Influence of BMI, Cigarette Smoking and Cryopreservation on Tyrosine Phosphorylation during Sperm Capacitation.

Capacitation involves tyrosine phosphorylation (TP) as a key marker. Lifestyle-related factors, such as obesity and smoking, are recognized for their adverse effects on semen quality and male fertility, yet the underlying mechanisms, including their potential impact on TP, remain unclear. Moreover, the effect of sperm cryopreservation on TP at the human sperm population level is unexplored. Flow cytometry analysis of global TP was performed on pre-capacitated, post-capacitated and 1- and 3-hours' incubated fresh and frozen-thawed samples from sperm donors (n = 40). Neither being overweight nor smoking (or both) significantly affected the percentage of sperm showing TP. However, elevated BMI and smoking intensity correlated with heightened basal TP levels (r = 0.226, p = 0.003) and heightened increase in TP after 3 h of incubation (r = 0.185, p = 0.017), respectively. Cryopreservation resulted in increased global TP levels after capacitation but not immediately after thawing. Nonetheless, most donors' thawed samples showed increased TP levels before and after capacitation as well as after incubation. Additionally, phosphorylation patterns in fresh and frozen-thawed samples were similar, indicating consistent sample response to capacitation stimuli despite differences in TP levels. Overall, this study sheds light on the potential impacts of lifestyle factors and cryopreservation on the dynamics of global TP levels during capacitation.

Looking at the Mental Health of Children and Adolescents with Cleft Lip and/or Palate through Neuroticism and Emotional Regulation Strategies: A Case-Controlled Observational Study.

Background: Children and adolescents with cleft lip and/or palate (CL/P) are at an increased risk of developing emotional disorders. This study aims to explore this question in greater depth by addressing three objectives: (1) the presence of neuroticism as an indicator of emotional symptomatology, (2) the use of adaptive and non-adaptive emotional regulation strategies, and (3) the relationship between these strategies and neuroticism. Methods: A case-control correlational methodology was employed, with 60 children and adolescents with CL/P (mean age = 12.80 years; 33 females) and 60 non-clinical equivalent children and adolescents. Results: The CL/P group has higher scores on neuroticism (t = -7.74; p ≤ 0.001, d Cohen = 1.43) and lower scores in almost all emotional regulation strategies. The presence of CL/P moderated the relationship between neuroticism and self-blame (Beta = -0.46, t = -2.81, p = 0.005), rumination (Beta = -0.49, t = -3.73, p < 0.001), catastrophizing (Beta = -0.61, t = -4.26, p < 0.001), and blaming others (Beta = -0.45, t = -2.84, p = 0.005). This model predicted a significant variance of neuroticism (all p < 0.005), which ranged from 39% to 41%. Conclusions: The CL/P group has worse mental health indicators. Particularly novel results about the CL/P group are the lower scores on regulation strategies (both adaptive and non-adaptive) and the fact that non-adaptive strategies contribute, contrary to their effect in the general population, to a decrease in neuroticism. It supports the need to incorporate mental health indicators in the diagnosis and treatment of children and adolescents with CL/P.

Sudden cardiac death triggered by minimal alcohol consumption in the context of novel PPA2 mutations in 2 unrelated families.

Biallelic variants in PPA2 gene cause a rare but lethal mitochondrial disorder. We describe the first four cases reported in Spain of PPA2 disease in two unrelated families. We have conducted a revision of the clinical history, necropsies, and postmortem genetic testing from probands, and clinical evaluation, genetic testing and blood transcript analysis in family members. All the cases harbored biallelic PPA2 variants in compound heterozygous status. Two brothers from family 1 suffered sudden death after a small first intake of alcohol in 2013 and 2022. The sister remains alive but affected with cardiomyopathy, extensive scar on cardiac imaging, and high sensitivity to alcohol intake. The three siblings carried PPA2 c.290A > G (p.Glu97Gly) novel missense variant and PPA2 c.513C > T (p.Cys171 = ) altering splicing site variant, both probably leading to mRNA degradation based on in-silico and transcript analyses. A teenager from family 2 suffered sudden death after a small intake of alcohol in 2018 and carried PPA2 c.683C > T (p.Pro228Leu) missense and PPA2 c.980_983del (p.Gln327fs) novel frameshift variant, both probably leading to abnormal protein structure. All cases were asymptomatic until adolescence. Furthermore, the sister in family 1 has survived as an asymptomatic adult. PPA2 disease can manifest as cardiac arrest in the young, especially after alcohol exposure. Our results show that PPA2 deficiency can be related to different pathogenicity mechanisms such as abnormal protein structure but also mRNA decay caused by synonymous or missense variants. Strict avoidance of alcohol consumption and early defibrillator implantation might prevent lethal arrhythmias in patients at risk.

Histological Typing in Patients With Cardiac Amyloidosis: JACC Review Topic of the Week.

Cardiac amyloidosis is increasingly recognized as a treatable form of heart failure. Highly effective specific therapies have recently become available for the 2 most frequent forms of cardiac amyloidosis: immunoglobulin light chain amyloidosis and transthyretin (ATTR) amyloidosis. Nevertheless, initiation of specific therapies requires recognition of cardiac amyloidosis and appropriate characterization of the amyloid type. Although noninvasive diagnosis is possible for ATTR cardiac amyloidosis, histological demonstration and typing of amyloid deposits is still required for a substantial number of patients with ATTR and in all patients with light chain amyloidosis and other rarer forms of cardiac amyloidosis. Amyloid histological typing can be performed using different techniques: mass spectrometry, immunohistochemistry, and immunoelectron microscopy. This review describes which patients require histological confirmation of cardiac amyloidosis along with when and how to type amyloid deposits in histologic specimens. Furthermore, it covers the characteristics and limitations of the different typing methods that are available in clinical practice.

Sequential-contingent strategy for fallopian tube evaluation using hysterosalpingo foam sonography: a cost analysis.

Background: Tubal pathology is the etiological factor in 20% of couples with female infertility. Historically, diagnostic evaluation of the fallopian tubes has been performed by means of hysterosalpingography (HSG). Hysterosonosalpingography with EXEM® foam is a recent technique with supposedly comparable effectiveness to HSG, but with better tolerability for patients. Methods: Tubal patency was assessed in patients who were candidates for intrauterine insemination (IUI) between January 2020 and July 2021. (I) Hysterosonosalpingography with EXEM® foam. (II) If the patient had both tubes patent, IUI was proposed. In case of inconclusive or pathological results, HSG was performed. (III) If the HSG concludes tubal patency, IUI is proposed. On the other hand, if it results in tubal obstruction, in vitro fertilization (IVF) is proposed. Results: Assessment of tubal patency by HSG showed concordance with previous Hysterosonosalpingography with EXEM® foam results in 45.5% (n=15) of cases of unilateral obstruction and 66.7% (n=6) of cases of bilateral obstruction. When we compare the total cost of HSG (€54,899.46) with the sum of patients who only required Hysterosonosalpingography with EXEM® foam together with those who underwent both tests (€45,575.96), it shows that the latter represents a cost benefit strategy (cost difference €9,323.50). Conclusions: Our results suggest a benefit of €9,323.50 when performing the contingent sequential strategy, in case of pathological or inconclusive results, compared to performing a systematic HSG in all patients. Patients with an obstructive hysterosalpingogram with EXEM® and a patent hysterosalpingogram will be the subgroup that will benefit the most, as IUI can be proposed instead of IVF. In addition, there is a benefit in terms of optimizing the medical procedure, hospital visits and speeding up the time to start assisted reproductive technology (ART).

Feasibility and safety of left bundle branch area pacing in cardiac amyloidosis. A single center experience.

BACKGROUND: Conventional right ventricle (RV) pacemaker stimulation has been associated with worse clinical outcomes in patients with cardiac amyloidosis (CA). Left bundle branch area pacing (LABPP) has been suggested as a promising alternative. We sought to assess the safety, feasibility, and outcomes of LABPP in patients with CA. METHODS: We retrospectively analyzed echocardiography and pacing parameters and clinical outcomes in 23 consecutive patients with CA and LBBAP implanted from June 2020 to October 2022. RESULTS: LBBAP was successfully performed in 22 over 23 patients (19 male, 78.6 ± 11.7 years, 20 ATTR, mean LVEF 45.5 ± 16.2%). After the procedure, 9 patients showed Qr pattern and 11 a qR pattern in V1 on ECG. Average procedure time was 67 ± 28 min. After 7.7 ± 5.2 months follow-up, no procedure-related complications had occurred. Although, a significant reduction in QRS width (p = .001) was achieved, we did not observe significant changes in LVEF and Nt ProBNP at 6 months of follow-up. Pacing parameters were stable during follow-up: LBB capture threshold and R wave amplitude were 1.0 ±  0.5 V and 10.6 ± 6.0 mV versus 0.8 ±  0.1 V, p = .21 and 10.6 ± 5.1 mV (p = .985) at follow up. CONCLUSION: LBBAP is safe and feasible pacing technique for patients with CA. LBBAP is associated with significant narrowing of QRSd without worsening in LVEF and Nt-proBNP.

PGT-HLA programmes for the cure of a sick sibling: clinical strategies for this challenging search.

The ultimate goal of a preimplantation genetic testing and human leukocyte antigen (PGT-HLA) matching programme is the birth of a healthy, HLA-compatible child for the treatment or cure of a sick sibling. Several authors have published successful cases of the births of children HLA-matched to siblings affected by different conditions and diseases. However, there are many reports of failed attempts. Couples seeking an HLA-matched sibling for their affected child look for positive outcomes in the shortest possible time. Nevertheless, there is no published consensus or guidelines with recommendations for these cases. Here, the authors aimed to analyse different approaches for these programmes, highlighting the most promising strategies for the families and fertility units. Furthermore, the authors mention a successful case of a PGT-HLA matching programme after a previous failed attempt following the strategies proposed. Which is the most cost-effective and time-efficient approach in a PGT-HLA matching programme?

Specific Cellular and Humoral Response after the Third Dose of Anti-SARS-CoV-2 RNA Vaccine in Patients with Immune-Mediated Rheumatic Diseases on Immunosuppressive Therapy.

OBJECTIVE: Data on cellular and humoral immunogenicity after the third dose of anti-SARS-CoV-2 vaccines in patients with immune-mediated rheumatic diseases (IMRDs) are scarce. Herein, we evaluated the adaptive immune response in IMRD patients treated with different immunosuppressive therapies (conventional synthetic disease-modifying antirheumatic drugs [csDMARDs], biological disease-modifying antirheumatic drugs [bDMARDs], and targeted synthetic disease-modifying antirheumatic drugs [tsDMARDs]) after the booster of the anti-SARS-CoV-2 vaccine to determine whether any drug reduced the vaccine's response. METHODS: A single-center prospective study was conducted, including patients presenting with IMRD and healthy controls (HC). Specific anti-SARS-CoV-2 interferon-gamma (IFN-γ) production was evaluated between 8-12 weeks after the third dose of the SARS-CoV-2 vaccine. In addition, anti-Spike IgG antibody titers were also measured. RESULTS: Samples were obtained from 79 IMRD patients (51 women, 28 men; mean age 57 ± 11.3 years old): 43 rheumatoid arthritis, 10 psoriatic arthritis, 14 ankylosing spondylitis, 10 undifferentiated spondyloarthritis, and 2 inflammatory bowel disease-associated spondyloarthritis (IBD-SpA). In total, 31 HC (mean age 50.9 ± 13.1 years old, 67.7% women) were included in the study. Post-vaccine results displayed positive T-cell immune responses in 68 out of 79 (86.1%) IMRD patients (82.3% of those without prior COVID-19). All HC and IMRDs patients had an antibody response against the SARS-CoV-2 receptor-binding domain; however, the HC response was significantly higher (median of 18,048 AU/mL) than in IMRDs patients (median of 6590.3 AU/mL, p < 0.001). MTX and leflunomide were associated with lower titers of IgG and IFN-γ responses. Among bDMARDs, adalimumab, etanercept, and guselkumab are associated with reduced cellular responses. CONCLUSION: Our preliminary data show that the majority of our IMRD patients develop cellular and humoral responses after the SARS-CoV-2 booster vaccination, emphasizing the relevance of vaccination in this group. However, the magnitude of specific responses was dependent on the immunosuppressive therapy administered. Specific vaccination protocols and personalized decisions about boosters are essential for these patients.

Menstrual blood-derived mesenchymal stromal cells: impact of preconditioning on the cargo of extracellular vesicles as potential therapeutics.

BACKGROUND: Mesenchymal stromal cells (MSCs) have been shown to exert their therapeutic effects through the secretion of broad spectrum of paracrine factors, including extracellular vesicles (EVs). Accordingly, EVs are being pursued as a promising alternative to cell-based therapies. Menstrual blood-derived stromal cells (MenSCs) are a type of MSC that, due to their immunomodulatory and regenerative properties, have emerged as an innovative source. Additionally, new strategies of cell priming may potentially alter the concentration and cargo of released EVs, leading to modification of their biological properties. In this study, we aimed to characterize the EVs released by MenSCs and compare their therapeutic potential under three different preconditioning conditions (proinflammatory stimuli, physioxia, and acute hypoxia). METHODS: MenSCs were isolated from five healthy women. Following culturing to 80% confluence, MenSCs were exposed to different priming conditions: basal (21% O2), proinflammatory stimuli (IFNγ and TNFα, 21% O2), physioxia (1-2% O2), and acute hypoxia (< 1% O2) for 48-72 h. Conditioned media from MenSCs was collected after 48 h and EVs were isolated by a combination of ultra-filtration and differential centrifugation. An extensive characterization ranging from nano-flow cytometry (nFC) to quantitative high-throughput shotgun proteomics was performed. Bioinformatics analyses were used to derive hypotheses on their biological properties. RESULTS: No differences in the morphology, size, or number of EVs released were detected between priming conditions. The proteome analysis associated with basal MenSC-EVs prominently revealed their immunomodulatory and regenerative capabilities. Furthermore, quantitative proteomic analysis of differentially produced MenSC-EVs provided sufficient evidence for the utility of the differential preconditioning in purpose-tailoring EVs for their therapeutic application: proinflammatory priming enhanced the anti-inflammatory, regenerative and immunomodulatory capacity in the innate response of EVs, physioxia priming also improves tissue regeneration, angiogenesis and their immunomodulatory capacity targeting on the adaptive response, while acute hypoxia priming, increased hemostasis and apoptotic processes regulation in MenSC-EVs, also by stimulating immunomodulation mainly through the adaptive response. CONCLUSIONS: Priming of MenSCs under proinflammatory and hypoxic conditions affected the cargo proteome of EVs released, resulting in different therapeutic potential, and thus warrants experimental exploration with the aim to generate better-defined MSC-derived bioproducts.

Predictors and outcomes of pacemaker implantation in patients with cardiac amyloidosis.

OBJECTIVE: We sought to investigate prevalence, incidence and prognostic implications of permanent pacemaker (PPM) implantation in patients with cardiac amyloidosis (CA), thereby identifying the predictors of time to PPM implantation. METHODS: Seven hundred eighty-seven patients with CA (602 men, median age 74 years, 571 transthyretin amyloidosis (ATTR), 216 light-chain amyloidosis (AL)) evaluated at two European referral centres were retrospectively included. Clinical, laboratory and instrumental data were analysed. The associations between PPM implantation and mortality, heart failure (HF) or a composite endpoint of mortality, cardiac transplantation and HF were analysed. RESULTS: 81 (10.3%) patients had a PPM before initial evaluation. Over a median follow-up time of 21.7 months (IQR 9.6-45.2), 81 (10.3%) additional patients (18 with AL (22.2%) and 63 with ATTR (77.8%)) underwent PPM implantation with a median time to implantation of 15.6 months (IQR 4.2-40), complete atrioventricular block was the most common indication (49.4%). Independent predictors of PPM implantation were QRS duration (HR 1.03, 95% CI 1.02 to 1.03, p<0.001) and interventricular septum (IVS) thickness (HR 1.1, 95% CI 1.03 to 1.17, p=0.003). The model to estimate the probability of PPM at 12 months and containing both factors showed a C-statistic of 0.71 and a calibration of slope of 0.98. CONCLUSIONS: Conduction system disease requiring PPM is a common complication in CA that affects up to 20.6% of patients. QRS duration and IVS thickness are independently associated with PPM implantation. A PPM implantation at 12 months model was devised and validated to identify patients with CA at higher risk of requiring a PPM and who require closer follow-up.

Specific Cellular and Humoral Immune Responses to the Neoantigen RBD of SARS-CoV-2 in Patients with Primary and Secondary Immunodeficiency and Healthy Donors.

Patients with antibody deficiency disorders, such as primary immunodeficiency (PID) or secondary immunodeficiency (SID) to B-cell lymphoproliferative disorder (B-CLPD), are two groups vulnerable to developing the severe or chronic form of coronavirus disease caused by SARS-CoV-2 (COVID-19). The data on adaptive immune responses against SARS-CoV-2 are well described in healthy donors, but still limited in patients with antibody deficiency of a different cause. Herein, we analyzed spike-specific IFN-γ and anti-spike IgG antibody responses at 3 to 6 months after exposure to SARS-CoV-2 derived from vaccination and/or infection in two cohorts of immunodeficient patients (PID vs. SID) compared to healthy controls (HCs). Pre-vaccine anti-SARS-CoV-2 cellular responses before vaccine administration were measured in 10 PID patients. Baseline cellular responses were detectable in 4 out of 10 PID patients who had COVID-19 prior to vaccination, perceiving an increase in cellular responses after two-dose vaccination (p < 0.001). Adequate specific cellular responses were observed in 18 out of 20 (90%) PID patients, in 14 out of 20 (70%) SID patients and in 74 out of 81 (96%) HCs after vaccination (and natural infection in some cases). Specific IFN-γ response was significantly higher in HC with respect to PID (1908.5 mUI/mL vs. 1694.1 mUI/mL; p = 0.005). Whereas all SID and HC patients mounted a specific humoral immune response, only 80% of PID patients showed positive anti-SARS-CoV-2 IgG. The titer of anti-SARS-CoV-2 IgG was significantly lower in SID compared with HC patients (p = 0.040), without significant differences between PID and HC patients (p = 0.123) and between PID and SID patients (p =0.683). High proportions of PID and SID patients showed adequate specific cellular responses to receptor binding domain (RBD) neoantigen, with a divergence between the two arms of the adaptive immune response in PID and SID patients. We also focused on the correlation of protection of positive SARS-CoV-2 cellular response to omicron exposure: 27 out of 81 (33.3%) HCs referred COVID-19 detected by PCR or antigen test, 24 with a mild course, 1 with moderate symptoms and the remaining 2 with bilateral pneumonia that were treated in an outpatient basis. Our results might support the relevance of these immunological studies to determine the correlation of protection with severe disease and for deciding the need for additional boosters on a personalized basis. Follow-up studies are required to evaluate the duration and variability in the immune response to COVID-19 vaccination or infection.

Live birth rates following individualized dosing algorithm of follitropin delta in a long GnRH agonist protocol.

PURPOSE: To explore the efficacy and safety of individualized follitropin delta dosing, based on serum anti-Müllerian hormone (AMH) concentration and bodyweight, in a long gonadotropin-releasing hormone (GnRH) agonist protocol. METHODS: Clinical outcomes after one treatment cycle are reported in women with AMH: 5-35 pmol/L. Oocytes were inseminated by intracytoplasmic sperm injection, blastocyst transfer was on Day 5 and remaining blastocysts were cryopreserved. Data collection included live births and neonatal health follow-up for all fresh/frozen transfers performed within one year after treatment allocation. RESULTS: In total, 104 women started stimulation, of whom 101 had oocyte recovery and 92 had blastocyst transfer. The average daily dose of follitropin delta was 11.0 ± 1.6 µg and the duration of stimulation was 10.3 ± 1.6 days. The mean number of oocytes was 12.5 ± 6.4, the mean number of blastocysts was 5.1 ± 3.4, and 85% had at least one good-quality blastocyst. Following mostly single blastocyst transfer (95%), the ongoing pregnancy rate was 43%, the live-birth rate was 43%, and the cumulative live-birth rate was 58% per started stimulation. There were 6 cases of early OHSS (5.8%) graded as mild (n = 3) and moderate (n = 3) and 6 cases of late OHSS (5.8%) graded as moderate (n = 3) and severe (n = 3). CONCLUSION: In this first evaluation of the individualized follitropin delta dosing in a long GnRH agonist protocol, the cumulative live-birth rate was high. A randomized trial comparing follitropin delta in a long GnRH agonist protocol versus in a GnRH antagonist protocol should provide further insight into the efficacy and safety of this treatment option. TRIAL REGISTRATION NUMBER: NCT03564509; June 21, 2018.

Menstrual blood-derived stromal cells: insights into their secretome in acute hypoxia conditions.

BACKGROUND: Despite constant advances in regenerative medicine, the closure of chronic wounds is still challenging. Therapeutic approaches using locally administered MSCs have been considered a promising option. However, the viability of these cells is seriously threatened by acute hypoxic stress linked to wound healing. In this work, we aimed to study the tolerance of Menstrual blood-derived stromal cells (MenSCs) to acute hypoxia and their therapeutic paracrine effect. METHODS: Isolated MenSCs were phenotypically characterized and evaluated in terms of proliferation, viability, and gene expression, under acute hypoxia (AH) compared with conventional cultured condition or normoxia (N). A step further, the secretome of MenSCs under acute hypoxia was analyzed with respect to their miRNAs content and by in vitro functional assays. For the analysis of differences between the two groups, Student's t-test was performed and one-way ANOVA and Tukey's multiple comparisons test for multiple groups were used. RESULTS: Our results revealed that the viability of MenSCs was not affected under acute hypoxia, although proliferation rate slowed down. Gene analysis revealed 5 up-regulated (BNIP3, ANGPTL4, IL6, IL1B, and PDK1) and 4 down-regulated genes (IDO1, HMOX1, ANGPTL2, and HGF) in AH compared to N. Global gene expression analysis revealed a decrease in the gene ontology functions of migration and wound response with respect to the normoxic condition. In contrast, functions such as angiogenesis were enriched under the AH condition. Regarding the secretome analysis, two miRNAs involved in angiogenic processes (hsa-miR-148a-3p and hsa-miR-378a-3p), were significantly up-expressed when compared to the normoxic condition, being MYC gene, the unique target of both. Functional assays on HUVECs revealed a potential pro-angiogenic capacity of MenSCs cultured in both oxygen conditions (N and AH) based on the wound closure and tube formation results of their released paracrine factors. However, when compared to normoxia, the paracrine factors of MenSCs under acute hypoxia slightly reduced the proliferation, migration, and in vitro wound closure of HUVECs. CONCLUSIONS: MenSC exhibited a good survival capacity under acute hypoxic conditions as well as beneficial properties applicable in the field of tissue regeneration through their secretome, which makes them a potential cell source for wound healing interventions.