## 3 Results and Discussion
### Methylation Changes and Exposure to Phthalates and Alternative Plasticizers  
Our group previously reported that the urinary concentrations of MEHP, MEHHP, 2 cx-MMHP, \(\underline{\gamma}\)MEHP-3, and \(\underline{\gamma}\)MEHP-5 were associated with risk of uterine leiomyoma, suggesting that phthalates could have the potent effects on pathogenesis of uterine leiomyoma [12]. Further associations between exposure to phthalates and alternative plasticizers and DNA methylation changes in two different tissues were analyzed as shown in Table 3. _ESR1_ methylation in leiomyoma was negatively associated with 2 cx-MMHP (\(\beta=-9.389\), 95% CI: \(-15.425\), \(-3.353\)) and MCPP (\(\beta=-10.670\), 95% CI: \(-20571\), \(0.769\)) after adjusting for confounding factors. However, 5 OXO-MEHTP, one of the alternatives, showed positive association with _ESR1_ methylation in leiomyoma (\(\beta=17.928\), 95% CI: \(6.420\), \(29.437\)). _PGR_ methylation in leiomyoma was significantly associated with MnBP (\(\beta=1.128\), 95% CI: \(0.055\), \(2.201\)), but negatively associated with \(\kappa\)-MINCH, another alternative metabolite (\(\beta=-1.220\), 95% CI: \(-2.210\), \(-0.230\)). In addition, _PGR_ methylation in myometrium was also positively associated with MEP (\(\beta=0.637\), 95% CI: \(0.093\), \(1.181\)) and MCPP (\(\beta=1.259\), 95% CI: \(0.250\), \(2.268\)). The epigenetic effect of phthalates was reported in several experimental and animal studies. One murine study showed that maternal exposure to DEHP caused testicular dysfunction via DNA hypermethylation, leading to increased expression of _DNA methyltransferases (Dnntts)_ and downregulated expression of _insulin-like hormone-3 (Inst3)_[42]. Exposure to phthalates mixture induced abnormal expression of miR-141-3p and miR-184 in rats, and their downregulated target genes were associated with prostate development and oncogenesis [43]. Another study found that MCF7 cells treated with dibutyl phthalate (DBP) or butyl benzyl phthalate (BBP) led to demethylation of promoter regions of  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline \multicolumn{2}{c}{**ESR1**} & \multicolumn{2}{c}{**PGR**} \\ \hline
**Phthalates and** & **Leiomyoma b ** & **Myometrium** & **Leiomyoma** & **Myometrium** \\ \hline MEHP & \(-4.267\)\((-9.432\), \(0.898\)) & \(2.773\)\((-3.1171\), \(8.663\)) & \(-0.330\)\((-1.381\), \(0.722\)) & \(0.566\)\((-0.080\), \(1.213\)) \\ MEOHHP & \(-4.797\)\((-1.1150\), \(1.574\)) & \(2.932\)\((-3.678\), \(9.541\)) & \(0.583\)\((-0.612\), \(1.777\)) & \(0.003\)\((-0.805\), \(0.812\)) \\ MEHHP & \(-4.797\)\((-12.060\), \(2.466\)) & \(2.010\)\((-5.541\), \(9.561\)) & \(0.726\)\((-0.612\), \(2.063\)) & \(-0.644\)\((-0.975\), \(0.846)\) \\
2 cx-MMHP & \(-9.389\)\((-1.5425\), \(-3.353\)) & \(2.086\)\((-5.374\), \(9.545)\) & \(-0.066\)\((-1.432\), \(1.301\)) & \(-0.222\)\((-1.117\), \(0.672)\) \\
5 cx-MEPP & \(-3.727\)\((-9.280\), \(1.735\)) & \(2.231\)\((-3.341\), \(7.923\)) & \(0.569\)\((-0.446\), \(1.585\)) & \(0.068\)\((-0  
_ESR1_[44]. In addition, phthalates are known to adversely affect estrogen signaling by interacting with ER\(\alpha\) and ER\(\beta\)[45]. Growth of uterine leiomyoma depends on ovarian steroids, estrogen and progesterone [46; 47]. However, there are very limited studies to determine the association between phthalates and expression of _ESR1_ and _PGR_ in relation to uterine leiomyoma. Our results showed that two phthalate metabolites (i.e., 2 cx-MMHP and MCPP) showed significant association with hypomethyllation of _ESR1_ in leiomyoma tissue, while MnBP was associated with hypermethylation of _PGR_, suggesting that phthalate metabolites might contribute to pathogenesis of uterine leiomyoma via increased expression of _ESR1_. A recent study by [PERSON] et al. [48] showed that mono-hydroxybutyl phthalate (MEBP) and MEHHP are significantly associated with overexpression of miR-10a-5p and miR-577 in leiomyoma patients.