and maternal H19
expression are closely regulated by genetic imprinting including numerous regulatory mechanisms such as allele-specific DNA loops, multiple enhancers, boundary elements and histone modifications.12, 13, 14
The human locus contains three DMRs: the DMR0 and DMR2 of the IGF2
gene and the H19
DMR. A notable discrepancy to the mouse Igf2–H19
locus is the absence of a human IGF2
DMR1. In human sperm from fertile and infertile men, only the methylation status of the 6th CTCF-binding site of the H19
DMR was previously analysed.5, 6, 7, 8
In this study, we report for the first time the methylation status of the two IGF2
DMRs (DMR0 and DMR2) and of the 3rd and 6th CTCF-binding sites of the H19
DMR in spermatozoa from men presenting normal and altered sperm parameters. Pyrosequencing is an exceedingly quantitative technology yielding accurate and highly reproducible information about methylation positions at a single-nucleotide resolution.9
In normozoospermic patients, a mean methylation level of 86.8±6.9% was found for these four regions. Taking into account the inter-assay variation, this suggests either a complete demethylation of ~10% of spermatozoa in normal human sperm or the existence of ~10% of unmethylated CpG at one position or another. In concordance with our results, a low proportion of unmethylated paternal haploid genome in normal human semen was previously reported.6, 7, 8
Moreover, for specific CpG positions inside the IGF2
DMR2 (CpG 7 and CpG 9), the methylation mean varied significantly displaying a specific methylation profile in this region and suggesting that the local chromatin architecture might influence the accessibility to methyltransferase activity. The systematic microscopic control of the spermatozoa preparation in our study eliminated the possibility of an eventual somatic cell contamination.
In this study, the IGF2
DMR0 was found highly methylated. The DMR0 may have an important role in the regulation of IGF2
imprinting control because this region is hypomethylated and associated with loss of imprinting in Wilms' tumours and colorectal cancers.15, 16, 17
The mouse Igf2
DMR0 was previously described as only maternally methylated in placenta and with a methylation of the two alleles in other tissues.13, 18
In humans, the DMR0 is differentially methylated in tissues as blood cells, placenta and kidney.16, 19
These two studies reported the methylation to be on the maternal allele in the DMR0. Our data provides evidence of a methylation of IGF2
DMR0 in spermatozoa. This is in concordance with the recent report of Murrell et al
analysing the DMR0 methylation status in growth disorders, cancers and normal tissues and suggesting that the active paternal IGF2
allele is methylated at the DMR0 in cis
methylation in normal individuals. In our study, the methylation pattern of DMR0 was similar in normal and abnormal sperms. The three other regions exhibited the expected DNA methylation profile corresponding to a paternal methylation imprint in normozoospermic patients.
The quantitative pyrosequencing analysis of normal sperm allowed us to define a threshold for the methylation status for each CpG position and to precisely detect sporadic or severe significant methylation defects at individual or consecutive CpG positions in sperm samples of infertile men.
In the samples with isolated abnormalities of spermatozoa morphology (T), a normal methylation profile of the four regions was only observed for 42% of patients. Thus, 58% of the sperm samples presented a significant loss of methylation at particular CpG positions for the IGF2
DMR2 that was associated in six men with a loss of methylation at some CpG positions in the 6th CTCF-binding site of the H19
DMR. To our knowledge, these methylation modifications were not previously reported in patients with teratozoospermia. No significant association was found between the loss of methylation and any specific morphological abnormality according to accurate and detailed analysis of sperm morphology.11
The loss of methylation in the IGF2
DMR2 and the 6th CTCF-binding site of the H19
DMR were accentuated in the samples with low-sperm concentrations (OAT-II to OAT-IV; 59% of patients). No methylation changes were observed for the IGF2
DMR0 or the 3rd CTCF-binding site, demonstrating that inside a ~100
kb locus, two regions with abnormal methylation profile and two regions without alterations can exist and highlighting the locus-specificity of the methylation loss associated with spermatogenesis failure.
Previous reports showed an abnormal paternal methylation imprint of the 6th CTCF-binding site of the H19
DMR, as well as GTL2
in a low proportion of patients with moderate and severe oligospermia.6, 7, 8
The same reports showed also a frequent gain of methylation in regions with a maternal methylation imprint (MEST/PEG1, LIT1
These results suggest a rise of methylation or a failure in erasure during germ-cell development. Nevertheless, the methylation profile of non-imprinted repetitive elements, such as long interspersed nucleotide elements (LINE1
) and ALU
element was normal7, 8
contradicting the reports of increased DNA methylation in poor quality human sperm.21
Our results support this hypothesis of strong methylation errors occurring at specific loci in sperm from infertile men without genome-wide alterations.
Although somatic cells were not observed by microscopic inspection of all spermatozoa preparations, a very improbable somatic contamination cannot be fully excluded. The expected methylation status of the somatic cells present in the ejaculate (round cells and leukocytes) is due to paternal methylated and maternal unmethylated alleles at the IGF2/H19 DMRs leading to a 50% of methylation that would be quantified by pyrosequencing. In our study, the methylation losses are from 10 to 80%. An undetectable somatic cell contamination by microscopy could not account for such a large reduction in methylation level consistent over the analysed subgroups.
The swim-up technique is the best available method for selecting spermatozoa and eliminates somatic cell contamination leading to a selection of highly mobile spermatozoa. Nevertheless, this technique could not be used in our study for preparation of sperm from OAT patients who have a dramatic reduction of the sperm motility. Moreover, the use of highly selected spermatozoa with a good motility would distort the methylation analysis. We therefore applied the described spermatozoa preparation protocol that can be applied to all patient groups.
The observed DNA methylation alterations could be due to failures in chromatin compaction events that are highly controlled by essential sperm-specific chromatin proteins.22
The haploid phase of spermatogenesis is marked by the expression of protamines facilitating the molecular remodelling and specific compaction of the male genome associated with gene silencing. However, the human sperm nucleus retains about 15% of the somatic histones.23
The nucleohistone and nucleoprotamine compartments form discrete structures that are at least partly sequence specific.23
The telomeric repetitive sequences are histone-rich, whereas the β-globulin and Alu sequences are protamine-enriched.24
gene retains the nucleohistone compartment, whereas the H19
gene is predominantly organised by a unique structural repeat condensing at least 20
kb of chromatin.25
The nucleohistone/nucleoprotamine partition of the sperm genome could support epigenetic information and operate in the imprinting establishment.
The observed alterations might also be related to less efficient DNA methyltransferases, especially DNMT3L and 3A. In mice, the Dnmt3L
and conditional Dnmt3A
KO result in azoospermia whereas Dnmt3a
KO disturbs the methylation imprinting.26, 27
It would therefore be interesting to measure the Dnmt3A/3L activities in human spermatozoa in case of oligozoospermia.
As the methylation mark apposition occurs at the spermatogonia stage,5
it could be hypothesised that the initial cause of the DNA methylation defects observed for the infertile men is either a failure of the resetting of methylation in a subset of adult spermatogonia or an alteration of the methylation maintenance in spermatocytes, spermatids or mature spermatozoa. Through testicular biopsies, it will be informative to collect spermatogonia, spermatocytes, spermatids and spermatozoa to compare the methylation status of the IGF2-H19
locus in men with or without infertility.
A possible link between ART and imprinting congenital disorders, such as Angelman, Prader-Willi and Beckwith-Wiedemann syndromes has been suggested by several groups.28, 29, 30
It is so far unclear at which stage(s) the epigenetic alterations could arise during the ART procedures. It has been argued that the use of spermatozoa from men with abnormal semen parameters and abnormal IGF2–H19
methylation status could affect imprinting. Two previous studies reported ART outcomes for men with methylation defect of their sperm: Kobayashi et al8
described a low percentage of pregnancies with several miscarriages in samples presenting abnormal imprinting. Benchaib et al31, 32
correlated a global sperm hypomethylation and low pregnancy rates without differences in fertilization rates or embryo quality. Using the methylation threshold concept, we could classify the 24 men who benefitted from ART into two groups, methylated and unmethylated. All the UM patients presented an oligozoosspermia, whereas the M patients had normal sperm characteristics or only abnormal sperm morphology. In our laboratory, no significant difference in rate fertilization was found between IVF and ICSI (64.5 versus
70%, respectively). Taking this information into account, it could be considered that the fertilization rate is expected in the UM group (63%) and increased in the M group (83%), whereas embryo quality and implantation rate were similar. Normal pregnancies, deliveries at term and birth of healthy newborns were obtained from men whose sperm had a drastic methylation loss. The delivery and ongoing pregnancy rate differed with a higher percentage in the group M but the difference was not significant. Larger studies are required to obtain sufficient statistical power to fully evaluate these potential associations.
Further studies might also address the relationship between the methylation status and the fertilization ability of individual spermatozoa.