Imprinted genes play an important role in fetal and placental development. Using quantitative bisulfite pyrosequencing assays, we determined the DNA methylation levels at two paternally methylated (H19 and MEG3) and four maternally methylated (LIT1, NESP55, PEG3, and SNRPN) imprinted regions in fetal muscle samples from abortions and stillbirths. Two of 55 (4%) spontaneous abortions and 10 of 57 (18%) stillbirths displayed hypermethylation in multiple genes. Interestingly, none of 34 induced abortions had extreme methylation values in multiple genes. All but two abortions/ stillbirths with multiple methylation abnormalities were male, indicating that the male embryo may be more susceptible to excess methylation. Hypermethylation of multiple imprinted genes is consistent with stochastic failures of the mechanism, which normally protects the hypomethylated allele from de novo methylation after fertilization. Two of six informative abortions/stillbirths with H19 hypermethylation revealed significant biallelic expression of the autocrine growth factor IGF2. In two other cases hypermethylation of MEG3 was associated with transcriptional down-regulation. We propose that primary epimutations resulting in inappropriate methylation and expression patterns of imprinted genes may contribute to both normal human variation and disease , in particular spontaneous pregnancy loss. Pregnancy loss is a common medical problem. Approximately 25% of reproductive-aged women attempting pregnancy have spontaneous abortions, and at least 10% of all clinical pregnancies are lost.1,2 The predominant cause of pregnancy loss in the first trimester are aneuploidies, mainly attributable to chromosome errors during maternal meiosis. 3,4 In addition, multiple other etiologies have been described, including single gene defects (ie, trombophilic mutations), endocrine and immunological factors, uterine anatomical abnormalities, environmental exposures, and infections. Despite modern diagnostics of chromosomal and nonchromosomal factors, a large proportion of cases remains unexplained.Epigenetic changes of the genetic information are not produced by changes in the DNA sequence itself, but by reversible modifications of DNA and chromatin structure. The most thoroughly studied epigenetic modification is methylation of CpG dinucleotides in cis-regulatory sequences, which is associated with specific histone modifications leading to a condensed chromatin structure and transcriptional repression. 5,6 Genomic imprinting is a parent-specific epigenetic modification in which allelespecific expression depends on male versus female germline transmission. Most imprinted genes display differentially methylated regions (DMRs), which are thought to function as imprinting control centers. 7,8 Parent-specific methylation patterns (genomic imprints) are established during gametogenesis. In the zygote and early embryo genome-wide demethylation waves erase most of these germline patterns, followed by de novo methylation and establishment of somatic methylation pattern...

Recently, several groups described the isolation of mouse spermatogonial stem cells (SSCs) and their potential to develop to embryonic stem cell (ESC)-like cells, so-called multipotent germline stem cells (mGSCs). We were the first to derive such mGSCs from SSCs isolated from adult mouse testis and, therefore, called these mGSCs multipotent adult germline stem cells (maGSCs). Here, we comparatively analyzed gene-specific and global DNA methylation profiles as well as the telomerase biology of several maGSC and male ESC lines. We show that undifferentiated maGSCs are very similar to undifferentiated male ESCs with regard to global DNA methylation, methylation of pluripotency marker gene loci, telomerase activity and telomere length. Imprinted gene methylation levels were generally lower in undifferentiated maGSCs than in undifferentiated male ESCs, but, compared with undifferentiated mGSCs derived by other groups, more similar to those of male ESCs. Differentiation of maGSCs increased the methylation of three of the four analyzed imprinted genes to almost somatic methylation patterns, but dramatically decreased global DNA methylation. Our findings further substantiate the pluripotency of maGSCs and their potential for regenerative medicine.

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