DNA methylation errors at imprinted loci after assisted conception originate in the parental sperm

Abstract

There is an increased prevalence of imprinting disorders, such as Beckwith-Wiedemann syndrome, associated with human assisted reproductive technologies (ART). Work on animal models suggests that in vitro culture may be the source of these imprinting errors. However, in this study we report that, in some cases, the errors are inherited from the father. We analyzed DNA methylation at seven autosomal imprinted loci and the XIST locus in 78 paired DNA samples. In seven out of seventeen cases where there was abnormal DNA methylation in the ART sample (41%), the identical alterations were present in the parental sperm. Furthermore, we also identified DNA sequence variations in the gene encoding DNMT3L, which were associated with the abnormal paternal DNA methylation. Both the imprinting errors and the DNA sequence variants were more prevalent in patients with oligospermia. Our data suggest that the increase in the incidence of imprinting disorders in individuals born by ART may be due, in some cases, to the use of sperm with intrinsic imprinting mutations.

Figure 1

Methylation profile of natural and ART conceptus samples at imprinted and non-imprinted loci. Aberrant methylation over the range of ±2SD (standard deviations) of the mean of the normal fertilization methylation profile is indicated by red circles. White circles and bar indicate the mean and SD. Paternally methylated loci, H19 and GTL2 (a); maternally methylated loci, PEG1, KCNQ1OT1(LIT1), ZAC, PEG3, SNRPN, XIST of XX karyotype (non-ART conceptus samples, n=19; ART, n=46) and XIST of XY karyotype (non-ART conceptus samples, n=19; ART, n=32) (b), non-imprinted repetitive elements, Alu and LINE1 (c).

Figure 2

DNA methylation analyses in sperm and ART-treated conceptuses revealed abnormal methylation at H19 and GTL2 DMRs. Genomic structure of the human DMRs of H19 (a). The extent of the region analyzed in this study and GenBank accession number are shown under the line. The horizontal arrows represent primer positions. Vertical arrowhead indicates the unique bisulfite PCR restriction enzyme site analyzed by TaqI. Arrow shows the unique C/A polymorphism at nucleotide 8008 in the amplified region of the H19 DMR. The vertical bars represent CpG sites. DNA methylation analyses by COBRA (b) and bisulfite-PCR sequencing (c) of genomic DNA prepared from the sperm and ART conceptus samples fertilized from the same sperm shown to have an abnormal pattern of DNA methylation at the human DMR of H19. Conceptus sample, DNA obtained from ART conceptus; sperm, sperm DNA from the male patient. In the COBRA assay, bisulfite-treated DNA was amplified by PCR and digested with restriction enzymes TaqI only cuts the PCR product if the site was originally methylated in the genome. In the bisulfite-PCR sequencing of ART conceptus and sperm DNA at H19 (cases 23, 28, 44, 50, 52 and 67), each row represents a unique methylation profile with an average of 20 clones sequenced for each sample. Closed and open circles represent methylated and unmethylated CpGs, respectively. Samples were heterozygous for a C/A polymorphism allowing differentiation between the two parental alleles. The results are summarized in Table 1. Genomic structure of human DMR of GTL2 (d). Vertical arrowhead indicates the unique bisulfite PCR restriction enzyme site analyzed by TaqI and arrow shows the unique G/A polymorphism at nucleotide 51436 in the amplified region of the GTL2 DMR. Like H19, DNA methylation analysis was performed for the human DMR of GTL2 by COBRA (e) and bisulfite-PCR sequencing (f) of genomic DNA (cases 28 and 57) was prepared from the ART conceptus shown to have an abnormal pattern and the matched parental sperm.

Figure 2

DNA methylation analyses in sperm and ART-treated conceptuses revealed abnormal methylation at H19 and GTL2 DMRs. Genomic structure of the human DMRs of H19 (a). The extent of the region analyzed in this study and GenBank accession number are shown under the line. The horizontal arrows represent primer positions. Vertical arrowhead indicates the unique bisulfite PCR restriction enzyme site analyzed by TaqI. Arrow shows the unique C/A polymorphism at nucleotide 8008 in the amplified region of the H19 DMR. The vertical bars represent CpG sites. DNA methylation analyses by COBRA (b) and bisulfite-PCR sequencing (c) of genomic DNA prepared from the sperm and ART conceptus samples fertilized from the same sperm shown to have an abnormal pattern of DNA methylation at the human DMR of H19. Conceptus sample, DNA obtained from ART conceptus; sperm, sperm DNA from the male patient. In the COBRA assay, bisulfite-treated DNA was amplified by PCR and digested with restriction enzymes TaqI only cuts the PCR product if the site was originally methylated in the genome. In the bisulfite-PCR sequencing of ART conceptus and sperm DNA at H19 (cases 23, 28, 44, 50, 52 and 67), each row represents a unique methylation profile with an average of 20 clones sequenced for each sample. Closed and open circles represent methylated and unmethylated CpGs, respectively. Samples were heterozygous for a C/A polymorphism allowing differentiation between the two parental alleles. The results are summarized in Table 1. Genomic structure of human DMR of GTL2 (d). Vertical arrowhead indicates the unique bisulfite PCR restriction enzyme site analyzed by TaqI and arrow shows the unique G/A polymorphism at nucleotide 51436 in the amplified region of the GTL2 DMR. Like H19, DNA methylation analysis was performed for the human DMR of GTL2 by COBRA (e) and bisulfite-PCR sequencing (f) of genomic DNA (cases 28 and 57) was prepared from the ART conceptus shown to have an abnormal pattern and the matched parental sperm.