Antagonist Pretreatment Research Articles

Impact of GnRH antagonist pretreatment on oocyte yield after ovarian stimulation: A retrospective analysis.

The study investigates whether a 3-day pretreatment course with a GnRH antagonist in the early follicular phase has an impact on the number of retrieved COCs in a GnRH antagonist stimulation protocol. This is a retrospective single center crossover study involving women who did not conceive after one GnRH antagonist stimulation cycle ("standard cycle") and proceeded with another GnRH antagonist stimulation cycle preceded by early administration of GnRH antagonist for 3 days ("pretreatment cycle") with fresh embryo transfer or frozen embryo transfer. 430 patients undergoing 860 cycles were included. The mean female age was 34.4 ± 4.8 years. Indications for fertility treatment included unexplained infertility (34.3%), male-factor infertility (33.3%), age (16.9%), PCOS (8.2%), tubal (4.7) and endometriosis (2.6%). All cycles were divided into two groups: group 1 (standard, 430 cycles) and group 2 (pretreatment, 430 cycles). The mean duration of stimulation was similar in both groups (10.3 vs 10.3 days, p = 0.28). The starting dose of gonadotropin (234.9 vs 196.8 IU, p<0.001), total amount of gonadotropin used (2419 vs 2020 IU, p<0.001), the total number of retrieved COCs (10 vs 7.8 p<0.001) and the number of mature oocytes (8 vs 5.8 p<0.001) were significantly higher in group 2 than in group 1. The Generalized estimating equation (GEE) regression analysis showed that the pretreatment strategy had a significant positive effect on the number of COCs (coefficient 2.4, p <0.001 after adjusting for known confounders (age, indication, stimulation dose, type, and duration of stimulation). In conclusion, A 3-day course of GnRH antagonist pretreatment increases the number of COCs obtained after ovarian stimulation.

O-010 Luteal estradiol versus follicular GnRH antagonist for ovarian follicular synchronization in POSEIDON poor responders: a multicentric randomized controlled trial

Abstract Study question In order to improve follicular synchronization, is pre-treatment with Luteal Estradiol (LE) more efficacious than Follicular GnRH Antagonist (FA) among poor responders? Summary answer Luteal Estradiol is better than Follicular GnRH Antagonist to improve maturation rates in Poor Ovarian Responders (POR) What is known already In poor responders, follicular asynchrony is a major obstacle in optimizing the pregnancy rates and leads to more cycle cancellations and increases the requirement of additional pool up cycles. It imposes significant financial burden on patients. ESHRE recommendations on FA and LE are conditional and require better quality studies. Several studies have evaluated the individual role of pre-treatment with LE or FA in synchronising the number of mature oocytes retrieved. There have been no studies comparing the efficacy of luteal estradiol versus follicular antagonist Study design, size, duration A randomised controlled trial was conducted at multiple centres of a private fertility clinic over 1year. Block randomization was performed using a Web-based randomization system.Allocation concealment was done using envelopes. 196 POR patients were randomized and 145 successfully completed the study. 80 patients(55.17%) received LE while 65 patients (44.83%) received FA. A per-protocol analysis was conducted. A subgroup analyses was done after excluding endometriosis and male-factor infertility as they could have been significant confounding factors. Participants/materials, setting, methods We randomised POR patients(POSEIDON criteria) into 2 arms, in their previous menstrual cycle. LE arm received oral Estradiol valerate 4 mg/day from 7days before expected menses to Day1 of menses. FA arm received Inj.Cetrorelix 0.25mg S.C from Day1 to Day5 of menses. The primary outcomes analysed were oocytes retrieved ,Follicular Output Rate(FORT), Follicle-to-Oocyte Index(FOI), Ovarian Sensitivity Index(OSI), maturation rate and MII /AFC. Secondary outcomes were cycle cancellation rates, fertilization rate, cleavage rate and blastulation rate. Main results and the role of chance There was a significant improvement in maturation rates with luteal phase pre-treatment using estradiol (LE) compared to follicular GnRH antagonist (FA) (0.72 ± 0.03 vs. 0.63 ± 0.03, p-value = 0.045). However, there were no statistically significant differences observed in the oocytes retrieved, Follicle-to-Oocyte Index (FOI), Follicular Output Rate (FORT), or Ovarian Sensitivity Index (OSI) between the two pre-treatment groups. These non-significant differences remained consistent among both expected (poseidon category 3 and 4) and unexpected (posiedon group 1 and 2) poor responders. Secondary outcomes, including cycle cancellation rates, fertilization rate, cleavage rate, and blastulation rate, did not show significant variations between the luteal estradiol (LE) and follicular GnRH antagonist (FA) pre-treatment groups. Subgroup analyses without endometriosis (N = 136) and severe male factor infertility (N = 122), revealed no significant differences between the two study groups in these specific subpopulations. Limitations, reasons for caution Limited generalizability due to small sample size, potential bias from lack of blinding, restricted insight into long-term outcomes, and patient heterogeneity are notable limitations in this study. Wider implications of the findings Among poor responders, luteal estradiol demonstrated a significant improvement in maturation rates, probably due to better follicle synchronicity at trigger, compared to follicular antagonist. It’s also more cost-effective and patient-friendly. However, further research with larger sample size and long-term clinical endpoints are needed to validate these findings and optimise clinical strategies. Trial registration number CTRI registration no. CTRI/2023/06/053760

P045 MMP12 stimulates colonic afferents, and nociceptors through the activation of PAR1

Abstract Background Abdominal pain is a cause of significant morbidity for people with colitis. We have previously shown that biopsy samples from people with Crohn’s disease (CD) are pro-nociceptive suggesting that peripheral nociceptor activation is a cause of pain in colitis. Furthermore, these effects were correlated with biopsy expression of macrophage metalloproteinase (MMP12), which was shown to stimulate mouse and human colonic nociceptors highlighting the potential utility of MMP12 inhibitors to act as visceral analgesics. Following on from this work we sought to determine the contribution of proteinase-activated receptor 1 (PAR1) to MMP12 mediated nociceptor activation. Methods MMP12 mediated sensory neuron activation: changes in the intracellular [Ca2+]i within thoracolumbar (T12-L5 spinal segments) dorsal root ganglia sensory neurons were examined in cells cultured overnight and loaded with Fluo-4-AM (30min) prior to imaging. Responses were measured to MMP12 or the PAR1 receptor agonists TRAP6 following pre-treatment with vehicle, the MMP12 inhibitor MMP408 or the PAR1 antagonist SCH79797. Afterwards cells were exposed to capsaicin (1 µM) followed by 50 mM KCl, to confirm the presence of nociceptors and neurons respectively by positive response. MMP12 mediated colonic nociceptor activation: electrophysiological recordings were made of lumbar splanchnic nerve activity and of the isolated colorectum (ex-vivo) perfused with carbogenated Krebs solution, and responses determined following application with MMP12 alone or in the presence of respective MMP12 or PAR1 inhibitors. All experiments were performed using tissue from male C57/B6 mice euthanised by rising concentration of CO2followed by exsanguination in accordance with Schedule 1 of the UK Animals Scientific Procedures act (1986). Results Application of MMP12 or TRAP6 produced a robust increase in [Ca2+]i levels in DRG sensory neurons classified as nociceptors by their co-sensitivity to capsaicin. These effects were significantly attenuated by pre-treatment with respective MMP12 or PAR1 inhibitors, with the response to MMP12 also being inhibited by PAR1 antagonist pre-treatment. Consistent with this pattern of nociceptor stimulation, the application of MMP12 also a robust activation of colonic afferents that was abolished by inhibition of MMP12 or PAR1 activity. Conclusion Findings demonstrate that MMP12 stimulates colonic afferents, and nociceptors through the activation of PAR1.

Fisetin, a dietary flavonoid, promotes transintestinal cholesterol excretion through the activation of PPARδ

Fisetin, a dietary polyphenol abundantly found in strawberries, exhibits a broad spectrum of health-promoting activities, including antihyperlipidemic effects. This study aimed to investigate the regulatory effect of fisetin on cholesterol elimination through novel transintestinal cholesterol excretion (TICE) pathway. A hypercholesterolemic mouse model and human colon epithelial cancer cell line Caco-2 were utilized to conduct the study. In hypercholesterolemic mice, fisetin (25 mg/kg) treatment reduced serum total cholesterol by 46.48% and significantly decreased lipid accumulation in the liver. Furthermore, fisetin administration led to a substantial increase in the fecal neutral sterol contents, including coprostanol, coprostanone, dihydrocholesterol, and cholesterol. Specifically, these sterol contents increased by approximately 224.20%, 151.40%, 70.40% and 50.72% respectively. The fluorescence intensity of 22-NBD-cholesterol in intestinal perfusion increased by 95.94% in fisetin group (25 mg/kg), indicating that fisetin stimulated TICE. In high cholesterol-induced Caco-2 cells, fisetin at a concentration of 30 μM reduced total cholesterol and free cholesterol by 37.21% and 45.30% respectively, stimulated cholesterol excretion, and inhibited cholesterol accumulation. Additionally, fisetin upregulated the gene and protein expression of cholesterol efflux transporters ABCG5/G8 and ABCB1, while downregulating the cholesterol uptake regulator NPC1L1. Furthermore, fisetin increased LDLR protein expression and decreased PCSK9 expression. Notably, fisetin significantly activated nuclear receptor PPARδ in Caco-2 cells. PPARδ antagonist pretreatment counteracted the regulatory effects of fisetin on TICE regulators, suggesting fisetin lowered cholesterol through enhancing TICE by activation of intestinal PPARδ. Fisetin could be used as functional dietarysupplement for eliminating cholesterol and reducing the incidence of cardiovascular diseases.

SAT375 Impact of Flutamide on Wake vs. Sleep LH Pulse Frequency in PCOS: An Interim Analysis

Abstract Disclosure: C.K. Matingou: None. S. Kim: None. M. Gilrain: None. C.M. Burt Solorzano: None. C.R. McCartney: None. Polycystic ovary syndrome (PCOS) is characterized by hyperandrogenism and ovulatory dysfunction. PCOS is also associated with luteinizing hormone (LH) excess, in part related to persistently high gonadotropin-releasing hormone (GnRH) pulse frequency. Furthermore, late pubertal girls with obesity and hyperandrogenemia did not exhibit sleep-associated decreases in LH (GnRH) pulse frequency when studied during the follicular phase, in contrast to non-hyperandrogenemic controls with or without obesity (JCEM 2014;99:2887-96). We therefore aimed to test two hypotheses: (1) sleep-associated reductions in LH pulse frequency are less pronounced in adult PCOS compared to late-follicular controls; and (2) sleep-associated reductions in LH pulse frequency are normalized in PCOS with 4 weeks of flutamide (androgen-receptor antagonist) pretreatment. To date, we have studied 5 women with PCOS (median age 30 y, BMI 42.6 kg/m2) and 5 ovulatory controls (median age 22 y, BMI 23.2 kg/m2). Subjects underwent two separate 16-hour frequent-blood-sampling studies to characterize pulsatile LH secretion, once after flutamide pretreatment (250 mg BID x 4 weeks) and once after placebo (PBO) pretreatment (BID for 4 weeks). In each study, 1500-2300 h and 2300-0700 h were designated the wake and sleep periods, respectively. The order of flutamide and PBO administration was randomized, and treatment allocation was switched after the first admission per a cross-over design. Controls were studied on cycle days 7-10, while those with PCOS were studied no earlier than cycle day 7. Serum progesterone concentrations were &amp;lt; 1.5 ng/dl for all admissions. With PBO pretreatment, participants with PCOS and controls exhibited the following: wake LH pulse frequency 1.19 (1.08-1.33) (median [interquartile range]) and 1.22 (0.97-1.27) pulses/h, respectively; sleep LH pulse frequency 0.93 (0.78-1.28) and 0.83 (0.78-0.99) pulses/h, respectively; and wake-to-sleep change in LH pulse frequency of -19.4 (-21.9 to +2.9) and -15.5 (-31.8 to -12.0) percent, respectively. For participants with PCOS, pretreatment with flutamide was associated with the following changes compared to PBO: wake LH pulse frequency +0.01 (-0.06 to +0.03) pulses/h; sleep LH pulse frequency +0.06 (+0.01 to +0.07) pulses/h; and wake-to-sleep LH pulse frequency change +5 (+7 to -12) percent. None of these differences were statistically significant by conservative nonparametric testing. We conclude that this interim analysis does not offer any indication that wake- and sleep-associated LH pulse frequencies are different between PCOS and late-follicular controls, nor does it suggest clear differences in wake-to-sleep changes in late-follicular LH pulse frequency between groups. Furthermore, this analysis does not seem to offer any indication that flutamide alone alters wake- and/or sleep-associated LH pulse frequencies in PCOS. Presentation Date: Saturday, June 17, 2023

A Brain Region-Dependent Alteration in the Expression of Vasopressin, Corticotropin-Releasing Factor, and Their Receptors Might Be in the Background of Kisspeptin-13-Induced Hypothalamic-Pituitary-Adrenal Axis Activation and Anxiety in Rats.

Previously, we reported that intracerebroventricularly administered kisspeptin-13 (KP-13) induces anxiety-like behavior and activates the hypothalamic-pituitary-adrenal (HPA) axis in rats. In the present study, we aimed to shed light on the mediation of KP-13's stress-evoking actions. The relative gene expressions of the corticotropin-releasing factor (Crf, Crfr1, and Crfr2) and arginine vasopressin (Avp, Avpr1a, and Avpr1b) systems were measured in the amygdala and hippocampus of male Wistar rats after icv KP-13 treatment. CRF and AVP protein content were also determined. A different set of animals received CRF or V1 receptor antagonist pretreatment before the KP-13 challenge, after which either an open-field test or plasma corticosterone levels measurement was performed. In the amygdala, KP-13 induced an upregulation of Avp and Avpr1b expression, and a downregulation of Crf. In the hippocampus, the mRNA level of Crf increased and the level of Avpr1a decreased. A significant rise in AVP protein content was also detected in the amygdala. KP-13 also evoked anxiety-like behavior in the open field test, which the V1 receptor blocker antagonized. Both CRF and V1 receptor blockers reduced the KP-13-evoked rise in the plasma corticosterone level. This suggests that KP-13 alters the AVP and CRF signaling and that might be responsible for its effect on the HPA axis and anxiety-like behavior.

Cannabidiol and mitragynine exhibit differential interactive effects in the attenuation of paclitaxel-induced mechanical allodynia, acute antinociception, and schedule-controlled responding in mice.

For many chemotherapy patients peripheral neuropathy is a debilitating side effect. Mitragyna speciosa (kratom) contains the alkaloid mitragynine (MG), which produces analgesia in multiple preclinical pain models. In humans, anecdotal reports suggest cannabidiol (CBD) may enhance kratom-related analgesia. We examined the interactive activity of MG and CBD in a mouse chemotherapy-induced peripheral neuropathy (CIPN) model. We also examined MG + CBD in acute antinociception and schedule-controlled responding assays, as well as examined underlying receptor mechanisms. Male and female C57BL/6J mice received a cycle of intraperitoneal (ip) paclitaxel injections (cumulative dose 32mg/kg). The von Frey assay was utilized to assess CIPN allodynia. In paclitaxel-naïve mice, schedule-controlled responding for food was conducted under a fixed ratio (FR)-10, and hot plate antinociception was examined. MG dose-relatedly attenuated CIPN allodynia (ED50 102.96mg/kg, ip), reduced schedule-controlled responding (ED50 46.04mg/kg, ip), and produced antinociception (ED50 68.83mg/kg, ip). CBD attenuated allodynia (ED50 85.14mg/kg, ip) but did not decrease schedule-controlled responding or produce antinociception. Isobolographic analysis revealed 1:1, 3:1 MG + CBD mixture ratios additively attenuated CIPN allodynia. All combinations decreased schedule-controlled responding and produced antinociception. WAY-100635 (serotonin 5-HT1A receptor antagonist) pretreatment (0.01mg/kg, ip) antagonized CBD anti-allodynia. Naltrexone (pan opioid receptor antagonist) pretreatment (0.032mg/kg, ip) antagonized MG anti-allodynia and acute antinociception but produced no change in MG-induced decreased schedule-controlled behavior. Yohimbine (α2 receptor antagonist) pretreatment (3.2mg/kg, ip) antagonized MG anti-allodynia and produced no change in MG-induced acute antinociception or decreased schedule-controlled behavior. Although more optimization is needed, these data suggest CBD combined with MG may be useful as a novel CIPN therapeutic.

Alveolar macrophage-derived secretory autophagosomes exacerbate inflammation in lipopolysaccharide-induced acute respiratory distress syndrome

Background: Acute respiratory distress syndrome (ARDS) has a high incidence among patients in intensive care units. Extracellular vesicles and particles released from activated alveolar macrophages (AMs) contribute to ARDS-associated lung injury; however, the mechanisms underlying the inflammatory process remain unclear. Purpose: This study investigated the effects and the mechanisms through which AM-derived secretory autophagosomes (SAPs) contribute to ARDS-associated lung injury. Methods: SAPs were isolated from cell culture supernatants of AMs treated or untreated with lipopolysaccharide (LPS) and intratracheally injected to determine whether SAP exacerbated lung injury in mice with ARDS. The SAP proteome was analysed using mass spectrometry. Lung fibroblasts were treated with SAPs and collected for transcriptome sequencing. A transwell assay was conducted to study the chemotactic activity of fibroblasts induced by SAPs. Results: We found that AMs contributed to ARDS-associated lung injury by releasing a novel type of pro-inflammatory vesicles named SAPs, which were characterised as double-membraned vesicles approximately 200 nm in diameter and light-chain-3 expression. Proteomic analysis of SAPs and gene ontology (GO) enrichment analysis revealed that LPS-SAPs contain several differentially expressed proteins involved in effecting an inflammatory response, including interleukin (IL)-1β. We confirmed this result by quantifying IL-1β in SAPs using ELISA. Moreover, administering SAPs from LPS-stimulated macrophages to mice with ARDS exacerbated lung injury, whereas IL-1 receptor antagonist (IL-1RA) pre-treatment significantly ameliorated SAP-induced lung injury. In addition, we conducted transcriptome sequencing of lung fibroblasts treated with SAPs to explore the effect of SAPs on fibroblasts in ARDS, which demonstrated that the expression of several chemokines (CXCL2, CXCL15, and so forth) significantly increased with SAP stimulation. Furthermore, the transwell assay indicated that SAPs could enhance the chemotactic activity of fibroblasts, suggesting that SAPs may exacerbate inflammation in ARDS by promoting neutrophil or monocyte recruitment. Conclusion: This is the first study to show that AM-derived SAPs contribute significantly to ARDS by secreting pro-inflammatory cytokines and promoting inflammatory cell recruitment by activating fibroblasts, which may be beneficial for therapeutic development of ARDS. National Natural Science Foundation of China (Grant numbers 81870066, 81670074, 81930058, 82270083 and 81971888), Six Talent Peaks Project in Jiangsu Province(Grant numbers TD-SWYY-003), and Social Development Specifc Projects of Jiangsu Province [Grant number BE2020786]. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Combined non-psychoactive Cannabis components cannabidiol and β-caryophyllene reduce chronic pain via CB1 interaction in a rat spinal cord injury model.

The most frequently reported use of medical marijuana is for pain relief. However, its psychoactive component Δ9-tetrahydrocannabinol (THC) causes significant side effects. Cannabidiol (CBD) and β-caryophyllene (BCP), two other cannabis constituents, possess more benign side effect profiles and are also reported to reduce neuropathic and inflammatory pain. We evaluated the analgesic potential of CBD and BCP individually and in combination in a rat spinal cord injury (SCI) clip compression chronic pain model. Individually, both phytocannabinoids produced dose-dependent reduction in tactile and cold hypersensitivity in male and female rats with SCI. When co-administered at fixed ratios based on individual A50s, CBD and BCP produced enhanced dose-dependent reduction in allodynic responses with synergistic effects observed for cold hypersensitivity in both sexes and additive effects for tactile hypersensitivity in males. Antinociceptive effects of both individual and combined treatment were generally less robust in females than males. CBD:BCP co-administration also partially reduced morphine-seeking behavior in a conditioned place preference (CPP) test. Minimal cannabinoidergic side effects were observed with high doses of the combination. The antinociceptive effects of the CBD:BCP co-administration were not altered by either CB2 or μ-opioid receptor antagonist pretreatment but, were nearly completely blocked by CB1 antagonist AM251. Since neither CBD or BCP are thought to mediate antinociception via CB1 activity, these findings suggest a novel CB1 interactive mechanism between these two phytocannabinoids in the SCI pain state. Together, these findings suggest that CBD:BCP co-administration may provide a safe and effective treatment option for the management of chronic SCI pain.

The Role of Intra-Amygdaloid Neurotensin and Dopamine Interaction in Spatial Learning and Memory.

Neurotransmitter and neuromodulator neurotensin (NT) has been proved to facilitate spatial and passive avoidance learning after microinjected into the rat central nucleus of amygdala (CeA). These previous studies of our laboratory also revealed that neurotensin-1 receptor (NTS1) is involved in the mentioned actions of NT. Extensive literature confirms the interaction between neurotensinergic and dopaminergic systems, and our research group also suppose that the mesolimbic dopaminergic system (MLDS) is involved in the spatial learning and memory-facilitating effect of NT in the CeA. In the present work, NT and dopamine (DA) interaction has been examined in the Morris water maze and passive avoidance tests. Rats received 100 ng NT, 5 µg dopamine D2 receptor antagonist sulpiride in itself, sulpiride as a pretreatment before NT or vehicle solution into the CeA. NT microinjection significantly decreased target-finding latency in the Morris water maze test and significantly increased entrance latency in the passive avoidance test, as was expected based on our previous findings. The DA D2 receptor antagonist pretreatment was able to inhibit both effects of NT. The results confirm the facilitatory effect of NT on spatial learning and memory and let us conclude that these actions can be exerted via the DA D2 receptors.

Structure-Activity Relationships for Psilocybin, Baeocystin, Aeruginascin, and Related Analogues to Produce Pharmacological Effects in Mice.

4-Phosphoryloxy-N,N-dimethyltryptamine (psilocybin) is a naturally occurring tertiary amine found in many mushroom species. Psilocybin is a prodrug for 4-hydroxy-N,N-dimethyltryptamine (psilocin), which induces psychedelic effects via agonist activity at the serotonin (5-HT) 2A receptor (5-HT2A). Several other 4-position ring-substituted tryptamines are present in psilocybin-containing mushrooms, including the secondary amine 4-phosphoryloxy-N-methyltryptamine (baeocystin) and the quaternary ammonium 4-phosphoryloxy-N,N,N-trimethyltryptamine (aeruginascin), but these compounds are not well studied. Here, we investigated the structure-activity relationships for psilocybin, baeocystin, and aeruginascin, as compared to their 4-acetoxy and 4-hydroxy analogues, using in vitro and in vivo methods. Broad receptor screening using radioligand binding assays in transfected cells revealed that secondary and tertiary tryptamines with either 4-acetoxy or 4-hydroxy substitutions display nanomolar affinity for most human 5-HT receptor subtypes tested, including the 5-HT2A and the serotonin 1A receptor (5-HT1A). The same compounds displayed affinity for 5-HT2A and 5-HT1A in mouse brain tissue in vitro and exhibited agonist efficacy in assays examining 5-HT2A-mediated calcium mobilization and β-arrestin 2 recruitment. In mouse experiments, only the tertiary amines psilocin, psilocybin, and 4-acetoxy-N,N-dimethyltryptamine (psilacetin) induced head twitch responses (ED50 0.11-0.29 mg/kg) indicative of psychedelic-like activity. Head twitches were blocked by 5-HT2A antagonist pretreatment, supporting 5-HT2A involvement. Both secondary and tertiary amines decreased body temperature and locomotor activity at higher doses, the effects of which were blocked by 5-HT1A antagonist pretreatment. Across all assays, the pharmacological effects of 4-acetoxy and 4-hydroxy compounds were similar, and these compounds were more potent than their 4-phosphoryloxy counterparts. Importantly, psilacetin appears to be a prodrug for psilocin that displays substantial serotonin receptor activities of its own.

Endothelin A (ETA) and Endothelin B (ETB) Receptor Subtypes Potentiate Epidermal Growth Factor (EGF)-Mediated Proliferation in Human Asthmatic Bronchial Airway Smooth Muscle.

BackgroundAsthma is a chronic disease characterized by chronic inflammation, reversible airway obstruction, airway hyperresponsiveness (AHR), and airway remodeling. One of the important features of asthma is airway remodeling, which plays a central role in airflow limitation. Airway remodeling involves numerous changes in the bronchial walls, including airway smooth muscle (ASM) cell hypertrophy and hyperplasia. Studies have shown that ASM hyperplasia in asthma is mediated by the increased production of mitogens. Endothelin-1 (ET-1) has been shown to induce proliferation and function as a co-mitogen in vascular and ASM. In patients with asthma, plasma and bronchoalveolar lavage fluid have been shown to have elevated ET-1 levels, which have been linked to airway remodeling and airflow obstruction in severe asthma. This study investigates the role of ET-1 in proliferation, the receptor subtype mediating its effect, and the signaling pathway.MethodologyNormal and asthmatic bronchial airway smooth muscle (BASM) cells were seeded into 5 × 103 cells/well. Cell proliferation was assayed using 5-bromo-2’-deoxyuridine (BrdU) incorporation. Confluent cells were treated with different concentrations of ET-1 in the presence or absence of the epidermal growth factor (EGF). Signaling pathways were explored using pretreatment of BASM with antagonists 15 minutes before ET-1/EGF stimulation.ResultsIn asthmatic BASM, ET-1 (0.1 nM) functions as a co-mitogen in the presence of EGF (10 nM), showing a significantly greater effect on asthmatic BASM proliferation compared with normal BASM. The ETA receptor antagonist BQ-123 (10-1,000 nM) significantly reduced the proliferative effect of ET-1/EGF on asthmatic BASM more than normal BASM. Moreover, the effect of ETB antagonist BQ-788 (1,000 nM) or pretreatment with the ETB agonist S6C (1-10 nM) followed by co-treatment with EGF in asthmatic BASM showed a small but significant decrease when pretreated with the inhibitor and increased with the agonist, thereby suggesting that the co-mitogenic effect of ET-1 is mainly via the activation of ETA receptors, with a small contribution by the ETB receptors in asthmatic BASM. Finally, pertussis toxin (PTX) pretreatment (25 and 50 ng/mL) showed that EGF and ET-1/EGF mitogenic and co-mitogenic signaling utilizes Gi/0-mediated transactivation by EGF and ET receptors, especially in asthmatic BASM, leading to the activation of Ras-ERK-PI3K pathways. Enhanced ERK and PI3K effects on proliferation suggested that these kinases modulate the co-mitogenic effect of ET-1 in asthmatic BASM. Enhanced cross-talk between ET and EGF receptors may be a potential mechanism contributing to airway remodeling in asthmatic BASM.ConclusionsET-1 enhances the mitogenic effect of EGF predominantly via the ETA receptor in asthmatic BASM with the activation of Ras, ERK, and PI3K. The cross-talk mechanism between ET and EGF receptors may be a potential therapeutic target to prevent the progression of airway remodeling in ASM in patients with asthma.

Pre-IVF treatment with a GnRH antagonist in women with endometriosis (PREGNANT): study protocol for a prospective, double-blind, placebo-controlled trial

IntroductionInfertility is a common complication of endometriosis. While in vitro fertilisation-embryo transfer (IVF) successfully treats endometriosis-associated infertility, there is some evidence that pregnancy rates may be diminished in women seeing...

Optimal timing of intervention in NSTE-ACS without pre-treatment

Abstract Introduction In patients (P) with non-ST segment elevation acute coronary syndromes (NSTE-ACS), an invasive strategy is recommended to reduce adverse outcomes. The optimal timing to perform coronary angiography (CA) remains undetermined, particularly in our era attending to the new European guidelines restricting pre-treatment (PT). Objective To evaluate the prognostic value of an early strategy (ES; &amp;lt;24h) versus a delayed strategy (DS; &amp;gt;24h) when no loading dose of a P2Y12 antagonist was given as PT in NSTE-ACS. Material and methods Retrospective analysis of P data admitted with NSTE-ACS at multicentric national registry between 2015–19. Compared demographic and clinical characteristics of P with an ES versus DS. A multivariate logistic regression was performed to evaluate predictor factors of in-hospital and 1-year endpoints. Survival was evaluated through Kaplan-Meier curve and Cox multivariate regression. Results 691P were included, mean age 64±11 years, 77.4% male. 59.2% performed CA as an ES. P proposed to a DS presented higher KK class, higher levels of creatinine and lower of hemoglobin. They also needed more frequently invasive (1.1 vs 0.7%, p=0.692) or non-invasive ventilation (1.8 vs 0.7%, p=282). A higher proportion of DS patients used the transfemoral access (5.5 vs 9.3%, p=0.058). On CA, 6.2% had normal coronary arteries, 49.2% 1-vessel disease and 45.1% multivessel disease. Revascularization was performed in 88.2%: PCI in 86.2%, CABG in 1.7% and both in 0.3%, with no significant differences. Pending CA, 98.4% were medicated with aspirin, 64.8% ticagrelor and 44% clopidogrel, with no differences. P proposed to an ES were more medicated with glycoprotein inhibitor (36.3 vs 26.4%, p=0.015) and non-fractioned heparin (6.4 vs 2.1%, p=0.01) and less with fondaparinux (56.2 vs 65.2%, p=0.017). A higher percentage of calcium-channel blockers (25.2 vs 11.7%, p&amp;lt;0.001) and nitrates (74.1 vs 53.3%, p&amp;lt;0.001) was observed in the DS. No difference was observed in beta-blockers (p=0.581). Discharge medication followed these tendencies. There was a trend to worse in-hospital outcomes in the DS regarding heart failure, shock, ventricular arrhythmias, cardiac arrest and death, although not significatively different, except for major bleeding (1.8 vs 0.2%, p=0.044). 1-year composite endpoint of mortality and cardiovascular rehospitalization occurred in 9.9%, with no difference between groups (p=0.181). Predictor factors, evaluated through Cox multivariate regression, were ejection fraction &amp;lt;50% (p=0.001), KK class &amp;gt;I (p=0.002) and nitrate prescription at discharge (p=0.001). A DS was not a predictor factor (p=0.812). Conclusion Our results are in accordance with available data. In P with higher-risk NSTE-ACS in the absence of P2Y12 antagonist PT, an ES was not associated with a reduction in the composite of global mortality and rehospitalization for cardiovascular causes. Funding Acknowledgement Type of funding sources: None.

Evaluation of GnRH antagonist pretreatment before ovarian stimulation in a GnRH antagonist protocol in normal ovulatory women undergoing IVF/ICSI: a randomized controlled trial

BackgroundSynchronization of follicles is key to improving ovulation stimulation with the gonadotropin-releasing hormone (GnRH) antagonist protocol. GnRH antagonist administration in the early follicular phase can quickly decrease gonadotrophin (Gn) levels and achieve downregulation before stimulation, which may improves synchronization. A previous small randomized controlled study (RCT) showed that pretreatment with a GnRH antagonist for 3 days before stimulation may increase oocyte retrieval but cannot increase the pregnancy rate. This study investigated whether the GnRH antagonist pretreatment protocol in ovulatory women can increase the synchronization of follicles and pregnancy outcomes compared with the conventional GnRH antagonist protocol.MethodsThis RCT included 136 normal ovulatory women undergoing in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI). Both groups were treated with recombinant follicle-stimulating hormone (r-FSH) and a flexible GnRH antagonist protocol. The women were randomized into two equal groups with or without GnRH antagonist administration from day 2 of the menstrual cycle for 3 days before stimulation. Our primary outcome was the number of retrieved oocytes. Secondary outcomes included the pregnancy rate and live birth rate.ResultsBoth groups had similar baseline characteristics. The number of retrieved oocytes in the study group was comparable to that in the control group (9.5 [8.0–13.0] vs. 11.0 [7.0–14.8], P = 0.469). There was no significant difference in the follicle size. The fertilization rate, number of good-quality embryos, implantation rate, pregnancy rate, ongoing pregnancy rate, live birth rate per embryonic transfer cycle, and miscarriage rate were similar between the two groups.ConclusionThis large RCT analysed GnRH antagonist pretreatment with the GnRH antagonist protocol applied to normal ovulatory women undergoing IVF/ICSI. The number of retrieved oocytes and pregnancy outcomes did not significantly vary.Trial registrationChinese Clinical Trial Registry, ChiCTR1800019730. Registered 26 November 2018.

Porphyromonas Gingivalis Infection Induces Synaptic Failure via Increased IL-1β Production in Leptomeningeal Cells.

Studies have reported that synaptic failure occurs before the Alzheimer's disease (AD) onset. The systemic Porphyromonas gingivalis (P. gingivalis) infection is involved in memory decline. We previously showed that leptomeningeal cells, covering the brain, activate glial cells by releasing IL-1β in response to systemic inflammation. In the present study, we focused on the impact of leptomeningeal cells on neurons during systemic P. gingivalis infection. The responses of leptomeningeal cells and cortical neurons to systemic P. gingivalis infection were examined in 15-month-old mice. The mechanism of IL-1β production by P. gingivalis infected leptomeningeal cells was examined, and primary cortical neurons were treated with P. gingivalis infected leptomeningeal cells condition medium (Pg LCM). Systemic P. gingivalis infection increased the expression of IL-1β in leptomeninges and reduced the synaptophysin (SYP) expression in leptomeninges proximity cortex in mice. Leptomeningeal cells phagocytosed P. gingivalis resulting in lysosomal rupture and cathepsin B (CatB) leakage. Leaked CatB mediated NLRP3 inflammasome activation inducing IL-1β secretion in leptomeningeal cells. Pg LCM decreased the expression of synaptic molecules, including SYP, which was inhibited by an IL-1 receptor antagonist pre-treatment. These observations demonstrate that P. gingivalis infection is involved in synaptic failure by inducing CatB/NLRP3 inflammasome-mediated IL-1β production in leptomeningeal cells. The periodontal bacteria-induced synaptic damage may accelerate the onset and cognitive decline of AD.

P–464 What is the optimal ovarian stimulation (OS) protocol for women who undergo planned oocyte cryopreservation (POC)?

Abstract Study question When repeated cycles of OS for planned oocyte cryopreservation using a standard GnRH antagonist protocol are required, can OS protocol modifications improve oocyte yield? Summary answer Compared to repeating a standard GnRH antagonist protocol, switching to a long GnRH agonist protocol for POC results in a higher number of cryopreserved oocytes. What is known already The total number of cryopreserved oocytes is a key parameter of POC programs because of its association with livebirth. A substantial proportion of women embarking on POC will undergo repeated cycles of OS to reach their desired target number of vitrified oocytes. According to recent guidelines, the GnRH antagonist protocol with GnRH agonist triggering is considered the first choice protocol for POC, because of its safety profile and convenience. However, in women with normal ovarian reserve, the long GnRH agonist protocol results in a higher number of oocytes retrieved. Evidence regarding the optimal protocol for POC is limited. Study design, size, duration This is a single-centre, retrospective cohort study including 283 women who had a first cycle for POC using a standard GnRH antagonist protocol and who requested a second OS cycle to increase their total number of vitrified oocytes for later use. The choice of protocol for the second cycle was left at the discretion of the reproductive medicine specialist. All OS cycles took place between January 2009 and December 2019 in a tertiary referral hospital. Participants/materials, setting, methods After ovarian reserve testing, the first cycle OS was performed using rFSH or HPhMG in a GnRH antagonist protocol. For the second cycle, a GnRH antagonist protocol with or without antagonist pretreatment, or a long GnRH agonist protocol was prescribed. The primary outcome was the number of mature oocytes (MII) vitrified per cycle. Cycle characteristics were compared. Data were assessed by generalized estimating equation (GEE) regression analysis adjusting for covariates. Main results and the role of chance In total, 226 (79.9%) women had a GnRH antagonist protocol and 57 (20.1%) had a long GnRH agonist protocol in their second OS cycle for POC. Overall, mean age was 36.6±2.4 years. The median (CI) number of mature oocytes vitrified after the second OS cycle was significantly higher than that after the first cycle [8 (5–11) vs. 7 (4–10), p &amp;lt; 0.001]. According to GEE multivariate regression, adjusting for relevant confounders, switching from a GnRH antagonist protocol in the first cycle to a long GnRH agonist protocol in the second cycle was the only significant predictor of the number of vitrified oocytes after the subsequent cycle (coefficient 1.59, CI 0.29–2.89, p-value = 0.017). Age, AFC, initial dose and type of gonadotropins did not predict the number of vitrified oocytes. None of the women developed moderate or severe OHSS. Similarly, of 174 women who underwent their first OS cycle with a standard GnRH antagonist protocol, 133 women (76.4%) had the same protocol for their second cycle and 41 women (23.6%) an additional three-day course of GnRH antagonist pretreatment. According to GEE multivariate regression, this protocol modification did not result in more mature oocytes available for vitrification (coefficient –0.25, CI –1.86–1.36, p-value = 0.76). Limitations, reasons for caution These data should be interpreted with caution because of the retrospective design and limited sample. Although more oocytes were obtained with a long GnRH agonist protocol we have no data on livebirth in women returning to use their oocytes to support the choice for a specific OS protocol for POC. Wider implications of the findings: Although oocyte yield in the context of POC is an important parameter that may be modulated by the choice of OS protocol, the ultimate outcome measure of a successful POC program is livebirth after oocyte vitrification. Future research of oocyte parameters reflecting oocyte quality is paramount. Trial registration number Not applicable

P–684 Impact of GnRH antagonist pretreatment on oocyte yield after ovarian stimulation: a retrospective analysis

Abstract Study question Does a 3-day pretreatment course with a GnRH antagonist in the early follicular phase increase the number of oocytes in a GnRH antagonist stimulation protocol? Summary answer The administration of 3 days of GnRH antagonist before starting ovarian stimulation in a GnRH antagonist protocol increases the number of COCs (Cumulus-Oocyte-Complexes). What is known already The GnRH antagonist protocol is characterized by higher gonadotropin and E2 serum levels at the start of ovarian stimulation (OS), compared with a long pituitary down regulation protocol. The unsuppressed FSH level at the start of a GnRH antagonist cycle allows the initial growth of follicles before addition of exogenous FSH, which may result in asynchrony of the follicular cohort. Menstrual administration of a GnRH antagonist can inhibit follicle growth and improve homogeneity of recruitable follicles. Previous studies showed a trend toward higher numbers of COCs and improved maturation and fertilization rates of retrieved oocytes. Study design, size, duration Retrospective single center crossover study, including consecutive women enrolled in an IVF program in a university hospital from January 2011 to December 2020. All women underwent one standard GnRH antagonist stimulation cycle (“standard cycle”) and one GnRH antagonist stimulation cycle preceded by early administration of GnRH antagonist for 3 days (“pretreatment cycle”). Women with basal progesterone levels &amp;gt;1.5ng/ml, and women undergoing oocyte freezing, oocyte donation or PGT were excluded. In total, 427 patients were included. Participants/materials, setting, methods Women were included when the pretreatment cycle occurred within a time interval of &amp;lt; 12 months following the start of stimulation in the standard cycle. The primary outcome was the total number of COCs. Main results and the role of chance The average female age was 35.1 ± 4.7 years. Indications for fertility treatment included unexplained infertility (34.3%), male-factor infertility (33.3%), age (16.9%), PCOS (8.2%) and endometriosis (2.6%). All cycles were divided into two groups: group 1 (standard, 427 cycles) and group 2 (pretreatment, 427 cycles). The mean duration of stimulation was similar in both groups (10.3 vs 10.3 days, p = 0.2). The starting dose of gonadotropin (196.8 vs 234.9IU, p &amp;lt; 0.001) and total amount of gonadotropin used (2000.7 vs 2415.2IU, p &amp;lt; 0.001) were higher in group 2. The total number of obtained COCs (6.2 vs 8.8 p &amp;lt; 0.001) and the number of mature oocytes (4.2 vs 6.4 p &amp;lt; 0.001) were significantly higher in group 2. The Generalized estimating equation (GEE) multivariate regression analysis showed that the pretreatment strategy had a significant positive effect on the number of COCs (coefficient 2.8, p value &amp;lt;0.001 after adjusting for the confounders age, indication of infertility, stimulation dose, type and total amount of gonatropins used). Limitations, reasons for caution Despite the large dataset, the presence of biases related to the retrospective study design cannot be excluded. Besides, the impact of GnRH pretreatment on pregnancy rate cannot be assessed because of the crossover design. Wider implications of the findings: A 3-day course of GnRH antagonist pretreatment increases the number of COCs obtained after OS. Furthermore, since the initiation of OS in a GnRH antagonist protocol relies on the occurrence of spontaneous menses, addition of three days of GnRH antagonist pretreatment may enhance scheduling flexibility without reducing efficacy. Trial registration number Not applicable

Abstract 2746: CD276 in tumor cells contributes to the M2 macrophage-rich immunosuppressive tumor microenvironment via upregulating the CCL2-CCR2 axis in ovarian cancer

Abstract [Objective] Increased infiltration of CD8+ T cells and decreased infiltration of M2 macrophages are favorable prognostic factors in high-grade serous ovarian cancer (HGSOC). CD276 (B7H3) is a transmembrane co-inhibitory protein belonging to the B7 family. CD276 expression is suppressed in CD8+ T cell-rich, PD-L1-high immune reactive tumors. This suggests that CD276 plays immunosuppressive roles in addition to acting as a molecular brake for countering the T cell activation. In this study, we aimed to elucidate the role of CD276 in HGSOC microenvironment toward M2 macrophages. [Methods] CD276 expression was evaluated in HGSOC clinical cases (n=62) by immunohistochemistry tests. The correlation between survival and the presence of tumor-infiltrating immune cells, including M2 macrophages, was assessed. CD276 knockout murine ovarian cancer cell lines (HM1 and ID8) were established and used for experiments. [Results] In HGSOC patients, higher CD276 expression associated with poor prognosis (p&amp;lt;0.001). CD276 expression positively correlated with the number of CD206+ M2 macrophages (Pearson's r=0.29, p=0.02), but not with that of CD8+ T cells. Although CD276 knockout did not affect in vitro proliferation of HM1 and ID8 cell lines, CD276 knockout tumors were less progressive than control tumors in immunocompetent mouse models (p&amp;lt;0.001). Lower infiltration of CCR2+/CD206+ M2 macrophages and higher infiltration of IFN-γ+ CD8+ T cells was observed in the knockout tumors (p&amp;lt;0.001 and P=0.02, respectively). The difference in progression between CD276 knockout tumors and control tumors was negated when tumor cells were inoculated into immunodeficient nude mice. RNA sequencing and protein analyses revealed decreased levels of CCL2, the ligand of CCR2, in CD276 knockout cells and tumors. In vitro, monocyte chemotaxis was reduced by CD276 knockout cells' supernatant (p&amp;lt;0.001), and the difference was partially negated with CCR2 antagonist pretreatment. Furthermore, CCR2 blocking in vivo reduced tumor-infiltrating M2 macrophages and tumor volume in the CD276 control (p=0.048 and p=0.02, respectively), but not in the CD276 knockout group, suggesting that CCL2-CCR2 axis-induced M2 macrophages partially contribute to CD276-mediated tumor progression. [Conclusion] CD276 contributes to an unfavorable immunosuppressive tumor microenvironment driven by M2 macrophages via the CCL2-CCR2 axis in HGSOC. Citation Format: Taito Miyamoto, Ryusuke Murakami, Masayo Ukita, Mana Taki, Koji Yamanoi, Kaoru Abiko, Ken Yamaguchi, Junzo Hamanishi, Takashi Kobayashi, Masaki Mandai. CD276 in tumor cells contributes to the M2 macrophage-rich immunosuppressive tumor microenvironment via upregulating the CCL2-CCR2 axis in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2746.

Corticotropin-Releasing Hormone from the Pontine Micturition Center Plays an Inhibitory Role in Micturition.

Lower urinary tract or voiding disorders are prevalent across all ages and affect >40% of adults over 40 years old, leading to decreased quality of life and high health care costs. The pontine micturition center (PMC; i.e., Barrington's nucleus) contains a large population of neurons that localize the stress-related neuropeptide, corticotropin-releasing hormone (CRH) and project to neurons in the spinal cord to regulate micturition. How the PMC and CRH-expressing neurons in the PMC control volitional micturition is of critical importance for human voiding disorders. To investigate the specific role of CRH in the PMC, neurons in the PMC-expressing CRH were optogenetically activated during in vivo cystometry in unanesthetized mice of either sex. Optogenetic activation of CRH-PMC neurons led to increased intermicturition interval and voided volume, similar to the altered voiding phenotype produced by social stress. Female mice showed a significantly more pronounced phenotype change compared with male mice. These effects were eliminated by CRH-receptor 1 antagonist pretreatment. Optogenetic inhibition of CRH-PMC neurons led to an altered voiding phenotype characterized by more frequent voids and smaller voided volumes. Last, in a cyclophosphamide cystitis model of bladder overactivity, optogenetic activation of CRH-PMC neurons returned the voiding pattern to normal. Collectively, our findings demonstrate that CRH from PMC spinal-projecting neurons has an inhibitory function on micturition and is a potential therapeutic target for human disease states, such as voiding postponement, urinary retention, and underactive or overactive bladder.SIGNIFICANCE STATEMENT The pontine micturition center (PMC), which is a major regulator of volitional micturition, is neurochemically heterogeneous, and excitatory neurotransmission derived from PMC neurons is thought to mediate the micturition reflex. In the present study, using optogenetic manipulation of CRH-containing neurons in double-transgenic mice, we demonstrate that CRH, which is prominent in PMC-spinal projections, has an inhibitory function on volitional micturition. Moreover, engaging this inhibitory function of CRH can ameliorate bladder hyperexcitability induced by cyclophosphamide in a model of cystitis. The data underscore CRH as a novel target for the treatment of voiding dysfunctions, which are highly prevalent disease processes in children and adults.