Reproductive toxicity of ritonavir in male: Insight into mouse sperm capacitation.

Since COVID-19 began in 2019, therapeutic agents are being developed for its treatment. Among the numerous potential therapeutic agents, ritonavir (RTV), an anti-viral agent, has recently been identified as an important element of the COVID-19 treatment. Moreover, RTV has also been applied in the drug repurposing of cancer cells. However, previous studies have shown that RTV has toxic effects on various cell types. In addition, RTV regulates AKT phosphorylation within cancer cells, and AKT is known to control sperm functions (motility, capacitation, and so on). Although deleterious effects of RTV have been reported, it is not known whether RTV has male reproduction toxicity. Therefore, in this study, we aimed to investigate the effects of RTV on sperm function and male fertility. In the present study, sperm collected from the cauda epididymis of mice were incubated with various concentrations of RTV (0, 0.1, 1, 10, and 100 µM). The expression levels of AKT, phospho-AKT (Thr308 and Ser473), and phospho-tyrosine proteins, sperm motility, motion kinematics, capacitation status, and cell viability were assessed after capacitation. The results revealed that AKT phosphorylation at Thr308 and Ser473 was significantly increased, and the levels of tyrosine-phosphorylated proteins (at approximately 25 and 100 kDa) were significantly increased in a dose-dependent manner. In addition, RTV adversely affected sperm motility, motion kinematics, and cell viability. Taken together, RTV may have negative effects on sperm function through an abnormal increase in tyrosine phosphorylation and phospho-AKT levels. Therefore, individuals taking or prescribing RTV should be aware of its reproductive toxicity.

The Bovine Seminal Plasma Protein PDC-109 Possesses Pan-Antiviral Activity.

Mammalian seminal plasma contains a multitude of bioactive components, including lipids, glucose, mineral elements, metabolites, proteins, cytokines, and growth factors, with various functions during insemination and fertilization. The seminal plasma protein PDC-109 is one of the major soluble components of the bovine ejaculate and is crucially important for sperm motility, capacitation, and acrosome reaction. A hitherto underappreciated function of seminal plasma is its anti-microbial and antiviral activity, which may limit the sexual transmission of infectious diseases during intercourse. We have recently discovered that PDC-109 inhibits the membrane fusion activity of influenza virus particles and significantly impairs viral infections at micromolar concentrations. Here we investigated whether the antiviral activity of PDC-109 is restricted to Influenza or if other mammalian viruses are similarly affected. We focused on Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), the etiological agent of the Coronavirus Disease 19 (COVID-19), thoroughly assessing PDC-109 inhibition with SARS-CoV-2 Spike (S)-pseudotyped reporter virus particles, but also live-virus infections. Consistent with our previous publications, we found significant virus inhibition, albeit accompanied by substantial cytotoxicity. However, using time-of-addition experiments we discovered a treatment regimen that enables virus suppression without affecting cell viability. We furthermore demonstrated that PDC-109 is also able to impair infections mediated by the VSV glycoprotein (VSVg), thus indicating a broad pan-antiviral activity against multiple virus species and families.

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.

4BNT162b2 mRNA COVID-19 vaccine and semen: What do we know?

BACKGROUND: The effects of an mRNA COVID-19 vaccine on spermatozoa parameters are not known. The aim of this study was to evaluate the effect of the BNT162b2 mRNA COVID-19 vaccine on human semen, comparing spermatozoa parameters before and after vaccine inoculation. MATERIALS AND METHODS: In this single-center prospective study, voluntary subjects who received mRNA vaccines from February to July 2021 were enrolled. The study population included male subjects aged between 18 and 45 years who completed the BNT162b2 mRNA COVID-19 vaccine cycle. All subjects were evaluated before the first dose of vaccine (T0) and after 3 months (T1) with semen analysis and further analysis of seminal plasma, including colorimetric determination of reactive oxygen metabolites (d-ROM test), electrolytes, and interleukin 6 (IL-6) assessment by enzyme-linked immunosorbent assay technology. RESULTS: The experimental sample included 47 subjects (age: 29.3 ± 6.0 years, range 24-32; body mass index: 23.15 ± 2.5 kg/m2 , range 19.2-28.0). All the subjects reported no systemic side effects. No significant differences were observed in any spermatozoa parameter between T0 and T1. A subanalysis was performed in oligoazoospermic and asthenozoospermic subjects, confirming the same results. Electrolyte analysis also showed no significant differences before and after vaccine inoculation. Finally, no significant differences were observed in T0, compared to T1 for the d-ROM test and IL-6. DISCUSSION AND CONCLUSION: In this study, no significant differences in spermatozoa parameters before and after vaccine inoculations were found. Furthermore, oxidative stress analysis,, the activity of the cell membrane, and IL-6, as a marker of inflammation, was not affected by the mRNA COVID-19 vaccine. These results suggest that this vaccine is safe for male semen quality.

Effects of acute severe acute respiratory syndrome coronavirus 2 infection on male hormone profile, ACE2 and TMPRSS2 expression, and potential for transmission of severe acute respiratory syndrome coronavirus 2 in semen of Asian men.

OBJECTIVE: To confirm if severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can be detected in semen of men with acute coronavirus disease 2019 and if their male hormone profile (testosterone, follicle-stimulating hormone, luteinizing hormone, sex hormone binding globulin, and free androgen index) is adversely affected during the acute phase of infection and any relation to the ACE2 and/or TMPRSS2 expression in human semen. DESIGN: Clinical study. SETTING: National University Hospital, Singapore. PATIENTS: Asian men aged 21-55 years who were admitted to National University Hospital, Singapore, with a laboratory-confirmed diagnosis of SARS-CoV-2 infection via nasopharyngeal swab in the acute phase of the infection, within 2-14 days of the development of symptoms or contact history, were recruited for the study. INTERVENTIONS: Blood was collected in the morning to assess the male hormone profile. Human semen were obtained by masturbation and sent to the molecular diagnostic laboratories to detect the presence of SARS-CoV-2 RNA and assess the ACE2 and TMPRSS2 expression. MAIN OUTCOME MEASURES: Male hormone profile level and expression of SARS-CoV-2 RNA, ACE2, and TMPRSS2 in human semen. RESULTS: A total of 63 men of Asian ethnicities agreed to participate in the study. Subsequently, 65% of recruited men had completely normal levels of male hormone profile. Moreover, 27% were noted to have higher luteinizing hormone levels between 6.6 and 16.1 IU/L (normal range, 0.8-6.1 IU/L), and 10% had higher follicle-stimulating hormone levels between 13.6 and 41.6 IU/L (normal range, 1.5-12.4 IU/L); all had normal testosterone levels. No SARS-CoV-2 RNAs were detected in all human semen. The ACE2 and TMPRSS2 expression was undetectable in 26 samples, whereas 23 samples only had a detectable TMPRSS2 expression and 4 only had an ACE2 expression. The remaining 3 expressed both ACE2 and TMPRSS2. CONCLUSIONS: Severe acute respiratory syndrome coronavirus 2 could not be found in the semen of a cohort of young to middle-aged Asian men with mild acute SARS-CoV-2 infection. However, there was a detectable expression of ACE2 and TMPRSS2 in semen, although not causal, and it may be correlated to changes in male hormone profiles and male age.

Male reproductive system inflammation after healing from coronavirus disease 2019.

BACKGROUND: There is evidence that, after severe acute respiratory syndrome coronavirus 2 infection, male reproductive function and semen quality may be damaged OBJECTIVES: To evaluate a panel of inflammatory mediators in semen in patients recovered from coronavirus disease 2019. MATERIAL AND METHODS: Sexually active men with previous severe acute respiratory syndrome coronavirus 2 infection and proven recovery from coronavirus disease 2019 were enrolled in a prospective cohort study. Clinical, uro-andrological data and semen specimens were prospectively collected. For previously hospitalized coronavirus disease 2019 patients, data on serum inflammatory markers were retrospectively collected. RESULTS: A total of 43 men were enrolled in the study. Of these, 32 men were normozoospermic, three were oligozoospermic, and eight were crypto-azoospermic. Serum inflammatory markers (procalcitonin and C-reactive protein) were analyzed in previously hospitalized patients both at admission and at peak of infection. Levels at admission were statistically significantly higher in patients resulting in crypto-azoospermic with respect to those resulting in normozoospermic (p = 0.05; p = 0.03 and p = 0.02, respectively) after healing. Seminal cytokine levels were similar among all groups. Interleukin-1ß and tumor necrosis factor-α levels were significantly negatively related to sperm total number and concentration, whereas interleukin-4 was correlated with sperm motility. DISCUSSION AND CONCLUSION: Negative correlations between interleukin-1ß and tumor necrosis factor-α and sperm number and the overall high levels of semen cytokines indicate a potential detrimental role of severe acute respiratory syndrome coronavirus 2 driven inflammation on spermatogenesis. Overall, our results indicate that male patients recovering from coronavirus disease 2019 deserve accurate follow-up for their fertility status.

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.

Assessment of SARS-CoV-2 in human semen-a cohort study.

OBJECTIVE: To investigate the presence of viral RNA in human semen of patients with severe acute-respiratory syndrome coronavirus 2 (SARS-CoV-2) and to evaluate its presence and relevance in semen parameters. DESIGN: Pilot cohort study. SETTING: University hospital. PATIENT(S): Thirty-four men were distributed as: 1) patients in convalescence (patients with confirmed SARS-CoV-2 infection in pharyngeal swab according to reverse-transcription polymerase chain reaction [RT-PCR] or antibodies); 2) negative control group (no antibodies); and 3) patients with an acute infection (detection of SARS-CoV-2 in pharyngeal swab). INTERVENTION: Semen and a blood sample were collected from each individual. MAIN OUTCOME MEASURE(S): Analysis of semen quality according to the World Health Organization standards. Detection of SARS-CoV-2 by RT-PCR in the native semen sample and after density gradient preparation. Confirmation of immunoglobulin (Ig) A und IgG antibodies in the blood. RESULT(S): Eighteen semen samples from recovered men were obtained 8-54 days after absence of symptoms, 14 from control subjects, and 2 from patients with an active COVID-19 infection. No RNA was detected by means of RT-PCR in the semen, including semen samples from two patients with an acute COVID-19 infection. Subjects with a moderate infection showed an impairment of sperm quality. CONCLUSION(S): A mild COVID-19 infection is not likely to affect testis and epididymis function, whereas semen parameters did seem impaired after a moderate infection. SARS-CoV-2 RNA could not be detected in semen of recovered and acute COVID-19-positive men. This suggests no viral transmission during sexual contact and assisted reproductive techniques, although further data need to be obtained.