DIRAS3 regulates autophagy in an endometriosis epithelial cell line.

RESEARCH QUESTION: What is the role of DIRAS3 in endometriosis pathogenesis? DESIGN: Prospective patient cohort study combined with experiments in the 12Z human endometriosis epithelial cell line model to determine the role of DIRAS3 in endometriosis. Endometrium and endometriosis lesion samples were collected from premenopausal women from 24 control and 40 endometriosis patients by laparoscopic surgery. The role of DIRAS3 in endometriosis was assessed by siRNA knockdown in 12Z cells followed by proliferation, apoptosis, invasion and autophagy assays. Autophagy was induced by serum starvation and the levels of autophagy determined by assessing changes in the expression levels and localization of autophagy marker proteins, such as LC3. RESULTS: DIRAS3 mRNA showed a large increase in expression in ectopic endometriosis lesions compared with endometrium from control patients, with expression largely localized to the epithelium. DIRAS3 knockdown in 12Z endometriosis epithelial cells caused a significant reduction in the number of proliferating cells (1.6-fold, adjusted P = 0.0007) and increased apoptosis (AnnexinV/7AAD double-positive cells +48%, P = 0.01), indicating an effect on cell proliferation. Induction of autophagy by serum starvation caused significant upregulation in DIRAS3 expression after 24 h (mRNA +2.4-fold [adjusted P = 0.017], protein +8.1-fold (adjusted P = 0.029), reduced LC3I/LC3II ratio (-2.2-fold, adjusted P = 0.044) and an increase in the number of double positive LC3/DIRAS3 puncta (+2.3-fold, P = 0.02). Knockdown of DIRAS3 in serum-starved cells led to a reduction in autophagy, indicated by an overall decrease in LC3 expression and significant increase in LC3I/LC3II ratio. CONCLUSIONS: DIRAS3 is highly upregulated in endometriosis lesions. Studies in an endometriosis epithelial cell line indicate that DIRAS3 facilitates cell survival in this context by inducing autophagy.

Does the Use of the "Proseek® Multiplex Inflammation I Panel" Demonstrate a Difference in Local and Systemic Immune Responses in Endometriosis Patients with or without Deep-Infiltrating Lesions?

Endometriotic lesions are able to infiltrate surrounding tissue. This is made possible partly by an altered local and systemic immune response that helps achieve neoangiogenesis, cell proliferation and immune escape. Deep-infiltrating endometriosis (DIE) differs from other subtypes through the invasion of its lesions over 5 mm into affected tissue. Despite the invasive nature of these lesions and the wider range of symptoms they can trigger, DIE is described as a stable disease. This elicits the need for a better understanding of the underlying pathogenesis. We used the "Proseek® Multiplex Inflammation I Panel" in order to simultaneously detect 92 inflammatory proteins in plasma and peritoneal fluid (PF) of controls and patients with endometriosis, as well as in particular patients with DIE, in order to gain a better insight into the systemically and locally involved immune response. Extracellular newly identified receptor for advanced gycation end-products binding protein (EN-RAGE), C-C motif Chemokine ligand 23 (CCL23), Eukaryotic translation initiation factor 4-binding protein 1 (4E-BP1) and human glial cell-line derived neurotrophic factor (hGDNF) were significantly increased in plasma of endometriosis patients compared to controls, whereas Hepatocyte Growth factor (HGF) and TNF-related apoptosis inducing ligand (TRAIL) were decreased. In PF of endometriosis patients, we found Interleukin 18 (IL-18) to be decreased, yet Interleukin 8 (IL-8) and Interleukin 6 (IL-6) to be increased. TNF-related activation-induced cytokine (TRANCE) and C-C motif Chemokine ligand 11 (CCL11) were significantly decreased in plasma, whereas C-C motif Chemokine ligand 23 (CCL23), Stem Cell Factor (SCF) and C-X-C motif chemokine 5 (CXCL5) were significantly increased in PF of patients with DIE compared to endometriosis patients without DIE. Although DIE lesions are characterized by increased angiogenetic and pro-inflammatory properties, our current study seems to support the theory that the systemic immune system does not play a major role in the pathogenesis of these lesions.

Comparison of a practice-based versus theory-based training program for conducting vacuum-assisted deliveries: a randomized-controlled trial.

PURPOSE: Vacuum-assisted deliveries (VAD) are complex procedures that require training and experience to be performed proficiently. We aimed to evaluate if a more resource intensive practice-based training program for conducting VAD is more efficient compared to a purely theory-based training program, with respect to immediate training effects and persistence of skills 4-8 weeks after the initial training. METHODS: In this randomized-controlled study conducted in maternity staff, participants performed a simulated low-cavity non-rotational vacuum delivery before (baseline test) and immediately after the training (first post-training test) as well as 4-8 weeks thereafter (second post-training test). The study's primary endpoint was to compare training effectiveness between the two study groups using a validated objective structured assessment of technical skills (OSATS) rating scale. RESULTS: Sixty-two participants were randomized to either the theory-based group (n = 31) or the practice-based group (n = 31). Total global and specific OSATS scores, as well as distance of cup application to the flexion point improved significantly from baseline test to the first post-training test in both groups (pall < 0.007). Skill deterioration after 4-8 weeks was only found in the theory-based group, whereas skills remained stable in the practice-based group. CONCLUSION: A practice-based training program for conducting VAD results in comparable immediate improvement of skills compared to a theory-based training program, but the retention of skills 4-8 weeks after training is superior in a practice-based program. Future studies need to evaluate, whether VAD simulation training improves maternal and neonatal outcome after VAD.

Is Chronic Endometritis Associated with Tubal Infertility? A Prospective Cohort Study.

STUDY OBJECTIVE: Chronic endometritis (CE), which often presents asymptomatically, is associated with recurrent pregnancy loss, recurrent implantation failure after in vitro fertilization, and endometriosis. Data connecting CE with fallopian tubal occlusion are limited. The aim was to assess a potential association of CE, defined by the presence of syndecan-1 (CD138)-positive plasma cells in endometrial tissue samples, with fallopian tube patency and other factors for infertility, including endometriosis, adenomyosis, and hydrosalpinges. DESIGN: Prospective, monocentral pilot study. SETTING: Tertiary care center. PATIENTS: A cohort of 100 women who were infertile was enrolled from July 2019 to December 2020. INTERVENTIONS: Hysteroscopy with endometrial biopsy and laparoscopy with chromopertubation. MEASUREMENTS AND MAIN RESULTS: CE was found in 13 women (13.0%) and was associated with endometriosis (p = .034) and unilateral/bilateral fallopian tube blockage (p = .013). In women with endometriosis, the mean number of CD138-positive cells was positively correlated with the revised American Society for Reproductive Medicine score (r = .302, p = .028). In a binary regression model, the presence of a hydrosalpinx on one or both sides (odds ratio 15.308; 95% confidence interval, 1.637-143.189; p = .017) and the finding of CE in the endometrial tissue sample (odds ratio 5.273; 95% confidence interval, 1.257-22.116; p = .023) were significantly associated with fallopian tubal occlusion. CONCLUSION: CE was significantly associated with blockage of the fallopian tubes and endometriosis. Endometriosis stage was associated with the number of CD138-positive cells in endometrial biopsies.

Performing central venous catheters in neonates and small infants undergoing cardiac surgery using a wireless transducer for ultrasound guidance: a prospective, observational pilot study.

BACKGROUND: Neonates and small infants with congenital cardiac disease undergoing cardiac surgery represent major challenges facing paediatric anaesthesia and perioperative medicine. AIMS: We here aimed to investigate the success rates in performing ultrasound (US) guided central venous catheter insertion (CVC) in neonates and small infants undergoing cardiac surgery, and to evaluate the practicability and feasibility of thereby using a novel wireless US transducer (WUST). METHODS: Thirty neonates and small infants with a maximum body weight of 10 kg and need for CVC before cardiac surgery were included in this observational trial and were subdivided into two groups according to their weight: < 5 kg and ≥ 5 kg. Cannulation success, failure rate, essential procedure related time periods, and complications were recorded and the clinical utility of the WUST was assessed by a 5-point Likert scale. RESULTS: In total, CVC-insertion was successful in 27 (90%) of the patients and the first attempt was successful in 24 (78%) of patients. Success rates of CVC were 80% < 5 kg and 100% ≥5 kg. Comparing the two groups we found a clear trend towards longer needle insertion time in patients weighing < 5 kg (33 [28-69] vs. 24 [15-37]s, P = .07), whereas, the total time for catheter insertion and the duration of the whole procedure were similar in both groups (199 [167-228] vs. 178 [138-234] and 720[538-818] vs. 660 [562-833]s. In total, we report 3 (10%) cases of local hematoma as procedure-related complications. Assessments of the WUST revealed very good survey results for all parameters of practicability and handling (all ratings between 4.5 and 5.0). CONCLUSION: Although difficulties in CVC-placement seem to relate to vessel size and patient's weight, US guided CVC-insertion represents a valuable, fast, and safe intervention in neonates and small children undergoing cardiac surgery. Using the WUST is feasible for this clinical application and may aid in efforts aiming to optimize perioperative care. TRIAL REGISTRATION: Wireless US-guided CVC placement in infants; Clinicaltrials.gov: NCT04597021 ; Date of Registration: 21October, 2020; retrospectively registered.

The Role of Long Non-Coding RNAs in Endometriosis.

Endometriosis is a chronic gynecological disorder affecting the quality of life and fertility of many women around the world. Heterogeneous and non-specific symptoms may lead to a delay in diagnosis, with treatment options limited to surgery and hormonal therapy. Hence, there is a need to better understand the pathogenesis of the disease to improve diagnosis and treatment. Long non-coding RNAs (lncRNAs) have been increasingly shown to be involved in gene regulation but remain relatively under investigated in endometriosis. Mutational and transcriptomic studies have implicated lncRNAs in the pathogenesis of endometriosis. Single-nucleotide polymorphisms (SNPs) in lncRNAs or their regulatory regions have been associated with endometriosis. Genome-wide transcriptomic studies have identified lncRNAs that show deregulated expression in endometriosis, some of which have been subjected to further experiments, which support a role in endometriosis. Mechanistic studies indicate that lncRNAs may regulate genes involved in endometriosis by acting as a molecular sponge for miRNAs, by directly targeting regulatory elements via interactions with chromatin or transcription factors or by affecting signaling pathways. Future studies should concentrate on determining the role of uncharacterized lncRNAs revealed by endometriosis transcriptome studies and the relevance of lncRNAs implicated in the disease by in vitro and animal model studies.

LINC01133 Inhibits Invasion and Promotes Proliferation in an Endometriosis Epithelial Cell Line.

Endometriosis is a common gynecological disorder characterized by ectopic growth of endometrium outside the uterus and is associated with chronic pain and infertility. We investigated the role of the long intergenic noncoding RNA 01133 (LINC01133) in endometriosis, an lncRNA that has been implicated in several types of cancer. We found that LINC01133 is upregulated in ectopic endometriotic lesions. As expression appeared higher in the epithelial endometrial layer, we performed a siRNA knockdown of LINC01133 in an endometriosis epithelial cell line. Phenotypic assays indicated that LINC01133 may promote proliferation and suppress cellular migration, and affect the cytoskeleton and morphology of the cells. Gene ontology analysis of differentially expressed genes indicated that cell proliferation and migration pathways were affected in line with the observed phenotype. We validated upregulation of p21 and downregulation of Cyclin A at the protein level, which together with the quantification of the DNA content using fluorescence-activated cell sorting (FACS) analysis indicated that the observed effects on cellular proliferation may be due to changes in cell cycle. Further, we found testis-specific protein kinase 1 (TESK1) kinase upregulation corresponding with phosphorylation and inactivation of actin severing protein Cofilin, which could explain changes in the cytoskeleton and cellular migration. These results indicate that endometriosis is associated with LINC01133 upregulation, which may affect pathogenesis via the cellular proliferation and migration pathways.

Investigating selected adhesion molecules as urinary biomarkers for diagnosing endometriosis.

RESEARCH QUESTION: Are selected cell adhesion molecules useful as urinary biomarkers for diagnosing endometriosis? DESIGN: Prospective, longitudinal study (the Endometriosis Marker Austria) in patients who underwent laparoscopic surgery for benign gynaecological pathologies. A total of 149 patients not receiving hormonal treatment for at least 3 months prior to recruitment were included and preoperative urine protein levels of soluble vascular adhesion molecule-1 (sVCAM-1), soluble intracellular adhesion molecule-1 (sICAM-1), E-selectin and P-selectin were measured using a magnetic bead-based multiplex assay, normalized to creatinine levels of each sample. Levels were correlated with endometriosis status, menstrual cycle phase, body mass index, cigarette smoking and severity and entity of the lesions. RESULTS: Urine levels of sVCAM-1, sICAM-1, E-selectin and P-selectin did not differ between women with (n = 84) and without (n = 65) endometriosis and among subgroups. Accordingly, receiver operating characteristic analysis to examine the value of using sVCAM-1, sICAM-1, E-selectin and P-selectin levels and sVCAM/sICAM ratio to diagnose endometriosis were not significant. Whether the serum sVCAM-1 levels correlated with the urine levels of the protein in the same women was also investigated, which revealed no significant correlations for sVCAM or sICAM. CONCLUSION: Although a previous study had suggested that serum sVCAM is a promising biomarker for diagnosing endometriosis, no significant differences were found in urine levels of sVCAM-1, sICAM-1, E-selectin and P-selectin between women with and without endometriosis. Other markers should be studied in an effort to establish a truly non-invasive urinary test for diagnosing endometriosis.

Allograft rejection is associated with development of functional IgE specific for donor MHC antigens.

BACKGROUND: Donor-specific antibodies of the IgG isotype are measured routinely for diagnostic purposes in renal transplant recipients and are associated with antibody-mediated rejection and long-term graft loss. OBJECTIVE: This study aimed to investigate whether MHC-specific antibodies of the IgE isotype are induced during allograft rejection. METHODS: Anti-MHC/HLA IgE levels were measured in sera of mice grafted with skin or heart transplants from various donor strains and in sera of kidney transplant patients with high levels of HLA IgG. Mediator release was triggered in vitro by stimulating basophils that were coated with murine or human IgE-positive serum, respectively, with specific recombinant MHC/HLA antigens. Kidney tissue samples obtained from organ donors were analyzed by using flow cytometry for cells expressing the high-affinity receptor for IgE (FcεRI). RESULTS: Donor MHC class I- and MHC class II-specific IgE was found on acute rejection of skin and heart grafts in several murine strain combinations, as well as during chronic antibody-mediated heart graft rejection. Anti-HLA IgE, including donor HLA class I and II specificities, was identified in a group of sensitized transplant recipients. Murine and human anti-MHC/HLA IgE triggered mediator release in coated basophils on stimulation with specific MHC/HLA antigens. HLA-specific IgE was not linked to atopy, and allergen-specific IgE present in allergic patients did not cross-react with HLA antigens. FcεRI+ cells were found in the human renal cortex and medulla and provide targets for HLA-specific IgE. CONCLUSION: These results demonstrate that MHC/HLA-specific IgE develops during an alloresponse and is functional in mediating effector mechanisms.

Enhanced expression of TACE contributes to elevated levels of sVCAM-1 in endometriosis.

STUDY QUESTION: Are increased sVCAM-1 and sICAM-1 levels associated with tumor necrosis factor-alpha-converting enzyme (TACE) activity in endometriosis? SUMMARY ANSWER: Here we provide the first functional evidence that induced TACE activity in human endometriotic epithelial cells is at least in part responsible for the enhanced release of sVCAM-1 from these cells. WHAT IS KNOWN ALREADY: We and others have shown that serum-soluble (s)VCAM-1 levels are significantly higher in women with endometriosis, compared to disease-free controls. Experimental evidence exists suggesting a role of sICAM-1 and sVCAM-1 in the pathogenesis of endometriosis. TACE was identified as the protease responsible for phorbol 12-myristate 13-acetate (PMA)-induced VCAM-1 release in murine endothelial cells. Additionally, it has recently been shown that TACE is upregulated in the endometrial luminal epithelium of the mid-secretory phase in infertile women. STUDY DESIGN, SIZE, DURATION: This study was conducted at the Tertiary Endometriosis Referral Center of the Medical University of Vienna. Samples from a total number of 97 women were collected between July 2013 and September 2014. PARTICIPANTS/MATERIALS, SETTING, METHODS: After complete surgical exploration of the abdominopelvic cavity, 49 women with histologically proven endometriosis and 48 endometriosis-free control women were enrolled. Each participating woman contributed only one sample of eutopic endometrium and normal peritoneum, and some of the women with endometriosis contributed samples of diverse types of endometriotic lesions (in total 52 ectopic samples). Among the 49 women with endometriosis, 36 matched samples of endometriotic lesions and corresponding eutopic endometrium were collected. In order to detect sVCAM-1 and TACE protein by ELISA, peritoneal fluid (PF) samples were collected from 44 cases and 32 controls during surgery. Expression of TACE mRNA was analyzed by qRT-PCR in 111 endometrium tissue samples (28 eutopic control samples, 33 eutopic samples from women with endometriosis, 50 ectopic samples from lesions) and 37 healthy peritoneum samples. Immunohistochemistry was performed in 123 tissue samples (39 eutopic control samples, 42 eutopic samples from women with endometriosis, 42 ectopic samples from lesions) and the relation between tissue TACE protein levels and sVCAM-1 secretion was examined. PMA-induced sVCAM-1 release, and TACE- and VCAM-1-transcripts or proteins were measured in an immortalized endometriotic epithelial cell line (11Z) pre-incubated either with TACE inhibitors or following TACE siRNA knockdown. MAIN RESULTS AND THE ROLE OF CHANCE: Here, we demonstrate that TACE protein is overexpressed in epithelium of tissue samples of both eutopic endometrium and ectopic lesions of women with endometriosis compared to disease-free controls (P < 0.001 both) and that the overexpression of the protein in the lesions is due to activation of TACE gene transcription (P < 0.001). Moreover, epithelial TACE protein was significantly higher in ectopic samples than in corresponding eutopic tissue of women with the disease (P < 0.001). High endometrial tissue TACE protein expression correlated with higher serum sVCAM-1 levels (P < 0.05) but not with sICAM-1 levels. Inhibition of TACE either by TACE inhibitors or by TACE siRNA knockdown resulted in decreased PMA-induced shedding of sVCAM-1 in vitro (P < 0.005 or P < 0.01, respectively), but the TACE inhibitors did not affect transcription of TACE or VCAM-1. Additionally, we observed an upregulation of TACE in proliferative endometrial epithelium of infertile (P < 0.005), compared to fertile women. TACE was increased in infertile women with endometriosis (P = 0.051) but not in infertile women without endometriosis. LIMITATIONS, REASONS FOR CAUTION: Albeit well characterized, our control population included women with other gynecologic diseases, which may have impacted the levels of sVCAM-1 and tissue TACE expression levels, e.g. benign ovarian cysts or uterine fibroids. Thus, the results of our analysis have to be interpreted carefully and in the context of the current experimental settings. WIDER IMPLICATIONS OF THE FINDINGS: The dysregulation of TACE substrate shedding represents a promising yet relatively unexplored area of endometriosis progression and could serve as a basis for the development of new treatments of the disease. STUDY FUNDING AND COMPETING INTEREST(S): This work was supported by the Ingrid Flick Foundation. The authors have no competing interests to declare.