key: cord-0747183-g6kag5ij authors: Nora, Holtmann; Philippos, Edimiris; Marcel, Andree; Cornelius, Doehmen; Dunja, Baston-Buest; Ortwin, Adams; Jan-Steffen, Kruessel; Petra, Bielfeld Alexandra title: Assessment of SARS-CoV-2 in human semen - a cohort study date: 2020-05-29 journal: Fertil Steril DOI: 10.1016/j.fertnstert.2020.05.028 sha: c7a1ccbf36656b156946ba870d37e448ffc07bd2 doc_id: 747183 cord_uid: g6kag5ij Abstract Objective To investigate the presence of viral RNA in human semen of severe-acute-respiratory syndrome coronavirus 2 (SARS-CoV-2) recovered and positive patients and to evaluate its presence and relevance on semen parameters. Design Pilot cohort study Setting University hospital Patients 34 adult males were distributed into a) patients in convalescence (patients with confirmed SARS-CoV-2 infection in pharyngeal swab by RT-PCR and/or antibodies), b) negative control group (no antibodies) and c) 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 measures Analysis of semen quality according to the WHO 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 Ig-G antibodies in the blood. Results 18 semen samples from recovered males were obtained 8 to 54 days after absence of symptoms, 14 samples from controls and 2 samples from patients with an active COVID-19 infection. No RNA was detected by 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 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 males. This suggests no viral transmission during sexual contact and assisted reproductive techniques (ART), however, further data need to be obtained. In December 2019 clusters of a novel type of pneumonia were reported in Wuhan City, Hubai Province, China (1) and defined by the World Health Organisation (WHO) as coronavirus disease 2019 in February 2020 (2). The severe acute respiratory syndrome 108 coronavirus 2 (SARS-CoV-2) was identified as the causing viral pathogen for the pandemic (3) . In order to constrain the world-wide outbreak of COVID-19, viral transmission pathways are intensively studied. So far, it is known that the coronavirus is predominantly transmitted 111 through respiratory droplets (4) . In addition, viral RNA has been detected in various biological samples, such as faeces, urine and blood (5). SARS-CoV-2 seems to have a high affinity binding capability to the angiotensin converting enzyme-2 (ACE-2) in human cells, 114 which is expressed in multiple organ systems, including the testis (6) . Although the testis are immunologically privileged in case of viremia, some viruses can cross the blood-testis barrier causing local inflammation of the testis (7) . The virus may persist after an acute infection as 117 for example human immunodeficiency virus (HIV) and can theoretically replicate within the male reproductive tract (8). Hence, viral RNA of primarily non-sexual transmitted diseases can be found in semen (9). 120 The presence of SARS-CoV-2 in the male reproductive tract may reduce male fertility through orchitis or spermatogonial stem cell infection and may have implications for sexual transmission and consequently for embryonic infection, miscarriage and congenital disease 123 (10) . In order to thoroughly advise couples with the acute desire for a child, information about the impact of COVID-19 on male reproductive function and viral seeding are needed. The aim of this study is to a) determine any possible implications of COVID-19 on male 126 semen parameters and b) analyse the semen for any presence of SARS-CoV-2 RNA in recovered men and males with an active COVID-19 infection. defined patients requiring hospitalisation with oxygen supply up to 6 litres to achieve more than 92% of peripheral oxygenation. The control group consisted of healthy volunteers with 153 no reported andrological pathology. A semen sample of each participant was obtained by masturbation and ejaculation directly into non-cytotoxic sterile containers. Freshly collected semen was liquefied for 30-60min at room temperature and processed within one hour of ejaculation for analysis of sperm 159 characteristics according to the criteria published by the WHO. Sperm morphology was not assessed due to safety concerns. Samples were homogenized, and 500µL were transferred to the tube for viral testing of the native sample. 162 To prepare the semen sample for the viral testing, the remaining semen was prepared in a 2step washing process modified according to the center's standards procedure for HIV or HEP C infected males. First, the semen was counted and filtered through a 30°C pre-warmed 165 90%/45% colloidal silica density gradient with 1.800rpm for 20min (SpermFilter®, Gynotec B.V., GC Malden, Netherlands) prepared with GM 501 SpermAir (SPA) sperm processing media (Gynemed, Lensahn, Germany). Second, the pellet was washed in 3ml pre-warmed 168 SPA with 2.300rpm for 10min and the resulting pellet resuspended in 500µl SPA, counted and transferred to the viral testing tube of the processed sperm. Detection of SARS-CoV-2 in semen Centrifugation of the native and processed sperm sample for 1min at 3.500rpm. RNA extraction was performed from 200µl supernatant using the EZ1 Virus Mini Kit v2. 174 (Qiagen®, Hilden, Germany) following the manufacturer's instructions. 60µl were eluted from 200µl starting material. 5µl of the eluate were tested in RT-PCR using the TaqMan®technique. A 113-base pair amplicon in the E-gene of SARS-CoV-2 was amplified and 177 detected, as described with minor modifications (11) . RT-PCR was performed with an ABI 7500 FAST sequence detector system (PE Applied Biosystems, Weiterstadt, Germany). The thermal protocol described was shortened to 40 cycles of 95°C. We used the LightMix®, We used a commercial anti-SARS-CoV-2 S1 IgG and IgA ELISA (IgG Cat. No. EI 2606- following sensitivity and specificity of the commercial anti-SARS-CoV-2 S1 IgG and IgA ELISA are indicated: IgG sensitivity increases from <10 days (30.3%) after start of symptoms to >21 days after start to ~94%. IgG specificity is high with ~99%. IgA sensitivity increases 195 from 51.5% <10 days after start of symptoms to 100% >21 days. IgA specificity is also high ~88%. Statistical analysis was performed using SPSS 23 and Mann-Whitney U test. Two-sided P values <0.5 were considered statistically significant. Table 1 ). 17 out of 18 recovered participants described symptoms, mainly fever (10 out of 18), cough, headache, ague, muscle pain, body ache, dyspnea and fatigue. 2 participants had anosmia and loss of taste. One participant reported testicular discomfort. 237 4 participants with a moderate course of disease were hospitalized due to high fever and dyspnea. None of them needed endotracheal intubation. However, 2 subjects received an antiretroviral therapy with lopinavir/ritonavir for 1 day in one case and for 3 days in the other 240 case. The third subject was given hydroxychloroquine and moxifloxacin. Other medication taken by some participants was paracetamol. The control group did not suffer from any symptoms related to COVID-19 in the past 8 243 Table 1) . 252 SARS-CoV-2 RNA could neither be detected in semen samples from recovered nor from acute infected subjects. The results of the sperm analysis are summarized in Table 2 . Patients with a moderate 258 infection have a statistical significant impairment of sperm quality (sperm concentration, total number of sperm per ejaculate, total number of progressive motility, total number of complete motility) in comparison with men recovered from a mild infection and the control group. We 261 divided the individuals in fever positive vs. fever negative regardless of their classification into mild and moderate and analyzed the semen accordingly as depicted in Table 3 . Although there were statistical significant differences regarding the volume, the complete motility and 264 the number of immotile sperms, the values were all still in the normal range (Table 3 ). ACE2 is the cell entry receptor for SARS-CoV-2 which is not only found in the respiratory system but also in the testis. This finding led to the hypothesis that the human testis and 270 therewith semen is a target for a SARS-CoV-2 infection which might increase the understanding of this rapidly spreading disease (12) . Furthermore, the investigation of semen samples regarding the presence of SARS-CoV-2 RNA is highly important since it was shown 273 before for several different viruses, that viremic patients can shed viruses into their semen (9). in the investigated semen samples (14) . The fact, that only very low titers of SARS-CoV-2 have been detected so far in non-respiratory sites like feces and urine specimens (5,15) additionally supports the hypothesis that SARS-CoV-2 shows only a minor risk of virus 285 shedding into the semen. Nevertheless, even a minor risk is not acceptable in the light of treating otherwise healthy couples for infertility reasons. Therefore, it is of tremendous importance to investigate particularly non-treated males' semen since many individuals 288 suffering from a mild form of COVID-19 might not even have associated their symptoms to an actual infection with SARS-CoV-2. Here, our study differs from the report of Song et al. showed though that 6 out of 38 males with a positive nasopharyngeal swap who still had 303 symptoms or stopped having symptoms 2-3 days before semen analysis presented with SARS CoV-2 in the semen (17) . On another note, it is of interest, that although it was described before in the literature that viral infections have a negative impact on semen parameters like 306 volume, number of spermatozoa and motility we could not detect a negative influence of the SARS-CoV-2 infection in respect of the aforementioned sperm count parameters in recovered subjects with mild symptoms. However, patients facing a moderate course of disease and 309 being in need of hospital care had a reduced sperm quality (Table 2) . On one hand this could be an effect of the infection with SARS-CoV-2 in association with the severity of the illness or due to a transitory higher viral load. On the other hand, the impaired male fertility in this 312 subgroup could be pre-existing. As these subjects were treated with lopinavir/ritonavir and hydroxychloroquin, an impact of this medication on sperm parameters is possible, however unlikely, since it was only applied for a few days. Moreover, there exists no evidence that 315 lopinavir/ritonavir or hydroxychloroquin have an impact on male fertility (18, 19) . Additionally it is noteworthy, that in general modifications of the sperm count due to trauma, injury or infection might be seen only after 3 months of time. Subsequently, another semen 318 analysis after the aforementioned time would be desirable. Our study has certain limitations. First, we investigated a relative small sample size. Second, sperm analysis of tested individuals performed before the outbreak of the pandemic was not obtained, limiting the 321 diagnosis of pre-existing male infertility. Third, we only analyzed 2 patients with an active COVID-19 infection, it will be necessary to ascertain our findings in a lager sample size. Finally, our preliminary results lack any data about long-term effects of SARS-CoV-2 on 324 male reproductive function. In summary, SARS-CoV-2 does not seem to have a short-term impact on male fertility in 327 patients with mild symptoms regarding sperm characteristics according to the WHO criteria. We found no evidence of SARS-CoV-2 shedding in semen of recovered males or patients with an acute COVID-19 infection after a recovery time of 32,7 days on average. Statistical analysis according to Mann-Whitney U Test for non parametric distribution dedicated by asterisk (* or **; *mild vs. moderate; **moderate vs. control) with P< .05. No statistically significant differences could be detected between mild and control. § : 2 patients with cryptozoospermia were excluded of the analysis according to WHO Statistical analysis according to Mann-Whitney U Test for non parametric distribution dedicated by asterisk (*) with P< .05 considered significant. *** different by trend 468 360 2. WHO Director-General's remarks at the media briefing on 2019-nCoV on 11 A pneumonia outbreak associated with a new coronavirus of probable bat origin Severe Acute Respiratory Syndrome: Historical, Epidemiologic, and Clinical Features First Case of 2019 Novel Coronavirus in the United States Structure analysis of the receptor binding of 2019-372 nCoV Structural, cellular and molecular aspects of immune privilege in the testis Zika virus in semen and spermatozoa The Breadth of Viruses in Human Semen Viruses in the mammalian male genital tract and their effects on 381 the reproductive system Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR scRNA-seq Profiling of Human Testes Reveals the Presence of the ACE2 Receptor, A Target for SARS-CoV-2 Infection in Spermatogonia Persistence and clinical relevance of Zika virus in the male genital 390 tract No evidence of SARS-CoV-2 in semen of males recovering from COVID-19 Absence of 2019 Novel Coronavirus in Semen and Testes of COVID-19 Patients In press Detectable HIV-1 in semen in individuals with very low blood viral loads Clinical Characteristics and Results of Semen 399 Tests Among Men With Coronavirus Disease Antivirals and Male Reproduction Paternal safety of anti-rheumatic medications 333 We would like to thank all individuals for their participation in our study. Additionally, we would like to thank Stephanie Engels and Stefanie van den Boom for technical assistance.