<p>Postmenopause is characterized by a permanent decline in estrogen levels, effecting various tissues and contributing to systemic aging. Reduced hormonal levels negatively impact vaginal health, leading to tissue atrophy, which can be alleviated with topical estrogen therapies. This study aimed to assess the effects of menopausal hormone deficiencies on DNA methylation in vaginal tissue and swabs, compared to buccal swabs, and evaluate the impact of local estrogen therapy on these changes. Samples from 126 women were categorized into three groups: premenopausal, postmenopausal without local estrogen therapy, and postmenopausal with local estrogen therapy. Postmenopausal women with urogenital atrophy exhibited elevated cytosine methylation (5-mC) compared to premenopausal women, whereas those receiving local estrogen therapy had 5-mC levels similar to premenopausal women. Additionally, 5-hydroxymethylcytosine (5-hmC) levels, a marker of demethylation, was also higher in the estrogen-treated group. The 5-mC/5-hmC ratio was notably elevated in women with urogenital atrophy. Significant differences in DNA methylation dynamics were observed in vaginal samples but not in buccal swabs among postmenopausal groups. Menopausal hormonal deficiencies lead to epigenetic changes in vaginal tissue, particularly increased cytosine methylation. Local estrogen therapy can mitigate these changes, providing insights into aging processes. Analyzing DNA methylation in vaginal swabs may serve as a non-invasive tool for monitoring age-related changes and assessing therapeutic efficacy. Further research is needed to understand these epigenetic modifications and their clinical implications.</p>