Left atrial appendage occlusion for recurrent stroke while on oral anticoagulation: a case series.

Background: Clinical practice guidelines recommend oral anticoagulation (OAC) for stroke prevention in selected patients with atrial fibrillation (AF). However, some patients still experience thrombo-embolic events despite adequate anticoagulation. The optimal management of these cases remains uncertain, leading to practice pattern variability. We present a series of three cases illustrating the use of left atrial appendage occlusion (LAAO) as an adjunctive stroke prevention strategy in AF patients with recurrent thrombo-embolic events despite adequate anticoagulation. Case summary: Case one describes an 89-year-old female on apixaban who presented with a thrombus and underwent successful mechanical thrombectomy. Left atrial appendage occlusion was performed, and no subsequent thrombo-embolic events were reported. Case 2 involves a 72-year-old female on full-dose apixaban who experienced recurrent strokes despite adequate anticoagulation. Thrombectomy was performed twice, and complications arose during LAAO. The patient was discharged on warfarin + clopidogrel and remained event-free at the six-month follow-up. Case 3 features an 88-year-old female on rivaroxaban who experienced recurrent cerebral ischaemic events and gastrointestinal bleeding. Left atrial appendage occlusion using an Amplatzer Amulet™ device was successful, and the patient remained event-free at the one-year follow-up. Discussion: This case series emphasizes the complexity of stroke prevention in AF patients and underscores the need for an individualized approach. Incorporating LAAO alongside OAC can provide additional stroke protection for patients with inadequate response to anticoagulation. Further randomized controlled trials are needed to evaluate the efficacy and safety of this approach. In light of the limited evidence available, these cases contribute to the growing body of knowledge on the potential role of LAAO in secondary stroke prevention in AF patients with recurrent thrombo-embolic events despite appropriate anticoagulation.

Unstable Angina Risk Stratification Using High-Sensitivity Cardiac Troponin and Coronary Angiography.

Patients formerly diagnosed with unstable angina (UA) are being reclassified as non-ST-elevation myocardial infarction with the widespread adoption of high-sensitivity troponin (hsTn) assays, leading to significant changes in the incidence and prognosis of UA. This study aimed to evaluate the value of hsTn and the presence of significant obstructive coronary artery disease (CAD) in the risk stratification of patients with UA. We conducted a retrospective, single-center study of 742 patients hospitalized for UA between 2016 and 2021. The primary end point of this study was all-cause mortality. The secondary outcome (major adverse cardiac events [MACEs]) was defined as a composite of nonfatal myocardial infarction (MI), hospitalization for heart failure (hHF), and repeated coronary angiography because of recurring UA (rUA) after the index event. The outcomes were assessed within 1 month, 1 year, and up to 5 years of follow-up. The average follow-up duration was 45 ± 24 months, and 37.2% (n = 276) of patients completed a 5-year follow-up. No in-hospital death was observed, and 6.9% of patients died during follow-up, which was more commonly a late event (>12 months). The composite secondary end point (MI+hHF+rUA) was observed in 16.7% of the patients. There were 3.2% nonfatal MI, 2.3% hHF, and 11.6% rUA during follow-up. We developed a risk model (UA mortality risk) using variables with the highest discriminatory power: age, hsTn, and ST-segment deviation. Our model performed well against the Global Registry of Acute Coronary Events and Thrombolysis in Myocardial Infarction risk scores in predicting death during follow-up. Obstructive CAD on coronary angiography was the only independent predictor of MACEs during follow-up. In conclusion, a contemporary cohort of patients with UA presented with favorable prognosis, particularly, within the first year after the index event. Nonsignificant increases in hsTn levels add to the risk stratification of patients with UA, and the presence of obstructive CAD was the only independent predictor of MACEs, highlighting the potential importance of assessing coronary anatomy.

Unstable Angina: Risk Stratification for Significant Coronary Artery Disease in The Era of High-Sensitivity Cardiac Troponin.

Introduction: High-sensitivity troponin (hsTn) has a very high diagnostic accuracy for myocardial infarction (MI), and patients who were formerly diagnosed with unstable angina (UA) are being reclassified as having NSTEMI in the era of hsTn. This paradigm shift has changed the clinical features of UA, which remain poorly characterized, specifically the occurrence of obstructive coronary artery disease (CAD) and the need for myocardial revascularization. The main purpose of this study was to clinically characterize contemporary UA patients, assess predictors of obstructive CAD, and develop a risk model to predict significant CAD in this population. Methods: We conducted a retrospective cohort study of 742 patients admitted to the hospital with UA. All patients underwent coronary angiography. The endpoint of the study was the presence of obstructive CAD on angiography. The cohort was divided into two groups: patients with significant coronary artery disease (CAD+) and those without CAD (CAD-). We developed a score (UA CAD Risk) based on the multivariate model and compared it with the GRACE, ESC, and TIMI risk scores using ROC analysis. Results: Obstructive CAD was observed on angiography in 53% of the patients. Age, dyslipidemia, troponin level, male sex, ST-segment depression, and wall motion abnormalities on echocardiography were independent predictors of obstructive CAD. hsTn levels (undetectable vs. nonsignificant detection) had a negative predictive value of 81% to exclude obstructive CAD. We developed a prediction model with obstructive CAD as the outcome (AUC: 0.60). Conclusions: In a contemporary UA cohort, approximately 50% of the patients did not have obstructive CAD on angiography. Commonly available cardiac tests at hospital admission show limited discrimination power in identifying patients at risk of obstructive CAD. A revised diagnostic and etiology algorithm for patients with UA is warranted.

Review: Unveiling the effect of beta-nerve growth factor on the reproductive function in llamas and cows.

The actions of the beta-nerve growth factor (ß-NGF) on the neuroendocrine and reproductive system have challenged classical views on the control of reproductive function. After endometrial absorption, ß-NGF triggers ovulation and promotes the development of functional corpora lutea in camelids. In this article, we review evidence showing that, in camelids, ß-NGF exerts its actions by acting in both the hypothalamus and the ovary. In the hypothalamus, ß-NGF may induce gonadotropin-releasing hormone (GnRH) release by interacting with neurons or glial cells expressing receptors for ß-NGF. The LH surge occurs under the influence of ovarian estradiol and requires the release of GnRH into the portal vessels to reach the pituitary gland. In the ovary, ß-NGF may be promoting the differentiation of follicular to luteal cells by modifying the steroidogenic profile of ovarian follicular cells in both camelids and ruminants. Although the mechanisms for these actions are largely undetermined, we aim to offer an update on the current understanding of the effects of ß-NGF controlling reproductive function in camelids and ruminants.

Administration of Beta-Nerve Growth Factor during the Preovulatory Stage Improves Endocrine and Luteal Function in Dairy Heifers.

The neurotrophin beta-nerve growth factor (NGF), which is present in the semen of different mammals, elicits potent ovulatory and luteotrophic actions in llamas following systemic administration. Here, we determine if purified NGF given intramuscularly (IM) during the preovulatory stage affects the corpus luteum (CL), hormone production, endometrial gene expression, and pregnancy rate of dairy heifers. Holstein-Friesian heifers were estrus-synchronized using estradiol benzoate (EB) plus an intravaginal progesterone (P4) device (DIB). After eight days, the device was removed and cloprostenol was given IM; the next day (day 9), heifers received EB IM plus one of the following: (i) 1 mg of NGF (NGF D9 group), (ii) 1 mg of NGF 32 h after EB (NGF D10 group), or (iii) phosphate buffer saline (control group). To measure pregnancy rates, heifers were treated similarly, then artificially inseminated with sexed semen 48-52 h after DIB removal, then an ultrasound was conducted 30 days after insemination. The females given NGF along with EB (NGF D9) showed significantly higher luteinizing hormone (LH) concentrations, larger CL vascular areas, and higher plasma P4 concentrations than the NGF D10 and control animals. Downregulation of the P4 receptor (PGR), and upregulation of both lipoprotein lipase (LPL) and Solute Carrier Family 6 member 14 (SLC6A14) endometrial genes, were detected in NGF D9 heifers. Furthermore, these heifers had a 10% higher pregnancy rate than the control group. We conclude that the higher P4 output, in response to the early NGF administration, led to the enhanced gene expression of transcripts related to uterine receptivity that may result in enhanced pregnancy rates.

Microcirculation function assessment in acute myocardial infarction: A systematic review of microcirculatory resistance indices.

Background: Up to 50% of acute myocardial infarction (MI) patients present with microvascular dysfunction, after a successful percutaneous coronary intervention (PCI), which leads to worse clinical outcomes. The main purpose of this study is to provide a critical appraisal of the emerging role of invasive microvascular resistance indices in the MI setting, using the index of microcirculatory resistance (IMR), hyperemic microvascular resistance (HMR) and zero-flow pressure (Pzf). Methods: We systematically explored relevant studies in the context of MI that correlated microcirculation resistance indices with microvascular dysfunction on cardiac magnetic resonance (CMR), microvascular dysfunction occurring in infarct related arteries (IRA) and non-IRA and its relation to clinical outcomes. Results: The microcirculation resistance indices correlated significantly with microvascular obstruction (MVO) and infarct size (IS) on CMR. Although HMR and Pzf seem to have better diagnostic accuracy for MVO and IS, IMR has more validation data. Although, both IMR and HMR were independent predictors of adverse cardiovascular events, HMR has no validated cut-off value and data is limited to small observational studies. The presence of microvascular dysfunction in non-IRA does not impact prognosis. Conclusion: Microvascular resistance indices are valuable means to evaluate microcirculation function following MI. Microvascular dysfunction relates to the extent of myocardial damage and clinical outcomes after MI. Systematic review registration: [https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42021228432], identifier [CRD42021228432].

3D printing for left atrial appendage closure: A meta-analysis and systematic review.

BACKGROUND: Three-dimensional printing (3D) has emerged as an alternative to imaging to guide left atrial appendage closure (LAAC) device sizing. AIMS: We assessed the usefulness of 3D printing compared to a standard imaging-only approach for LAAC. METHODS: We identified studies comparing an imaging-only with a 3D printing approach in LAAC. A fixed-effects meta-analysis was performed targeting a co-primary endpoint of disagreement in device sizing and leaks. RESULTS: Eight studies that assigned 283 participants to an imaging-only approach and 3D printing approach (145 patients) were included. 3D printing significantly reduced the risk of the co-primary endpoint (risk raio (RR) = 0.19; 95% confidence interval (CI) 0.09-0.37), with consistency across the studies (I2 = 0%). Individually, both device size disagreements [RR 0.13 (95% CI 0.06-0.29), P < 0.001] and leaks [RR 0.24 (95% CI 0.09-0.64) P = 0.004] were reduced under a 3D printing modeling strategy. CONCLUSION: Compared with an imaging-only strategy, 3D printing is associated with reduction in device size disagreements and leaks.

Oxytocin as a potential pharmacological tool to combat obesity.

The neuropeptide oxytocin (OT) has emerged as an important anorexigen in the regulation of food intake and energy balance. It has been shown that the release of OT and activation of hypothalamic OT neurons coincide with food ingestion. Its effects on feeding have largely been attributed to limiting meal size through interactions in key regulatory brain regions governing the homeostatic control of food intake such as the hypothalamus and hindbrain in addition to key feeding reward areas such as the nucleus accumbens and ventral tegmental area. Furthermore, the magnitude of an anorexigenic response to OT and feeding-related activation of the brain OT circuit are modified by the composition and flavor of a diet, as well as by a social context in which a meal is consumed. OT is particularly effective in reducing consumption of carbohydrates and sweet tastants. Pharmacologic, genetic, and pair-feeding studies indicate that OT-elicited weight loss cannot be fully explained by reductions of food intake and that the overall impact of OT on energy balance is also partly a result of OT-elicited changes in lipolysis, energy expenditure, and glucose regulation. Peripheral administration of OT mimics many of its effects when it is given into the central nervous system, raising the questions of whether and to what extent circulating OT acts through peripheral OT receptors to regulate energy balance. Although OT has been found to elicit weight loss in female mice, recent studies have indicated that sex and estrous cycle may impact oxytocinergic modulation of food intake. Despite the overall promising basic research data, attempts to use OT in the clinical setting to combat obesity and overeating have generated somewhat mixed results. The focus of this mini-review is to briefly summarize the role of OT in feeding and metabolism, address gaps and inconsistencies in our knowledge, and discuss some of the limitations to the potential use of chronic OT that should help guide future research on OT as a tailor-made anti-obesity therapeutic.

Seminal plasma nerve growth factor signaling on the reproductive physiology of female llamas.

The ovulation mechanism is one of the fascinating physiological processes in reproductive biology in mammals. From the reproductive point of view, the species have been classified as spontaneous or induced ovulators. Although the release of GnRH followed by the preovulatory LH surge is shared between both types of ovulation, the stimulus to initiate GnRH release varies between both categories. In spontaneous ovulators, ovulation depends on the systemic concentration of ovarian steroids, however, in induced ovulators, different stimuli such as copulation, environmental, and social cues can facilitate or induce ovulation regardless of the increases in systemic estradiol concentration. In this review, we document evidence that a male-derived protein is the main factor responsible for inducing ovulation and also modulating the ovarian function in the domestic South American camelid, the llama. The neurotrophin beta-Nerve Growth Factor (ß-NGF) is the principal factor present in the semen of llamas responsible for inducing ovulation in this species. After the intrauterine deposit of semen during mating, ß-NGF is absorbed through the endometrium to reach the circulatory system, where it reaches the hypothalamus and stimulates GnRH release. The potential site of action of this neurotrophin at the brain has not been elucidated, however, hypotheses are raised that the factor may cross the blood-brain barrier and stimulate upstream neuronal networks that lead to the stimulation of GnRH-secreting neurons. It is possible that ß-NGF could be sensed at the median eminence without crossing the blood-brain barrier. Finally, it has been observed that this factor is not only a powerful stimulator of ovulation but also has a luteotrophic effect, resulting in the development of a corpus luteum capable of secreting more progesterone when compared to other ovulation-stimulating analogues.

Transbasilic Approach for Percutaneous Closure of an Atrial Septal Defect.

Atrial septal defect (ASD) closure is indicated in the presence of a significant left-to-right shunt. In the case of an interrupted inferior vena cava (IVC), the standard percutaneous approach can be troublesome. The authors report a case of a 55-year-old female patient with an ostium secundum ASD with a significant left-to-right shunt at rest (Qp/Qs, 1.6). The cardiac computed tomography scan showed an interrupted IVC above the renal veins. To our knowledge, this is the first case in the literature where the transbasilic peripheral vein approach for ASD closure was used.

Chorion Alterations in Eyed-Stage Salmonid Eggs Farmed in La Araucanía, Chile: A Retrospective Study.

The chorion is the primary envelop that protects the fish embryo against mechanical actions, pathogens, and abrupt changes in physical and chemicals conditions of the incubation medium. During embryo development, chorion alterations are not rare, but the occurrence of these is scarcely reported. Increased frequency of chorion alterations can result in increased embryo mortality and thus decreased reproductive performance and losses for fish farms. In this study, we characterize different chorion alterations observed in samples collected over 14 years from 12 salmon and trout farms located in the region of La Araucanía in southern Chile, which sent live eyed-stage embryos ('eyed-eggs') for quality analysis to our laboratory. We found soft chorion as the most common alteration observed, being present in the whole 14-year series analyzed in Atlantic Salmon (Salmo salar) and affecting up to 35.0% of the samples examined in a year. This alteration also affected up to 20.0 and 5.7% of Coho Salmon (Oncorhynchus kisutch) and Rainbow Trout (Oncorhynchus mykiss) samples analyzed in a year, respectively. We also found an increase of other chorion alterations, including perforated and white-spotted chorion in Atlantic and Coho Salmon, in the last 8 years. Among the three species, Rainbow Trout exhibited fewer chorion alterations. As the embryonated eggs analyzed here were obtained from broodstocks maintained under standard industrial conditions, these alterations might be linked to changes in environmental conditions affecting the incubation water that need to be further investigated.

Translation and cross-cultural adaptation of the "Quality of Life in Epilepsy (QOLIE-31-P)" Questionnaire for Chile.

PURPOSE: To report the translation and cross-cultural adaptation of the Quality of Life in Epilepsy (QOLIE-31-P) Questionnaire in patients with epilepsy (PWE) in Chile. METHODS: Translation from the original and posterior back-translation was performed by independent translators, two in each step. The final consensual translation was modified for the Chilean context and assessed by cognitive interviews with 12 PWE from Chile's public healthcare system, selected by quotas. RESULTS: Main changes made to the original questionnaire were the addition, in some items, of an alternative, indicating the nonexistence of limitations produced by epilepsy, examples to clarify some questions, and some minor wording modifications. There was no addition or deletion of items. CONCLUSION: A culturally adapted version of the QOLIE-31-P questionnaire was obtained in conditions to be assessed psychometrically in a sample of PWE in Chile.

Non-vitamin K antagonist oral anticoagulation versus left atrial appendage occlusion for primary and secondary stroke prevention after cardioembolic stroke.

INTRODUCTION: This study aimed to evaluate the performance of non-vitamin K antagonist oral anticoagulation (NOAC) in patients with previous stroke and non-valvular atrial fibrillation (AF) compared with left atrial appendage occlusion (LAAO) in primary and secondary stroke prevention settings. METHODS: This was a prospective, single-center, non-randomized cohort study of 302 consecutive patients with non-valvular AF and at high risk for stroke. Two treatment strategies were compared: LAAO (n=91) and long-term treatment with NOAC (n=149). The primary outcome was the composite endpoint of death, stroke and major bleeding. Propensity score and cause-of-death analyses were performed to compare outcomes. RESULTS: In a mean follow-up of 13 months, there were 30 deaths (LAAO 8.8% vs. NOAC 14.8%), five strokes (LAAO 1.1% vs. NOAC 2.7%) and six major bleeds (LAAO 1.1% vs. NOAC 3.4%). There was a non-significant trend for a lower incidence of the primary endpoint in the LAAO group (11.0% vs. 20.9%; HR 0.42, 95% CI 0.17-1.05, p=0.064). Considering only secondary prevention LAAO patients (34.1% of the LAAO group), there was also a non-significant lower incidence of the primary endpoint (LAAO 6.5% vs. 20.9%; HR 0.30, 95% CI 0.07-1.39, p=0.12). While about a fifth of LAAO patients stopped antiplatelet treatment six months after device implantation due to recurrent minor bleeding, no adverse cardiovascular event or major bleeding occurred in this subset of patients. CONCLUSION: In this registry-based study, LAAO was a reasonable alternative to NOAC for the prevention of a composite endpoint of all-cause mortality, stroke and major bleeding in patients at high risk for stroke.

Non-vitamin K antagonist oral anticoagulation versus left atrial appendage occlusion for primary and secondary stroke prevention after cardioembolic stroke.

INTRODUCTION: This study aimed to evaluate the performance of non-vitamin K antagonist oral anticoagulation (NOAC) in patients with previous stroke and non-valvular atrial fibrillation (AF) compared with left atrial appendage occlusion (LAAO) in primary and secondary stroke prevention settings. METHODS: This was a prospective, single-center, non-randomized cohort study of 302 consecutive patients with non-valvular AF and at high risk for stroke. Two treatment strategies were compared: LAAO (n=91) and long-term treatment with NOAC (n=149). The primary outcome was the composite endpoint of death, stroke and major bleeding. Propensity score and cause-of-death analyses were performed to compare outcomes. RESULTS: In a mean follow-up of 13 months, there were 30 deaths (LAAO 8.8% vs. NOAC 14.8%), five strokes (LAAO 1.1% vs. NOAC 2.7%) and six major bleeds (LAAO 1.1% vs. NOAC 3.4%). There was a non-significant trend for a lower incidence of the primary endpoint in the LAAO group (11.0% vs. 20.9%; HR 0.42, 95% CI 0.17-1.05, p=0.064). Considering only secondary prevention LAAO patients (34.1% of the LAAO group), there was also a non-significant lower incidence of the primary endpoint (LAAO 6.5% vs. 20.9%; HR 0.30, 95% CI 0.07-1.39, p=0.12). While about a fifth of LAAO patients stopped antiplatelet treatment six months after device implantation due to recurrent minor bleeding, no adverse cardiovascular event or major bleeding occurred in this subset of patients. CONCLUSION: In this registry-based study, LAAO was a reasonable alternative to NOAC for the prevention of a composite endpoint of all-cause mortality, stroke and major bleeding in patients at high risk for stroke.

Identification of peripheral oxytocin-expressing cells using systemically applied cell-type specific adeno-associated viral vector.

Oxytocin is primarily synthesised in the brain and is widely known for its role in lactation and parturition after being released into the blood from the posterior pituitary gland. Nevertheless, peripheral tissues have also been reported to express oxytocin. Using systemic injection of a recombinant adeno-associated virus vector, we investigated the expression of the green fluorescent protein Venus under the control of the oxytocin promoter in the gastrointestinal tract, pancreas and testes of adult rats. Here, we confirm that the vector infects oxytocin neurones of the enteric nervous system in ganglia of the myenteric and submucosal plexuses. Venus was detected in 25%-60% of the ganglia in the myenteric and submucosal plexuses identified by co-staining with the neuronal marker PGP9.5. Oxytocin expression was also detected in the islets of Langerhans in the pancreas and the Leydig cells of the testes. Our data illustrate that peripheral administration of the viral vector represents a powerful method for selectively labelling oxytocin-producing cells outside the brain.

Iatrogenic atrial septal defect after percutaneous left atrial appendage closure: a single-center study.

There is conflicting evidence regarding the significance of iatrogenic atrial septal defects (iASDs) after transseptal puncture during percutaneous cardiac interventions. To study the clinical outcome of iASD after percutaneous left atrial appendage occlusion (LAAo). Single-center, retrospective study of 70 consecutive patients who underwent percutaneous LAAo between May 2010 and August 2017, and subsequent transesophageal echocardiography (TEE) at 1 month. The sample population was divided into two groups: A (with iASD, 22 (37%) patients) and B (no iASD, 44 (63%) patients). Procedures were guided either by TEE (36 patients (54%)) or intracardiac echocardiography (ICE) from the left atrium (30 patients (46%)). The primary end point was presence of iASD at 1 month, and secondary end points included mortality, hospital admission due to heart failure (HF), and right atrium (RA) size during follow-up. 70 patients were included in this study and the prevalence of iASD at 1 month was 37%. The use of ICE was associated with iASD (adjusted odds ratio, 3.79; 95% CI 1.27-11.34). The presence of iASD was not associated with adverse events (mortality, 15.4% vs 20.5%; P = 0.60; HF hospitalizations, 7.7% vs 13.6%, P = 0.45; and RA area, 24.8 ± 7.0 cm2 vs 22.2 ± 6.8 cm2, P = 0.192). At 1-month follow-up after LAAo, iASD was present in one third of patients, but was not associated with clinical outcomes. The use of ICE was associated with a higher risk of short-term iASD.

Determinants and prognostic implication of periprocedural myocardial injury after successful recanalization of coronary chronic total occlusion.

Periprocedural myocardial injury (PMI) has been generally associated with major adverse cardiac events (MACE), however, limited studies addressed its clinical implications following chronic total occlusion (CTO) percutaneous coronary intervention (PCI). To evaluate the determinants and prognostic implication of PMI following CTO-PCI. Retrospective single-centre study of 125 consecutive patients undergoing CTO-PCI was attempted between December 2013 and December 2017. Angiographic success was achieved in 115 patients (92.0%) and cTn-I values were obtained 12-24 h following PCI. PMI was defined as an elevation of cTn-I above 5 times the 99th-percentile upper reference limit. Baseline demographic, clinical, angiographic and procedural characteristics were compared. Multivariate analysis was performed to determine the predictors of PMI and the correlates of PMI and 1-year MACE, a composite of all-cause death, non-fatal myocardial infarction, and target lesion revascularization. Overall, mean age was 67 ± 17 years; 25 patients (21.7%) were female; and PMI occurred in 41 patients (35.7%). Multivessel coronary artery disease (MVD) (odds ratio [OR], 3.41; 95% confidence interval [CI], 1.09-10.67; p = 0.04) and procedural complications (a composite of iatrogenic coronary artery dissection/haematoma or perforation) (OR, 19.08; 95% CI, 3.77-96.65; p < 0.01) predicted PMI. Significant collateralization (Rentrop 3) (hazard ratio, [HR], 0.19; 95% CI, 0.06-0.64; p < 0.01) and procedural complications (HR, 8.86; 95% CI, 2.66-29.46; p < 0.01) were independently associated with 1-year MACE, while PMI was not (p = 0.26). In this contemporary cohort, PMI following successful CTO-PCI was a common finding and was predicted by MVD and procedural complications. PMI was not independently associated with 1-year MACE.

ß-NGF Stimulates Steroidogenic Enzyme and VEGFA Gene Expression, and Progesterone Secretion via ERK 1/2 Pathway in Primary Culture of Llama Granulosa Cells.

The beta-nerve growth factor (ß-NGF) from llama seminal plasma exerts ovulatory and luteotrophic effects following intramuscular or intrauterine infusion in llamas and alpacas. In this study, we investigate the in vitro effect of llama ß-NGF on the expression of genes involved in angiogenesis and progesterone synthesis as well as progesterone release in preovulatory llama granulosa cells; we also determine whether these changes are mediated via the ERK1/2 signaling pathway. From adult female llamas, we collected granulosa cells from preovulatory follicles by transvaginal ultrasound-guided follicle aspiration; these cells were pooled and incubated. After 80% confluence, the cultured granulosa cells were treated with ß-NGF, ß-NGF plus the MAPK inhibitor U0126, or luteinizing hormone, and the abundance of angiogenic and steroidogenic enzyme mRNA transcripts were quantified after 10 and 20 h by RT-qPCR. We also quantified the progesterone concentration in the media after 48 h by radioimmunoassay. We found that application of ß-NGF increases the abundance of mRNA transcripts of the vascular endothelial growth factor (VEGFA) and the steroidogenic enzymes cytochrome P450 side-chain cleavage (P450scc/CYP11A1), steroidogenic acute regulatory protein (STAR), and 3ß-hydroxysteroid dehydrogenase (HSD3B1) at 10 and 20 h of treatment. Application of the MAPK inhibitor U0126 resulted in downregulation of the genes encoding these enzymes. ß-NGF also enhanced progesterone synthesis, which was prevented by the prior application of the MAPK inhibitor U0126. Finally, western blot analysis confirmed that ß-NGF activates the ERK1/2 signaling pathway. In conclusion, our results indicate that ß-NGF exerts direct luteotropic effects on llama ovarian tissue via the ERK 1/2 pathway.