Estado antioxidante del líquido folicular en mujeres sometidas a estimulación ovárica: implicaciones en la maduración folicular y en la fertilidad

257 p.La infertilidad es un problema sociológico que afecta cada vez a más parejas, que acuden a las técnicas de reproducción asistida (RA) como medida terapéutica. Una de las fases iniciales de la RA es la estimulación ovárica controlada para poder obtener un número elevado de ovocitos viables para ser fecundados. Estos tratamientos suponen para la mujer un estrés psicológico añadido a las repercusiones físicas del tratamiento. Es, por tanto, fundamental que los tratamientos sean efectivos. En este contexto se enmarca esta tesis doctoral, es decir, en la búsqueda de biomarcadores de disfunción y de fertilidad que permitan mejorar a medio/largo plazo las técnicas de RA. OBJETIVOS. El objetivo de este trabajo es caracterizar el estado antioxidante del líquido folicular de folículos A) en distinto grado de maduración (pequeños y grandes), y B) de mujeres fértiles (Donantes) y subfértiles (Pacientes), sometidas a un ciclo de estimulación ovárica controlada, de forma que a partir de los resultados puedan establecerse biomarcadores de maduración folicular y de fertilidad. METODOLOGÍA. Se han determinado en el líquido folicular bioquímicos y marcadores del estado antioxidante del líquido folicular. En particular se ha determinado: 1) actividad antioxidante total, 2) niveles de ¿-tocoferol, ¿-tocoferol y óxido nítrico, y 3) actividades de los enzimas antioxidantes: SOD, catalasa, GPx, GST y de la familia PON. Se han comparado estos parámetros entre folículos pequeños y grandes para cada mujer y entre los dos grupos poblacionales. Se han cuantificado en el líquido folicular las proteínas PON1, PON2 y PON3. Se han estudiado las posibles asociaciones entre los parámetros analizados y parámetros de fertilidad. Se ha desarrollado una metodología para la determinación de biomarcadores de lesión oxidativa proteínica.RESULTADOS Y CONCLUSIONES. Existen diferencias significativas del estado antioxidante del líquido folicular entre folículos grandes y pequeños, subrayándose la importancia de mantener un estado reducido del líquido folicular para la correcta maduración del folículo. Diversos marcadores del estado antioxidante del líquido folicular se asocian a parámetros de fertilidad tras un ciclo de estimulación ovárica controlada. En particular, las actividades Se-GPx, y actividades del sistema PON se correlacionan positivamente con el número de ovocitos totales y maduros en el grupo de Donantes. La actividad PON3 en muestras de Donantes es mayor que de Pacientes. Se sugiere un papel de las proteínas de la familia PON en la fertilidad. Existen diferencias en el tipo de lesión oxidativa de proteínas del líquido folicular entre Donantes y Pacientes, presentando las Pacientes un mayor daño proteínico derivado de procesos de glicoxidación. Finalmente, se han visto diferencias significativas en el grado de lesión oxidativa proteínica entre ciclos productivos y no productivos

Estado antioxidante del líquido folicular en mujeres sometidas a estimulación ovárica: implicaciones en la maduración folicular y en la fertilidad

257 p.La infertilidad es un problema sociológico que afecta cada vez a más parejas, que acuden a las técnicas de reproducción asistida (RA) como medida terapéutica. Una de las fases iniciales de la RA es la estimulación ovárica controlada para poder obtener un número elevado de ovocitos viables para ser fecundados. Estos tratamientos suponen para la mujer un estrés psicológico añadido a las repercusiones físicas del tratamiento. Es, por tanto, fundamental que los tratamientos sean efectivos. En este contexto se enmarca esta tesis doctoral, es decir, en la búsqueda de biomarcadores de disfunción y de fertilidad que permitan mejorar a medio/largo plazo las técnicas de RA. OBJETIVOS. El objetivo de este trabajo es caracterizar el estado antioxidante del líquido folicular de folículos A) en distinto grado de maduración (pequeños y grandes), y B) de mujeres fértiles (Donantes) y subfértiles (Pacientes), sometidas a un ciclo de estimulación ovárica controlada, de forma que a partir de los resultados puedan establecerse biomarcadores de maduración folicular y de fertilidad. METODOLOGÍA. Se han determinado en el líquido folicular bioquímicos y marcadores del estado antioxidante del líquido folicular. En particular se ha determinado: 1) actividad antioxidante total, 2) niveles de ¿-tocoferol, ¿-tocoferol y óxido nítrico, y 3) actividades de los enzimas antioxidantes: SOD, catalasa, GPx, GST y de la familia PON. Se han comparado estos parámetros entre folículos pequeños y grandes para cada mujer y entre los dos grupos poblacionales. Se han cuantificado en el líquido folicular las proteínas PON1, PON2 y PON3. Se han estudiado las posibles asociaciones entre los parámetros analizados y parámetros de fertilidad. Se ha desarrollado una metodología para la determinación de biomarcadores de lesión oxidativa proteínica.RESULTADOS Y CONCLUSIONES. Existen diferencias significativas del estado antioxidante del líquido folicular entre folículos grandes y pequeños, subrayándose la importancia de mantener un estado reducido del líquido folicular para la correcta maduración del folículo. Diversos marcadores del estado antioxidante del líquido folicular se asocian a parámetros de fertilidad tras un ciclo de estimulación ovárica controlada. En particular, las actividades Se-GPx, y actividades del sistema PON se correlacionan positivamente con el número de ovocitos totales y maduros en el grupo de Donantes. La actividad PON3 en muestras de Donantes es mayor que de Pacientes. Se sugiere un papel de las proteínas de la familia PON en la fertilidad. Existen diferencias en el tipo de lesión oxidativa de proteínas del líquido folicular entre Donantes y Pacientes, presentando las Pacientes un mayor daño proteínico derivado de procesos de glicoxidación. Finalmente, se han visto diferencias significativas en el grado de lesión oxidativa proteínica entre ciclos productivos y no productivos

Influence Of Oxygen Partial Pressure On The Characteristics Of Human Hepatocarcinoma Cells

Most of the in vitro studies using liver cell lines have been performed under atmospheric oxygen partial pressure (21% O-2). However, the oxygen concentrations in the liver and cancer cells are far from this value. In the present study, we have evaluated the influence of oxygen on 1) the tumor cell lines features (growth, steadystate ROS levels, GSH content, activities of antioxidant enzymes, p66 Shc and SOD expressions, metalloproteinases secretion, migration, invasion, and adhesion) of human hepatocellular carcinoma cell lines, and b) the response of the cells to an oxidant stimulus (aqueous leaf extract of the V. baccifera plant species). For this purpose, three hepatocarcinoma cell lines with different p53 status, HepG2 (wild-type), Huh7 (mutated), and Hep3B (deleted), were cultured (6-30 days) under atmospheric (21%) and more physiological (8%) pO(2). Results showed that after long-term culturing at 8% versus 21% O-2, the cellular proliferation rate and the steady-state levels of mitochondrial O-2-were unaffected. However, the intracellular basal ROS levels were higher independently of the characteristics of the cell line. Moreover, the lower pO(2) was associated with lower glutathione content, the induction of p66 Shc and Mn-SOD proteins, and increased SOD activity only in HepG2. This cell line also showed a higher migration rate, secretion of active metalloproteinases, and a faster invasion. HepG2 cells were more resistant to the oxidative stress induced by V. baccifera. Results suggest that the longterm culturing of human hepatoma cells at a low, more physiological pO(2) induces antioxidant adaptations that could be mediated by p53, and may alter the cellular response to a subsequent oxidant challenge. Data support the necessity of validating outcomes from studies performed with hepatoma cell cultures under ambient O-2.This work was supported by research grants from the Basque Government (Department of Education, Universities and Research, ref. IT687-13), and University of the Basque Country, UPV/EHU (CLUMBER UFI11/20 and pre-doctoral and post-doctoral grants to J.T.). We thank Jose Antonio Lopez for his valuable technical assistance with cell cultures. Technical and human support provided by SGIKer (UPV/EHU, MICINN, GV/EJ, ESF) is gratefully acknowledged

Analysis of Protein Oxidative Modifications in Follicular Fluid from Fertile Women: Natural Versus Stimulated Cycles

Oxidative stress is associated with obstetric complications during ovarian hyperstimulation in women undergoing in vitro fertilization. The follicular fluid contains high levels of proteins, which are the main targets of free radicals. The aim of this work was to determine specific biomarkers of non-enzymatic oxidative modifications of proteins from follicular fluid in vivo, and the effect of ovarian stimulation with gonadotropins on these biomarkers. For this purpose, 27 fertile women underwent both a natural and a stimulated cycle. The biomarkers, glutamic semialdehyde (GSA), aminoadipic semialdehyde (AASA), N-epsilon-(carboxymethyl)lysine (CML), and N-epsilon-(carboxyethyl)lysine (CEL), were measured by gas-liquid chromatography coupled to mass spectrometry. Results showed that follicular fluid contained products of protein modifications by direct metal-catalyzed oxidation (GSA and AASA), glycoxidation (CML and CEL), and lipoxidation (CML). GSA was the most abundant biomarker (91.5%). The levels of CML amounted to 6% of the total lesions and were higher than AASA (1.3%) and CEL (1.2%). In the natural cycle, CEL was significantly lower (p < 0.05) than in the stimulated cycle, suggesting that natural cycles are more protected against protein glycoxidation. These findings are the basis for further research to elucidate the possible relevance of this follicular biomarker of advanced glycation end product in fertility programs.This research was funded by the Basque Government, Department of Education, Universities and Research (project ref. IT687-13 and predoctoral grant to I.P.) and Department of Economic Development and Competitiveness, SPRI (refs. IG-20130001214 and IG-2014 0000837), University of the Basque Country UPV/EHU (ref. GIU16/62), and Fundacion Jesus Gangoiti (grant to S.M.)

Immune response and reactogenicity after immunization with two-doses of an experimental COVID-19 vaccine (CVnCOV) followed by a third-fourth shot with a standard mRNA vaccine (BNT162b2): RescueVacs multicenter cohort study

Background: There is no evidence to date on immunogenic response among individuals who participated in clinical trials of COVID-19 experimental vaccines redirected to standard national vaccination regimens. Methods: This multicentre, prospective controlled cohort study included subjects who received a COVID-19 experimental vaccine (CVnCoV)(test group, TG) - and unvaccinated subjects (control group, CG), selected among individuals to be vaccinated according to the Spanish vaccination program. All study subjects received BNT162b2 as a standard national vaccination schedule, except 8 (from CG) who received mRNA-1273 and were excluded from immunogenicity analyses. Anti-RBD antibodies level and neutralising titres (NT50) against G614, Beta, Mu, Delta and Omicron variants were analysed. Reactogenicity was also assessed. Findings: 130 participants (TG:92; CG:38) completed standard vaccination. In TG, median (IQR) of anti-RBD antibodies after first BNT162b2 dose were 10740·0 BAU/mL (4466·0-12500) compared to 29·8 BAU/mL (14·5-47·8) in CG (p <0·0001). Median NT50 (IQR) of G614 was 2674·0 (1865·0-3997·0) in TG and 63·0 (16·0-123·1) in CG (p <0·0001). After second BNT162b2 dose, anti-RBD levels increased to ≥12500 BAU/mL (11625·0-12500) in TG compared to 1859·0 BAU/mL (915·4-3820·0) in CG (p <0·0001). NT50 was 2626·5 (1756·0-5472·0) and 850·4 (525·1-1608·0), respectively (p <0·0001). Variant-specific (Beta, Mu, Omicron) response was also assessed. Most frequent adverse reactions were headache, myalgia, and local pain. No severe AEs were reported. Interpretation: Heterologous BNT162b2 as third and fourth doses in previously suboptimal immunized individuals elicit stronger immune response than that obtained with two doses of BNT162b2. This apparent benefit was also observed in variant-specific response. No safety concerns arose.This work is partially funded by Institute of Health Carlos III (Instituto de Salud Carlos III − ISCIII −), (grants PI19CIII/00004 −JA- and PI21CIII/00025 −MPO, JG-), and COVID-19 FUND (grants COV20/00679 −MPO- and COV20/00072 −JA-) and CIBERINFEC, co-financed by the European Regional Development Fund (FEDER) “A way to make Europe”. Instituto de Salud Carlos III is a Spanish public body assigned to the Ministry of Science and Innovation that manages and promotes public clinical research related to public health. The authors thank Esther Prieto, MD (cited with consent) for editorial assistance and writing support (funded by the Research Foundation of HCSC).S

Safety and immunogenicity of the protein-based PHH-1V compared to BNT162b2 as a heterologous SARS-CoV-2 booster vaccine in adults vaccinated against COVID-19 : a multicentre, randomised, double-blind, non-inferiority phase IIb trial

A SARS-CoV-2 protein-based heterodimer vaccine, PHH-1V, has been shown to be safe and well-tolerated in healthy young adults in a first-in-human, Phase I/IIa study dose-escalation trial. Here, we report the interim results of the Phase IIb HH-2, where the immunogenicity and safety of a heterologous booster with PHH-1V is assessed versus a homologous booster with BNT162b2 at 14, 28 and 98 days after vaccine administration. The HH-2 study is an ongoing multicentre, randomised, active-controlled, double-blind, non-inferiority Phase IIb trial, where participants 18 years or older who had received two doses of BNT162b2 were randomly assigned in a 2:1 ratio to receive a booster dose of vaccine-either heterologous (PHH-1V group) or homologous (BNT162b2 group)-in 10 centres in Spain. Eligible subjects were allocated to treatment stratified by age group (18-64 versus ≥65 years) with approximately 10% of the sample enrolled in the older age group. The primary endpoints were humoral immunogenicity measured by changes in levels of neutralizing antibodies (PBNA) against the ancestral Wuhan-Hu-1 strain after the PHH-1V or the BNT162b2 boost, and the safety and tolerability of PHH-1V as a boost. The secondary endpoints were to compare changes in levels of neutralizing antibodies against different variants of SARS-CoV-2 and the T-cell responses towards the SARS-CoV-2 spike glycoprotein peptides. The exploratory endpoint was to assess the number of subjects with SARS-CoV-2 infections ≥14 days after PHH-1V booster. This study is ongoing and is registered with , . From 15 November 2021, 782 adults were randomly assigned to PHH-1V (n = 522) or BNT162b2 (n = 260) boost vaccine groups. The geometric mean titre (GMT) ratio of neutralizing antibodies on days 14, 28 and 98, shown as BNT162b2 active control versus PHH-1V, was, respectively, 1.68 (p < 0.0001), 1.31 (p = 0.0007) and 0.86 (p = 0.40) for the ancestral Wuhan-Hu-1 strain; 0.62 (p < 0.0001), 0.65 (p < 0.0001) and 0.56 (p = 0.003) for the Beta variant; 1.01 (p = 0.92), 0.88 (p = 0.11) and 0.52 (p = 0.0003) for the Delta variant; and 0.59 (p ≤ 0.0001), 0.66 (p < 0.0001) and 0.57 (p = 0.0028) for the Omicron BA.1 variant. Additionally, PHH-1V as a booster dose induced a significant increase of CD4 + and CD8 + T-cells expressing IFN-γ on day 14. There were 458 participants who experienced at least one adverse event (89.3%) in the PHH-1V and 238 (94.4%) in the BNT162b2 group. The most frequent adverse events were injection site pain (79.7% and 89.3%), fatigue (27.5% and 42.1%) and headache (31.2 and 40.1%) for the PHH-1V and the BNT162b2 groups, respectively. A total of 52 COVID-19 cases occurred from day 14 post-vaccination (10.14%) for the PHH-1V group and 30 (11.90%) for the BNT162b2 group (p = 0.45), and none of the subjects developed severe COVID-19. Our interim results from the Phase IIb HH-2 trial show that PHH-1V as a heterologous booster vaccine, when compared to BNT162b2, although it does not reach a non-inferior neutralizing antibody response against the Wuhan-Hu-1 strain at days 14 and 28 after vaccination, it does so at day 98. PHH-1V as a heterologous booster elicits a superior neutralizing antibody response against the previous circulating Beta and the currently circulating Omicron BA.1 SARS-CoV-2 variants in all time points assessed, and for the Delta variant on day 98 as well. Moreover, the PHH-1V boost also induces a strong and balanced T-cell response. Concerning the safety profile, subjects in the PHH-1V group report significantly fewer adverse events than those in the BNT162b2 group, most of mild intensity, and both vaccine groups present comparable COVID-19 breakthrough cases, none of them severe. HIPRA SCIENTIFIC, S.L.U

Risk Factors for COVID-19 in Inflammatory Bowel Disease: A National, ENEIDA-Based Case–Control Study (COVID-19-EII)

(1) Scant information is available concerning the characteristics that may favour the acquisition of COVID-19 in patients with inflammatory bowel disease (IBD). Therefore, the aim of this study was to assess these differences between infected and noninfected patients with IBD. (2) This nationwide case-control study evaluated patients with inflammatory bowel disease with COVID-19 (cases) and without COVID-19 (controls) during the period March-July 2020 included in the ENEIDA of GETECCU. (3) A total of 496 cases and 964 controls from 73 Spanish centres were included. No differences were found in the basal characteristics between cases and controls. Cases had higher comorbidity Charlson scores (24% vs. 19%; p = 0.02) and occupational risk (28% vs. 10.5%; p < 0.0001) more frequently than did controls. Lockdown was the only protective measure against COVID-19 (50% vs. 70%; p < 0.0001). No differences were found in the use of systemic steroids, immunosuppressants or biologics between cases and controls. Cases were more often treated with 5-aminosalicylates (42% vs. 34%; p = 0.003). Having a moderate Charlson score (OR: 2.7; 95%CI: 1.3-5.9), occupational risk (OR: 2.9; 95%CI: 1.8-4.4) and the use of 5-aminosalicylates (OR: 1.7; 95%CI: 1.2-2.5) were factors for COVID-19. The strict lockdown was the only protective factor (OR: 0.1; 95%CI: 0.09-0.2). (4) Comorbidities and occupational exposure are the most relevant factors for COVID-19 in patients with IBD. The risk of COVID-19 seems not to be increased by immunosuppressants or biologics, with a potential effect of 5-aminosalicylates, which should be investigated further and interpreted with caution

Nationwide COVID-19-EII Study : Incidence, Environmental Risk Factors and Long-Term Follow-Up of Patients with Inflammatory Bowel Disease and COVID-19 of the ENEIDA Registry

We aim to describe the incidence and source of contagion of COVID-19 in patients with IBD, as well as the risk factors for a severe course and long-term sequelae. This is a prospective observational study of IBD and COVID-19 included in the ENEIDA registry (53,682 from 73 centres) between March-July 2020 followed-up for 12 months. Results were compared with data of the general population (National Centre of Epidemiology and Catalonia). A total of 482 patients with COVID-19 were identified. Twenty-eight percent were infected in the work environment, and 48% were infected by intrafamilial transmission, despite having good adherence to lockdown. Thirty-five percent required hospitalization, 7.9% had severe COVID-19 and 3.7% died. Similar data were reported in the general population (hospitalisation 19.5%, ICU 2.1% and mortality 4.6%). Factors related to death and severe COVID-19 were being aged ≥ 60 years (OR 7.1, 95% CI: 1.8-27 and 4.5, 95% CI: 1.3-15.9), while having ≥2 comorbidities increased mortality (OR 3.9, 95% CI: 1.3-11.6). None of the drugs for IBD were related to severe COVID-19. Immunosuppression was definitively stopped in 1% of patients at 12 months. The prognosis of COVID-19 in IBD, even in immunosuppressed patients, is similar to that in the general population. Thus, there is no need for more strict protection measures in IBD

Risk Factors for COVID-19 in Inflammatory Bowel Disease: A National, ENEIDA-Based Case&ndash;Control Study (COVID-19-EII)

(1) Scant information is available concerning the characteristics that may favour the acquisition of COVID-19 in patients with inflammatory bowel disease (IBD). Therefore, the aim of this study was to assess these differences between infected and noninfected patients with IBD. (2) This nationwide case&ndash;control study evaluated patients with inflammatory bowel disease with COVID-19 (cases) and without COVID-19 (controls) during the period March&ndash;July 2020 included in the ENEIDA of GETECCU. (3) A total of 496 cases and 964 controls from 73 Spanish centres were included. No differences were found in the basal characteristics between cases and controls. Cases had higher comorbidity Charlson scores (24% vs. 19%; p = 0.02) and occupational risk (28% vs. 10.5%; p &lt; 0.0001) more frequently than did controls. Lockdown was the only protective measure against COVID-19 (50% vs. 70%; p &lt; 0.0001). No differences were found in the use of systemic steroids, immunosuppressants or biologics between cases and controls. Cases were more often treated with 5-aminosalicylates (42% vs. 34%; p = 0.003). Having a moderate Charlson score (OR: 2.7; 95%CI: 1.3&ndash;5.9), occupational risk (OR: 2.9; 95%CI: 1.8&ndash;4.4) and the use of 5-aminosalicylates (OR: 1.7; 95%CI: 1.2&ndash;2.5) were factors for COVID-19. The strict lockdown was the only protective factor (OR: 0.1; 95%CI: 0.09&ndash;0.2). (4) Comorbidities and occupational exposure are the most relevant factors for COVID-19 in patients with IBD. The risk of COVID-19 seems not to be increased by immunosuppressants or biologics, with a potential effect of 5-aminosalicylates, which should be investigated further and interpreted with caution