In order to investigate the role of the nuclear factor kB (NFKB) pathway on gene expression in the eutopic endometrium in endometriosis, and in particular of interleukin-6 (IL6), we evaluated RELA, IkB kinase (CHUK), NFKBIA and IL6 expressions and NFKB DNA binding in eutopic endometrium from women with endometriosis. Eutopic endometrium was obtained from 37 women with endometriosis and 42 fertile women during laparoscopy. We analysed RELA, CHUK, NFKBIA and IL6 mRNA levels (RT-PCR); RELA, CHUK and NFKBIA proteins and p-NFKBIA/NFKBIA ratio (western blot); and NFKB binding (DNA shift assay) and IL6 concentration (ELISA) in endometrial explants. Our results indicate that mRNA and cytoplasmic proteins of RELA and CHUK exhibit constant levels in normal endometrium during the menstrual cycle. A dramatic increase (P!0.05) in NFKBIA mRNA expression, RELA nuclear presence and the mRNA and the protein of IL6 during late secretory phase was also observed in this tissue. By contrast, in eutopic endometrium from endometriosis patients, a decrease (P!0.05) in IL6 mRNA and protein (61%), NFKBIA mRNA (46%), p-NFKBIA/NFKBIA ratio (42%), RELA nuclear stromal (68%) and CHUK (48%) proteins were found exclusively during the late secretory phase compared with normal endometrium. In conclusion, the canonical activation of NFKB pathway is deregulated and may have reduced transcriptional function affecting NFKBIA and IL6 expression, genes related local proinflammatory processes. These molecular alterations observed during the late secretory phase in eutopic endometrium from endometriosis patients constitute a NFKB system dysfunction, suggesting that NFKB could be an important factor in endometriosis aetiology.

Background: Endometriosis is a common gynaecological disorder characterized by the presence of endometrial tissue outside of the uterus. The fragments in normal menstruation are composed of necrotic and living cells, which do not survive in ectopic locations because of programmed cell death. The aim of this study was to evaluate if the balance between cell proliferation and apoptosis is changed in eutopic endometrium from women with endometriosis throughout the menstrual cycle by studying bax (proapoptotic), c-myc (regulator of cell cycle) and TGF-beta1 (involved in cell differentiation) genes.

Endometriosis is a benign gynecological pathology in which immune system deregulation may play a role in its initiation and progression. In endometriotic lesions, intercellular adhesion molecule-1 (ICAM1) is released from the cell membrane by proteolytic cleavage of its extracellular domain, a process that coincides with increased expression and proteolytic activity of metalloproteinases such as MMP1 and MMP9. The objective of our study was to investigate the association between MMP1 and MMP9 activities and ICAM1 cleavage mediated by tumor necrosis factor (TNF) in eutopic endometrial stromal cells from women with and without (control) endometriosis during culture. The RNA was evaluated by RT-PCR, and the protein was determined by western blot (ICAM1, MMP1), casein or gelatin zymographies (secreted active MMP1 or MMP9 respectively), ELISA (soluble ICAM1 (sICAM1)), and fluorescence assay (secreted active MMP1). Under basal conditions, proMMP9 dimer and MMP9 were higher in endometriosis cell cultures. In stromal cultures derived from control women and those with endometriosis, TNF augmented the intracellular proMMP1 (1.2-fold in control stromal cells) and ICAM1 (1.4-and 1.9-fold), greatly increased MMP1 and proMMP9 levels, and the sICAM1 concentration (2.3-and 4.3-fold) in their media compared with basal levels. The combination of TNF and MMP9 increased the sICAM1 concentration 14-fold in the endometriosis cell media, whereas GM6001 inhibited the stimulatory effect of TNF in both cell cultures. The deregulation of MMP9, and the TNF participation in the MMP1 and proMMP9 secretions, in the MMP9 expression and in the expression and cleavage of ICAM1 may contribute to the pathophysiology of this disease.

The role of the tumor necrosis factor (TNF) system in endometriosis is not completely clear and therefore was the focus of this study. In eutopic endometria obtained from women with (n = 41) and without (controls; n = 34) endometriosis during laparoscopy, the subcellular location and the percentages for TNF, TNFR1, TNFR2 and CD45 positive-cells were determined (immunohistochemistry); the protein concentration (ELISA) of the soluble receptors (sTNFR1 and sTNFR2) were measured in 4h-explants culture media and the TNF concentration was performed in ex vivo endometrial homogenates. The TNF, TNFR1 and TNFR2 mRNAs were analyzed by real-time PCR. In control endometria, TNF, TNFR1 and sTNFR1 proteins increased during the late secretory phase. In endometriosis endometria, the protein highest level of TNFR1 was reached during the early and the mid secretory phases and of sTNFR1 concentration during the proliferative phase; however, during the late secretory phase, both TNFR1 and sTNFR1 protein levels were reduced compared to the control endometria (p < 0.05). TNFR2 was scarcely immunodetected in some CD45-positive stromal cells. The TNFand CD45-positive cell percentages, the TNF and sTNFR2 concentrations, and TNF and TNFR2 mRNAs were similar in control and endometriosis endometria. Although TNFR1 mRNA was highly expressed, no significant differences were found between control and endometriosis endometria. In summary, this study establishes that TNF and TNFR1 protein expressions and the sTNFR concentrations in eutopic endometria from women with and without endometriosis are dependent on the menstrual cycle. The differences on the TNFR1 and sTNFR1 protein pattern between both groups, mainly during the mid and late secretory phases may play a role in the lower implantation rates observed in women with endometriosis, and also could be related to the altered cell death, which favor the augmented cell viability facilitating the characteristic endometrial implantation and growth outside the uterus in this disease.