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. 2011 Apr;84(4):801-15.
doi: 10.1095/biolreprod.110.086181. Epub 2010 Sep 23.

Unique transcriptome, pathways, and networks in the human endometrial fibroblast response to progesterone in endometriosis

L Aghajanova  1 , K Tatsumi, J A Horcajadas, A M Zamah, F J Esteban, C N Herndon, M Conti, L C Giudice
Affiliations

Unique transcriptome, pathways, and networks in the human endometrial fibroblast response to progesterone in endometriosis

L Aghajanova et al. Biol Reprod. 2011 Apr.

Abstract

Eutopic endometrium in endometriosis has molecular evidence of resistance to progesterone (P(4)) and activation of the PKA pathway in the stromal compartment. To investigate global and temporal responses of eutopic endometrium to P(4), we compared early (6-h), intermediate (48-h), and late (14-Day) transcriptomes, signaling pathways, and networks of human endometrial stromal fibroblasts (hESF) from women with endometriosis (hESF(endo)) with hESF from women without endometriosis (hESF(nonendo)). Endometrial biopsy samples were obtained from subjects with and without mild peritoneal endometriosis (n = 4 per group), and hESF were isolated and treated with P(4) (1 μM) plus estradiol (E(2)) (10 nM), E(2) alone (10 nM), or vehicle for up to 14 days. Total RNA was subjected to microarray analysis using a Gene 1.0 ST (Affymetrix) platform and analyzed by using bioinformatic algorithms, and data were validated by quantitative real-time PCR and ELISA. Results revealed unique kinetic expression of specific genes and unique pathways, distinct biological and molecular processes, and signaling pathways and networks during the early, intermediate, and late responses to P(4) in both hESF(nonendo) and hESF(endo), although a blunted response to P(4) was observed in the latter. The normal response of hESF to P(4) involves a tightly regulated kinetic cascade involving key components in the P(4) receptor and MAPK signaling pathways that results in inhibition of E(2)-mediated proliferation and eventual differentiation to the decidual phenotype, but this was not established in the hESF(endo) early response to P(4). The abnormal response of this cell type to P(4) may contribute to compromised embryonic implantation and infertility in women with endometriosis.

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Figures

FIG. 1.
FIG. 1.
Experimental design. Treatment groups were similar for hESFnonendo and hESFendo. For analysis of microarray and QRT-PCR data, all groups were normalized to t = 0, and then the normalization was conducted within each time-group: E2P4 was normalized to E2, which was normalized to the vehicle control, with the resulting data reflecting the pure P4 response.
FIG. 2.
FIG. 2.
Clustering and cluster trees. A) Principal component analysis of hESFnonendo and hESFendo at t = 0, 6, and 48 h and at 14 days, treated with or without E2 and P4. PCA was applied to all samples that were characterized by the gene expression of all probes on a Gene 1.0 ST platform. Blue, no endometriosis samples; red, endometriosis samples. B) Hierarchical clustering analysis of hESFnonendo and hESFendo at t = 0, 6, and 48 h and at 14 days, treated with or without (control [c]) estrogen (E2), and progesterone (P4), using only the profiles of significantly regulated genes. The heat map represents relative expression levels of genes in the hESF samples: each horizontal line represents a single gene, and each column represents a single sample. The relative expression of each gene is color coded as high (red) or low (blue) or no change (yellow). Groups: A, E2 14-Day endo; B, c 14-Day endo; C, E2 48-h endo; D, c 48-h endo; E, E2P4 48-h endo; F, E2P4 14-Day endo; G, E2 48-h nonendo; H, c 48-h nonendo; I, E2P4 48-h nonendo; J, E2 14-Day nonendo; K, c 14-Day nonendo; L, E2P4 14-h nonendo; M, E2 6-h endo; N, E2P4 6-h endo; O, E2 6-h nonendo; P, E2P4 6-h nonendo; Q, c 6-h nonendo; R, t = 0 endo; S, t = 0 nonendo; T, c 6-h endo.
FIG. 3.
FIG. 3.
Validation of microarray gene expression profiling by QRT-PCR. Right column indicates fold change expression of genes in the microarray data set in the present study. Left column indicates QRT-PCR validation of microarray data of gene expression in hESFnonendo and hESFendo treated with or without E2 and P4 for 6 h, 48 h, and 14 days (n = 4 in each group). Y axis displays the fold change of expression in hESF treated withP4 and E2, relative to E2 and normalized to the vehicle control and t = 0 at each time point. *, Significance accepted at P ≤ 0.05. #, Significance accepted at P = 0.05. Error bars represent means ± SEM.
FIG. 3.
FIG. 3.
Validation of microarray gene expression profiling by QRT-PCR. Right column indicates fold change expression of genes in the microarray data set in the present study. Left column indicates QRT-PCR validation of microarray data of gene expression in hESFnonendo and hESFendo treated with or without E2 and P4 for 6 h, 48 h, and 14 days (n = 4 in each group). Y axis displays the fold change of expression in hESF treated withP4 and E2, relative to E2 and normalized to the vehicle control and t = 0 at each time point. *, Significance accepted at P ≤ 0.05. #, Significance accepted at P = 0.05. Error bars represent means ± SEM.
FIG. 4.
FIG. 4.
Validation of microarray gene expression profiling by ELISA. IGFBP1 and PRL protein secretion in CM from hESFnonendo and hESFendo treated with or without E2 and P4 for 14 days (n = 4 in each group), normalized to the total RNA level. *, Significance accepted at P ≤ 0.05. Error bars represent means ± SEM.
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