Table 1.
Summary of blastocysts divided among experimental procedures.
Table 2.
PCR primers and parameters for bisulfite mutagenesis.
Table 3.
Expression primers and PCR parameters.
Fig 1.
Imprinted DNA methylation profiles of control blastocysts.
Black dots represent methylated CpGs, white dots represent unmethylated CpGs. The SNP location is indicated by an arrowhead. “MAT” represents the maternal oocyte-contributed allele; “PAT” represents the paternal sperm-contributed allele. A) Diagram of the SNRPN ICR amplified region analyzed on human chromosome 15q11.2 (GenBank: U41384) in control blastocysts, a 371 bp region consisting of 24 assessable CpGs and a G/A SNP (Rs220029). The SNRPN ICR is maternally methylated. Approximately 50% methylation is expected, derived from one MAT allele and one PAT allele. B) Diagram of the H19 ICR amplified region analyzed on human chromosome 11p15.5 (GenBank: AF087017) in control blastocysts, a 170 bp region consisting of 14 assessable CpGs and a A/C SNP (Rs2071094). The H19 ICR is paternally methylated. Approximately 50% methylation is expected, derived from one MAT allele and one PAT allele. C) Diagram of the KCNQ1OT1 ICR amplified region analyzed on human chromosome 11p15.5 (GenBank: U90095) in control blastocysts, a 265 bp region consisting of 22 assessable CpGs and a G/A SNP (Rs56134313). The KCNQ1OT1 ICR is maternally methylated. Approximately 50% methylation is expected, derived from one MAT allele and one PAT allele. D) Summary chart of percent methylation in all control blastocysts at the SNRPN (n = 20), H19 (n = 20), and KCNQ1OT1 (n = 20) ICRs. Each white diamond represents the methylation percentage for one individual control blastocyst per ICR. Average percent methylation for each cohort is indicated above each gene. Methylation dot diagrams for individual control blastocysts can be found in S1 Fig, and a summary can be found in S2 Table.
Fig 2.
Imprinted DNA methylation profiles of trisomy blastocysts.
Black dots represent methylated CpGs, white dots represent unmethylated CpGs. The SNP location is indicated by an arrowhead. “MAT” represents the maternal oocyte-contributed allele; “PAT” represents the paternal sperm-contributed allele. A) Diagram of the SNRPN ICR amplified region analyzed in trisomy 15 blastocysts. Approximately 66% methylation is expected with an additional MAT allele. Approximately 33% methylation is expected with an additional PAT allele. B) Diagram of the H19 ICR amplified region analyzed in trisomy 11 blastocysts. Approximately 33% methylation is expected with an additional MAT allele. Approximately 66% methylation is expected with an additional PAT allele. C) Diagram of the KCNQ1OT1 ICR amplified region analyzed in trisomy 11 blastocysts. Approximately 66% methylation is expected with an additional MAT allele. Approximately 33% methylation is expected with an additional PAT allele. D) Summary chart of percent methylation in all trisomy 15 blastocysts at the SNRPN (n = 20), H19 (n = 5), and KCNQ1OT1 (n = 5) ICRs. Black diamonds represent blastocysts with presumable gain of the third chromosome 15 from the oocyte, grey diamonds represent blastocysts with presumable gain of the third chromosome 15 from the sperm. White diamonds represent the methylation percentage for the ICRs on diploid chromosome 11. Average percent methylation for each cohort is indicated. E) Summary chart of percent methylation in all trisomy 11 blastocysts at the SNRPN (n = 5), H19 (n = 20), and KCNQ1OT1 (n = 20) ICRs. Black diamonds represent blastocysts with presumable gain of the third chromosome 11 from the oocyte, grey diamonds represent blastocysts with presumable gain of the third chromosome 11 from the sperm. White diamonds represent the methylation percentage for the ICR on diploid chromosome 15. Average percent methylation for each cohort is indicated. Methylation dot diagrams for individual trisomy blastocysts and diploid ICRs can be found in S2 and S4 Figs, and a summary can be found in S2 Table.
Fig 3.
Imprinted DNA methylation profiles of monosomy blastocysts.
Black dots represent methylated CpGs, white dots represent unmethylated CpGs. The SNP location is indicated by an arrowhead. “MAT” represents the maternal oocyte-contributed allele; “PAT” represents the paternal sperm-contributed allele. A) Diagram of the SNRPN ICR amplified region analyzed in monosomy 15 blastocysts. Hypomethylation (0–25%) is expected with the presence of only the PAT allele. Hypermethylation (75–100%) is expected with the presence of only the MAT allele. B) Diagram of the H19 ICR amplified region analyzed in monosomy 11 blastocysts. Hypermethylation (75–100%) is expected with the presence of only the PAT allele. Hypomethylation (0–25%) is expected with the presence of only the MAT allele. C) Diagram of the KCNQ1OT1 ICR amplified region analyzed in monosomy 11 blastocysts. Hypomethylation (0–25%) is expected with the presence of only the PAT allele. Hypermethylation (75–100%) is expected with the presence of only the MAT allele. D) Summary chart of percent methylation in all monosomy 15 blastocysts at the SNRPN (n = 20), H19 (n = 5), and KCNQ1OT1 (n = 5) ICRs. Black diamonds represent blastocysts with presumable loss of a chromosome 15 from the oocyte, grey diamonds represent blastocysts with presumable loss of a chromosome 15 from the sperm. White diamonds represent the methylation percentage for the ICRs on diploid chromosome 11. Average percent methylation for each cohort is indicated. E) Summary chart of percent methylation in all monosomy 11 blastocysts at the SNRPN (n = 5), H19 (n = 25), and KCNQ1OT1 (n = 25) ICRs. Black diamonds represent blastocysts with presumable loss of a chromosome 11 from the oocyte, grey diamonds represent blastocysts with presumable loss of a chromosome 11 from the sperm. White diamonds represent the methylation percentage for the ICR on diploid chromosome 15. The grey dotted box highlights the six blastocysts with unexpected KCNQ1OT1 methylation profiles. Average percent methylation for each cohort is indicated. Methylation dot diagrams for individual monosomy blastocysts and diploid ICRs can be found in S3 and S4 Figs, and a summary can be found in S2 Table.
Fig 4.
Summary of relative expression for imprinted genes on Chromosome 15 and Chromosome 11 in individual blastocysts.
A) Relative gene expression of SNRPN and UBE3A in control blastocysts (n = 10) compared to trisomy 15 (n = 10) and monosomy 15 (n = 10) blastocysts. B) Relative gene expression of H19, KCNQ1OT1 and CDKN1C in control blastocysts (n = 10) compared to trisomy 11 (n = 10) and monosomy 11 (n = 10) blastocysts. Relative expression between control and aneuploid groups was normalized to the housekeeping gene PPIA (7p13) using the Relative Expression Software Tool (REST) to determine an expression ratio that was tested for significance by a Pair Wise Fixed Reallocation Randomization Test. Differences were considered to be statistically significant when p<0.05, Star “*” denotes statistical significance.