Oxidative Stress Markers and Sperm DNA Fragmentation in Men Recovered from COVID-19.

SARS-CoV-2 negatively affects semen characteristics, impairs various biochemical processes in seminal fluid and within spermatogenic cells ultimately leading to male fertility decline. However, the distinct mechanisms, in particular, the role of oxidative stress on the consequences of coronavirus infection, have not been well investigated, which is the purpose of the present study. The standard semen parameters, its pro- and antioxidant system state, as well as the level of sperm DNA fragmentation, were assessed in 17 semen samples of men five months after the coronavirus infection and in 22 age-matched control patients. We determined that the DNA fragmentation rate negatively correlated with the period after coronavirus recovery, as well as seminal fluid superoxide dismutase activity and uric acid level. It was demonstrated that COVID-19 is not always associated with increased DNA fragmentation, allowing them to be considered as two independent factors. Thus, the most significant changes were noted in the samples of men after COVID-19 and abnormal TUNEL results: increased round cell number, decreased seminal fluid's nitrotyrosine level, and total antioxidant capacity and Zn, as well as an increased 8-hydroxy-2'-deoxyguanosine level within spermatozoa. The data obtained indicate that increased DNA fragmentation and diminished semen quality in men can be the result of an imbalance in semen pro- and antioxidant components after COVID-19.

Cellular and Molecular Mechanisms of In Vivo and In Vitro SARS-CoV-2 Infection: A Lesson from Human Sperm.

Despite the major target of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, being the respiratory system, clinical evidence suggests that the male reproductive system may represent another viral target organ. Revealing the effect of SARS-CoV-2 infection on testis and sperm is a priority for reproductive biology, as well as for reproductive medicine. Here, we confirmed that the SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2) is highly expressed on human testis and ejaculated sperm; moreover, we provide evidence for the expression of the co-receptors transmembrane protease/serine (TMPRSS2), Basigin (BSG), and Catepsin L (CTSL). Human sperm were readily infected, both in vivo and in vitro, by SARS-CoV-2, as demonstrated by confocal and electron microscopy. The demonstration that the seminiferous epithelium and sperm support SARS-CoV-2 viral replication suggests the possibility that the spermatogenetic process may be detrimentally affected by the virus, and at the same time, supports the need to implement safety measures and guidelines to ensure specific care in reproductive medicine.

ACE2 Receptor and Its Isoform Short-ACE2 Are Expressed on Human Spermatozoa.

Angiotensin-converting enzyme 2 (ACE2) is a protein widely expressed in numerous cell types, with different biological roles mainly related to the renin-angiotensin system. Recently, ACE2 has been in the spotlight due to its involvement in the SARS-CoV-2 entry into cells. There are no data available regarding the expression of ACE2 and its short-ACE2 isoform at the protein level on human spermatozoa. Here, protein expression was demonstrated by western blot and the percentage of sperm displaying surface ACE2 was assessed by flow cytometry. Immunocytochemistry assays showed that full-length ACE2 was mainly expressed in sperm midpiece, while short ACE2 was preferentially distributed on the equatorial and post-acrosomal region of the sperm head. To our knowledge, this is the first study demonstrating the expression of protein ACE2 on spermatozoa. Further studies are warranted to determine the role of ACE2 isoforms in male reproduction.

PAMAM-calix-dendrimers: Synthesis and Thiacalixarene Conformation Effect on DNA Binding.

A convenient method for the synthesis of the first generation PAMAM dendrimers based on the thiacalix[4]arene has been developed for the first time. Three new PAMAM-calix-dendrimers with the macrocyclic core in cone, partial cone, and 1,3-alternate conformations were obtained with high yields. The interaction of the obtained compounds with salmon sperm DNA resulted in the formation of the associates of the size up to 200 nm, as shown by the UV-Vis spectroscopy, DLS, and TEM. It was demonstrated by the CD method that the structure of the DNA did not undergo significant changes upon binding. The PAMAM-calix-dendrimer based on the macrocycle in cone conformation stabilized DNA and prevented its degradation.

Effects of moderate COVID-19 infection on semen oxidative status and parameters 14 and 120 days after diagnosis.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus causing coronavirus disease 2019 (COVID-19). Because COVID-19 is a multisystem infection, there are some concerns regarding its possible effects on male fertility. This study aimed to investigate the effects of COVID-19 on semen oxidative status and parameters 14 and 120 days after diagnosis in patients presenting with moderate infection (defined as respiratory symptoms, with or without fever, with Spo2 <93% and >90% and lung involvement <50%). Semen samples were obtained from 20 participants at two time points: the first sample on Day 14 and the second on Day 120 after diagnosis. Semen parameters (sperm concentration, motility, morphology, and viability) were evaluated, as were levels of seminal reactive oxygen species (ROS), malondialdehyde (MDA), total antioxidant capacity (TAC) and sperm DNA fragmentation. Semen parameters, including sperm motility and DNA integrity, improved at 120 days after the COVID-19 diagnosis relative to values at 14 days. In addition, ROS and MDA levels were significantly reduced in patients 120 days after infection, and TAC increased at 120 days compared with 14 days (during the acute stage of infection). In conclusion, the present study shows that the detrimental effects of COVID-19 on sperm properties caused by oxidative stress decrease up to Day 120 after diagnosis.

COVID-19 and male reproductive function: a prospective, longitudinal cohort study.

The existing evidence suggests that the human reproductive system may be potentially vulnerable to COVID-19 infection. However, little is known about the virus-host interaction of COVID-19 in sperm cells. We are the first to address the connection between changes in multiple seminal biomarkers and reproductive function in male patients recovering from COVID-19. In a prospective longitudinal cohort study, seminal ACE2 activity, markers of inflammation and oxidative stress, apoptotic variables, and semen quality parameters were evaluated at 10-day intervals for a maximum follow-up time of 60 days among male patients with laboratory-confirmed COVID-19 (n = 84) and healthy controls (CON; n = 105). At the baseline and the subsequent follow-ups, the COVID-19 group revealed significantly higher levels of seminal plasma ACE2 enzymatic activity, IL-1ß, IL-6, IL-8, IL-10, TGF-ß, TNF-α, IFN-α, IFN-γ, ROS, caspase-8, caspase-9, and caspase-3 activity as well as lower levels of SOD activity than those in the CON group (P < 0.05). These perturbations tended to persist over time and were correlated with significant impairments in semen volume, progressive motility, sperm morphology, sperm concentration, and the number of spermatozoa. We provide the direct experimental evidence that the male reproductive system could be targeted and damaged by the COVID-19 infection. These findings go beyond our current understanding of the disease, suggesting that the reproductive function of the patients recovering from the disease should be precisely followed and evaluated to detect and avoid more serious reproductive problems in the future, as they may develop a transient state of male subfertility like those with oligoasthenoteratozoospermia.

Relationship between COVID-19 and the male reproductive system.

OBJECTIVE: The objective of this review is to provide currently available information on the potential effects of coronavirus disease 2019 (COVID-19) on male fertility. MATERIALS AND METHODS: This is a mini-review. Due to the similarity between the COVID-19 and severe acute respiratory syndrome (SARS) virus, we searched for the following keywords: "SARS-CoV, male reproductive system, infertility, COVID-19, SARS-CoV-2, and orchitis". By reviewing and analyzing the literature, we analyzed the influence of temperature on sperm, the expression of angiotensin-converting enzyme 2 (ACE2) in the testes, and the impact of SARS-CoV-2 on the male reproductive system. RESULTS: SARS-CoV-2 enters the body through the ACE2 receptor. The high expression of ACE2 on the surface of spermatogonia and supporting cells in the testes, as well as the immune response caused by COVID-19, can lead to testicular spermatogenesis dysfunction and reduced sperm count. CONCLUSIONS: COVID-19 infection can affect male reproductive function, and standard treatment strategies should be established in time to help male patients infected with COVID-19.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and its effect on gametogenesis and early pregnancy.

SARS-CoV-2 infection and pregnancy has been the topic of hundreds of publications over the last several months; however, few studies have focused on the implications of infection in early pregnancy and reproductive tissues. Here, we analyzed available evidence pertaining to SARS-CoV-2 infection, in early pregnancy, and in reproductive tissues. We searched PubMed and Embase databases in accordance with guidelines of Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) for publications from inception to June 4, 2020. Four reviewers screened titles and abstracts and obtained full-text articles for analysis. Sixty-two studies were included in the review. Biological plausibility for infection with SARS-CoV-2 exists in testis, ovaries, and placenta as they express ACE2 receptor activity. In males, SARS-CoV-2 infection could lead to functional abnormalities leading to spermatogenic failure and male infertility. In females, an alteration of the ACE2 cascade via SARS-CoV-2 infection could lead to impairment in important follicular and luteal processes. There is also evidence of significant placental pathology in SARS-CoV-2 infection, but it is unclear what effects there may be for early pregnancy, though available data suggest less severe effects compared to other respiratory virus outbreaks. Further investigation is needed regarding SARS-CoV-2 in reproductive function and early pregnancy.

The ACE2 expression in Sertoli cells and germ cells may cause male reproductive disorder after SARS-CoV-2 infection.

The serious coronavirus disease-2019 (COVID-19) was first reported in December 2019 in Wuhan, China. COVID-19 is an infectious disease caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2). Angiotensin converting enzyme 2(ACE2) is the cellular receptor for SARS-CoV-2. Considering the critical roles of testicular cells for the transmission of genetic information between generations, we analyzed single-cell RNA-sequencing (scRNA-seq) data of adult human testis. The mRNA expression of ACE2 was expressed in both germ cells and somatic cells. Moreover, the positive rate of ACE2 in testes of infertile men was higher than normal, which indicates that SARS-CoV-2 may cause reproductive disorders through pathway activated by ACE2 and the men with reproductive disorder may easily to be infected by SARS-CoV-2. The expression level of ACE2 was related to the age, and the mid-aged with higher positive rate than young men testicular cells. Taken together, this research provides a biological background of the potential route for infection of SARS-CoV-2 and may enable rapid deciphering male-related reproductive disorders induced by COVID-19.

COVID-19 and human spermatozoa-Potential risks for infertility and sexual transmission?

As COVID-19 infections wreak havoc across the globe, attention has rightly been focused on the vital organ systems (lung, kidney and heart) that are vulnerable to viral attack and contribute to the acute pathology associated with this disease. However, we should not lose sight of the fact that COVID-19 will attack any cell type in the body expressing ACE2 - including human spermatozoa. These cells possess the entire repertoire of receptors (AT1R, AT2R, MAS) and ligand processing enzymes (ACE1 and ACE2) needed to support the angiotensin signalling cascade. The latter not only provides COVID-19 with a foothold on the sperm surface but may also promote integration, given the additional presence of a range of proteases (TMPRSS2, TMPRSS11B, TMPRSS12, furin) capable of promoting viral fusion. This article reviews the roles played by these various cellular constituents in maintaining the vitality of human spermatozoa and their competence for fertilization. The reproductive consequences of a viral attack on these systems, in terms of fertility and the risk of sexual transmission, are currently unknown. However, we should be alive to the possibility that there may be reproductive consequences of COVID-19 infection in young males that go beyond their capacity to survive a viral attack.

Could SARS-CoV-2 affect male fertility?

We performed this systematic review to evaluate the possibility of an impact of SARS-CoV-2 infection on male fertility. SARS-CoV-2 enters the cells with the help of ACE2; therefore, testicular expression of ACE2 was analysed from transcriptome sequencing studies and our unpublished data. Literature suggested that SARS-CoV-1 (2002-2004 SARS) had a significant adverse impact on testicular architecture, suggesting a high possibility of the impact of SARS-CoV-2 as well. Out of two studies on semen samples from COVID-19 affected patients, one reported the presence of SARS-CoV-2 in the semen samples while the other denied it, raising conflict about its presence in the semen samples and the possibility of sexual transmission. Our transcriptome sequencing studies on rat testicular germ cells showed ACE expression in rat testicular germ cells. We also found ACE2 expression in transcriptome sequencing data for human spermatozoa, corroborating its presence in the testicular germ cells. Transcriptome sequencing data from literature search revealed ACE2 expression in the germ, Sertoli and Leydig cells. The presence of ACE2 on almost all testicular cells and the report of a significant impact of previous SARS coronavirus on testes suggest that SARS-CoV-2 is highly likely to affect testicular tissue, semen parameters and male fertility.