Prevention of Uterine Fibroids: molecular mechanisms and potential clinical application

Abstract

Uterine fibroids (UFs; leiomyoma) are the most common benign neoplastic threat to women worldwide, exacting an immense personal burden on female health and a monetary expense to the healthcare system estimated in the hundreds of billions of dollars every year globally. With no long-term non-invasive treatment option currently available to treat UFs, deeper insights regarding tumor etiology are the key for developing newer therapies. Accordingly, in this review, we discuss new mechanistic paradigm to explain UF tumor development through an exquisite model involving developmental reprogramming of myometrial stem cells due to early life endocrine disruptors exposure, inflammation, fibrosis, DNA damage, and eventually tissue stiffness. Further, we propose to utilize shear wave elastography as a potential screening tool for the early identification of women at risk for developing UFs who can benefit from several simple preventive strategies, including the consumption of natural compounds such as vitamin D and green tea as a safe fertility friendly non-hormonal modality to delay or even arrest or reverse UF progression.

1. Introduction

Uterine fibroids (UFs; leiomyomas) are benign monoclonal neoplasms of the myometrium (MM) and represent the most common tumor in women worldwide [1]. UFs occur in 70-80% of women overall and are clinically manifest in 25-50% by age of 50 [2]. Although benign, these tumors are nonetheless associated with significant morbidity; they are the primary indicator of hysterectomy, and a major source of gynecologic and reproductive dysfunction, ranging from profuse menstrual bleeding and pelvic pain to infertility, recurrent miscarriage, and pre-term labor. Accordingly, the annual health care costs associated with UFs have been estimated around $34 billion. UFs thus represent a significant societal health and financial burden [1, 2]. An increasing body of evidence supports the notion that UFs originate from aberrant stem cells in myometrium. Despite the remarkable advances made in defining the cellular hierarchy in other hormonally regulated tissues, such as breast [3] and prostate [4], little is known about the cellular origin and initial steps in UFs tumorigenesis [1, 2, 5, 6]. UFs arise from the genetic transformation of a single myometrial (MM) stem cell (SC) into a tumor-initiating cell (UF SC) that seeds and sustains UF growth through asymmetric cell divisions and monoclonal expansion. Heretofore, the genetic drivers dominantly responsible for SC transformation have been identified. The most prevalent among these, accounting for ~70% of UFs, are somatic mutations in the MED12 subunit of the multiprotein RNA polymerase II transcriptional Mediator [1, 7, 8]. However, the molecular basis by which MED12 mutations drive UF formation is largely unknown.

2. Molecular mechanisms underlying uterine fibroids pathogenesis.

2.1. Endocrine disrupting chemicals exposure

Like many diseases, there is ample evidence that both environmental exposures and genetic alterations contribute to UF pathogenesis. Our group and others have shown that early life environmental exposures to endocrine-disrupting compounds (EDCs) during critical periods of development can increase UF risk partly by inducing developmental reprogramming of the epigenome [9] due to the epigenomic plasticity inherent in progenitor cells and developing tissues. This includes exposures to EDCs such as phthalates, Bisphenol A (BPA) plasticizers, diethylstilbestrol (DES), genistein (GEN), Perfluoroalkyl substances (PFAS) and Parabens in personal care products [10]. Walker et al have established that EDC exposures can reprogram the epigenome to increase the risk for reproductive tract tumors, including UFs [11, 12]. In the Eker rat model, developmental EDC exposure in the early neonatal stage increases the incidence, multiplicity, and size of UFs in adult rats [13]. Importantly, in women, early life exposure to EDCs has also been identified as a risk factor for the adult onset of UFs [14, 15]. Minority communities are particularly at risk for hazardous environmental exposures due to low socioeconomic status and poor quality of life [16], an intriguing parallel to the disproportionate risk for this disease in women of color [17].

The widespread application of phthalate plasticizers in consumer products such as personal care products has led to problems in women's reproductive health. Phthalates are a class of multi-functional chemicals divided into two main groups based on their molecular weight. Parent phthalates can migrate out of products and enter the human body to form metabolites, which can be considered a common biomarker detected in nearly all reproductive-aged women [18]. Di-(2-Ethylhexyl) phthalate (DEHP) is a common plasticizer in medical products. In vitro and in vivo exposure to DEHP reduced apoptosis and increased cell viability, proliferation, and collagen production throughout the induction of HIF-1α, COX-2, PCNA, and BLC2 expression [19, 20]. A systematic review by Fu et al. involved nine studies that reported DEHP was positively associated with the risk of UFs [21]. Various tissues metabolize DEHP to more bioactive metabolites, including mono-(2-ethyl-5-hexyl) phthalate (MEHP), mono-(2-ethyl-5-oxohexyl) phthalate (MEOHP), mono-(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), and mono-(2-ethyl-5-1carboxypentyl) phthalate (MECPP). MEHHP, a critical DEHP metabolite, promotes UFs cell survival by increasing cellular tryptophan uptake, kynurenine production, and aryl hydrocarbon receptor (AHR) pathway activation [22]. Zota et al. reported that Black women were at risk of exposure to parabens, and phthalates via beauty products such as hair relaxers and vaginal douches [23]. Lee et al. demonstrated significant associations between high urinary concentrations of DEHP metabolites, parabens and their metabolites in women with gynecological disorders [24]. Moreover, exposure to BPA increased UF odds, in a dose-response positive relationship [25]. BPA, detected in human blood and urine, was able to promote the proliferation of human UFs cells via nonclassical membrane-anchored estrogen receptors (ERs), such as G protein-coupled receptor to activate downstream pathways [26, 27]. Also, BPA activated downstream PI3K/AKT signaling pathways to promote the UFs cell proliferation [28] and contribute to UF growth [29]. Finally, a study has investigated the correlation between benign gynecological diseases and PFAS exposure, Hammarstrand et al. reported that exposure to high levels of PFAS in drinking water was associated with a possible increased risk of UFs [30].

2.2. Inflammation

Effects of EDCs on tumor development are extended to induce inflammation, DNA damage, and epigenetic alteration, which impact cell proliferation and apoptosis signaling and lead to UF growth [21]. Our preliminary data showed that myometrium stem cells (MMSCs) isolated from adult rats (5 months of age, pro-fibroid stage) exposed neonatally to DES, are epigenetically reprogrammed, and the transcriptome profiling of these adult rat MMSCs displayed a distinct pro-inflammatory related expression pattern compared to Vehicle (VEH) exposed MMSCs. Furthermore, we noticed that the production of key inflammatory cytokines TNF-α and IL-6 were also increased in DES-MMSCs as compared to VEH-MMSCs [31-34]. Inflammatory cells and cytokines in the local tissue microenvironment can coordinate a milieu of pro-inflammatory responses, which can act in an autocrine and/or paracrine manner on both healthy and unhealthy cells. Thus, the composition of the inflammatory microenvironment has a pivotal influence on the risk of disease development and progression [35]. In UFs, recent studies showed that higher numbers of macrophages are found inside and close to UFs as compared to the more distant myometrium [36-38]. Notably, our group has shown that several key pro-inflammatory mediators, including IL-1β, TNFα, INFγ, and NF-κB are overexpressed in UFs tissues and isolated stem cells as compared to corresponding normal MM samples (MyoN) [39]. Moreover, MMSCs from women with UFs (at risk myometrium, MyoF) showed an activated pro-inflammatory profile compared to normal MMSCs from women without UF (MyoN), highlighting the role of inflammation in UF pathogenesis [40]. Notably, EDC exposure levels and associated burden of disease and costs were higher in low socioeconomic minority communities such as non-Hispanic Blacks ($56.8 billion; 16.5% of total costs) and Mexican Americans ($50.1 billion; 14.6%) compared with their proportion of the total population (12.6% and 13.5%, respectively) [41]. Racial/ethnic disparities in UF can be attributed at least partly to that and necessary actions/policies need to be taken to address such disparities.

2.3. Obesity

Obesity is also an important risk factor for UFs, likely working via induction of chronic inflammation in the uterus [42]. Epidemiological studies have shown inconsistent results regarding obesity and UF risk, however subjects criteria were different [43]. Our group and others have shown that adipocytes enhanced human UF cell proliferation, inflammation, and fibrosis [44, 45], The latter study showed that UFs from obese patients expressed more TNF-α, MCP-1, phospho-NF-κB p65, VEGF-A and TGFβ3 than UF tissues from lean patients. In addition, a large cross-sectional study showed a positive correlation between African ancestry with high body mass index (BMI) in increasing the risk of UFs [46]. Interestingly, Caloric restriction and major weight loss have effectively reduced inflammatory markers in several organs [47, 48]. However, the genesis of this proinflammatory environment in the human myometrium, which leads to development of UFs, remains unknown.

2.4. Extracellular matrix accumulation and tissue stiffness

UFs are associated with a high extracellular matrix (ECM) accumulation, which justifies the stiff characteristics of these tumors [49]. ECM is composed of a network of macromolecules, primarily collagens, proteoglycans, elastin, and cell-binding glycoproteins in disorganized pattern [50]. ECM not only plays a role as a cell scaffold but also is a reservoir of growth factors and bioactive molecules. For example, the pleiotropic cytokine TGF-β is stored in the ECM in an inactive form and requires activation to exert its biological actions, which include the expression of ECM component [51]. Interestingly, TGF-β overexpression has been related to the development of UFs [52], suggesting that it is an important factor contributing to the excessive ECM accumulation that characterized these tumors. Importantly, an aberrant ECM expression can influence immune cell behavior, thereby actively contributing to immune responses [53]. In addition, ECM is crucial for the conversion of mechanical cues into biochemical signals, a process called mechanotransduction [54]. Mechanical stretch on integrins, which function as transmembrane receptors for ECM components, activates FAK and Src family kinases [55]. Activation of FAK initiates actin polymerization and activation of RhoA, which interacts with ROCK and activates the ERK/p38 MAPK-signaling cascade, resulting in changes in gene expression [56]. It is also important to highlight the bidirectional interaction between cells and their microenvironment, where cells sense the mechanical force from the recently produced ECM and activate mechanical signaling that will contribute to the secretion of various ECM components, which in turn will continue remodeling the ECM. This phenomenon of continuous interactions is referred to as dynamic reciprocity [57]. Remarkably, cells respond differentially to ECM stiffness levels, cell geometry, and cell density to control cell proliferation and differentiation [55]. It has been observed a novel link between mechanical signals and genomic stability since cells that undergo nuclear deformation and nuclear envelope opening, showed DNA double-strand breaks and activation of DNA repair machineries [58]. Our preliminary data showed that MMSCs from rats exposed neonatally to EDC had a significant decrease in nucleotide excision repair (NER) capacity, a DNA repair mechanism, capacity compared to normal VEH-MMSCs, Interestingly, when VEH-MMSCs were exposed to exogenous TGF-β1, we found that NER capacity was statistically decreased in comparison with the control group, highlighting the role that TGF- β family members can play in UF development through ECM remodeling, inflammation and DNA repair impairment [59].

2.5. Ethnic disparity

The most important and frequently reported risk factor for UFs is race, disproportionately impacting black women, who experience increased tumor burden, earlier and more severe symptoms [1]. However, UF is not the only disease that affects ethnicities differentially, many fibroproliferative diseases, including keloids scars and scleroderma, are more prevalent in individuals of African ancestry as compared to individuals of European ancestry [60]. It has been proposed that this disparity is due to the selection for an enhanced Th2 response that confers resistance to diseases caused by helminthic worms prevalent in sub-Saharan Africa, and concurrently increases susceptibility to fibrosis due to the profibrotic action of Th2 cytokines [61]. Notably, we recently showed, using transcriptomic analysis, that at-risk myometrium (MyoF) demonstrated a unique signature related to the ethnicity where ECM-related signaling pathways were among the top enriched pathways. Briefly, we observed that among the top 15 overrepresented Reactome pathways in Black over White MyoN, most of them were related to ECM signaling, suggesting its importance regarding the underlying increased prevalence and burden of UF in Black women. In addition, the overrepresented analysis of the top enriched ECM-related pathways on Black vs White in MyoF over MyoN comparison showed that Black MyoF over MyoN comparison is more enriched in pathways related to ECM than White MyoF vs MyoN, suggesting that ECM signaling is related to the transition from normal myometrium to myometrium at-risk in Black patients [62]. We believe that myometrium stiffness plays a role in UF pathogenesis and partly explains the ethnic disparity in UF incidence and severity (unpublished data). Figure 1 summarizes the molecular mechanisms that we believe are underlying UF pathogenesis in both human and corresponding UF rat model. Given the importance of ECM remodeling and accumulation in UF pathophysiology, several compounds have been studied as medical treatments that target ECM in these tumors [56]. Therefore, further knowledge of how ECM and mechanotransduction influence UF cell behavior may unravel the UF ethnic disparity mystery and contribute to the development of therapies for this condition.

Figure 1:

Molecular mechanisms underlying Uterine Fibroid development in human (right) and in the Eker rat animal model (left). EDCs: Endocrine-disrupting chemicals; MED12: Mediator Complex Subunit 12, Tsc-2: Tuberous sclerosis complex subunit 2

3. Shear Wave Elastography and proposed clinical application.

The phrase “prevention is better than cure” rings true in modern-day medicine. Preventive medicine is associated with a reduction in morbidity and mortality, particularly in health conditions such as substance abuse, mental health, oral and dental health, infectious diseases, and chronic diseases [63]. Preventive medicine also decreases healthcare costs which are reported at a total cost of $3.7 trillion for chronic diseases and is expected to increase as the population ages [64]. UFs may not be traditionally thought of by many as a chronic disease because it is a condition that affects individuals with a uterus and is variable in its presentation even within the affected cohort [65, 66]. However, a chronic disease, according to the center of disease control and prevention (CDC), is broadly defined as a health condition that lasts up to or greater than 1 year and requires ongoing medical attention or limits activities of daily living or both [67]. By this definition, UF is a chronic disease [67, 68]. In a 2013 Global Burden of Disease (GBD) study on chronic diseases, 171 million patients globally were estimated to have chronic fibroid symptoms. In 2019, ~ 1.4 million disability-adjusted life years were attributed to UFs by The Institute for Health Metrics and Evaluation (IHME) [65].

According to the American College of Obstetricians and Gynecologists (ACOG), preventive medicine in women’s health plays a central role in improving the health and quality of life of patients through the delivery of services such as screening and prevention of disease, family planning, nutrition and exercise, immunizations, infections disease exposure and management, psychological well-being, and behavioral health. Given the established impact of symptomatic UF on patients’ health, its economic burden, and the clear benefit of preventive medicine in healthcare, the development of screening and prevention tools for UF disease deserves concerted effort and attention. Furthermore, the disproportionate impact of UF on Black individuals provides the opportunity to address this health disparity through the implementation of screening and prevention tools.

Studies show that human tissues’ mechanical properties (i.e., stiffness) measured by ultrasound elastography (USE) correlate with histological characteristics including uterine diseases [69-73]. Additionally, research shows that USE can improve sensitivity and specificity in the diagnosis of many diverse pathologies [74], thus aiding physicians in treatment planning [75]. USE, specifically strain elastography which measures stiffness in a semi-quantitative manner, in which a ratio is used to describe the stiffness of a structure relative to adjacent structures, has been utilized sparingly in gynecology, albeit providing some proof of its conceptual use in gynecologic conditions. For example, a few studies investigating UFs and myometrial stiffness showed a difference in UF stiffness compared to normal myometrium [76, 77]. Shear wave elastography (SWE) is an objective quantitative ultrasound elastography technique which can depict the stiffness of anatomic structures to assist in their detection and characterization [78]. It offers an advantage over USE because it is less user dependent, more objective, and reliable and yields an absolute measure of tissue stiffness. To date, there are 3 published studies that focus on SWE in the evaluation of uterine pathologies – UF and adenomyosis. Two studies used histopathology as a final diagnosis [79, 80] and one used magnetic resonance imaging (MRI) for the diagnosis of adenomyosis [81]. Two studies showed that the myometrial stiffness of uteri with adenomyosis and leiomyoma was significantly higher than normal myometrium with no abnormalities [78, 79]. The third study showed that the stiffness of adenomyosis was higher than normal myometrium [80].

The consistency of these findings in the above studies is exciting because they suggest that SWE may provide an option for preventive management of UF. Based on our preliminary findings as discussed above, we hypothesize that at-risk-myometrium (MyoF) is stiffer than normal non-UF myometrium (MyoN) prior to the development of UF. Based on this theory, SWE may provide an ability to identify patients with uteri that are primed for UF development based on myometrial stiffness. Early identification of myometrial stiffness can provide a window of opportunity to implement interventions and treatment plans to modify and potentially prevent UF growth (Figure 2). Young females may undergo initial screening at an early age, perhaps age 21. An earlier age may be determined based on family history or risk factors such as early menarche, ethnicity, and BMI etc. If stiff myometrium is determined by SWE, the patient may be categorized as high risk for UF development. Healthcare provider (HCP) may then make recommendations to modify behavior, diet and lifestyle based on available data on the risk factors and contributory causes that foster UF growth. These may include decreasing red meat intake, decreasing alcohol consumption, use of Vitamin D supplementation, incorporating of Green Tea Extract into the diet, increasing physical activity to maintain a healthy weight, avoidance of exogenous hormonal sources, avoidance of environmental pollutants and EDCs, including phthalates, ozone pollution, etc. [1, 82, 83]. Notably, it is well-known that human behaviors are notoriously difficult to change which might make lifestyle modification challenging as well as education of limiting EDC exposure especially in deprived communities.

Figure 2:

Proposed application of shear wave elastography (SWE) as a screening tool for identification of women at risk of UF development and corresponding preventive measures to be taken. MyoN: normal non-fibroid myometrium; MyoF: at risk-myometrium, UF: uterine fibroid, EGCG: Epigallocatechin Gallate.

Furthermore, since significant differences in stiffness have been observed ex-vivo between UFs and leiomyosarcomas [84] and myometrium [85], in vivo between adenomyosis, myometrium [86] and some UF phenotypes [87], and a large variability in stiffness has been observed in unclassified UFs [87-89], we may see significant differences in SWE tissue stiffness between different UF types (soft, firm, solid, hard). Also, since significant reductions in UF sizes have been noted with use of some of the new GnRH analog therapies for the treatment of symptomatic UFs e.g., Relugolix [90], significant changes in UF stiffness measured by SWE may also be observable. This theory is supported by Athanasiou et al. [91], who correlated the reduction of breast tissue stiffness measured by SWE, in patients who responded to chemotherapy. Also, change in stiffness after interventional radiology has been correlated with improvements in patient outcomes [92]. Therefore, it may be reasonable to expect that significant differences in baseline tissue stiffness measured by SWE will be observed between women who do and do not respond to medical therapy. If this logic follows, then not only would we be able to prevent UFs growth using SWE, but we may also be able to predict which patient will respond to medical therapy and who will not. The ability to do this in a timely fashion will lead to less disappointment, decreased healthcare costs, and will prevent loss of valuable time. Also, timely intervention with other alternative treatment options (procedural or surgical) can be pursued without unnecessary delays.

4. Conclusion

In conclusion, current treatment options for UFs are primarily surgical or radiological. They range from hysterectomy or myomectomy to minimally invasive options, including uterine artery embolization (UAE) and magnetic resonance-guided focused ultrasound (MRgFUS). However, the deleterious impact of these procedures on reproductive function is either clear (hysterectomy) or controversial (UAE, MRgFUS), rendering such options unsuitable for women who wish to retain future fertility. Likewise, medical therapies designed to blunt the stimulatory effects of gonadal steroid hormones on UF growth are contraindicated in women actively pursuing a pregnancy and are otherwise approved only for short-term use due to long-term safety concerns. Accordingly, no long-term noninvasive treatment option currently exists for UFs, and deeper mechanistic insights into tumor etiology will be key to developing newer therapies for treatment or preferably prevention of UF. Hence, we propose using SWE as a screening tool with subsequent prevention strategies for women at risk of UF development. Moreover, we could use SWE to monitor response to medical therapy in patients with symptomatic UFs.