5-Aza-2′-deoxycytidine Leads to Reduced Embryo Implantation and Reduced Expression of DNA Methyltransferases and Essential Endometrial Genes

Abstract

BackgroundThe DNA demethylating agent 5-aza-2′-deoxycytidine (5-aza-CdR) incorporates into DNA and decreases DNA methylation, sparking interest in its use as a potential therapeutic agent. We aimed to determine the effects of maternal 5-aza-CdR treatment on embryo implantation in the mouse and to evaluate whether these effects are associated with decreased levels of DNA methyltransferases (Dnmts) and three genes (estrogen receptor α [Esr1], progesterone receptor [Pgr], and homeobox A10 [Hoxa10]) that are vital for control of endometrial changes during implantation.Methods and Principal FindingsMice treated with 5-aza-CdR had a dose-dependent decrease in number of implantation sites, with defected endometrial decidualization and stromal cell proliferation. Western blot analysis on pseudo-pregnant day 3 (PD3) showed that 0.1 mg/kg 5-aza-CdR significantly repressed Dnmt3a protein level, and 0.5 mg/kg 5-aza-CdR significantly repressed Dnmt1, Dnmt3a, and Dnmt3b protein levels in the endometrium. On PD5, mice showed significantly decreased Dnmt3a protein level with 0.1 mg/kg 5-aza-CdR, and significantly decreased Dnmt1 and Dnmt3a with 0.5 mg/kg 5-aza-CdR. Immunohistochemical staining showed that 5-aza-CdR repressed DNMT expression in a cell type–specific fashion within the uterus, including decreased expression of Dnmt1 in luminal and/or glandular epithelium and of Dnmt3a and Dnmt3b in stroma. Furthermore, the 5′ flanking regions of the Esr1, Pgr, and Hoxa10 were hypomethylated on PD5. Interestingly, the higher (0.5 mg/kg) dose of 5-aza-CdR decreased protein expression of Esr1, Pgr, and Hoxa10 in the endometrium on PD5 in both methylation-dependent and methylation-independent manners.ConclusionsThe effects of 5-aza-CdR on embryo implantation in mice were associated with altered expression of endometrial Dnmts and genes controlling endometrial changes, suggesting that altered gene methylation, and not cytotoxicity alone, contributes to implantation defects induced by 5-aza-CdR.

Epigenetic regulations through DNA methylation and hydroxymethylation: clues for early pregnancy in decidualization

“…Enhanced expression of DNMT1 and DNMT3A in decidua, together with the fact that 5-aza-dC significantly prohibits the maintenance of decidualization and aberrantly upregulates two hyper-methylated genes ( Bcl3 and Slc16a3 ), strongly supports an essential role of this epigenetic regulation for successful postimplantation decidual development (Figure 4). The negative effects of 5-aza-dC on decidualization is also supported by another study using a mouse model (68). Recently, appreciable differential methylation on a single chromatin is uncovered comparing fertile and infertile decidua from genetically different mice (73).…”

“…In our pilot study using a mouse model, application of 5-aza-dC while establishing receptivity (days 3–4) seems to have minimal impact on embryo attachment and the number of implantation sites, however, the proliferation of local stromal cells is comprised profoundly, indicating the predominant adverse effects in the postimplantation uterus (67). Another study administrating 5-aza-dC immediately after fertilization (day 1) through day 4 shows opposing effects on receptivity and moderate reduction in the expression of DNMTs (68), possibly due to increased chronic cytotoxicity by extended drug treatment. It is also possible that untested and potentially harmful effects on hormone production are at fault, as DNA methylation of crucial genes involves corpus lutea formation (52).…”

Epigenetic regulation of the placental HSD11B2 barrier and its role as a critical regulator of fetal development

“…This was associated with a cell specific reduction in mRNA levels and an increase in the methylation levels of many genes involved in endometrial change, as well as a decrease in stromal cell proliferation and differentiation. 55 Furthermore, intravenous infusion of 5-AZA in patients with metastatic lung carcinomas induced hematopoiesis toxicity, 56 thus limiting their use in pregnant women. Given these significant problems and challenges associated with their use, the administration of pharmacological epigenetic modifiers to modify the placental epigenome is not feasible at the present time.…”

Tris(1,3-dichloro-2-propyl)phosphate Induces Genome-Wide Hypomethylation within Early Zebrafish Embryos

“…3 After rapid erasure of maternal and paternal methylation marks following fertilization, zygotic genome activation, tissue-specific gene expression, and normal somatic development of mammalian and zebrafish embryos are dependent on steady, de novo genome-wide cytosine methylation by DNA methyltransferases (DNMT) during the MZT. 4,5 Therefore, alterations in de novo DNA methylation marks, via impacts on DMNT activity or methyl donor concentrations, during early embryonic development have the potential to change the trajectory of normal development and, if inherited, persist across multiple generations. 6 …”