The Interplay between DNA Methylation, Hydroxymethylation, and Steroid Hormone Signalling in Endometrial Biology and Endometriosis

Steroid hormones coordinate endometrial function through their respective receptors, with tightly regulated signalling across the menstrual cycle. Epigenetic mechanisms, including DNA methylation and hydroxymethylation, are increasingly recognised as key regulators of hormonal signalling, with growing evidence that this relationship is bidirectional; steroid hormones can also influence the epigenetic landscape. While aberrant DNA methylation is implicated in the dysregulation of hormone signalling in endometrial pathologies such as endometriosis, the role of hydroxymethylation and its mediators, Ten-Eleven Translocation (TET) enzymes, remains comparatively underexplored. Emerging evidence suggests TET enzymes may contribute to endometriosis pathogenesis, yet their specific roles in the endometrium remain unclear. Therefore, this thesis aimed to investigate the hormonal regulation of steroid hormone receptors and TETs in endometrial stromal cell lines derived from the eutopic endometrium of individuals with and without endometriosis (eESCs and ESCs). Hormonal regulation, proliferation, and expression of TET enzymes and steroid hormone receptors were examined in ESCs and eESCs following prolonged hormonal treatment. Short-term TETs and hormone receptor dynamics were explored in hormone-treated endometrial tissue explants and through inhibition of TET activity in stromal cell lines. Gene and protein expression were analysed by qRT-PCR, ddPCR, and western blotting. Several protocols designed to distinguish cytosines modified by methylation and hydroxymethylation in DNA were trialled for targeted sequencing of steroid hormone receptor promoters. ESCs and eESCs displayed distinct growth dynamics. eESCs exhibited reduced proliferation and lower PR, AR, and IGFBP1 mRNA expression, suggesting impaired hormone responsiveness. Estrogen receptor levels were largely unaffected by treatment. Inhibition of TET activity reduced cell viability and in eESCs led to reduced expression of TET3, ERα, PR, and AR, particularly with 17β-estradiol. Endometrial explants expressed androgen steroidogenic enzymes, supporting the functional relevance of androgen signalling in the endometrium. DNA treatment and sequencing protocols were optimised to inform the design of a novel, locus-specific approach for detecting 5-hydroxymethylcytosine in endometrial DNA. These findings demonstrate a close relationship between TET enzymes and steroid hormone receptors in endometrial stromal cells, suggesting coordinated regulation of epigenetic and hormonal signalling. This work highlights the potential role of hydroxymethylation in endometrial hormone responsiveness and its contribution to endometriosis pathogenesis.