Non-endometriosis Patients Research Articles

A delicate redox balance between iron and heme oxygenase-1 as an essential biological feature of endometriosis

Recent studies have focused on the role of oxidative stress, which may be implicated in the development, progression, and pathophysiology of endometriosis. The aim of this study is to investigate the redox balance of endometriosis by simultaneously measuring iron-related compounds (total iron, heme iron, free iron, oxyhemoglobin [oxyHb], methemoglobin [metHb] and 8-hydroxy-2-deoxy guanosine [8-OHdG]) and antioxidants (bilirubin, heme oxygenase-1 [HO-1] and total antioxidant capacity [TAC]). This study includes 236 histopathologically confirmed cases (178 cases of endometriosis and 58 cases of non-endometriosis). Cyst fluid samples were collected from patients admitted to the Department of Gynecology, Nara Medical University, Kashihara, Japan, for surgery. Age at diagnosis (p <0.001), the maximum diameter of the cyst (p <0.001) and CA125 levels (p <0.001) differed significantly between the two groups. Total iron, heme iron, free iron, metHb, and oxyHb were markedly higher in endometriosis compared to non-endometriosis. Bilirubin, HO-1 and TAC were significantly higher in endometriosis patients compared with those from non-endometriosis patients. In endometriosis, total iron showed a positive correlation with HO-1 (r, 0.518, p = 0.001), but there were no antioxidants that correlated with iron in non-endometriosis. Iron and HO-1 did not correlate with age or tumor size. HO-1 may regulate the delicate balance of iron-induced oxidative stress in endometriotic cyst fluid.

Luaran Transfer Embrio Simpan Beku pada Pasien Endometriosis Pasca Operasi dan Non Endometriosis yang Menjalani IVF di Klinik Permata Hati RSUP Dr. Sardjito

Background: Endometriosis is a chronic condition that is influenced by the hormone estrogen which affects women of childbearing age, and is associated with pelvic pain and infertility. In Vitro Fertilization (IVF) is currently the most efficient assisted reproductive technology and its high success rate is often done for infertility therapy in women associated with endometriosisObjective: The aim of this study is to determine whether postoperative endometriosis affected pregnancy outcomes in patients underwent frozen embryo transfer in IVF / ICSI programs.Method: This Research is done with a retrospective cohort design. The data was taken from medical records, research subjects who met the inclusion and exclusion criteria. The research data was collected, processed and analyzed using SPSS 23. Univariate, bivariate and multivariate data analysis was carried out to determine the effect between variablesResult: There were 458 research subjects in this study. Endometriosis patients were 119 subjects (26%). 57 subjects were categorized as minimum-mild endometriosis (47.9%) and moderate-severe subjects as many as 62 subjects (52.1%). The biochemical pregnancy rate (36.31%) and clinical pregnancy (29.4%) in patients with endometriosis was slightly higher than in non-endometriosis. But statistically it did not affect success rate of achieving biochemical (p = 0.428; RR 0.89; 95% CI: 0.71-1.24) and clinical pregnancy (p = 0.535; RR 0.883; 95% CI: 0.63- 1.22). The rate of miscarriage in postoperative endometriosis patients was higher than non-endometriosis patients (88.6% vs 80.7%) but was not statistically significant (p = 0.294; RR 1.69; 95% CI: 0.61-4.67) . Biochemical and clinical pregnancies were significantly affected by age, infertility, endometrial thickness, embryo age and embryo quality. The incidence of miscarriage was affected by the ovarian stimulation protocol.Conclusion: Endometriosis post operative statistically has no effect on pregnancy outcomes in the IVF / ICSI cycle with frozen embryo transfer compared with another cause of infertility .Keywords:Endometriosis, In Vitro Fertilization, Clinical pregnancy, biochemical pregnancy, miscarriage

Higher IL-1beta level in the follicular liquid of endometriosis compared with non-endometriosis patients

Objective: To ascertain the IL-1b level in the follicular liquid of endometriosis sufferers compared with that of non-endometriosis sufferers.Materials and Methods: The ELISA method, for detecting the IL-1b, is taken from follicular fluid of endometriosis as well as non-endometriosis patients that diagnosed according to the ASRM criteria.Results: The 54 subjects were dominantly endometriosis &lt;34 year old (88.9%). The IL-1b levels of endometriosis patients and non-endometriosis patients were 82.86 ± 30.15 and 56.05 ± 23.27 respectively (p=0.01). The ROC value of IL-1b was found at a cut-off point of 26 (with the IL-1b cut-off point of 65.16). The discriminating value of AUC (area under curve) was 59.3%.Conclusion: It was concluded that the IL-1b level in endometriosis is higher than that in non- endometriosis.

Cellular Phenotyping of Endometriosis at the Single Cell Level Highlights Complex Heterogeneity

Study Objective To understand how endometriosis cells work and interact with the surrounding cells by looking at cellular components and biomarkers. Design In this DoD funded study, both eutopic and peritoneal ectopic tissue were collected from patients with stage III-IV endometriosis. An unbiased single cell transcriptomic (scRNAseq) approach was used to comprehensively identify all the cellular components of endometriosis lesions and their microenvironment. Setting Tissue samples were obtained in the operating room and brought to the Laboratory for its fresh processing RNA sequencing Patients or Participants Women between 18-50 who were preoperatively diagnosed with stage III or IV endometriosis were consented prior to their scheduled laparoscopic surgery. Tissue banked samples of eutopic endometrium from “non-endometriosis patients” were used for comparison. Interventions Peritoneal endometriosis lesions and eutopic endometrium were collected during surgery from patients with stage III and IV endometriosis. These tissue samples were then used to generate scRNAseq data using a droplet based RNAseq platform (10x Genomics). Matched eutopic endometrium was also analyzed for comparison. Measurements and Main Results Our unbiased single cell approach allowed us to capture multiple cell types from ectopic, adjacent ectopic, and eutopic endometrium. We identified the major cell types composing the ectopic endometrium and its microenvironment, as well as in the adjacent peritoneum. Cell types identified within the microenvironment included T cells, B cells, macrophages, endothelial, fibroblast, muscle cells and pericytes. We identified endometrial epithelial cells, in both ectopic and matched eutopic tissues, and their single-cell differential expression analysis revealed substantial differences in their gene expression and main differences emerged from the macrophage populations. Conclusion Precise determination of the heterogeneous cellular composition of endometriosis lesion allows for characterization of important key players, such as epithelial cells and macrophages among others, for lesion formation and evolution. With this detailed approach, we hope to establish a list of potential biomarkers, a much needed effort for endometriosis diagnosis and treatment.

Analysis of DNA methylation and its correlation with mRNA expression of epidermal growth factor receptor encoding for cytoskeleton regulating protein in peritoneal endometriosis tissue

It has been known that the EGFR have the role for regulation the cytoskeleton activity and its expression increased in endometriosis tissue. The aim of this study was to evaluate the DNA methylation of the EGFR gene that might cause the alteration of its mRNA expression in peritoneal endometriosis tissue. Samples were peritoneal endometriosis tissue from 20 endometriosis patients and 20 female of non-endometriosis patients. The DNA methylation of the EGFR gene was analyzed by the method of Methylation Specific PCR and ImageJ software, while its expression of mRNA were analyzed by the method of qRT-PCR. The DNA methylation in the EGFR gene in peritoneal endometriosis tissues increased compared to normal endometrial tissues (peritoneal endometriosis tissue = 56%, normal endometriosis tissue = 19%). The expression of mRNA EGFR gene in endometriosis peritoneal tissues was 1.341 fold increased relative to normal endometrium. There is no significant correlation between the DNA methylation with expression of mRNA EGFR (p = 0.947 and r = -0.016). Increasing of EGFR mRNA expression in endometriosis tissue that was not caused by alteration of its DNA methylation, have to play a role in the pathogenesis of endometriosis.

MicroRNA-451 is downregulated in the follicular fluid of women with endometriosis and influences mouse and human embryonic potential

BackgroundPrevious work demonstrated that there are numerous miRNAs in human follicular fluids, some of which are associated with reproductive diseases. In the current study, we sought to determine whether microRNAs (miRNAs) in the follicular fluid (FF) are differentially expressed between women with and without endometriosis and to uncover the association of miRNAs with the oocyte and embryonic development potential.MethodsFF was harvested from 30 women with endometriosis and 30 women without who underwent in vitro fertilization treatment at the University Hospital between February and December 2016. The FF samples were subjected to miRNA profiling and validation via quantitative reverse transcription polymerase chain reaction analysis. Mouse/human metaphase-I (MI) oocytes were harvested and micro-injected with an miR-451 inhibitor, and the effects of miR-451 knockdown on Wnt/WNT signalling genes were investigated.ResultsOocyte number, fertilization rate, and number of available embryos were decreased significantly in women with endometriosis relative to those without endometriosis. Hsa-miR-451 in FF was downregulated in endometriosis patients relative to control subjects (P < 0.01). Moreover, the proportions of mouse/human MI oocytes that developed into 2-pronuclei (2PN), 2-cell, 8–10-cell and blastocyst-stage embryos were affected by miR-451 knockdown in mouse/human oocytes. Components of the Wnt signalling pathway were aberrantly expressed in the mouse/human oocytes and embryos in the miR-451 inhibitor-injected group.ConclusionsmiR-451 was downregulated in FF samples from endometriosis patients and was modestly effective in distinguishing endometriosis patients from non-endometriosis patients. miR-451 downregulation in mouse and human oocytes affected pre-implantation embryogenesis by suppressing the Wnt signalling pathway. This miRNA might serve as a novel biomarker of oocyte and embryo quality in assisted reproductive treatment.

Roles of cell migration and invasion mediated by Twist in endometriosis.

To investigate the roles of cell migration and invasion mediated by Twist in endometriosis. The protein levels and locations of Twist, N-cadherin and E-cadherin were measured by Western blot and immunohistochemistry in ectopic endometrium and eutopic endometrium of ovarian endometriosis as well as normal endometrium of nonendometriosis patients. The messenger RNA (mRNA) expressions of Twist, N-cadherin and E-cadherin in these tissues were measured by quantitative reverse transcription polymerase chain reaction. Stable overexpression of Twist in eutopic endometrial stromal cells was transfected with a plasmid-mediated delivery system. The protein and mRNA expressions of N-cadherin and E-cadherin were detected by western blot and reverse transcription polymerase chain reaction. The changes of migration and invasion of endometrial stromal cells were explored by transwell. Levels of protein and mRNA of Twist and N-cadherin showed the highest expression in ectopic endometrium of ovarian endometriosis, while lowest in normal endometrium of nonendometriosis patients. On the contrary, the expression of E-cadherin showed highest in normal endometrium of nonendometriosis patients. The overexpression of Twist after transfection significantly upregulated the protein and mRNA expression of N-cadherin, while downregulated the protein and mRNA expression of E-cadherin. There is significant difference between groups. For transwell, the overexpression of Twist in eutopic endometrial stromal cell significantly promoted cell migration and invasion. Twist might be related with the increase of migration and invasion in endometrial stromal cells, mediated by epithelial-to-mesenchymal transition.

Lipoxin A4 inhibits the invasion and migration of endometrial stromal cells by down-regulating NF-κB signaling-mediated autophagy

Objective: To investigate whether the suppressive effects of lipoxin A4 (LXA4) on endometriosis are mediated by the regulation of autophagic activity, and to further explore the actual molecular mechanism. Methods: (1) Eutopic and ectopic endometria were obtained from 13 patients with endometriosis, and 10 eutopic endometria collected from non-endometriosis patients were used as control. The expression of the autophagy-related biochemical markers [microtubule-associated protein 1 light chain 3 (LC3) and p62] were detected by western blot. Levels of LXA4 in the biopsies were measured by ELISA. (2) Primary human endometrial stromal cells (ESC) were isolated and cultured in vitro from eutopic endometria of infertility patients with endometriosis. After treatment with exogenous LXA4 or autophagy inhibitor 3-methyladenine (3-MA) or autophagy inducer rapamycin, cell migration and invasion were evaluated by transwell assay, and autophagy was detected by western blot. (3) ESC were treated with LXA4, the gene expressions of nuclear factor kappa B (NF-κB) etc. were examined by quantitative real-time PCR, and the activation of NF-κB signaling was detected by western blot. (4) ESC were incubated with 10 μmol/L NF-κB inhibitor BAY11-7080, the autophagic activation was detected by western blot. Results: (1) Autophagy-related marker, LC3-Ⅱ and LC3-Ⅱ/LC3-Ⅰ ratio, showed a significant up-regulation in ectopic lesions of endometriosis compared with eutopic endometria of affected or healthy women (all P<0.05) . However, the LXA4 level significantly decreased in ectopic tissue (P<0.05) . There was a significant negative correlation between LXA4 concentration and relative expression of LC3-Ⅱ in ectopic lesions (r= -0.780, P=0.002) . (2) 10 and 100 nmol/L exogenous LXA4 could significantly down-regulate the LC3-Ⅱ protein expression and up-regulate the p62 protein expression (all P<0.05) . LXA4 markedly inhibited the invasion and migration of ESC (P<0.05) ;while the reactivation of autophagy by rapamycin almost reversed the anti-invasion and anti-migration effects of LXA4. (3) After LXA4 treatment, the expression level of NF-κB gene significantly decreased (P<0.05) . Furthermore, the results of western blot analysis showed that the nuclear translocation of NF-κB p65 was markedly down-regulated under LXA4 treatment (P<0.05) . (4) The NF-κB inhibitor BAY11-7080 markedly suppressed the autophagic activation of LXA4 (P<0.05) . Conclusion: LXA4 could inhibit the invasion and migration of ESC by down-regulating the NF-κB signaling-mediated autophagy.

Peritoneal fluid cytokines related to endometriosis in patients evaluated for infertility

Our aim was to characterize peritoneal cytokine profiles in patients with infertility, with and without endometriosis, to illuminate potential differences in immune profiles that may reflect mechanistic differences between these two patient populations. Cross-sectional study. University hospital and research center. Women undergoing laparoscopy for infertility investigation (n = 107). Peritoneal fluid was collected during surgery. Clinical characteristics were registered preoperatively. We determined the concentration of 48 different cytokines from the peritoneal fluid with multiplex immunoassays. Associations between cytokines and clinical findings were assessed with logistic regression and partial least squares discriminant analyses (PLS-DA). Concentrations of SCGF-β, IL-8, HGF, and MCP-1 were significantly higher, while IL-13 was significantly lower in the endometriosis group compared with the group without endometriosis. Multiple stepwise logistic regression identified a combination of SCGF-β, IL-13, and G-CSF concentrations that predicted the presence of endometriosis with 86% sensitivity and 67% specificity. PLS-DA identified a class of 11 cytokines (SCGF-β, HGF, IL-13, MCP-1, CTACK, MCP-3, M-CSF, LIF, IL-5, IL-9, and IFN-a2) that were more characteristic of endometriosis than nonendometriosis patients. By combining univariate and multivariate analyses, profiles of cytokines more likely to be enriched or depleted in infertility patients with endometriosis compared with those without endometriosis were identified. These findings may inform future analyses of pathophysiological mechanisms of endometriosis in infertile patients, including dysregulated Th1/Th2 response and mobilization and proliferation of hematopoietic stem cells.

Expression of p27 and Jun activation domain-binding protein 1 in endometriosis.

The goal of this study was to investigate differential expression of the cell cycle signaling proteins p27 and Jun activation domain-binding protein 1 (Jab1) in benign ovarian cysts of patients with endometriosis compared to controls. Ovarian tissues of 26 endometriosis and 28 non-endometriosis patients were used to evaluate expression of p27 and Jab1 by immunohistochemistry. There are no differences in clinical characteristics between the endometriosis and non-endometriosis group. In the endometriosis group, CD10, Jab1 and p27 were positive in 50, 48.3 and 57.7 % of cases, respectively, compared to 14.3, 17.9 and 50 %, respectively, in the non-endometriosis group. Jab1 and p27 were positive in 69.2 and 84.6 %, respectively, of the definite endometriosis cases. These results demonstrate that Jab1 expression was increased in endometriosis, while p27 expression was similar in the endometriosis and non-endometriosis groups.

Hysterosalpingo-contrast-sonography (HyCoSy) in the assessment of tubal patency in endometriosis patients

ObjectiveTubal patency in women with endometriosis has traditionally been evaluated by laparoscopy. The aim of this study was to investigate the accuracy of hysterosalpingo-contrast-sonography (HyCoSy) in the assessment of tubal patency in these women. Study designA retrospective study was conducted at Physiopathology of Human Reproduction Unit. Infertile women who underwent HyCoSy and then a laparoscopy (dye test) within 6 months from the HyCoSy were included. Tubal patency was assessed by HyCoSy and the findings were compared with the results of laparoscopy, which was considered the gold standard for assessment of tubal patency. Sensitivity, specificity, positive and negative predictive values (PPV, NPV) and positive and negative likelihood ratios (Lh+, Lh−) were calculated including the 95% confidence interval (CI). ResultsA total of 1452 women underwent HyCoSy and 126 of them received a laparoscopy within 6 months from the HyCoSy. Of the 126 women, 42 (33.3%) had a diagnosis of pelvic endometriosis and 84 (66.7%) had no endometriosis. In the endometriosis population, HyCoSy showed a sensitivity, specificity, PPV, NPV, Lh+ and Lh− of 85% (95% CI 62–96), 93% (95% CI 82–97), 81% (95% CI 58–94), 94% (95% CI 84–98), 12.6 (95% CI 4.8–33) and 0.15 (95% CI 0.05–0.4) respectively. In the non-endometriosis group, HyCoSy showed a sensitivity, specificity, PPV, NPV, LR+ and LR− of 85% (95% CI 65–95), 93% (95% CI 87–96), 71% (95% CI 53–85), 97% (95% CI 92–99), 13.2 (95% CI 6.9–25) and 0.15 (95% CI 0.06–0.3) respectively. The diagnostic accuracy of HyCoSy was 91% in the endometriosis group and 92% in the non-endometriosis patients. ConclusionsHyCoSy showed high accuracy in evaluating tubal patency in infertile non-endometriosis women and in those affected by endometriosis.

Silencing of cofilin-1 gene attenuates biological behaviours of stromal cells derived from eutopic endometria of women with endometriosis

Eutopic endometria with endometriosis (EMs) differ dramatically from normal endometria, physiologically and biochemically, yet the pathogenesis of EMs remains unclear. Cofilin-1 (CFL1), a critical modulator of the actin cystoskeleton, is associated with tumour progression, cell motility, cell adhesion, cell invasion and angiogenesis. Although eutopic endometria with EMs exhibit many malignant-like behaviours and a higher expression of CFL1 than normal endometria, the effects of CFL1 on the pathogenesis of EMs are unknown. The aim of this study was to explore the role of CFL1 expression in proliferation, apoptosis, adhesion, invasion, angiogenesis and ultrastructure of endometrial cells. We isolated and cultured stromal cells derived from the eutopic endometria of 30 patients with advanced ovarian EMs (ESCs, Stromal Cells of eutopic endometria in Endometriosis patients) and 30 control patients without EMs (NSCs, Stromal Cells of eutopic endometria in Non-endometriosis patients), and evaluated their proliferation, apoptosis, adhesion, invasion and expression of markers of adhesion, invasion and angiogenesis in vitro. In addition, these functions were examined after short hairpin RNA (shRNA) was used to silence the CFL1 gene in ESCs, and pEGFP-N1-CFL recombinant plasmid was transiently transfected into NSCs to up-regulate CFL1 expression. Under basal conditions, CFL1 mRNA and protein were overexpressed in ESCs. Proliferation, adhesion, invasion and markers of adhesion, invasion and angiogenesis were enhanced in ESCs compared with NSCs; in contrast, the apoptosis rate was lower in ESCs than in NSCs. Silencing the CFL1 gene in ESCs markedly attenuated proliferation, adhesion, invasion and expression of the markers, but enhanced apoptosis. Conversely, up-regulation of CFL1 in NSCs increased proliferation, adhesion, invasion and expression of the markers but reduced apoptosis. The overexpression of CFL1 in ESCs is associated with enhanced proliferation, adhesion, invasion and angiogenesis and reduced apoptosis in EMs. These malignant-like behaviours of ESCs in EMs can be attenuated by inducing CFL1 gene silencing with shRNA interference.

Endometriosis-induced alterations in mouse metaphase II oocyte microtubules and chromosomal alignment: a possible cause of infertility

To examine the effect of peritoneal fluid (PF) of patients with endometriosis on the cytoskeleton of metaphase II oocytes and correlate the results with the stage of endometriosis and the duration of infertility. Prospective-controlled study. Center for reproductive medicine at a tertiary-care hospital. Women with endometriosis (n=23) and tubal ligation/reversal (n=15). Peritoneal fluid obtained from 38 women (23 with endometriosis and 15 tubal ligation/reversal) after laparoscopy. Four hundred metaphase II oocytes were used: 165 frozen metaphase II oocytes were incubated in the PF of patients with endometriosis, 135 oocytes incubated in the PF of nonendometriosis patients (control subjects) and 100 oocytes incubated in human tubal fluid (HTF) media. Spindle abnormalities (microtubule and chromosomal) were evaluated by confocal imaging. In the endometriosis group, the cytoskeleton had a higher frequency of abnormal meiotic spindle and chromosomal misalignment (score ≥3), indicating severe damage compared with the control groups. The proportions of abnormalities in microtubule and chromosome alterations in endometriosis (67.9% and 63.6%, respectively) were significantly higher than for oocytes incubated with PF of the nonendometriosis group (24.4% and 14.8%) as well as the HTF group (13% and 13%). Oocyte cytoskeleton damage positively correlated with the duration of infertility and the stage of endometriosis. Alteration of oocyte cytoskeleton might be one of the causes of poor oocyte quality in patients with endometriosis.

Stage of endometriosis does not affect intrauterine insemination outcome

OBJECTIVE: There is evidence that endometriosis can and does decrease fertility. Treatment options include medical therapy, surgical intervention, and assisted reproduction. The relative value of ART based on the stages of endometriosis, however, is untested. The aim of this study was to evaluate the effects of different stages of endometriosis on the outcome of intrauterine insemination (IUI).DESIGN: Retrospective study.MATERIALS AND METHODS: The study group consisted of 114 women with laparoscopically diagnosed endometriosis undergoing IUI between 2003-2005 and 458 non-endometriosis patients (control group) including tubal, ovulatory, male, cervical and unexplained infertility. A total of 1596 cycles were observed and the number of IUI cycles per patient ranged from 1 to 6. Ovulation induction was done using clomiphine citrate (CC), controlled ovarian hyperstimulation by pure FSH and/or HMG or both CC+FSH. 10,000 IU of hCG was given when the dominant follicle reached maturity. A single IUI was performed 36 h after hCG administration. The primary outcomes were: clinical pregnancy rate, miscarriage rate and the number of cycles needed to achieve pregnancy.RESULTS: Women with endometriosis required significantly higher gonadotropin doses compared with non-endometriosis women p<0.001. Patients with endometriosis showed lower pregnancy rates (10.2% vs. 22.0% per cycle) as well as higher miscarriage rates (30.0% vs. 11.2% per cycle) than non-endometriosis patients (p = 0.001). Mean number of IUI cycles per patient were comparable among the 4 stages in non-endometriosis and endometriosis patients.Table 1Endometriosis and IUI outcomesVariableNon-endometriosisEndometriosisP-valueGonadotropin (days)9.63.3<0.001Cycles/patient (mean)2.812.690.89Cycles/pregnant patient2.872.960.85Pregnancy/patient/per cycle) (%)266/ 458 (58.1 and 22.0%)47/ 114 (41.2 and 14.3%)0.001Miscarriage/ patient/cycle) (%)126/ 458 (27.5 and 11.1%)64/ 114 (56.1% and 30.0%)0.001P<0.05 was considered significant for hCG dose using the Wilcoxon rank sum test and for pregnancy and miscarriage rates per patient using chi-square test. Open table in a new tab OBJECTIVE: There is evidence that endometriosis can and does decrease fertility. Treatment options include medical therapy, surgical intervention, and assisted reproduction. The relative value of ART based on the stages of endometriosis, however, is untested. The aim of this study was to evaluate the effects of different stages of endometriosis on the outcome of intrauterine insemination (IUI). DESIGN: Retrospective study. MATERIALS AND METHODS: The study group consisted of 114 women with laparoscopically diagnosed endometriosis undergoing IUI between 2003-2005 and 458 non-endometriosis patients (control group) including tubal, ovulatory, male, cervical and unexplained infertility. A total of 1596 cycles were observed and the number of IUI cycles per patient ranged from 1 to 6. Ovulation induction was done using clomiphine citrate (CC), controlled ovarian hyperstimulation by pure FSH and/or HMG or both CC+FSH. 10,000 IU of hCG was given when the dominant follicle reached maturity. A single IUI was performed 36 h after hCG administration. The primary outcomes were: clinical pregnancy rate, miscarriage rate and the number of cycles needed to achieve pregnancy. RESULTS: Women with endometriosis required significantly higher gonadotropin doses compared with non-endometriosis women p<0.001. Patients with endometriosis showed lower pregnancy rates (10.2% vs. 22.0% per cycle) as well as higher miscarriage rates (30.0% vs. 11.2% per cycle) than non-endometriosis patients (p = 0.001). Mean number of IUI cycles per patient were comparable among the 4 stages in non-endometriosis and endometriosis patients. P<0.05 was considered significant for hCG dose using the Wilcoxon rank sum test and for pregnancy and miscarriage rates per patient using chi-square test.

Correlation between DNA damage of oocytes incubated in the peritoneal fluid of endometriosis, stage of disease and duration of infertility

OBJECTIVE: The objective of this study was to correlate the DNA damage of oocytes incubated in the peritoneal fluid of endometriosis with the stage of the disease and duration of infertility. DESIGN: Prospective controlled in vitro study. MATERIALS AND METHODS: 300 Mature frozen metaphase II mouse oocytes were divided into four groups. Group I: 87 oocytes incubated in the PF of patients with mild endometriosis; group II: 93 oocytes incubated in the PF of patients with severe endometriosis; group III: 90 oocytes incubated in the PF of non-endometriosis patients; i.e. tubal ligation/ reversal (controls) and group IV: 30 oocytes incubated inHTF. Oocytes were divided equally and incubated for 30 min, 1.5 and 4 h. Oocytes were fixed and TUNEL staining was done for DNA damage and oocytes were examined by fluorescent microscopy. TUNEL positive staining indicated damaged DNA, while weakly positive or negative indicated no damage. RESULTS: Oocytes that were incubated in PF of patients with endometriosis showed significantly more DNA damage compared with control at 1.5 and 4 h (p<0.001, respectively). Oocytes incubated in PF from patients with severe endometriosis showed more damage compared with mild endometriosis patients at all time points (p<0.001, respectively). Duration of infertility was associated with the degree of damage 44% (0 - 2y) 76% (3 - 4y), and 100% damage at 5+ y of infertility respectively after 4 h incubation (p = 0.022 and 0.006 for 0 - 2 y vs. 3-4 y and 5+ y, respectively). Mild or severe damage was observed in all oocytes at 4 h.table 1DNA damage and endometriosisDNA DamageControl HTFControl TubalEndometriosisIncubation30 min9 (90.0%)26 (86.7%)48 (80.0%)Weakly +1 (10.0%)4 (13.3%)7 (11.7%)Positive0 (0%)0 (0%)5 (8.3%)1.5 hoursNegative9 (90.0%)27 (90.0%)9 (15.0%)Weakly +1 (10.0%)3 (10.0%)15 (25.0%)Positive0 (0%)0 (0%)36 (60.0%)4 hoursNegative9 (90.0%)24 (80.0%)0 (0%)Weakly +1 (10.0%)6 (20.0%)21 (35.0%)Positive0 (0%)0 (0%)39 (65.0%) Open table in a new tab CONCLUSIONS: The extent of DNA damage depends on the duration the oocytes are exposed to the peritoneal fluid. This may lead to failure of fertilization or embryonic development. Stage of endometriosis affects the oocyte quality. Selecting oocytes that are most likely to develop is crucial for in vitro fertilization in management of endometriosis related infertility.

Differentially expressed genes in human endometrial endothelial cells derived from eutopic endometrium of patients with endometriosis compared with those from patients without endometriosis

The pathogenesis of endometriosis remains poorly defined. The aberrant angiogenesis that occurs in eutopic endometrium may play a role in the lesion formation and survival. The difference in gene expression profile between human endometrial endothelial cells (HEECs) from eutopic endometria of patients with and without endometriosis would be a factor that affects the occurrence of endometriosis. To explore the difference, we performed in vitro culture and identified the endothelial origin, as well as observed growth features, of HEECs from the two different sources. We also identified their differences in gene expression profiles by combined suppression subtractive hybridization (SSH) with Genechip, and confirmed the results by quantitative reverse transcription-polymerase chain reaction. The HEECs derived from endometriosis patients exhibited a potent survival ability in vitro compared with those from non-endometriosis patients. In the HEECs from EM patients, an altered secretion pattern of extracellular matrix (ECM) components and up-regulation of GREM1 were found. These findings may be used to interpret the remarkable change of phenotype in HEECs from endometriosis patients. The synergistic action of these differentially expressed genes is to promote cell proliferation and concomitantly to inhibit apoptosis. Among the up-regulated ECM genes, TSP2 was the only one which exhibits the capacity to suppress angiogenesis; it may therefore function as an antagonist to the aberrant angiogenesis and may confine its extent and severity. It may be postulated that differential regulation of some of these genes in eutopic HEECs plays a facilitating role during the peritoneal vascularization of ectopic endometrial lesions by enhancing angiogenic activity via a paracrine effect.

Serum interleukin-6 levels are elevated in women with minimal–mild endometriosis

There is a need for a reliable marker of endometriosis, especially in early stages of peritoneal disease during which imaging is not effective. The use of serum interleukin (IL)-6 as a marker is controversial. To readdress the matter, patients undergoing laparoscopy were prospectively evaluated for serum IL-6 levels. A total of 119 women 31 years old who underwent laparoscopy were divided into groups: control patients (n = 38) with no pathologic findings; endometriosis sufferers (n = 47) with minimal-mild (MM, n = 11) or moderate-severe (MS, n = 36) endometriosis; uterine myomas (n = 13) and benign ovarian pathologies (n = 21). Blood was drawn on cycles days 5-12 and stored for subsequent analysis of IL-6 and carbohydrate antigen (CA)-125 levels. Serum IL-6 levels were significantly (P = 0.002) higher in women with MM endometriosis (29.4 9.0 pg/ml) than in controls (15.7 9.3 pg/ml). When all the non-endometriosis patients were grouped together (n = 72) and serum IL-6 (17.8 12.1 pg/ml) compared with MS (n = 36; 17.6 10.3 pg/ml) and MM (n = 11; 29.4 9.0 pg/ml) endometriosis significantly (P < 0.01) higher levels in MM endometriosis were observed as compared to the other two groups. Serum Ca-125 levels were significantly (P < 0.01) elevated in MS endometriosis. A serum IL-6 threshold of 25.75 pg/ml afforded a sensitivity of 75% and specificity of 83% in the diagnosis of MM endometriosis. Sensitivity and specificity for CA-125 in the diagnosis of MS endometriosis, using 35 IU/ml as the cut-off value, were 47% and 97%, respectively. IL-6 is a reliable non-invasive marker of MM endometriosis, whereas Ca-125 is of use as a marker of severe cases.

High density of small nerve fibres in the functional layer of the endometrium in women with endometriosis

Endometriosis is a common gynaecological disease and is frequently associated with recurrent and serious pelvic pain such as dysmenorrhoea and dyspareunia, but the mechanisms by which these symptoms are generated are not well understood. Histological sections of endometrial tissue were prepared from endometrial curettings and hysterectomies performed on women with endometriosis (n=25 and n=10, respectively) and without endometriosis (n=47 and n=35, respectively). These were stained immunohistochemically for the highly specific polyclonal rabbit anti-protein gene product 9.5 (PGP9.5) and monoclonal mouse anti-neurofilament protein (NF) to demonstrate both myelinated and unmyelinated nerve fibres. Small nerve fibres were identified throughout the basal and functional layers of the endometrium in all endometriosis patients, but were not seen in the functional layer of the endometrium in any of the women without endometriosis (P<0.001). NF-immunoreactive nerve fibres were present in the basal layer in all endometriosis patients but not in non-endometriosis patients, with one exception (P<0.001). Small nerve fibres detected in the functional layer in all women with endometriosis may have important implications for understanding the generation of pain in these patients. The presence of nerve fibres in an endometrial biopsy may be a novel surrogate marker of clinical endometriosis.

Is granulocyte colony-stimulating factor level predictive for human IVF outcome?

We investigated granulocyte colony-stimulating factor (G-CSF) in human reproduction. From a total sample of 93 patients, we analysed in group 1 (n = 82) the level of G-CSF and estradiol (E(2)) in serum and follicular fluid (FF) on day of follicular puncture (FP). Furthermore, in response to ovarian stimulation, G-CSF levels in serum were compared between low (n = 11), moderate (n = 53) and high (n = 18) response patients. In group 2 (n = 23) serum for G-CSF assessment was collected throughout menstrual cycle until gestation. Group 3 (n = 11) patients with endometriosis were assessed for G-CSF in serum and FF on day of FP without further differentiation. G-CSF in FF was higher than in serum (P < 0.01). G-CSF in serum increased from low through moderate to high response (P < 0.001); pregnancy rates were 0, 24.5 and 33.5% respectively. G-CSF in serum increased throughout stimulation, reached a peak with ovulation induction (P = 0.01) and decreased until embryo transfer (P=0.001). G-CSF level only in pregnant patients (n = 11) increased from embryo transfer to implantation to gestation (P = 0.005). In endometriosis patients G-CSF in serum and FF was lower than in non-endometriosis patients (P < or = 0.03) and corresponded with low response patients. G-CSF is involved in follicle development and may be a predictor of IVF outcome.

Mechanism of elevated vascular endothelial growth factor levels in peritoneal fluids from patients with endometriosis.

In order to investigate the mechanism of elevated vascular endothelial growth factor (VEGF) in peritoneal fluids from patients with endometriosis, macrophages were recovered from peritoneal fluids obtained at the time of diagnostic laparoscopy from infertile women with endometriosis (EMT group, n=20) and without endometriosis (control group, n=20). Macrophages were cultured in vitro. The VEGF levels of peritoneal fluid and the supernatant of macrophages culture were determined by enzyme linked immunoassay (ELISA). Meanwhile, the eutopic (n=20) and ectopic endometrium (n=20) from endometriosis patients, and normal edometrium (n=20) from non-endometriosis patients were obtained for the analysis of VEGF expression by labeled Streptavidin Biotin (LSAB). It was found that VEGF levels in peritoneal fluid and macrophages culture supernatant were significantly higher in EMT group than in control group (P<0.01). In normal endometrium, VEGF showed a cyclic changes and similar in eutopic and ectopic endometrium from patients with endometriosis. There was no difference in the intensity of VEGF in endometrium between two groups within each menstrual phase. It is suggested that altered VEGF production by peritoneal macrophages and ectopic endometrium secretion may contribute to the elevated VEGF levels in the peritoneal fluid of patients with endometriosis.