Spatial and pregnancy-related changes in the protein, amino acid, and carbohydrate composition of bovine oviduct fluid

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. Knowledge of how the biochemical composition of the bovine oviduct is altered due to the oviduct anatomy or the presence of an embryo is lacking. Thus, the aim of this study was to assess the effect of (І) oviduct anatomy and (ІІ) embryo presence on oviductal fluid (OF) protein, amino acid, and carbohydrate composition. Cross-bred beef heifers (n = 19) were synchronized and those in standing estrus were randomly allocated to a cyclic (non-bred) or pregnant (artificially inseminated) group. All heifers were slaughtered on Day 3 after estrus. The oviducts ipsilateral to the corpus luteum from each animal were isolated, straightened and cut, separating ampulla and isthmus. Each portion was flushed with 500 μµl of PBS enabling recovery of the oocyte/embryo. Recovered unfertilized oocytes (cyclic group) and embryos (8-cell embryos; pregnant group) were located in the isthmus of the oviduct. Samples of flushing medium from the isthmus and ampulla were used for proteomic (n = 2 per group), amino acid (n = 5), and carbohydrate (n = 5) analysis. For proteomic analysis, total protein from cyclic and pregnant samples were labelled with different cyanine fluorescent probes and separated according to the isoelectric point using immobilized pH gradient strips (pH 3–10, 17 cm, Protean® IEF cell system, Bio Rad). Second dimension was performed in a polyacrylamide gel (12%) in the presence of SDS using a Protean II XL system (Bio Rad). Images were obtained with a Typhoon 9410 scanner and analyzed with Progenesis SameSpots software v 4.0. Amino acid content in the OF was determined by high performance liquid chromatography (HPLC). Glucose, lactate, and pyruvate were quantified using microfluorometric enzyme-linked assays. For the proteomic assessment, the results of the image analysis were compared by ANOVA. For both amino acid and carbohydrate analyses, statistical analysis was carried out by 2-way ANOVA with the Holm-Sidak nonparametric post hoc analysis. On Day 3 post-estrus, OF composition varied based on (І) anatomical region, where isthmic metabolites were present in lower (i.e., lactate, glycine, and alanine) or higher (i.e., arginine) concentrations compared to the ampulla; and (ІІ) embryo presence, which was correlated with greater, arginine, phosphoglycerate kinase 1, serum albumin, α-1-antiproteinase and IGL@ protein concentrations. In conclusion, data indicate that the composition of bovine OF is anatomically dynamic and influenced by the presence of an early embryo

Análisis transcriptómico y proteómico del oviducto y útero bovino en la fase periovulatoria

En el período cercano a la ovulación o periovulatorio el tracto genital femenino ofrece el microentorno necesario para el transporte e interacción de los gametos, hasta culminar con una fecundación exitosa. En la especie bovina, el espermatozoide debe permanecer en el tracto femenino para obtener su capacidad fecundante; las moléculas y mecanismos que participan en dotar de esta capacidad a los espermatozoides, no están del todo esclarecidos. Por ello, es de suma importancia conocer estos mecanismos a fondo para desarrollar nuevas técnicas de reproducción asistida que mejoren la capacidad fecundante del gameto masculino. Nuestros objetivos fueron identificar los diferentes mecanismos moleculares presentes en el tracto genital femenino implicados en el proceso de maduración del espermatozoide, tras su depósito, transporte y permanencia en el periodo previo a la ovulación, así como analizar las similitudes y diferencias en la expresión del transcriptoma y proteoma del útero y oviducto bovino. Para identificar la expresión del transcriptoma y la expresión génica diferencial, utilizamos el análisis de micromatrices. Las muestras de epitelio oviductal, de tejido endometrial y de los fluidos oviductales (FOBs) y uterinos (FUBs) procedieron de seis novillas sincronizadas hormonalmente en la fase periovulatoria. Para la identificación de proteínas contenidas en FOB y FUB utilizamos las técnicas de electroforesis bidimensional, DiGE y espectrometría de masas (MS/MS). Nuestros resultados del análisis de micromatrices, indicaron que un total de 5509 genes son expresados diferencialmente en el oviducto y útero bovino en la fase periovulatoria. Así, también observamos que la expresión génica entre el oviducto y endometrio no es idéntica, si bien tienen una amplia similitud donde comparten expresión de genes que intervienen en sintetizar proteínas relacionadas con la unión, respuesta al estímulo, en los mecanismos de defensa y de protección ante el estrés oxidativo. También se comprobó la existencia de vías específicas para cada uno de los tejidos estudiados. Así, en el oviducto destacan la expresión de genes relacionados con actividad catalítica, del choque térmico y en endometrio destacan la presencia de transcritos relacionados con la adhesión focal y la modificación de la matriz extracelular. Se ha detectado la expresión de hasta 175 genes que intervienen en los procesos de secreción. Identificamos 47 proteínas por MS/MS, procedentes de geles bidimensionales-DiGE, de las cuales 29 son comunes para FOB y FUB. Estas proteínas intervienen en proceso biológicos generales como la comunicación celular, la respuesta al estrés y procesos catabólicos. Observamos que 9 de ellas fueron exclusivas del FOB destacando algunas proteínas de la familia de las anexinas implicadas en los mecanismos de unión, así como en el establecimiento del reservorio oviductal. Otras 5 proteínas fueron identificadas como únicas de FUB involucradas en actividades catalíticas y de glicolisis. SUMMARY During the period upcoming to ovulation or peri-ovulatory phase, the female genital tract provides the microenvironment required for transport and interaction of gametes, culminating in a successful fertilization. In the bovine species, the sperm must remain in the female tract to obtain fertilizing capacity, the molecules and mechanisms involved in providing this capability to sperm, is not entirely clarified. It is therefore important to investigate these mechanisms thoroughly to develop new assisted reproductive techniques that improve the fertilizing capacity of the male gamete. Our aims were to identify the different molecular mechanisms present in the female genital tract involved in the maturation of sperm, after insemination, transport and permanence in the peri-ovulatory phase and analyze the similarities and differences in the expression of the transcriptome and proteome of bovine uterus and oviduct. To identify transcriptome expression and differential gene expression, we used microarray analysis. The sample of oviductal epithelium, endometrial tissue and oviductal fluids (FOBs) and uterine (FUBs) proceeded from six heifers hormonally synchronized in the peri-ovulatory phase. For the identification of protein contained in FUB and FOB bimensional electrophoresis techniques, DIGE and mass spectrometry (MS / MS) were used. Our results of the microarray analysis indicated that a total of 5509 genes were differentially expressed in the bovine oviduct and uterus in the peri-ovulatory phase. Thus, we also observed that gene expression in the oviduct and endometrium is not identical, have similarity, they share expression of genes involved in synthesizing proteins referring to binding, response to stimuli, mechanisms of defense and protection against oxidative stress. We also confirmed the existence of specific pathways for each of the tissues analyzed. In the oviduct, the expressed gene are related to catalytic activity and heat shock; however, in the endometrium there are gene expression associated with focal adhesion, and modification of the extracellular matrix. Up to 175 genes that are expressed are involved in the secretion pathways. We identified 47 proteins by MS / MS from two-dimensional gels and DiGE. A number of 29 proteins are common between FOB and FUB and are involved in general biological processes as cellular communication, stress response and catabolic processes. We identified 9 proteins that are specific for the FOB. It is important to emphasize the proteins of the annexin family. These proteins are involved in the binding mechanism and in the establishment of the sperm reservoir. Other five proteins were specific for the FUB and are involved in catalytic activities and glycolysis

Global attitudes in the management of acute appendicitis during COVID-19 pandemic: ACIE Appy Study

Background: Surgical strategies are being adapted to face the COVID-19 pandemic. Recommendations on the management of acute appendicitis have been based on expert opinion, but very little evidence is available. This study addressed that dearth with a snapshot of worldwide approaches to appendicitis. Methods: The Association of Italian Surgeons in Europe designed an online survey to assess the current attitude of surgeons globally regarding the management of patients with acute appendicitis during the pandemic. Questions were divided into baseline information, hospital organization and screening, personal protective equipment, management and surgical approach, and patient presentation before versus during the pandemic. Results: Of 744 answers, 709 (from 66 countries) were complete and were included in the analysis. Most hospitals were treating both patients with and those without COVID. There was variation in screening indications and modality used, with chest X-ray plus molecular testing (PCR) being the commonest (19\ub78 per cent). Conservative management of complicated and uncomplicated appendicitis was used by 6\ub76 and 2\ub74 per cent respectively before, but 23\ub77 and 5\ub73 per cent, during the pandemic (both P < 0\ub7001). One-third changed their approach from laparoscopic to open surgery owing to the popular (but evidence-lacking) advice from expert groups during the initial phase of the pandemic. No agreement on how to filter surgical smoke plume during laparoscopy was identified. There was an overall reduction in the number of patients admitted with appendicitis and one-third felt that patients who did present had more severe appendicitis than they usually observe. Conclusion: Conservative management of mild appendicitis has been possible during the pandemic. The fact that some surgeons switched to open appendicectomy may reflect the poor guidelines that emanated in the early phase of SARS-CoV-2

Reduction of cardiac imaging tests during the COVID-19 pandemic: The case of Italy. Findings from the IAEA Non-invasive Cardiology Protocol Survey on COVID-19 (INCAPS COVID)

Background: In early 2020, COVID-19 massively hit Italy, earlier and harder than any other European country. This caused a series of strict containment measures, aimed at blocking the spread of the pandemic. Healthcare delivery was also affected when resources were diverted towards care of COVID-19 patients, including intensive care wards. Aim of the study: The aim is assessing the impact of COVID-19 on cardiac imaging in Italy, compare to the Rest of Europe (RoE) and the World (RoW). Methods: A global survey was conducted in May–June 2020 worldwide, through a questionnaire distributed online. The survey covered three periods: March and April 2020, and March 2019. Data from 52 Italian centres, a subset of the 909 participating centres from 108 countries, were analyzed. Results: In Italy, volumes decreased by 67% in March 2020, compared to March 2019, as opposed to a significantly lower decrease (p &lt; 0.001) in RoE and RoW (41% and 40%, respectively). A further decrease from March 2020 to April 2020 summed up to 76% for the North, 77% for the Centre and 86% for the South. When compared to the RoE and RoW, this further decrease from March 2020 to April 2020 in Italy was significantly less (p = 0.005), most likely reflecting the earlier effects of the containment measures in Italy, taken earlier than anywhere else in the West. Conclusions: The COVID-19 pandemic massively hit Italy and caused a disruption of healthcare services, including cardiac imaging studies. This raises concern about the medium- and long-term consequences for the high number of patients who were denied timely diagnoses and the subsequent lifesaving therapies and procedures

Impact of COVID-19 on Diagnostic Cardiac Procedural Volume in Oceania: The IAEA Non-Invasive Cardiology Protocol Survey on COVID-19 (INCAPS COVID)

Objectives: The INCAPS COVID Oceania study aimed to assess the impact caused by the COVID-19 pandemic on cardiac procedure volume provided in the Oceania region. Methods: A retrospective survey was performed comparing procedure volumes within March 2019 (pre-COVID-19) with April 2020 (during first wave of COVID-19 pandemic). Sixty-three (63) health care facilities within Oceania that perform cardiac diagnostic procedures were surveyed, including a mixture of metropolitan and regional, hospital and outpatient, public and private sites, and 846 facilities outside of Oceania. The percentage change in procedure volume was measured between March 2019 and April 2020, compared by test type and by facility. Results: In Oceania, the total cardiac diagnostic procedure volume was reduced by 52.2% from March 2019 to April 2020, compared to a reduction of 75.9% seen in the rest of the world (p&lt;0.001). Within Oceania sites, this reduction varied significantly between procedure types, but not between types of health care facility. All procedure types (other than stress cardiac magnetic resonance [CMR] and positron emission tomography [PET]) saw significant reductions in volume over this time period (p&lt;0.001). In Oceania, transthoracic echocardiography (TTE) decreased by 51.6%, transoesophageal echocardiography (TOE) by 74.0%, and stress tests by 65% overall, which was more pronounced for stress electrocardiograph (ECG) (81.8%) and stress echocardiography (76.7%) compared to stress single-photon emission computerised tomography (SPECT) (44.3%). Invasive coronary angiography decreased by 36.7% in Oceania. Conclusion: A significant reduction in cardiac diagnostic procedure volume was seen across all facility types in Oceania and was likely a function of recommendations from cardiac societies and directives from government to minimise spread of COVID-19 amongst patients and staff. Longer term evaluation is important to assess for negative patient outcomes which may relate to deferral of usual models of care within cardiology