3 Pregnancy complications and placental abnormalities in the study and control cases. Type of conception ICSI-AGA Case 8 ICSI-AGA Case 9 ICSI-AGA Case 10 ICSI-AGA Case 16 ICSI-SGA Case 4 and 5 ICSI-SGA Case 6 and 7 IVF-AGA Case 13 IVF-SGA Case 2 IVF-SGA Case 19 ICSI-AGA Case 17 ICSI-AGA Case 23 NC-SGA Case 1
Pregnancy/placental complication 47,XY þ21; heart malformation Maternal bleeding at 6 months Transverse arrest Maternal cholestasis Marginal placental previa Velamentous cord insertion Spontaneous rupture of the membranes Asymmetric IUGR Cardiac anomaly Postpartum hemorrhage Diet-controlled gestational diabetes Severe preeclampsia, severe asymmetrical IUGR
Note: ICSI ¼ intracytoplasmic sperm injection; NC ¼ natural conception; AGA ¼ appropriate for gestational age; SGA ¼ small for gestational age; IUGR ¼ intrauterine growth restriction. Chan Wong. H19 ICR1 methylation in ART placentas. Fertil Steril 2011.
Fertility and Sterility
2526.e3
2524
In this study, we compared DNA methylation at the H19 ICR1 in placentas conceived by ART and natural conception. We also determined whether aberrant methylation is particularly associated with a small for gestational age (SGA) birth, because H19 and IGF2 are associated with fetal growth and placental development. Women who had conceived by IVF (n ¼ 32) or ICSI (n ¼ 45) were mainly recruited from the University of British Columbia (UBC) Centre of Reproductive Health (Vancouver, British Columbia, Canada), Genesis Fertility Clinic (Vancouver), and Pacific Centre for Reproductive Medicine (Burnaby, British Columbia, Canada). Women who have conceived naturally (NC, n ¼ 12) were recruited from the BC Women’s Hospital and Health Centre in Vancouver. Each case was designated as appropriate for gestational age (IVFAGA, ICSI-AGA, and NC-AGA) or small for gestational age (IVF-SGA, ICSI-SGA, and NC-SGA). For NC-SGA, the patients also displayed intrauterine growth restriction consisting of either: 1. amniotic fluid index of <50 mm; 2. reduced, reversed, or absent diastolic flow in the umbilical artery; or 3. uterine artery notching at 2224þ6 weeks of gestation. SGA is defined as birth weight below the 10th percentile for gestational age. Reference charts from the Canadian Perinatal Surveillance System were used (18). This study was approved by the UBC Research Ethics Boards. The placenta was obtained after delivery. Chorionic villi were sampled at two sites and DNA was extracted. Methylation-sensitive single nucleotide primer extension (MS-SNuPE) was used to determine methylation at two CpG sites (C10 and C12) of the H19 ICR1, as described by Sievers et al. with minor modifications to the polymerase chain reaction protocol (19). Anthropometric data were compared, including maternal age, birth weight, and gestational age (Supplemental Table 1). There were no significant differences in maternal age between each pregnancy group (analysis of variance, P¼.4). However, gestational age and birth weight were significantly different (P<.0001 and
Fertility and Sterility Vol. 95, No. 8, June 30, 2011 Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/$36.00 doi:10.1016/j.fertnstert.2011.05.047
P¼.0006, respectively; analysis of variance). Bonferroni’s post hoc test revealed that for gestational age, NC-SGA was significantly different from NC-AGA, ICSI-AGA, and IVF-AGA (P<.05, for all groups). In most comparisons, birth weight was lower in SGA compared to AGA groups. However, there were no significant differences in birth weight between NC-AGA and IVF-SGA, and between IVFSGA and IVF-AGA. The frequencies of SGA in IVF and ICSI pregnancies were not significantly different (P¼.80, Fisher’s exact test). For each sample, methylation values at C10 and C12 were correlated (r ¼ 0.70, P<.0001). The intra-placental mean methylation values were also correlated (r ¼ 0.72, P<.0001). Given these results, subsequent analyses of DNA methylation comparing each pregnancy group were done using the mean of C10 and C12 at both placental sites. Mean SD methylation values in IVF-AGA (n ¼ 25), IVFSGA (n ¼ 7), ICSI-AGA (n ¼ 32), and ICSI-SGA (n ¼ 13) groups were 45.52% 4.86%, 47.25% 5.77%, 45.64% 6.06%, and 42.73% 4.39%, respectively (Fig. 1). Mean SD methylation values in the control groups, NC-AGA (n ¼ 7) and NC-SGA (n ¼ 5), were 44.68% 4.18% and 44.63% 3.60%, respectively (Fig. 1). No significant difference was detected between mean methylation across all sample groups (P¼.49, analysis of variance). There was also no significant differences in the variance of the mean of each sample group using the Bartlett’s test. In all samples, there was no correlation between birth weight and mean methylation (r ¼ 0.021, P¼.9). There was also no correlation between gestational age and mean methylation (r ¼ 0.0063, P¼.96). Because mean methylation was not significantly different between assisted and natural conceptions, all samples were combined to generate a mean SD of 45.16% 6.83%. Hypomethylation was then defined as a mean value below 31.5% using a cut-off of 2 standard deviations. Hypomethylation was defined as below 31.7% for C10 and 33.2% for C12. One case with mean methylation of less than 31.5% was observed from the ICSI-AGA group (Supplemental Table 2). However, hypomethylation was confined to one placental site, affecting both C10 and C12 (18.63% and 14.32%, respectively). There were eight cases that had at least one CpG hypomethylated, which was observed in IVF and ICSI cases (n ¼ 6) as well as natural conception cases (n ¼ 2) (Supplemental Table 2). The frequency of hypomethylation, occurring in at least one CpG, in IVF and ICSI placentas was not significantly greater than that of natural conception placentas (P¼.24, Fisher’s exact test). Methylation analyses of umbilical cord blood were available from seven ART cases. There was no significant correlation between mean methylation values in the cord blood samples compared to placental tissue (r ¼ 0.54, P¼.21). Perinatal complications were recorded at delivery (Supplemental Table 3). One NC-AGA case had polyhydramnios. One NC-SGA had severe preeclampsia. Velamentous cord insertion was observed in two ICSI-SGA cases, in which one displayed hypomethylation in the placenta. One ICSI-AGA case displayed marginal placenta previa. Further, the newborn of one ICSI-AGA case had mosaic trisomy 21 (46,XY/47,XY þ21; 9/20) and a heart malformation. Aside from one case, hypomethylation was not observed in ART cases that had perinatal complications. Overall, we did not observe any significant differences in H19 ICR1 methylation between ART and natural conception placentas.
Fertility and Sterility
FIGURE 1 Percent methylation at H19 ICR1 in the placenta between pregnancy groups. Each data point represents the mean methylation of C10 and C12 of the H19 ICR1 in two sampled sites of one placenta. The mean methylation for each sample group is represented by the bar in each respective column. No significant differences in mean methylation between groups were observed, P¼ .49.
Chan Wong. H19 ICR1 methylation in ART placentas. Fertil Steril 2011.
This is similar to a recent report in which 10 loci were investigated in different tissues (16). However, significantly higher methylation in MEST was observed in maternal and cord blood in IVF pregnancies (16). In another study, hypomethylation at KvDMR1 was observed in peripheral blood samples in both IVF and ICSI pregnancies (20). Using bead-arrays, differences in DNA methylation at imprinted and nonimprinted loci were detected in IVF placentas and cord blood (15). Significant differences in expression were also detected in cord blood (CEBPA and COPG2) and placenta (SERPINF1 and MEST) (15). Aberrant DNA methylation at the ICR1 was also more frequent in ART conceptions (17). However, significantly lower expression of both H19 and IGF2 were detected in the placenta, which did not correspond to changes in ICR1 methylation (17). We did not observe a difference in methylation in SGA pregnancies conceived by IVF or ICSI, compared with natural conception. In one other study, altered imprinting at six loci including H19/ IGF2 was also not observed in ICSI-SGA pregnancies compared with term-born AGA natural conceptions (21). In natural conceptions, the H19 ICR1 was reported to be significantly less methylated in intrauterine growth restriction placentas compared with normal birth weight placentas (22). Decreased expression of IGF2 and altered expression of other imprinted genes were also observed in placentas from SGA or intrauterine growth restriction natural pregnancies (23). However, in fetal blood, no differences in methylation at IGF2, GNASAS, INSIGF, and LEP were observed between preterm SGA and AGA pregnancies of natural conception (24). In this study, it is likely that hypomethylation at H19 ICR1 did not affect pregnancy or placental complications. Abnormal cord insertion and placenta previa have been associated with ART, but their effects on fetal growth restriction are unclear (25–28).
2525
We also observed that hypomethylation can be restricted to one placental site or one CpG site. It is unknown whether hypomethylation at a single CpG significantly affects expression of the imprinted allele. However, it has been postulated that the placenta is labile to epigenetic changes compared with the fetus (8). This may also be a mechanism of adapting placental physiology to changes in bioavailability of nutrients during pregnancy. We did not observe a correlation in methylation between placenta and cord blood. Katari et al. reported higher mean methylation in cord blood (15). However, methylation in the placenta has been reported to be either higher or lower than maternal or cord blood, depending on which loci (16). At H19, significantly lower methylation in the placenta was reported (16). Nonetheless, differences in methylation between placenta and cord blood have been
suggested to be due to higher plasticity of genomic imprinting in extra-embryonic tissues (16). Overall, we are the first to report on SGA pregnancies derived from IVF and ICSI. However, the effect of ART or infertility on DNA methylation at imprinted genes remains inconclusive or confined to specific genes. It is important for future studies to relate epigenetic status of imprinted genes with transcriptional activity to determine the full clinical effect of epimutations in ART pregnancies. Acknowledgments: The authors thank all the research participants in this study. They further thank the UBC Centre of Reproductive Health, Genesis Fertility Clinic, Pacific Centre for Reproductive Medicine, and other clinics in Canada for their assistance in recruitment. The authors appreciate the technical assistance of WPR’s lab.
REFERENCES 1. Schieve LA, Meikle SF, Ferre C, Peterson HB, Jeng G, Wilcox LS. Low and very low birth weight in infants conceived with use of assisted reproductive technology. N Engl J Med 2002;346: 731–7. 2. Pinborg A. IVF/ICSI twin pregnancies: risks and prevention. Hum Reprod Update 2005;11:575–93. 3. DeBaun MR, Niemitz EL, Feinberg AP. Association of in vitro fertilization with Beckwith-Wiedemann syndrome and epigenetic alterations of LIT1 and H19. Am J Hum Genet 2003;72:156–60. 4. Maher ER, Afnan M, Barratt CL. Epigenetic risks related to assisted reproductive technologies: epigenetics, imprinting, ART and icebergs? Hum Reprod 2003;18:2508–11. 5. Orstavik KH, Eiklid K, van der Hagen CB, Spetalen S, Kierulf K, Skjeldal O, et al. Another case of imprinting defect in a girl with Angelman syndrome who was conceived by intracytoplasmic semen injection. Am J Hum Genet 2003;72:218–9. 6. Halliday J, Oke K, Breheny S, Algar E, J Amor D. Beckwith-Wiedemann syndrome and IVF: a casecontrol study. Am J Hum Genet 2004;75:526–8. 7. Adamson GD, de Mouzon J, Lancaster P, Nygren KG, Sullivan E, Zegers-Hochschild F. International Committee for Monitoring Assisted Reproductive Technology. World collaborative report on in vitro fertilization, 2000. Fertil Steril 2006;85: 1586–622. 8. 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. 9. 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. 10. Borghol N, Lornage J, Blachere T, Sophie Garret A, Lefevre A. Epigenetic status of the H19 locus in human oocytes following in vitro maturation. Genomics 2006;87:417–26. 11. Boissonnas CC, Abdalaoui HE, Haelewyn V, Fauque P, Dupont JM, Gut I, et al. Specific
2526
Chan Wong et al.
12.
13.
14.
15.
16.
17.
18.
19.
epigenetic alterations of IGF2-H19 locus in spermatozoa from infertile men. Eur J Hum Genet 2010;18:73–80. 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. 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. 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. Katari S, Turan N, Bibikova M, Erinle O, Chalian R, Foster M, et al. DNA methylation and gene expression differences in children conceived in vitro or in vivo. Hum Mol Genet 2009;18:3769–78. 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;47:371–6. Turan N, Katari S, Gerson LF, Chalian R, Foster MW, Gaughan JP, et al. Inter- and intraindividual variation in allele-specific DNA methylation and gene expression in children conceived using assisted reproductive technology. PLoS Genet 2010;6:e1001033. Kramer MS, Platt RW, Wen SW, Joseph KS, Allen A, Abrahamowicz M, et al. A new and improved population-based Canadian reference for birth weight for gestational age. Pediatrics 2001;108:E35. Sievers S, Alemazkour K, Zahn S, Perlman EJ, Gillis AJ, Looijenga LH, et al. IGF2/H19 imprinting analysis of human germ cell tumors (GCTs) using the methylation-sensitive singlenucleotide primer extension method reflects the origin of GCTs in different stages of primordial germ cell development. Genes Chromosomes Cancer 2005;44:256–64.
H19 ICR1 methylation in ART placentas
20. Gomes MV, Huber J, Ferriani RA, Amaral Neto AM, Ramos ES. Abnormal methylation at the KvDMR1 imprinting control region in clinically normal children conceived by assisted reproductive technologies. Mol Hum Reprod 2009;15:471–7. 21. Kanber D, Buiting K, Zeschnigk M, Ludwig M, Horsthemke B. Low frequency of imprinting defects in ICSI children born small for gestational age. Eur J Hum Genet 2009;17:22–9. 22. Bourque DK, Avila L, Penaherrera M, von Dadelszen P, Robinson WP. Decreased placental methylation at the H19/IGF2 imprinting control region is associated with normotensive intrauterine growth restriction but not preeclampsia. Placenta 2010;31:197–202. 23. McMinn J, Wei M, Schupf N, Cusmai J, Johnson EB, Smith AC, et al. Unbalanced placental expression of imprinted genes in human intrauterine growth restriction. Placenta 2006;27:540–9. 24. Tobi EW, Heijmans BT, Kremer D, Putter H, Delemarre-van de Waal HA, Finken MJ, et al. DNA methylation of IGF2, GNASAS, INSIGF and LEP and being born small for gestational age. Epigenetics 2011;6:171–6. 25. Daniel Y, Schreiber L, Geva E, Amit A, Pausner D, Kupferminc MJ, et al. Do placentae of term singleton pregnancies obtained by assisted reproductive technologies differ from those of spontaneously conceived pregnancies? Hum Reprod 1999;14:1107–10. 26. Romundstad LB, Romundstad PR, Sunde A, von During V, Skjaerven R, Vatten LJ. Increased risk of placenta previa in pregnancies following IVF/ ICSI; a comparison of ART and non-ART pregnancies in the same mother. Hum Reprod 2006;21:2353–8. 27. Jackson RA, Gibson KA, Wu YW, Croughan MS. Perinatal outcomes in singletons following in vitro fertilization: a meta-analysis. Obstet Gynecol 2004;103:551–63. 28. Ogueh O, Morin L, Usher RH, Benjamin A. Obstetric implications of low-lying placentas diagnosed in the second trimester. Int J Gynaecol Obstet 2003;83:11–7.
Vol. 95, No. 8, June 30, 2011
SUPPLEMENTAL TABLE 1 Mean maternal age, gestational age, and birth weight of each pregnancy group. Pregnancy
n
Maternal age, y (mean ± SD)
Gestational age at birth, wk (mean ± SD)
Birthweight, g (mean ± SD)
IVF-AGA IVF-SGA ICSI-AGA ICSI-SGA NC-AGA NC-SGA
25 7 32 13 7 5
36.4 5.1 37.8 3.2 35.0 4.8 34.5 0.71 33.0 4.9 32.7 5.5
39.1 1.6 37.6 1.7 39.4 1.8 35.5 2.1 38.8 3.3 34.0 5.6
3352.6 581.2 2534.5 315.1 3374.5 542.5 2413.8 448.4 3463 263.7 1866 1036
Note: ICSI ¼ intracytoplasmic sperm injection; NC ¼ natural conception; AGA ¼ appropriate for gestational age; SGA ¼ small for gestational age. Chan Wong. H19 ICR1 methylation in ART placentas. Fertil Steril 2011.
Fertility and Sterility
2526.e1
SUPPLEMENTAL TABLE 2 DNA hypomethylation at individual CpGs (C10 and C12) of the ICR1 at two sites of each placenta. Site 1 Type of conception IVF-AGA (n ¼ 1) Case 17 IVF-SGA (n ¼ 1) Case 2 ICSI-AGA (n ¼ 1) Case 7 ICSI-SGA (n ¼ 3) Case 3 Case 4 Case 7 NC-AGA (n ¼ 1) Case 3 NC-SGA (n ¼ 1) Case 4
Site 2
C10
C12
C10
C12
Mean
31.28a
37.07
40.97
39.6
37.23
37.48
25.19a
60.06
30.89a
39.38
37.91
42.77
18.63a
14.32a
28.41a
31.51a 36.69 29.37a
28.13a 38.1 25.93a
51.03 32.98 46.4
54.63 29.77a 47.77
41.33 34.39 37.37
42.44
38.5
32.68
29.97a
35.9
38.40
39.26
37.14
30.11a
36.23
Note: Hypomethylation at C10 and C12 is defined as a value below 31.7% and 33.2% methylation, respectively. Hypomethylation overall is defined as a value below 31.5% methylation. Hypomethylation was observed in all sample groups, IVF-AGA, IVF-SGA, ICSI-AGA, ICSI-SGA, NC-AGA, and NC-SGA. ICSI ¼ intracytoplasmic sperm injection; NC ¼ natural conception; AGA ¼ appropriate for gestational age; SGA ¼ small for gestational age. a Hypomethylated. Chan Wong. H19 ICR1 methylation in ART placentas. Fertil Steril 2011.
2526.e2
Chan Wong et al.
H19 ICR1 methylation in ART placentas
Vol. 95, No. 8, June 30, 2011
SUPPLEMENTAL TABLE 3 Pregnancy complications and placental abnormalities in the study and control cases. Type of conception ICSI-AGA Case 8 ICSI-AGA Case 9 ICSI-AGA Case 10 ICSI-AGA Case 16 ICSI-SGA Case 4 and 5 ICSI-SGA Case 6 and 7 IVF-AGA Case 13 IVF-SGA Case 2 IVF-SGA Case 19 ICSI-AGA Case 17 ICSI-AGA Case 23 NC-SGA Case 1
Pregnancy/placental complication 47,XY þ21; heart malformation Maternal bleeding at 6 months Transverse arrest Maternal cholestasis Marginal placental previa Velamentous cord insertion Spontaneous rupture of the membranes Asymmetric IUGR Cardiac anomaly Postpartum hemorrhage Diet-controlled gestational diabetes Severe preeclampsia, severe asymmetrical IUGR
Note: ICSI ¼ intracytoplasmic sperm injection; NC ¼ natural conception; AGA ¼ appropriate for gestational age; SGA ¼ small for gestational age; IUGR ¼ intrauterine growth restriction. Chan Wong. H19 ICR1 methylation in ART placentas. Fertil Steril 2011.
Fertility and Sterility
2526.e3