Endocrine-disrupting chemicals and uterine fibroids.

HHS Public Access Author manuscript Author Manuscript

Fertil Steril. Author manuscript; available in PMC 2017 September 15. Published in final edited form as: Fertil Steril. 2016 September 15; 106(4): 967–977. doi:10.1016/j.fertnstert.2016.08.023.

Endocrine disrupting chemicals and uterine fibroids Tiffany A. Katz, Ph.D., Texas A&M University Health Science Center, Institute of Biotechnology, Center for Translational Cancer Research, Houston TX

Author Manuscript

Qiwei Yang, Ph.D., Augusta University, Medical College of Georgia, Department of Obsetetrics and Gynecology, Augusta GA Lindsey S. Treviño, Ph.D., Texas A&M University Health Science Center, Institute of Biotechnology, Center for Translational Cancer Research, Houston TX Cheryl L. Walker, Ph.D.; A.T.S.; F.A.A.A.S., and Texas A&M University, Health Science Center, Institute of Biotechnology, Center for Translational Cancer, Research, Houston TX Ayman Al-Hendy, M.D.; Ph.D. Augusta University, Medical College of Georgia, Department of Obsetetrics and Gynecology, Augusta GA

Author Manuscript

Abstract

Author Manuscript

Uterine fibroids are the most frequent gynecologic tumor, affecting 70–80% of women over their lifetime. Although these tumors are benign, they can cause significant morbidity and may require invasive treatments such as myomectomy and hysterectomy. Many risk factors for these tumors have been identified, including environmental exposures to endocrine disrupting chemicals (EDCs), e.g. genestein, diethylstilbestrol etc. Uterine development may be a particularly sensitive window to environmental exposures, as some perinatal EDC exposures have been shown to increase tumorigenesis in both rodent models and human epidemiological studies. While the mechanisms by which EDC exposures may increase tumorigenesis are still being elucidated, epigenetic reprogramming of the developing uterus is an emerging hypothesis. Given the remarkably high incidence of uterine fibroids, and their significant impact on women’s health, understanding more about how prenatal exposures to EDCs (and other environmental agents) may increase fibroid risk could be key to developing prevention and treatment strategies in the future.

Corresponding: [email protected], Phone: 706-721-3833, Address: 1120 15th St., Augusta, GA 30912. Co-corresponding: [email protected], Phone: 713-677-7440, Address: 2121 W. Holcombe Blvd, Houston, TX 77030. Conflict of interest: None of the authors have a financial relationship with a commercial entity with an expressed interest in the subject-matter of this manuscript. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Katz et al.

Page 2

Author Manuscript

Keywords Uterine fibroids; Endocrine disrupting chemicals; Developmental reprogramming; Epigenome; Myometrial stem cells; Tumor-initiating cells

1. General overview 1.1. Uterine fibroids: The Human Disease

Author Manuscript

Uterine fibroids (UFs) affect 70–80% of women during their lifetime, and are the leading indication for hysterectomies in premenopausal women, with over 200,000 per year in the US (1–4) at a cost of up to 34 billion dollars each year (5). Medical management and uterine preserving procedures are available to treat this disease, but a lack of comprehensive data comparing treatment options leads most practitioners to resort to the classic treatment of hysterectomy (6). Although UFs are benign tumors, they can cause a variety of symptoms such as pain, bleeding, and bladder dysfunction as well as complications leading to infertility, miscarriage and other reproductive disorders (7–10). Arising in the myometrial layer of the uterus, UFs are hormonally responsive to estradiol and progesterone as well as other steroid hormones, and regress after menopause (11). The cause of uterine fibroids is largely unknown but there are established risk factors: increased age up to menopause, increased BMI, nulliparity, early age at menarche, family history, and African American ethnicity (11,12). While many of the risk factors for this disease are hormone-related, the mechanism underlying the increase in risk for African American women compared to Caucasians remains to be identified (1,2,13–16).

Author Manuscript

The incidence of UFs is likely underestimated, as only 20–50% of women with fibroids will develop symptoms (1,17). Data on latency for development of these tumors is limited, although the time of onset is thought to be 10–15 years earlier for African American women (1,12,18). Ultrasound has been used to find clinically undiagnosed UFs (1,18–20), but only one study has found fibroid free women prospectively to ascertain the timing of fibroid onset (21). The Study of Environment, Lifestyle, and Fibroids (SELF) was designed to investigate the timing of UFs onset. The SELF study enrolled 1696 African American women aged 23– 34 without UFs in the Detroit area from 2010 to 2012 and plans to follow up every 20 months with ultrasound assessment to determine more precisely the onset of UFs (21).

Author Manuscript

In addition to hormones, these lesions are influenced by genetic aberrations as well as growth factor signaling pathways. Translocations in the high mobility group genes HMGA1 and HMGA2 have been described, possibly influencing fibroblast growth factor (FGF) pathway activity and resulting in increased tumor size (22,23). The Mediator Complex Subunit 12 (MED12) gene has been reported to be mutated in UFs in multiple cohorts of women, including American, South African, and Finnish studies (24–28). There are also genetic factors that predispose to these tumors as part of heritable cancer syndromes, including mutations in fumarate hydratase (FH) seen in patients with hereditary leiomyomatosis and renal cancer (HLRCC) (29,30). FH is an enzyme in the TCA cycle and

Fertil Steril. Author manuscript; available in PMC 2017 September 15.

Katz et al.

Page 3

Author Manuscript

acts as a classical tumor suppressor, with mutations in FH significantly increasing risk of fibroids in the uterus and other tissues in patients with HLRCC (31). 1.2 Endocrine disrupting chemicals and uterine fibroids

Author Manuscript

As defined by the National Institute of Environmental Health Sciences, endocrine disrupting chemicals (EDCs) are “chemicals that interfere with the body’s endocrine system and produce adverse developmental, reproductive, neurological and immune effects.” The endocrine system is comprised of glands that are distributed throughout the body and synthesize the hormones that are released in the circulatory system to regulate development, physiological processes and homeostatic functions. These glands include, but are not limited to, the hypothalamus, pituitary, thyroid and reproductive organs. EDCs can be natural or man-made, such as pharmaceuticals, plasticizers, dioxins, polychlorinated biphenyls (PCBs), organochlorines, polyfluoroalkyls (PFOAs), phthalates and pesticides. Although the route of exposure is dependent on the individual EDC, common routes of exposure in humans are ingestion, inhalation and dermal absorption. Importantly, EDCs can exhibit non-monotonic dose response curves and low doses of EDCs can produce a pathophysiological effect (32).

Author Manuscript

Numerous EDCs have been shown to interact with nuclear receptors to exert their actions in target tissues (32). EDC binding to nuclear receptors can alter hormonal functions by mimicking naturally occurring hormones in the body, blocking the endogenous hormone from binding or interfering with the production or regulation of hormones and/or their receptors. An individual EDC may interact with more than one receptor, and multiple EDCs can interact with the same receptor, highlighting the complexity of in the response of animals and humans to environmental EDC exposures. For example, the xenoestrogen bisphenol A (BPA) has been shown to bind and activate estrogen receptor (ER) (33,34), estrogen-related receptor gamma (ERRγ) (35) and pregnane X receptor (PXR) (36). In addition to BPA, a variety of other EDCs (i.e. diethylstilbestrol (DES), PCBs, PFOA, and phthalates) can also bind to ERs (33,37–40). Liganded nuclear hormone receptors that function as transcription factors interact with DNA, producing what are termed “genomic” effects that regulate gene transcription, but are also capable of action outside the nucleus via what is termed “non-genomic signaling” (41,42). EDCs are also capable of inducing both genomic and non-genomic signaling: both BPA and DES, for example, have been shown to activate nongenomic signaling pathways through the ER (43–48). Regardless of the mode of action, EDCs have been linked to several adverse health outcomes including diabetes, obesity, cardiovascular disease, reproductive tract disorders, and neurodevelopmental disorders (32).

Author Manuscript

2. Pathogenesis of Uterine Fibroids 2.1 Chromosomal alterations and MED12 mutations In contrast to mutations that are inherited from a parent and present throughout a person’s life in virtually every cell in the body, somatic mutations associated with clonal tumors such as fibroids occur in a single progenitor cell in tissues, from which the tumor arises. Most of uterine fibroid cases are sporadic in nature, however few rare familial syndromes are


Katz et al.

Page 4

Author Manuscript

associated with development of UFs. In these syndromes, genetic alterations occur in the FH, COL4A5 and COL4A6 genes (49). In sporadic tumors, several recurrent genetic aberrations have been identified, including deletions in 7q, trisomy of chromosome 12, reciprocal translocation of chromosome 12, and monosomy of chromosome 22, among others (50–52). However, these genetic alterations occur at relatively low frequency.

Author Manuscript

In contrast, MED12 somatic gene mutations have been found in sporadic UFs with a very high frequency (70–80%) (25). The MED12 gene encodes one of the components of the Mediator complex, which consists of 26 subunits that bridge DNA regulatory sequences to the RNA polymerase II initiation complex. The Mediator complex is highly conserved in eukaryotes, and can participate in transcriptional repression or act as a positive co-regulator (25,53). Pathway analysis in MED12-deficient UFs demonstrated that focal adhesion, extracellular matrix receptor interaction, and Wnt signaling are substantially altered in these tumors (25). Subsequent studies have confirmed the important role of MED12 mutation in UFs (24,25,28,54–56), making MED12 somatic mutations the most widely detected DNA mutation in human fibroid lesions. 2.2 Nuclear Hormone Receptors 2.2.1 Estrogen Receptors—A striking feature of UFs is their dependency on the ovarian steroids estrogen and progesterone (50,57). Both clinical and experimental data suggest that estrogen stimulates the growth of UFs during reproductive years. Regression is seen after menopause, and continuous gonadotropin-releasing hormone (GnRH) agonist treatment inhibits UF growth by decreasing ovarian hormone production. Estrogens, such as 17βestradiol, exert their biological effects on target cells, including myometrial cells, through the activation of ERs.

Author Manuscript

The cellular effects of estrogen are the result of a combination of non-genomic and genomic actions via membrane and nuclear-ER-mediated signaling pathways (42). There are two classes of ERs, ERα and ERβ, and both are capable of rapid membrane signaling (i.e. nongenomic signaling) (58–61). These non-genomic effects are independent of gene transcription or protein synthesis and involve steroid-induced modulation of cytoplasmic or cell membrane-bound regulatory proteins. These signaling cascades include extracellular signal-related kinase/mitogen-activated protein kinases (ERK/MAPK), p38/MAPK, the phosphatidylinositol 3-kinase/Akt (PI3K/Akt), phospholipase C/Protein Kinase C (PLC/ PKC), and cAMP/Protein Kinase A (cAMP/PKA) (62–66). In addition, steroid hormone receptor modulation of other cell membrane-associated molecules, such as ion channels and G-protein-coupled receptors (e.g., GPR30) has also been reported (67,68).

Author Manuscript

The more classical pathway for ligand-activation of ER occurs in the nucleus, and both ER subtypes have a DNA-binding domain and can function as transcription factors to regulate gene expression by binding to specific estrogen response elements (i.e. genomic mechanism) (69) (Table 1). In the absence of hormone, ERs are largely located in the cytosol. When estrogen binds to the receptor, a cascade of events is triggered, starting with the migration of the receptor from the cytosol into the nucleus, dimerization of the receptor, and subsequent binding of the receptor dimer to estrogen response elements. The DNA/receptor complex


Katz et al.

Page 5

Author Manuscript

then recruits other proteins that are responsible for transcriptional activation of repression, which eventually alters target gene expression. 2.2.2 Progesterone Receptors—Progesterone is an endogenous steroid hormone involved in the menstrual cycle and pregnancy; therefore, it is essential in female reproduction. Similar to estrogen, progesterone exerts its effects through progesterone receptor (PR), a member of the steroid hormone nuclear receptor superfamily. There are two PR isoforms, PR-A and PR-B, which are identical except for an additional 165 amino acids present only in the PR-B isoform (70). Similar to ERs, PRs signal through both genomic and non-genomic pathways (71–76). The ability of PR isoforms to target distinct promoters and modulate the expression of diverse downstream genes is influenced in a cell- and contextspecific manner, including the functional interaction of PRs with other transcriptional factors, post-translational modifications, etc. (72).

Author Manuscript

In UFs, progesterone regulates many targets, which may play an important role in UF pathogenesis (Table 1) (77–79). A direct functional link between progesterone and UF development was shown in a mouse xenograft model, where fibroid tumor growth was associated with administration of estrogen plus progesterone, but not with estrogen administration alone. Moreover, fibroid tumor size was significantly decreased in response to progesterone withdrawal using the PR antagonist mifepristone (RU486) (80,81). As a result of these and other studies, several selective progesterone receptor modulators (SPRMs) have been shown in clinical trials to inhibit UF growth (49,50,82–87).

3. Stem Cells and the Origin of Uterine Fibroids Author Manuscript

UFs are monoclonal tumors that arise from the uterine smooth muscle tissue (50,88). Somatic mutations in genes such as MED12 support clonality and indicate that each fibroid lesion originates from the transformation of a single cell (89–97). Furthermore, tumorinitiating stem cells (TICs) derived from UFs carry MED12 mutations seen in UFs (50). Interestingly, distinct MED12 mutations have been detected in different UF lesions in the same uterus (25), consistent with the emergence of each MED12 mutation as an independent, somatic clonal event. Recently, in an animal model of MED12 deficiency, mutations in this gene were shown to act as a driver promoting development of UFs and genomic instability (98). 3.1. Myometrial stem cells

Author Manuscript

Tissue-specific stem cells have been identified in a variety of tissues and organs (99). The presence of a myometrial stem cell (MSC) population in the uterus was first shown in 2007 in mouse myometrium and non-pregnant human myometrium, using 5-bromo-2′deoxyuridine and sorting of a side population, respectively (89,100). In contrast to the main population of myometrial cells, the side population of MSCs is capable of generating functional human myometrial tissues efficiently when transplanted into the uteri of severely immunodeficient mice (89). In addition, MSCs from the differentiated myometrial cell population, exhibit high levels of OCT4 expression (an embryonic stem cell marker) and very low expression of ER, PR and smooth muscle cell-specific markers including smoothelin and calponin (89). Moreover, MSCs prefer a hypoxic environment for survival Fertil Steril. Author manuscript; available in PMC 2017 September 15.

Katz et al.

Page 6

Author Manuscript

and growth, which is consistent with known uterine physiology: during pregnancy mechanical stretching of the uterine wall induces hypoxia in the myometrium that could promote MSC proliferation (101), which is required to support the growing fetus and enlarging uterus. However, the connection of this observation to future development of UFs is not clear at the moment.

Author Manuscript

Recently, MSCs expressing Stro1 and CD44 markers were identified (102). Using Stro-1/ CD44 surface markers, the stem cells from adjacent myometrium and human fibroid tissues were isolated (102). The undifferentiated status of isolated cells was confirmed by expression of the ABCG2 transporter, as well as expression of additional stem cell markers OCT4, NANOG, and GDB3, and the low expression of steroid receptors ERα and PRA/PR-B. Mesodermal cell origin was established by the presence of typical mesenchymal markers (CD90, CD105, and CD73) and absence of hematopoietic stem cell markers (CD34, CD45), and confirmed by the ability of these cells to differentiate in vitro into adipocytes, osteocytes, and chondrocytes. The functional capability of these cells to form myometriumlike lesions was established in a xenotransplantation mouse model. The injected cells labeled with superparamagnetic iron oxide were tracked by both magnetic resonance imaging and fluorescence imaging, thus demonstrating the regenerative potential of Stro-1/CD44 stem cells in vivo (102). 3.2. Tumor (fibroid) initiating cells (TICs)

Author Manuscript

Several studies have been performed to identify tumor-initiating cells (TICs) in UFs (89,90,94,95). One principal difference between MSCs and TICs at the DNA level is that MED12 mutations are found only in UF TICs, but not normal myometrium or MSCs (90). TICs derived from UFs have stem cell characteristics, and are proliferative, give rise to tumors in vivo (90,95) and comprise~1% of UF cells, (99). Similar to MSCs, TICs express low levels of ER and PR, although they are paradoxically dependent on estrogen/ progesterone for growth (90,102). This suggests important paracrine interactions may occur between TICs and surrounding myometrial cells that mediate estrogen/progesterone induced UF growth (91). In a cell co-culture system, with both UF TICs and mature myometrial cells, treatment with estrogen/progesterone results in secretion of WNT ligands, which selectively induce nuclear translocation of β-catenin in UF TICs, eventually leading to the growth and proliferation of TICs (91). These observations suggest a role for WNT/β-catenin signaling in response to estrogen/progesterone, similar to the aberrant WNT/β-catenin/ GSK-3 axis reported to be involved in the formation and maintenance of other TICs (103).

4. Environmental Exposures and Uterine Fibroids Author Manuscript

4.1 Epidemiological data The effect of developmental exposure to DES on UF development later in life has been investigated (104–108). Two large prospective studies found a positive association between developmental DES exposure and UF risk (104,107). In the Nurses Health Study II 11,831 cases of UFs were diagnosed in 1.3 million person-years of follow-up (over 20 years), making this the largest prospective study investigating the influence of prenatal DES exposure on UFs. In this study prenatal exposure to DES increased risk for UFs by 13% in


Katz et al.

Page 7

Author Manuscript

women over 35 years of age (107). Additionally, exposure during the first trimester of gestation was the most hazardous with an increased risk of 21% compared to unexposed women. The second study to find a positive association between DES exposure and UFs is the NIEHS Uterine Fibroid Study, in which 1364 DES exposed or unexposed women aged 35–49 in the Washington DC area were screened for UFs (104). In this study large fibroids were more common in those exposed to prenatal DES. The odds ratio for Caucasian women exposed to DES was 2.4, which was even higher for large fibroids. Unfortunately, there were not enough exposed AA women to make meaningful comparisons (104). Another study used a subset of the NIEHS Sister Study cohort to evaluate the risk for UFs after prenatal exposures in 3,534 AA women aged 35–59. In this study the strongest factors associated with increased risk of UFs were DES exposure, maternal or gestational diabetes, and monozygotic twins with RR of 2.02, 1.54, and 1.94 respectively (106).

Author Manuscript Author Manuscript

However, not all data were concordant. When a larger portion of NIEHS Sister Study data was evaluated the association between DES and UFs was less clear. This study included 19,972 Caucasian women in a prospective analysis and found five early life factors to be associated with greater than 20% increased risk for UFs: prenatal DES exposure, gestational diabetes, pregnancy diabetes (mothers who were diabetic prior to pregnancy), soy formula, and gestational age at birth. The strongest associations were in women whose mothers had pregnancy diabetes and in those who were born more than a month early. DES exposure resulted in an adjusted relative risk (RR) of 1.42, and gestational diabetes and soy formula had similar RR of 1.28 and 1.25 respectively. Although there is a discrepancy in this study: when separating out women who reported to have ‘definitely’ or ‘probably’ been exposed to DES, women who were definitely exposed showed no increased risk, while those who identified as being probably exposed had a RR of 2.07. This study also reported that experiencing menarche under the age of 10 or 11 resulted in RR of 1.54 and 1.32, respectively, and being poor had a RR of 1.24 (105). Another study of 85 UF cases also failed to find an association between prenatal DES and UF risk (108). In contrast to the human epidemiological data, experimental studies have shown an unequivocal link between DES exposure and UFs. 4.2 Experimental Data from Animal Models

Author Manuscript

4.2.1 The Eker Rat model for Uterine Fibroids—Several genetically engineered mouse and rat models have been developed for UFs, although to date, only one, the Eker rat, has been used in experimental studies to explore the linkage between environmental exposures and UFs. The Eker rat develops spontaneous UFs at a 65% incidence, due to a germline retroviral insertion in the tuberous sclerosis complex 2 (Tsc2) tumor suppressor gene (109,110). The resulting tumors have a similar presentation as seen in women, occurring with high frequency often in multiples, and are histologically welldifferentiated and benign (111). The UFs that develop in Eker rats are hormone responsive, express ERα and PR, and as seen in women, parity is protective (112,113). Furthermore, several UFderived cell lines were established from these tumors, the most widely used being the ELT-3 cell line (114,115). Selective estrogen receptor modulators (SERMs), specifically tamoxifen and raloxifene, have also been shown to inhibit growth of UF cells isolated from Eker rats (116) as well as incidence and size of UFs that develop in these animals (117). There is


Katz et al.

Page 8

Author Manuscript

evidence that SERMs may be efficacious in women as well, as studies in both premenopausal (118) and postmenopausal (119) women demonstrate UF regression after raloxifene treatment. Aberrant expression of HMGA occurs in UFs in women and in the Eker rats (120), and both are stimulated to grow by insulin-like growth factor-I (IGF-I) (121). In women, TSC2 is expressed in normal uterus and fibroids, and although not well studied, defects in this tumor suppressor have been reported in UFs (122).

Author Manuscript

4.2.2 Mouse models—Interestingly, while heterozygous mice carrying a defective Tsc2 allele do not develop UFs, uterine-specific Tsc2 deletion driven by a PR-cre recombinase (Tsc2fl;PRcre) leads to development of aggressive myometrial tumors (123). 100% of Tsc2fl;PRcre mice develop myometrial thickening by 12 weeks of age and mesenchymal tumors, which express ER and PR, by 24 weeks of age. That tumors were driven by loss of Tsc2, which functions to suppress the mechanistic target of rapamycin (mTORC1/S6) pathway, was shown by blocking mTOR activity with rapamycin, which prevented the dramatic enlargement of the uterus. Ovariectomy also prevented uterine enlargement, which was rescued by administration of estrogen or estrogen + progesterone. The estrogendependence of these tumors was confirmed by treatment with the aromatase inhibitor letrozole, which abolished uterine overgrowth and S6 activation (123). Another group has designed a uterine-specific Tsc2 deleted mouse model by crossing Tsc2fl mice with antiMullerian hormone receptor type 2 (Amhr2)-cre mice (124). These mice also developed myometrial enlargement but the hyperplasia observed did not progress to tumors. This hyperplasia was also driven by mTORC1/S6 activation as rapamycin treatment attenuated the uterine enlargement (124).

Author Manuscript

As mentioned previously, mutations in MED12 are found in 70–80% of UFs in women. A conditional Med12 mutation mouse model expressing a Med12 missense variant (c. 131G>A), the principle mutation found in human UFs, has recently been developed (98). These animals were developed by crossing Med12 variant knock-in mice with Amhr2-cre mice. By 12 weeks of age 80% of females had developed UFs (98). Inconsistent with epidemiological and Eker rat model data, these animals developed more UFs when repeatedly bred.

Author Manuscript

4.2.3 Experimental Data Linking EDC Exposure to Uterine Fibroids— Developmental exposure to DES has been shown to influence susceptibility to UFs later in life in animal models (125–128). As previously mentioned, in the Eker rat model, the Tsc2EK mutation increases susceptibility to develop spontaneous UFs with age. When exposed to DES neonatally, tumor incidence significantly increases as does multiplicity and tumor size (129). Eker rats without the Tsc2 mutation did not develop any tumors even if they were exposed to DES neonatally. These data imply that reprogramming due to DES exposure acts by increasing the penetrance of a Tsc2 mutation (129). Experimental studies in Eker rats have identified a window of susceptibility to environmental exposures that coincides with key periods of myometrial development. Postnatal day (PND) 3–12, when the inner circular myometrium is differentiating and the uterine glands are developing, has been shown to be a critical window of susceptibility for promotion of uterine fibroids by DES (130). During this time the developing uterus is Fertil Steril. Author manuscript; available in PMC 2017 September 15.

Katz et al.

Page 9


normally protected from estrogens, which are bound by circulating steroid hormone binding proteins, such as alpha-feto protein. This allows differentiation to proceed largely independent of steroid hormone exposure until much later in development, when alpha-feto protein production ceases and is cleared by the liver, which occurs around postnatal day 17 in rats (131). Since DES, and other xenoestrogens do not bind alpha-feto protein, even low dose exposures to these estrogenic chemicals can be very potent, and lead to adverse effects (132). Using the Eker rat model, the Walker group showed that exposure to DES during PND 3–5 or 10–12 increased tumor incidence from 63% in unexposed animals to 95% and 100% respectively, whereas a later exposure did not (130). Of interest, estrogen responsive genes were reprogrammed by DES exposure during this critical time period. In the myometrium of 5–6 month old rats (10 months before tumor development), Calbindin D9K and PR expression fluctuated as expected with estrus cycles in the unexposed animals but remained high during all stages of the estrus cycle in the animals that had been exposed neonatally to DES (130). Other reprogrammed genes were subsequently identified in the adult myometrium of neonata DES-exposed rats including Dio2, Gdf10, Car8, Gria2, and Mmp3, all of which exhibited an exaggerated response to estrogen as a consequence of this reprogramming (133). Exposure to the phytoestrogen genistein during PND 10–12 also reprogrammed expression of several of these genes, and like DES, increased tumor incidence and multiplicity (134).. These data suggest that developmental DES exposure may have an influence on UF risk later in life, although additional studies are needed to determine the impact of other EDCs on UF risk.


4.2.4 Developmental Reprogramming of the Epigenome—One mechanism by which early life EDC exposures may exert their effects is by reprogramming the developing epigenome (135–137). DES, genistein, and the plasticizer BPA have been shown to exhibit tissue-specific patterns of developmental reprogramming and/or promotion of UFs. All three EDCs act as ER ligands, and induce ER-mediated gene transcription. However, only DES and genistein induce non-genomic ER signaling to activate PI3K/AKT in the developing uterus. Activation of PI3K/AKT signaling has been shown to induce phosphorylation of the histone methyltransferase enhancer of zeste homolog 2 (EZH2), which represses EZH2 activity and reduces levels of the histone 3 lysine 27 trimethyl (H3K27me3) repressive mark on chromatin. Furthermore, DES and genistein, but not BPA, caused estrogen-responsive genes in the adult myometrium to become reprogrammed, and hyper-responsive to hormone (133). Importantly, this pattern of EZH2 engagement to decrease H3K27 methylation correlated with the effect of these xenoestrogens on tumorigenesis. Developmental reprogramming by DES and genistein promoted development of UFs, increasing tumor incidence and multiplicity, whereas BPA did not (134). These data show that environmental estrogens have distinct effects in the developing uterus that determines their ability to engage the epigenetic regulator EZH2, decrease levels of the repressive epigenetic histone H3K27 methyl mark on chromatin during developmental reprogramming, and promote uterine tumorigenesis (134). Altered DNA methylation patterns have also been observed in UFs from both rodents and humans. In two human studies global DNA methylation was assessed in paired UF and myometrial samples (138,139). One study found reduced DNA methylation in UFs while the


Katz et al.

Page 10


other found that 62% of differentially methylated sites were hypermethylated. Interestingly, although the first study found the genome to be hypomethylated there was increased mRNA expression of both DNMT1 and DNMT3a (139). In both studies when interrogating individual differentially methylated regions an inverse relationship was found between DNA methylation and gene expression. Three tumor suppressors, KLF11, DLEC1, and KRT19 were hypermethylated at the promoter and displayed reduced gene expression in UFs (138). These studies indicate that DNA methylation is abnormally regulated in human UF and may play a role in UF etiology and development. An early study in rodents investigated neonatal exposures and reprogramming of DNA methylation in animals treated with DES during PND 1–5, with DNA methylation surveyed at PND 17 (pre-pubertal), and PND 21 and PND 30 (post-puberty) (140). Lactoferrin is an estrogen responsive gene that is known to be reprogrammed as a result of neonatal DES exposure. At PND 21 and 30, 2 out of 5 CpG sites in the promoter just upstream of the estrogen response element were demethylated in animals exposed to DES during PND 1–5 compared to unexposed controls. This postpubertal DES induced demethylation was dependent on ovarian hormones: DES-exposed mice that were ovariectomized did not exhibit this demethylation (140).

Author Manuscript

This emerging field of epigenome reprogramming by EDCs and its influence on UF development is in its infancy, but clearly warrants additional investigation. While animal studies have identified EDC-induced epigenetic alterations in both histone and DNA methylation in UFs, similar studies in human UFs and translation of these findings to the human disease has been lacking. Furthermore, because most environmental exposures occur at very low EDC levels, and exposures to some EDCs are nearly ubiquitous, establishing causal linkages between environmental EDC exposures and the human disease presents a significant challenge for epidemiologists. Additional mechanistic studies to identify epigenetic biomarkers of exposure to specific EDCs holds promise in this regard, but such biomarkers are not currently available. Since the field has not focused on identifying EDCspecific epigenetic changes that could be used in such translational studies, this would be an important direction to consider in the future.

5. Concluding remarks

Author Manuscript

UFs are an important disease of reproductive age women, and as hormone-dependent tumors, are potentially promoted by exposure to hormone-active environmental agents such as EDCs. When EDC exposures occur during crucial times of uterine development, alterations in cell signaling pathways, including estrogen signaling, and the epigenome may increase susceptibility to develop UFs in adulthood. Despite the high prevalence and major impact of UFs on women’s health, the precise mechanism underlying EDC-dependent effects on MSCs that are the progenitors for these tumors are not adequately understood: virtually nothing is known about how EDCs impact the number or pathophysiology of these cells to promote tumorigenesis. Delineating the effects of EDC exposure and underlying mechanisms by which they, or other environmental exposures, promote MSC progression to UFs, including the role of epigenetic alterations and acquisition of mutations in genes such as MED12, will be key to the development of new interventions to prevent and treat this important disease of women.


Katz et al.

Page 11

Author Manuscript

Acknowledgments This work was supported in part by an Augusta University Startup package, the Augusta University Intramural Grants Program (QY), the National Institutes of Health grant HD04622811 (to AA) and the National Institute of Environmental Health Sciences (RC2ES018789, P30ES023512 and ES023206), the Cancer Prevention Research Institute of Texas (RP120855) and the Welch Foundation (BE-0023, Houston, TX) to CLW.

References

Author Manuscript Author Manuscript Author Manuscript

1. Baird DD, Dunson DB. Why is parity protective for uterine fibroids? Epidemiology. 2003; 14:247– 250. [PubMed: 12606893] 2. Eltoukhi HM, Modi MN, Weston M, Armstrong AY, Stewart EA. The health disparities of uterine fibroid tumors for African American women: a public health issue. American journal of obstetrics and gynecology. 2014; 210:194–199. [PubMed: 23942040] 3. Farquhar CM, Naoom S, Steiner CA. The impact of endometrial ablation on hysterectomy rates in women with benign uterine conditions in the United States. Int J Technol Assess Health Care. 2002; 18:625–634. [PubMed: 12391955] 4. Mauskopf J, Flynn M, Thieda P, Spalding J, Duchane J. The economic impact of uterine fibroids in the United States: a summary of published estimates. J Womens Health (Larchmt). 2005; 14:692– 703. [PubMed: 16232101] 5. Cardozo ER, Clark AD, Banks NK, Henne MB, Stegmann BJ, Segars JH. The estimated annual cost of uterine leiomyomata in the United States. American journal of obstetrics and gynecology. 2012; 206:211, e211–219. [PubMed: 22244472] 6. Segars JH. Uterine Fibroid Research: A Work in Progress. Reprod Sci. 2014; 21:1065–1066. [PubMed: 25138824] 7. Hart R, Khalaf Y, Yeong CT, Seed P, Taylor A, Braude P. A prospective controlled study of the effect of intramural uterine fibroids on the outcome of assisted conception. Hum Reprod. 2001; 16:2411–2417. [PubMed: 11679530] 8. Segars JH, Parrott EC, Nagel JD, Guo XC, Gao X, Birnbaum LS, Pinn VW, Dixon D. Proceedings from the Third National Institutes of Health International Congress on Advances in Uterine Leiomyoma Research: comprehensive review, conference summary and future recommendations. Human reproduction update. 2014; 20:309–333. [PubMed: 24401287] 9. Sheiner E, Bashiri A, Levy A, Hershkovitz R, Katz M, Mazor M. Obstetric characteristics and perinatal outcome of pregnancies with uterine leiomyomas. The Journal of reproductive medicine. 2004; 49:182–186. [PubMed: 15098887] 10. Surrey ES, Lietz AK, Schoolcraft WB. Impact of intramural leiomyomata in patients with a normal endometrial cavity on in vitro fertilization-embryo transfer cycle outcome. Fertility and sterility. 2001; 75:405–410. [PubMed: 11172848] 11. Flake GP, Andersen J, Dixon D. Etiology and pathogenesis of uterine leiomyomas: a review. Environmental health perspectives. 2003; 111:1037–1054. [PubMed: 12826476] 12. Laughlin SK, Schroeder JC, Baird DD. New directions in the epidemiology of uterine fibroids. Seminars in reproductive medicine. 2010; 28:204–217. [PubMed: 20414843] 13. Moore AB, Flake GP, Swartz CD, Heartwell G, Cousins D, Haseman JK, Kissling GE, Sidawy MK, Dixon D. Association of race, age and body mass index with gross pathology of uterine fibroids. The Journal of reproductive medicine. 2008; 53:90–96. [PubMed: 18357799] 14. Peddada SD, Laughlin SK, Miner K, Guyon JP, Haneke K, Vahdat HL, Semelka RC, Kowalik A, Armao D, Davis B, Baird DD. Growth of uterine leiomyomata among premenopausal black and white women. Proceedings of the National Academy of Sciences of the United States of America. 2008; 105:19887–19892. [PubMed: 19047643] 15. Viswanathan M, Hartmann K, McKoy N, Stuart G, Rankins N, Thieda P, Lux LJ, Lohr KN. Management of uterine fibroids: an update of the evidence. Evid Rep Technol Assess (Full Rep). 2007:1–122. [PubMed: 18288885]


Katz et al.

Page 12

Author Manuscript Author Manuscript Author Manuscript Author Manuscript

16. Weiss G, Noorhasan D, Schott LL, Powell L, Randolph JF Jr, Johnston JM. Racial differences in women who have a hysterectomy for benign conditions. Womens Health Issues. 2009; 19:202– 210. [PubMed: 19447324] 17. Baird DD. Invited commentary: uterine leiomyomata-we know so little but could learn so much. American journal of epidemiology. 2004; 159:124–126. [PubMed: 14718212] 18. Laughlin SK, Baird DD, Savitz DA, Herring AH, Hartmann KE. Prevalence of uterine leiomyomas in the first trimester of pregnancy: an ultrasound-screening study. Obstetrics and gynecology. 2009; 113:630–635. [PubMed: 19300327] 19. Borgfeldt C, Andolf E. Transvaginal ultrasonographic findings in the uterus and the endometrium: low prevalence of leiomyoma in a random sample of women age 25–40 years. Acta obstetricia et gynecologica Scandinavica. 2000; 79:202–207. [PubMed: 10716301] 20. Eskenazi B, Warner M, Samuels S, Young J, Gerthoux PM, Needham L, Patterson D, Olive D, Gavoni N, Vercellini P, Mocarelli P. Serum dioxin concentrations and risk of uterine leiomyoma in the Seveso Women’s Health Study. American journal of epidemiology. 2007; 166:79–87. [PubMed: 17443023] 21. Baird DD, Harmon QE, Upson K, Moore KR, Barker-Cummings C, Baker S, Cooper T, Wegienka G. A Prospective, Ultrasound-Based Study to Evaluate Risk Factors for Uterine Fibroid Incidence and Growth: Methods and Results of Recruitment. J Womens Health (Larchmt). 2015; 24:907– 915. [PubMed: 26334691] 22. Markowski DN, Bartnitzke S, Belge G, Drieschner N, Helmke BM, Bullerdiek J. Cell culture and senescence in uterine fibroids. Cancer genetics and cytogenetics. 2010; 202:53–57. [PubMed: 20804922] 23. Markowski DN, von Ahsen I, Nezhad MH, Wosniok W, Helmke BM, Bullerdiek J. HMGA2 and the p19Arf-TP53-CDKN1A axis: a delicate balance in the growth of uterine leiomyomas. Genes, chromosomes & cancer. 2010; 49:661–668. [PubMed: 20544840] 24. Makinen N, Heinonen HR, Moore S, Tomlinson IP, van der Spuy ZM, Aaltonen LA. MED12 exon 2 mutations are common in uterine leiomyomas from South African patients. Oncotarget. 2011; 2:966–969. [PubMed: 22182697] 25. Makinen N, Mehine M, Tolvanen J, Kaasinen E, Li Y, Lehtonen HJ, Gentile M, Yan J, Enge M, Taipale M, Aavikko M, Katainen R, Virolainen E, Bohling T, Koski TA, Launonen V, Sjoberg J, Taipale J, Vahteristo P, Aaltonen LA. MED12, the mediator complex subunit 12 gene, is mutated at high frequency in uterine leiomyomas. Science. 2011; 334:252–255. [PubMed: 21868628] 26. Makinen N, Vahteristo P, Butzow R, Sjoberg J, Aaltonen LA. Exomic landscape of MED12 mutation-negative and -positive uterine leiomyomas. International journal of cancer Journal international du cancer. 2014; 134:1008–1012. [PubMed: 23913526] 27. Makinen N, Vahteristo P, Kampjarvi K, Arola J, Butzow R, Aaltonen LA. MED12 exon 2 mutations in histopathological uterine leiomyoma variants. Eur J Hum Genet. 2013; 21:1300– 1303. [PubMed: 23443020] 28. McGuire MM, Yatsenko A, Hoffner L, Jones M, Surti U, Rajkovic A. Whole exome sequencing in a random sample of North American women with leiomyomas identifies MED12 mutations in majority of uterine leiomyomas. PloS one. 2012; 7:e33251. [PubMed: 22428002] 29. Tomlinson IP, Alam NA, Rowan AJ, Barclay E, Jaeger EE, Kelsell D, Leigh I, Gorman P, Lamlum H, Rahman S, Roylance RR, Olpin S, Bevan S, Barker K, Hearle N, Houlston RS, Kiuru M, Lehtonen R, Karhu A, Vilkki S, Laiho P, Eklund C, Vierimaa O, Aittomaki K, Hietala M, Sistonen P, Paetau A, Salovaara R, Herva R, Launonen V, Aaltonen LA, Multiple Leiomyoma C. Germline mutations in FH predispose to dominantly inherited uterine fibroids, skin leiomyomata and papillary renal cell cancer. Nature genetics. 2002; 30:406–410. [PubMed: 11865300] 30. Toro JR, Nickerson ML, Wei MH, Warren MB, Glenn GM, Turner ML, Stewart L, Duray P, Tourre O, Sharma N, Choyke P, Stratton P, Merino M, Walther MM, Linehan WM, Schmidt LS, Zbar B. Mutations in the fumarate hydratase gene cause hereditary leiomyomatosis and renal cell cancer in families in North America. American journal of human genetics. 2003; 73:95–106. [PubMed: 12772087] 31. Stewart L, Glenn GM, Stratton P, Goldstein AM, Merino MJ, Tucker MA, Linehan WM, Toro JR. Association of germline mutations in the fumarate hydratase gene and uterine fibroids in women


Katz et al.

Page 13


with hereditary leiomyomatosis and renal cell cancer. Arch Dermatol. 2008; 144:1584–1592. [PubMed: 19075141] 32. Gore AC, Chappell VA, Fenton SE, Flaws JA, Nadal A, Prins GS, Toppari J, Zoeller RT. EDC-2: The Endocrine Society’s Second Scientific Statement on Endocrine-Disrupting Chemicals. Endocrine reviews. 2015; 36:E1–E150. [PubMed: 26544531] 33. Kuiper GG, Lemmen JG, Carlsson B, Corton JC, Safe SH, van der Saag PT, van der Burg B, Gustafsson JA. Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor beta. Endocrinology. 1998; 139:4252–4263. [PubMed: 9751507] 34. Sengupta S, Obiorah I, Maximov PY, Curpan R, Jordan VC. Molecular mechanism of action of bisphenol and bisphenol A mediated by oestrogen receptor alpha in growth and apoptosis of breast cancer cells. British journal of pharmacology. 2013; 169:167–178. [PubMed: 23373633] 35. Matsushima A, Kakuta Y, Teramoto T, Koshiba T, Liu X, Okada H, Tokunaga T, Kawabata S, Kimura M, Shimohigashi Y. Structural evidence for endocrine disruptor bisphenol A binding to human nuclear receptor ERR gamma. Journal of biochemistry. 2007; 142:517–524. [PubMed: 17761695] 36. Sui Y, Ai N, Park SH, Rios-Pilier J, Perkins JT, Welsh WJ, Zhou C. Bisphenol A and its analogues activate human pregnane X receptor. Environmental health perspectives. 2012; 120:399–405. [PubMed: 22214767] 37. Benninghoff AD, Bisson WH, Koch DC, Ehresman DJ, Kolluri SK, Williams DE. Estrogen-like activity of perfluoroalkyl acids in vivo and interaction with human and rainbow trout estrogen receptors in vitro. Toxicological sciences : an official journal of the Society of Toxicology. 2011; 120:42–58. [PubMed: 21163906] 38. Henry ND, Fair PA. Comparison of in vitro cytotoxicity, estrogenicity and anti-estrogenicity of triclosan, perfluorooctane sulfonate and perfluorooctanoic acid. Journal of applied toxicology : JAT. 2013; 33:265–272. [PubMed: 21935973] 39. Jobling S, Reynolds T, White R, Parker MG, Sumpter JP. A variety of environmentally persistent chemicals, including some phthalate plasticizers, are weakly estrogenic. Environmental health perspectives. 1995; 103:582–587. [PubMed: 7556011] 40. Korach KS, Metzler M, McLachlan JA. Estrogenic activity in vivo and in vitro of some diethylstilbestrol metabolites and analogs. Proceedings of the National Academy of Sciences of the United States of America. 1978; 75:468–471. [PubMed: 272664] 41. Trevino LS, Wang Q, Walker CL. Hypothesis: Activation of rapid signaling by environmental estrogens and epigenetic reprogramming in breast cancer. Reprod Toxicol. 2015; 54:136–140. [PubMed: 25554384] 42. Yang Q, Diamond MP, Al-Hendy A. Early Life Adverse Environmental Exposures Increase the Risk of Uterine Fibroid Development: Role of Epigenetic Regulation. Frontiers in pharmacology. 2016; 7:40. [PubMed: 26973527] 43. Ge LC, Chen ZJ, Liu HY, Zhang KS, Liu H, Huang HB, Zhang G, Wong CK, Giesy JP, Du J, Wang HS. Involvement of activating ERK1/2 through G protein coupled receptor 30 and estrogen receptor alpha/beta in low doses of bisphenol A promoting growth of Sertoli TM4 cells. Toxicology letters. 2014; 226:81–89. [PubMed: 24495410] 44. Prins GS, Hu WY, Shi GB, Hu DP, Majumdar S, Li G, Huang K, Nelles JL, Ho SM, Walker CL, Kajdacsy-Balla A, van Breemen RB. Bisphenol A promotes human prostate stem-progenitor cell self-renewal and increases in vivo carcinogenesis in human prostate epithelium. Endocrinology. 2014; 155:805–817. [PubMed: 24424067] 45. Sheng ZG, Zhu BZ. Low concentrations of bisphenol A induce mouse spermatogonial cell proliferation by G protein-coupled receptor 30 and estrogen receptor-alpha. Environmental health perspectives. 2011; 119:1775–1780. [PubMed: 21813366] 46. Bulayeva NN, Watson CS. Xenoestrogen-induced ERK-1 and ERK-2 activation via multiple membrane-initiated signaling pathways. Environmental health perspectives. 2004; 112:1481–1487. [PubMed: 15531431] 47. Dong S, Terasaka S, Kiyama R. Bisphenol A induces a rapid activation of Erk1/2 through GPR30 in human breast cancer cells. Environmental pollution. 2011; 159:212–218. [PubMed: 20875696]


Katz et al.

Page 14


48. Li X, Zhang S, Safe S. Activation of kinase pathways in MCF-7 cells by 17beta-estradiol and structurally diverse estrogenic compounds. The Journal of steroid biochemistry and molecular biology. 2006; 98:122–132. [PubMed: 16413991] 49. Stewart EA, Laughlin-Tommaso SK, Catherino WH, Lalitkumar S, Gupta D, Vollenhoven B. Uterine fibroids. Nature reviews Disease primers. 2016; 2:16043. 50. Bulun SE. Uterine fibroids. The New England journal of medicine. 2013; 369:1344–1355. [PubMed: 24088094] 51. Yang Q, Mas A, Diamond MP, Al-Hendy A. The Mechanism and Function of Epigenetics in Uterine Leiomyoma Development. Reprod Sci. 2015 52. Islam MS, Protic O, Stortoni P, Grechi G, Lamanna P, Petraglia F, Castellucci M, Ciarmela P. Complex networks of multiple factors in the pathogenesis of uterine leiomyoma. Fertility and sterility. 2013; 100:178–193. [PubMed: 23557758] 53. Donner AJ, Szostek S, Hoover JM, Espinosa JM. CDK8 is a stimulus-specific positive coregulator of p53 target genes. Molecular cell. 2007; 27:121–133. [PubMed: 17612495] 54. Halder SK, Laknaur A, Miller J, Layman LC, Diamond M, Al-Hendy A. Novel MED12 gene somatic mutations in women from the Southern United States with symptomatic uterine fibroids. Molecular genetics and genomics : MGG. 2014 55. Markowski DN, Bartnitzke S, Loning T, Drieschner N, Helmke BM, Bullerdiek J. MED12 mutations in uterine fibroids--their relationship to cytogenetic subgroups. International journal of cancer Journal international du cancer. 2012; 131:1528–1536. [PubMed: 22223266] 56. Kampjarvi K, Park MJ, Mehine M, Kim NH, Clark AD, Butzow R, Bohling T, Bohm J, Mecklin JP, Jarvinen H, Tomlinson IP, van der Spuy ZM, Sjoberg J, Boyer TG, Vahteristo P. Mutations in Exon 1 highlight the role of MED12 in uterine leiomyomas. Human mutation. 2014; 35:1136– 1141. [PubMed: 24980722] 57. Moravek MB, Yin P, Ono M, St Coon J, Dyson MT, Navarro A, Marsh EE, Chakravarti D, Kim JJ, Wei JJ, Bulun SE. Ovarian steroids, stem cells and uterine leiomyoma: therapeutic implications. Human reproduction update. 2015; 21:1–12. [PubMed: 25205766] 58. Wong CW, McNally C, Nickbarg E, Komm BS, Cheskis BJ. Estrogen receptor-interacting protein that modulates its nongenomic activity-crosstalk with Src/Erk phosphorylation cascade. Proceedings of the National Academy of Sciences of the United States of America. 2002; 99:14783–14788. [PubMed: 12415108] 59. Hofmeister MV, Damkier HH, Christensen BM, Olde B, Fredrik Leeb-Lundberg LM, Fenton RA, Praetorius HA, Praetorius J. 17beta-Estradiol induces nongenomic effects in renal intercalated cells through G protein-coupled estrogen receptor 1. American journal of physiology Renal physiology. 2012; 302:F358–368. [PubMed: 21993891] 60. Bjornstrom L, Sjoberg M. Mechanisms of estrogen receptor signaling: convergence of genomic and nongenomic actions on target genes. Mol Endocrinol. 2005; 19:833–842. [PubMed: 15695368] 61. Bjornstrom L, Sjoberg M. Signal transducers and activators of transcription as downstream targets of nongenomic estrogen receptor actions. Mol Endocrinol. 2002; 16:2202–2214. [PubMed: 12351686] 62. Improta-Brears T, Whorton AR, Codazzi F, York JD, Meyer T, McDonnell DP. Estrogen-induced activation of mitogen-activated protein kinase requires mobilization of intracellular calcium. Proceedings of the National Academy of Sciences of the United States of America. 1999; 96:4686–4691. [PubMed: 10200323] 63. Incerpi S, D’Arezzo S, Marino M, Musanti R, Pallottini V, Pascolini A, Trentalance A. Short-term activation by low 17beta-estradiol concentrations of the Na+/H+ exchanger in rat aortic smooth muscle cells: physiopathological implications. Endocrinology. 2003; 144:4315–4324. [PubMed: 12959986] 64. Klinge CM, Blankenship KA, Risinger KE, Bhatnagar S, Noisin EL, Sumanasekera WK, Zhao L, Brey DM, Keynton RS. Resveratrol and estradiol rapidly activate MAPK signaling through estrogen receptors alpha and beta in endothelial cells. The Journal of biological chemistry. 2005; 280:7460–7468. [PubMed: 15615701]


Katz et al.

Page 15


65. Marino M, Galluzzo P, Ascenzi P. Estrogen signaling multiple pathways to impact gene transcription. Current genomics. 2006; 7:497–508. [PubMed: 18369406] 66. Simoncini T, Hafezi-Moghadam A, Brazil DP, Ley K, Chin WW, Liao JK. Interaction of oestrogen receptor with the regulatory subunit of phosphatidylinositol-3-OH kinase. Nature. 2000; 407:538– 541. [PubMed: 11029009] 67. Prossnitz ER, Arterburn JB. International Union of Basic and Clinical Pharmacology. XCVII G. Protein-Coupled Estrogen Receptor and Its Pharmacologic Modulators Pharmacological reviews. 2015; 67:505–540. 68. Kelly MJ, Qiu J, Ronnekleiv OK. Estrogen modulation of G-protein-coupled receptor activation of potassium channels in the central nervous system. Annals of the New York Academy of Sciences. 2003; 1007:6–16. [PubMed: 14993035] 69. Burns KA, Korach KS. Estrogen receptors and human disease: an update. Archives of toxicology. 2012; 86:1491–1504. [PubMed: 22648069] 70. Kastner P, Krust A, Turcotte B, Stropp U, Tora L, Gronemeyer H, Chambon P. Two distinct estrogen-regulated promoters generate transcripts encoding the two functionally different human progesterone receptor forms A and B. The EMBO journal. 1990; 9:1603–1614. [PubMed: 2328727] 71. Singh M, Su C, Ng S. Non-genomic mechanisms of progesterone action in the brain. Frontiers in neuroscience. 2013; 7:159. [PubMed: 24065876] 72. Patel B, Elguero S, Thakore S, Dahoud W, Bedaiwy M, Mesiano S. Role of nuclear progesterone receptor isoforms in uterine pathophysiology. Human reproduction update. 2015; 21:155–173. [PubMed: 25406186] 73. Boonyaratanakornkit V, Scott MP, Ribon V, Sherman L, Anderson SM, Maller JL, Miller WT, Edwards DP. Progesterone receptor contains a proline-rich motif that directly interacts with SH3 domains and activates c-Src family tyrosine kinases. Molecular cell. 2001; 8:269–280. [PubMed: 11545730] 74. Dressing GE, Goldberg JE, Charles NJ, Schwertfeger KL, Lange CA. Membrane progesterone receptor expression in mammalian tissues: a review of regulation and physiological implications. Steroids. 2011; 76:11–17. [PubMed: 20869977] 75. Faivre EJ, Daniel AR, Hillard CJ, Lange CA. Progesterone receptor rapid signaling mediates serine 345 phosphorylation and tethering to specificity protein 1 transcription factors. Mol Endocrinol. 2008; 22:823–837. [PubMed: 18202149] 76. Migliaccio A, Piccolo D, Castoria G, Di Domenico M, Bilancio A, Lombardi M, Gong W, Beato M, Auricchio F. Activation of the Src/p21ras/Erk pathway by progesterone receptor via cross-talk with estrogen receptor. The EMBO journal. 1998; 17:2008–2018. [PubMed: 9524123] 77. Shimomura Y, Matsuo H, Samoto T, Maruo T. Up-regulation by progesterone of proliferating cell nuclear antigen and epidermal growth factor expression in human uterine leiomyoma. The Journal of clinical endocrinology and metabolism. 1998; 83:2192–2198. [PubMed: 9626159] 78. Yamada T, Nakago S, Kurachi O, Wang J, Takekida S, Matsuo H, Maruo T. Progesterone downregulates insulin-like growth factor-I expression in cultured human uterine leiomyoma cells. Hum Reprod. 2004; 19:815–821. [PubMed: 15033949] 79. Tsigkou A, Reis FM, Lee MH, Jiang B, Tosti C, Centini G, Shen FR, Chen YG, Petraglia F. Increased progesterone receptor expression in uterine leiomyoma: correlation with age, number of leiomyomas, and clinical symptoms. Fertility and sterility. 2015; 104:170–175. e171. [PubMed: 26006736] 80. Whitaker LH, Williams AR, Critchley HO. Selective progesterone receptor modulators. Current opinion in obstetrics & gynecology. 2014; 26:237–242. [PubMed: 24950125] 81. Ishikawa H, Ishi K, Serna VA, Kakazu R, Bulun SE, Kurita T. Progesterone is essential for maintenance and growth of uterine leiomyoma. Endocrinology. 2010; 151:2433–2442. [PubMed: 20375184] 82. Wagenfeld A, Saunders PT, Whitaker L, Critchley HO. Selective progesterone receptor modulators (SPRMs): progesterone receptor action, mode of action on the endometrium and treatment options in gynecological therapies. Expert opinion on therapeutic targets. 2016:1–10.


Katz et al.

Page 16


83. Olejek A, Olszak-Wasik K, Czerwinska-Bednarska A. Long-term intermittent pharmacological therapy of uterine fibroids - a possibility to avoid hysterectomy and its negative consequences. Prz Menopauzalny. 2016; 15:48–51. [PubMed: 27095959] 84. Puchar A, Luton D, Koskas M. Ulipristal acetate for uterine fibroid-related symptoms. Drugs Today (Barc). 2015; 51:661–667. [PubMed: 26744741] 85. Nelson AL. Investigational hormone receptor agonists as ongoing female contraception: a focus on selective progesterone receptor modulators in early clinical development. Expert Opin Investig Drugs. 2015; 24:1321–1330. 86. Donnez J, Arriagada P, Donnez O, Dolmans MM. Current management of myomas: the place of medical therapy with the advent of selective progesterone receptor modulators. Current opinion in obstetrics & gynecology. 2015; 27:422–431. [PubMed: 26536207] 87. Donnez J, Tomaszewski J, Vazquez F, Bouchard P, Lemieszczuk B, Baro F, Nouri K, Selvaggi L, Sodowski K, Bestel E, Terrill P, Osterloh I, Loumaye E, Group PIS. Ulipristal acetate versus leuprolide acetate for uterine fibroids. The New England journal of medicine. 2012; 366:421–432. [PubMed: 22296076] 88. Linder D, Gartler SM. Glucose-6-phosphate dehydrogenase mosaicism: utilization as a cell marker in the study of leiomyomas. Science. 1965; 150:67–69. [PubMed: 5833538] 89. Ono M, Maruyama T, Masuda H, Kajitani T, Nagashima T, Arase T, Ito M, Ohta K, Uchida H, Asada H, Yoshimura Y, Okano H, Matsuzaki Y. Side population in human uterine myometrium displays phenotypic and functional characteristics of myometrial stem cells. Proceedings of the National Academy of Sciences of the United States of America. 2007; 104:18700–18705. [PubMed: 18003928] 90. Ono M, Qiang W, Serna VA, Yin P, St Coon J, Navarro A, Monsivais D, Kakinuma T, Dyson M, Druschitz S, Unno K, Kurita T, Bulun SE. Role of stem cells in human uterine leiomyoma growth. PloS one. 2012; 7:e36935. [PubMed: 22570742] 91. Ono M, Yin P, Navarro A, Moravek MB, St Coon J, Druschitz SA, Serna VA, Qiang W, Brooks DC, Malpani SS, Ma J, Ercan CM, Mittal N, Monsivais D, Dyson MT, Yemelyanov A, Maruyama T, Chakravarti D, Kim JJ, Kurita T, Gottardi CJ, Bulun SE. Paracrine activation of WNT/betacatenin pathway in uterine leiomyoma stem cells promotes tumor growth. Proceedings of the National Academy of Sciences of the United States of America. 2013; 110:17053–17058. [PubMed: 24082114] 92. Galvez BG, Martin NS, Salama-Cohen P, Lazcano JJ, Coronado MJ, Lamelas ML, AlvarezBarrientes A, Eiro N, Vizoso F, Rodriguez C. An adult myometrial pluripotential precursor that promotes healing of damaged muscular tissues. In Vivo. 2010; 24:431–441. [PubMed: 20668309] 93. Zhou S, Yi T, Shen K, Zhang B, Huang F, Zhao X. Hypoxia: the driving force of uterine myometrial stem cells differentiation into leiomyoma cells. Medical hypotheses. 2011; 77:985– 986. [PubMed: 21903341] 94. Chang HL, Senaratne TN, Zhang L, Szotek PP, Stewart E, Dombkowski D, Preffer F, Donahoe PK, Teixeira J. Uterine leiomyomas exhibit fewer stem/progenitor cell characteristics when compared with corresponding normal myometrium. Reprod Sci. 2010; 17:158–167. [PubMed: 19805552] 95. Mas A, Cervello I, Gil-Sanchis C, Faus A, Ferro J, Pellicer A, Simon C. Identification and characterization of the human leiomyoma side population as putative tumor-initiating cells. Fertility and sterility. 2012; 98:741–751. e746. [PubMed: 22633281] 96. Maruyama T, Ono M, Yoshimura Y. Somatic stem cells in the myometrium and in myomas. Seminars in reproductive medicine. 2013; 31:77–81. [PubMed: 23329640] 97. Mas A, Cervello I, Gil-Sanchis C, Simon C. Current understanding of somatic stem cells in leiomyoma formation. Fertility and sterility. 2014; 102:613–620. [PubMed: 24890270] 98. Mittal P, Shin YH, Yatsenko SA, Castro CA, Surti U, Rajkovic A. Med12 gain-of-function mutation causes leiomyomas and genomic instability. The Journal of clinical investigation. 2015; 125:3280–3284. [PubMed: 26193636] 99. Ono M, Bulun SE, Maruyama T. Tissue-specific stem cells in the myometrium and tumor-initiating cells in leiomyoma. Biology of reproduction. 2014; 91:149. [PubMed: 25376230]


Katz et al.

Page 17


100. Szotek PP, Chang HL, Zhang L, Preffer F, Dombkowski D, Donahoe PK, Teixeira J. Adult mouse myometrial label-retaining cells divide in response to gonadotropin stimulation. Stem Cells. 2007; 25:1317–1325. [PubMed: 17289934] 101. Shynlova O, Dorogin A, Lye SJ. Stretch-induced uterine myocyte differentiation during rat pregnancy: involvement of caspase activation. Biology of reproduction. 2010; 82:1248–1255. [PubMed: 20181619] 102. Mas A, Nair S, Laknaur A, Simon C, Diamond MP, Al-Hendy A. Stro-1/CD44 as putative human myometrial and fibroid stem cell markers. Fertility and sterility. 2015; 104:225–234. e223. [PubMed: 25989979] 103. Benoit YD, Guezguez B, Boyd AL, Bhatia M. Molecular Pathways: Epigenetic Modulation of Wnt/Glycogen Synthase Kinase-3 Signaling to Target Human Cancer Stem Cells. Clinical cancer research : an official journal of the American Association for Cancer Research. 2014 104. Baird DD, Newbold R. Prenatal diethylstilbestrol (DES) exposure is associated with uterine leiomyoma development. Reprod Toxicol. 2005; 20:81–84. [PubMed: 15808789] 105. D’Aloisio AA, Baird DD, DeRoo LA, Sandler DP. Association of intrauterine and early-life exposures with diagnosis of uterine leiomyomata by 35 years of age in the Sister Study. Environ Health Perspect. 2010; 118:375–381. [PubMed: 20194067] 106. D’Aloisio AA, Baird DD, DeRoo LA, Sandler DP. Early-life exposures and early-onset uterine leiomyomata in black women in the Sister Study. Environ Health Perspect. 2012; 120:406–412. [PubMed: 22049383] 107. Mahalingaiah S, Hart JE, Wise LA, Terry KL, Boynton-Jarrett R, Missmer SA. Prenatal diethylstilbestrol exposure and risk of uterine leiomyomata in the Nurses’ Health Study II. American journal of epidemiology. 2014; 179:186–191. [PubMed: 24142917] 108. Wise LA, Palmer JR, Rowlings K, Kaufman RH, Herbst AL, Noller KL, Titus-Ernstoff L, Troisi R, Hatch EE, Robboy SJ. Risk of benign gynecologic tumors in relation to prenatal diethylstilbestrol exposure. Obstetrics and gynecology. 2005; 105:167–173. [PubMed: 15625159] 109. Kobayashi T, Hirayama Y, Kobayashi E, Kubo Y, Hino O. A germline insertion in the tuberous sclerosis (Tsc2) gene gives rise to the Eker rat model of dominantly inherited cancer. Nature genetics. 1995; 9:70–74. [PubMed: 7704028] 110. Yeung RS, Xiao GH, Jin F, Lee WC, Testa JR, Knudson AG. Predisposition to renal carcinoma in the Eker rat is determined by germ-line mutation of the tuberous sclerosis 2 (TSC2) gene. Proceedings of the National Academy of Sciences of the United States of America. 1994; 91:11413–11416. [PubMed: 7972075] 111. Walker CL, Hunter D, Everitt JI. Uterine leiomyoma in the Eker rat: a unique model for important diseases of women. Genes, chromosomes & cancer. 2003; 38:349–356. [PubMed: 14566855] 112. Burroughs KD, Fuchs-Young R, Davis B, Walker CL. Altered hormonal responsiveness of proliferation and apoptosis during myometrial maturation and the development of uterine leiomyomas in the rat. Biology of reproduction. 2000; 63:1322–1330. [PubMed: 11058535] 113. Walker CL, Cesen-Cummings K, Houle C, Baird D, Barrett JC, Davis B. Protective effect of pregnancy for development of uterine leiomyoma. Carcinogenesis. 2001; 22:2049–2052. [PubMed: 11751438] 114. Hodges LC, Hunter DS, Bergerson JS, Fuchs-Young R, Walker CL. An in vivo/in vitro model to assess endocrine disrupting activity of xenoestrogens in uterine leiomyoma. Annals of the New York Academy of Sciences. 2001; 948:100–111. [PubMed: 11795388] 115. Howe SR, Gottardis MM, Everitt JI, Goldsworthy TL, Wolf DC, Walker C. Rodent model of reproductive tract leiomyomata. Establishment and characterization of tumor-derived cell lines. The American journal of pathology. 1995; 146:1568–1579. [PubMed: 7539981] 116. Fuchs-Young R, Howe S, Hale L, Miles R, Walker C. Inhibition of estrogen-stimulated growth of uterine leiomyomas by selective estrogen receptor modulators. Molecular carcinogenesis. 1996; 17:151–159. [PubMed: 8944075] 117. Walker CL, Burroughs KD, Davis B, Sowell K, Everitt JI, Fuchs-Young R. Preclinical evidence for therapeutic efficacy of selective estrogen receptor modulators for uterine leiomyoma. Journal of the Society for Gynecologic Investigation. 2000; 7:249–256. [PubMed: 10964025]


Katz et al.

Page 18


118. Jirecek S, Lee A, Pavo I, Crans G, Eppel W, Wenzl R. Raloxifene prevents the growth of uterine leiomyomas in premenopausal women. Fertility and sterility. 2004; 81:132–136. [PubMed: 14711556] 119. Palomba S, Orio F Jr, Russo T, Falbo A, Tolino A, Lombardi G, Cimini V, Zullo F. Antiproliferative and proapoptotic effects of raloxifene on uterine leiomyomas in postmenopausal women. Fertility and sterility. 2005; 84:154–161. [PubMed: 16009171] 120. Hunter DS, Klotzbucher M, Kugoh H, Cai SL, Mullen JP, Manfioletti G, Fuhrman U, Walker CL. Aberrant expression of HMGA2 in uterine leiomyoma associated with loss of TSC2 tumor suppressor gene function. Cancer research. 2002; 62:3766–3772. [PubMed: 12097287] 121. Howe SR, Pass HI, Ethier SP, Matthews WJ, Walker C. Presence of an insulin-like growth factor I autocrine loop predicts uterine fibroid responsiveness to tamoxifen. Cancer research. 1996; 56:4049–4055. [PubMed: 8752178] 122. Wei J, Chiriboga L, Mizuguchi M, Yee H, Mittal K. Expression profile of tuberin and some potential tumorigenic factors in 60 patients with uterine leiomyomata. Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc. 2005; 18:179–188. 123. Prizant H, Sen A, Light A, Cho SN, DeMayo FJ, Lydon JP, Hammes SR. Uterine-specific loss of Tsc2 leads to myometrial tumors in both the uterus and lungs. Mol Endocrinol. 2013; 27:1403– 1414. [PubMed: 23820898] 124. Kaneko-Tarui T, Commandeur AE, Patterson AL, DeKuiper JL, Petillo D, Styer AK, Teixeira JM. Hyperplasia and fibrosis in mice with conditional loss of the TSC2 tumor suppressor in Mullerian duct mesenchyme-derived myometria. Molecular human reproduction. 2014; 20:1126–1134. [PubMed: 25189766] 125. Branham WS, Zehr DR, Chen JJ, Sheehan DM. Uterine abnormalities in rats exposed neonatally to diethylstilbestrol, ethynylestradiol, or clomiphene citrate. Toxicology. 1988; 51:201–212. [PubMed: 3176028] 126. Brody JR, Cunha GR. Histologic, morphometric, and immunocytochemical analysis of myometrial development in rats and mice: II. Effects of DES on development. The American journal of anatomy. 1989; 186:21–42. [PubMed: 2782287] 127. Newbold RR, Jefferson WN, Grissom SF, Padilla-Banks E, Snyder RJ, Lobenhofer EK. Developmental exposure to diethylstilbestrol alters uterine gene expression that may be associated with uterine neoplasia later in life. Molecular carcinogenesis. 2007; 46:783–796. [PubMed: 17394237] 128. Newbold RR, Moore AB, Dixon D. Characterization of uterine leiomyomas in CD-1 mice following developmental exposure to diethylstilbestrol (DES). Toxicologic pathology. 2002; 30:611–616. [PubMed: 12371671] 129. Cook KE, Jenkins SM. Pathologic uterine torsion associated with placental abruption, maternal shock, and intrauterine fetal demise. American journal of obstetrics and gynecology. 2005; 192:2082–2083. [PubMed: 15970905] 130. Cook JD, Davis BJ, Goewey JA, Berry TD, Walker CL. Identification of a sensitive period for developmental programming that increases risk for uterine leiomyoma in Eker rats. Reproductive sciences. 2007; 14:121–136. [PubMed: 17636224] 131. Branham WS, Sheehan DM. Ovarian and adrenal contributions to postnatal growth and differentiation of the rat uterus. Biology of reproduction. 1995; 53:863–872. [PubMed: 8547482] 132. Sheehan DM, Branham WS, Medlock KL, Olson ME, Zehr DR. Uterine responses to estradiol in the neonatal rat. Endocrinology. 1981; 109:76–82. [PubMed: 7238415] 133. Greathouse KL, Cook JD, Lin K, Davis BJ, Berry TD, Bredfeldt TG, Walker CL. Identification of uterine leiomyoma genes developmentally reprogrammed by neonatal exposure to diethylstilbestrol. Reprod Sci. 2008; 15:765–778. [PubMed: 19017814] 134. Greathouse KL, Bredfeldt T, Everitt JI, Lin K, Berry T, Kannan K, Mittelstadt ML, Ho SM, Walker CL. Environmental estrogens differentially engage the histone methyltransferase EZH2 to increase risk of uterine tumorigenesis. Mol Cancer Res. 2012; 10:546–557. [PubMed: 22504913] 135. Trevino LS, Wang Q, Walker CL. Phosphorylation of epigenetic “readers, writers and erasers”: Implications for developmental reprogramming and the epigenetic basis for health and disease. Prog Biophys Mol Biol. 2015; 118:8–13. [PubMed: 25841987]


Katz et al.

Page 19

Author Manuscript

136. Walker CL, Ho SM. Developmental reprogramming of cancer susceptibility. Nature reviews Cancer. 2012; 12:479–486. [PubMed: 22695395] 137. Wong RL, Walker CL. Molecular pathways: environmental estrogens activate nongenomic signaling to developmentally reprogram the epigenome. Clinical cancer research : an official journal of the American Association for Cancer Research. 2013; 19:3732–3737. [PubMed: 23549878] 138. Navarro A, Yin P, Monsivais D, Lin SM, Du P, Wei JJ, Bulun SE. Genome-wide DNA methylation indicates silencing of tumor suppressor genes in uterine leiomyoma. PloS one. 2012; 7:e33284. [PubMed: 22428009] 139. Yamagata Y, Maekawa R, Asada H, Taketani T, Tamura I, Tamura H, Ogane J, Hattori N, Shiota K, Sugino N. Aberrant DNA methylation status in human uterine leiomyoma. Mol Hum Reprod. 2009; 15:259–267. [PubMed: 19218588] 140. Li S, Washburn KA, Moore R, Uno T, Teng C, Newbold RR, McLachlan JA, Negishi M. Developmental exposure to diethylstilbestrol elicits demethylation of estrogen-responsive lactoferrin gene in mouse uterus. Cancer research. 1997; 57:4356–4359. [PubMed: 9331098]

Author Manuscript Author Manuscript Author Manuscript Fertil Steril. Author manuscript; available in PMC 2017 September 15.

Katz et al.

Page 20


Figure 1. Developmental exposure to EDCs induces genetic/epigenetic abnormalities in myometrial stem cells leading to UF development

Environmental factors including EDCs induce genetic/epigenetic alterations, thereby leading to the conversion of myometrial stem cells into tumor initiating cells, eventually giving rise to the formation of uterine fibroids.

Author Manuscript Author Manuscript Fertil Steril. Author manuscript; available in PMC 2017 September 15.

Katz et al.

Page 21

Table 1

Author Manuscript

Identified targets of signaling pathways in uterine fibroids

Author Manuscript Author Manuscript Author Manuscript

Pathways

Targets

Type of samples

Ref

Estrogen

ER

fibroid tissues

Al-Hendy A et al J Clin Endocrinol Metab, 2015

Estrogen

P53

fibroid cells

Gao Z et al. J. Clin. Endocrinol Metab, 2002

Estrogen

c-fos, c-jun

fibroid tissues

Gustavsson I et al Mol Hum Reprod, 2000

Estrogen

connexin

fibroid tissues

Andersen J. et al. Am J Obstet Gynecol, 1993

Estrogen

PDGF, PI3K, MAPK, PLCr

fibroid cells

Barbarisi A et al, J Cell Physiol. 2001

Estrogen

PKCα, ERK

fibroid cells

Nierth-Simpson, EN, Endocrinology, 2009

Estrogen

PPARr, PTEN

fibroid tissues and cells

Jeong YJ et al, Int. J. Oncol, 2011

Estrogen

IGF-1, Myb, MAPK

fibroid cells

Swartz CD, et al, Mol. Hum Reprod 2005

Progesterone

PR-A, PR-B

fibroid tissues

Tsigkou A et al Fertil Steril 2015; Al-Hendy A et al J Clin Endocrinol Metab 2015

Progesterone

BCL-2

fibroid tissues

Matsuo H et al. J. Clin Endocrinol Metab. 1997

Progesterone

AKT

fibroid cells

Hoekstra AV et al J. Clin. Endocrinol Metab, 2009

Progesterone

IGF-1, EGF

fibroid cells

Yamada T, et al Human Reprod 2004

Progesterone

LAT2

fibroid cells

Luo X. et al J. Clin Endocrinol Metab, 2009

Progesterone

KLF11

fibroid cells

Yin P. et al Cancer Res, 2010

Progesterone

EGF

fibroid tissues

Shimomura Y et al. J Clin. Endocrinol Metab, 1998

Estrogen/progesterone

β-catenin, Axin2

Fibroid stem cells

Ono M et al, PNAS 2013


Wnt 11, Wnt16

fibroid cells

Ono M et al, PNAS 2013


miR-29b, collagen I α1

fibroid tissues

Wang T et al, Gen Chromosomes cancer, Marsh EE et al Fertil Steril 2008.

MED12 mutation

Wnt4, WISP1, β-catenin

fibroid tissues

Makinen N et al Science, 2011, Al-Hendy A et al J Clin Endocrinol Metab 2016

Vitamin D

VDR, MMP-2, MMP-9, TIMP-2

fibroid cells

Halder SK et al Hum Reprod, 2013

Vitamin D

TGF-β signaling, Smad2

fibroid cells

Halder SK et al J Clin Endocrinol Metab 2011

Vitamin D

ER, PR-A, PR-B


Al-Hendy A et al J Clin Endocrinol Metab 2015

Vitamin D

Wnt4, Wisp1, FEN1, mTOR

fibroid cells

Al-Hendy A et al J Clin Endocrinol Metab 2016

PDGF

PCNA, collagen α1

fibroid cells

Liang M et al, Cancer Biol Ther, 2006

TGF-β3

PDGF, PDGF receptor

fibroid cells

Wolanska M et al Eur J Obstet Gynecol Reprod Bio. 2007

TGF-β3

fibronectin

fibroid cells

Arici, Fertil Steril, 2000

TGF-β3

collagens

fibroid cells

Salama SA, Fertil Steril, 2012

IGF-1

BCL-2

IGF-1

AKT

IGF-1

MAPK

fibroid tissues

Yu L et al Mol Med 2008

Retinoic acid

RABP1, RABP2, ALDH1, ADH1


Zaitseva M, et al. Hum Reprod 2008

Gao Z et al J Clin Endocrinol Metab 2001 Peng L et al Fertil Steil 2009


Fibroids (uterine myomatosis, leiomyomas).

Uterine fibroids: current perspectives.

Aromatase inhibitors for uterine fibroids.

Uterine fibroids and current clinical challenges.

Ulipristal acetate in uterine fibroids.

Uterine fibroids associated with infertility.

Uterine fibroids: clinical manifestations and contemporary management.

Pathogenesis of endometriosis and uterine fibroids.

EMAS position statement: management of uterine fibroids.

Amniotic fluid embolism after uterine artery embolization for uterine fibroids.

New hysteroscopic techniques for submucosal uterine fibroids.

Thermal ablative treatment of uterine fibroids.

Overcoming the challenges of studying uterine fibroids.

Overview of latest evidence on uterine fibroids.

Current evidence on uterine embolization for fibroids.

Elective cesarean hysterectomy for uterine fibroids.

Uterine fibroids: pathogenesis and interactions with endometrium and endomyometrial junction.

Knowledge of, Perception of, and Attitude towards Uterine Fibroids among Women with Fibroids in Lagos, Nigeria.

Number and size of uterine fibroids and obstetric outcomes.

Blood cadmium and volume of uterine fibroids in premenopausal women.

Clinical Efficacy and Complications of Uterine Artery Embolization in Symptomatic Uterine Fibroids.

Case series: Pregnancy Outcome in Patients with Uterine Fibroids.

[Treatment of uterine fibroids using high-intensity ultrasound].

Alterations in uterine hemodynamics caused by uterine fibroids and their impact on in vitro fertilization outcomes.