We previously demonstrated that 17beta hydroxysteroid dehydrogenase type 2, the enzyme that inactivates estradiol to estrone, is expressed in luteal eutopic endometrium in response to progesterone but not in simultaneously biopsied peritoneal endometriotic tissue. This molecular evidence of progesterone resistance, together with the clinical observation of resistance of endometriosis to treatment with progestins, led us to determine the levels of progesterone receptor (PR) isoforms PR-A and PR-B in eutopic endometrial and extra-ovarian endometriotic tissues. It was proposed that progesterone action on target genes is mediated primarily by homodimers of PR-B, whereas the truncated variant PR-A acts as a repressor of PR-B function. Immunoprecipitation, followed by Western blot analysis, was performed to detect bands specific for PR-A and PR-B in paired samples of endometriotic and eutopic endometrial tissues simultaneously biopsed from 18 women undergoing laparoscopy during various phases of the menstrual cycle. PR-B was present in 17 of 18 eutopic endometrial samples, and its level increased in the preovulatory phase, as expected, whereas PR-A was detected in all samples (n = 18) with a similar, but less prominent, cyclic variation in its levels. In endometriotic samples, however, no detectable PR-B could be demonstrated, whereas PR-A was detected in all samples (n = 18), albeit in much lower levels and without any cyclic variation in contrast with the eutopic endometrium. Levels of PR-A and PR-B in endometriotic and eutopic endometrial tissues were determined and compared after normalization to total protein and estrogen receptor-alpha levels. Using RNase protection assay, we also demonstrated indirectly that only PR-A transcripts were present in endometriotic tissue samples (n = 8), whereas both PR-A and PR-B transcripts were readily detectable in all eutopic endometrial samples (n = 8). This was indicative that failure to detect PR-B protein in endometriotic tissues is due to the absence of PR-B transcripts. We conclude that progesterone resistance in endometriotic tissue from laboratory and clinical observations may be accounted for by the presence of the inhibitory PR isoform PR-A and the absence of the stimulatory isoform PR-B.

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Aberrant aromatase expression in stromal cells of endometriosis gives rise to conversion of circulating androstenedione to estrone in this tissue, whereas aromatase expression is absent in the eutopic endometrium. In this study, we initially demonstrated by Northern blotting transcripts of the reductive 17beta-hydroxysteroid dehydrogenase (17betaHSD) type 1, which catalyzes the conversion of estrone to 17beta-estradiol, in both eutopic endometrium and endometriosis. Thus, it follows that the product of the aromatase reaction, namely estrone, that is weakly estrogenic can be converted to the potent estrogen, 17beta-estradiol, in endometriotic tissues. It was previously demonstrated that progesterone stimulates the inactivation of 17beta-estradiol through conversion to estrone in eutopic endometrial epithelial cells. Subsequently, 17betaHSD type 2 was shown to catalyze this reaction, and its transcripts were detected in the epithelial cell component of the eutopic endometrium in secretory phase. Because 17beta-estradiol plays a critical role in the development and growth of endometriosis, we studied 17betaHSD-2 expression in endometriotic tissues and eutopic endometrium. We demonstrated, by Northern blotting, 17betaHSD-2 messenger ribonucleic acid (RNA) in all RNA samples of secretory eutopic endometrium (n=12) but not in secretory samples of endometriotic lesions (n=10), including paired samples of endometrium and endometriosis obtained simultaneously from eight patients. This messenger RNA was not detectable in any samples of proliferative eutopic endometrium or endometriosis (n=4) as expected. Next, we confirmed these findings by demonstration of immunoreactive 17betaHSD-2 in epithelial cells of secretory eutopic endometrium in 11 of 13 samples employing a monoclonal antibody against 17betaHSD-2, whereas 17betaHSD-2 was absent in paired secretory endometriotic tissues (n=4). Proliferative eutopic endometrial (n=8) and endometriotic (n=4) tissues were both negative for immunoreactive 17betaHSD-2, except for barely detectable levels in 1 eutopic endometrial sample. Finally, we sought to determine whether deficient 17betaHSD-2 expression in endometriotic tissues is due to impaired progesterone action in endometriosis. We determined by immunohistochemistry the expression of progesterone and estrogen receptors in these paired samples of secretory (n=4) and proliferative (n=4) eutopic endometrium and endometriosis, and no differences could be demonstrated. In conclusion, inactivation of 17beta-estradiol is impaired in endometriotic tissues due to deficient expression of 17betaHSD-2, which is normally expressed in eutopic endometrium in response to progesterone. The lack of 17betaHSD-2 expression in endometriosis is not due to alterations in the levels of immunoreactive progesterone or estrogen receptors in this tissue and may be related to an inhibitory aberration in the signaling pathway that regulates 17betaHSD-2 expression.

No abstract

In stromal cells of endometriosis, marked levels of aromatase P450 (P450arom) mRNA and activity are present and can be vigorously stimulated by (Bu)2cAMP or PGE2 to give rise to physiologically significant estrogen biosynthesis. Since eutopic endometrial tissue or stromal cells lack P450arom expression, we studied the molecular basis for differential P450arom expression in endometriosis and eutopic endometrium. First, we demonstrated by rapid amplification of cDNA 5'-ends that P450arom expression in pelvic endometriotic lesions is regulated almost exclusively via the alternative promoter II. Then, luciferase reporter plasmids containing deletion mutations of the 5'-flanking region of promoter II were transfected into endometriotic stromal cells. We identified two critical regulatory regions for cAMP induction of promoter II activity: 1) a-214/-100 bp proximal region responsible for a 3.7-fold induction, and 2) a -517/ -214 distal region responsible for potentiation of cAMP response up to 13-fold. In the -214/-100 region, we studied eutopic endometrial and endometriotic nuclear protein binding to a nuclear receptor half-site (NRHS, AGGTCA) and an imperfect cAMP response element (TGCACGTCA). Using electrophoretic mobility shift assay, cAMP response element-binding activity in nuclear proteins from both endometriotic and eutopic endometrial cells gave rise to formation of identical DNA-protein complexes. The NRHS probe, on the other hand, formed a distinct complex with nuclear proteins from endometriotic cells, which migrated at a much faster rate compared with the complex formed with nuclear proteins from eutopic endometrial cells. Employing recombinant proteins and antibodies against steroidogenic factor-1 (SF-1) and chicken ovalbumin upstream promoter transcription factor (COUP-TF), we demonstrated that COUP-TF but not SF-1 bound to NRHS in eutopic endometrial cells, whereas SF-1 was the primary NRHS-binding protein in endometriotic cells. In fact, COUP-TF transcripts were present in both eutopic endometrial (n = 12) and endometriotic tissues (n = 8), whereas SF-1 transcripts were detected in all endometriotic tissues (n = 12), but in only 3 of 15 eutopic endometrial tissues. Moreover, we demonstrated a dose-dependent direct competition between SF-1 and COUP-TF for occupancy of the NRHS, to which SF-1 bound with a higher affinity. Finally, overexpression of SF-1 in eutopic endometrial and endometriotic cells strikingly potentiated baseline and cAMP-induced activities of -517 promoter II construct, whereas overexpression of COUP-TF almost completely abolished these activities. In conclusion, COUP-TF might be one of the factors responsible for the inhibition of P450arom expression in eutopic endometrial stromal cells, which lack SF-1 expression in the majority (80%) of the samples; in contrast, aberrant SF-1 expression in endometriotic stromal cells can override this inhibition by competing for the same DNA-binding site, which is likely to account for high levels of baseline and cAMP-induced aromatase activity.

Aberrant aromatase expression in stromal cells of endometriosis gives rise to conversion of circulating androstenedione to estrone in this tissue, whereas aromatase expression is absent in the eutopic endometrium. In this study, we initially demonstrated by Northern blotting transcripts of the reductive 17beta-hydroxysteroid dehydrogenase (17betaHSD) type 1, which catalyzes the conversion of estrone to 17beta-estradiol, in both eutopic endometrium and endometriosis. Thus, it follows that the product of the aromatase reaction, namely estrone, that is weakly estrogenic can be converted to the potent estrogen, 17beta-estradiol, in endometriotic tissues. It was previously demonstrated that progesterone stimulates the inactivation of 17beta-estradiol through conversion to estrone in eutopic endometrial epithelial cells. Subsequently, 17betaHSD type 2 was shown to catalyze this reaction, and its transcripts were detected in the epithelial cell component of the eutopic endometrium in secretory phase. Because 17beta-estradiol plays a critical role in the development and growth of endometriosis, we studied 17betaHSD-2 expression in endometriotic tissues and eutopic endometrium. We demonstrated, by Northern blotting, 17betaHSD-2 messenger ribonucleic acid (RNA) in all RNA samples of secretory eutopic endometrium (n=12) but not in secretory samples of endometriotic lesions (n=10), including paired samples of endometrium and endometriosis obtained simultaneously from eight patients. This messenger RNA was not detectable in any samples of proliferative eutopic endometrium or endometriosis (n=4) as expected. Next, we confirmed these findings by demonstration of immunoreactive 17betaHSD-2 in epithelial cells of secretory eutopic endometrium in 11 of 13 samples employing a monoclonal antibody against 17betaHSD-2, whereas 17betaHSD-2 was absent in paired secretory endometriotic tissues (n=4). Proliferative eutopic endometrial (n=8) and endometriotic (n=4) tissues were both negative for immunoreactive 17betaHSD-2, except for barely detectable levels in 1 eutopic endometrial sample. Finally, we sought to determine whether deficient 17betaHSD-2 expression in endometriotic tissues is due to impaired progesterone action in endometriosis. We determined by immunohistochemistry the expression of progesterone and estrogen receptors in these paired samples of secretory (n=4) and proliferative (n=4) eutopic endometrium and endometriosis, and no differences could be demonstrated. In conclusion, inactivation of 17beta-estradiol is impaired in endometriotic tissues due to deficient expression of 17betaHSD-2, which is normally expressed in eutopic endometrium in response to progesterone. The lack of 17betaHSD-2 expression in endometriosis is not due to alterations in the levels of immunoreactive progesterone or estrogen receptors in this tissue and may be related to an inhibitory aberration in the signaling pathway that regulates 17betaHSD-2 expression.

Estrogen is the most important known factor that stimulates the growth of endometriosis. Estrogen delivery to endometriotic implants was classically viewed to be only via the circulating blood in an endocrine fashion. We recently uncovered an autocrine positive feedback mechanism, which favored the continuous production of estrogen and prostaglandin (PG)E 2 in the endometriotic stromal cells. The enzyme, aromatase, is aberrantly expressed in endometriotic stromal cells and catalyzes the conversion of C 19 steroids to estrogens, which then stimulate cyclooxygenase-2 to increase the levels of PGE 2 . PGE 2 , in turn, is a potent inducer of aromatase activity in endometriotic stromal cells. Aromatase is not expressed in the eutopic endometrium. Aromatase expression in endometriosis and its inhibition in eutopic endometrium are controlled by the competitive binding of a stimulatory transcription factor, steroidogenic factor-1, and an inhibitory factor, chicken ovalbumin upstream promotertranscription factor to a regulatory element in the aromatase P450 gene promoter. In addition, we find that endometriotic tissue is deficient in 17β-hydroxysteroid dehydrogenase type 2, which is normally expressed in eutopic endometrial glandular cells and inactivates estradiol-17β to estrone. This deficiency is another aberration that favors higher levels of estradiol-17β in endometriotic tissues in comparison with the eutopic endometrium. The clinical relevance of local aromatase expression in endometriosis was exemplified by the successful treatment of an unusually aggressive form of recur- Endocrine-Related Cancer (1999) 6 293-301 rent endometriosis in a postmenopausal woman using an aromatase inhibitor.

Although treatment of one unusually aggressive case of postmenopausal endometriosis with an aromatase inhibitor has been strikingly successful, large clinical trials are required to establish whether aromatase inhibitors will have a significant role in the medical management of endometriosis. Introduction of aromatase inhibitors into the treatment of endometriosis underscores the importance of basic research leading to the development of novel strategies in reproductive disorders. It was shown earlier that aromatase activity was not detectable in normal endometrium. Aromatase, however, is expressed inappropriately in endometriosis and stimulated by prostaglandin E2. Aromatase activity gives rise to local biosynthesis of oestrogen, which, in turn, stimulates prostaglandin E2 production, thus establishing a positive feedback cycle. This favours accumulation of oestrogen and prostaglandins in endometriosis, which is an inflammatory disorder dependent on oestrogen for growth.