key: cord-0777374-vi90gdlq authors: Fraietta, Renato; de Carvalho, Renata Cristina; Camillo, Jacqueline; Groner, Matheus Ferreira; Truzzi, José Carlos Cezar Ibanhez; Petkov, Camila Nogueira; Barradas, Valéria; Homsi, Carolina; Bellei, Nancy; Oehninger, Sergio title: SARS‐CoV‐2 is not found in human semen during mild COVID‐19 acute stage date: 2021-10-24 journal: Andrologia DOI: 10.1111/and.14286 sha: 9ab3edfab83116ca60dbae61d2f1b32f574ffbab doc_id: 777374 cord_uid: vi90gdlq The aim of this study was to verify the presence of SARS‐CoV‐2 in the seminal sample of men during the acute phase of COVID‐19. A prospective study was performed with inclusion of twenty‐two men diagnosed with COVID‐19 through RT‐PCR from pharyngeal smear samples and who were in the acute phase of infection. These men were evaluated regarding medical history and physical examination. Furthermore, seminal samples of each men were collected 7, 14 and 21 days after the infection was confirmed. The sample were used for seminal analysis, as well as for the presence of SARS‐CoV‐2 using RT‐PCR technique. In addition, cell culture was performed with subsequent repetition of the analysis of viral presence. None of the semen samples collected was positive for the detection of the virus that causes COVID‐19. Most of the men evaluated had a mild condition and the loss of smell was the most frequent symptom. There were no significant changes in seminal parameters within the period of study. Based on our pilot data, patients with a mild form of COVID‐19 in the acute stage of the disease are unlikely to have SARS‐CoV‐2 in semen. Coronavirus disease 2019 (COVID-19) is a viral disease that mainly affects the respiratory system of those infected . People with this condition can be classified into five categories of severity, namely: asymptomatic, individuals with a positive diagnosis for the disease but without symptoms; mildindividuals with symptoms such as fever, cough, sore throat but not shortness of breath, dyspnoea or abnormal chest images; moderatepatients who, during clinical or imaging examination, present characteristics of lower respiratory disease and oxygen saturation ≥94%; severe-those who had oxygen saturation <94%, a ratio between arterial oxygen partial pressure and the fraction of inspired oxygen <300 mm/Hg, respiratory rate >30 breaths/minute or pulmonary infiltrates >50%; critical-those individuals who have respiratory failure, septic shock and/or multiple organ dysfunction (NIH, 2021) . In addition, COVID-19 seems to go through 3 different phases: early infection phase that lasts for 5-7 days, pulmonary phase and hyperinflammation phase, and these last two phases can be observed between the 7th and 15th day after the onset of the disease (Siddiqi & Mehra, 2020) . The infectious agent responsible for this new disease is Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) . This virus has a spherical morphology and it is believed that, to infect the cell, it uses mechanisms similar to SARS-CoV . In a canonical pathway, the viral spike (S) protein binds to the cellular receptor angiotensin-converting enzyme 2 (ACE 2) to promote the fusion of the viral and cellular membranes; in order for the whole process to happen, it is necessary the activation (priming) of the S, which is given by the transmembrane protease serine 2 (TMPRSS2) (Hoffmann et al., 2020) . In a noncanonical mechanism, human coronaviruses can use other host proteins with or without the presence of ACE2 to aid the viral entry (Colaco et al., 2021) . These cellular components (ACE2 and TMPRSS2) have been found in considerable expression in lung cells (Lukassen et al., 2020) and nasal epithelial cells (Sungnak et al., 2020) ; such characteristic may be the justification for the main symptoms of COVID-19 to be fever, cough, shortness of breath and fatigue (Centers for Disease Control and Prevention (CDC), 2020). Because this disease also generates symptoms that go beyond the respiratory system, such as vomiting and diarrhoea (Centers for Disease Control and Prevention (CDC), 2020; Gupta et al., 2020) , other tissues were analysed and receptors that facilitate viral interaction were also found in them (Zou et al., 2020) . Thus, the male reproductive system is one of the places of vulnerability for this virus. The prostate, an organ with great expression of the TMPRSS2 gene in its epithelial cells, has a small percentage of cells that coexpress TMPRSS2 and ACE2, indicating that there is a fragility to the COVID-19 virus, but with a low risk . Still, there are reports that one of the symptoms of this infection may be orchialgia (Pan et al., 2020) , which could indicate some action of this virus on the testicles. It is known that the cell receptor used by SARS-CoV-2 is present in the somatic and germ cells of the testicles, and the protein responsible for the priming of the viral S appears only in the germ cells . However, when analysing the coexpression of the cell receptor (ACE2) and the virus activation protein (TMPRSS2), a rare presence of the two genes was observed together (Pan et al., 2020; Stanley et al., 2020) . These results raise doubts about the interference of SARS-CoV-2 in male fertility and its presence in the seminal sample. There are a few studies in the literature that sought to analyse whether SARS-CoV-2 is or is not present in the seminal sample of infected men; however, these assessments were made on samples from individuals already in the recovery phase, or in the acute phase of the disease without identifying the period of infection (days of diagnosis) Holtmann et al., 2020; Pan et al., 2020; Song, Wang, et al., 2020) . Thus, an early and evolutionary assessment of the presence and interference of the virus that causes COVID-19 in semen is important. This study aimed to verify the presence of SARS-CoV-2 in the seminal sample of men during the acute phase of COVID-19. This prospective study was conducted from May to November 2020 and involved twenty-two male volunteers aged between 23 and 31 years old who sought the university hospital with suspicion of COVID-19. After positive results (RT-PCR) in pharyngeal smear samples for SARS-CoV-2, they were invited to participate in the study. The informed consent form was obtained from each participant and was approved by the Sao Paulo Federal University (UNIFESP) research ethics committee (CAAE 31916820.8.0000.5505). All men answered a routine questionnaire to obtain the clinical history and underwent a physical examination, performed by a urologist specialised in infertility. They also performed semen collection for seminal analysis and detection of SARS-CoV-2. For viral detection (RT-PCR), an aliquot of semen was transferred to a tube containing lactate ringer in a 1:1 ratio. The remaining sample was then centrifuged at 2,000 rpm for 30 min and the seminal plasma was frozen at −80°C for subsequent cell culture. The detection of SARS-CoV-2 RNA by RT-PCR was performed according to the IFU for the GeneFinder™ COVID-19 Plus RealAmp Kit (OSANG Healthcare Co., Ltd.) (GeneFinder, 2020; Zymo Reserch, 2020) . RNA was isolated from the subject's semen using the Quick- A value of up to 40 cycles was defined as a positive test, and a value of 40 cycles or more was defined as a negative test (value defined according to the manufacturer). Seminal plasma samples from two patients were subjected to cell culture to check for viral replicative capacity. For the direct isolation of SARS-CoV-2, a cell line of VERO E6 (African green monkey kidney) was used, as described by Matsuyama et al., 2020 (Matsuyama et al., 2020 . The seminal plasma was diluted with culture medium and filtered through a disposable filter (0.22 nm) for inoculation. Subsequently, viral amplification was performed on VERO E6 cell line as previously described by Hoffman et al, 2020 (Hoffmann et al., 2020) . After demonstrating viral replication by checking the cytopathic effect formation within 96 hr of culture, the viral titre was determined by the amount of infectious viral particles capable of forming plaque (PFU/ml). Subsequently, molecular detection of SARS-CoV-2 in the culture supernatant was performed using the RT-PCR test. Initially, the procedures of descriptive statistics were applied. The symptoms were described and evaluated according to the frequency they were developed by the volunteers. For seminal evaluation, a graph was used to show the amount of motile morphologically normal spermatozoa (concentration × volume × progressive motility × morphology) for each patient in each collection period. Moreover, statistical analysis was performed with these parameters (SPSS software). Firstly, the Shapiro-Wilk test was applied to verify the normality of the data; those variables that did not show normal distribution were transformed by logarithm. In addition, the Mauchly teste was used to analyse sphericity. Then, the General Linear Model test for repeated measures was applied; p was considered significant when it was <0.05. Of the 22 volunteers, 20 were classified as having mild infection and underwent home treatment with symptomatic drugs and oximetry control, and two required hospitalisation, one with a moderate condition who sought medical care complaining of asthenia, myalgia, cough, anosmia and haemoptysis, being admitted to the ICU due to hemodynamic instability and another with a severe form of the disease with an imaging exam suggestive of a bacterial process and an oxygen saturation of 90% at the time of hospitalisation but without the need for O 2 support. All patients collected at least one seminal sample that was used to verify the presence of the virus. In samples that underwent cell culture with subsequent application of RT-PCR, the virus was not detected either. The frequency of symptoms, presence of testicular pain, need for treatment and need for hospitalisation is shown in Table 2 . Regarding the seminal analysis, 8 patients were excluded, of which 6 did not return for the subsequent analyses, 1 was azoospermic, and 1 had extremely low-sperm concentration, making it impossible to evaluate all semen variables. No significant difference was found between the seminal parameters when analysed on days 7, 14 and 21 after the initial diagnosis (Table S1 ). The evolution of the total number of motile morphologically normal spermatozoa in each patient can be followed in Figure S1 . The concern about the involvement of the male genital system in relation with COVID-19 by SARS-CoV-2 has been based on several aspects previously mentioned. Initial studies (Pan et al., 2020; Song, Wang, et al., 2020) evaluated semen in recovered patients, with no virus found in the sample analysed. Although other more recent studies reaffirm this finding Holtmann et al., 2020; Paoli et al., 2020) , suspicion of the presence and possible transmissibility through semen was maintained during the acute phase of the disease. A study ) evaluated a total of 38 patients, 15 of whom were in the acute phase of the disease and found the presence of the virus in the semen in 4 (26%) of those analysed during the initial phase, and in another 2 (8%) of those already recovered, (with 2 and 3 days of recovery). It is important to mention that this study did not describe the severity of the patients, but we can predict that, because they are hospitalised, they would potentially be classified between moderate to critical cases. Another important issue is the lack of description of the seminal collection process, which may pose risks of contamination of the sample by viruses not present in the semen. A recent research evaluated 15 men and the virus was present in 1 subject (Machado et al., 2021) . Another study (Rawlings et al., 2020) Seminal evaluation of these patients was also performed weekly during the 3-week follow-up. There was no difference between the total number of motile morphologically normal spermatozoa analysed in this period. Another article also evaluated the seminal pattern of patients after 26-34 days of symptoms, finding no evidence of seminal change in these patients . It is known that a complete spermatogenesis cycle occurs around 60 days (Misell et al., 2006) and that the presence of any acute febrile syndrome in this period can interfere with sperm production (Carlsen al., 2003) , thus, a longer-term follow-up can determine whether COVID-19 interferes with the fertile potential of these men. We consider the limitations of this study to be the relatively low number of patients evaluated and the representativeness of only mild cases; however, we can also consider that the inclusion of only patients with mild symptoms is also an advantage as it allowed us to characterise a more homogeneous population. Therefore, based on our pilot data, patients with a mild form of COVID-19 in the acute stage of the disease are unlikely to have SARS-CoV-2 in semen. 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Andrologia, 00, e14286