Aberrant paternal imprint methylation in the ART-treated conceptus
We first examined the methylation status of seven imprinted loci and the XIST DMR in DNA samples prepared from 78 XY karyotyped ART conceptuses and 38 non-ART conceptuses using COBRA (). In women, the XIST DMR is DNA methylated on one X chromosome whereas the other chromosome is unmethylated. In men, who have only one X chromosome, the single locus is normally methylated. Abnormal methylation was taken as being over the range of ±2SD (standard deviations) of the mean of the methylation in the normal fertilization products. Among the 78 ART-treated cases, we found 8 cases of aberrant paternal methylation (H19, 6 cases; GTL2, 2 cases; ). We confirmed the results of the COBRA assay by sequencing (). We examined global DNA methylation of the non-imprinted repetitive elements Alu and LINE1. There were no detectable differences between ART and normal samples (; ). Loss of DNA methylation was specific to the imprinted loci.
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. (more ...)
Abnormal methylation profiles detected by bisulfite PCR sequence in sperm and ART conceptus DNA
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 (more ...)
Aberrant maternal imprint methylation in the ART-treated conceptus
We found 12 cases of aberrant maternal methylation in the ART samples (PEG1, 1 case; KCNQ1OT1, 4 cases; ZAC, 1 case; PEG3, 1 case; XIST, 5 cases; ). The methylation errors were predominantly found at the H19 and KCNQ1OT1 DMRs, which commonly show alterations in DNA methylation in BWS patients. Five cases with an XX female karyotype were found with a methylation defect at the XIST locus but not at the autosomal DMRs. No samples showed complete imprint erasure.
In total, we identified 17 fetal samples with imprint methylation errors. Among them, a completely unmethylated pattern at a single locus was found in 14 cases and a mosaic pattern of aberrant methylation was identified at more than one loci in 3 cases.
Seven cases (21.8%) were the products of fertilization by ICSI, four cases (26.6%) were the products of IVF, two cases (11.8%) were involved a cryopreserved embryo and three cases (13.0%) were involved artificial insemination with husband's sperm (AIH). We also identified errors in three samples from our control group. We identified methylation errors in both the ICSI/IVF (n=39) and non-ICSI/IVF (n=39) groups indicating that ICSI was not responsible for these changes. There was no significant difference in the maternal ages between the ICSI/IVF and non-ICSI/IVF groups; 34.3±4.3 and 35.0±3.7 years old, respectively.
Relationship between aberrant methylation in sperm and conceptus after ART
We compared the quality of the parental sperm sample in the 17 cases where we identified imprinting errors. Seven samples were severely oligospermic (50.0%), four showed moderate oligospermia (25.0%) and six appeared normal (8.6%). There was an increased frequency in visible sperm abnormalities in the father associated with imprint errors in the conceptuses in line with our previous findings.19
Using DNA polymorphisms at the H19 and GTL2 loci, we found seven cases where the precise DNA methylation error was present in the father sperm and the conceptuses (; ), suggesting that the methylation errors were preexisting and not a consequence of the ART. We also identified four sperm samples (cases 13, 50, 53 and 70) that showed abnormal DNA methylation that was not present in the conceptus.
Mutation analyses of DNMT3A and DNMT3L
The consequences of Dnmt3a
deficiency in mice are loss of imprints and oligospermia, so we next examined 136 infertile men for gene mutations using SSCP and DNA sequencing. This group included the 7 male patients with an abnormal imprint in both the conceptuses and the sperm, 24 patients for which we previously reported an abnormal sperm imprint19
and an additional 105 patients who had undergone some fertility treatment where we had not identified a difference in DNA methylation. Of these, 27 had severe oligospermia, 30 had moderate oligospermia and 79 appeared normal.
We identified two sequence differences in DNMT3L that were predicted to alter the amino-acid sequence, one within exon 2 and one within the methylation enzyme activation motif IV in exon 10 (). The individual homozygous for the exon 2 polymorphism (518G/G) had DNA methylation errors at H19 and GTL2 and moderate oligospermia. Three heterozygous individuals (518G/A) showed normal methylation although one had severe oligospermia. Two individuals homozygous for the exon 10 alteration (1316G/G) had aberrant methylation of paternal and maternal imprinted loci and severe oligospermia. Of the twelve individuals that were heterozygous for this polymorphism (1316G/A), four showed abnormal methylation whereas eight appeared normal. They all had normal sperm or only moderate oligospermia.
Summary of the DNMT3L and DNMT3A sequence alterations identified in infertile men with abnormal imprint methylation in the sperm
We also identified sequence differences that were predicted to result in nonsense mutations. One was a 787C to T conversion in exon 5 located in the PHD domain. Six individuals homozygous for this alteration had abnormal methylation at some paternal and maternal loci and four also had severe oligospermia. The three heterozygous individuals had normal-appearing sperm and no detectable methylation errors. One individual homozygous for a nonsense variant (937C–T) in exon 6, also located in a PHD domain, had abnormal methylation at PEG3 in his sperm and three of the eleven individuals heterozygous for this sequence alteration had abnormally methylated sperm but none had oligospermia.
Of the seven male patients with an abnormal imprint in both the ART conceptuses and the sperm, two cases had variations in DNMT3L (case 50, 787C/C in exon 5 and case 28, 518G/G in exon 2), which could suggest that these variants affect the function of DNMT3L.
We identified three types of DNMT3A sequence alterations but these did not correlate with either an abnormal imprint or abnormalities in the number of sperm ().
In conclusion, we report seven cases where imprinting mutations in ART conceptuses match those present in the parental sperm and in two of these cases, the father also carried an alteration in the sequence of the DNMT3L gene. These findings suggest that the increased prevalence of imprinting disorders linked to ART does not originate solely through the process of ART but, in a significant number of cases, these alterations in DNA methylation preexist in the father's germ line. We may be selecting these cases because they are also linked with a low sperm count.