During mammalian embryo implantation and development, DNA methylation undergoes dramatic reprogramming that is crucial for the development of both the embryo and the maternal endometrium [23
]. In the present study, we showed that DNMT1 and DNMT3A were expressed in human villous trophoblast and glandular epithelial cells of decidua. DNMT1 was significantly down-regulated in the villous of women with EPL, but not in the decidua. DNMT3A expression was not significantly changed in the EPL group compared to the control group. The global DNA methylation level was significantly lower in the EPL villous than in villous from the controls. These findings suggest that insufficient embryonic maintenance methylation is associated with abnormal embryonic development in human early pregnancy loss. Evidence from in vitro
and in vivo
studies confirmed the importance of maintenance DNA methylation in embryo implantation and development. To the best of our knowledge, this is the first report describing defects in DNA maintenance methylation in the pathogenesis of EPL.
In the present study, we first demonstrated that DNMT1 and DNMT3A proteins were expressed in the nuclei of the villous trophoblast and glandular epithelium of normal human decidua. DNMT3B, a de novo
methyltransferase, was detected with low expression in decidual tissue, but not in villous (data not shown). The most likely explanation is that DNMT3B is not expressed in extra-embryonic lineages of post-implantation embryos [27
]. The expression of both DNA methyltransferases suggests that DNMT1 and DNMT3A may be DNA methylation regulatory enzymes at the feto-maternal interface during early embryo development. Subsequent semiquantitative analysis supported a pathological role of DNMT1 in EPL. De novo
methyltransferase DNMT3A expression had no evident relationship with EPL. A possible reason might be that de novo
methylation mainly happens during epigenetic reprogramming in the gametes and preimplantation embryo [28
]. After embryo implantation, the methylation is maintained by DNMT1 [30
] and there is no noticeable de novo
methylation change in the post-differentiated human villous [31
For the global methylation assay, we used the Methylamp™ Global DNA Methylation Quantification Ultra Kit. This commercial product for global methylation assay is commonly used [32
]. The assay uses a 5-methylcytosine antibody to distinguish methylated from unmethylated cytosine. These assays are influenced significantly by mCpG density so that more highly methylated regions are detected better than regions of low methylation density. These methods do not offer accurate quantitative results; however, they do allow comparisons between groups. The methylation levels in the EPL group and the control group were measured at the same time with the same kit to decide whether there is a relative difference of DNA methylation level between the two groups. Our results show that, compared with the control group, global DNA methylation was significantly reduced in the EPL group both in the patient samples and in the animal model.
The alterations in the expression of DNMT1 and global demethylation in villous of the EPL women suggest that aberrant maintenance DNA methylation may be involved in the pathogenesis of human EPL. It was reported that mouse embryos lacking Dnmt1 showed genome-wide demethylation and knockout of Dnmt1 in embryos led to a complete loss of methylation at both paternally and maternally methylated differential methylation regions (DMRs) [17
]. Mice deficient in the maintenance of genomic imprints exhibited significant developmental delays in multiple organ systems [33
]. Since demethylation of the genome decreases cell proliferation [15
] and the aborted villous has less cell proliferation and more cell apoptosis than normal villous [34
], insufficient DNA methylation may influence cell proliferation, migration and invasion, thus affecting embryonic implantation and post-implantation development [35
Implantation is the first and crucial step in governing reproductive outcomes [36
]. Fundamental to this process are dynamic and precisely ordered molecular and cellular events, driven by the combination of embryo and host-receptive-endometrium to facilitate the establishment of the maternal-fetal interface. Regulation of DNA methylation plays an important role in this process [35
]. In our in vitro
experiment of embryo implantation, the reduction of the implantation rate may also be caused by abnormal trophoblastic cell proliferation, migration and invasion. In addition, the expression of LIF by Ishikawa cells in the presence of a DNA methylation inhibitor did not significantly change, testifying to the acceptance of our embryonic adhesion/implantation model. LIF is one of the crucial cytokines in the endometrium or at the maternal-fetal interface [37
]. The steady expression of LIF in the presence of a DNA methylation inhibitor suggests that the disturbance of maintenance methylation may not influence the endometrial receptivity for embryos.
The findings of our mouse experiment confirm that disturbance of maintenance DNA methylation plays an important role in the pathogenesis of EPL. Female mice that received a uterine injection of a DNA methylation inhibitor presented typical symptoms of EPL with low implantation and pregnancy rates, and the embryos in the treated uterine horn showed global DNA demethylation. The number of fetuses in the treated uterine horn was less than the control horn, and the aborted fetuses and placentas in the treated horn had incomplete development of organs and obscured tissue structures. It has been shown that knockdown of DNMT1 results in decreased cell viability [38
]. Further research indicated that DNMT1 had an intimate relationship with DNA replication and the cell cycle, since inhibition of DNA methyltransferase interferes with DNA replication, and DNMT1 depletion triggers intra-S-phase cell cycle arrest [39
]. In the realm of organism development, DNA methylation also has long-term effects on the development of the fetus and placenta. Non-specific demethylation reagents have been found to cause growth retardation, malformation, fetal lethality and abnormal tissue structure in the placenta [40
]. Thus, we speculated that the poor formation of the embryonic organs and tissues in our study might be induced by the demethylation of the promoters of important developmental genes, thereby resulting in poor conditions for pregnancy. However, a thorough study of the mechanisms underlying the effect of DNA demethylation on early pregnancy loss is needed.