**3. Evidence of progesterone resistance in women with endometriosis**

The uterus is composed of heterogeneous cell types which undergo synchronous waves of proliferation and differentiation in response to cyclical changes in estrogen (E2) and progesterone levels. The spatiotemporal expression of PRs in epithelial, stromal and myometrial cellular compartments are under the control of estrogen, the primary endometrial mitogen. Progesterone appears to exert proliferative function or induce differentiation depending on the cell type; on epithelia, progesterone, acting via stromal PRs, inhibits estrogen-driven cell proliferation and on stroma progesterone appears to orchestrate a more complex pattern of proliferation and differentiation.

While the actions of progesterone are critical to the establishment and maintenance of pregnancy, approximately one third of women with endometriosis also present with infertility. For some women, the loss of tubal patency, the modification of the pelvis and the inflammation associated with condition appear to be causally related to the presentation of infertility. However, the characterization of the eutopic endometrium from women with endometriosis has also revealed many defects, including altered patterns of angiogenesis, dyssynchrony with the window of implantation as well as ultra structural abnormalities which may contribute to the infertility. Supporting this, isolated endometrial stromal fibroblasts from women with endometriosis do not appear to undergo a normal decidualisation response (Aghajanova et al., 2010), suggesting an impairment of the progesterone-mediated differentiation programme. Specific alterations in the expression molecular markers of endometrial receptivity have also been widely documented, especially the integrin αvβ3, certain steroid hormone receptors and HOXA10 gene expression, the latter of which has been identified from a genome wide association study of moderate to severe endometriosis as a potential candidate disease locus with proximity to the 7p15.2 SNP association (Painter et al., 2011).

Early studies also pointed to alteration in the normal pathways of estrogen metabolism in ectopic endometrial tissue; specifically expression of 17βHSD-2, the enzyme responsible for

Progesterone Resistance and Targeting

from a molecular understanding of the protein and condition.

**4. Discovery of small molecule modulators of PR function** 

have been characterised with selectivity for PR-A over PR-B or vice versa.

specialist evaluation (Ioffe et al., 2009; Mutter et al., 2008; Williams et al., 2007).

antagonist/agonist activity, such as J-867 (Table 1).

the Progesterone Receptors: A Therapeutic Approach to Endometriosis 161

al. (2004) first suggested a reduced risk of endometriosis associated with the T allele of the +331 variant and because of increased production of PR-B, this variant was suggested to reduce the risk of endometriosis. However the original findings suggesting that PR-B is not expressed in ectopic lesions have not had broad replication and Treloar et al. (2005) found no association with endometriosis and this variant either in a large study which included more than 900 families. Another putative functional variant in the *PGR* gene is termed the PROGINS allele. Cells prepared from the eutopic endometria of women carrying the PROGINS allele appear to respond with greater proliferative capacity to estradiol and progesterone, supporting the contention that the PROGINS polymorphism enhances the endometriosis phenotype (D'Amora et al., 2009). However while several studies have suggested that the variant increases susceptibility to endometriosis (De Carvalho et al., 2007; Lattuada et al., 2004; Wieser et al., 2002), others have not found an association (Govindan et al., 2007; Treloar et al., 2005; van Kaam et al., 2007). Therefore taken together, there is only modest supporting evidence of altered progesterone receptor expression, and progesterone resistance in contributing to endometriosis susceptibility and disease symptoms. Notwithstanding this, the current clinical utility of progesterone receptor agonists and anticipated benefit of PRAs, outweighs the confidence in PR as a therapeutic target attained

The identification of drug-like, potent and selective PR antagonists has been challenging. As well as being highly lipophilic, the ligand binding sites between homologous NHRs are highly conserved and PR has the highest sequence homology with GR, AR, MR and ER (Figure 1). Early classes of anti-progestagens were poorly selective, yet some, such as gestrinone, still found clinical utility in the treatment of endometriosis (Cornillie et al., 1986; Coutinho, 1982). Furthermore, the ligand binding domains of PR-A and PR-B are identical and yet several in vitro and in vivo lines of evidence suggest that the effects of progesterone on transcriptional activation and repression by PR-A and PR-B are different (Conneely et al., 2001; Tung et al., 2006). To date, however, there are no agonist or antagonist agents that

The development of selective and safe steroidal PRAs has been challenging, both due to reported hepatoxicity as well as potential dose-limiting anti-glucocorticoid effects, due to lack of selectivity (Robertson et al., 1999). More recently, an additional concern has emerged following histological evaluations of subjects dosed for more than 3 months on steroidal PRAs. The endometrium of these individuals undergoes a characteristic cystic histological change which may be difficult to distinguish from endometrial hyperplasia without

Mifepristone/RU-486, the founding member of the steroidal class of PRAs, was originally produced by Roussel-Uclaf and licensed for use for medical abortion and as an emergency contraceptive. The in vivo pharmacokinetic/pharmacodynamic profile of RU-486 is challenging to model as RU-486 generates a large number of pharmacologically active metabolites (Heikinheimo et al., 1987). Since its identification, >100 related analogues, principally by modifying the C-11 and/or C-17 positions of the steroid ring, have been synthesized and have shown all degrees of anti-progestagenic activity. The pharmacological profile of these examples range from pure antagonists, such as RU-486 to those with mixed

the conversion of estradiol to estrone appears to be reduced compared with the eutopic compartment (Bulun et al., 2010; Zeitoun et al., 1998). As 17βHSD-2 is a progesterone response gene, one group has published evidence to suggest that this may be due to apparent reduction in PR levels and especially in PR-B in ectopic tissue (Attia et al., 2000; Wu et al., 2006). However, this signpost to progesterone resistance has had relatively little formal observational replication; indeed others have not been able to find evidence of alteration in the PR-A/PR-B ratio (Bergqvist & Ferno, 1993; Igarashi et al., 2005). Furthermore, decrease in PR-A mRNA and an increase in the PR-B to PR-A ratio and total PR protein levels have been detected in eutopic samples obtained from a murine endometriosis model (Lee et al., 2009). PR expression has also been found to be similarly unaltered in the eutopic endometrium of baboons with experimentally induced endometriosis compared with baseline (Fazleabas et al., 2003).

Microarray studies performed on isolated cells, eutopic/ectopic tissue biopsies and cells excised by laser capture microdissection from patients have been revealed several pathways of altered gene expression. For instance Kao et al (2002; 2003) collected biopsies from eutopic endometrium from normal women and women with endometriosis at days 8-10 after the mid-cycle LH surge and performed a microarray analysis to identify differentially expressed genes. Whilst a formal analysis of progesterone-response genes, whose expression was either elevated or suppressed in diseased versus normal tissue, was not undertaken, the expression of several progesterone response genes, including Dickkopf-1 and glycodelin, was suppressed in diseased tissues samples. The suppression of the Dickkopf-1 response has been supported by more recent studies of progesterone response in isolated human endometrial stromal fibroblasts from normal women and women with endometriosis (Aghajanova et al., 2011). In studies comparing ectopic and eutopic gene expression obtained from women with endometriosis one of the key observational fingerprints was an impairment in the normal progesterone response, especially the expression of PR, IHH, FOXO1A and Cyp26A1 amongst others (Burney et al., 2007). Relatively few genes have been qualified as progesterone responsive by testing with a PRA and this has limited larger data assignments in published microarray data. Even when microarray analysis was performed on RNA extracted from human endometrial explants cultured in the presence of E2/P4 and treated with RU-486, only a small population of gene expression were differentially regulated (Catalano et al., 2003). Of these, JAK1 and JNK1 appeared to be down regulated in the presence of RU-486. These observations are intriguing as JNK activity is unregulated in women with endometriosis (Uz et al., 2011) and in a *Scid* mouse experimental model of endometriosis, JNK inhibitor treatment reduces disease burden (Altan et al., 2008).

Taken together, while the data support the notion that there is an abnormal progesterone response in eutopic and ectopic endometrial compartments in women with endometriosis, it is not clear whether this is a direct effect, causally associated with infertility, or could be used for diagnostic purposes. One of the key gaps is that many of these genes have not been formally tested to be directly PR mediated, and therefore lack the qualification of alteration.

Another, somewhat controversial, observation linked with the molecular basis of progesterone resistance has been revealed from studying functional polymorphisms in the promotor region of PR. A putative functional polymorphism in the PR promoter (+331C/T; rs10895068), creates an additional TATA box that provides a unique transcriptional start site and favours increased production of PR-B relative to PR-A (De Vivo et al., 2002). Berchuck et

the conversion of estradiol to estrone appears to be reduced compared with the eutopic compartment (Bulun et al., 2010; Zeitoun et al., 1998). As 17βHSD-2 is a progesterone response gene, one group has published evidence to suggest that this may be due to apparent reduction in PR levels and especially in PR-B in ectopic tissue (Attia et al., 2000; Wu et al., 2006). However, this signpost to progesterone resistance has had relatively little formal observational replication; indeed others have not been able to find evidence of alteration in the PR-A/PR-B ratio (Bergqvist & Ferno, 1993; Igarashi et al., 2005). Furthermore, decrease in PR-A mRNA and an increase in the PR-B to PR-A ratio and total PR protein levels have been detected in eutopic samples obtained from a murine endometriosis model (Lee et al., 2009). PR expression has also been found to be similarly unaltered in the eutopic endometrium of baboons with experimentally induced

Microarray studies performed on isolated cells, eutopic/ectopic tissue biopsies and cells excised by laser capture microdissection from patients have been revealed several pathways of altered gene expression. For instance Kao et al (2002; 2003) collected biopsies from eutopic endometrium from normal women and women with endometriosis at days 8-10 after the mid-cycle LH surge and performed a microarray analysis to identify differentially expressed genes. Whilst a formal analysis of progesterone-response genes, whose expression was either elevated or suppressed in diseased versus normal tissue, was not undertaken, the expression of several progesterone response genes, including Dickkopf-1 and glycodelin, was suppressed in diseased tissues samples. The suppression of the Dickkopf-1 response has been supported by more recent studies of progesterone response in isolated human endometrial stromal fibroblasts from normal women and women with endometriosis (Aghajanova et al., 2011). In studies comparing ectopic and eutopic gene expression obtained from women with endometriosis one of the key observational fingerprints was an impairment in the normal progesterone response, especially the expression of PR, IHH, FOXO1A and Cyp26A1 amongst others (Burney et al., 2007). Relatively few genes have been qualified as progesterone responsive by testing with a PRA and this has limited larger data assignments in published microarray data. Even when microarray analysis was performed on RNA extracted from human endometrial explants cultured in the presence of E2/P4 and treated with RU-486, only a small population of gene expression were differentially regulated (Catalano et al., 2003). Of these, JAK1 and JNK1 appeared to be down regulated in the presence of RU-486. These observations are intriguing as JNK activity is unregulated in women with endometriosis (Uz et al., 2011) and in a *Scid* mouse experimental model of

endometriosis, JNK inhibitor treatment reduces disease burden (Altan et al., 2008).

Taken together, while the data support the notion that there is an abnormal progesterone response in eutopic and ectopic endometrial compartments in women with endometriosis, it is not clear whether this is a direct effect, causally associated with infertility, or could be used for diagnostic purposes. One of the key gaps is that many of these genes have not been formally tested to be directly PR mediated, and therefore lack the qualification of alteration. Another, somewhat controversial, observation linked with the molecular basis of progesterone resistance has been revealed from studying functional polymorphisms in the promotor region of PR. A putative functional polymorphism in the PR promoter (+331C/T; rs10895068), creates an additional TATA box that provides a unique transcriptional start site and favours increased production of PR-B relative to PR-A (De Vivo et al., 2002). Berchuck et

endometriosis compared with baseline (Fazleabas et al., 2003).

al. (2004) first suggested a reduced risk of endometriosis associated with the T allele of the +331 variant and because of increased production of PR-B, this variant was suggested to reduce the risk of endometriosis. However the original findings suggesting that PR-B is not expressed in ectopic lesions have not had broad replication and Treloar et al. (2005) found no association with endometriosis and this variant either in a large study which included more than 900 families. Another putative functional variant in the *PGR* gene is termed the PROGINS allele. Cells prepared from the eutopic endometria of women carrying the PROGINS allele appear to respond with greater proliferative capacity to estradiol and progesterone, supporting the contention that the PROGINS polymorphism enhances the endometriosis phenotype (D'Amora et al., 2009). However while several studies have suggested that the variant increases susceptibility to endometriosis (De Carvalho et al., 2007; Lattuada et al., 2004; Wieser et al., 2002), others have not found an association (Govindan et al., 2007; Treloar et al., 2005; van Kaam et al., 2007). Therefore taken together, there is only modest supporting evidence of altered progesterone receptor expression, and progesterone resistance in contributing to endometriosis susceptibility and disease symptoms. Notwithstanding this, the current clinical utility of progesterone receptor agonists and anticipated benefit of PRAs, outweighs the confidence in PR as a therapeutic target attained from a molecular understanding of the protein and condition.
