(iv)
The number of spermatozoa with DNA fragmentation is increased.
(v) Infection with SARS-COV-2 may lead to potential fertility issues.
J o u r n a l P r e -p r o o f
Coronaviruses (COVs) are a group of positive single stranded RNA viruses, and they belong to order Nidovirale, family Coronaviridae and sub family Coronavirinae [1, 2] . COVs are crownlike in morphology and have up to 26-32 genome kilobases [3] . These viruses have been shown to infect animals (birds), and human, and as such, they have up to four identified genera, namely, the alpha (α)-COV, beta (β)-COV, gamma (γ) -COV and delta (δ)-COV. The αand β-COVs are the prominent human Coronaviruses (HCOV), while the γ-and δ-COVs are normally seen in birds [4] . Till date, seven COVs (2-α and 5-β) have been identified in human, including HCOV-229E, HCOV-OC43, severe acute respiratory syndrome (SARS-COV), HCOV-NL63, HCOV-HKU1, Middle East respiratory syndrome (MERS-COV) and the most recently discovered severe acute respiratory syndrome-2 (SARS-COV-2).
The first human COV to be identified was the HCOV-229E strain, which was isolated from the nasal discharge of patients with upper respiratory tract infection in the 1960s [5] . A year later, Mcintosh et al. reported the presence of HCOV-OC43 from the organ culture of respiratory tract infected patients [6] . Patients infected with both HCOV-229E and HCOV-OC43 presented with common cold symptoms, such as headache, sneezing, malaise, and sore-throat. HCOV-OC43 was later found to possess neurotropism in both in vitro cultured cells and infected mice [7, 8] . SARS on the other hand was first reported in 2002, as patients infected with SARS-COV presented with respiratory distress, cough, fever, headache, myalgia, malaise, and chills [9] .
In 2003, the SARS epidemic affected about 26 countries with more than 8000 cases and over 700 deaths [10] . The transmission occurs primarily from person to person, and the incubation time ranges from 3 to 14 days, which corresponds to the peak of viral load in the respiratory J o u r n a l P r e -p r o o f discharge and stool [10] . While the SARS epidemic was still ongoing, HCOV-NL63 and HCOV-HKU1 were discovered. In 2004, HCOV-NL63 was first isolated from the nasal discharge of a seven-month-old suffering from respiratory tract infection [11] . In 2005, Woo et al. detected a strain of HCOV-HKU1 from a 71-year-old man with pneumonia and bronchiolitis [12] .
Another β-HCOV is the MERS-COV which was first reported in Saudi Arabia in 2012. Since the first reveal, about 27 countries have disclosed cases of MERS [13] . About 35% of the reported patients infected with MERS-COV died. The common MERS symptoms include cough, fever, and shortness of breath and sometimes pneumonia [13] .
Seven years after the outbreak of MERS, Wuhan city of China reported the emergence of SARS-COV-2 [14] . In March 2020, the World Health Organization (WHO) declared the outbreak as a global pandemic, given its severity and rapid spread. As of September 14, 2020, more than 29 million cases have been reported, with over 900,000 deaths globally. Symptoms of SARS-COV-2 include fever, dry cough, tiredness, headache and sore throat, amongst others.
Since the discovery of SARS-COV-2 and its implication to cause coronavirus disease 2019 , studies have investigated the epidemiology, aetiology, clinical features, clinical treatment, and prevention of the disease. Studies have shown SARS-COV-2 to be present in saliva, respiratory fluids, blood, urine, and faeces. The extent of organ damage outside of the respiratory and cardiovascular system is still clinically unpredictable.
Despite that findings regarding the presence of SARS-COV-2 in the semen or the testis are limited and controversial, concerns relating to transmission through this route should not be overlooked [15] . The reason for this concern is that Ebola virus was reported to remain in the semen samples of patients up to nine months post recovery [16] [17] [18] . Similarly, Zika virus was also reported to be sexually transmitted [19] .
Hence, this review aimed to analyse the literature regarding (i) the effects of previously identified human coronaviruses on male fertility (ii) the impact of Covid-19 on male fertility and (iii) the implication for general health in terms of infection and transmission.
A literature search of studies published on HCOVs until September 2020, was carried out using
PubMed. To look at the impact of these HCOVs on male fertility, each HCOV strain was meshed with the following terms, "semen", "sperm", "testis", "male reproduction" and "male fertility", and searched for on PubMed (e.g, "HCOV-2299E and semen", "HCOV-229E and sperm"). The search was limited to full length articles published in English. The results of the search terms as of September 18 2020 are illustrated in Figure 1 . The vetting process includes:
(a) The mesh terms were searched for. This include "HCOV-2299E and semen", HCOV-229E and sperm", HCOV-229E and testis", HCOV-229E and male reproduction", and HCOV-229E and male fertility". The remaining HCOVs followed the same sequence as HCOV-229E.
(b) The titles of articles were screened, and if title conforms with the mesh term, the abstract was read.
(c) If abstract correlates with the aim of the current review, the whole article was read. 
The common denominator in the route of transmission/ infection of the previously identified HCOVs (HCOV-229E, HCOV-OC43) is through respiratory droplets and fomites, with SARS-COV and MERS-COV being transmitted through respiratory droplets, fomites and faecal-oral routes [20] . Very limited studies suggested the plausibility of transmission through the semen and what the general health implication is, when present in the testis.
After the isolation of HCOV-229E, it was reported to cause bronchiolitis and mild infection/inflammation of the upper respiratory tract in few patients [21] . The only study that investigated the impact of HCOV-229E on reproduction accessed the maternal vaginal and respiratory mucus during labour and the new-born gastric sample, of mother-child couples.
The authors reported the presence of HCOV-229E in three samples and hence suggested the possibility of vertical transmission during infection [22] .
HCOV-OC43 is not only present in the upper respiratory tract, and consequently cause the inflammation thereof, it was also implicated to play a role in neurological diseases, as findings have shown that this virus invades the central nervous system [23] [24] [25] . Dubé et al. reported that one of the processes involved in the neuropathogenesis of HCOV-OC43 is the neuron-toneuron propagation of this virus which is aided by axonal transport [26] . Keyaerts et al. showed that HCOV-OC43 infection in new-born mice can be treated with chloroquine acquired transplacentally or through maternal milk [27] . From these findings, the following can be suggested ( and consequently leading to diverse pathologies [28] . Some of the pathological findings reported, include severe degeneration of the renal distal tubule epithelium, hyaline membrane formation, exudation of fibrin fluid and damage to the aveoli of the lungs [29, 30] .
Regarding the presence and the impact of SARS-COV on male reproduction, findings remain controversial. Ding et al. reported that SARS-COV was not detected in the testis [28] , while Xu et al. not only showed its presence in the testis, they also reported its role in orchitis [31] .
Their results showed that the testes of patients who died from SARS exhibited germ cell destruction, absence of spermatozoa in the lumen of the seminiferous tubule, thickened basement membrane and leukocyte infiltration. It was suggested that the adverse impact seen may be as a result of (i) high fever that occurs in SARS and (ii) the treatment of SARS with steroids which could affect spermatogenesis [32] . Additionally, Zhao et al. reported that SARS-COV was found in the testicular epithelial cells and Leydig cells using electron microscopy [33] . These studies agreed on the extensive inflammatory response with cytokines release accompanying SARS infections. Hence, it can be suggested that these microvascular changes could provoke obstructive azoospermia on short term and psychological effect of the illness could result in endocrine disturbances.
This collectively suggests that even if SARS-COV is not present in the semen, it can negatively impact spermatogenesis, and ultimately impair male fertility.
During the SARS epidemic, an additional two strains of HCOV were identified (HCOV-NL63 and HCOV-HKU1) [11, 12] . HCOV-NL63 was identified as the key pathogen responsible for croup in children [34] [35] [36] , while Infection with HCOV-HKU1 has been shown to be associated with pneumonia and bronchiolitis [12] . However, no report was found on the impact of both HCOV-NL63 and HCOV-HKU1 on male reproduction.
Seven years after the discovery of HCOV-HKU1, MERS-COV, another HCOV that is known to cause severe upper respiratory tract infection, and other pathological conditions were identified. Studies from different parts of the world have shown the susceptibility of pregnant women to MERS-COV infection [37] [38] [39] [40] . In Saudi Arabia, a group of authors reported a still birth in a woman infected with MERS-COV at 5 month of gestation [37] , while a woman died after giving birth in the United Arab Emirates [39] . In Korea however, although a 39-year-old pregnant woman was infected with MERS-COV, she delivered and reached full recovery [40] .
To the best of our knowledge, no human study linking MERS-COV to male fertility has been reported. However, a recent study conducted by Hemida et al. reported that MERS-COV was detected in the seminal plasma from camels [41] .
Looking at the findings that SARS-COV was detected in the testis and the consequent testicular architectural alteration, and its role in orchitis, together with the findings that MERS-COV was found in the seminal plasma of dromedary camels, it is not unreasonable to believe that (i) SARS-COV-2 may also cause testicular dysfunction and (ii) SARS-COV-2 may be plausibly transmitted through the semen.
In comparison to the other two pathogenic COVs (SARS-COV and MERS-COV), studies on SARS-COV-2 are quite numerous (Figure 1 ). This is due to the rapid and harsh spread of the virus, and how this has affected the global mortality and morbidity rates. Many key discoveries have been made with respect to the epidemiology, diagnosis, cause, and treatment of Covid-19. Studies have shown SARS-COV-2 to be present in body fluids and faeces [42] . Findings regarding its presence in the semen and/or the male reproductive tract at large are controversial. This section of the review will highlight original studies that have: including two from the 23 recovered patients and four from the acutely infected patients [43] . is or are any pathological changes in the testis of Covid-19 patients [52] . After accessing the testicular tissues of twelve patients, they reported the absence of SARS-COV-2 in the testis of 90% of the cases. However, the testes of all the patients exhibited significant seminiferous tubular injury, reduced Leydig cell number, swelling of the Sertoli cells and mild lymphocytic inflammation. Zhang et al. also reported the absence of SARS-COV-2 in the prostatic secretion of ten covid-19 patients [53] . The sera of these patients, however, showed increased levels of inflammatory biomarkers.
From the studies available, SARS-CoV-2 has exhibited relevant characteristic factors supporting the views that COVID survivors might develop sexual and reproductive health issues on the long run. This is partly because the protein that enables SARS-COV-2 entry into the cell, angiotensin-converting enzyme 2 (ACE 2), is widely distributed in the testis, including spermatogonia, the Leydig and Sertoli cells. Hikemet et al., reported that amongst over 150 investigated cell types, which correspond to human tissues and organs, the expression of ACE 2 was higher in some tissues/organs, including the male reproductive organs [54] . Viral entry requires binding of spike (S) glycoproteins of SARS-COV-2 to the host receptor. Upon entry, the host proteases, such as transmembrane serine protease 2 (TMPRSS2) cleaves the viral S protein to stimulate a conformational change, to allow fusion of the viral and host cell membranes [55, 56] . Represented in Figure 2 is the summary of SARS-COV-2 entry into the cell. Zhen et al. also reported the distribution of ACE 2 in the testes, and that it was highly expressed in the testes of infertile men compared to the control [57] . This suggests that SAR-COV-2 may cause male reproductive disorder and that men with reproductive issues may be more susceptible to infection.
On the other hand, the proportion of mortality by sex is higher in men. Hence, SARS-CoV-2 causes a more severe outcome in men, which is a disadvantage compared to women [58] . It is, therefore, vital to determine the factors involved in these differences.
Bringing all the findings together, although most of the studies reported the absence of SARS-COV-2 in the semen and prostatic secretions, as well as testicular tissues, which may reduce the possibility of transmission through this route, it is evident that (i) there is testicular injury and inflammatory infiltration (ii) viral orchitis may occur, as patients experienced scrotal discomfort (iii) there is altered semen parameters and (iv) the number of spermatozoa with DNA fragmentation is increased. These results collectively suggest that infection with SARS-COV-2 may lead to potential fertility issues.
The question to be asked at this point is how these adverse effects are exerted. Although the mechanism (s) involved is largely unknown, studies have implicated the main receptor ACE 2 to which SARS-COV-2 binds to play a role. subunits, namely S1 and S2 subunits. Binding of S1 leads to a conformational change in S2, hence the primary function of S1 is to bind, while that of S2 is to fuse. For viral entry, the Nterminal of S (S1) binds ACE2 receptor. Binding of S1 to ACE2 leads to a conformational change in S2. During the conformational change, viral protein cleavage occurs, which is mediated by receptor transmembrane protease serine 2 (TMPRSS2). Following protein cleavage, fusion between viral and cellular membrane occurs, which subsequently result in the entry of the virus into the cell, thereby releasing its content.