A 2-step remote TUNEL approach for sperm DNA fragmentation assessment. Analysis in donors and patients.

OBJECTIVES: Infertility is a disease of the male or female reproductive systems. Male reproductive workup is based on routine semen analysis, although of limited value. The 2021 WHO Manual incorporated Sperm DNA Fragmentation (SDF) assessment, and highlighted the need for individual laboratories to define suitable thresholds. This study aimed to present an alternative to address this issue, determine an SDF cut-off value with fertile donors, and characterize SDF in a patient cohort and their relationship with semen parameters. STUDY DESIGN: A service unit was established to remotely perform TUNEL assay in a 2 step-process. Semen samples were received at andrology laboratories, subjected to routine semen analysis (WHO, 2010), partially processed and transported to the service unit for SDF evaluation. Using this setting, studies were done in fertile donors (n = 15) to define the cut-off value, and in men undergoing infertility workup (n = 318). RESULTS: A cut-off value of 9.17 % was determined with the fertile donor cohort. With this cut-off, a 64.46 % abnormal SDF incidence was determined in the patient cohort. SDF negatively correlated with sperm number, vitality and motility, and positively with abnormal morphology and male age (P < 0.05). TUNEL-positive cases depicted lower sperm quality and higher male age (P < 0.05). A similar abnormal SDF incidence was determined among patients with semen abnormalities. Asthenozoospermic and ≥40 years patient samples depicted higher (P < 0.05) SDF than those of the general population. SDF incidence was also high in normozoospermic patients. CONCLUSIONS: Using a 2-step remote approach with a standardized procedure and an SDF cut-off value established with fertile donors, high SDF incidence in semen samples depicting normal and abnormal quality were identified in men consulting for infertility, highlighting the relevance of its evaluation as part of the male fertility workup.

Impact of heat waves on semen quality: A retrospective study in Argentina between 2005 and 2023.

Heat waves, defined as periods with daily temperatures surpassing the historical average for a specific region, have become more frequent worldwide in recent years. Previous studies have reported a negative association between temperature and semen quality, but the focus has mainly been on Asian and European populations. The study included 54,926 men (18-60 years) undergoing routine semen analysis between 2005 and 2023 at CEUSA-LAEH andrology unit, in Buenos Aires, Argentina. Hourly temperature readings were provided by the Servicio Meteorológico Nacional. R programming (R Studio v2022.07.2) was used to define heat waves, calculate key characteristics, visualize results, and perform statistical tests at the IBYME laboratory. During the period studied, a total of 124 days had heat waves (defined after at least 3 consecutive days with 32.3 °C and 22 °C). Men exposed to heat waves during spermatogenesis exhibited lower sperm number (concentration and count; P < 0.0001) and decreased normal morphology (percentage of normal sperm and normal motile count; P < 0.05) compared to those not exposed. These differences were most pronounced between semen samples from years with several heat waves (2013, 2023) and none (2005, 2007, 2016), displaying 4-5 times higher fold changes (P < 0.05). Further analysis employing multiple regression revealed a significantly negative association between semen quality and heat wave length, suggesting that a prolonged exposure may be more detrimental than an acute exposure. Subsequent analysis focusing on prolonged exposure (≥6-days heat wave) during spermatogenesis revealed a negative (P < 0.05) association between early exposure (spermatocytogenesis: 64-90 days prior semen collection) and semen quality. This study underscores the negative association between early exposure to heat waves during sperm development and semen quality, raising concerns about its possible association with the worldwide declining male fertility. A comprehensive collaborative approach is crucial, involving global governmental policies, sustainable practices, and coordinated efforts across scientific, healthcare, and policy domains.

Glass wool column filtration for stallion sperm selection: A comparative analysis with the single-layer colloid centrifugation.

Glass wool column filtration (GWCF) selects human, bull, boar, dog and buffalo spermatozoa, but reports in the horse are scarce. Single-layer colloid centrifugation with Androcoll-E™ is currently the standard procedure to select good-quality equine sperm. This study was designed to assess GWCF (50 and 75 mg columns; GWCF-50 and GWCF-75, respectively) efficacy to select good-quality sperm from fresh and frozen-thawed equine semen, and to compare its performance with Androcoll-E™ colloid centrifugation. Percentage total motile (TM), progressively motile (PM), morphologically normal (MN), osmotically competent (HOS+) and acrosome-intact/osmotically competent (AI/HOS+) sperm were determined. In studies done with fresh semen samples (n = 17), suspensions subjected to GWCF-50 showed an improvement (p < .05) in PM and HOS+ sperm after selection. With GWCF-75, an increase (p < .05) in PM, MN and HOS+ sperm was observed. Results with GWCF were comparable or better than with Androcoll-E™ selection. Sperm recovery was similar between procedures for all semen parameters. Total sperm count recovery was lower after GWCF-75 (GWCF-50 = 60.0; GWCF-75 = 51.0; Androcoll-E™ = 76.0 million sperm; median; p = .013), but results on total progressive sperm count were similar (GWCF-50 = 23.0; GWCF-75 = 27.0; Androcoll-E™ = 24.0 million sperm; median; p = .3850). Using frozen-thawed semen samples (n = 16), an improvement (p < .05) in TM, PM, NM, HOS+ and AI/HOS+ sperm was observed in GWCF-75 filtrates. Results were comparable to Androcoll-E™ centrifugation, except HOS+ that increased (p < .05) only after GWCF-75. Recovery was comparable for all parameters in frozen samples. GWCF is a simple and low-cost procedure that selects equine sperm with a quality comparable to colloid centrifugation with Androcoll-E™.

Association between meteorological variables and semen quality: a retrospective study.

Spermatogenesis is a temperature-dependent process, and high summer temperatures have been linked to lower sperm concentration and count. However, reports describing the association between other meteorological variables and semen quality are scarce. This study evaluated the association between semen quality and temperature, humidity, pressure, apparent temperature (AT), temperature-humidity index (THI), simplified wet-bulb global temperature (sWBGT), and sunshine duration. Semen samples were obtained at the Laboratorio de Andrología y Reproducción (LAR, Argentina), from men undergoing routine andrology examination (n=11657) and computer-assisted sperm analysis (n=4705) following WHO 2010 criteria. Meteorological variables readings were obtained from the Sistema Meteorológico Nacional. Sperm quality parameters were negatively affected in summer when compared to winter. Additionally, there was a significant decrease in sperm kinematics between winter and spring. Branch and bound variable selection followed by multiple regression analysis revealed a significant association between semen quality and meteorological variables. Specifically, changes in sunshine duration and humidity reinforced the prognosis of semen quality. Highest/lowest sunshine duration and humidity quantiles resulted in decreased sperm concentration, count, motility, vitality and membrane competence, nuclear maturity, and sperm kinematics associated to highest sunshine duration and lowest humidity. Findings from this report highlight the relevance of environmental studies for predicting alterations in male reproductive health associated to variations in meteorological variables, especially considering the current climate changes around the planet due to global warming and its consequences for human health.

Epithelial and Neural Cadherin in Mammalian Fertilization: Studies in the Mouse Model.

Successful mammalian fertilization requires a well-orchestrated sequence of molecular events leading to gamete fusion. Since this interaction involves Ca2+-dependent adhesion events, the participation of the Ca+2-dependent cell-cell adhesion proteins Epithelial (E-cad) and Neural (N-cad) cadherin is envisaged. We have previously reported the expression of E-cad and N-cad in human gametes and showed evidence of their involvement in sperm-oocyte adhesion events leading to fertilization. To overcome ethical limitations associated with the use of human gametes in fertilization-related studies, the mouse has been selected worldwide as the experimental model for over 4 decades. Herein, we report a detailed study aimed at characterizing the expression of E-cad and N-cad in murine gametes and their involvement in murine fertilization using specific antibodies and blocking peptides towards both adhesion proteins. E-cad and N-cad protein forms, as well as other members of the adhesion complex, specifically ß-catenin and actin, were identified in spermatozoa, cumulus cells and oocytes protein extracts by means of Western immunoblotting. In addition, subcellular localization of these proteins was determined in whole cells using optical fluorescent microscopy. Gamete pre-incubation with anti-E-cad (ECCD-1) or N-cad (H-63) antibodies resulted in decreased (p < 0.05) In Vitro Fertilization (IVF) rates, when using both cumulus-oocytes complexes and cumulus-free oocytes. Moreover, IVF assays done with denuded oocytes and either antibodies or blocking peptides against E-cad and N-cad led to lower (p < 0.05) fertilization rates. When assessing each step, penetration of the cumulus mass was lower (p < 0.05) when spermatozoa were pre-incubated with ECCD-1 or blocking peptides towards E-cad or towards both E- and N-cad. Moreover, sperm-oolemma binding was impaired (p < 0.0005) after sperm pre-incubation with E-cad antibody or blocking peptide towards E-cad, N-cad or both proteins. Finally, sperm-oocyte fusion was lower (p < 0.05) after sperm pre-incubation with either antibody or blocking peptide against E-cad or N-cad. Our studies demonstrate the expression of members of the adherent complex in the murine model, and the use of antibodies and specific peptides revealed E-cad and N-cad participation in mammalian fertilization.

Impact of age, clinical conditions, and lifestyle on routine semen parameters and sperm kinematics.

OBJECTIVE: To assess the impact of aging on routine semen and computer-assisted sperm analysis (CASA) motility parameters according to the current World Health Organization guidelines; and to evaluate the effect of obesity and lifestyle (alcohol consumption, cigarette smoking) in older men's semen. DESIGN: Blind cross-sectional study. SETTING: Research laboratory and andrology and reproduction laboratory. PATIENT(S): A population of 11,706 men. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Semen analysis: routine (semen volume, sperm concentration and count, motility, vitality, morphology, hypo-osmotic swelling test, round and peroxidase-positive cell concentration) and CASA (straight-line velocity, curvilinear velocity, average path velocity, linearity, straightness, beat cross frequency, wobble, amplitude of lateral head displacement, and mean angular displacement) parameters; and body mass index. RESULT(S): A negative correlation was found between age and routine semen parameters: volume, sperm count, motility, vitality, total motile spermatozoa and normal-motile spermatozoa, round cell concentration, and hypo-osmotic swelling test values. Several CASA variables (straight-line velocity, curvilinear velocity, average path velocity, beat cross frequency, amplitude of lateral head displacement, and mean angular displacement) were also negatively affected. Using 40 years as a cut-off value, significant differences in most parameters correlated to age. In a selected subpopulation of men unexposed to known fertility-compromising factors, the same evaluations were performed, finding some parameters still decreased. Although obesity exerted a significant deleterious effect on older patients' semen quality, alcohol consumption and cigarette smoking mildly affected it. CONCLUSION(S): Male aging, with the contribution of unhealthy conditions, are paramount effectors of sperm quality deterioration.

Incidence of Sperm Surface Autoantibodies and Relationship with Routine Semen Parameters and Sperm Kinematics.

PROBLEM: Antisperm antibodies (ASA) are associated with male subfertility. However, results on sperm surface autoantibodies are controversial, the relationship between ASA and semen parameters (WHO, 2010) is unknown, and data on ASA and sperm kinematics are scarce. METHOD OF STUDY: A retrospective study carried out in men undergoing routine semen analysis (WHO 2010), ASA evaluation (direct SpermMAR(™) (IgG) test), and computer-assisted sperm analysis (CASA). RESULTS: A 2.6% and a 5.9% incidence of ASA-positive cases were found (cut-off 50% and 10%, respectively; n = 7492). ASA-positive samples had lower (P < 0.0001) sperm concentration, count, motility, and hypo-osmotic swelling (HOS) test score. HOS results did not correlate with sperm vitality in normozoospermic samples with high ASA levels. In unselected samples, ASA-positive samples (cut-off 50%) showed decreased sperm kinematics (VSL, VAP, LIN, ALH, STR, BCF, WOB), but in normozoospermic samples, ASA-positive and ASA-negative subgroups had similar CASA results. CONCLUSIONS: ASA evaluation is highly relevant in full semen assessment.

Tyrosyl-DNA-phosphodiesterase I (TDP1) participates in the removal and repair of stabilized-Top2α cleavage complexes in human cells.

Tyrosyl-DNA-phosphodiesterase 1 (TDP1) is a DNA repair enzyme that removes irreversible protein-linked 3' DNA complexes, 3' phosphoglycolates, alkylation damage-induced DNA breaks, and 3' deoxyribose nucleosides. In addition to its extended spectrum of substrates, TDP1 interacts with several DNA damage response factors. To determine whether TDP1 participates in the repair of topoisomerase II (Top2) induced DNA lesions, we generated TDP1 depleted (TDP1kd) human tumoral cells. We found that TDP1kd cells are hypersensitive to etoposide (ETO). Moreover, we established in a chromatin context that following treatment with ETO, TDP1kd cells accumulate increased amounts of Top2α cleavage complexes, removing them with an altered kinetics. We also showed that TDP1 depleted cells accumulate increased γH2AX and pS296Chk1 signals following treatment with ETO. Similarly, cytogenetics analyses following Top2 poisoning revealed increased amounts of chromatid and chromosome breaks and exchanges on TDP1kd cells in the presence or not of the DNA-PKcs inhibitor NU7026. However, the levels of sister chromatid exchanges were similar in both TDP1kd and control non-silenced cell lines. This suggests a role of TDP1 in both canonical non-homologous end joining and alternative end joining, but not in the homologous recombination repair pathway. Finally, micronucleus analyses following ETO treatment revealed a higher frequency of micronucleus containing γH2AX signals on TDP1kd cells. Together, our results highlight an active role of TDP1 in the repair of Top2-induced DNA damage and its relevance on the genome stability maintenance in human cells.

Falso seudoaneurisma del ventrículo izquierdo / False Left Ventricle Pseudoaneurysm

El ventrículo izquierdo puede presentar abultamientos patológicos, que se denominan aneurisma, seudoaneurisma o divertículo. Los aneurismas son los más frecuentes y su etiología más común es la enfermedad coronaria. Presentamos un paciente de 16 años, derivado al Hospital Escuela de Corrientes con diagnóstico de accidente cerebrovascular. Por eco-Doppler color, resonancia magnética y cinecoronariografía se diagnosticó seudoaneurisma ventricular izquierdo. El examen histológico demostró la presencia de fibras musculares cardíacas, por lo que se concluyó que se trataba de un aneurisma ventricular izquierdo. Esta discordancia entre el diagnóstico por imágenes y la anatomía patológica fue descripta por Mackenzie con el nombre de falso seudoaneurisma.

The left ventricle can show pathological bulges that are called aneurysm, pseudoaneurysm or diverticle. Aneurysms are the most frequent and their most common etiology is coronary disease. We present a 16 years old patient, referred to the Hospital Escuela de Corrientes with diagnosis of stroke. Diagnosis by color Doppler echo, magnetic resonante and cinecoronariography was left ventricle pseudoaneurysm. Histological examination showed the presence of cardiac muscular fibers, thereby confirming left ventricle aneurysm. This disagreement between the imaging and the histological diagnoses was described by Mackenzie et al as "false pseudoaneurysm".