Endometriosis: Pathogenesis and Treatment 2014 Ed.

11. Aromatase Expression in Endometriosis and Its Significance

Hiroshi Ishikawa1 and Makio Shozu 

(1)

Department of Reproductive Medicine, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan

Makio Shozu

Email: shozu@faculty.chiba-u.jp

Abstract

Endometriosis is a chronic inflammatory disease frequently observed in the ovary, pelvic peritoneum, and rectovaginal septum. The growth and progression of an endometriotic lesion depends on a sex steroid, estrogen. Aromatase, a key enzyme in estrogen biosynthesis, is highly expressed in the endometriotic tissue, resulting in in situ production of estrogen that, in addition to endocrine estrogen from the ovary, may contribute to the etiology and progression of endometriosis. The aberrant expression of aromatase together with the elevated expression of 17β-hydroxysteroid dehydrogenase type 1 and the absence of 17β-hydroxysteroid dehydrogenase type 2 observed in the endometriotic tissue would contribute to an increase in the tissue concentration of estrogen. Aromatase expression is regulated at multiple levels, from the transcription of CYP19A1 and epigenetic codes to posttranslational modification and degradation of the protein. Among the multiple promoters of CYP19A1, the most proximal promoter PII is the most active in endometriosis and is regulated by cAMP, prostaglandin E2, steroidogenic factor-1, and possibly the end product estrogen. Hypomethylation of CpG islands on CYP19A1observed in the endometriotic tissue may contribute to the upregulation of aromatase expression.

Similar to spontaneous menopause, inhibition of in situ estrogen biosynthesis may regress endometriosis. The use of aromatase inhibitors (AIs), which selectively inhibit aromatase activity in human tissues, is a possible treatment for inhibiting local estrogen biosynthesis in endometriosis. AIs have been used as monotherapy or in combination therapies with progestins, oral contraceptive pills, and gonadotropin-releasing hormone agonists to reduce endometriosis-related pain in premenopausal women.

Keywords

AromataseAromatase inhibitorEstrogenLetrozole

11.1 Introduction

Endometriosis is defined as the proliferation of the endometrial gland-like epithelium and surrounding stroma outside the uterus. It is frequently observed in the pelvic peritoneum, ovary, and rectovaginal septum. In total, 6–10 % of women of reproductive age are suffering from endometriosis-related pain, i.e., menorrhagia, dyspareunia, and chronic pelvic pain. Moreover, endometriosis may cause infertility. Although considerably rare, ovarian endometrioma can transform and give rise to ovarian cancer during long-term management of the disease.

Endometriosis becomes symptomatic in the reproductive age and is self-limited after spontaneous menopause. Similar to spontaneous menopause, long-term inhibition of ovulation using progestins, oral contraceptive pills (OCPs), and gonadotropin-releasing hormone (GnRH) agonists alleviates the symptoms. On the other hand, estrogen use in postmenopausal women induces symptomatic regrowth of endometrial lesions. These findings suggest that the progression of endometriosis is closely associated with estrogen as well as ovulation.

The ovary synthesizes estrogens de novo from cholesterol and supplies most of the circulating estrogens in women of reproductive age. In addition to the ovary, a small amount of estrogens can be synthesized in situ at peripheral fat, bones, the skin, the brain, and vessels, which are unable to synthesize estrogen from cholesterol de novo but are able to convert circulating androgens to estrogens [12].

There are 2 rate-limiting steps in estrogen biosynthesis. The first step is the mobilization of cholesterol across the mitochondrial membrane into the mitochondria. This step is mediated by the steroidogenic acute regulatory protein (StAR) expressed in the mitochondria. The second step is the enzymatic reaction converting androgens to estrogens by aromatase. Aromatase is a member of the cytochrome P450 superfamily and is expressed in the endoplasmic reticulum. Aromatase is a unique enzyme that can catalyze aromatization. The rate of estrogen synthesis depends on these 2 steps in the ovary, where estrogen is synthesized de novo. On the other hand, the synthesis depends only on the second step in peripheral organs. In these peripheral organs, expression levels of aromatase determine the production rate of estrogen as long as circulating androgen is supplied [3].

Estrogen plays essential roles in reproduction, bone development, and epiphyseal closure in puberty, bone mineral metabolism, and possibly cognitive function. In addition to the physiological roles, recent studies have focused on the pathological roles and revealed that the overproduction of estrogen in situ causes diseases. Breast cancer tissues express a high level of aromatase, and the resulting in situ estrogen promotes cancer cell growth in postmenopausal women. Endometriosis is another example in which the aberrant expression of aromatase is involved in disease pathogenesis. The inhibition of in situ estrogen synthesis using an aromatase inhibitor (AI) is therefore a potential choice of treatment, similar to that seen in the case of breast cancer.

Here, we review the aromatase expression in endometriotic tissues and the mechanisms underlying the aberrant expression. Following this, we discuss the pathophysiological significance of in situ estrogen in endometriotic tissues and of aromatase as a molecular target treatment for refractory endometriosis.

11.2 Aromatase Genes and Their Expression in Endometriotic Tissues

11.2.1 Genomic Structure of CYP19A1

Aromatase is a unique enzyme responsible for the conversion of androgens to estrogens in humans. Aromatase is encoded by CYP19A1 on chromosome 15q22, which spans approximately 132 kb and is composed of at least 9 alternative first exons and their 9 downstream coding exons (exons 2–10). Each first exon contains a unique 5′-untranslated sequence and a unique upstream promoter sequence. Each promoter possesses multiple transcriptional regulatory element-binding sites, which enable tissue-specific and promoter-specific regulation of aromatase expression.

The transcription of aromatase begins from 1 of the 9 first exons, is extended to exon 10, and ends at 1 of the 2 poly A signals located at the end of the 3′-untranslated region of exon 10. The resulting primary transcripts give rise to mRNA by splicing all introns. Because there are 9 different first exons, at least 9 different mRNAs are formed; however, all of these encode the same aromatase protein because the protein coding sequence is located in the shared exon 2 and its downstream.

11.2.2 Regulation of Aromatase Expression

11.2.2.1 Expression of Aromatase and In Situ Estrogen

Aromatase is highly expressed in ovarian granulosa cells of Graafian follicles and in placental cytotrophoblasts. Aromatase is also expressed in Sertoli cells, neurons, adipose stromal cells, vascular smooth muscle cells, skin fibroblasts, bone osteoblasts and osteoclasts, hepatocytes, and uterine myometrium. More recently, aromatase expression has been reported in the stomach, lung, colon, and macrophages, albeit at a low level [47]. Almost all estrogen receptor (ER)-positive cells express aromatase, although the expression levels vary.

In addition to normal tissues, aromatase is expressed in some pathological conditions such as breast cancer, endometriosis, uterine fibroids, and endometrial cancer. Estrogens synthesized in situ within these peripheral organs can bind to ERs of their own cells or those of neighboring cells and stimulate proliferation, thereby playing a role in disease pathogenesis. The biological actions of this in situ estrogen are more potent than those expected from a comparable amount of endocrine estrogen, possibly because in situ estrogen directly acts on neighboring cells with neither dilution into the circulating volume nor binding to proteins that interfere with the hormonal action.

11.2.2.2 Tissue-Specific Regulation of Aromatase

As described above, aromatase expression is regulated in a tissue-specific manner in multiple tissues by the alternate use of multiple promoters. For example, aromatase transcription is driven in the ovary by the most proximal promoter PII, which is regulated by cAMP downstream of a gonadotropin, follicle-stimulating hormone (FSH). The transcription in the brain is driven by promoter 1f. A further upstream promoter, I.4, drives the transcription in adipose tissues. In these promoters, specific steroid hormones are essential for expression: testosterone and glucocorticoid for promoters 1f and I.4, respectively. Local factors such as growth factors and cytokines also play regulatory roles in these promoters. For the placenta, promoter I.1 is a predominant promoter, while all other promoters are transcribed at very low levels. Promoter I.1 is quite different from other promoters in terms of its structure and function: it does not have a nuclear half site for binding of the NR5A (SF1) and permits constitutive expression, while others strictly downregulate basal expression.

The promoter-specific regulation is realized by relatively short upstream segments of promoters (less than approximately 500 bp), where tissue-specific enhancers and regulatory cis-elements possibly exist. Thus, a part of genomic DNA containing these short sequences mimics the physiological regulation by factors such as FSH, steroid hormones, cytokines, and prostaglandins. Using transgenic mice, it has been shown that these short sequences are capable of tissue-specific expression in the ovary and placenta [8]. However, the absolute level of expression of these transgenes (promoter constructs) is generally lower than that observed in vivo, suggesting that undiscovered enhancers exist outside the promoter region. Another explanation has recently been reported for the low level of in vitro expression compared with that of in vivo expression. Estrogen as a product of aromatase binds to the TSPYL5 promoter and induces the expression of TSPYL5, which in turn is integrated into the transcriptional machinery of aromatase and efficiently enhances its expression [9]. This explains the reason why expression levels of aromatase demonstrated by promoter constructs that lack estrogen synthesizing activity are generally low.

11.2.2.3 Promoter Switching

Physiological expression of aromatase in human tissues, except for the placenta, is basically downregulated, and upregulation occurs only when appropriate stimuli exist. In comparison, it has been known that high levels of aromatase are constitutively expressed in pathological conditions of the breast. For example, aromatase is highly expressed in adipose tissues surrounding breast tumors in postmenopausal women [10]. Similarly, aberrant expression of aromatase has been confirmed in other pathological conditions such as endometriosis, uterine fibroids, endometrial cancer, and lung cancer [31113].

Promoter switching is a possible event underlying the aberrant expression of aromatase in pathological conditions. This was initially described in breast adipose tissues of women with or without breast cancer [14]. The normal breast adipose tissue of cancer-free women expresses low levels of aromatase by the predominant use of promoter I.4, whereas the adipose tissue surrounding breast tumors expresses high levels of aromatase by the predominant use of promoters PII and I.3. A possible explanation for “promoter switching” is that cancer-derived or cancer-prone local factors drive aromatase expression. Consistent with this, local factors drive aromatase expression. A promoter construct composed of tandem sequences of mini-promoters of CYP19A1 mimics promoter switching in the breast [15].

11.2.3 CYP19A1 Polymorphisms and Risk of Endometriosis

Endometriosis is 6–7 times more frequent among first-degree relatives and is presumed to have a multifactorial inheritance. Several studies have reported polymorphisms of CYP19A1 in endometriosis, including dysequilibrium for both breast cancer and endometrial cancer. As shown in Table 11.1, the polymorphisms of CYP19A1 have been associated with the risk of endometriosis; however, the results are controversial [1624].

Table 11.1

Association between CYP19A1 gene polymorphisms and the risk of endometriosis

Nucleotide polymorphisms

Location

Author

Cases

Range of age (mean ± SD)

Control

Range of age (mean ± SD)

Staging of cases (number of subjects)a

Results

rs 10046 C1558T

3′ untranslated region of Exon 10

Szczepanska et al.

115

20–39

197

19–39

I (59), II (56)

No associationb

Lamp et al.

150

18–45 (32.1 ± 6.1)

199

30–50 (39.8 ± 5.3)

I (53), II (39), III (36), IV (22)

No association

Vietri et al.

104

22–45 (36.8 ± 6.7)

86

18–48 (37.8 ± 5.1)

Undescribed

Genotype CC was overpresented in cases (48.1 % vs. 30.2 %)

Hur et al.

224

Undescribed

188

Undescribed

III to IV (224)

No association

Huber et al.

32

(52.3 ± 5.4)

790

(34.6 ± 7.0)

I (0), II (21), III (10), IV (1)

No association

rs700519 C > T, Arg264Cys

Exon7

Wang et al.

300

(34.3 ± 6.9)

337

(52.2 ± 4.2)

I (7), II (52), III (165), IV (76)

No association

Tsuchiya et al.

79

20–45

59

20–45

I (21), II (10), III (23), IV (25)

No association

Huber et al.

32

(52.3 ± 5.4)

790

(34.6 ± 7.0)

I (0), II (21), III (10), IV (1)

No association

rs2236722 T > C, Trp39Arg

Exon2

Wang et al.

300

(34.3 ± 6.9)

337

(52.2 ± 4.2)

I (7), II (52), III (165), IV (76)

No association

Hur et al.

224

Undescribed

188

Undescribed

III to IV (224)

No association

240 A > G, Val80

At codon80 in exon3

Vietri et al.

104

22–45 (36.8 ± 6.7)

86

18–48 (37.8 ± 5.1)

Undescribed

Genotype AA was overpresented in cases (50.0 % vs. 32.6 %)

Hur et al.

224

Undescribed

188

Undescribed

III to IV (224)

No association

3 bp insertion/deletion

50 bp upstream from the (TTTA)n tract in intron4

Lamp et al.

150

18–45 (32.1 ± 6.1)

199

30–50 (39.8 ± 5.3)

I (53), II (39), III (36), IV (22)

No association

Kado et al.

140

24–48 (36.3 ± 8.1)

177

(63.8 ± 6.1)

I to II (32), III to IV (108)

Del/Del was frequently observed in cases

rs1004982

Intron

Trabert et al.

256

18–49

567

Matched to cases

Undescribed

Increased risk of endometriosis

rs18700479

Intron

Increased risk of endometriosis

rs936307

Intron

Increased risk of endometriosis

(TTTA) repeat number

Intron4

Lamp et al.

150

18–45 (32.1 ± 6.1)

199

30–50 (39.8 ± 5.3)

I (53), II (39), III (36), IV (22)

No significant association between [TTTA] repeat <7 and [TTTA] repeat 8–13

Hur et al.

224

Undescribed

188

Undescribed

III to IV (224)

No significant association between [TTTA] repeat <7 and [TTTA] repeat 8–13

Kado et al.

140

24–48 (36.3 ± 8.1)

177

(63.8 ± 6.1)

I to II (32), III to IV (108)

No significant association between [TTTA] repeat <7 and [TTTA] repeat 8–13

Arvanitis et al.

275

21–37 (27.2 ± 3.2)

346

26–53 (34.5 ± 7.4)

Undescribed

[TTTA] 10 allele increased risk by 4.99 times for endometriosis

aThe staging was assessed by the revised American Society for Reproductive Medicine score

bNo statistically significant association was observed among different genotypes

Recent genome-wide association studies (GWAS) identified genetic variants in multiple loci that have been associated with endometriosis. These single-nucleotide polymorphisms (SNPs) have never been identified by traditional candidate gene strategies for specific diseases. GWAS-identified endometriosis-associated SNPs have ethnic differences [25]. No SNPs on chromosome 15q22, where CYP19A1maps, have been identified by GWAS.

11.2.4 Epigenetic Regulation of Aromatase

An epigenetic change is defined as heritable changes in gene expression that do not represent changes in the DNA sequence. DNA methylation and histone modification are the most often explored epigenetic mechanisms. Transcriptional regulation by noncoding RNAs, particularly micro RNAs (miRNAs), is another distinct epigenetic mechanism.

11.2.4.1 DNA Methylation of CYP19A1

Changes in the methylation status of CpG islands of CYP19A1 have been reported in endometriosis [26]. A CpG island located approximately 70 kb downstream from exon 1.1 of CYP19A1 is hypomethylated in endometriosis (stromal cells obtained from ovarian chocolate cysts) but hypermethylated in the eutopic endometrium (stromal cells obtained from disease-free women). The methyl-CpG-binding proteins MBD1 and MeCP2, which contain an amino-terminal methyl-CpG-binding domain and a carboxy-terminal transcriptional repression domain, bind to the hypermethylated region of the CpG island in eutopic tissues, which in turn may suppress aromatase expression in the eutopic endometrium [27].

The treatment of endometrial stromal cells with 5-aza-deoxycytidine (an irreversible inhibitor of DNA methyltransferase1, which is essential for maintaining the methylation status of genomic DNA) induces aromatase expression in endometrial stromal cells obtained from disease-free women. The demethylation of the CpG islands in CYP19A1 may be relevant to the upregulation of aromatase [28], although other explanations are possible because 5-aza-deoxycytidine alters the expression level of a broad spectrum of genes that may indirectly or directly affect aromatase expression.

11.2.4.2 Histone Modification

Histone modifications affect the chromatin structure and the subsequent interaction of transcription factors with their response elements in the promoters. The acetylation of histone H3 and histone H4 activates transcription by loosening the chromatin structure and allowing the recruitment of transcription factors to their response elements. In contrast, trimethylation of lysine at sites 9 and 27 on histone H3 inactivates transcription by causing the chromatin to become more condensed. Histone modifications affect Cyp19a1 mRNA expression in rat granulosa cells [29]. We found that the acetylation of H3 and H4 at the promoter I.4 region occurs during the induction of aromatase expression by dexamethasone in breast cancer cell lines. No histone modification has been reported for aromatase regulation in endometriosis till date.

11.2.4.3 Regulation of Aromatase by miRNA

miRNAs are short noncoding RNAs that act by targeting partially complementary sequences within mRNAs. They consist of 19–25 nucleotides and commonly exist in the 3′-untranslated region of the target genes. Traditionally, miRNAs negatively regulate the transcription of their target genes. A single miRNA may target many genes, and each of them may in turn be regulated by different miRNAs [30].

The transcriptional repression of aromatase mRNA by miRNAs has been reported in mammalian ovarian tissues (miR-503 and miR-378 directly inhibit aromatase, miR-224 and miR-383 indirectly inhibit aromatase), trophoblast differentiation (miR-19b and miR-106a downregulate aromatase), and endometrial cancer (miR98 represses aromatase) [3133]. There has been no report on miRNA regulation of aromatase mRNA in endometriosis.

11.2.5 Posttranslational Regulation of Aromatase

In addition to the mRNA level, aromatase activity is regulated at the protein level. It has been reported that phosphorylation and dephosphorylation of amino acids in the aromatase protein alter the enzymatic activity [3435].

Recently we found a novel mechanism underlying posttranslational regulation by autophagy [36]. Insulin-like growth factor-1 enhances aromatase activity over 50 % as early as 1 h in THP-1 myeloid leukemia cells through the inhibition of autophagy. A part of the newly synthesized aromatase protein is continuously transported to the lysosome and is degraded when aromatase protein synthesis is increased.

Another mechanism underlying posttranslational regulation has been proposed. A series of transfection experiments revealed that mRNA stability and protein translation efficiency vary among the 5′-noncoding sequence of exon 1 of aromatase [37]. The 5′-noncoding sequence of exons I.3 and I.4 contains the cis-acting elements responsible for the modulation of aromatase levels.

No study has reported the posttranslational regulation of aromatase in endometriosis where aromatase is highly expressed and translated.

11.3 Aromatase Expression in Endometriosis

11.3.1 Aberrant Biosynthesis of Estrogen in Endometriosis

There is unequivocal clinical evidence that the development and progression of endometriosis depends on ovarian estrogen. In addition to endocrine estrogen, numerous studies have shed light on the role of estrogen synthesized by endometriotic cells in situ. Although there have been no reports on direct measurements of the concentration of estrogen in the endometriotic tissues, comprehensive accumulated data have indicated the overproduction of estrogen in situ and its role in the pathogenesis in endometriotic tissues.

The endometriotic tissue expresses higher levels of aromatase than the eutopic endometrium, and it efficiently converts androstenedione to estrogen [3842]. Moreover, endometriotic tissues do not inactivate estradiol by conversion to estrone because of the reduced expression of 17β-hydroxysteroid dehydrogenase (17β-HSD) type 2, whereas the eutopic endometrial epithelium does [4344]. Thus, endometriosis has more available estrogen, which possibly stimulates disease progression. Interestingly, the eutopic endometrium of women with endometriosis overexpresses aromatase before actual implantation to the abdominal cavity. This supports the notion that the overexpression of aromatase in the endometrium is a pathogenic factor that causes ectopic implantation and growth of endometrial tissues in regurgitating menstrual blood [45]. This remains to be determined in future.

The diagnostic use of the aberrant expression of aromatase has been examined. A Japanese group collected endometrial biopsy specimens from patients and immunostained the specimens for aromatase. Receiver operating characteristic curve analysis revealed a cut value of 20 H-scores with 91 % sensitivity, 100 % specificity, 100 % positive predictive value, and 72 % negative predictive value to distinguish the eutopic endometrium from women with or without endometriosis [46].

In addition to a high aromatase activity and reduced expression of 17β-HSD type 2, the expression of 17β-HSD type 1 is higher in the endometriotic tissues than in the eutopic endometrium. 17β-HSD type 1 converts estrone, a primary estrogen synthesized from androstenedione, to estradiol, an active form of estrogen, thereby enhancing the action of estrogen as a product. Compared with the eutopic endometrium, the aberrant expression of aromatase, the elevated expression of 17β-HSD type 1, and the reduced expression of 17β-HSD type 2 in the endometriotic tissue collectively give rise to elevated local levels of estradiol [47]. Another study reported that compared with the normal endometrium, aromatase, 17β-HSD types 1 and 7 (but not type2), sulfatase, and ERβ were statistically significantly upregulated, while ERα was significantly downregulated in ovarian endometrioma. There were no significant differences in 17β-HSD type 2, sulfotransferase, and progesterone receptors A and B gene expression [48]. Aromatase and 17β-HSD type1 mRNA levels were extremely low in the normal human endometrium, while mRNAs for 17β-HSD types 2 and 4, expressed sequence tags, and sequence-tagged sites were readily detectable [49].

Endometriotic tissues express a complete set of steroidogenic enzymes to synthesize estrogen: cholesterol side-chain cleavage enzyme (P450scc), 3β-hydroxysteroid dehydrogenase 2 (HSD3B2), 17-hydroxylase/17,20-lyase (P450c17), and aromatase. The rate-limiting steps in estrogen synthesis are cholesterol mobilization into the side-chain cleavage enzyme (the initial step) and aromatization (the last step). Both these steps are enhanced in endometriotic tissues: StAR, which facilitates the entry of cytosolic cholesterol into the mitochondria, is overexpressed in endometriotic tissues compared with the normal endometrium. A master transcription regulator for steroidogenesis, steroidogenic factor 1 (SF1 or Ad4BP), also called NR5A1, is expressed at a higher level in endometriotic tissues than in endometrial tissues [39]. This explains the overexpression of a series of, if not all, steroidogenic enzymes in endometriotic tissues.

Prostaglandin E2 (PGE2) is another gateway regulator of the aberrant expression of steroidogenic enzymes in endometriotic tissues. Inflammatory endometriotic tissues express a high level of cyclooxygenase-2 (COX-2) and synthesize substantial amounts of PGE2, which in turn induces a series of steroidogenic enzymes, resulting in the production of progesterone, estrone, and estradiol. Estrogen in turn enhances the expression of COX-2. Thus, PGE2 and estrogen form a different cycle along with their synthesizing enzymes [5051]. Increased PGE2 and estrogens synergistically stimulate the progression of endometriosis [39].

Compared with the normal and eutopic endometrium, the expression of ERα and ERβ is increased in endometriotic tissues [52]. Predominant expression of ERα mRNA compared with that of ERβ mRNA has been reported [53]. In addition, the downregulation of ERα and upregulation of ERβ in ovarian endometrioma compared with those in the normal endometrium have been reported [54]. A role of ERβ in the regulation of ERα in endometriotic stromal cells has been elucidated. ERβ knockdown significantly increased ERα mRNA and protein levels in endometriotic stromal cells, whereas ERβ overexpression decreased ERα mRNA and protein levels. High levels of ERβ suppress ERα expression and the response to estradiol in both endometrial and endometriotic stromal cells via binding to classic and nonclassic DNA motifs in alternatively used ERα promoters [55]. Estrogen is believed to have a strong effect on endometriotic tissues through binding of ERα and ERβ.

11.3.2 Regulation of Aromatase In Situ in Endometriosis

The overexpression of aromatase mRNA has been confirmed in endometriotic tissues and endometriotic stromal cells isolated and cultured from ovarian endometrioma. The level of the transcript correlates with the 17β-estradiol-producing activity in vitro in endometriotic stromal cells [33941]. The aromatase activity of endometriotic stromal cells is stimulated by the peritoneal fluid, tumor necrotizing factor-α, and interleukin-6 [56].

Macrophage migration inhibitory factor (MIF), a major pro-inflammatory and growth-promoting factor expressed in active endometriotic lesions, enhances the aromatase activity of endometriotic cells by posttranscriptional mRNA stabilization of aromatase. The resulting aromatase increases in situ estrogen, which in turn upregulates MIF expression. Accordingly, MIF and aromatase produce a positive feedback loop to enhance their mutual expression and promote endometriosis. Whether miRNA-mediated regulation is involved in this reciprocal enhancement between MIF and aromatase remains unclear. The inhibition of endogenous MIF may be a therapeutic candidate for endometriosis [57].

As described earlier, COX-2 is another regulator of aromatase expression for reciprocal enhancement in endometriotic tissues. Compared with the eutopic endometrium, COX-2 is overexpressed in endometriotic tissues of healthy women, enhancing PGE2 production in situ. PGE2 induces aromatase, and the resulting estrogen in turn stimulates COX-2 expression. This may prove to be another mechanism underlying the transcriptional enhancement of aromatase [39].

SF1 is a master transcription factor of steroidogenic enzymes, as explained earlier. SF1 induces the transcription of StAR, an essential protein in the initiation of steroidogenesis via cholesterol translocation into the mitochondria. SF1 binds to nuclear half sites of promoter PII of CYP19A1 and induces aromatase expression in endometriotic tissues. SF1 efficiently induces the transcription from promoter PII in cooperation with the transcriptional cAMP response element-binding protein [42]. In comparison, the chicken ovalbumin upstream promoter transcription factor (COUP-TF) alternatively binds to the nuclear half sites of promoter PII in the eutopic endometrium. This may explain why aromatase is constitutively suppressed in the eutopic endometrium, particularly in healthy women.

Why does the transcription factor switch from COUP-TF to SF1 in endometriotic tissues? A CpG island located upstream of SF1 promoter is highly methylated in the normal eutopic endometrium but unmethylated in endometriotic tissues. Thus, cancelation of silencing by genomic methylation seems to be a major mechanism underlying the reactivation of SF1 in endometriotic tissues, resulting in an overexpression of aromatase [58].

Many factors that may contribute to the induction and subsequent maintenance of aromatase expression in endometriosis have been reported. Some of these interact with each other and reciprocally increase expression. The contribution of each gene to the pathogenesis of endometriosis remains to be determined.

11.4 Aromatase Inhibitor (AIs)

11.4.1 AIs

AIs were originally developed for the treatment of breast cancer. By the 1990s, it had been revealed that breast cancer tissues synthesize estrogen in situ, which promotes breast cancer cell growth. After the molecular cloning of aromatase in the late 1990s, the aberrant expression of aromatase was identified as a cause of the overproduction of estrogen in situ. AIs were then developed as a new endocrine treatment of breast cancer. Initial trials showed that AIs reduced the level of estrogen in situ and suppressed the progression of cancer in postmenopausal women [5960]. Subsequent large-scale studies confirmed the therapeutic advantages over tamoxifen and reported the reduction of cancer development in the contralateral breast, suggesting a cancer preventive effect [61]. The role of in situ estrogen in pathology was first exemplified in breast cancer, and this established aromatase as a molecular target for endocrine therapy.

AIs are now widely used for the treatment of ER-positive breast cancer in postmenopausal women. The use of AIs shows a survival benefit compared with that of other endocrine therapies in women with advanced breast cancer [62].

11.4.2 Pharmacology

The functional aromatase enzyme complex is composed of 2 polypeptides: aromatase cytochrome P450 and NADPH-cytochrome P450 reductase. The former is a product of CYP19A1, a single gene on chromosome 15q22, which binds to androgens and hydroxylates them twice at the C19 position located between the A and B rings, resulting in aromatization of the A ring. The latter is a flavoprotein, ubiquitously distributed in most cells. NADPH-cytochrome P450 reductase confers an electron from NADP to aromatase for the enzymatic reaction.

AIs are classified into three generations according to the history of development (Table 11.2). The first-generation inhibitor aminoglutethimide is not selective to aromatase and causes a “medical adrenalectomy,” leading to lethargy, skin rashes, and nausea. The second-generation inhibitors include fadrozole and formestane, which are more selective than aminoglutethimide. A risk of glucocorticoid suppression is reduced but still exists. The third-generation AIs include letrozole, anastrozole, and exemestane (6-methylenandrost-1, 4-diene-3, 17-dione). These are more selective and potent; thus, they are excellent for use in clinical practice [6364]. The former two are nonsteroidal, while the latter is steroidal. Both types bind to a substrate-binding site of aromatase as a false substrate. The next step in the binding differs between the 2 types of compounds. A steroidal compound forms an unbreakable complex with the aromatase protein, and the enzymatic activity of the aromatase is thus permanently blocked once the binding occurs. This may lead to accelerated degradation of the aromatase protein [65]. Thus, enzymatic activity does not recover even if all unattached inhibitors are removed, and the enzymatic activity can only be restored by new enzyme synthesis [66]. In contrast, nonsteroidal compounds can dissociate from aromatase, resulting in recovery of the enzymatic activity. The difference in inhibitory mechanisms between steroidal and nonsteroidal AIs is expected to provide rationale for alternate treatments for patients with breast cancer who are resistant or refractory to other types of AIs [67].

Table 11.2

Three generations of aromatase inhibitors

Generation

Aromatase inhibitor

Nonsteroidal

Steroidal

First

Aminoglutethimide

 

Second

Fadrozole

Formestane

Third

Letrozole anastrozole

Exemestane

The third-generation AIs can decrease the local concentration of estradiol by 97–99 %. The therapeutic dose of AIs for breast cancer significantly reduces circulating estrogen levels in postmenopausal women. In comparison, the inhibition of estradiol synthesis in premenopausal women is not sufficient, with only a 20–30 % reduction in circulating estrogen levels [6869]. This less effective suppression in premenopausal women is because of androgen availability for nonsteroidal inhibitors: androgen as a substrate is present at micromolar concentrations in the ovary (follicle), while circulating androgen at nanomolar concentrations is the only source of androgen for extraglandular (peripheral) estrogen synthesis. Competitive binding is insufficient in androgen-rich follicles, resulting in insufficient inhibition.

Another mechanism underlying the insufficient suppression observed in premenopausal women is the compensatory increase in FSH. The suppression of estrogen synthesis causes an increase in FSH secretion, which stimulates follicular development and aromatase expression and eventually restores estradiol secretion to some extent [70].

11.4.3 Side Effects of AIs

AIs cause undesired health issues related to the presence or absence of estrogen suppression. Bone loss and fracture are the most important issues, particularly in postmenopausal women who use AIs for more than 6 months. Concomitant use of bisphosphonate is recommended for women who have additional risk factors for fracture [71]. Compared with the use of tamoxifen, long-term use of AIs increases the odds of cardiovascular disease [72]. Other side effects associated with the use of AIs are hot flushes, headache, joint stiffness or pain, leg cramps, arthralgia, nausea, and diarrhea. All of them are generally mild and are therefore tolerable.

It has been reported that AIs + norethindrone acetate or other OCs prescribed for the treatment of endometriosis cause bone mineral loss, as seen in the case of breast cancer. The change is significant but not symptomatic. This is partly because of the shorter duration of therapy compared with that for patients with breast cancer and partly because of the bone protective action of concomitantly used sex steroids [64].

11.4.4 AIs for the Treatment of Endometriosis

In most cases, the progression of endometriotic lesions and endometriosis-related symptoms can be controlled by the use of various types of medications, surgery, or a combination of both. Thus, trials of AIs are limited to patients with severe pain, including deep pareunia and chronic pain, who are refractory to established treatment options. The use of AIs in endometriosis was initially reported for postmenopausal women with refractory endometriosis of the rectovaginal space [73]. The therapeutic effect is now tested in premenopausal women suffering from refractory alleviation of endometriosis-related pain.

As described earlier, the application of AIs to premenopausal women leads to an elevation in FSH levels and subsequent follicular development, which increases estradiol, thereby potentially nullifying the therapeutic effect. Thus, additional agents to control ovarian activation are required. To this end, progesterone or progestins, OCs, and GnRH agonists have been tested. All of these are used in the conventional therapy for endometriosis.

11.4.4.1 AI Monotherapy

A few reports have provided evidence of the efficacy of AI monotherapy for the treatment of endometriosis-related pain in premenopausal women. In these studies, vaginally administered anastrozole (0.25 mg/day) combined with oral elemental calcium (1.2 g/day) and cholecalciferol (800 IU/day) for 6 months improved rectovaginal endometriosis-related dysmenorrhea [74].

A prospective randomized clinical trial has been conducted to compare the effect of short-term letrozole and a GnRH agonist (triptorelin) versus case control on the pregnancy rate and recurrence of endometriosis after laparoscopic surgery [75]. The overall pregnancy and recurrence rate of the letrozole group (2.5 mg/day for 2 months) were similar to those of the triptorelin group. The pregnancy and recurrence rate among the 3 groups were as follows: 23.4 and 6.4 % in the letrozole group, 27.5 and 5 % in the triptorelin group, and 28.1 and 5.3 % in the control group, respectively.

Another case report has revealed the efficacy and side effects of AIs in the treatment of premenopausal patients with endometriosis-related chronic pelvic pain refractory to conventional treatment. Four premenopausal patients were treated with either anastrozole (1 mg/day) combined with alendronate (10 mg/day) or letrozole (2.5 mg/day) combined with calcium (1.5 g/day) and vitamin D (800 U/day) for 6 months, and marked improvement of pelvic pain was observed in all the patients without significant hormonal change and bone mineral loss [76]. The most common side effect was irregular bleeding with anastrozole and joint pains with letrozole.

Thus, AI monotherapy for endometriosis-related pain is tolerable for a short duration, if the prevention of bone mineral loss is adequate.

11.4.4.2 Combination of AIs with Progesterone or Progestins

A prospective noncomparative study on the efficacy of letrozole + norethindrone acetate for endometriosis-related chronic pelvic pain has been conducted [77]. This study included 10 women who were refractory to conventional medication for endometriosis-related pain. The diagnosis of endometriosis was confirmed by laparoscopic biopsy. The combined use of letrozole (2.5 mg/day), norethindrone acetate (2.5 mg/day), calcium citrate (1,250 mg/day), and vitamin D (800 IU/day) for 6 months relieved the pain in 9 of 10 women. The authors concluded that letrozole is a candidate for the medical management of refractory endometriosis-related pain.

Two refractory cases with severe pain were treated with the cyclic administration of anastrozole combined with progesterone. One treatment course consisted of 21 days of treatment with anastrozole (1 mg/day), progesterone (200 mg/day), and calcitriol (1,25-dihydroxyvitamin D3; 0.5 μg/day), followed by 7 days without treatment. In addition, rofecoxib (a selective COX-2 inhibitor; 12.5–50 mg/day) was continuously administered. A six-course treatment resulted in a rapid, progressive reduction in pain, and the remission lasted over 24 months after treatment [78].

Another large-scale, prospective, open-label, nonrandomized trial including 82 women with pain caused by rectovaginal endometriosis has been conducted. Patients received either norethisterone acetate (2.5 mg/day) alone or a combination of letrozole (2.5 mg/day), norethisterone acetate (2.5 mg/day), elemental calcium (1,000 mg/day), and vitamin D3 (880 IU/day) for 6 months. Both regimens were similarly effective for dysmenorrhea; however, the efficacy differed between the 2 regimens with respect to chronic pelvic pain and deep dyspareunia. The reduction rate of the intensity of the pain scale in the combination therapy was 1.4–1.5-fold larger than that in therapy with norethisterone acetate alone [79]. A carryover effect was not observed in both regimens in terms of pain relief. Side effects, including joint pain (n = 5) and myalgia (n = 5), hair loss (n = 1), and decreased libido (n = 1), were only reported for the combination therapy (n = 41). However, withdrawal due to side effects was not statistically different between the combination group (n = 4) and the norethisterone acetate group (n = 3).

Overall, the use of AIs enhances the therapeutic efficacy of progesterone/progestins with regard to pelvic pain and deep dyspareunia that are refractory to established treatments. However, the improvement in the pain relapses soon after the completion of treatment, and the side effects, albeit minor, are increased. For extended treatment, great caution is required regarding side effects related to long-term use of AIs, such as bone loss and arthritis, as reported in case of long-term AI monotherapy for breast cancer.

11.4.4.3 Combination of AIs with OCPs

The efficacy of AIs for refractory endometriosis has been also examined in combination with OCPs. A phase 2 prospective open-label trial was conducted on 15 premenopausal women with documented refractory endometriosis and chronic pelvic pain [80]. The use of anastrozole (1 mg/day) and 1 tablet of ethinyl estradiol (20 μg/day)/levonorgestrel (0.1 mg/day) daily for 6 months significantly relieved the pain. Side effects were mild, and no adverse effects on major organs were observed.

The efficacy of AIs + OCPs for premenopausal patients with ovarian endometriomas and chronic pelvic pain, who were previously treated with surgery and medication with an unsatisfactory result, has been reported. Five women received letrozole (2.5 mg/day), desogestrel (0.15 mg/day), ethinyl estradiol (0.03 mg/day), calcium (1,200 mg/day), and vitamin D (800 IU/day) daily for 6 months. Ovarian endometriomas disappeared and the pain was relieved in all 5 women. Loss of bone mineral density was not observed [81]. This result is excellent; however, interpretation is limited because of the small number of patients and the fact that the study was nonrandomized and did not include controls. In addition, the primary target of the treatment was ovarian endometrioma and not rectovaginal endometriosis.

Further randomized trials are necessary to elucidate the benefits of a combination of AIs with OCPs.

11.4.4.4 Combination of AIs with GnRH Agonists

One prospective randomized trial examined whether the addition of anastrozole to goserelin is superior to goserelin alone in terms of adjuvant therapy after conservative surgery [82]. Forty women with stage IV (the revised American Society for Reproductive Medicine score > 40) severe endometriosis received either goserelin (3.6 mg/4 weeks) alone or goserelin + anastrozole (1 mg/day). Compared with goserelin alone, 6 months of treatment with goserelin + anastrozole significantly increased the pain-free interval (1.7 months versus >2.4 months) and decreased symptom recurrence rates until 2 years (35 % versus 7.5 %). The postmenopausal quality of life and bone mineral density at 2 years after medical therapy remained unaffected.

These studies were designed to evaluate the efficacy of AIs combined with progestins or GnRH agonists and actually suggested that the addition of AIs is beneficial for pain relief but may cause unfavorable side effects. Therefore, which is better for combination with AIs in terms of risk and benefit: progestins or GnRH agonists? A randomized prospective study was designed to compare the efficacy and tolerability of AIs combined with either progestin or a GnRH agonist [83]. Women with rectovaginal endometriosis were treated with letrozole (2.5 mg/day) for 6 months and were randomized to also receive either oral norethisterone acetate (2.5 mg/day) or triptorelin (11.25 mg every 3 months). The reduction in the intensity of nonmenstrual pelvic pain and deep dyspareunia did not differ between the 2 medications, whereas the interruption of treatment and bone mineral loss were more frequent and severe and patient satisfaction was therefore lower in the triptorelin group. AIs possibly enhance the efficacy of conventional medicines for endometriosis in premenopausal women; however, they may cause intolerable side effects.

11.4.5 AIs for the Treatment of Infertility in Women with Endometriosis

In 2001, a new application of letrozole for ovulation induction in clomiphene citrate (CC)-resistant anovulatory women was reported [84]. The administration of letrozole in the early follicular phase would release the pituitary/hypothalamic axis from estrogenic negative feedback and increase FSH, somewhat similar to the mechanisms of CC.

The clinical features of AIs as ovulation-inducing medicines differ from those of CC. CC causes thinning of the endometrium during the therapeutic cycle and decreases the cervical mucus, whereas AIs do not. This negative effect on the endometrium explains the reason why the pregnancy rate of CC-treated women is somewhat lower than that expected from the ovulation rate. AIs do not have the negative effects on the endometrium. A reason for the difference is the half-life of the medicine: CC, particularly an isomer of CC existing in the medicine, lasts longer in the body and exerts an antiestrogenic action for a longer period than letrozole: the half-lives are 5–7 days and 45 h for CC and letrozole, respectively. In addition, CC increases circulating estrogen, which binds to ER and accelerates its degradation. Thus, it takes time to recover estrogen responsibility through de novo synthesis of ER after CC is eliminated from the blood. In comparison, AIs do not induce ER degradation and are rapidly eliminated from the body after the cessation of administration. Thus, the endometrium maintains its sensitivity to estrogen and quickly recovers to the level of untreated cycles after the cessation of medication, whereas estrogen levels are decreased to half to one-third of the levels observed in natural cycles. This supports the absence of any direct antiestrogenic effects of letrozole on the endometrium [85].

Despite the lack of a negative effect on the endometrium, meta-analysis does not support that AIs achieve higher pregnancy rates than CC for women with polycystic ovary syndrome. Meta-analyses of 6 randomized controlled trials comparing letrozole with CC demonstrated that letrozole improved the ovulation rate per patient, without a statistically significant difference in the ovulation rate per cycle or pregnancy, live birth, multiple pregnancy, or miscarriage rates [86]. Treatment with letrozole for unexplained infertility is almost equally effective to that with CC; however, it may have some advantages in terms of low serum estradiol levels, the pregnancy rate per cycle, and the abortion rate [708788]. There is a report on the efficacy of AIs for infertility in women with endometriosis: the pregnancy rate with letrozole was the same as that with CC alone in an intrauterine insemination program for women with minimal to mild endometriosis who did not achieve pregnancy after 6–12 months following laparoscopic treatment [89].

The use of AIs for ovulation induction is an off-label use and the safety has not been established. A concern about teratogenicity was reported in an abstract in 2005; however, there have been no additional studies published since then. In contrast, a large multicenter retrospective study showed that compared with CC, AIs do not increase congenital cardiac anomalies [90].

11.4.6 AIs for the Treatment of Endometriosis in Postmenopausal Women

Endometriosis spontaneously resolves after menopause; however, endometriosis may progress and cause symptoms, albeit rare. Such patients have been treated with surgery, including hysterectomy, at earlier ages and experience recurrent endometriosis-related pain. AIs have been reported to be successful in the treatment of postmenopausal women with endometriosis [9192]. As expected, no additional medication is required to prevent FSH increases during AIs use.

11.5 Conclusions

Endometriotic tissues overexpress aromatase and synthesize estrogen in situ, which may play roles in pathogenesis and progression by enhancing auto-implantation, proliferation, and angiogenesis. Researchers propose several mechanisms underlying this overexpression, namely switching of the transcriptional regulators from COUP-TF to SF1; overexpression of COX-2, MIF, and cytokines; and changes in the methylation status of CpG islands in CYP19A1.

Clinical trials of AIs have been conducted and have revealed that AIs reduce endometriosis-related pain, particularly in recurrent cases. The effectiveness supports the notion that locally synthesized estrogen plays a role in the progression of endometriosis, unlike anticipated. Short-term use of AIs (less than 6 months) either as monotherapy or in combination with other medications for ovulation inhibition reduced endometriosis-related pain without significant severe side effects. Future studies will be required to confirm these conclusions.

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