Evaluation of Sperm DNA Fragmentation Using Two Different Methods: TUNEL via Fluorescence Microscopy, and Flow Cytometry.

Background and Objectives: Sperm DNA fragmentation refers to any break in one or both of the strands of DNA in the head of a sperm. The most widely used methodologies for assessing sperm DNA fragmentation are the sperm chromatin structure assay (SCSA), the sperm chromatin dispersion assay (SCD), the single-cell gel electrophoresis assay (SCGE-comet), and the terminal-deoxynucleotidyl-transferase (TdT)-mediated dUTP nick end labelling (TUNEL) assay. The aim of this study was to compare the efficiency and sensitivity of the analysis of sperm DNA fragmentation using TUNEL via fluorescence microscopy, and flow cytometry. Materials and Methods: Semen samples were collected and analyzed for standard characteristics using light microscopy, and for sperm DNA fragmentation using both TUNEL via fluorescence microscopy, and flow cytometry. Results: There were no significant differences in the values of the sperm DNA fragmentation index (DFI) obtained when the analysis was performed using TUNEL or flow cytometry (p = 0.543). Spearman's correlation analysis revealed a significant negative correlation between sperm motility (%) and sperm DNA fragmentation (p < 0.01), as well as between sperm concentration and sperm DNA fragmentation (p < 0.05). The Mann-Whitney U test showed no significant difference in the DFI among couples with repeated implantation failure (RIF) and miscarriages (p = 0.352). Conclusions: Both methods (TUNEL via fluorescence microscopy, and flow cytometry) have a high efficiency and sensitivity in accurately detecting sperm DNA fragmentation, and can be effectively used to assess male fertility.

Sepsis-induced coagulopathy in preterm neonates with Gram-positive sepsis presents with hypercoagulation and reduced platelet activation compared with healthy preterm neonates.

Background: Neonatal sepsis is frequently accompanied by coagulopathy and thrombocytopenia attributed to the cross-link between inflammation and coagulation. However, sepsis-induced coagulopathy and platelet function in septic preterm neonates remain to be elucidated. In addition, there is no robust evidence for a causal relationship between thrombocytopenia and bleeding in preterm neonates with sepsis. Objective: This single-center prospective cohort study aimed to assess sepsis-induced coagulopathy and platelet function in preterm neonates during sepsis. Methods: We included 25 preterm neonates with Gram-positive sepsis born at gestational age 24 + 1 to 34 + 3 and studied in comparison to 30 healthy counterparts. Coagulation was assessed using conventional coagulation tests (CCTs) and rotational thromboelastometry (ROTEM). Platelet function was evaluated by flow cytometry. The study was conducted at 3-time points, at 1st, at 2nd to 3rd, and at 5th to 7th day of sepsis, respectively. Results: Compared with healthy controls, neonates with Gram-positive sepsis present in ROTEM a hypercoagulable state; a higher maximum clot firmness (MCF) and higher amplitudes of intrinsic rotational thromboelastometry (INTEM) (INTEM MCF: median, 71; P .004 and INTEM A10: median, 67; P .005, respectively), extrinsic rotational thromboelastometry (EXTEM) (EXTEM MCF: median, 70; P .02 and EXTEM A10: median, 67; P .02, respectively), and rotational thromboelastometry assay for fibrin formation (FIBTEM) (FIBTEM MCF: median, 25; P < .001 and FIBTEM A10: median, 23; P .002, respectively). Conversely, CCTs exhibited hypocoagulation. Thrombocytopenia in preterm neonates with Gram-positive sepsis is not associated with an increased bleeding risk. In Gram-positive sepsis, platelets display increased glycoprotein (GP) surface receptors' expression (GPIb: median, 2.8; P .03, GPIIb: median, 3.1; P .004, and GPIIIa: median, 3.9; P .008, respectively) and reduced activation (P-selectin: median, 1; P < .001). A higher expression of platelets GP and improved degranulation capacity were recorded in patients in higher gestational age groups of >32 weeks of gestation. Platelet GPIb expression is age-dependent in healthy neonates. Conclusion: Neonatal Gram-positive sepsis is characterized by a progressive hypercoagulation along with increased GP expression, reduced platelet activation, and thrombocytopenia without bleeding. Platelet GP expression and degranulation capacity are age-dependent among neonates with sepsis. Platelet GP expression is age-dependent among healthy counterparts.

Differences among Indian and European pemphigus patients based on demographics, clinical parameters and propensity for relapse: results of a prospective bicontinental cohort study.

Limited studies have explored pemphigus variations among different ethnic groups residing in their respective geographical locations. This bicontinental study aimed to compare clinical and immunological parameters in Indian and European pemphigus patients in complete remission, off therapy, or on minimal therapy. 105 patients (India, n= 75; Bulgaria, n=15; Greece, n=15) with pemphigus vulgaris (PV) or pemphigus foliaceous (PF) in complete remission on minimal therapy (n=64) or complete remission off therapy (n=41) were recruited. Demographic, clinical, and immunological parameters were compared. Indian patients were significantly younger, the maximal disease severity during the preceding active disease phase was significantly lower, and treatment duration until complete remission was significantly shorter, compared to European patients. European patients had significantly higher anti-Dsg3 serum levels and higher IgG positivity rate based on direct immunofluorescence microscopy at baseline. Furthermore, European patients revealed higher CD19, CD19+ CD27+ cell counts, compared with patients from India. Of note, none of the European patients (n=30) relapsed within the study period, in contrast to 29/75 (38.6%) Indian patients. Treatment strategies differed significantly between the two cohorts, with more frequent utilization of rituximab to achieve remission in the Indian cohort, while prednisolone was more widely used for maintaining remission in the European cohort. The observed heterogeneity of pemphigus among patients of different ethnicities in terms of demographics, clinical parameters, and propensity for relapse may be due to genetic background or different treatment strategies.

Effects of Different Drug Therapies and COVID-19 mRNA Vaccination on Semen Quality in a Man with Ankylosing Spondylitis: A Case Report.

Background and Objectives: Ankylosing spondylitis (AS) is a condition that affects 0.1% to 0.5% of the adult population. The aim of this case report was to investigate the possible effects of the drugs taken for treatment of AS as well as mRNA vaccination for COVID-19 on semen quality by performing a highly detailed analysis. Materials and Methods: Sperm characteristics were examined by light microscopy, DNA fragmentation (DFI) was analysed by flow cytometry and morphology was evaluated by transmission electron microscopy (TEM). Results: Semen analysis under therapy with (1) celecoxib and sulphasalazine showed: concentration 47 million/mL, 53% progressive motility, 7% normal morphology and 9.6% DFI, (2) Golimumab and before mRNA Vaccination showed: concentration 108 million/mL, 82% progressive motility, 1% normal morphology and 7.6% DFI, and (3) Golimumab and after 3 doses of mRNA Vaccination showed: concentration 142 million/mL, 85% progressive motility, 1% normal morphology and 6.8% DFI. TEM revealed head, neck and tail abnormalities, as well as the presence of cells with incomplete spermiogenesis white cells and phagocytes in the sample under therapy with celecoxib and sulphasalazine. Golimumab treatment lead to an increased incidence of elongated heads but in general reduced inflammation as no white cells were evident in TEM. Conclusion: The anti-inflamatory drugs celecoxib and sulphasalazine had no adverse effect on sperm quality as all parameters were within normal limits and the patient achieved under that treatment 2 pregnancies following natural conception that lead to the birth of a healthy boy and girl respectively. Anti-TNFa treatment with Golimumab exerted a negative effect on morphology but not on concentration, motility and DFI. After 3 doses of mRNA Vaccination, sperm concentration increased while motility, morphology and DFI remained similar to the values before vaccination suggesting no negative effect of the mRNA vaccine for COVID-19 on sperm quality.

Altered Expression of TLR2 and TLR4 on Peripheral CD14+ Blood Monocytes in Children with Urinary Tract Infection.

Urinary tract infection (UTI) is the second most common bacterial infection, after otitis media, in infants and children. The mechanisms of disease susceptibility and the role of immunity in the pathogenesis of UTI in children have been evaluated. In recent years, Toll-Like Receptors (TLRs) have been recognized as specific components of the innate immune system constituting important mediators in host immune recognition. The aim of the present study was to determine ΤLR2 and TLR4 expression during the acute phase of UTI in infants and children by measuring the CD14/TLR2 and CD14/TLR4 expression on monocytes. We also attempted to compare the TLRs expression with the immunological status of the patients to healthy children. The study group consisted of 60 children (36 females and 24 males) and the control group included 60 age-matched pediatric subjects (27 females and 33 males). In our study, no antibody deficiency was found either in the children with UTI or in healthy subjects. There might be a connection between low IgA, IgG, and IgG subclasses serum levels and UTI as there was a statistically significant difference between patients and healthy children. A higher expression of CD14/TLR2 was revealed in patients (90,07%) compared to controls (85,48%) as well as CD14/TLR4 in patients (90,53%) compared to controls (87,25%) (statistically significant difference, p < 0,05). The results of this study could provide new understanding of UTIs' pathogenesis in children.

Cardiac complications and immunophenotypic profile of infectious mononucleosis syndrome in children.

OBJECTIVE: To investigate cardiac complications in infectious mononucleosis patients and to associate them with biochemical and immunological parameters, as well as with spleen ultrasound findings. DESIGN: Cross-sectional study with follow-up. SETTING: Tertiary care pediatric unit, in the city of Thessaloniki, Greece. PARTICIPANTS AND INTERVENTIONS: Twenty-five children (15 boys, aged 1-11.6 years) suffering from infectious mononucleosis were studied during the acute phase and after 3-6 months. Cardiac evaluation comprised of electrocardiogram, echocardiogram, and measurement of creatine phosphokinase, creatine phosphokinase cardiac isoenzyme, and troponin levels. Biochemical and immunological tests included serum transaminases, serum amylase, CD3+/CD8+ T-lymphocytes subpopulation and CD4+/CD8+ T-lymphocytes ratio. RESULTS: During acute phase, all children had splenomegaly and normal serum amylase values. 17 patients had elevated serum transaminases. Percentages of CD3+/CD8+ T-lymphocytes subpopulation were elevated and CD4+/CD8+ ratio was decreased in all patients. Echocardiography revealed mild pericardial effusion in 13 patients (10/21 with Epstein-Barr infection, 3/4 with cytomegalovirus infection), but none presented with myocarditis. Four out of these 13 patients also had markedly elevated liver enzymes, 10/13 had significant splenomegaly and 12/13 presented very low CD4+/CD8+ T-lymphocytes ratio. Pericardial effusion demonstrated a statistically significant association solely with very low CD4+/CD8+ T-lymphocytes ratio (<0.5). Repetition of laboratory tests 3-6 months post-discharge detected persistent mild pericardial effusion in five patients, along with decreased CD4+/CD8+ ratio in 1/5. CONCLUSIONS: In infectious mononucleosis syndrome, asymptomatic pericardial effusion could be associated with very low CD4+/CD8+ ratio (<0.5). Further studies would extend and confirm such an association.

Regulatory Τ-cell differentiation between maternal and cord blood samples in pregnancies with spontaneous vaginal delivery and with elective cesarian section.

PROBLEM: The immunological mechanisms preventing fetal antigenic rejection during normal pregnancy and the extent to which the type of delivery influences lymphocyte reactions are elusive. METHOD OF STUDY: Maternal peripheral blood and neonatal umbilical cord blood (CB) was collected upon labor after vaginal delivery or cesarian section. Leukocytes were analyzed with flow cytometry, focusing on regulatory and γ/δ T-cells. RESULTS: In CB from neonates delivered by vaginal delivery, natural killer cells were increased. On the other hand, in maternal blood, γ/δ T-cells were increased, and activated T-cells (cluster of differentiation [CD]4+/25(dim) /122+ cells) were decreased. Moreover, maternal blood presented increased levels of T regulatory cell subsets like CD4+/25(high) /45RO+, CD4+/25(high) /DR+, CD4+/25(high) /CD38+ and CD4+/25(high) /71+. In CB, CD19+, CD4+/25(high) /45RA+ and CD4+/25(high) /122+ cells were increased. CONCLUSION: The effect of delivery type on lymphocyte immunophenotype was minimal. Mothers' and neonates' lymphocyte subsets differed significantly. Mothers' phenotype comprised significantly of lymphocytes involved in tolerance (memory and activated regulatory T-cells, γ/δ T-cells).

CD4/CD8 T-cell ratio in peritoneal dialysis effluents predicts the outcome of peritonitis in patients undergoing continuous ambulatory peritoneal dialysis.

This study aimed to clarify the role of peritoneal T-lymphocytes in peritoneal immune defense mechanisms. This study was designed to examine the changes in T-cell subpopulations during peritonitis in patients treated with continuous ambulatory peritoneal dialysis (CAPD). Our observations were correlated to responses to treatment and with outcomes. The present study was carried out in 20 patients (8 males, 12 females) under CAPD. Peritonitis was diagnosed according to the criteria defined by the Ad Hoc Advisory Committee on Peritonitis Management. Peritoneal dialysate effluent (PDE) samples were collected from our patients, and lymphocyte subsets (CD2+, CD3+, CD3+/4+, CD3+/8+, CD3-/16+56+, CD4/CD8 ratio) were quantitated by using monoclonal antibodies. CD4/CD8 ratio was measured every day during peritonitis until the patients had completely recovered. The serial measurements of the CD4/CD8 ratio made in the PDE during peritonitis followed two patterns: the first pattern was characterized by a progressive increase in the CD4/CD8 ratio. The CD4/CD8 ratios on days 5, 6, and 7 were significantly higher than those on day 1 (P < 0.05). Overall, the patients who exhibited pattern 1 had favorable clinical courses. The second pattern was characterized by high initial CD4/CD8 ratios, which progressively decreased significantly (P < 0.05). This second pattern was associated with a delayed clinical response to treatment. Symptoms and signs of peritonitis persisted beyond 72 h. The pattern of the CD4/CD8 ratio in PDE may determine the outcome of peritonitis in CAPD patients.

Adiponectin and insulin resistance in childhood obesity.

OBJECTIVES: To measure adiponectin serum levels in Greek children and adolescents and correlate them with body fat and insulin resistance. PATIENTS AND METHODS: Forty-six obese prepubertal children (19 M, 27 F) and 34 obese adolescents (17 M, 17 F) ages 9.33 +/- 1.57 and 13.6 +/- 1.42 years, respectively, and 43 matched control individuals were studied. Body mass index standard deviation score and percent body fat were measured by bioelectric impedance analysis. Fasting indices of insulin resistance (HOMA-IR and fasting glucose-to-insulin ratio) were calculated for all participants. Indices of insulin resistance derived from oral glucose tolerance tests were estimated in obese participants. Adiponectin was measured by enzyme-linked immunosorbent assay. RESULTS: (MEAN +/- SD):: Adiponectin serum levels were significantly lower in obese participants than in nonobese participants (8.11 +/- 3.80 vs 11.81 +/- 4.98 microg/mL, P < 0.001), in obese children than in nonobese children (8.86 +/- 3.86 vs 13.08 +/- 5.48 microg/mL, P < 0.001), in obese adolescents than in nonobese adolescents (7.04 +/- 3.43 vs 10.47 +/- 4.10 microg/mL, P = 0.002), and in obese adolescent boys than in obese adolescent girls (5.87 +/- 3.52 vs 8.31 +/- 3.16 microg/mL, P = 0.042). There were significant correlations between adiponectin and age, body mass index, body mass index standard deviation score, homeostasis model assessment for insulin resistance, and fasting glucose-to-insulin ratio. Adiponectin correlated with percent body fat after adjustment for sex. Adiponectin correlated significantly with several indices of insulin resistance, such as the areas under the curves for glucose and insulin, whole-body insulin sensitivity index, glucose 120', and insulin 30', in obese participants. CONCLUSIONS: Adiponectin was significantly lower in obese participants than in nonobese participants in general, and it correlated significantly with fasting indices of insulin resistance and with indices derived from oral glucose tolerance tests. It is worthwhile to further investigate the option of applying a simple measurement of serum adiponectin as a screening tool before applying more time-consuming techniques in young obese individuals.

Candidal overgrowth in diabetic patients: potential predisposing factors.

This study was designed to investigate the potential factors that influence the prevalence of the oral carriage of Candida species in patients with type II diabetes mellitus. One hundred and twenty-eight diabetic patients (68 males and 60 females, mean age 54 +/- 7 years) were sequentially enrolled along with 84 (44 males and 40 females mean age 52 +/- 8 years) healthy subjects. Samples were obtained by swabbing the oral mucosa of all participants. Yeast isolates were identified by germ tube test, with API 32 ID system, and by chlamydospore production on 'cornmeal' Tween-80 agar. Candida spp. was recovered from the oral cavity of 64% of the diabetic group, in contrast to 40% of the control group. Candida albicans was the most frequently isolated species in both groups. Potential etiologic factors such as xerostomia, dentures, age, gender and diabetes on oral carriage of Candida spp. were evaluated. The oral carriage of Candida spp. was significantly higher in 'diabetic' patients compared with the healthy subjects but it seems that parameters such as xerostomia, dentures, age, gender and glycemic control cannot be directly associated with Candida growth in the oral cavity in the presence of diabetes.

Comparison of salivary IgA and systemic IgA and IgG antibodies to Saccharomyces cerevisiae in HIV-infected subjects.

Our objective was to investigate the concentrations of IgA and IgG antibodies to Saccharomyces cerevisiae in whole saliva and serum samples from HIV-infected patients and to compare them with the corresponding antibody values of healthy controls. A cross-sectional design was used. The test group consisted of 23 HIV-infected male individuals, aged 20-41 years old, free of any other systemic disease. Twenty healthy subjects aged 27-43 years old served as controls. Whole unstimulated saliva and blood were collected from all subjects. IgA concentrations in saliva and IgA and IgG concentrations in serum were measured by solid-phase enzyme-linked immunosorbent assay. Salivary antibody concentrations were calculated by reference to a pooled standard saliva obtained from 10 healthy males with high levels of anti-S. cerevisiae antibody activity. Total IgA and IgG concentrations were measured by nephelometry / tholocymetry assay. No significant difference was observed in salivary specific IgA and serum specific IgG levels to S. cerevisiae, while serum specific IgA were significantly lower in HIV infected patients compared to control group. Opportunistic infections due to S. cerevisiae, although rare, cannot be dismissed. This yeast can show a potential virulence in debilitated patients, therefore, further extensive investigation should be considered.