Using Advanced In Vitro Models to Investigate the Adverse Effects of Methotrexate on the Developing Epigenome

Abstract

Drug exposure during pregnancy has the potential to impact three generations in one: the mother (F0), the embryo (F1), and the future grandchildren in the form of developing gametes (F2). Recently, the effect of pharmaceutical drugs on the epigenome has come to light with more novel drugs being designed to target the epigenome. The epigenome is vital for successful development, therefore, it is important to understand the risk of exposure to current and novel drugs during pregnancy and their potential impact on the developing epigenome. This thesis outlines a proof-of-concept study detailing the use of in vitro models of embryogenesis and gametogenesis to investigate the impact of drug exposure on the developing epigenome. The folate antagonist methotrexate (MTX) was used at lower doses, equivalent to doses for treatment of rheumatoid arthritis, to interrogate the impact of perinatal exposure on the developing epigenome in vitro. Treatment of mouse embryonic stem cells (mESC) and epiblast-like cells with low-dose MTX, as a proxy for exposure in early embryonic development, resulted in a significant downward trend in the global average of CpG methylation, as well as hypomethylation and hypermethylation of CpGs genome-wide and at imprinted DMRs. Similarly, treatment of primordial germ cell-like cells, derived from the in vitro gametogenesis protocol as a proxy for the developing germline, also exhibited hypomethylation of imprinted DMRs and indicated an increased risk of reduced germline specification during development impacting offspring fertility and the health of future generations. Intriguingly, MTX treatment was shown to significantly increase global H3K27me3 levels in mESCs suggesting that MTX may impact the wider epigenome and highlighting the importance of investigating the impact of drug exposure on the developing epigenome. In conclusion, this thesis indicates that there is no safe dose in which MTX can be administered during pregnancy without potential harm to the developing embryo and its epigenome. Use of in vitro models of embryogenesis and gametogenesis are advised for future hazard testing of drugs and an emphasis on interrogating the teratogenic mechanisms of epigenetic perturbation is proposed.