Epididymis Response Partly Compensates for Spermatozoa Oxidative Defects in snGPx4 and GPx5 Double Mutant Mice

We report here that spermatozoa of mice lacking both the sperm nucleaus glutathione peroxidase 4 (snGPx4) and the epididymal glutathione peroxidase 5 (GPx5) activities display sperm nucleus structural abnormalities including delayed and defective nuclear compaction, nuclear instability and DNA damage. We show that to counteract the GPx activity losses, the epididymis of the double KO animals mounted an antioxydant response resulting in a strong increase in the global H2O2-scavenger activity especially in the cauda epididymis. Quantitative RT-PCR data show that together with the up-regulation of epididymal scavengers (of the thioredoxin/peroxiredoxin system as well as glutathione-S-transferases) the epididymis of double mutant animals increased the expression of several disulfide isomerases in an attempt to recover normal disulfide-bridging activity. Despite these compensatory mechanisms cauda-stored spermatozoa of double mutant animals show high levels of DNA oxidation, increased fragmentation and greater susceptibility to nuclear decondensation. Nevertheless, the enzymatic epididymal salvage response is sufficient to maintain full fertility of double KO males whatever their age, crossed with young WT female mice

Impact du stress oxidant sur la qualité nucléaire spermatique

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Impact of slow freezing on sperm nuclear quality and telomere length

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La supplémentation en hypotaurine des milieux de préparation et de congélation améliore la qualité fonctionnelle et nucléaire spermatique

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Détection de la maladie résiduelle du sarcome d’Ewing dans le tissu ovarien et testiculaire par une méthode sensible et spécifique

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Prostasomes: inhibitors of capacitation and modulators of cellular signalling in human sperm

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Détection de la maladie résiduelle du sarcome d’Ewing dans le tissu ovarien et testiculaire par une méthode sensible et spécifique

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Quelle est la place de l’automatisation du spermogramme-spermocytogramme dans un laboratoire de Biologie de la Reproduction: performance et validation de méthode de l’automate du SQA-Vision®

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Validation of a SARS-CoV-2 RT-PCR assay: a requirement to evaluate viral contamination in human semen

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An accurate and reliable screening for SARS-CoV-2 in human sperm samples by RT-PCR: A requirement to evaluate the viral contamination risk during SARS-CoV-2 pandemic

International audienceStudy question:How to ensure a reliable and accurate detection of SARS-CoV-2 in seminal plasma and spermatozoa fractions of human sperm samples? Summary answer:This RT-PCR assay showed high sensibility, repeatability and reproducibility for SARS-CoV-2 detection in seminal plasma and spermatozoa fractions, with a detection limit of 17 genomes/reaction. What is known already:SARS-CoV-2 pandemic brings numerous concerns, such as the safety of gametes for patients undergoing assisted reproductive technologies, fertility preservation or sperm donation. Transient viremia and expression of SARS-CoV-2 receptors in testis and accessory glands bring the question of the presence of the virus in sperm samples. Moreover, the contamination during sperm collection may be possible. The few available studies about this issue mostly showed the absence of SARS-CoV-2 detection in semen of COVID-19 patients, except one reported study. All these studies performed SARS-CoV-2 detection with RT-PCR assays approved for naso-pharyngeal swabs, without a process specifically validated for semen fractions. Study design, size, duration:Method validation was conducted between July 2020 and January 2021. SARS-CoV-2 direct detection was performed according to the French Society of Microbiology guidelines (SFM). Repeatability (n=6), reproducibility (n=3), limit of quantification (n=2) and of detection (n=6) were evaluated in seminal plasma (SP) and spermatozoa samples isolated after density gradient centrifugation and cryopreserved. In addition, variability of the whole analytical method efficiency was evaluated in samples of men with normal (n=6) or altered sperm parameters (n=6).Participants/materials, setting, methods:Samples were surplus semen obtained from men undergoing routine semen analysis after granting informed consent. Assays were performed on SP and frozen spermatozoa fractions. After automated RNA extraction (MGISP-960, MGI-Tech®), real-time RT-PCR was performed using the one-step multiplex TaqPath COVID-19 kit (ThermoFisher®) targeting three viral regions (ORF1, nucleocapsid-N and spike-S proteins). An exogenous internal control was added before RNA extraction. Positive samples and dilution ranges were prepared with a standard (SARS-CoV-2 inactivated virus, QnosticTM Randox®). Main results and the role of chance:RT-PCR assay applied for human sperm samples has been previously validated and is routinely used for SARS-CoV-2 detection in naso-pharyngeal swabs. We evaluated the efficiency of RNA extraction and RT-PCR for SARS-CoV-2 detection in semen fractions. The qualitative and quantitative performance of the whole analytical method was validated with an accuracy profile for SP and spermatozoa fractions. Overall, for repeatability, the standard deviation (SD) of the cycle threshold (Ct) was lower than 0.40 for the strong positive sample and 0.50 for the low positive one. An exception was observed for the S target of the low positive SP samples (SD=3) which was consistent with S being the less sensitive target of the assay. For reproducibility, SD of the Ct was lower than 0.30 for the strong positive sample and 0.80 for the low positive, except for the S target of the low positive (SD=1.5). The linearity range was determined for N target, the most sensitive target of the RT-PCR assay. It layed between 5200 and 52 SARS-CoV-2 genomes/reaction. The limit of detection of the RT-PCR assay was 17 viral genomes/reaction. Equal efficiency of the assay was observed for SP and spermatozoa independently of semen parameters (normal and altered sperm parameters). Limitations, reasons for caution:Our detection method was validated for the whole process: RNA extraction (reagents and system), RT-PCR (reagents and thermocycler QuantStudio 5TM) and for both SP and frozen spermatozoa fractions. Variability might be observed with a different extraction system or a different type of biological sample.Wider implications of the findings:This validated RT-PCR assay enables accurate and reliable screening of SARS-CoV-2 in SP and spermatozoa fractions, mandatory to investigate the presence of the virus in semen samples of patients undergoing assisted reproductive techniques, fertility preservation or sperm donation, and to ensure viral safety in the cryobanking process during covid-19 pandemic