Endometriosis Endometrium Research Articles

Identification of endometriosis‐related genes by representational difference analysis of cDNA

Accumulated evidence reveals that abnormally expressed genes in eutopic endometrium of endometriosis play a critical role in the pathogenesis of endometriosis. Identification of endometriosis-related genes for further revealing the pathogenesis of endometriosis and offering the basis for developing the molecular-targeted diagnosis and therapy of endometriosis. Forty women with endometriosis and forty control women without endometriosis during their secretory phase were selected for this study. cDNA representational difference analysis (cDNA-RDA) was performed to screen the up-regulated genes in eutopic endometrium samples of endometriosis (n = 10) compared with the controls (n = 10). To validate the results, MAT2A, the most abundantly expressed gene, was selected to detect mRNA and protein levels between eutopic endometrium of endometriosis (n = 40) and controls (n = 40) using immunohistochemistry, real-time fluorescent quantitative PCR and Western blotting. Ten up-regulated genes were identified in eutopic endometria of endometriosis compared with controls. Among these genes, COX-2, BRAF, NRAS and CFL1 have already been reported to be associated with the endometriosis in previous studies. MAT2A, SEPT9, ATAD3A and CADM2 have been reported to be involved in other diseases but not in endometriosis. NAA15 and CCDC21 have not reported in any diseases. Further study showed that MAT2A protein was localised in both endometrial glandular and stromal cells. Compared with controls, the mRNA and protein levels of MAT2A were significantly higher in eutopic endometrium of endometriosis (P < 0.05). cDNA-RDA can be used to effectively identify the endometriosis-related genes, which can provide novel experimental data and further understand the pathogenesis of endometriosis.

Expression of DNMT1, DNMT3a, and DNMT3b in eutopic endometrium

To examine the expression of DNMT1, DNMT3a, and DNMT3b in the eutopic and ectopic endometrium in women with endometriosis. RT-PCR and real-time RT-PCR were used to examine the expression of DNMT1, DNMT3a, and DNMT3b in the eutopic and ectopic endometrium in 20 women with endometriosis and the endometrium in 20 women without endometriosis. Immunofluorescene staining was used to detect the expression of DNMT1 in these tissues. The expression levels of DNMT1, DNMT3a, and DNMT3b were significantly lower in the ectopic endometrium and eutopic endometrium than those of the control endometium (P<0.05). The changes in the ectopic endometium compared with the control endometium were 0.44, 0.12, and 0.27 folds for DNMT1, DNMT3a, and DNMT3b, respectively, and these in the eutopic endometrium were 0.27, 0.13, and 0.15 folds for DNMT1,DNMT3a, and DNMT3b, respectively. The expression level of DNMT1, DNMT3a, and DNMT3b between the ectopic endometrium and eutopic endometrium was not significantly different (P>0.05 ). Immunofluorescence staining that DNMT1 protein level significantly decreased in the ectopic endometrium and eutopic endometrium of endometriosis patients. Decreased expression levels of DNMT1, DNMT3a, and DNMT3b in the ectopic endometrium and eutopic endometrium may play a role in patients with abnormal epigenetics which may lead to endometriosis.

Expression and significance of HOXA10 gene in the eutopic and ectopic endometrium of endometriosis

To investigate the expression and clinical significance of HOXA10 gene in the eutopic and ectopic endometrium of endometriosis. Mehtods Between Jan.2009 to Aug.2010, 30 patients with endometriosis undergoing laparoscopic surgery in Maternal and Children's Hospital of Foshan. Eutopic and ectopic endometrium were obtained. In the mean time, 30 patients with benign ovary cyst or tubal infertility undergoing laparoscopic surgery were selected as controls. Their uterine endometrium were obtained real-time fluorescent quantitation, western blot and immunohistochemistry technique were used to detect mRNA and protein expression of HOXA10 gene in the eutopic endometrium group, ectopic endometrium group and control group. The mRNA and protein expression of HOXA10 gene were 0.61 ± 0.07 and 0.47 ± 0.05 in the eutopic endometrium of endometriosis, 0.64 ± 0.06 and 0.50 ± 0.05 in ectopic endometrium of endometriosis, which were significantly lower than 1.22 ± 0.14 and 1.42 ± 0.14 in control group (P < 0.01). However, the mRNA and protein expression of HOXA 10 between eutopic and ectopic endometrium of endometriosis did not reach statistical difference (P > 0.05). The expression of HOXA10 in eutopic and ectopic endometrium of endometriosis were decreased by immunohistochemistry staining. The lower expression of HOXA10 gene in the eutopic and ectopic endometrium of endometriosis might be associated with pathogenesis and infertility of endometriosis.

Epigenetic inactivation of SPOCK2 in the malignant transformation of ovarian endometriosis

To investigate epigenetic inactivation of SPOCK2 gene in the malignant transformation of ovarian endometriosis (EM) by comparing the methylation status and protein expression of SPOCK2 gene in the malignant tissues, ectopic endometria and the eutopic endometria of endometriosis. From Jan. 2005 to Jan. 2011, 22 paraffin-embedded specimens diagnosed as malignant transformation of ovarian endometriosis (EAOC) including 11 cases with ovarian endometrioid carcinoma, 8 cases with clear cell carcinoma, 2 cases with serous cystadenocarcinoma and 1 case with mucous cystoadenocarcinoma matched with 22 cases with ovarian endometriosis and 16 cases with normal endometrium form cervical intraepithelial neoplasia(CIN) patients as controls in Department of Obstetrics and Gynecology of Shengjing Hospital. Twenty-two malignant tissues, 15 ectopic endometria and 10 eutopic endometria were captured by microdissection in EAOC group; 22 ectopic endometria and 17 eutopic endometria were captured in EM group; 22 endometrium were captured in the NE group. The methylation statue of SPOCK2 was determined by combined bisulfite restriction analysis, and the protein expression of SPOCK2 was evaluated by immunohistochemistry. (1) Methylation of SPOCK2: in the EAOC group, the frequency of SPOCK2 hypermethylation in malignant tissue was 45% (10/22), which was significantly higher than 1/15 in the ectopic endometrium (P < 0.05). There was no statistical difference of the frequency of SPOCK2 hypermethylation in ectopic endometrium in the EAOC group (1/15) and EM group (5%, 1/22) (P > 0.05). (2) SPOCK2 protein: the loss rate of SPOCK2 was 44% (11/22) in malignant tissue in EAOC group, which were significantly higher than 2/15 of in ectopic endometrium of EAOC (P < 0.05). However, there was no remarkable difference in loss rate of SPOCK2 protein between ectopic endeometrium of EAOC and endometrium of EM [2/15 vs. 5% (1/22), P > 0.05]. No significantly difference in loss rate of SPOCK2 in eutopic endometrium was observed among three groups (P > 0.05). (3) The abnormal methylation of SPOCK2 could lead to loss expression of protein (P < 0.05). Epigenetic inactivation of SPOCK2 gene is involved in the malignant transformation of ovarian endometriosis.

Vascular endothelial growth factor expression up-regulated by endometrial ischemia in secretory phase plays an important role in endometriosis

Primary human endometrial cells were exposed to hypoxia preconditioning (HPC), HPC-hypoxia, and hypoxia conditions, and then endometrial tissue treated with ischemia preconditioning (IPC) was transplanted onto the chick embryo chorioallantoic membrane to investigate the role of slight ischemia of endometrium in the pathologic process of endometriosis. IPC up-regulated vascular endothelial growth factor expression and decreased apoptosis of endometrial cells, thus facilitating the endometrial fragments' ectopic implantation.

Altered expression of activin, cripto, and follistatin in the endometrium of women with endometrioma

To evaluate the expression pattern of activin A, activin receptors, and activin modulators messenger RNA (mRNA) in the eutopic endometrium of patients with endometriosis at different phases of the menstrual cycle and to evaluate the mRNA expression of the same proteins in endometriomas during the menstrual cycle. Prospective study. University hospital. Women with and without endometriosis. Samples of endometrial and endometriotic tissue from women with endometrioma (n=48), and endometrial samples from women without endometriosis (controls) (n=48). Quantification of activin A, activin B, activin receptor II, nodal, cripto, inhibin α, and follistatin expression by real-time reverse-transcriptase polymerase chain reaction (RT-PCR). The eutopic endometrium of patients with endometriosis showed [1] higher activin A mRNA expression in the proliferative phase and a lack of late secretory phase peak, [2] a lack of endometrial cycle-related variations of cripto and inhibin α mRNA expression, and [3] an inverse expression pattern of follistatin mRNA. Endometriomas showed similar variations in the expression of activin-related protein mRNA during the menstrual cycle as eutopic endometrium. The disturbed expression of endometrial activin A, cripto (activin receptor antagonist), and follistatin (activin-binding protein) suggests a dysfunction of the activin pathway in endometriosis. Endometriomas showed similar changes of activin-related proteins during the menstrual cycle, which supports a common biology for eutopic and ectopic endometrium in endometriosis.

Proteolytic tailoring of the heat shock protein 70 and its implications in the pathogenesis of endometriosis

To investigate the mechanism underlying the appearance of a 20-kd HSP70 fragment and its consequences in the ectopic endometrium of endometriosis patients. Experimental study. Research institute and obstetrics and gynecology clinic. Participants with (n = 18) and without (n = 20) endometriosis. None. Reverse-transcription polymerase chain reaction, protease assays, and in silico tools were used to investigate the origin of the 20-kd HSP70 fragment. Immunocolocalization studies were carried out to determine whether subtilisin/kexin isozyme 1 (SKI-1) and HSP70 are colocalized. Expression and localization of surrogate markers of inflammation, such as nuclear factor NF-κB and interleukin IL-6 were examined by immunoblotting and in situ studies. HSP70 is posttranslationally processed into a 20-kd fragment by SKI-1, a protease of the subtilisin family, in ectopic endometrium (ECE). Immunocolocalization studies revealed spatial proximity of SKI-1 and HSP70 in ECE. Furthermore, ECE demonstrated nuclear localization of the transcription factor, NF-κB and high expression of its target protein, IL-6. This study hints at the possible mechanisms underlying the trimming of HSP70 in ECE and also at the role of proteases in the pathogenesis of endometriosis. The possible repercussions of HSP70 fragmentation include dysregulation of key regulatory proteins, resulting in the escalation of inflammatory events in endometriotic lesions.

Expression of oct-4 and c-kit antigens in endometriosis

The objective of this study was to test the expression of the oct-4 and c-kit, both markers of stem cells, in the ectopic endometrial tissue of endometriotic lesions of women with severe endometriosis. Our findings show that ectopic epithelial cells express oct-4 and c-kit and this suggests that the ectopic endometrium in endometriosis has a stem cell origin and could explain the possible progression to ovarian cancer.

Uterine Lymphatic and Blood Micro-Vessels in Women with Endometriosis through the Menstrual Cycle

Purpose Endometriosis is a common disease, associated with persistent and severe symptoms including infertility and pain, however, pathogenesis remains poorly understood. It has been hypothesized that fragments of viable endometrial tissue shed at menstruation reach the peritoneal cavity and other distant sites by retrograde menstruation and dissemination into the lymphatic system. In this study, uterine lymphatic and blood micro-vessel populations were compared in women with and without endometriosis during the menstrual cycle. Methods Paraffin-embedded hysterectomy specimens from premenopausal women with histologically normal endometrium (37 control and 42 endometriosis) were obtained. Immunohistochemical staining was performed with antibodies for D2–40 (lymphatic endothelium), CD31 (pan-endothelial marker), and endoglin (activated endothelial cells in angiogenesis). Lymphatic (LVD) and blood (BVD) micro-vessel density were quantified with an automated cellular imaging system using color and morphometric properties to identify micro-vessels. Results Subtle but significant differences in uterine BVD and LVD were detected in endometriosis. LVD was significantly increased in basal layer endometrium of endometriosis patients during the proliferative phase (mean ± SD = 54.3 ± 20.1 vs. 41.4 ± 9.9, p = 0.025). Endoglin-positive BVD was increased in the subepithelial region of endometrium in endometriosis during the secretory phase (19.3 ± 16.6 vs. 6.4 ± 8.2, p = 0.038). Conclusions This report for the first time demonstrates that endometrial LVD is altered in women with endometriosis and supports changes in BVD in these women. These alterations are likely to contribute to pathogenesis of endometriosis, through lymphatic spread and increased angiogenic potential of shed endometrial fragments.

Unique Transcriptome, Pathways, and Networks in the Human Endometrial Fibroblast Response to Progesterone in Endometriosis

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.

Inhibitory effect on estrogen production and its influence on invasive abilityof human endometrial cells of endometriosis by medicated serum of SLW

To study the inhibitory effect of medicated serum of SLW on estrogen production and to approach on the key mechanism of SLW in inhibiting the invasion ability of endometrial cells of endometriosis. First, the model of eutopic primary cultured endometrial cells of endometriosis and hysteromyoma in vitro was successfully established. Taking that of endometrial cells of hysteromyoma as control, the secretion level of E2 of endometrial cells in the culture media supernatant at different time point with the treatment of high, middle and low dose of SLW serum was detected by electrochemiluminescence immunoassay, the activities of matrix metalloproteinase-2, 9 (MMP-2, 9) were detected by gelatinase zymography assay, and the expression of tissue inhibitor of metalloproteinase-1, 2 (TIMP-1, 2) protein was observed by immunofluorescence. After the optimal time for SLW to inhibit invasion ability of endometrial cells was identified based on time-effect relationship, another endometrial cells were divided into six groups: hysteromyoma endometrium group, eutopic endometrium of endometriosis group, eutopic endometrium of endometriosis + middle dose of SLW serum group, eutopic endometrium of endometriosis + middle dose of SLW serum + E2 group, eutopic endometrium of endometriosis + anastrozole serum group , and eutopic endometrium of endometriosis + E2 group. The activities of MMP-2, 9 and the expression of TIMP-1, 2 protein were detected according to the optimal time point. The secretion level of E2 of eutopic endometrium in endometriosis could be decreased by SLW, which showed the dependence of time and concentration. The result of gelatinase zymography assay and immunofluorescence respectively showed that along with the time the activities of MMP-2, 9 of eutopic endometrial cells of endometriosis were significantly higher than those of hysteromyoma at the same time point (P < 0.01). After 48 hours, with the treatment of middle dose of SLW serum, the activities of MMP-2, 9 of eutopic endometrial cells of endometriosis could be decreased (P < 0.01) while the expression of TIMP-1, 2 protein could be increased obviously (P < 0.01). The malignant invasion ability improved by SLW of eutopic endometrial cells of endometriosis was partly recruited by add-back E2 treatment. There was no significant difference in the activity of MMP-2 and the expression of TIMP-1, 2 protein between eutopic endometrium of endometriosis + middle dose of SLW serum + E2group and untreated group. The behavior of invasion of endometrial cells of endometriosis could be deteriorated treated by E2 as contrast to anastrozole, a specific aromatase inhibitor. SLW could decrease the secretion of E2 so as to inhibit the invasion of the eutopic endometrial cells of endometriosis.

Detection of mitochondrial biomarkers in eutopic endometria of endometriosis using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry

To detect specific mitochondrial proteins in eutopic endometrial samples from women with and without endometriosis and to build diagnostic models. Eutopic endometrial samples from women with endometriosis (excluding adenomyosis) and women with benign indications as control were studied by using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry protein-chip technology. After finding the biomarkers, the diagnostic model was evaluated and validated by leave-one cross-validation. Collaborative investigation in an academic research environment. Twenty-four patients with endometriosis (excluding adenomyosis) and 29 patients with benign indications as control. Surgical excision of eutopic endometrial biopsy of patients with endometriosis and controls. Mitochondrial protein expression. Seventy-eight qualified mitochondrial protein peaks were detected, ten of them had a significant difference. Three combined potential biomarkers, with mass-to-charge ratios (m/z) of 15,334, 15,128, and 16,069, were found, and the diagnostic system distinguished endometriosis from control samples with a specificity of 86.2% and a sensitivity of 87.5%. We discovered potential mitochondrial biomarkers of eutopic endometrium in endometriosis and set up a diagnostic model. Further identification of the proteins we found will help to explain pathology, new diagnoses, and therapeutic approaches for endometriosis.

Relationship between cofilin-1 expression and implantation capacity in eutopic endometrium of patient with endometriosis

To investigate the relationship between expression of cofilin-1 and in vivo implantation capacity of eutopic endometrium of endometriosis. Eutopic endometrium of 20 cases with stage III or IV endometriosis were obtained by laparoscopic or laparotomy surgery (endometriosis group) matched with 20 cases of eutopic endometrium from patients with cervical cancer in situ (control group) in Department of Obstetrics and Gynecology, ShengJing Hospital. All cases' eutopic endometrium were collected and injected into abdominal cavity of nude mice to establish endometriosis animal model, then the successful rate of animal model and volume of endometriosis lesion were calculated. The expression and positive rate of cofilin-1 protein were measured by western blot and immunohistochemistry staining. The mean volume of endometriosis lesions (2.38+/-0.22) mm3 in endometriosis group was significantly bigger than (0.36+/-0.08) mm3 in control group (P<0.05). The successful rate of establishing endometriosis model was 95% (19/20) in endometriosis group and 5% (1/20) in control group, which reached statistical difference (P<0.05). The expression and positive rate of cofilin-1 protein in eutopic endometrium and the expression of cofilin-1 protein in endometriosis lesion of animal model were 0.82+/-0.06, 90% (18/20), 0.85+/-0.03 and 0.21+/-0.03, 20% (4/20), 0.22+/-0.02 in control group, which reached statistical difference (P<0.05). The successful rate of establishing endometriosis model with positive cofilin-1 in endometrium 86% (19/22) was significantly higher than 6% (1/16) of model's endometrium with negative cofilin-1 expression (P<0.05). Implanting capacity and cofilin-1 expression level of eutopic endometrium of endometriosis were more intensive than that of normal endometrium. High cofilin-1 expression was probably related with implanting capacity of eutopic endometrium of endometriosis.

Identification biomarkers of eutopic endometrium in endometriosis using artificial neural networks and protein fingerprinting

Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) protein chip array technology was used to detect biomarkers of eutopic endometrium in endometriosis patients. Five potential biomarkers (6,898 m/z, 5,891 m/z, 5,385 m/z, 6,448 m/z, and 5,425 m/z) were found.

Expression of nonsteroidal anti-inflammatory drug-activated gene 1 (NAG-1) on the eutopic endometrium of the patients with endometriosis

OBJECTIVE: Altered regulation of apoptosis in eutopic endometrium could contribute to survival of endometriotic cell and developed endometriosis. NAG-1, member of transforming growth factor – β (TGF- β) superfamilies, concerns in induction of cell apoptosis. We hypothesized that decreased expression of NAG-1 of eutopic endometrium may be involved in the pathophysiology of endometriosis and investigated the expression of NAG-1 in eutopic endometrium of endometriosis and normal endometrium.

Endometrium from women with endometriosis shows increased proliferation activity

Examination of 631 infertile women, including 434 women with endometriosis and 197 without endometriosis, revealed an increasing incidence of endometrial polyps, especially in the test subjects with endometriosis, and advanced stages of endometriosis. Expression of Ki-67 and Bcl-2 proteins and MTT assay results were significantly higher in endometrium of patients with endometriosis than in patients without endometriosis. These showed increased proliferation activity and cell proliferation markers not only clinically but also pathologically.

Distribution of nerve fibers in endometrium and its clinical significance in adenomyosis

To investigate nerve fibers distribution in endometrium of adenomyosis and their relationship with dysmenorrhea. Endometrial tissue was sampled from 74 hysterectomy specimens including 32 cases with adenomyosis and 42 cases with uterine fibroids. Two-step Envision immunohistochemical staining was used to detect distribution of nerve fibers in endometrium. Highly specific polyclonal rabbit anti-protein gene product 9.5 (PGP9.5) and monoclonal mouse anti-neurofilament protein (NF) were used to demonstrate both myelinated and unmyelinated nerve fibers in endometrium in women with adenomyosis and uterine fibroids. The positive rate of PGP9.5 immunoreactive nerve fibers in the functional layer of endometrium of pain patients were with 64% (14/22) in adenomyosis and 67% (10/15) in uterine fibroids. And their density were 0.6 (0 - 9.4)/mm(2) and 0.6 (0 - 6.0)/mm(2) without reaching statistical difference (P > 0.05). No expression of NF could be detected in the functional layer of endometrium of adenomyosis and uterine fibroids. There were no PGP9.5 immunoreactive nerve fibers in the functional layer of endometrium in non-pain women with adenomyosis and uterine fibroids. Moreover, No NF immunoreactive nerve fibers in the functional layer of endometrium were shown in non-pain patients with adenomyosis and uterine fibroids. PGP9.5 immunoreactive nerve fibers and the nerve density in the basal layer of endometrium were 64% (14/22), 1.1 (0 - 12.0)/mm(2) in pain adenomyosis and 50% (5/10), 0.6 (0 - 3.0)/mm(2) in non-pain adenomyosis. NF immunoreactive nerve fibers and the density in the basal layer of endometrium were 23% (5/22), (0 - 0.6)/mm(2) in pain adenomyosis and 20% (2/10), (0 - 1.0)/mm(2) in non-pain adenomyosis. PGP9.5 immunoreactive nerve fibers and the nerve density in the basal layer of endometrium were 80% (12/15) and 1.6 (0 - 10.0)/mm(2) in pain fibroids and 44% (12/27), 0 (0 - 5.0)/mm(2) in non-pain fibroids. NF immunoreactive nerve fibers and the nerve density in the basal layer of endometrium were 40% (6/15), 0 (0 - 0.4)/mm(2) in pain fibroids and 15% (4/27), 0 (0 - 1.0)/mm(2) in non-pain fibroids. There was no statistical different PGP9.5 and NF immunoreactive nerve fibers distribution in basal layer of endometrium between pain adenomyosis and pain fibroids or between non-pain adenomyosis and non-pain fibroids (all P > 0.05). However, PGP9.5 immunoreactive nerve fibers density in basal layer of endometrium was higher in pain adenomyosis and fibroids when compared with non-pain adenomyosis and fibroids (P < 0.05). PGP9.5 immunoreactive nerve fibers might confer the occurrence of pelvic pain, however, NF immunoreactive nerve fibers may not involved in the pathogenesis of pain.

Prokineticin 1 mRNA expression in the endometrium of healthy women and in the eutopic endometrium of women with endometriosis

To examine prokineticin 1 (PROK1) mRNA expression in eutopic endometrial glands obtained from patients with or without endometriosis, to investigate the presence of additional endometrial abnormalities in women with endometriosis. Prospective laboratory study. University hospital. Twelve control women and 12 patients affected by endometriosis in the secretory phase of the menstrual cycle. Endometrial specimens were obtained from women affected (cases) or not (control group) by endometriosis. Endometrial glands were freshly isolated from endometrial biopsies. PROK1 mRNA expression levels by real-time polymerase chain reaction analysis. PROK1 mRNA was detectable in 4 of 12 (33%) samples obtained from women affected by endometriosis, whereas 10 of 12 (83%) samples obtained from normal women were positive for PROK1 detection by real-time polymerase chain reaction. Moreover, detectable PROK1 mRNA levels were 10 times lower in samples obtained from women with endometriosis than in samples obtained from control women. PROK1 is a newly discovered angiogenic factor implicated in the vascular function of peri-implantation endometrium and early pregnancy. An altered expression of PROK1 could be one of the several biochemical abnormalities characterizing eutopic endometrium in endometriosis.

Nuclear factor κB pathway and interleukin-6 are affected in eutopic endometrium of women with endometriosis

In order to investigate the role of the nuclear factor kappaB (NFKB) pathway on gene expression in the eutopic endometrium in endometriosis, and in particular of interleukin-6 (IL6), we evaluated RELA, IkappaB 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.

Expression of Annexin-1 in patients with endometriosis

Annexin-1 was identified as an endometriosis-related protein by comparative proteomics in previous study. As an endogenous anti-inflammatory mediator, Annexin-1 has been shown to regulate the immune response, cell proliferation and apoptosis. To investigate whether Annexin-1 is involved in the pathogenesis of endometriosis, we examined the expression of Annexin-1 in eutopic endometrium of women with or without endometriosis, and detected its expression in peritoneal fluids of those with endometriosis. Eutopic endometrium samples from twenty-five women with endometriosis and those from sixteen age-matched women without endometriosis were collected. Peritoneal fluids were obtained from ten patients with endometriosis. The expression of Annexin-1 protein in eutopic endometrium was detected by immunohistochemistry and Western blotting, and mRNA detected by real-time PCR. Annexin-1 protein in the peritoneal fluids was detected by Western blotting. Annexin-1 mRNA and protein were overexpressed in eutopic endometrium of endometriosis without significant differences between the proliferative and secretory phase. Immunohistochemistry showed that Annexin-1 protein was expressed mainly in endometrial glandular cells throughout the menstrual cycle. Annexin-1 protein was detected in the peritoneal fluids of all the ten patients with endometriosis. Annexin-1 is overexpressed in eutopic endometrium and presents in the peritoneal fluids of patients with endometriosis, and may play a role in the pathogenesis of endometriosis.