Role of interleukin-1ß in nerve growth factor expression, neurogenesis and deep dyspareunia in endometriosis.

STUDY QUESTION: Does interleukin-1ß (IL-1ß) play a role in promoting nerve growth factor expression, neurogenesis and deep dyspareunia in endometriosis? SUMMARY ANSWER: IL-1ß directly stimulates nerve growth factor (NGF) expression in endometriosis and is associated with local neurogenesis around endometriosis and more severe deep dyspareunia. WHAT IS KNOWN ALREADY: Local nerve density around endometriosis (using the pan-neuronal marker PGP9.5) is associated with deep dyspareunia in endometriosis, mediated in part by NGF expression. STUDY DESIGN, SIZE, DURATION: This in vitro study included endometriotic tissue samples from 45 patients. PARTICIPANTS/MATERIALS, SETTING, METHODS: This study was conducted in a university hospital affiliated research institute and included 45 women with surgically excised deep uterosacral/rectovaginal endometriosis (DIE, n = 12), ovarian endometriomas (OMA, n = 14) or superficial peritoneal uterosacral/cul-de-sac endometriosis (SUP, n = 19). Immunolocalisation of IL-1ß, IL-1 receptor type 1 (IL-1R1), NGF and PGP9.5 in endometriotic tissues was examined by immunohistochemistry (IHC), and the intensity of IHC staining in the endometriotic epithelium and stroma was semi-quantitatively evaluated using the Histoscore method (H-score). For each case, deep dyspareunia was pre-operatively rated by the patient on an 11-point numeric rating scale (0-10). In addition, primary endometriosis stromal cells were isolated and cultured from surgically excised endometriosis. These cells were treated with IL-1ß alone or in combination of Anakinra (an inhibitor of IL-1R1), small inference RNA (siRNA) against IL-1R1, siRNA against c-FOS or NGF neutralising antibody. The mRNA and protein levels of target genes (NGF and c-FOS) were assessed by reverse-transcription qPCR and western blot/ELISA, respectively. Furthermore, immunofluorescent microscopy was used to examine the neurite growth of rat pheochromocytoma PC-12 cells, as an in vitro model of neurogenesis. MAIN RESULTS AND THE ROLE OF CHANCE: For IHC, IL-1ß expression in the endometriosis epithelium was significantly associated with more severe deep dyspareunia (r = 0.37, P = 0.02), higher nerve fibre bundle density around endometriosis (r = 0.42, P = 0.01) and greater NGF expression by the endometriosis epithelium (r = 0.42, P = 0.01) and stroma (r = 0.45, P = 0.01). In primary endometriosis stromal cells, treatment with exogenous IL-1ß significantly increased the mRNA and protein levels of NGF and c-FOS. Pre-treatment with Anakinra, siRNA against IL-1R1, or siRNA against c-FOS, each attenuated IL-1 ß-induced increases of NGF expression. In addition, supernatants from IL-1ß treated endometriosis stromal cells significantly stimulated PC-12 neurite growth compared to controls, and these effects could be attenuated by pre-treatment with NGF neutralising antibody or Anakinra. LARGE-SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: We did not have data from cultures of endometriosis glandular epithelium, due to the known difficulties with primary cultures of this cell type. WIDER IMPLICATIONS OF THE FINDINGS: Our study revealed a mechanism for deep dyspareunia in endometriosis, whereby IL-1ß stimulates NGF expression, promoting local neurogenesis around endometriosis, which in turn leads to tender pelvic anatomic sites and thus deep-hitting dyspareunia. There may also be potential for drug targeting of IL-1ß and/or NGF in the management of endometriosis-associated pain. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by grants from the Canadian Institutes of Health Research (MOP-142273 and PJT-156084). P.Y. is also supported by a Health Professional Investigator Award from the Michael Smith Foundation for Health Research. MB has financial affiliations with Abbvie and Allergan. Otherwise, there are no conflicts of interest to declare.

Nerve Bundle Density and Expression of NGF and IL-1ß Are Intra-Individually Heterogenous in Subtypes of Endometriosis.

Endometriosis is a gynecological disorder associated with local inflammation and neuroproliferation. Increased nerve bundle density has been attributed to increased expression of nerve growth factor (NGF) and interleukin-1ß (IL-1ß). Immunohistochemical analysis was carried out on 12 patients presenting with all three anatomic subtypes of endometriosis (deep, superficial peritoneal, endometrioma) at surgery, with at least two surgically excised subtypes available for analysis. Immunolocalization for nerve bundle density around endometriosis using protein gene product 9.5 (PGP9.5), as well as NGF and IL-1ß histoscores in endometriosis epithelium/stroma, was performed to evaluate differences in scores between lesions and anatomic subtypes per patient. Intra-individual heterogeneity in scores across lesions was assessed using the coefficient of variation (CV). The degree of score variability between subtypes was evaluated using the percentage difference between mean scores from one subtype to another subtype for each marker. PGP9.5 nerve bundle density was heterogenous across multiple subtypes of endometriosis, ranging from 50.0% to 173.2%, where most patients (8/12) showed CV ≥ 100%. The percentage difference in scores showed that PGP9.5 nerve bundle density and NGF and IL-1ß expression were heterogenous between anatomic subtypes within the same patient. Based on these observations of intra-individual heterogeneity, we conclude that markers of neuroproliferation in endometriosis should be stratified by anatomic subtype in future studies of clinical correlation.

Interleukin-1ß and plasminogen activating system members in endometriotic stromal cell migration/invasion.

OBJECTIVE: To study the role of interleukin (IL)-1ß and the plasminogen activating (PA) system members in endometriotic stromal cell (ESC) migration/invasion. DESIGN: Primary cultures of ESCs. SETTING: Tertiary referral center for endometriosis and pelvic pain. PATIENT(S): Patients with surgically excised endometriosis. INTERVENTION(S): Interleukin-1ß stimulation of primary cultures of ESCs and knockdown of the PA system members urokinase plasminogen activator (uPA), uPA receptor, and plasminogen activator inhibitor-1 (PAI-1). MAIN OUTCOME MEASURE(S): Invasion/migration assays. RESULT(S): In primary cultures, IL-1ß-stimulated ESC production of the PA system members uPA, uPA receptor, and PAI-1. Interleukin-1ß also enhanced ESC migration and invasion, and these effects were inhibited by the IL-1 receptor-1 antagonist anakinra. Knockdown of each of the 3 PA system members also inhibited ESC migration and invasion. Knockdown of these PA system members further attenuated the impact of IL-1ß on migration and invasion, suggesting that they mediated the promigration and proinvasion effects of IL-1ß. To supplement the cell culture work, immunohistochemistry was performed on tissue sections of endometriotic epithelium/stroma: uPA, PAI-1, and IL-1ß histoscores were not found to be correlated with each other. CONCLUSION(S): In primary cultures of ESCs, IL-1ß induces migration and invasion, which is mediated by PA system members and inhibited by the drug anakinra. However, the immunohistochemistry expression of IL-1ß, urokinase plasminogen inhibitor-1, and PAI-1 were not correlated, suggesting other regulatory mechanisms for PA system members. Inhibition of IL-1ß (e.g., with anakinra) may have potential as a novel treatment approach for the migration/invasion of endometriosis.

Assessing the Impact of COVID-19 Vaccines on Sickle Cell Anaemia Patients: A Comparative Analysis of Biochemical and Haematological Parameters.

The coronavirus disease 2019 (COVID-19) vaccines have been developed to help prevent the spread of the virus infections. The COVID-19 vaccines, including Pfizer, Moderna, and AstraZeneca, have undergone rigorous testing and have demonstrated both safety and effectiveness. Extensive evidence supports their effectiveness in preventing severe illness, hospitalization, and mortality associated with COVID-19 infection. The administration of COVID-19 vaccines can directly affect hematological and biochemical parameters, with reported cases showing an association with thrombosis and thrombocytopenia. Therefore, it was hypothesized that COVID-19 vaccines may also influence hematological and biochemical markers in sickle cell patients. This study aimed to investigate the side effects of COVID-19 vaccines on sickle cell patients, providing a comprehensive evaluation of hematological and biochemical parameters. To our knowledge, this is the first study of its kind conducted in Saudi Arabia. The study included the evaluation of Pfizer and Oxford-AstraZeneca vaccines in sickle cell patients, measuring key parameters. Our findings revealed varying impacts of both vaccines on the ALT, AST, and CRP levels. Notably, CRP and ALT exhibited potential as indicators for renal disease, diabetes, and arthritis. However, further investigations are necessary to uncover the underlying mechanisms that drive these observed differences and comprehend their clinical implications for this vulnerable patient population. The unique nature of our study fills a crucial research gap and underscores the need for additional research in this area.

Standardized protocol for quantification of nerve bundle density as a biomarker for endometriosis.

Introduction: We propose a standardized protocol for measurement of nerve bundle density in endometriosis as a potential biomarker, including in deep endometriosis (DE), ovarian endometriomas (OMA) and superficial peritoneal endometriosis (SUP). Methods: This was a prospective cohort of surgically excised endometriosis samples from Dec 1st 2013 and Dec 31st 2017 at a tertiary referral center for endometriosis in Vancouver, BC, Canada. Surgical data were available from linked patient registry. Protein gene product 9.5 (PGP9.5) was used to identify nerve bundles on immunohistochemistry. PGP9.5 nerve bundles were counted visually. To calculate nerve bundle density, PGP9.5 nerve bundle count was divided by the tissue surface area (total on the slide). All samples were assessed using NHS Elements software for semi-automated measurement of the tissue surface area. For a subset of samples, high power fields (HPFs) were also counted as manual measurement of the tissue surface area. Intraclass correlation was used to assess intra observer and inter observer reliability. Generalized linear mixed model (GLMM) with random intercepts only was conducted to assess differences in PGP9.5 nerve bundle density by endometriosis type (DE, OMA, SUP). Results: In total, 236 tissue samples out of 121 participants were available for analysis in the current study. Semi-automated surface area measurement could be performed in 94.5% of the samples and showed good correlation with manually counted HPFs (Spearman's rho = 0.781, p < 0.001). To assess intra observer reliability, 11 samples were assessed twice by the same observer; to assess inter observer reliability, 11 random samples were blindly assessed by two observers. Intra observer reliability and inter observer reliability for nerve bundle density were excellent: 0.979 and 0.985, respectively. PGP9.5 nerve bundle density varied among samples and no nerve bundles could be found in 24.6% of the samples. GLMM showed a significant difference in PGP9.5 nerve bundle density between the different endometriosis types (X2 = 87.6, P < 0.001 after adjusting for hormonal therapy, with higher density in DE and SUP in comparison to OMA). Conclusion: A standardized protocol is presented to measure PGP9.5 nerve bundle density in endometriosis, which may serve as a biomarker reflecting local neurogenesis in the endometriosis microenvironment.

Evaluate the side effect associated with COVID-19 vaccine on adolescents in Riyadh, Saudi Arabia: A cross-section study.

OBJECTIVES: To investigate the side effects of Pizer- BioNTech mRNA (BNT162b2) and Spikevax (mRNA- 1273) Coronavirus disease 2019 (COVID-19) vaccines on adolescents in Riyadh, Saudi Arabia. METHODS: A cross-sectional study using an online questionnaire was carried out among COVID-19 vaccine adolescent recipients in Riyadh, Saudi Arabia. After receiving at least one dose of each vaccine, general and demographic data were collected, and vaccine-related side effects were evaluated. RESULTS: The final sample consisted of 604 participants with a majority age group of 16-17 years old. Approximately 89.1% of the study participants were female. Most participants reported pain at the injection site (85.1% 1st dose, 79.8% 2nd dose), feeling tired, and headache (58.6% 1st dose, 64.2% 2nd dose). Moreover, we found that patients who took the first dose and had a chronic disease had 2.4 times higher odds of having menstrual disorder (females) than non-chronic disease patients (p=0.03) and 4.5 times higher odds of exhibiting breathing congestion (p=0.01). In addition, patients with chronic disease had 2.4 times higher odds of exhibiting muscle and joint pain and dizziness than non-chronic disease patients (p=0.01, p=0.02). Males were less likely to have dizziness after the first dose than females (OR=0.26, p=0.01). CONCLUSION: This study investigates the adverse effects of COVID-19 vaccines among adolescents in Riyadh. As a result, this study creates a database to inform people about the risk of experiencing side effects based on their gender, age, and the vaccine type; more investigation is needed to better understand the link between risk factors and the development of adverse effects.

Immunoreactivity of Kisspeptin and Kisspeptin Receptor in Eutopic and Ectopic Endometrial Tissue of Women With and Without Endometriosis.

Endometriosis is characterized by the presence of ectopic endometrial tissues. Mechanisms of tissue dissemination in endometriosis may be similar to those involved in tumor metastasis. We hypothesize that dysregulation of kisspeptin (KISS1), a metastasis suppressor in endometrial carcinoma, may contribute to the pathogenesis of endometriosis. In this study, we characterized the immunoreactivity of kisspeptin and its receptor, KISS1R, in eutopic and ectopic endometrial tissue of women with and without endometriosis, in proliferative and secretory menstrual cycle phases. Immunohistochemistry was performed using KISS1 and KISS1R antibodies on samples from women with (n = 35) and without (n = 14) endometriosis. Samples from women with endometriosis included eutopic endometrium (n = 20) samples, superficial endometriotic implants (SUP, n = 10) deep infiltrating endometriotic implants (DIE, n = 15), and ovarian endometriomas (OMA, n = 15). Immunoreactivity was quantified using histoscores. KISS1 and KISS1R immunoreactivity was significantly lower in eutopic endometrial stroma of women with versus without endometriosis, regardless of the menstrual cycle phase (P = 0.001 and P = 0.015 respectively). In endometriotic implants, KISS1 levels were significantly lower in both glandular and stromal components of DIE (P < 0.01) and OMA (P < 0.01) compared to SUP. KISS1R immunoreactivity was lower in the glandular component of OMA (P = 0.035) compared to SUP. KISS1 and KISS1R levels are lower in eutopic endometrial stroma from women with versus without endometriosis, consistent with a role for decreased KISS1 expression in the pathogenesis of endometriosis. As deeply invasive lesions showed lower KISS1 levels than superficial lesions, downregulation of KISS1 levels may contribute to implant invasiveness.

ARID1A Mutations Promote P300-Dependent Endometrial Invasion through Super-Enhancer Hyperacetylation.

Endometriosis affects 1 in 10 women and is characterized by the presence of abnormal endometrium at ectopic sites. ARID1A mutations are observed in deeply invasive forms of the disease, often correlating with malignancy. To identify epigenetic dependencies driving invasion, we use an unbiased approach to map chromatin state transitions accompanying ARID1A loss in the endometrium. We show that super-enhancers marked by high H3K27 acetylation are strongly associated with ARID1A binding. ARID1A loss leads to H3K27 hyperacetylation and increased chromatin accessibility and enhancer RNA transcription at super-enhancers, but not typical enhancers, indicating that ARID1A normally prevents super-enhancer hyperactivation. ARID1A co-localizes with P300 at super-enhancers, and genetic or pharmacological inhibition of P300 in ARID1A mutant endometrial epithelia suppresses invasion and induces anoikis through the rescue of super-enhancer hyperacetylation. Among hyperactivated super-enhancers, SERPINE1 (PAI-1) is identified as an essential target gene driving ARID1A mutant endometrial invasion. Broadly, our findings provide rationale for therapeutic strategies targeting super-enhancers in ARID1A mutant endometrium.

Plasminogen activator inhibitor-1 (PAI-1) expression in endometriosis.

PURPOSE: Deep infiltrating endometriosis (DIE) is defined as an endometriotic lesion penetrating to a depth of >5 mm and is associated with pelvic pain, but the underlying mechanisms are unclear. Our objective is to investigate whether plasminogen activator inhibitor-1 expression (PAI-1) in endometriotic tissues is increased in women with DIE. METHODS: In this blinded in vitro study, immunohistochemistry and Histoscore were used to examine the expression of PAI-1 in glandular epithelium (GECs) and stroma (SCs) in a total of 62 women: deep infiltrating uterosacral/rectovaginal endometriosis (DIE; n = 13), ovarian endometrioma (OMA; n = 14), superficial peritoneal uterosacral/cul-de-sac endometriosis (SUP; n = 23), uterine (eutopic) endometrium from women with endometriosis (UE; n = 6), and non-endometriosis eutopic endometrium (UC; n = 6). The following patient characteristics were also collected: age, American Fertility Society stage, hormonal suppression, phase of menstrual cycle, dysmenorrhea score and deep dyspareunia score. RESULTS: PAI-1 expression in GECs and SCs of the DIE group was significantly higher than that of SUP group (p = 0.01, p = 0.01, respectively) and UE group (p = 0.03, p = 0.04, respectively). Interestingly, increased PAI-1 expression in GECs and SCs was also significantly correlated with increased dysmenorrhea (r = 0.38, p = 0.01; r = 0.34, p = 0.02, respectively). CONCLUSIONS: We found higher expression of PAI-1 in DIE, and an association between PAI-1 and worse dysmenorrhea.