New Insights into Molecular Pathogenesis of Uterine Fibroids: From the Lab to a Clinician-Friendly Review

Abstract

Uterine fibroids (UFs) (also known as leiomyomas or myomas) are the most common form of benign uterine tumors, affecting 70–80% of women over their lifetime. Although uterine fibroids (UFs) are benign, these lesions cause significant morbidity and represent a major public health concern in reproductive age women. It has been hypothesized that leiomyomas arise from clonal proliferation of a single myometrial cell due to an initial genetic insult. However, these early cytogenetic alterations are insufficient for tumor development. In recent years, many advances have been made in the understanding of molecular mechanisms underlying the pathogenesis of uterine fibroids, and aberrations in several complex signaling pathways have shown to be involved in myoma development. In addition, most of these altered signaling cascades converge in a summative way, making the understanding of myoma biology even more complex. In this chapter, we focus on integrating this new knowledge in a simpler way to make it friendly to the general gynecologist.

1. Introduction

Uterine fibroids (UFs) (also known as leiomyomas or myomas) are the most common form of benign uterine tumors, affecting 70–80% of women over their lifetime [1]. Although uterine fibroids (UFs) are benign, these lesions cause significant morbidity and represent a major public health concern in reproductive age women [2]. Leiomyomas are monoclonal tumors that arise from uterine smooth muscle (i.e., the myometrium) [3]. Histologically, leiomyomas are composed of disordered smooth muscle cells, vascular smooth muscle cells, fibroblasts, myofibroblasts and are rich in extracellular matrix (ECM) [4, 5, 6]. Despite their high prevalence, the exact pathophysiology of uterine myomas is still unknown, although ethnicity-related data seem to indicate that the prevalence is about three times higher in African-American women. However, some other factors, such as early menarche, nulliparity, heredity, obesity, diabetes, hypertension, exposure to diethylstilbestrol (DES), air pollution, and dietary factors like high-fat diet, alcohol, and vitamin D deficiency, may also be involved in its incidence and development [5, 7, 8].

In recent years, many advances have been made, allowing us to understand the molecular mechanisms underlying the pathogenesis of UFs. The role of myometrial stem cells as tumor-initiating cells (TICs), the contribution of an abnormal ECM to tumor biology, the science of solid-state signaling in growth factors (GFs) metabolism and related pathways, the influence of epigenetic factors, steroid hormones and their receptors, the impact of myofibroblastic differentiation on inflammation and repair processes, and myometrial ischemia in tumor environment, have allowed a better understanding of myoma development and its clinical manifestations, such as abnormal uterine bleeding, dysmenorrhea, and infertility. In this chapter, we focus on integrating this new knowledge in a simpler way to make it friendly to the general gynecologist. However, it is important for better understanding of this chapter that many of the described molecular processes converge, link, and overlap.

2. Myometrial stem cells and myoma development

Somatic stem cells (SSCs) are a subset of cells residing in normal adult tissues that, through asymmetric division, retain their ability to self-renew while producing daughter cells that go on to differentiate and play a role in tissue regeneration and repair. Likewise, TICs are a subset of cells within a tumor cell population, which, also through asymmetric division, retain the ability to reconstitute tumors [9]. Several studies have described the presence of putative SSCs in myometrium (myometrial stem cells) and leiomyomas (leiomyoma stem cells). It has been suggested that several myometrial pathologies, such as UFs, can be attributed to the dysregulation of these SSCs, or that they might have derived from differentiated myometrial cells that acquire stem-like features (TICs) [9, 10, 11, 12]. It is hypothesized that leiomyomas originate from somatic mutations in myometrial stem cells which transform them to fibroid progenitor cells (leiomyoma stem cells, TICs), resulting in progressive loss of growth regulation [10, 12]. The tumor grows as genetically abnormal clones of cells derived from a single fibroid progenitor cell (in which the original mutation took place). Although fibroids are clonal in origin, considerable heterogeneity exists, and fibroids vary greatly in size, location, and appearance even within the same uterus. Interestingly, multiple myomas within the same uterus are not clonally related; each myoma arises independently [13]. In addition, fibroids from the same woman grow at different rates, with some regressing, despite a uniform hormonal milieu [14]. Moreover, fibroid characteristics, such as size, location, and ultrasonographic blood flow, do not correlate with fibroid growth, uterine bleeding, or any other symptomatology [14, 15], and therefore, further research is needed to identify biological or molecular factors that determine fibroid behavior.

The cellular composition of clonal fibroids is heterogeneous, including smooth muscle cells, vascular smooth muscle cells, fibroblasts, and myofibroblasts [3]. These phenotypically different clones exhibit differential expression of fibroid-associated genes: CRABP2 (encoding cellular retinoic acid-binding protein 2), PGR (encoding progesterone receptor B), and TGFBR2 (encoding TGF-β3 receptor 2) [4, 16]. These different gene profiles help to explain the heterogeneity of fibroid biology and clinical type.

Although the understanding of human myometrial stem cells is still limited, it is theorized that their population remains undifferentiated in its niche and under the right conditions can contribute to either physiologic (pregnancy) or pathologic processes (leiomyoma) [11].

It has been hypothesized that uterine hypoxia, aberrant methylation, or abnormal estrogen signaling could play a critical role in the transformation of a myometrial stem cell into a TIC [17, 18].

Despite a distinguishing feature of UFs being their dependency on the estrogen and progesterone to grow, leiomyoma stem cells comprised 1% of all tumor cells and they have very low sex steroid hormone receptor levels [9].

Ono et al. [19] studied the biological behavior of leiomyoma stem cells under different conditions, with the use of the side population technique. They concluded that even if leiomyoma stem cells are necessary for in vivo growth of UFs, their low estrogen and progesterone receptor levels, and their inability to grow and survive in a steroid hormone-only enrichment culture, suggest that other factors than steroid hormones are necessary for leiomyoma stem cell survival. Interestingly, leiomyoma stem cells had tumorigenic capacity under estrogen and progesterone stimulation when inoculated together with differentiated myometrial cells, demonstrating an indirect paracrine effect of steroid hormones on leiomyoma stem cells via the mature (differentiated) neighboring cells (normal myometrial or leiomyoma cells) [19]. Furthermore, leiomyoma stem cells were able to differentiate into uterine leiomyoma cells, gain their same potential of proliferation, and express steroid hormone receptors after coculture with mixed myometrial cells [9, 19].

In summary, it has been proposed that a single myometrial stem cell goes through tumorigenic transformation following a cellular insult (genetic hit) and gives rise to daughter leiomyoma stem cell (TIC), which proliferate, undergo self-renewal, and clonally expand in response to steroid hormones via paracrine signaling from surrounding differentiated myometrial and leiomyoma cells.

It has been demonstrated that paracrine activation of the wingless-type (Wnt)/β-catenin pathway mediated by estrogen and progesterone has a critical role in enhancing the growth and proliferation of leiomyoma stem cells [19]. The presence of steroid hormones stimulates the secretion of Wnt ligands from mature myometrium or leiomyoma cells. This induces the nuclear translocation of β-catenin in leiomyoma stem cells, leading to the expression of genes with a critical role in UFs growth and development. This pathway can stimulate the expression of transforming growth factor-β3 (TGF-β3), which induces fibronectin (an ECM protein) expression, cell proliferation, and extracellular matrix accumulation [4, 20] (see below). Then, the development of clinical disease is dependent on a paracrine mechanism and a multistep process from transformation to the fibroid progenitor through to growth acceleration. Even though some cell-related events have been identified, the exact cell of origin of uterine leiomyomas remains unknown.

3. The conception of UFs as a fibrotic/inflammatory disease: the role of myofibroblastic transformation

It is well accepted that menstruation, ovulation, and parturition represent physiological injury that triggers an inflammatory reaction of the uterus, then, demanding tissue repair and remodeling. It has been hypothesized that UFs could be the consequence of an improper inflammatory response and fibrosis development mediated by myofibroblasts [6, 17, 21, 22].

A myofibroblast is a cell phenotype that has the characteristics of a fibroblast and smooth muscle cells and is characterized by the expression of α-smooth muscle actin (α-SMA); therefore, it is a nonmuscle contracting cell [23, 24]. Myofibroblasts drive connective tissue remodeling by combining ECM synthesizing features of fibroblasts with the cytoskeletal characteristics of contractile smooth muscle cells and have been observed in several fibrotic diseases, including systemic sclerosis, glomerulosclerosis, idiopathic pulmonary fibrosis, and liver cirrhosis [24, 25]. Chronic inflammation plays a critical role in fibrosis and tumorigenesis. The pathophysiology of UFs is the same as that of other fibrotic conditions, in which an injury triggers normally quiescent cells to dedifferentiate into a myofibroblast-like, more proliferative phenotype [25].

Myofibroblasts are activated by inflammation, tissue injury, mechanical forces, hypoxia, and oxidative stress. Once activated, myofibroblasts proliferate and produce ECM proteins in the process of wound healing. After finishing this role in tissue repair, these specialized cells lose their contractile activity, decrease α-SMA expression, and disappear by apoptosis [6]. On the other hand, their inappropriate function has been shown to cause fibrosis, creating a collagenous and stiff scar, such as how it occurs in keloids [17, 26]. In the uterus, myofibroblastic transformation could occur from smooth muscle cells, connective tissue fibroblasts, vascular smooth muscle cells, or direct myofibroblastic differentiation of SSCs [17, 27]. Furthermore, the presence of myofibroblasts in UFs has been well documented [6]; then, UFs can be considered as a fibrotic disorder, sharing several characteristics with those of keloids [17].

Cellular transformation into a myofibroblastic phenotype is key to the establishment and progression of fibrogenesis [28]. Several studies have documented a pivotal role of inflammation and myofibroblastic transformation in the pathogenesis of UFs, and it is beginning to be accepted that local chronic inflammation generates a microenvironment that fosters the development of UFs [22]. The consideration of UFs as a chronic inflammatory disease is supported by the presence of high expression of inflammatory cytokines in fibroids, including interleukin (IL)-1, IL-6, IL-13, IL-15, tumor necrosis factor (TNF)-α, granulocyte-macrophage colony-stimulating factor (GM-CSF), and erythropoietin [28]. Moreover, Protic et al. [22] showed the presence of many inflammatory cells, such as macrophages, mast cells, and leukocytes, inside UFs and their surrounding tissues when compared to autologous myometrium distant from the tumor.

Moreover, a recent study by Orciani et al. [29] showed a distinct cytokine expression pattern related to chronic inflammation in leiomyoma progenitor cells (leiomyoma stem cells) that could favor a microenvironment suitable for leiomyoma onset and development. In addition, the authors hypothesized that this upregulated cytokine profile could play a role in adverse obstetric outcomes, including infertility, spontaneous miscarriage, and preterm delivery in women with UFs.

Myofibroblastic differentiation can be triggered by multiple cell pathways, environmental cues, physical factors, such an ECM tension, and a variety of inflammatory molecules including cytokines, steroid hormones, and growth factors released locally by adjacent resident cells [6]. Growth factors are usually carried in the ECM. They are stimulated and then released by mechanical stress or proteolytic cleavage. Then, they migrate to bind to membrane receptors [28]. This phenomenon leads to activation of the intracellular complexes that migrate to the nucleus, thus promoting the transcription or repression of target genes that are involved in fibrosis [30].

The central player of myofibroblastic differentiation is transforming growth factor-β (TGF-β). Transforming growth factor-β1 (TGF-β1) activates fibroblasts by starting α-SMA synthesis leading to myofibroblastic differentiation [31]. This molecular marker, which is not present in a fibroblast, is characteristic of the new myofibroblastic phenotype and stimulates the contractile properties of myofibroblasts [32]. Expression of α-SMA is a highly regulated process and depends on the presence of TGF-β1 and Activin-A [25].

Although most women experience causes of uterine inflammation, such as reproductive events, they all do not have UFs. This observation suggests that the initiation of myoma development does not solely depend on inflammation. There are other factors that may influence the risk of developing UFs under the chronic inflammatory condition.

4. Genetic aberrations and myoma

A genetic predisposition to UFs appears to be present as a family predisposition has been shown [33]. There is about a 2.5-fold increased risk of developing myomas in first-degree relatives of women with these tumors. Moreover, the diagnosis of UFs is almost twice in monozygotic twins compared to dizygotic twins [34]. Cytogenetically, most of the UFs (60%) are chromosomally normal [35]. However, several recurrent genetic aberrations, related to a variety of tumor-promoting functions, such as DNA repair, apoptosis regulation, modulation of epithelial-mesenchymal transition, and formation of fibroid-like tissues, have been identified in uterine fibroids [5]. Different rates of growth can reflect the different chromosomal abnormalities present in individual tumors. Current research has revealed the existence of four subgroups of UFs, depending on somatic mutations or chromosomal alterations [4]:

4.1 Somatic mutation in the mediator complex subunit 12 gene (MED12)

Somatic mutation in the mediator complex subunit 12 gene (MED12) is the most frequent mutation found in uterine myomas (70%) and it has been identified only in leiomyoma stem cells but not in normal myometrial stem cells [9, 36]. Therefore, it has been hypothesized that at least one genetic hit may transform a myometrial stem cell into a TIC, which then interacts with the surrounding myometrium to give rise to a UF [5]. However, the role of these mutations is not fully understood. Distinct MED12 somatic mutations have been detected in different fibroid lesions in the same uterus, but not in all [10, 13]. MED12 gene encodes one of the components of the mediator complex, which is a group of 26 proteins that work together to regulate gene activity. Mediator complex acts as a transcriptional regulator that bridges DNA regulatory sequences to the RNA polymerase II initiation complex. MED12 protein is part of several chemical signaling pathways involving cell growth, migration, and differentiation. Aberrant function of MED12 contributes to tumorigenesis, and it is believed that MED12 mutation is a driver for stimulating the development of uterine fibroids and genomic instability [10]. In addition, MED12 deficiency inhibits TGF-β signaling by reducing activation of Wnt/β-catenin pathway [37, 38, 39, 40]. MED12 mutations have been suggested as a causal agent of UFs [36]; however, direct supportive cause and effect evidence remain lacking.

4.2 Chromosomal rearrangements in high mobility group AT-hook 2 (HMGA2) gene

Chromosomal rearrangements in high mobility group AT-hook 2 (HMGA2) gene have been identified in 7.5–10% of UFs, and these are the second most common genetic alterations in these tumors [41]. HMGA protein functions as an architectural factor and contains structural DNA-binding domains and may act as a transcription-regulating factor. With few exceptions, HMGA2 is expressed in human only during early development and is reduced to undetectable levels of transcription in adult tissues. The expression of HMGA2 in adult tissues is associated with modulation of epithelial-mesenchymal transition, mesenchymal differentiation, tumor formation, and certain characteristic cancer-promoting mutations, such as inactivation of p53-induced apoptosis. Overexpression of HMGA2 in myometrial cells may lead to abnormal growth of the myometrial stem cell niche, resulting in fibroid-like tissues [10]. It has been postulated that MED12 and HMGA2 mutations were mutually exclusive in fibroids [42], suggesting distinct tumorigenic pathways. However, a recent study reported simultaneous alterations in both genes in 50% of the tumors. The authors concluded that HMGA2 and MED12 alterations frequently coexist in UFs [43].

Although alterations in MED12 and HMGA2 have been postulated as the initial insult leading to unregulated cell growth, and there is evidence that such alterations support leiomyoma stem-cell self-renewal and cell proliferation, it is unclear whether these genetic alterations cause the transformation of a myometrial stem cell or simply support already existing leiomyoma stem-progenitor cells.

4.3 Hereditary leiomyomatosis and renal cell carcinoma

Most cases of uterine myomas are sporadic in nature; however, there are a few rare familial disorders that are accompanied by the development of uterine fibroids through genetic disorders. Hereditary leiomyomatosis and renal cell carcinoma are autosomal dominant syndromes with both cutaneous and uterine leiomyomas [44]. The risk of renal cell carcinoma and that of leiomyosarcoma are increased in this syndrome. The gene involved is fumarate hydratase (FH), coding for an enzyme involved in Krebs cycle. FH acts as a classic tumor suppressor; thus, inactivating mutations of FH significantly increase the risk of fibroids in the uterus and other tissues. Mutations in FH are mutually exclusive to MED12 and HMGA2 mutations [45].

4.4 The Alport syndrome-diffuse leiomyomatosis association

The Alport syndrome-diffuse leiomyomatosis association can be defined as a hereditary disease of type IV collagen combining features of Alport syndrome (hematuric nephropathy, deafness, and ocular abnormalities) and diffuse leiomyomatosis of the respiratory, gastrointestinal, and genitourinary tracts. The disease is caused by mutations of both the COL4A5 and COL4A6 genes [46].

Other recurrent genetic aberrations and chromosomal rearrangements have been identified in sporadic fibroids, such as trisomy of chromosome 12, deletions in 7q, monosomy of chromosome 22, and RAD51 gene (a DNA repair protein), among others [10, 11]. These alterations might have a common origin through a single genomic event that results in multiple chromosomal breaks and random reassembly (the so-called chromothripsis). It has been proposed that tumorigenesis occurs when tissue-specific tumor-promoting changes are formed through these events [11].

Although the aforementioned cytogenetic alterations are thought to start tumorigenesis via clonal proliferation, they are considered insufficient for tumor development. Therefore, alterations in several complex signaling pathways have been postulated to play an additional role in the pathogenesis of UFs.

5. Signaling pathways involved in myoma growth and development

Several complicated cellular processes, involving different signaling pathways, are thought to play a pivotal role in the pathogenesis of UFs. Currently available knowledge has identified that several of these pathways converge/interconnect/overlap in a summative way; they can act as signal integrators and incorporate inputs from other molecular pathways. This “crosstalk” between signaling pathways makes the understanding of myoma biology, and the development of targeted therapies even more difficult. The physiology and detailed description of these pathways are beyond the scope of this chapter; however, they have been well described elsewhere [47, 48].

5.1 Receptor tyrosine kinases

Receptor tyrosine kinases (RTKs) are cell-surface growth factor receptors. Generally, when growth factors bind to RTK, receptor phosphorylation (activation) leads to downstream activation of several pathways. Therefore, RTKs are important regulators of important cellular processes, such as differentiation, proliferation, and survival. There is a growing body of evidence for the role of RTKs in pathophysiology of UFs, mediating cell-to-cell interactions, and the development of an adequate tumoral microenvironment by increasing expression of the RTK downstream effectors: the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR pathways) [47, 48]. Interestingly, previous data show that RTKs are also intermediate factors for sex steroid effects since estrogens upregulate RTKs, suggesting a crosstalk between both pathways in UFs [49, 50, 51].

5.2 MAPK/ERK pathway

The MAPK/ERK pathway (also known as the Ras/Raf/MEK/ERK pathway) is a chain of proteins that communicate signals from surface-cell receptors to the DNA in the nucleus. This pathway regulates several critical cell processes, such as cell proliferation, survival, and apoptosis. They included several subfamilies, such as extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs), and p38 mitogen-activated protein kinases (p38 MAPK), which have been linked to leiomyoma biology [47]. The binding of growth factors to their receptors (RTKs) leads to a cascade of molecular events that include sequential phosphorylation (activation) of Ras, Raf, MEK, and ERK proteins. In turn, ERK activates several transcription factors. This pathway has been shown to be upregulated in UFs. Moreover, it has complex and bidirectional interaction with steroid hormone-induced signals. For example, estrogen can activate this pathway through G-protein-coupled receptors and growth factors can modulate the response to steroids of MAPK/ERK through effects of MAPK/ERK on transcriptional activity of steroid receptors [47].

5.3 PI3K/Akt/mTOR pathway

The phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway is another RTK-ligand activated intracellular signaling pathway important in regulating the cell cycle. Therefore, it is directly related to cellular quiescence, proliferation, and longevity. In addition to RTK-ligand binding, PI3K can be activated by G-protein-coupled receptors and membrane-bound steroid receptors. To date, several studies have demonstrated aberrant PI3K/Akt/mTOR signaling in UFs [47].

5.4 Smad protein signaling

Smads are intracellular proteins that transmit signals from several cell membrane receptors to the nucleus. They are the main signal transducers for receptors of the TGF-β superfamily, including Activin-A, which are critically important for regulating cell development and growth [52]. In vertebrates, eight Smads have been identified and named Smad1 to Smad8 and they are divided in three distinct subtypes: receptor-regulated Smads (R-Smads), common partner Smads (Co-Smads), and inhibitory Smads (I-Smads). Smads exert their function by forming heterotrimers of two R-Smads and one Co-Smad that act as a transcription factor. Smad4 is the only Co-Smad described in humans. Upon ligand binding to Smad-coupled receptors, two R-Smads are phosphorylated and then heterotrimerize with one common Co-Smad (Smad4). The resulting complex translocates to the nucleus to act as a transcription factor for target genes [52]. There is evidence that UFs demonstrate aberrant Smad signaling, and that several molecules with a pivotal role in leiomyoma pathophysiology, including TGF-β, Activin-A, and myostatin, converge on Smads to regulate cellular proliferation and ECM formation at the transcriptional level [47, 52].

5.5 Wnt/β-catenin pathway

The wingless-type integration site (Wnt) family are a group of signal transduction pathways involved in several cellular functions, such as cell proliferation and differentiation, apoptosis, cell migration, and stem cell maintenance in adults. β-Catenin is a dual protein involved in the regulation and coordination of cell-cell adhesion and gene transcription. β-Catenin acts as an intracellular signal transducer in the Wnt signaling pathway [53].

Wingless-type proteins function as ligands and bind to specific receptors. When Wnt binds to its receptor, a stable β-catenin detaches from the β-catenin destruction complex and is free to translocate to the nucleus to alter gene transcription by acting as a transcriptional coactivator (the so-called canonical Wnt pathway). However, in the absence of Wnt activation, the unstable β-catenin binds to the protein destruction complex and is degraded through ubiquitination [53].

The activation of canonical Wnt pathway (β-catenin-dependent) stimulates the differentiation of resting fibroblasts into myofibroblasts and has a key role in fibrotic processes, such as myoma development [54]. In a recent article, El Sabeh et al. [53] reviewed the role of Wnt/β-catenin signaling pathway in uterine leiomyoma biology. Interestingly, evidence suggests that MED12 mutations, which seem to drive fibroid formation (see above), are implicated in the regulation of the Wnt/β-catenin pathway. It was shown that β-catenin targeted the MED12 to activate transcription. Moreover, the inhibition of the β-catenin/MED12 interaction suppressed β-catenin activation in response to Wnt signaling [39]. Since MED12 was shown to be essential for canonical Wnt signaling and MED12 limits β-catenin-dependent growth during mouse embryonic development, it has been postulated that MED12 mutations resulting in absent or defective MED12 can lead to a β-catenin pathway-dependent growth [53]. Indeed, fibroids with MED12 mutations have increased levels of Wnt4 ligand compared with those without these mutations [38]. Similarly, in a recent study using an immortalized human uterine myometrial smooth muscle cell line, the overexpression of mutant MED12 resulted in increased expression of Wnt4 and β-catenin when compared to the cells with the overexpression of wild-type MED12 [55]. Likewise, estrogen and the interaction of Wnt4/β-catenin pathway along with TGF-β might explain the enhanced growth observed in fibroids with MED12 mutations [4]. Furthermore, it has been shown that TGF-β activates canonical (β-catenin-dependent) Wnt signaling and that activation of Wnt canonical signaling is required for TGF-β-mediated fibrosis [54]. In this context, Wnt pathway could provide a putative mechanism by which myometrial stem cells stimulate transformation into TICs.

As previously described, paracrine activation of the Wnt/β-catenin pathway, mediated by estrogen and progesterone, induces the nuclear translocation of β-catenin in leiomyoma stem cells [19], leading to the expression of genes with a critical role in UFs growth and development. Therefore, it has been accepted that sex steroid-induced proliferation in UFs is modulated, at least in part, through Wnt expression by mature cells and its paracrine response to β-catenin signaling in leiomyoma stem cells.

In addition, Wnt/β-catenin pathway has several crosstalks with other signaling pathways involved in UFs biology, such as PI3K/Akt/mTOR, Ras/Raf/MEK/ERK, insulin-like growth factor (IGF), and sex steroid pathways [53].

5.6 Other pathways

Aberrations in other pathways have been described in UFs, such as peroxisome proliferator-activated receptors (PPARs) and retinoic acid signaling pathways [47, 48]. Briefly, both pathways are involved in cell growth, development, and differentiation by binding nuclear receptors and finally regulating gene transcription. Interestingly, retinoic acid receptors (RARs and RXRs) act as heterodimers for several nuclear receptors, including vitamin D receptor (VDR), while PPARs act as a thyroid hormone receptor [47]. PPARs form heterodimers with RXRs to regulate gene transcription. Interestingly, retinoids might be linked to ethnic heterogeneity observed in UFs since black women have different expression profiles for RARs and RXRs compared with white women [56]. There is evidence that both pathways have an inhibitory role in leiomyoma cell proliferation. Of note, peroxisome proliferator-activated receptor gamma (PPARγ) stimulation leads to inhibition of estrogen-mediated gene expression [47].

In summary, molecular events in myoma biology are regulated through a complex network of interconnected and convergent, intracellular, extracellular, and intercellular pathways. Each pathway does not work on its own but it is a complicated network of molecular processes, although several signal transduction pathways converge into a final pathway.

6. Epigenetic modulation in myoma development

Epigenetics is defined as all heritable changes in gene expression (phenotype) that are not coded in the DNA sequence. Therefore, these changes are mediated by altered gene expression [41]. Unlike genetic changes, epigenetic changes are reversible and do not change the DNA sequence, but they can change how the DNA sequence is “read.” Epigenetic changes affect gene expression by silencing (switching off), switching on, and stabilizing genes. Environment and behaviors, such as diet and exercise, can result in epigenetic changes. Epigenetic changes affect gene expression in different ways. In humans, three main epigenetic mechanisms play a role in modulating gene expression in fibroid formation:

6.1 DNA methylation and demethylation

DNA methyltransferases (DNMTs) are enzymes that catalyze the transfer of a methyl group to DNA. Typically, methylation in the promoter region is associated with suppression of gene expression. By contrast, demethylation tends to activate gene transcription [57]. Li et al. [58] found global DNA hypomethylation and decreased expression of DNMTs (DNMT3A and DMT3B) in uterine fibroids when compared to the adjacent myometrium, suggesting an epigenetic mechanism in leiomyoma development. A potential link between estrogen receptor-α gene hypomethylation and uterine fibroid formation has also been postulated [59]. Other studies have investigated the role of DNA hypermethylation in silencing tumor suppressor genes in the pathophysiology of uterine fibroids [60, 61]. Navarro et al. [61] found 55 genes with differential promoter methylation and concomitant differences in messenger RNA (mRNA) expression in uterine leiomyoma versus normal myometrium. Eighty percent of the identified genes showed an inverse relationship between DNA methylation status and mRNA expression in uterine leiomyoma tissues, and most genes (62%) displayed hypermethylation associated with gene silencing. Interestingly, they found hypermethylation, mRNA repression, and protein expression of known tumor suppressor genes in uterine leiomyomas, suggesting a possible functional role of promoter DNA methylation-mediated gene silencing in the pathogenesis of UFs [61].

6.2 Histone modification

Histone modification is the second most important epigenetic factor that has a critical role in the regulation of gene expression. Histones are proteins that act as spools around which DNA winds to create structural units called nucleosomes. Histones prevent DNA from becoming tangled and protect it from DNA damage. Histones can be modified in many ways in their N-terminal tail, including acetylation, phosphorylation, and methylation, among others. Histone methylation can determine either activation or repression of gene transcription; instead, histone acetylation determines gene activation [17]. Wei et al. [62] studied the histone deacetylase 6 (HDAC6) expression and its pathogenic role in uterine leiomyoma development. They found a regular pattern of increasing HDCA6 and estrogen receptor-α-gene expression in leiomyoma samples.

6.3 Noncoding RNA (MicroRNA)

MicroRNA (miRNA) are a novel class of small nonprotein coding, single-stranded RNAs that regulate a high number of biological processes and their related pathways, such as gene expression via gene silencing with either translational repression or degradation of mRNAs. However, more recent studies have demonstrated that some miRNAs can upregulate target genes by directly binding to their promoter [57]. Current research has proposed the role of alterations in miRNA levels in tumorigenesis. In a recent comprehensive review by Ciebiera et al. [63], the current knowledge about the role of different miRNA families in the biology of UFs is summarized. In this paper, the authors provide updated information about microRNA families and their predicted target genes that are significantly dysregulated in uterine leiomyomas compared with normal myometrium. This dysregulation affects several leiomyoma-involved signaling pathways, including MAPK/ERK, Wnt, janus kinase (JAK)-signal transducer and activator of transcription (STAT), and TGF-β [57]. The implications of such alterations affect several processes, such as tumor senescence, angiogenesis, inflammation, ECM accumulation, cell proliferation, and apoptosis [63].

Peng et al. [64] demonstrated the interaction between HMGA2 and lethal-7 (let-7) miRNAs. The repression of HMGA2 via let-7 s inhibited cellular proliferation in vitro. In the same report, microRNA profiling analysis revealed that let-7 s were significantly dysregulated in uterine leiomyomas: high in small leiomyomas (<3 cm) and lower in large leiomyomas (>10 cm). The authors hypothesized that Let-7-mediated repression of HMGA2 mechanism can be an important molecular event in leiomyoma growth.

The microRNA-21 (miR-21) family has been implicated in excessive ECM formation by stopping the Smad7 protein, an inhibitory Smad that inhibits TGF-β pathway [48]. Furthermore, the expression of transforming growth factor beta receptor 2 (TGFβR2) is the target of miR-21 in UFs, so it may mediate its biological activities by binding to TGF-β receptors [63].

The importance of the miR-29 family is one of the best known in UFs since the members of this family target several ECM-related genes. The overexpression of miR-29 s decreases the production of ECM compounds, and its expression is lower in UFs compared to normal myometrium. Interestingly, several observations have confirmed that sex steroids can downregulate miR-29 s and, as a result, upregulate collagen expression [65, 66].

A detailed description of miRNA families and their biological actions involved in UFs pathophysiology is beyond the scope of this chapter and can be found elsewhere [63, 67].

7. Growth factors

Growth factors (GFs) are proteins or peptides, produced locally by smooth muscle cells and fibroblasts and secreted into extracellular space, that take part in several cellular events, like proliferation, growth, ECM synthesis, and angiogenesis, important for leiomyoma growth [68]. GFs act over short distances either in an autocrine and/or paracrine manner and exert most of their effects on target cells by interacting with specific cell-surface receptors, with subsequent signaling transmission via signal transduction systems in the cell [28, 30]. Therefore, overexpression of either GFs or their receptors plays an important role in tumorigenesis. Aberration of several growth factors and their receptors or signaling pathways has been implicated in myoma pathophysiology, such as TGF-β, Activin-A, myostatin, epidermal growth factor (EGF), heparin-binding EGF (HB-EGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), fibroblast growth factor 1 (FGF-1), and fibroblast growth factor 2 (FGF-2) [68]. All the above-cited GFs are ligands of RTKs and activate two critical signaling cascades such as MAPK/ERK and PI3K/Akt/mTOR pathways [48]. On the other hand, members of the TGF-β superfamily (Activin-A and myostatin) act through serine/threonine kinase receptors/Smad pathway [30], whereas TGF-β is able to activate both MAPK/ERK and serine/threonine kinase receptors/Smad pathway [17]. In this chapter, we will discuss the most researched topics and those considered the most relevant for myoma development.

7.1 Transforming growth factor-β (TGF-β)

Transforming growth factor-β are peptides that regulate ECM production as well as cell growth and differentiation and it has three different isoforms (TGF-β1, transforming growth factor-β2 (TGF-β2), and TGF-β3). It is produced by many cell types, including macrophages and myofibroblasts. TGF-β signaling has been found to be involved in diseases with abnormal ECM production and fibrosis development and there is a growing body of evidence for a role of abnormal TGF-β signaling pathways in UFs [69, 70]. Moreover, expression of TGF-β is stimulated by the Wnt/β-catenin pathway, and several studies have demonstrated a crosstalk between Wnt/β-catenin and TGF-β pathways [5, 37, 53, 54]. TGF-β shows bimodal effects on cell proliferation, induces elevated production of ECM-related genes, and decreases production of ECM degradation-related genes [17]. In addition, it can either inhibit or stimulate the proliferation of human smooth muscle cells in a dose-dependent manner. At low concentrations, TGF-β induces cell proliferation by stimulating autocrine PDGF secretion, whereas it induces the opposite effect at higher concentrations via downregulation of the PDGF receptor and by direct inhibition [17, 30]. Interestingly, the most studied pathway of myofibroblast formation is TGF-β1-dependent differentiation from fibroblasts [71]. TGF-β is induced by mechanical tension and induced α-SMA expression. The expression of ECM proteins, fibronectin and fibromodulin (a collagen-binding protein), is also stimulated by TGF-β [70]. It has been shown that TGF-β receptor type 2 and 1(TGFβR2 and TGFβR1, respectively) expression is elevated in leiomyoma compared to myometrium [68]. The most common isoform found in mesenchymal cells is TGF-β3, and studies have shown that the TGF-β3 level is three-to fivefold increased in fibroids as compared with normal myometrium [69]. Moreover, it has been shown that TGF-β3 induces collagen and versican expression in leiomyoma cells and directly stimulates myometrial and leiomyoma cell proliferation [68, 70]. Although most studies on the role of TGF-β in UFs pathophysiology have focused on its role in fibrosis and ECM production, TGF-β is also related to UFs pathogenesis through its angiogenic effects [72].

From the clinical point of view, TGF-β3 regulation of bone morphogenetic proteins (BMPs), another ligand member of the TGF-β family, has been implicated in myoma-related symptoms, such as defective endometrial decidualization, impairment of endometrial receptivity, infertility, and excessive uterine bleeding due to suppression of the expression of local anticoagulant factors (such as plasminogen inhibitor activator 1, antithrombin III, and thrombomodulin) [37, 73]. TGF-β1 and TGF-β3 are considered as key players in excessive ECM accumulation and fibrosis observed in UFs, and both molecules exert their functions, such as myofibroblastic transformation, ECM remodeling, regulation of the inflammatory response and fibrosis promotion, at least in part, through activation of the MAPK/ERK and Smad 2/3 signaling pathways [17, 68].

7.2 Activin-A

Activin-A, a growth factor member of the TGF-β superfamily, has been proven to have an inflammatory and profibrotic role via binding to type II (activin receptor type IIA (ActRIIA), activin receptor type IIB (ActRIIB)) and type I (type I activin receptor (ActRIB), activin receptor-like kinase 4 or ALK4) [25, 68]. Activin-A has been shown to be more highly expressed in leiomyomas than normal myometrium, whereas the levels of its receptors (ActRIIA, ActRIIB, ALK4) remain unchanged [68]. A recent review postulates that Activin-A could be produced by the endometrium and myometrium as a response to physiological inflammation processes such as ovulation, menstruation, or parturition and that it can exert its actions by an autocrine/paracrine mechanism [25]. Other published study by the same authors confirmed the presence of Activin-A positive macrophages inside uterine leiomyoma tissue, demonstrating another source of Activin-A in UFs [22]. In addition, in vitro studies have shown that stimulation of primary myometrial and leiomyoma cells with TNF-α results in increased mRNA expression levels of Activin-A in these cells. Besides, it has been shown that local production of Activin-A by myometrial and endometrial cells favors the macrophage proinflammatory phenotype via positive feedback mechanism and that such mechanism could support the maintenance of an inflammatory environment inside the leiomyoma tissue [22, 25]. It has been hypothesized that Activin-A plays a central role in coordinating inflammation and myofibroblastic transition in myoma development and growth. Activin-A increases mRNA levels of several ECM components, including type I collagen, fibronectin, and versican, in leiomyomas compared to adjacent myometrium. Furthermore, Activin-A activates Smad 2/3 signaling pathway regulating gene transcription and contributing to fibrosis. Like TGF-β, Activin-A may also activate non-Smad pathways [25, 52].

7.3 Fibroblast growth factors

Two members of the FGF family have been implicated in myoma biology, particularly inducing angiogenesis. FGF-1, also known as acidic fibroblast growth factor (aFGF), and FGF-2, also known as basic fibroblast growth factor (bFGF) [30, 68]. Leiomyomas show an increased protein expression of both FGF-1 and FGF-2 compared with normal myometrium. Of note, FGF-2 was primarily bound to the ECM of myometrium and fibroids. This observation suggests that the enhanced growth of leiomyomas may be due, in part, to the presence of large amounts of FGF-2 that are stored in the ECM of these tumors [68]. There is evidence that VEGF acts in synergy with FGFs and can release bFGF from the ECM [41]. Furthermore, FGF receptor 1 (FGFR-1) and 2 (FGFR-2) expressions are increased in leiomyoma compared with adjacent myometrium. Interestingly, FGFR-1 expression is suppressed in early luteal phase in normal women, but not in women with leiomyoma-related bleeding [74]. These findings support the role of the FGF-2 ligand-receptor system in the pathogenesis of leiomyoma-related bleeding. Interestingly, despite their higher concentration, leiomyoma cells are less responsive to FGF-2 mitogenic effect compared to normal myometrial cells [68].

8. Endocrine-disrupting chemicals and fibroids

Endocrine-disrupting chemicals (EDCs) are chemicals that intervene with the endocrine system and may lead to reproductive, immune, and neurological adverse effects. Environmental exposure during critical periods of development alters the programming of normal physiologic responses, therefore, causing the rate of development of the disease to increase later in life, also known as developmental programming [10, 75]. Interaction of EDCs with nuclear receptors can change hormone function by mimicking hormone function, blocking the endogenous hormone from binding, or interfering with the production or regulation of hormones and/or their receptors. An individual EDC can interact with more than one receptor, and several EDCs can interact with the same receptor, highlighting the complex response to environmental EDC exposure [10]. Uterine development may be a particularly sensitive window to environmental exposures, as some perinatal EDC exposures have been shown to increase tumorigenesis in both experimental and human epidemiologic studies. There is a positive association between developmental diethylstilbestrol (DES) and UFs risk [75]. In addition, perinatal exposure to genistein and DES has shown to increase the penetrance and growth of UFs in animal studies [75]. The mechanisms by which EDC exposures may increase tumorigenesis are still being elucidated, but DNA instability, epigenetic mechanisms, reprogramming of the developing uterus estrogen-responsive gene expression, and stem-cell disruption that could change uterine sensitivity to steroid hormones in adulthood are emergent hypothesis [10, 75].

9. Extracellular matrix in uterine fibroids

It is universally accepted that a myriad of chemical signaling molecules has been identified in UFs and that sex hormones orchestrate the complex systems leading to fibroid development. However, these chemical and hormonal signals are only a part of the biological mechanism of myoma development, and these signals alone do not fully account for fibroid development and growth.

Excessive ECM accumulation is considered critical for myoma development and appears to play a crucial role in the formation of the bulky and stiff structure, and their associated symptoms such as excessive uterine bleeding and pelvic pain or pressure [6]. This accumulative process is due to an imbalance between synthesis and dissolution.

Most of the ECM components are secreted by fibroblasts and myofibroblasts; it has been suggested that both quantity and topology of the ECM are altered in UFs compared with the myometrium [6, 76]. Current knowledge supports the fact that the stiffness of fibroid tissue has a direct effect on the growth of the tumor through the induction of fibrosis. Fibrosis has two characteristics: (I) resistance to apoptosis leading to the persistence of fibroid cells and (II) secretion of collagen and other components of the ECM such as proteoglycans (PGs) by those cells leading to abundant disposition of highly crosslinked, disoriented, and often widely dispersed collagen fibrils [77].

In UFs, ECM accumulation is affected by growth factors (TGF-β, Activin-A, and PDGF), cytokines (TNF-α), steroid hormones (estrogen and progesterone), and miRNAs [6]. Furthermore, ECM serves as a reservoir for those growth factors and cytokines and often activation of these factors occurs in the ECM. In addition to growth factors’ sequestration, excessive ECM contributes to initiate solid-state signaling [77].

Solid-state signaling, also called mechanotransduction, occurs when mechanical stress, such as osmotic pressure, shear stress, surface tension, and tensional forces, triggered by the fibroid growth, is converted to biochemical cell signals [76, 77]. Mechanotransduction is reported to have a significant role in both embryogenesis and tumorigenesis by regulating signaling pathways and gene expression [76]. The ECM and the cell communicate with each other and adapt to mechanical stimuli by an ongoing bidirectional process known as dynamic reciprocity. This process is facilitated with the help of specialized molecules such as ion channels, surface receptors, integrins, and the cytoskeleton [76, 77]. The activation of downstream mechanical signaling pathways can alter gene expression, leading to changes in ECM density, composition, and organization that ultimately affect cell shape and contractility. Mechanical signaling appears to play a crucial role in cell adhesion, migration, proliferation, and survival as well as inflammation and fibrosis [6]. Interestingly, it has been suggested that mechanotransduction system would be able to transfer signal faster than the diffusion-based system [77].

Mechanotransduction occurs at the sites of focal adhesions where heterodimeric integrins (α and β) serve as a molecular bridge between ECM and intracellular molecules/cytoskeleton, acting as mechanosensors [6, 77]. Integrins, especially integrin β1, act as transmembrane adhesion receptors that couple extracellular matrix stresses to the intracellular cytoskeleton to influence their integrity and growth. Integrin β1 has been found to be overexpressed in leiomyoma cells compared to myometrial cells [76]. When activated, integrin β guides the reorganization and polymerization of the actin filaments, further generating mechanical stress. Biochemical pathways downstream of integrin aggregation include the activation of cytoplasmic tyrosine kinases, such as focal adhesion kinase (FAK), Rho family of GTPases (Rho GTPases)/Rho-associated kinases (ROCK)/ERK/p38 MAPK signaling cascade, involved in gene expression, cell cycle regulation, and ECM remodeling [6, 76, 77, 78].

The imbalance in the deposition and degradation of an abnormal and fibrotic ECM increases mechanical stress transmitted to cells [76]. An important concept is that reciprocity is an indispensable characteristic of mechanosensitive cells. Cells sense the mechanical force from a newly generated altered ECM and further activate mechanical signaling that results in altered cell behavior and ECM remodeling [76, 77]. It has been demonstrated that UFs cells respond to the increased stress by changes in the organization of the intracellular structures, fibrosis, and deposition of excess ECM.

Therefore, fibroid cells exist in a state of increased mechanical stress due to excessive ECM and fibrosis; however, they respond inadequately, with an abnormal orientation of actin fibers, abnormal collagen, angular shape, and distortion of the nuclear envelop. These findings highlight the fact that mechanical signaling is altered in UFs [77, 78].

In a recent study, Ko et al. [79] showed that primary fibroid cells expressed higher levels of β-catenin (see above) when cultured on stiffer surfaces, highlighting the biomechanical cues influencing β-catenin expression. Interestingly, β-catenin signaling occurred independently of MED12 mutations, but was instead induced by the ECM stiffness, probably through the integrin-FAK pathway [79]. While mechanotransduction has been suggested as an important signaling pathway in UFs, the interaction between mechanotransduction and Wnt/β-catenin signaling pathways in UFs is not clearly elucidated.

The normal myometrium is composed of smooth muscle cells separated by ECM. The ECM is composed of collagen, proteoglycans, glycosaminoglycans, and elastin. Several studies comparing the differences between the normal myometrium and fibroids have demonstrated differences in ECM composition [6, 17].

Collagens are the major component of ECM that contributes to the stability and maintenance of structural integrity of the tissues. In addition to their role in wound healing and fibrosis, collagens are known to regulate cell migration, proliferation, differentiation, and survival by signaling through cell-surface receptors, such as integrins. Several collagen alterations, such as abnormal fibril structure and orientation, have been identified in UFs [17, 76, 77]. Likewise, leiomyoma cells have an abnormal expression of collagen subtypes, with overexpression of type I, III, and V collagens compared to the adjacent myometrium [17, 76]. It has been demonstrated that the expression of collagen in leiomyoma cells is regulated by TGF-β [76]. Moreover, Koohestani et al. [80] demonstrated a direct effect of ECM collagens on the proliferation of leiomyoma smooth muscle cells through interplay between the collagen matrix and the PDGF-stimulated MAPK/ERK pathway.

Proteoglycans are another important constituent of the ECM. They are composed of a protein covalently bound to negatively charged glycosaminoglycans. Proteoglycans have been recognized not only to play a part in providing shape and biomechanical strength of tissues, but also to exhibit direct and indirect cell signaling properties. PGs interact with other ECM components, such as collagen, fibronectin, laminin, growth factors (such as TGF-β), and cytokines and through these interactions have shown to have a role in cell proliferation and differentiation [76, 77].

Versican is a large chondroitin sulfate proteoglycan that plays an important role in cell migration, adhesion, proliferation, and inflammation. The expression of versican was reported to be elevated in UFs. This increase could contribute to disorganization of ECM, increased tumor bulk, and stiffness of the fibroid, which would eventually lead to increased mechanical stress [76]. It was further demonstrated that versican expression is regulated by TGF-β3 [76]. Decorin is a small dermatan sulfate proteoglycan that regulates matrix assembly by binding to fibronectin and collagen via its core protein whose presence has been correlated with fibroid size [77]. Available evidence suggests that UFs contain less decorin than normal myometrial tissue [6]; however, it has a modified structure in UFs compared to normal myometrium (higher molecular weight). These features of decorin could contribute to increase osmotic pressure within the fibroid tissue [77]. Since decorin acts as an antagonist of TGF-β signaling, this increased TGF-β signaling may promote fibrosis [6].

Dermatopontin is an extracellular protein that binds to decorin and is involved in the formation of collagen fibrils. The expression of dermatopontin is decreased in myoma compared to normal myometrium [17]. Interestingly, in humans, both keloids and leiomyoma show a decreased expression of dermatopontin building a molecular link between these two fibrotic conditions [17]. There is evidence that ECM accumulation in UFs is due to an imbalance between synthesis and dissolution, like disordered wound healing observed in keloid formation.

Overall, the abnormal collagen, along with excess deposition of hydrophilic proteoglycans, not only contributes to the increase in bulk of the fibroids but also contributes to their stiffness. Paradoxically, UFs can increase in volume by up to 138% in 6 months but have a low mitotic index [4]; therefore, this accelerated growth is thought to be due to changes in the regulation of ECM components rather than cell proliferation. Leiomyoma cells exist in a state of hyperosmolarity and respond to the osmotic stress by regulating the expression of osmotic stress genes. The expression of such genes is increased in UFs, demonstrating the critical role of water homeostasis and osmotic stress in increasing fibroid stiffness [76, 77].

Other factor that plays an important role in myoma growth and regression is ECM remodeling process. This process is regulated by the combined action of matrix metalloproteinases (MMPs) that are responsible for the degradation of ECM, and the tissue inhibitors of MMPs (TIMPs) that are the physiological regulators of MMPs. The available evidence suggests that a dysregulation of MMPs and TIMPs could play a critical role in the development of a more fibrous ECM in UFs. Notably, several forms of MMPs and TIMPs are expressed differentially in UFs and normal myometrium [6]. The current research suggests that MMPs participate in other physiological processes, such as cell migration, differentiation, angiogenesis, and apoptosis [6]. Moreover, they can directly or indirectly affect the functions of various cytokines that play roles in inflammation and repair processes including interferon-β (IFN-β) and TGF-β1, among others. Likewise, MMPs enable proteolytic release of several growth factors with mitogenic properties, such as FGFs and TGF-β [6]. Interestingly, TGF-β is significantly elevated in UFs, and studies have shown a TGF-β-induced fibrotic transformation of normal myometrial cells along with decrease in the expression of MMPs [6].

Moore et al. [81] investigated the interactions between human uterine leiomyoma cells and uterine leiomyoma-derived fibroblasts, and their importance in cell growth and ECM protein production using a coculture system. They showed that uterine leiomyoma-derived fibroblasts can stimulate uterine leiomyoma cell proliferation and enhance the production of ECM proteins with elevated levels of collagen type I, several growth factors, such as platelet-derived growth factor (PDGF)-A, PDGF-B, vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), TGF-β1, TGF-β3, activation of receptor tyrosine kinases (RTKs) of the mentioned growth factor ligands, and TGF-β receptor signaling. These data support that leiomyoma growth may be mediated in part by autocrine and/or paracrine mechanisms and highlight the importance of interactions between fibroid tumor cells and ECM fibroblasts in vivo in promoting growth factor synthesis, activating signaling pathways, and then stimulating tumor cell proliferation and production of ECM proteins [81].

10. The role of hypoxia and angiogenesis in UFs biology

The exact trigger that initiates the fibrotic process in UFs remains unclear. It has been proposed that hypoxia may play a role in triggering uterine stem cells transformation into TICs [17, 28] supporting this hypothesis. Ono et al. [82] reported that myometrial stem cells were able to differentiate into mature myometrial cells only under hypoxic conditions in vitro, suggesting that hypoxia may be the driving force behind their growth and transformation into leiomyoma cells. It is widely accepted that tumor environment in UFs is severally hypoxic [72, 83]. Several studies have confirmed that UFs have an abnormal vasculature. UFs have less vascular density than surrounding myometrium, and this decreased vasculature likely accounts for the severe hypoxia in fibroid tissue [72]. Recent studies have shown that UF is relatively avascular, in contrast to the peri-fibroid tissue [84]. UFs are surrounded by a dense myometrial vascular network. It has been suggested that this vascular capsule region corresponds to the vascular peripheral rim seen on ultrasound [15], and to the plane of tissue dissection during myomectomy.

Normally, angiogenic process involved an interaction between the blood vessels themselves and their surrounding ECM. Ultimately, angiogenesis depends on the balance between angiogenic factors and their inhibitors [72]. To date, it is believed that the unique vascular architecture of UFs, consisting of an avascular surrounded by a well-vascularized capsule, is most likely due to an angiogenic imbalance. Intriguingly, it is well known that the expression of several angiogenic factors is dysregulated in UFs, including EGF, HB-EGF, VEGF, bFGF, PDGF, TGF-β, and adrenomedullin [72]. There are several possible explanations for the diminished fibroid vasculature despite an increased presence of angiogenic factors, including an aberrant hypoxia response, lack of response to angiogenic promoters and overexpression of angiogenic inhibitors [72].

Tissue hypoxia is a potent stimuli for angiogenesis leading to up-regulation of hypoxia-inducible factor-Iα (HIF-Iα), which drives the expression of angiogenic factors and orchestrates the tissue adaptions to hypoxia [72]. Interestingly, HIF-Iα has not been identified in UFs [85]. Therefore, although leiomyoma tissues are hypoxic, and UFs feature down-regulation of key molecular regulators of the hypoxia response. The mechanism responsible for the lack of HIF-Iα up-regulation, which underlies the aberrant hypoxic response in UFs, remains unclear. In addition, the low vascular density observed in UF seems to be the consequence of an inadequate angiogenic response due to this altered hypoxic response [72]. Remarkably, Carmeliet et al. [86] reported that the growth of HIF-Iα-deficient tumors was not retarded but accelerated, owing to decreased hypoxia-induced apoptosis and increased stress-induced proliferation. Taken together, HIF-Iα down-regulation may explain the diminished vasculature and the continuous growth in spite of this.

On the other hand, the lack of response to angiogenic promoters could be attributed to genetic aberrations or abnormal DNA methylation observed in UFs [60]. Alternatively, an anti-angiogenic gene expression profile has been shown in UFs compared with adjacent myometrium [87].

It has been suggested that the vascularized capsule surrounding the avascular fibroid may be due to the stimulatory effects of angiogenic factor secretion by the tumor on the surrounding normal myometrium. Interestingly, Wei et al. [88] described a VEGF concentration gradient in the fibroid, with a steady increase in VEGF expression from the central zone to the periphery of the fibroid. The increased angiogenesis and vascular density observed in the normal myometrium surrounding the fibroid may account for menorrhagia in women with UFs and their tendency to bleed during myomectomy [72].

Other vascular abnormalities have been described in UFs, such as lack of smooth muscle cells and arterial spiraling when compared to normal myometrium, suggesting a vascular maturation defect. In addition, the angiogenic environment within the tumor may also suppress vessel maturation [72].

11. Vitamin D and its role in myoma biology

Vitamin D is a group of fat-soluble secosteroids (a steroid molecule with one ring open). In human biology, the most important compound in this group is vitamin D3 (cholecalciferol). Vitamin D3 is obtained from sunlight and dietary sources. By itself vitamin D3 is inactive. It is converted to its active form by two hydroxylations: the first in the liver, by the enzymes of the cytochrome P450 superfamily, CYP2R1 or CYP27A1, to form 25-hydroxycholecalciferol (calcifediol, 25-OH vitamin D3). The second hydroxylation occurs mainly in the kidney through the action of the CYP27B1 to convert 25-OH vitamin D3 into 1,25-dihydroxycholecalciferol (calcitriol, 1,25-OH2 vitamin D3), the active form of vitamin D3 [89, 90]. On the other hand, the enzyme CYP24A1 catalyzes hydroxylation reactions that lead to 1,25-OH2 vitamin D3 degradation [91].

Calcitriol acts a lipid-soluble hormone and exerts its biological actions by the activation of VDR in the nucleus. Once VDR is activated, it forms a heterodimer complex with the RXR (a retinoic acid receptor, see above). This heterodimer complex then binds to specific target DNA sequence known as the vitamin D response elements (VDREs) to modulate the expression of target genes, involved in the regulation of cell proliferation, differentiation, angiogenesis, DNA repair, and apoptosis [90]. In addition, calcitriol can bind other cell-surface receptors through second messenger pathways, exerting nongenomic actions [89, 90].

Several studies have found an association between vitamin D deficiency and the increased risk of developing UFs. It is well known that African-American women are at increased risk of developing UFs, and studies have revealed that black females have lower serum levels of vitamin D as compared with white females [89]. Moreover, an inverse relationship between vitamin D serum levels and severity of UFs has been described in this population [92]. Therefore, it can be hypothesized that these factors could lead to a loss of vitamin D functions that could explain, at least in part, why African-American females have increased incidence of UFs compared with other ethnic groups [89]. To further support this hypothesis, genomic studies have demonstrated several ethnic-related nucleotide polymorphisms, in VDR and different genes involved in vitamin D metabolism and its serum concentration, to be associated with UFs [89, 93].

In UFs, vitamin D3 inhibits the growth of both leiomyoma and myometrial cells in a concentration-dependent manner, and its serum concentration is inversely correlated with UFs size. Vitamin D3 acts an antifibrotic factor through inhibition of TGF-β3-induced fibrosis in leiomyoma cells [89, 94]. Moreover, in vitro studies have demonstrated that vitamin D3 also inhibits proliferation and induces apoptosis in cultured myoma cells through the downregulation of several genes involved in cell cycle regulation, and by suppression of both expression and activity of catechol-O-methyltransferase (COMT), an enzyme that modulates the biological effects of estrogens and plays a role in myoma formation [95]. Additionally, vitamin D3 suppresses the action of MMP, increases VDR and TIMPs, reduces the expression of ECM proteins, and downregulates sex hormone receptors and their coactivators, in a concentration-dependent manner [89, 96, 97].

There is evidence that UFs express reduced levels of VDR compared with myometrium, and that leiomyoma tissues showed inverse correlation between the upregulated estrogen and progesterone receptor and VDR levels [90]. A recent study by Othman et al. [91] evaluated tissue levels of active vitamin D and the expression of CYP27B1 and CYP24A1 in UFs [91]. The authors confirmed that leiomyoma cells not only contain lower level of vitamin D3 compared to myometrium, but also that uterine tissue expresses extrarenal CYP27B1. Therefore, these findings confirm that both myoma or normal myometrium tissues can produce their own active vitamin D3, and that such locally produced vitamin D3 modulates cell functions through paracrine/autocrine action. In the same study, the authors found that CYP24A1 was upregulated in UFs [91]. This mechanism not only enables UFs to degrade vitamin D3 and suppress its antitumor effects, but also to sustain a tumor microenvironment of hypovitaminosis D.

In summary, physiological actions of vitamin D are driven by the regulation of multiple cellular pathways, including proliferation, apoptosis, DNA repair, ECM deposition, and signaling. Its action in UFs cells is strictly correlated with the ability of vitamin D to control VDR expression. Moreover, dysregulation of vitamin D metabolizing enzymes observed in UFs could locally regulate vitamin D action.

In addition to its antiestrogenic/antiprogesteronic effect, VDR activation is able to suppress other relevant signaling cascades in myoma biology, such as Wnt/β-catenin, mTOR, and TGF-β pathways [47, 48, 98, 99].

12. Ovarian steroid hormones and pathogenesis of uterine fibroids

The pivotal role of ovarian steroid hormones in the pathogenesis of uterine fibroids is supported by epidemiological, clinical, and experimental evidence [100]. The fact that fibroids occur during the reproductive years and regress after menopause indicates a growth dependent on ovarian hormones [100]. Although there is no evidence of abnormal levels of circulating sex steroids in women with fibroids, tissue estrogen levels are higher in patients with UFs [101]. These findings provide some clues that tissue receptor expression, abnormal signaling, and other estrogen-related aberrations play a role in UFs development and growth.

The effects of estradiol and progesterone are interrelated and involve the mediation of receptors, transcription factors, kinase proteins, growth factors, and numerous autocrine and paracrine factors [100, 101]. As mentioned above, myometrial stem cells express very low levels of estrogen and progesterone receptors; therefore, to maintain the growth ratio, these cells require paracrine factors released from the surrounding myometrial cells expressing abundantly sex steroid receptors (crosstalk with Wnt/β-catenin) [19, 100]. Sex steroids lead to tumor expansion by stimulating a modest rate of cellular proliferation and the production of copious amount of ECM [6, 102]. Furthermore, intricate networks of postreceptor signaling can also be activated by alternative pathways that bypass the hormone-receptor complex, thus allowing hormone-like effects by nonhormonal mediators [48].

12.1 Estrogens

Estrogens are a key element of UFs pathogenesis. Estrogens elicit its physiological effects on the target cells by binding to estrogen receptors (ERs). ERs are currently classified into nuclear (classical) and membrane-bound (mERs). Nuclear ERs are subdivided in estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ). On the other hand, mERs are localized at the plasma membrane and are subdivided in α, β, and G-protein-coupled estrogen receptor 1 (GPER1). GPER1 is also known as G-protein-coupled receptor 30 (GPR30). All ERs are expressed in fibroids following the differentiation of fibroid progenitor cells [47, 101, 102].

Estrogen-dependent signaling pathways can be classified as genomic and nongenomic. The genomic (transcriptional) pathway is mainly mediated by nuclear ERs. In this pathway, the estrogen-ER complex binds estrogen response elements (EREs) of DNA to regulate gene transcription. Conversely, nongenomic pathway is mainly mediated by mERs and GPER1 and works in a similar way to GFs receptors: rapidly activating downstream signaling cascades, including MAPK/ERK, PI3/Akt/mTOR and phospholipase C (PLC)/inositol trisphosphate (IP3)/calcium, among others. Furthermore, there is evidence of crosstalk between rapid estrogen signaling and GFs signaling through RTKs [101].

In addition to activation from estrogen binding, ERα is also activated by the MAPK pathway and via other kinases. Therefore, estrogen-bound ERα induces GFs expression, which can then stimulate the MAPK pathway and further activate ERα in an autocrine fashion. This feedback by MAPK on ERs is an example of the complexity and interconnectedness of signaling pathways in UFs [101, 102]. Interestingly, rapid activation of MAPK pathway by estrogen in primary myoma cells was related to estrogen-induced PDGF secretion and it was proposed that PDGF, alone or associated with other GFs, was the main GF involved in the proliferation response of leiomyoma cells to estrogen stimulation.

Estrogen upregulates the expression of several genes involved in UFs pathogenesis, including growth factors and their receptors (VEGF, PDGF, IGF-I, TGF-β), collagens, GPR1, and ERs [17, 101, 103].

Likewise, estrogen sensitizes the tissue to progesterone by promoting expression of progesterone receptor, providing a microenvironment in which progesterone can induce fibroid growth [100, 101, 102]. Conversely, estrogen downregulates Activin-A and myostatin expression [17]. Importantly, it has been shown that many of these processes occur only in UFs cells but not in normal myometrium; therefore, it has been postulated that this differential effect can be a contributing factor in UFs pathobiology [101].

Interestingly, it has been shown that even if estrogen downregulates EGF expression, it upregulates the expression of its receptors (epidermal growth factor receptors (EGFRs)). Interestingly, estrogen activation of GPR1 induces MMP activation and release of HB-EGF which binds and activates upregulated EGFRs. This transactivation represents an important integrator of estrogen and GF cellular signaling [101].

Other actions of estrogen include the inactivation of several tumor suppressor genes, such as p53, providing insight into tumor transformation mechanisms in UFs and how they are closely linked to estrogen signaling [17, 101].

Estrogen receptors are abundantly expressed in uterine myomas, which ensure considerable responsiveness to the circulating estrogen. Whether UFs are richer in ERs than surrounding myometrium is still debatable, with some studies showing such a difference, whereas others do not [17, 100]. However, there is an enhanced response to estrogen stimulation by fibroid cells compared to normal myometrium.

Moreover, studies have demonstrated that ethnic-related polymorphisms, involving ERs [104, 105] and elements of estrogen signaling and metabolism, such as aromatase (CYP19A1) [106, 107] and COMT [108], are related to the increased leiomyoma risk in different ethnic groups.

Uterine fibroids are exposed to estrogen not only through ovarian steroidogenesis, but also through local conversion of androgens by aromatase within the tumors themselves. Interestingly, a quantifiable increase in aromatase expression has been found in African-American women [106]. Therefore, UFs are capable of producing enough estrogen to sustain their own growth. Furthermore, UFs have remarkably levels of aromatase compared with adjacent myometrium [102].

12.2 Progesterone

Progesterone action is required for full development and proliferation of myoma cells, and available data suggest that progesterone might be the primary hormone driving the growth of UFs. Estrogen is also necessary, but not sufficient for myoma growth [102]. Ishikawa et al. [109] showed that estrogen/ERα regulates progesterone receptor expression and that estrogen alone is not a mitogen in vivo. These findings suggest a more permissive role for estrogen acting via induction of PR expression, and thereby allowing UFs responsiveness to progesterone (estrogen-progesterone crosstalk) [47].

In UFs, progesterone actions are mediated by nuclear progesterone receptors A and B (PR-A and PR-B) [47, 102]. Several studies have demonstrated increased expression of both PR-A and PR-B in UFs compared to normal myometrium, and that it seems to be even higher according to women’s age and number of tumors [100]. Interestingly, an overexpression of PR-B has been found on UFs surface, suggesting that the predominant expression of PR-B in this part reveals an activated phenotype for progestational proliferation related to myoma growth [17]. Furthermore, studies have demonstrated that mitotic activity in UFs is significantly higher in secretory (progesterone-dominant) phase of the menstrual cycle [47]. Conversely, PRs expression is lower in women experiencing severe bleeding and dysmenorrhea [100]. Ligand-bound PR binds to DNA and regulates transcription of several target genes. In addition to this transcriptional pathway, progesterone can activate rapid signaling pathways (nongenomic). Activated PRs activate MAPK/ERK signaling pathway. Moreover, it has been shown that progesterone can rapidly activate PI3/Akt pathway inducing UFs growth [110].

Progesterone influences myoma growth by the interaction with GFs (progesterone-GFs signaling crosstalk), and other genes involved in cell proliferation, including upregulation of TGF-β1, TGF-β3, EGF and expression of B-cell lymphoma 2 (Bcl-2) oncogene [17, 47, 110]. On the other hand, progesterone inhibits IGF-I, TNF-α, and decorin expression in myoma cells. Since decorin inhibits TGF-β activity, its reduced levels in leiomyoma may enhance TGF-β-mediated ECM deposition [6, 17].

Finally, there is much more to be learned in terms of how progesterone promotes proliferation, the repertoire of genes involved, and how progesterone and GFs signaling pathways crosstalk in leiomyomas.

13. Conclusions

The hallmarks of uterine fibroid development and growth are clonal expansion of an aberrant myometrial stem cell and excessive deposition of extracellular matrix.

Although alterations in MED12 and HMGA2 have been postulated as the initial insult leading to unregulated cell growth, it is unclear whether these genetic alterations cause the transformation of a myometrial stem cell or simply support already existing leiomyoma stem-progenitor cells.

Extracellular matrix acts as a reservoir of growth factors and protects them from degradation. In addition, sex steroids increase ECM production by regulating expression and activity of growth factors. These processes are tightly regulated through a complex network of interconnected signaling pathways.

We have discussed the putative role of inflammation and an abnormal hypoxia response in myoma development and the role of myofibroblasts as the central player in those aberrant processes.

Even though some cell-related events have been identified, the exact cell of origin and the exact molecular processes involved in uterine leiomyomas formation remain unknown.

Conflict of interest

The authors declare no conflict of interest.