key: cord-265221-qtkwciym
authors: Bahadur, Gulam; Acharya, Santanu; Muneer, Asif; Huirne, Judith; Łukaszuk, Mariusz; Doreski, Pablo Alexis; Homburg, Roy
title: SARS-CoV-2: diagnostic and design conundrums, and the male factor infertility
date: 2020-06-03
journal: Reprod Biomed Online
DOI: 10.1016/j.rbmo.2020.05.014
sha: 
doc_id: 265221
cord_uid: qtkwciym

The question on whether SARS-CoV-2 (Severe acute respiratory syndrome-related coronavirus (SARS-CoV or SARS-CoV-2, Covid-19) can be harboured in testes and/or the semen is currently unanswered. It is essential to understand the limitations of both antibody and real time polymerase chain reaction (RT-PCR) tests in interpreting SARS-CoV-2 data in relation to semen and testicular tissues analyses without appropriate controls. Here we critically analyse the evidence so far and the possible implications. The diagnostic test limitations posed in both sampling and testing methodologies, their validation, and relevancy in interpreting data are also highlighted.

raising equal concerns for embryo and fetal development (Colaco et al., 2020) .In males, ACE2 receptor sites have been reported in testicular tissue which then have the capability to harbour SARS-CoV-2 virus and eventual shedding into the semen and hence its implication in sexual transmission, early pregnancy or early in utero embryonic development. This paper concerns the available information on SARS-CoV-2 and the male factors.

As SARS-CoV-2 is a novel strain of virus, there is little information on the mechanism by which it seems to invade the respiratory system and relies on information from the previous SARS or influenza studies. Therefore, SARS-CoV-2 viral RNA in a specimen cannot be directly interpreted as a potential for disease transmission and infection. Furthermore, viral RNA can be detected long after the disappearance of the infectious virus. In the most recent reports the viral load data is absent. Viral culture to evaluate viral virulence and activity are so far absent in clinical practice because of the relatively long turn-around time and its low sensitivity for virus detection. The virus can be neutralised by the immune system by lysing their envelope or aggregating virus particles, thereby preventing subsequent infection. However, the nucleic acid remains and is degraded over time. The presence of nucleic acid in PCR alone cannot be used to define viral shedding or infection potential as experienced with other viral diseases such as SARS-CoV, Middle East respiratory syndrome (MERS) coronavirus, influenza virus, Ebola virus, Zika virus (Atkinson, 2020) .

The main SARS-Cov-2 entry into cells appears to be via the viral spike (S) protein attaching to the Angiotensin Converting Enzyme 2 (ACE2) and employing the cellular serine protease (TMPRSS2) (Hoffmann et al., 2020) . Both ACE2 and TMPRSS2 are also present in the testis and therefore testicular infection and subsequent sexual transmission is gaining interest amongst scientists. Studies analysing SARS-CoV-2 in seminal fluid or testicular biopsies have so far lacked appropriate controls and patients suffered from predominantly mild infections and tested several weeks after the infection, thereby increasing the complexity of result interpretation. A study conducted in 34 men, 25-75 days after mainly mild initial SARS-Cov-2 infection, showed that there was an absence of virus using RT-PCR in the semen (Pan F et al., 2020) . This may be causally reassuring (Eisenberg ML., 2020) but such cases cannot be generalised to those suffering severe infections and also does not account for the limitations of PCR test interpretation described earlier.

In contrast, of 38 men providing semen samples, 23 men (60.5%) had clinically recovered, whilst 15 men (39.5%) were at the acute stage of infection. SARS-CoV-2 in semen from 6 patients (15.8%) was positive, including 4 of 15 patients (26.7%) who were in the acute stage of infection. However, 2 men (8.7%) were recovering, which is particularly noteworthy . Other viruses such as Zika, HIV and CMV are found in semen and it is possible for viral shedding to occur, if the viral loads due to the severity of infection are high. This is especially true if the bloodtestes/deferens/epididymis barriers are damaged due to systemic local inflammation. It is unlikely that SARS-CoV-2 could replicate in the male reproductive tract Considering SARS-CoV-2 infected male patients, is there a possibility that a virus with a size of 70-90 nm may breach the blood-testis barrier? Furthermore, is the mechanism of filtering out SARS-CoV-2 so complex and efficient that ACE2 could be absorbing the entire SARS-CoV-2 viruses, in which case should we be analysing the alterations to the ACE-2 receptor sites instead? In particular, this applies to the testes, where immune privilege may protect the immunogenic germ cells from some systemic viral infections. However, a number of viruses e.g. mumps, HIV, HHV8, Ebola, Zika are confirmed to have traversed the blood-testis barrier to elicit an immune response within the testicle (Salam et al., 2017; Zhao et al., 2014) . In the case of mumps, men can develop orchitis which is associated with testicular atrophy and azoospermia (Philips J., 2006) . There is also a report relating to SARS infection and orchitis (Xu et al. 2006) . However, the mere presence of viruses in a semen sample does not mean that the disease is sexually transmitted (Feldmann., 2018) .

On a practical level, this may have an impact for cryopreserving semen for oncology patients and highlights the need to resume fertility services. For men with older female partners, age dependent subfertility may become irreversible if clinics remain closed except for emergency oncology patients. For this reason, a measured approach is suggested to resume fertility services (Tesarik J, 2020; HFEA, 2020) . Likewise, if ovarian stimulation and fertilization were to occur, freezing the resulting embryos and delaying embryo transfer may be another strategy. The use of highly secure devices and segregated cryovessels is suggested while acknowledging the risks associated with couriering cryopreserved samples between clinics (Yakass and Woodward, 2020) . The general problem of recruiting sperm donors in light of the incubation for SAR-Cov-2 also raises uncertainties. Currently the 2-week suspension from the end of symptoms is suggested for donors showing respiratory symptoms or those who have returned from high-risk areas, seems not to be based on any particular follow-up studies or latency period for SARS-Cov-2 (La Marca et al., 2020) . No data exists on semen quality following a SARS-CoV-2 infection but it is likely the stress period will increase cortisol levels and depress sperm quality similar to the effects of febrile systemic illnesses (Chan et al., 2020) . It is important to establish under controlled experiments whether the testis could harbour SARS-Cov-2 and its dormancy period before drafting policies on reproductive health (Cardona-Maya., 2020), while risk managing patients and staff (Anifandis et al., 2020) .

Against this backdrop are the numerous publications concerning SARS-Cov-2 and reproductive health in non-peer reviewed MedRxiv platform (https://www.medrxiv.org/) which cautions readers about its use to guide clinical practice or health-related behaviours, while urging news media to refrain from reporting these.

The non-peer reviewed papers suggest that there is no clinical evidence on whether SARS-CoV-2 infection can affect male gonadal function. In a study of 81 reproductive-aged men with SARS-CoV-2, the serum luteinizing hormone (LH) was significantly increased, but the ratio of testosterone (T) to LH and the ratio of follicle stimulating hormone (FSH) to LH were dramatically decreased . There were no semen parameters reported alongside this, whilst stress and corticosteroid therapy may also affect the hypothalamic-pituitary-gonadal axis separately. The question on whether SARS-CoV-2 can directly infect the testes or the male genital tract and be sexually transmitted from mildly infected males has been considered (Song C., 2020) . In this study, the RT-PCR result was negative in 11 semen and 1 testicular biopsy specimens from a deceased 67-year-old. While they concluded that there is no evidence of sexual transmission of SARS-CoV-2 from males there is no data to allow this statement with the serious limitations of interpretation raised earlier. For example, these studies should have included PCR results from respiratory tissues or ocular fluids in optimally infected cases most likely to show viral presence. Also no SARS-CoV-2 was detected in expressed prostatic secretion (EPS) of 18 confirmed Covid-19 infected patients and 5 strongly suspected cases but absent semen analyses. They comment on the safety of sexual intercourse and virus transmission remaining unknown and yet there is no data to show (Quan et al., 2020) . Furthermore, some papers take a leap into the unknown by analysing vaginal fluids from 35 female patients for SARS-CoV-2 as a mode of sexual transmission and where 42.9% of partners were subsequently infected, completely disregards the lack of social distancing (Cui et al., 2020) . All these studies lack adequate controls, quality of infected males well away from the infected period. We have further highlighted the limitation of interpretation from the diagnostic tests. Despite the warnings on using these data, it is the appearance in the media which seems impossible to correct.

Standard testing kits for the public have so far used non-validated procedures for antibodies of which there may be up to a 50% false negative rate (Blanchard S, 2020) . In hospital and research establishments testing of bodily fluids and tissues is expected to be more sensitive but these too have not been validated for diagnosis of SARS-CoV-2 infection. Caution therefore needs to be exercised in accepting the validity of a SARS-CoV-2 infection test (FDA1), while the predictive values of a positive (or negative) test result is dependent on the prevalence of antibody-positive individuals in a given population as well as the sensitivity and specificity of the test. In other words, the predictive value of a serological test may be higher where there is widespread infection. To minimize false positive results, the serological tests must specifically be able to identify antibodies against SARS-CoV-2 and should not "cross-react" with other antigens or similar respiratory viruses (FDA2). Different types of clinical specimens from infected SARS-CoV-2 individuals tested by RT-PCR have shown a high degree of respiratory tissue preference; broncho-alveolar lavage fluid (93%), sputum (72%), nasal swabs (63%), pharyngeal swabs (32%), faeces (29%), blood (1%) and urine (0%) . Controlled data on specificity and sensitivity of tissue specific tests with `spiking' are absent. This limited report provides a glimmer of hope for the blood transfusion sector but equally raises questions about offering diagnostic SARS-CoV-2 testing in blood samples against swab tests.

Overall, little confidence exists in the clinical application of serological antibody tests showing heterogeneous assay performance. In SARS-CoV-2 RT-PCR-positive individuals, seropositivity showed time dependence and peaked at 81.8-100.0% in samples taken >20 days after the onset of symptoms. Test specificity ranged from 84.3-100.0% in pre-COVID-19 specimens and IgM detection was more variable than IgG, with optimal detection when both IgM and IgG results were combined. Agreement between two ELISAs and ten lateral flow assays ranged from 75.8-94.8%. Improving serological tests will require evaluations of the full spectrum of SARS-CoV-2 infections, from asymptomatic individuals or mild infection to severe disease, and later convalescence (Whitman et al., 2020) .

In contrast, RT-PCR which is used extensively in diagnostic virology and expected to give a precise result is lacking in several key aspects. The SARS-CoV-2 infection validation has been against previous 2002/03 SARS viral strains and aided by the use of synthetic nucleic acid technology (Corman et al., 2020) . Other SARS-CoV RNA has been used as a positive control. Whilst the molecular assay sensitivity for SARS-coronavirus (SARS-CoV) is too low for early stages of the infection, the sensitivity improves with the severity of infection to optimize the test . These types of tests normally take months rather than weeks to validate. While work is in progress to improve the diagnostic tests, the limits of using the tests along with the sample type, fluid or tissue, sampling technique, severity of the infection and above all using appropriate controls need to be factored in the results. RT-PCR swab kits which give a positive result does not necessarily mean that infection exists, as the virus is not known to be alive and active. RT PCR sensitivity in symptomatic patients is approximately 60-95% while good sampling collection and virus transfer is key to false negatives.

Whilst population testing targets preoccupies the political-public arena, little attention is paid to critically appraise the underlying inaccurate diagnostic science, highly commercialised without complete validation. This false reassurance constitutes a major risk factor to the public through free movement of virus releasing spreaders. Therefore, the best policy is to presume that all patients are infected and to use the best available infection prevention and PPE possible during this pandemic. The limitations of tests also cast doubt on the many publications coming through as to how to interpret a negative test result. To understand the tests, the public needs to understand the underlying science.

The design and validation for SARS-CoV-2 RT-PCR testing was framed close genetic relatedness to the 2003 SARS-CoV virus and aided by the use of synthetic nucleic acid technology (Corman et al., 2020) . This was further improved with the availability of the SARS-CoV-2 genome sequence and incorporating variant viruses from an animal reservoir to theoretically ensure broad sensitivity. Sampling problems contribute to false negatives and multiple site sampling may improve the test sensitivity . The rapid introduction of new diagnostic tests and controls during this evaluation study may add to the uncertainty of measurements. In non-pregnant patients falsenegative results ranged from 17-63% for SARS-CoV-2 RT-PCT (Kelly et al., 2020) . There was no clear gold standard test available. Diagnostic test characteristics included sensitivity and specificity, while positive and negative predictive values of SARS-CoV-2 RT-PCR assays are difficult to determine. SARS-CoV-2 RT-PCR false-negative testing has major clinical implications, especially in pregnant women and in men with suspicion of severe/critical SARS-CoV-2infection and for the research reports emerging. Two individuals, with a history of exposure to SARS-CoV-2-infected patients showed positive results on RT-PCR a day before onset , thereby suggesting infected individuals can be infectious before they become symptomatic. Finally, the timing of sampling needs to be understood to ensure optimal detection is possible. Unlike the earlier SARS virus peaking at 10 days, the SARS-CoV-2 peaks at around 5-6 days after symptom onset. Sputum samples generally showed higher viral loads than throat swab samples, while in the same two patients viral RNA was not detected in urine or stool samples , raising the question whether severity of illness would alter detection at other sites and whether adequate viral spiking controls were performed to rule out sensitivity of testing for these sites. Clearly as many as 3-5 repeat SARS-CoV2 RT-PCR testing may be required for a positive result .

The WHO recommends two negative SARS-CoV-2 RNA PCR tests, at least 24 h apart, before discharge although this is not widely practiced (WHO, 2020) . Prolonged presence of SARS-CoV-2 RNA may suggest the potential for viral replication to occur. The incubation time for SARS-CoV-2 also remains unclear to provide guidance to patients and clinics, especially when fertility services are to resume.

Due to the limitations in the accuracy of the diagnostic methods, every man with infertility and having a semen test and all women should be treated as potentially positive and infective even in case of negative results. SARS-CoV-2 has brought unique challenges to the global reproductive healthcare community finding newer ways to risk manage patients (Anifandis etal 2020)

Unravelling the mechanism of SARS-CoV-2 entry into semen and the testes would help us assess the early impact on male reproductive function. Equally, the impact of SARS-CoV-2 related stress and fever manifesting in depressed semen quality can not be underestimated and also possible intergenerational effects (Chan et al., 2020) . The limited and poorly reported small studies in seminal plasma and the testes lack adequate controls to establish evidence-based guidelines for the public nor are they able to produce guidance on sexual practices or reproduction, given the lack of social distancing appears a major risk. The main weaknesses of reports relate to poorly validated diagnostic procedures, lack of controls, mildly infected males tested several weeks later along with the fact that vital respiratory tissue most likely to harbour the viral RNA has never been reported alongside as well as the lack of control analyses.

The proof of SARS-CoV-2 shedding into semen or in the testes is still not established and there is a lack of good quality evidence. The findings are consistent with all reports so far. Therefore, no firm conclusions can be drawn on sexual transmission of SARS-CoV-2 but it makes sense to use barrier protection to minimise STD generally. The degree of SARS-CoV-2 detection uncertainty seems to have evaded public scrutiny, while media reporting from mainly non-peer reviewed reports is unacceptable. Further studies are required at all levels to help improve the quality of evidence to understand the effects of SARS-CoV-2 on male fertility and the health of the testes.

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