Defects in imprinting and genome-wide DNA methylation are not common in the in vitro fertilization population  Verity F. Oliver, Ph.D., Harriet L. Miles, B.M., B.S., M.D., Wayne S. Cutfield, M.B.Ch.B., D.C.H., F.R.A.C.P., Paul L. Hofman, M.D., Jackie L. Ludgate, B.Sc., Ian M. Morison, M.B.Ch.B., Ph.D.  Fertility and Sterility  Volume 97, Issue 1, Pages 147-153.e7 (January 2012) DOI: 10.1016/j.fertnstert.2011.10.027 Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Graph of mean methylation by MSQ-PCR at the imprinted genes H19, KCNQ1OT1, IGF2, and SNRPN. Each data point represents the mean percentage methylation for one individual from a minimum of two separate MSQ-PCR runs. The horizontal line represents the mean percentage methylation within each group, and the dotted line represents the borders of the normal range of methylation for each loci. Fertility and Sterility 2012 97, 147-153.e7DOI: (10.1016/j.fertnstert.2011.10.027) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Bisulfite sequencing results for the four informative MSQ-PCR outliers at H19, KCNQ1OT1, or SNRPN. A closed circle represents a methylated CpG site, and an open circle represents an unmethylated CpG site. A gray circle represents a CpG site altered by a polymorphism. The informative SNP locations (used for inferring parental transmission of the allele) and the MSQ-PCR site and results are indicated above the sequence. The numbers at the bottom refer to consecutive CpGs within the amplicon. The genotype at informative SNPs is shown to the left of the sequence. An asterisk marks sequences from apparent PCR hybrids. Where multiple clones of a given methylation haplotype were observed, this was noted on the right. (A) H19. IVF 1 showed hypermethylation (73%) of H19 CpG15 by MSQ-PCR; there is no evidence of this by bisulfite sequencing; instead one CG allele shows hypomethylation at CpG15. Three probable PCR hybrids are shown (see Results section). Control 1 showed hypermethylation (74%) of CpG15 by MSQ-PCR. Sequences of both the CA allele and the CG allele indicate various intermediate levels of methylation. Several alleles could be an artefact of PCR hybrids. On average, control 1 showed 50% methylation. (B) KCNQ1OT1. Only one sequence from control 2 showed hypomethylation of the CpG8 MSQ-PCR site, but this sequence appears to be a PCR hybrid. (C) SNRPN. IVF 2 showed hypermethylation (68%) at CpG16 by MSQ-PCR. The bisulfite sequencing results indicate normal methylation at this site. Fertility and Sterility 2012 97, 147-153.e7DOI: (10.1016/j.fertnstert.2011.10.027) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Mean methylation by MSQ-PCR of satellite 2 repetitive DNA (see Fig. 1 for legend description). Fertility and Sterility 2012 97, 147-153.e7DOI: (10.1016/j.fertnstert.2011.10.027) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 1 The MSQ-PCR results grouped by media type used on the developing embryo. Each data point represents the mean percentage of methylation for one individual. The horizontal line indicates the mean percentage of methylation for each group. Two media brands were used: Medicult and Scandinavian Science IVF (ScandIVF). The results are shown for (A) H19, (B) KCNQ1OT1, (C) SNRPN, (D) IGF2, and (E) satellite 2 repeat DNA. Only SNRPN indicated a statistically significant difference between the ScandIVF and the Medicult groups (P=.0006). Fertility and Sterility 2012 97, 147-153.e7DOI: (10.1016/j.fertnstert.2011.10.027) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 2 The MSQ-PCR methylation percentage plotted against the birth weight standard deviations scores (SDS) for individuals in both the IVF and the naturally conceived control groups. Red circles show data from control individuals, and blue squares show data from IVF individuals. The results are shown for (A) H19, (B) KCNQ1OT1, (C) SNRPN, (D) IGF2, and (E) satellite 2 repeat DNA. There was no relationship between birth weight SDS and methylation at any of the genes examined. Fertility and Sterility 2012 97, 147-153.e7DOI: (10.1016/j.fertnstert.2011.10.027) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 3 The MSQ-PCR methylation percentage plotted against the midparental height-corrected SDs for individuals in both the IVF and the naturally conceived control groups. See Supplemental Figure 2 for legend details. The results are shown for (A) H19, (B) KCNQ1OT1, (C) SNRPN, (D) IGF2, and (E) satellite 2 repeat DNA. There was no relationship between midparental height-corrected SDs and methylation at any of the genes examined. Fertility and Sterility 2012 97, 147-153.e7DOI: (10.1016/j.fertnstert.2011.10.027) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions