INTRACELLULAR SARS-COV-2 FOUNDIN THE SPERMATOZOA OF CONVALESCENT MEN WITH NEGATIVE PCR SEMEN SAMPLES REVEALED BY ELECTRON MICROSCOPY

INTRODUCTION AND OBJECTIVE: SARS-CoV-2 can invade different testicular cell types, such as spermatogonia, spermatids, Sertoli, and Leydig cells. We investigated the viral presence inside the sperm of negative PCR infected men up to 3 months after discharge from the hospital. METHODS: This cross-sectional study included 13 of a 26 moderate-to-severe SARS-CoV-2 infected men cohort (mean 34.3 ± 6.5 years;range: 21-50 years old). Patients were enrolled 30 to 90 days after the diagnosis. Semen samples were obtained by masturbation and processed within one hour according to WHO guidelines. All patients were PCR negative for the virus in the ejaculate. Samples were liquefied for 30 min at room temperature in 0.1M phosphate buffer before centrifuging at 500 g for 10 min. The supernatant was removed, and pellets were fixed in 2,5% v/v glutaraldehyde in 0.1M phosphate buffer for 2h at 4°C, post-fixed in 1% OsO4 for 1h at 4°C, stained overnight in 1% aqueous uranyl acetate. Then, the pellets were dehydrated sequentially in 30%, 70%, and 100% ethanol and embedded in epoxy resin. Ultrathin sections (70nm) were obtained in an ultramicrotome, collected on nickel grids, and double-stained by uranyl acetate and lead citrate. Micrographs were obtained with a Jeol JEM 1010 electron microscope (Tokyo, Japan, 80 kV). RESULTS: We identified viruses inside spermatozoa in 9/13 patients up to 90 days after discharge from the hospital. Moreover, in all 13 men, a type of DNA-based extracellular traps, probably in a cfDNAdependent manner, like described in the COVID-19 systemic inflammatory response. FIGURE: High magnification electron micrograph of a spermatozoon with the nucleus (nu) displaying the typical condensed chromatin. The remained cytoplasm contains several viral particles (ranging in diameter from 90 to 110 nm). The inset corresponds to a higher magnification of the boxed area containing two virions, showing the SARS-CoV-2 characteristics: viral envelope (white arrowhead), nucleocapsids (black arrowhead), and spike-like projections (white arrow). CONCLUSIONS: Although SARS-CoV-2 is not found in the infected men's semen, it was intracellularly present in the spermatozoa. The potential implications for assisted conception should be addressed. (Figure Presented).

ELEVATED ROS LEVELS AND DNA FRAGMENTATION IN SPERM FROM CONVALESCENT MEN IN SARS-COV-2 INFECTION

INTRODUCTION AND OBJECTIVE: Current evidence has proven the systemic nature of COVID19, including its involvement in the male reproductive tract. We aimed to investigate seminal parameters of moderate-to-severe COVID-19 men during the convalescence phase. METHODS: This cross-sectional study included 18 to 50-yearold men with confirmed moderate-to-severe COVID-19. Patients were enrolled 15 to 45 days after the diagnosis. After a urologist's initial clinical evaluation, semen samples were obtained by masturbation and processed within one hour. Semen analysis was performed using the World Health Organization (WHO) manual (6th edition). Sperm function tests were conducted in an andrology laboratory, including Reactive oxygen species (ROS), DNA fragmentation, lipid peroxidation, and Creatine Kinase (CK) analysis. An essential endocrine evaluation was performed. Patients with a history of disorders that could impair testicular function were excluded. A group of pre-vasectomy baseline samples was used as a control group. Statistical analysis was performed using R version 4.0.5. One-tailed and paired T-tests were used for comparisons between groups. RESULTS: The sample size was 26 men (mean 34.3±6.5 years;range: 21-50 years). Sperm concentration (mean 38.74±32, P <0.01) and total motile count (mean 55.3±66.8, P <0.01) were significantly reduced in the COVID-19 group. The DNA fragmentation (mean 41.1±29.2) and ROS (mean 4.84±8.7) were significantly higher in post-infection patients. Other parameters such as WHO/ Kruger morphology and progressive motility were also reduced in the disease group, albeit not statistically significant. Total testosterone (mean 409.2±201.2) was lower in the convalescent men. All semen samples were negative for SARS-CoV-2 using the PCR analysis. CONCLUSIONS: Our findings indicate that male reproductive injury can be a relevant component of SARS-CoV-2 systemic infection. High DNA fragmentation and ROS, hallmarks of tissue injury, might signal a direct testicular involvement. The morphological and functional damage could represent significant impairment of the male reproductive health if persistent after convalescence.

CRYOPRESERVED SEMINAL SAMPLES FROM PATIENTS WITH ACUTE COVID-19 HAVE THE WORST POST-THAW QUALITY WHEN COMPARED TO OTHER ANDROLOGICAL DISEASES

OBJECTIVE: To evaluate the effect of COVID-19 in sperm cryopreservation processes, including functional parameters evaluated pre-cryopreservation and post-thaw, and to compare post-thaw results from COVID-19 patients to samples from others systemic and andrological Disease MATERIALS AND METHODS: In this cross-sectional study, 37 semen samples of male patients aged 18 to 45 years at Division of Urology, Department of Surgery, Hospital das Clinicas of the University of Sao Paulo or at Androscience- Science and Innovation Center in Andrology, High-Complex Clinical and Research Andrology Laboratory, were initially recruited from April 2020 to April 2021. Patients were categorized as acute COVID-19 (n=15), confirmed by RT-PCR (COVID-19 group), and healthy individuals with normozoospermic semen samples (n=22;Control group). Were evaluated seminal parameters, cryosurvival rates (%), mitochondrial activity (%;3,30 -diaminobenzidine stain), reactive oxygen species levels (ROS;chemiluminescent technique) and DNA fragmentation (%;SCSA method) in precryopreservation and post-thaw samples. Samples were cryopreserved by the slow freezing technique. A complementary retrospective study was performed comparing post-thawed samples from COVID-19 group with data from patients with others male diseases: Male infertility (n=35);Severe infertility (n=62), caused severe oligozoospermia, grade 3 varicocele, gonadal dysgenesis, testicular nodule, testicular hypotrophy;testicular cancer (n=55);and other malignant diseases (leukemia, lymphoma, sarcoma, multiple myeloma;n=30). Was used T-test to statistical analysis (p<0.05). RESULTS: Macroscopy analysis of COVID-group revealed abnormal viscosity in 53.33%, semen volume = 4.50 ± 1.72 ml and pH = 8.13 ± 0.23. COVID-19 fresh samples demonstrated mean of progressive motility = 29.07±16.83%, sperm morphology = 2.07±1.58%, and DNA fragmentation index = 42.91±33.38%. Cryopreservation decreased progressive motility (to 5.39±7.92%;p=0.02), sperm vitality (70.46±8.50 vs. 72.20±23.27;p=0.042) and ROS (0.516±0.978 vs. 4.393±9.956 x 104 cpm;p=0.018). When we compared with cryopreserved normozoospermic samples, there was observed a significant difference in HDS (p=0.002). Cryosurvival rate from COVID-19 samples was 19.93;19.71%, and had significant difference when compared with severe infertility (40.16;31.05%;p=0.003), and other malignant diseases (53.14;28.55%, <0.001). CONCLUSIONS: Seminal samples from patients with COVID-19 showed reduced fertile potential, especially when compared to the reference values. In the comparisons performed with samples from patients with different andrological diagnoses, common in the specialized andrology laboratory routine, we can suggest that samples from patients with the acute form of COVID-19 had the worst quality, with low cryosurvival rates. This information contribute to the conduct of these patients during assisted reproduction routines and preservation of male fertility. IMPACT STATEMENT: It will contribute to conducts in the cryopreservation of sperm in patients with acute COVID-19.

The European economic recovery plan, a historic breakthrough: Sustainability without funding is unsustainable

While the Covid-19 pandemic continues to rage in Europe, complicated by the appearance of new variants forcing many countries to lock down their populations again and restrict economic activity, the prospects for surmounting the health - and consequent economic - crisis are growing bleaker. Unlike what happened in the previous crisis of 2008, the European Union has, very fortunately, prioritized solidarity in its attempt to deal with that crisis by adopting a European Recovery Plan. As Catherine Vieilledent and Jean-Francois Drevet see it, this represents a historic breakthrough: at last the EU is providing itself with the necessary funds as an economic and political entity. This article describes the content of the Recovery Plan, the various instruments mobilized to enable the member states to sustain and revive their economies, and shows how it marks a major change in the history of the building of the European Community. With a common budget unblocked, the possibility of borrowing, and the return to using its own resources, the EU is changing its scope and affording itself some powerful long-term tools for action. This is major progress, attesting concretely to the Union's usefulness to its citizens, which could give the whole European project a new chance to reposition itself on the world stage. © 2021 Futuribles. All rights reserved.

2020, a good year for Europe?

In this first European column written in 2021 (the January-February issue having hit the newsstands in late December, the previous one went to press in late November), Jean-Francois Drevet offers his assessment of what the year 2020 brought for the European Union. Though it was a year marked by the Covid-19 crisis and Brexit, paradoxically the EU did see some advances. Drevet's column details these in four main areas: the Economic Recovery Plan, the negotiations on the UK's exit from the EU, the management of migration issues, and security policy. On the first two, the Union was able, most encouragingly, to present a strong, determined face. By contrast, progress on the last two was more laboured, though these are areas not to be neglected if the EU's image on the global stage is to be burnished and its position strengthened. © 2021 Futuribles. All rights reserved.

The Grasshopper, the Ant and European Indebtedness

The hard-won European agreement of 21 July underwriting the principle of a joint debt package is an important event in the history of European construction. In response to the economic crisis produced by the Covid-19 pandemic, the member states eventually agreed to a collective recovery plan. While politically essential, the agreement nonetheless raises many concerns and questions about the way the joint loan will eventually be repaid. After decades of tight fiscal discipline, closely overseen by Germany, many EU member states will probably allow their indebtedness to rise, in order to limit the social damage. This is worrying but, re-reading the economic history of the continent, as Jean-François Drevet does in this column, we can see that states (including Germany) have defaulted on their debts for many years now and have always found solutions that averted disaster. This look back at history is meant to be reassuring, but also stimulating: given so much past ingenuity, we should go forward and invest the money in this debt package without too many reservations. © 2020 Futuribles. All rights reserved.