Inflammatory status influences aromatase and steroid receptor expression in endometriosis

Abstract

Aberrant up-regulation of aromatase in eutopic endometrium and implants from women with endometriosis has been reported. Aromatase induction may be mediated by increased cyclooxygenase-2 (COX-2). Recently, we demonstrated that progesterone receptor (PR)-A and PR-B serve an antiinflammatory role in the uterus by antagonizing nuclear factor kappaB activation and COX-2 expression. PR-C, which antagonizes PR-B, is up-regulated by inflammation. Although estrogen receptor alpha (ERalpha) is implicated in endometriosis, an antiinflammatory role of ERbeta has been suggested. We examined stage-specific expression of aromatase, COX-2, ER, and PR isoform expression in eutopic endometrium, implants, peritoneum, and endometrioma samples from endometriosis patients. Endometrial and peritoneal biopsies were obtained from unaffected women and those with fibroids. Aromatase expression in eutopic endometrium from endometriosis patients was significantly increased compared with controls. Aromatase expression in endometriosis implants was markedly increased compared with eutopic endometrium. Aromatase mRNA levels were increased significantly in red implants relative to black implants and endometrioma cyst capsule. Moreover, COX-2 expression was increased in implants and in eutopic endometrium of women with endometriosis as compared with control endometrium. As observed for aromatase mRNA, the highest levels of COX-2 mRNA were found in red implants. The ratio of ERbeta/ERalpha mRNA was significantly elevated in endometriomas compared with endometriosis implants and eutopic endometrium. Expression of PR-C mRNA relative to PR-A and PR-B mRNA was significantly increased in endometriomas compared with eutopic and control endometrium. PR-A protein was barely detectable in endometriomas. Thus, whereas PR-C may enhance disease progression, up-regulation of ERbeta may play an antiinflammatory and opposing role.

Figure 1

Aromatase/CYP19 mRNA transcripts containing untranslated exon IIa (CYP19 IIa) are up-regulated in eutopic endometrium of women with endometriosis, reaching highest levels in the proliferative phase of the menstrual cycle. Aromatase expression is manyfold higher in endometriosis implants than in eutopic endometrium among women with minimal-mild (I-II) and moderate-severe (III-IV) stage disease. A, CYP19 IIa mRNA levels were analyzed in endometrium from unaffected women (control), women with endometriosis, and those with uterine fibroids using qRT-PCR. Relative levels of CYP19 IIa mRNA were calculated by normalizing against h36B4 mRNA. Data, expressed as arbitrary units, are the mean ± sem of values from eight control samples, 12 eutopic endometrial samples, and seven fibroid samples. *, Significantly different from the others at P = 0.029. B, CYP19 IIa mRNA levels were analyzed in endometrium from unaffected women (control), women with endometriosis, and those with uterine fibroids at proliferative and secretory phases of the menstrual cycle and after medical suppression with D-MPA treatment. Relative levels of CYP19 IIa mRNA were calculated by normalizing against h36B4 mRNA. Data, expressed as arbitrary units, are the mean ± sem of values from the number of subjects indicated on the abscissa. C, CYP19 IIa mRNA levels were analyzed in eutopic endometrium and endometriosis implants using qRT-PCR, as described above. The ratios of mRNA levels in implants relative to eutopic endometrium are plotted according to the endometriosis revised American Society of Reproductive Medicine stage. Data are the mean ± sem of values from the number of subjects indicated on the abscissa.

Figure 2

Aromatase expression is higher in red endometriosis implants than in black implants and endometriomas, does not change according to menstrual phase, and is suppressed by D-MPA treatment. Visually normal peritoneum exhibits increased aromatase expression in endometriosis. A, Gonad-specific exon IIa-containing (CYP19 IIa) mRNA levels were analyzed in red and black implants and in endometrioma cyst wall determined to be free of ovarian tissue. B, CYP19 IIa mRNA levels were analyzed in endometriosis implants according to the menstrual cycle phase or with D-MPA treatment. C, CYP19 IIa mRNA levels were analyzed in biopsies of visually normal peritoneum from unaffected subjects (control) and from women with stage I-II and stage III-IV disease. Control value was 52.4 ± 16.5 arbitrary units, which cannot be visualized on this scale. For all tissues studied, CYP19 IIa mRNA was analyzed by qRT-PCR. Relative levels of CYP19 IIa mRNA were calculated by normalizing against h36B4 mRNA. Data, expressed as arbitrary units, are the mean ± sem of values from the number of subjects indicated on the abscissa.

Figure 3

Aromatase protein expression is up-regulated in eutopic endometrium and implants from women with endometriosis. A, Immunoblot of aromatase protein in three samples of control endometrial biopsies (EMB) (3635, 3712, and 3352) and in eutopic endometrium (EU) and a red implant (IMP) from a patient with stage I endometriosis. C–E, Aromatase immunostaining of red implant (C), eutopic endometrium (D), and black implant (E) from a patient with dysmenorrhea and stage I endometriosis. B, Nonimmune IgG control staining of the red implant section in C, Note immunostaining (dark red) of both stromal and epithelial cells in red and black endometriosis implants. Eutopic endometrium in late proliferative phase (D) shows intense epithelial staining, whereas the immunostaining in stroma is less intense. Aromatase immunostaining of a eutopic endometrial sample in secretory phase (F) shows lack of immunostaining in the epithelial cells with positive staining in the stroma. In an endometrioma cyst wall (G), diffuse immunostaining (dark red) of the stromal and epithelial cells can be seen. In all of these sections, epithelial (glandular) elements are indicated by a thick blue arrow, and stromal cells are indicated by a thin green arrow. Normal uterine visceral peritoneal sample (H) stained for aromatase serves as a negative control. Human placental sections were used as a positive control (G) (thick blue arrow indicates the syncytiotrophoblast). Aromatase immunostaining is indicated by the dark red color, and counterstaining with hematoxylin is in blue.

Figure 4

COX-2 mRNA expression is highest in red implants and reflects the pattern of aromatase expression. COX-2 mRNA levels were analyzed by qRT-PCR in endometrial biopsies (EMB) from unaffected subjects (control EMB), from women with uterine fibroids (fibroid EMB), and endometriosis (eutopic EMB), in red and black endometriotic implants and in endometrioma. Relative levels of COX-2 mRNA were calculated by normalizing against h36B4 mRNA. Data, expressed as arbitrary units, are the mean ± sem of values from the number of subjects indicated on the abscissa.

Figure 5

The ERβ to ERα mRNA expression ratio is increased in endometriosis implants as compared with control and eutopic endometrium and is even further up-regulated in endometriomas. ERα and ERβ mRNA levels were analyzed by qRT-PCR in endometrial biopsies (EMB) from unaffected subjects (control EMB) and from women with endometriosis (eutopic EMB) and with fibroids (fibroid EMB), in red and black peritoneal implants, and in endometriomas. Relative levels of ERα and ERβ mRNA were calculated by normalizing against h36B4 mRNA, and the ratios of ERβ to ERα mRNA were calculated. Data are the mean ± sem of ERβ/ERα in tissues from the number of subjects indicated on the abscissa. *, Significantly different (P = 0.008) from all others; +, significantly different (P = 0.013) from each other.

Figure 6

Immunostaining of ERβ is increased in endometriosis. A, Human prostate counterstained with hematoxylin after incubation with nonimmune IgG (negative control); B, human prostate ERβ immunostaining in brown is mostly in the epithelial cells and is both nuclear and cytoplasmic; C, secretory endometrium from a control patient with mostly negative epithelial and stromal ERβ immunostaining; only some light cytoplasmic staining can be seen; D, eutopic endometrium from a patient with endometriosis at late proliferative phase with intense cytoplasmic and faint nuclear staining in the epithelial cells and some faint staining in stroma as well; E, endometrioma cyst capsule showing intense nuclear immunostaining of stromal cells and epithelial cells; immunostaining in the epithelial cells is both nuclear and cytoplasmic; F, endometrioma cyst capsule from another patient, with intense staining present in nuclei and cytoplasm of the glandular elements and some sporadic staining also observed in the stroma; G, red endometriosis implant, showing diffuse and intense staining of the epithelial and immediate subepithelial layers and sporadic and faint immunostaining observed in the stromal cells; H, black endometriosis implant, with intense ERβ immunostaining seen in both epithelial and stromal cells; I, visually and histologically normal peritoneum from a patient with stage II endometriosis, with some mild ERβ immunostaining evident only in the endothelial cells (black arrow), as expected. In all sections, thick blue arrows indicate epithelial compartment, and thin green arrows indicate the stromal cells.

Figure 7

Expression of PR-C mRNA relative to PR-A and PR-B mRNAs is markedly increased in endometriosis implants and endometrioma cyst capsule, as compared with normal and eutopic endometrium. PR-A protein expression is absent in an endometrioma biopsy sample. A, Relative levels of PR-A, PR-B, and PR-C mRNAs were analyzed in control endometrial biopsies (EMB), eutopic EMB, endometriosis implants, and endometriomas by qRT-PCR using specific primers for PR-B, PR-A plus PR-B, and PR-A plus PR-B plus PR-C. The percentage of each PR isoform mRNA relative to total PR expression was calculated. Data are the mean ± sem of percentage of PR isoform mRNA for the number of subjects indicated on the abscissa. Statistical differences are discussed in the text. B, Immunoblot of PR protein expression in an endometrial biopsy from eutopic endometrium and endometrioma cyst wall from the same subject. Note lack of PR-A band in endometrioma sample.

Figure 8

Immunostaining for PR isoforms reveals absence of PR-A protein in endometrioma. A–C, Control endometrium from late proliferative phase. A, PR-A immunostaining (in brown) is localized primarily in nucleus. Both epithelial and stromal cells stain positively for PR-A; B, PR-B staining is both nuclear and cytoplasmic and somewhat more intense in cytoplasm of epithelial cells; C, immunostaining with sc-539 (total PR) antibody reveals intense cytoplasmic staining in the epithelial cells and some scattered nuclear and cytoplasmic staining in stroma. D–F, Secretory-phase eutopic endometrium from a patient with endometriosis. D, PR-A immunostaining is more intense in the nuclei of the stromal cells compared with the epithelium; E, PR-B is present in both stromal and epithelial cell nuclei, with more intense staining observed in stromal cells; F, total PR immunostaining with sc-539 reveals intense nuclear staining in both stroma and epithelia. Intense cytoplasmic staining is observed in the epithelial cells. G–I, Red endometriosis implant. G, PR-A immunostaining is localized within the stromal cells of the implant; H, PR-B staining, although mostly in the stroma, also can be observed in the epithelial cells; I, total PR immunostaining is intense, both in the stromal and epithelial cells. J–L, Endometrioma cyst capsule. J, PR-A immunostaining is barely detectable, both in epithelial and the stromal cells of the endometrioma; K, epithelial cells and some stromal cells in the same endometrioma stain well for PR-B; L, nuclear and cytoplasmic total PR immunostaining are present both in epithelial and stromal cells.