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. 2009 Jan;80(1):105-14.
doi: 10.1095/biolreprod.108.070300. Epub 2008 Sep 24.

Steroidogenic enzyme and key decidualization marker dysregulation in endometrial stromal cells from women with versus without endometriosis

L Aghajanova  1 , A Hamilton, J Kwintkiewicz, K C Vo, L C Giudice
Affiliations

Steroidogenic enzyme and key decidualization marker dysregulation in endometrial stromal cells from women with versus without endometriosis

L Aghajanova et al. Biol Reprod. 2009 Jan.

Abstract

Identification of mechanisms underlying endometriosis pathogenesis will facilitate understanding and treatment of infertility and pain associated with this disorder. Herein, we investigated the expression of steroidogenic pathway enzymes and key decidualization biomarkers in endometrial tissue and in eutopic endometrial stromal fibroblasts (hESFs) from women with vs. those without endometriosis, and subsequently treated in vitro with 8-bromo-cAMP (8-Br-cAMP) or progesterone (P4). Real-time quantitative PCR, immunohistochemistry, ELISA, and radiometric aromatase activity assay were used. The results demonstrate significantly increased (14.5-fold; P=0.037) expression of aromatase in eutopic endometrium of women with disease. In 8-Br-cAMP-treated hESF from eutopic endometrium of women with endometriosis, the balance in estradiol (E2) and P4 biosynthetic and metabolizing enzymes is disturbed (decreased HSD3B1 and HSD17B2, and increased HSD17B1 and aromatase), with the equilibrium being shifted towards an E2-enriched milieu. However, hESF from the same group of women treated with P4 did not demonstrate such responsiveness. Lower expression of IGFBP1 and prolactin mRNA and protein was observed in hESF from women with vs. those without endometriosis in response to 8-Br-cAMP, but not P4, suggesting a blunted response of these decidual biomarkers to activation of the PKA pathway in eutopic endometrium in women with disease. The dichotomy of 8-Br-cAMP regulation of select steroidogenic enzymes leading to an enriched E2 milieu within the endometrium and a blunted response of decidual biomarkers to this decidualizing agent of hESF from women with endometriosis suggests resistance to full decidualization of the stromal fibroblasts and mechanisms underlying implantation failure and the pathophysiology of this disorder.

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Figures

FIG. 1.
FIG. 1.
Expression of mRNA for steroidogenic pathway enzymes in tissue biopsies of eutopic MSE from women with endometriosis (n = 5) expressed as fold change to expression in MSE from women without endometriosis (n = 5), as revealed by real-time RT-PCR. *Significance accepted at P ≤ 0.05 (Mann-Whitney test). Error bars represent ± SEM.
FIG. 2.
FIG. 2.
A) Expression of mRNA for steroidogenic pathway enzymes in human endometrial stromal cells from eutopic endometrium from women with (n = 7) and without endometriosis (n = 5) treated with 0.5 mM 8-Br-cAMP for 96 h, expressed as fold change to the expression in 96-h no-treatment controls, as revealed by real-time RT-PCR. *Significance accepted at P ≤ 0.05 (Mann-Whitney test). Error bars represent ± SEM. B) Expression of IGFBP1 and PRL mRNA in endometrial stromal fibroblasts (hESFs) from women with (n = 7) and without endometriosis (n = 5) decidualized in vitro with 0.5 mM 8-Br-cAMP for 96 h, expressed as fold change to the expression in 96-h no-treatment controls (left side of B) and expression of IGFBP1 and PRL mRNA in hESFs from women with (n = 20) and without endometriosis (n = 7) decidualized in vitro for 14 days with P4, expressed as fold change to the expression in 14 days no hormone controls (right side of B), as revealed by real-time RT-PCR. The y-axis is presented as a log scale. *Significance accepted at P ≤ 0.05 (Mann-Whitney test). Error bars represent ± SEM. C) Levels of secreted IGFBP1 and PRL in culture medium from cultured hESFs from women with (n = 7) and without (n = 5) endometriosis decidualized in vitro with 0.5 mM 8-Br-cAMP for 96 h. *Significance accepted at P ≤ 0.05 (two-tailed type 3 Student t-test). Error bars represent ± SEM.
FIG. 3.
FIG. 3.
Representative picture of HSD3B immunostaining of MSE from women without endometriosis (n = 4) (A), with endometriosis (n = 4) (B), nondecidualized (C) and decidualized with 8-Br-cAMP (D) hESFs from women without endometriosis (n = 4), as well as nondecidualized (E) and decidualized with 8-Br-cAMP (F) hESFs from women with endometriosis (n = 4). Immunostaining of placental tissue at 20-wk gestational age, which was used as a positive control (G), demonstrates the expression of HSD3B protein in syncytiotrophoblast cells of placental villi. Cytotrophoblasts lying underneath the syncytial layer do not contain detectable levels of this enzyme and serve as an endogenous negative control. Original magnification ×200.
FIG. 4.
FIG. 4.
Representative picture of HSD17B2 immunostaining of MSE from women without endometriosis (n = 4) (A), with endometriosis (n = 4) (B), nondecidualized (C) and decidualized with 8-Br-cAMP (D) hESFs from women without endometriosis (n = 4), as well as nondecidualized (E) and decidualized with 8-Br-cAMP (F) hESFs from women with endometriosis (n = 4). HSD17B2 antibody in placental sections cross-reacted with endothelial cells around the perivascular capillary network (G). Negative control samples did not show immunoreactivity: H (hESFs) and I MSE. Original magnification ×200.
FIG. 5.
FIG. 5.
Aromatase (CYP19A1) activity of endometrial stromal fibroblasts (hESFs) from women with (n = 4) and without endometriosis (n = 4) treated in vitro with or without 8-Br-cAMP for 96 h, with subsequent incubation with 12.5 nM of [1β-3H]androstenedione for 16 h. Results are expressed as picomoles of [3H]H2O formed in 16 h/mg total protein. Cells from each patient were run in triplicates. *Significance accepted at P < 0.05 (one-way ANOVA with the Tukey-Kramer Multiple comparisons test). Error bars represent ± SEM.
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