Estudio de biomarcadores en sangre periférica en mujeres con fallo reproductivo recurrente

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Medicina, leída el 18-07-2022El fallo reproductivo recurrente engloba dos entidades clínicas bien diferenciadas como son los abortos de repetición (AR) y el fallo de implantación recurrente (FIR). El AR se define como la pérdida de dos o más gestaciones antes de las 24 semanas de edad gestacional, mientras que para FIR no existe un consenso internacional en su definición, pero la más aceptada sería la no consecución del embarazo en mujeres menores de 40 años, tras haber transferido al menos 4 embriones de buena calidad, en un mínimo 3 transferencias (incluyendo tanto las realizadas en fresco como las de embriones criopreservados). Ambas patologías comparten algunas etiologías como pueden ser: causas anatómicas, genéticas, endocrinas, entre otras. Pero, en un gran número de pacientes, su origen permanece desconocido pudiendo alcanzar hasta un 50% en las pacientes con AR. El sistema inmunitario ha cobrado en los últimos años una vital importancia dentro de poder explicar un alto grado de pacientes que previamente permanecían como de causa desconocida. Se ha estudiado extensamente en los últimos 20 años el papel que las células NK tanto en número como en función, juegan en estas dos patologías. Asimismo, los monocitos, células clave también para el desarrollo del embarazo, han cobrado más importancia en los últimos años, aunque su estudio se ha centrado en su papel en otras patologías, principalmente en preeclampsia. Inicialmente se pensaba que un estado proinflamatorio durante el embarazo podría ser perjudicial para el correcto desarrollo del mismo...Recurrent reproductive failure encompasses two well-differentiated entities: recurrent pregnancy loss (RPL) and recurrent implantation failure (RIF). RPL is defined as the loss of two or more pregnancies before 24 weeks of gestational age, while for RIF, there is no international consensus on its definition, but the most accepted would be the non-achievement of pregnancy in women under 40 years of age, after having transferred at least four good quality embryos, in a minimum of 3 transfers (including both those made fresh and those of cryopreserved embryos). Both pathologies share some etiologies, such as anatomical, genetic, and endocrine causes, among others. Therefore, in many patients, its origin remains unknown and can reach up to 50% in patients with RPL. In recent years, the immune system has gained vital importance in explaining a high degree of patients who previously remained as of unknown cause. The role that NK cells play in these two pathologies, both in number and function, has been extensively studied in the last 20 years. Likewise, monocytes, also essential cells for the development of pregnancy, have become more important in recent years, although their study has focused on their role in other pathologies, mainly preeclampsia. Initially, it was thought that a pro-inflammatory state during pregnancy could be detrimental to its proper development...Fac. de MedicinaTRUEunpu

Non-criteria obstetric antiphospholipid syndrome: how different is from Sidney criteria? A single-center study

This study aims to compare the demographic characteristics, clinical features, serology, and fetal-maternal outcomes between women with obstetric antiphospholipid syndrome (APS) and those with non-criteria (NC)-APS and seronegative (SN)-APS. Two-hundred and sixty-three women with APS obstetric morbidity ever pregnant were included. Of those, 66 met the APS classification criteria, 140 were NC-APS, and 57 were SN-APS. Patients with other autoimmune diseases were excluded. Adverse pregnancy outcomes (APO) included early pregnancy loss, fetal death, preeclampsia, abruptio placentae, and preterm birth. The mean age of the study group was 33.6 ± 5.3 years, and patients were followed up for 129.5 ± 81.9 months. In the NC-APS group, 31 (22.1%) did not fulfill clinical and serological criteria (Subgroup A), 49 (35%) did meet clinical but not serologic criteria (Subgroup B), and 60 (42.9%) fulfilled the serologic criteria but not the clinical ones (Subgroup C). The cardiovascular risk burden was higher in the APS group, due to a higher proportion of smoking. Patients with criteria APS received more intensive treatment than patients in the other study groups. The addition of standard of care (SoC) treatment significantly improved live birth and decreased APO in all groups. Significant clinical differences were observed between the study groups. However, when treated with SoC, fetal-maternal outcomes were similar, with a significant improvement in live births and a decrease in APO. Risk stratification in patients with obstetric morbidity associated with APS can help individualize their treatment

Validation of a Quick Flow Cytometry-Based Assay for Acute Infection Based on CD64 and CD169 Expression. New Tools for Early Diagnosis in COVID-19 Pandemic

Objectives: Several parameters aid in deciphering between viral and bacterial infections; however, new tools should be investigated in order to reduce the time to results and proceed with an early target-therapy. Validation of a biomarker study, including CD64 and CD169 expression, was conducted. Material and Methods: Patients with active SARS-CoV-2 infection (ACov-2), bacterial infection (ABI), healthy controls, and antiretroviral-controlled chronic HIV infection were assessed. Whole blood was stained and, after lysing no-wash protocol, acquired by flow cytometry. The median fluorescence intensity (MFI) of CD64 and CD169 was measured in granulocytes, monocytes, and lymphocytes. The CD64 MFI ratio granulocytes to lymphocytes (CD64N) and CD169 MFI ratio monocytes to lymphocytes (CD169Mo) were evaluated as biomarkers of acute bacterial and viral infection, respectively. Results: A CD64N ratio higher than 3.3 identified patients with ABI with 83.3 and 85.9% sensitivity and specificity, with an area under the curve (AUC) of 83.5%. In contrast, other analytic or hematological parameters used in the clinic had lower AUC compared with the CD64N ratio. Moreover, a CD169Mo ratio higher than 3.3 was able to identify ACov-2 with 91.7 and 89.8 sensitivity and specificity, with the highest AUC (92.0%). Conclusion: This work confirms the previous data of CD64N and CD169Mo ratios in an independent cohort, including controlled chronic viral HIV infection patients as biomarkers of acute bacterial and viral infections, respectively. Such an approach would benefit from quick pathogen identification for a direct-therapy with a clear application in different Health Care Units, especially during this COVID pandemic.This work was partially supported by Instituto de Salud Carlos III Grant: FIS (COV20/0170

Innate and adaptive immune assessment at admission to predict clinical outcome in covid-19 patients

During the COVID-19 pandemic, many studies have been carried out to evaluate different immune system components to search for prognostic biomarkers of the disease. A broad multiparametric antibody panel of cellular and humoral components of the innate and the adaptative immune response in patients with active SARS-CoV-2 infection has been evaluated in this study. A total of 155 patients were studied at admission into our center and were categorized according to the requirement of oxygen therapy as mild or severe (the latter being those with the requirement). The patients with severe disease were older and had high ferritin, D-dimer, C-reactive protein, troponin, interleukin-6 (IL-6) levels, and neutrophilia with lymphopenia at admission. Moreover, the patients with mild symptoms had significantly increased circulating non-classical monocytes, innate lymphoid cells, and regulatory NK cells. In contrast, severe patients had a low frequency of Th1 and regulatory T cells with increased activated and exhausted CD8 phenotype (CD8+CD38+HLADR+ and CD8+CD27-CD28-, respectively). The predictive model included age, ferritin, D-dimer, lymph counts, C4, CD8+CD27-CD28-, and non-classical monocytes in the logistic regression analysis. The model predicted severity with an area under the curve of 78%. Both innate and adaptive immune parameters could be considered potential predictive biomarkers of the prognosis of COVID-19 disease.Funding: This work was partially supported by the Cantabrian Government, grant number 2020UIC22-PUB-001, and by Instituto de Salud Carlos III, grant number COV20/00170

Immune Assessment of BNT162b2 m-RNA-Spike Based Vaccine Response in Adults

Vaccine efficacy is based on clinical data. Currently, the assessment of immune response after SARS-CoV-2 vaccination is scarce. A total of 52 healthcare workers were immunized with the same lot of BNT162b2 vaccine. The immunological response against the vaccine was tested using a T-specific assay based on the expression of CD25 and CD134 after stimulation with anti-N, -S, and -M specific peptides of SARS-CoV-2. Moreover, IgG anti-S2 and -RBD antibodies were detected using ELISA. Furthermore, the cell subsets involved in the response to the vaccine were measured in peripheral blood by flow cytometry. Humoral-specific responses against the vaccine were detected in 94% and 100% after the first and second doses, respectively. Therefore, anti-S T-specific responses were observed in 57% and 90% of the subjects after the first and second doses of the vaccine, respectively. Thirty days after the second dose, significant increases in T helper 1 memory cells (p < 0.001), peripheral memory T follicular helper (pTFH) cells (p < 0.032), and switched memory (p = 0.005) were observed. This study describes the specific humoral and cellular immune responses after vaccination with the new mRNA-based BNT162b2 vaccine. A mobilization of TFH into the circulation occurs, reflecting a specific activation of the immune system.Funding: This work was partially supported by the Cantabrian Government, grant number 2020UIC22-PUB-001, and from Instituto de Salud Carlos III, grant number COV20/00170

COVID-19 mRNA Based Vaccine Immune-Response Assessment in Nursing Home Residents for Public Health Decision

Nursing home residents (NHR) have been targeted as a vaccination priority due to their higher risk of worse outcome after COVID-19 infection. The mRNA-based vaccine BTN2b2 was first approved in Europe for NHRs. The assessment of the specific vaccine immune response (both humoral and cellular) at long term in NHRs has not been addressed yet. A representative sample of 624 NHR subjects in Northern region of Spain was studied to assess immune response against full vaccination with BTN2b2. The anti-S1 antibody levels and specific T cells were measured at two and six months after vaccination. 24.4% of NHR had a previous infection prior to vaccination. The remaining NHR were included in the full vaccination assessment group (FVA). After two months, a 94.9% of the FVA presented anti-S1 antibodies, whereas those seronegative without specific cellular response were 2.54%. At long-term, the frequency of NHR within the FVA group with anti-S1 antibodies at six months were 88.12% and the seronegative subjects without specific cellular response was 8.07%. The cellular immune assays complement the humoral test in the immune vaccine response assessment. Therefore, the cellular immune assessment in NHRs allows for the fine tuning of those seronegative subjects with potential competent immune responses against the vaccine

Immunophenotyping of peripheral blood monocytes could help identify a baseline pro-inflammatory profile in women with recurrent reproductive failure

11 p.-4 fig.-4 tab.Recurrent pregnancy loss (RPL) and recurrent implantation failure (RIF) are two well-defined clinical entities, but the role of the monocytes in their pathophysiology needs to be clarified. This study aimed to evaluate the role of the three monocyte subsets (classical, intermediate, and non-classical) and relevant cytokines/chemokines in a cohort of RPL and RIF women to better characterize a baseline proinflammatory profile that could define inflammatory pathophysiology in these two different conditions. We evaluated 108 non-pregnant women: 53 RPL, 24 RIF, and 31 fertile healthy controls (HC). Multiparametric flow cytometry was used to quantify the frequency of surface chemokine receptors (CCR2, CCR5, and CX3CR1) on the monocyte subsets. Cytokines were assessed in plasma samples using a multiplex assay. The CX3CR1+ and CCR5+ intermediate monocytes were significantly higher in RPL and RIF compared to HC. A significant positive correlation was observed between CX3CR1+ intermediate monocytes and IL-17A (P = .03, r = 0.43). The Boruta algorithm followed by a multivariate logistic regression model was used to select the most relevant variables that could help define RPL and RIF: in RPL were CX3CR1 non-classical monocytes, TGF-β1, and CCR5 intermediate monocytes; in RIF: CCR5 intermediate monocytes and TGF-β3. The combination of these variables could predict RPL and RIF with 90 % and 82 %, respectively. Our study suggests that a combination of specific blood monocyte subsets and cytokines could aid in identifying RPL and RIF women with a pro-inflammatory profile. These findings could provide a more integrated understanding of these pathologies. Further investigation and validation in independent cohorts are warranted.The project received a research grant from the Carlos III Institute of Health, Ministry of Economy and Competitiveness (Spain), awarded on the 2016 call under the Health Strategy Action 2016–2017, within the National Research Program oriented to Societal Challenges, within the Technical, Scientific and Innovation Research National Plan 2013–2016, with reference PI16/01428, and was co-supported by The European Regional Development Fund (ERDF).Peer reviewe

Non-Criteria Obstetric Antiphospholipid Syndrome: How Different Is from Sidney Criteria? A Single-Center Study

This study aims to compare the demographic characteristics, clinical features, serology, and fetal&ndash;maternal outcomes between women with obstetric antiphospholipid syndrome (APS) and those with non-criteria (NC)-APS and seronegative (SN)-APS. Two-hundred and sixty-three women with APS obstetric morbidity ever pregnant were included. Of those, 66 met the APS classification criteria, 140 were NC-APS, and 57 were SN-APS. Patients with other autoimmune diseases were excluded. Adverse pregnancy outcomes (APO) included early pregnancy loss, fetal death, preeclampsia, abruptio placentae, and preterm birth. The mean age of the study group was 33.6 &plusmn; 5.3 years, and patients were followed up for 129.5 &plusmn; 81.9 months. In the NC-APS group, 31 (22.1%) did not fulfill clinical and serological criteria (Subgroup A), 49 (35%) did meet clinical but not serologic criteria (Subgroup B), and 60 (42.9%) fulfilled the serologic criteria but not the clinical ones (Subgroup C). The cardiovascular risk burden was higher in the APS group, due to a higher proportion of smoking. Patients with criteria APS received more intensive treatment than patients in the other study groups. The addition of standard of care (SoC) treatment significantly improved live birth and decreased APO in all groups. Significant clinical differences were observed between the study groups. However, when treated with SoC, fetal&ndash;maternal outcomes were similar, with a significant improvement in live births and a decrease in APO. Risk stratification in patients with obstetric morbidity associated with APS can help individualize their treatment

Does Adjusted Global Antiphospholipid Syndrome Score (aGAPSS) Predict the Obstetric Outcome in Antiphospholipid Antibody Carriers? A Single Center Study

The adjusted Global Antiphospholipid Syndrome (APS) Score (aGAPSS) is a tool proposed to quantify the risk for antiphospholipid antibody (aPL)-related clinical manifestations. However, aGAPSS has been validated mainly for thrombotic events and studies on APS-related obstetric manifestations are scarce. Furthermore, the majority of them included patients with positive aPL and diferent autoimmune diseases. Here, we assess the utility of aGAPSS to predict the response to treatment in aPL carriers without other autoimmune disorders. One-hundred and thirty-seven women with aPL ever pregnant were included. Sixty-fve meet the APS classifcation criteria, 61 had APS-related obstetric manifestations, and 11 were asymp tomatic carriers. The patients? aGAPSS risk was grouped as low (<6, N=73), medium (6?11, N=40), and high risk (?12, N=24). Since vascular risk factors included in the aGAPSS were infrequent in this population (<10%), the aGAPSS score was mainly determined by the aPL profle. Overall, the live birth rate was 75%, and 37.2% of the patients had at least one adverse pregnancy outcome (APO). When considering patients according to the aGAPSS (high, medium, and low risk), no signifcant diferences were found for pregnancy loss (29.2%, 25%, and 21.9%) or APO (33.3%, 47.5%, and 32.9%). In the present study, including aPL carriers without other autoimmune diseases, aGAPSS is not a valuable tool to identify patients at risk for obstetric complications despite treatment. In these patients with gestational desire, in addition to the aPL profle, other pregnancy-specifc factors, such as age or previous obstetric history, should be consideredPregnancyAntiphospholipid syndromeAntiphospholipid antibodiesGAPSSScoreNon-criteria obstetric manifestation