Researchers found that SARS-CoV-2 testicular tropism is higher than previously thought

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Brazilian And American Study Discovers That The Testes Of Males Could Be Viral Sanctuaries For The SARS-CoV-2 Virus.

Alarming findings from a new study by researchers from Universidade Federal de Minas Gerais-Brazil, University of Texas Medical Branch-USA, University of Pittsburgh School of Medicine-USA, Faculdade de Medicina de São Jose do Rio Preto-Brazil, Universidade Estadual Paulista-Brazil and Clínica MF Fertilidade Masculina-Brazil shows that the SARS-CoV-2 infects, replicates, elevates angiotensin II and activates immune cells in human testes. These findings also implicate that the human male testes is perfect reservoir for the SARS-CoV-2 virus.

The study findings were published on a preprint server and are currently being peer reviewed.

https://www.medrxiv.org/content/10.1101/2022.02.05.22270327v1

Since the testis displays one of the highest expressions of Angiotensin Converting Enzyme 2 (ACE2) receptors, which mediate the cellular entry of SARS-CoV-2, and current data suggest that men are more affected than women(1), deep testicular evaluations of patients affected by COVID-19 is imperative.

Previous studies present discordant results concerning SARS-CoV-2 detection in testicular parenchyma through RT-PCR (2–5)and the extent of testicular damage caused by the virus(5). Moreover, many questions about viral infection remain unexplored, such as the viral replication, route of infection, and the identity of infected cells.

Although some testicular alterations promoted by SARS-CoV-2 infection were previously demonstrated(2–5), the players of testicular pathogenesis in COVID-19 remain unknown. Thus, studying the cellular, enzymatic, hormonal, and critical gene alterations in the testes of COVID-19 patients should contribute to a better understanding of SARS-CoV-2 biology and its possible impact on testes and male fertility. Recently, Edenfield and Easley(6) published a perspective article in Nature Reviews Urology urging the need to unveil the potential mechanisms involving SARS-CoV-2’s entry and pathophysiological effects on affected subjects.

We used different methods to detect SARS-CoV-2 in the testis parenchyma of patients deceased with COVID-19 and investigated the virus infective and replicative capacities. We also revealed the cellular and molecular alterations in human testicular pathophysiology and correlated the findings with the patients’ clinical data, unveiling potential mechanisms underlying the observed alterations


SARS-CoV-2 reliable detection in testes

We first tested the testicular tissue using a conventional RT-qPCR protocol for SARS-CoV-2 and only patient #8 was positively detected (cycle threshold – CT=38).

To improve detection and reduce interference of intrinsic tissue factors, we performed a cDNA synthesis using SARS-CoV-2 specific viral primers. The RT-qPCR revealed the virus presence in 10 of 11 patients (Table 2). Previous studies presented conflicting results regarding the detection of SARS-CoV-2 RNA in the testes(2–5).

To confirm the genetic data, we used a nano-designed sensor (which employs localized surface plasmon resonance – LSPR)(11) to detect the SARS-CoV-2 Spike (S)- protein and the nucleocapsid (N)-protein (Supplemental Fig.1a-q). S-protein was found in all, while N-protein was observed in nine patients (Table 2 and Supplemental Fig.1m- q).

Prominent S-protein immunolabeling was evidenced in testes of all COVID-19 patients (Supplemental Fig.2), especially in patient #8 (positively detected by all methodologies). These findings suggest that the SARS-CoV-2 tropism for testes is higher than previously thought and that conventional RT-qPCR protocol may only detect infected testes with a higher viral load. Our data suggest that more sensitive techniques are required for the reliable detection of SARS-CoV-2 (even in a low viral titer) in testes.

Main infected cells in testis parenchyma

Several infected monocytes/macrophages (CD68+) were detected surrounding blood vessels and migrating to the parenchyma (IF and TEM data), suggesting that these cells might be delivering SARS-CoV-2 to testis (Fig.1a-c, arrows), contributing to infection of testicular cells. Infected macrophages were confirmed through double immunofluorescence and TEM (Fig.1d-g).

Monocytes/macrophages were also found inside the tubular compartment, indicating a possible route for viral spreading inside the seminiferous tubules (Fig.1h). Conversely, chymase positive mast cells were not labeled for S-protein (Supplemental Fig.3a-b).

Most S-protein labeling was identified inside the seminiferous tubules, mainly in germ cells (Fig.1 i-m). While we show germ cells positive for the S-protein, other authors showed positivity for the N-protein(4). In some areas, spermatogonial cells displayed intense S-protein labeling compared to spermatocytes and spermatids (Fig.1n, red arrowhead).

Nevertheless, SARS-CoV-2 S-protein was detected in spermatogonia, spermatocytes and spermatids (Fig.1o-t). Patients who died up to 20 days after intensive care unit (ICU) admission presented the highest mean fluorescence index in the testis (Supplemental Fig.2a). Germ cells infected with SARS-CoV-2 increase the concerns of potential sexual transmission, reinforced by the detection of SARS-CoV-2 RNA in semen of patients who suffered the severe form of COVID-19(17).

Some Sertoli, Leydig (highly express ACE2(18)), and peritubular myoid cells also presented viral particles in their cytoplasm (TEM data), albeit with lower S-protein immunolabelling intensity (Fig. 2a-c’, arrowheads and insets). However, many of these cells did not show obvious viral particles in the cytoplasm (TEM data) (Fig. 2e-j).

Viral replication and viability

TEM analyses identified a viral replication factory in macrophages, which are known to express ACE2 and TMPRSS2(19), and in spermatogonial cells, which highly express TMPRSS2(18) (Fig.3a-f). In both cell types, we observed SARS-CoV-2 replication complexes with the formation of convoluted membranes (replication membranous webs, RMW) containing double-membrane vesicles (DMV) and Endoplasmic Reticulum Golgi Intermediate Complex (ERGIC) showing new virions (Fig.3c-f). These morphological features are in accordance with SARS-CoV-2 replication in the cell cytoplasm(20).

The testicular immune privilege prevents the autoimmune attack of haploid germ cells, but it also allows viruses to escape immunosurveillance(21). The “macrophage paradox”, described in SARS-CoV-2-induced lung damages(19) may also apply to the testes. Thus, although macrophages combat viral infections, they can act as Trojan horses(19), facilitating viral entrance and replication in testis.

While it is possible that the testis can be infected by direct invasion caused by viremia, our results point that infected monocytes/macrophages migrating (e.g., from lungs) may also be actively transporting the virus into the testes(22). Identifying viral access to the testis and the local of replication are relevant because testicular immune tolerance may hamper the viral clearance from the human body, like what is observed for other viruses(21).

In a BSL-3 lab, we exposed VERO CCL-81 cell cultures to testicular homogenates from an infected patient and found that SARS-CoV-2 was infective and able to replicate in these cells. This data was confirmed by immunohistochemistry, the cytopathic effect in the infected VERO cell culture and conventional RT-qPCR (Fig.3g-j), indicating that we were not observing just fragments of viral particles.

Previous research indicated that SARS-CoV-2 negativity in RT-qPCR tests (at least in two consecutive assays) usually occurs between 6-12 days from the time of onset

of symptoms(23). However, according to our data, the virus remains viable for a longer period within the testis. Indeed, SARS-CoV-2 was detected in the testis of patient #1, who died 26 days after symptoms’ onset (Fig.3k). Thus, our findings suggest that the testes may serve as sanctuaries for SARS-CoV-2, maintaining infective viruses for extended periods. Testis environment can even be related to the delayed viral clearance in men compared to women(24). Furthermore, the testes may not be neglected in evaluating the patients’ clinical condition because it is a site of viral replication and consequently a source of viral load.

Testis fibrosis and vascular alterations

Testicular fibrosis was commonly observed in the testis parenchyma of COVID- 19 patients. Collagen fibers increased progressively (Fig.6o-u), showing high amounts of type I and III collagen in testis parenchyma (Fig.6t-u, Supplemental Fig.5e-h). Further, the expression levels of Col3a were highly augmented in the testes of phase I patients (Fig.6e). It is known that Angiotensin II, activated mast cells, and activated macrophages stimulate collagen synthesis by fibroblasts(50–52).

Expressions of the angiogenic factors VEGF and PDGFA and small vessel volumetric proportion (CD31+) increased in the testis of COVID-19 patients (Fig.6e). Immature blood vessels (TEM and Immunohistochemistry data; CD31+ cells) were observed inside the tunica propria, suggesting an angiogenic process (Fig,6v and Supplemental Fig.5i-m). Red blood cells outside the testicular vasculature were frequently observed in the testes of COVID-19 patients (Fig, 6w-α, arrows). First phase patients presented many red blood cells inside the seminiferous tubule lumen and intratesticular rete testis, possibly due to a reduction in tight epithelial junctions (Supplemental Fig.5n). Some thrombi were detected inside the vascular system (Supplemental Fig.5o).

Furthermore, Endothelin 1/2/3, a protein involved in blood-vessel vasoconstriction, was highly expressed in the first phase patients but reduced in others

(Supplemental Fig.5q-t). The triad, angiotensin II, activated mast cells, and activated macrophages can increase vascular permeability(31,53,54).

Concerning the study limitation, we should highlight important points of the current study. Notably, the chronology of testicular pathogeny was based on the presence of advanced germ cells in testis parenchyma and the time of ICU admission. Although our histological and molecular alterations reinforced this conceptual classification, the data of all individuals were also presented in a single group. Moreover, only severely ill patients, who died from COVID-19, were included in the study.

We should mention that recent data on semen demonstrate that patients recovered from COVID-19 reestablish their sperm quality after three months of the infection(54). Not all COVID-19 patients studied presented the same comorbidities, and Controls were not submitted to the medications used for COVID-19 patients.

On the other hand, we managed to harvest and process the testicles on the same day as the death occurred, in contrast with many cadaver studies with long organ collection delays, which may have compromised the precise histology, detection of virion particles, and perception of peptides, mRNA and hormones fluctuations.

CONCLUSIONS

Herein, we used different and sensitive methods to detect SARS-CoV-2 proteins, RNA, and virus particles in the testis. In figure 7, we hypothesized the potential viral, cellular, and molecular mechanisms of infection and damage by SARS-CoV-2 in testes of non-vaccinated and severely ill patients.

A direct influence of SARS-CoV-2 in testicular cells might deregulate ACE2, elevating the levels of angiotensin II, a potent pro-inflammatory and angiogenic peptide. Angiogenic and inflammatory factors might induce the infiltration and activation of mast cells and macrophages. Therefore, the

deleterious effects evidenced in the testes of COVID-19 patients, i. e. fibrosis, vascular alteration, inflammation, tunica propria thickening, Sertoli cell barrier loss, germ cell apoptosis, and inhibition of Leydig cells, may be associated with elevated angiotensin II and activation responses of mast cells and macrophages (Fig. 7a).

A protein-protein (or gene-gene) network constructed from the STRING database (Fig. 7b) shows the interaction of genes associated with angiotensin II regulation, activation of mast cells and macrophages, and testis tubular and intertubular compartment alterations.

Our multidisciplinary findings might contribute to a better understanding of SARS-CoV-2 tropism, biology, and impact on testes and male fertility. This is the first study that shows:

1) the high SARS-CoV-2 tropism to the testis;

2) one mode of SARS- CoV-2 entrance in testes;

3) SARS-CoV-2 preferred infection and replication in spermatogonia and macrophages;

4) that the virus remains infective after a long infection period in the testes (viral reservoir);

5) high levels of angiotensin II and activated mast cells and macrophages are critical players in promoting all testicular alterations;

6) the more extended severe condition, the lower the number of surviving germ cells;

7) fluctuation in several essential testicular genes;

8) that the intratesticular testosterone levels are 30 times reduced in testes of COVID-19 patients;

9) the prevalent types of collagen present in SARS-CoV-2 mediated testicular fibrosis;

10) the fluctuation of vasoconstrictive peptides in testes of COVID-19 critically ill patients.

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