## 3 Results and Discussion
### Methylation Changes and Exposure to Phthalates and Alternative Plasticizers
The consequent target genes of phthalates-associated miRNAs are linked to multiple fibroid-related processes including angiogenesis, apoptosis, and proliferation of connective tissues [48]. This study supports our findings suggesting that epigenetic alteration is associated with phthalates exposure and uterine leiomyoma development. Furthermore, EDC, including phthalates, are known to induce oxidative stress and its associated burden such as DNA methylation changes (i.e., hypermethylation and hypomethyllation). EDC-induced reactive oxygen species (ROS) interact with androgen and ERs, leading to sex-hormone pathologies [49]. Thus, phthalates-generated ROS induce methylation changes in _ESR1_; this, in turn, may cause estrogen pathologies and uterine fibroids. There is little data related to phthalates and _PGR_ expression; a few mechanistic, preclinical, and clinical studies reported that phthalates interfere with normal function of PR. DEHP treatment inhibited progesterone secretion from human luteal cells [50] and decreased secretion of progesterone in mice [51]. One study on the structural binding mechanism of DEHP and its metabolites with PR found that phthalates induced dysfunction of progesterone signaling and adverse effects due to their high binding affinity [52]. Our results also showed that phthalates seemed to inhibit _PGR_ expression in leiomyoma; however, additional studies are needed to confirm this. In this study, we also analyzed alternative plasticizer metabolites in leiomyoma patients and examined the association between exposed levels of alternative plasticizers and methylation of _ESR1_ and _PGR_ in leiomyoma. Interestingly, alternative metabolite 5 OXO-MEHTP had an opposing association with _ESR1_ methylation compared to phthalate metabolites, while cx-MINCH showed another opposing association with _PGR_ methylation in leiomyoma. DINCH and DEHTP have been used as the substitute for DEHP, but their metabolites' effects on methylation changes in _ESR1_ and _PGR_ are opposed to those of DEHP metabolites. These differences in biological activity at the molecular level may be associated with different structures and physical properties with different chemodynamics. Furthermore, DINCH showed significant impacts on steroidogenesis, testicular development, and function in some studies [53; 54], and an in vitro study by [PERSON] et al. showed that DINCH metabolites had a weak osteogenic property [55]; these results support our results. However, further findings, including in vivo and in vitro should be done to confirm the correct epigenetic mechanism. There are some limitations in our study; the relatively small sample size with large variation in disease status may restrict the statistical power of the results. We also obtained only one spot urine sample from each of the cases, which would be only moderately predictive for long-term exposure, and exposure misclassification cannot be fully ruled out. Some of the study specimens accompany endometriosis and adenomyosis, which may be associated with phthalates exposure. Therefore, further study for only leiomyoma cases is needed. Despite these limitations, this study, to our knowledge, is the first to report methylation changes of _ESR1_ and _PGR_ in related to uterine leiomyoma and exposure to phthalates and alternative plasticizers.