High SARS-CoV-2 tropism and activation of immune cells in the testes of non-vaccinated deceased COVID-19 patients

  1. Guilherme M. J. Costa
  2. Samyra M. S. N. Lacerda
  3. André F. A. Figueiredo
  4. Natália T. Wnuk
  5. Marcos R. G. Brener
  6. Lídia M. Andrade
  7. Gabriel H. Campolina-Silva
  8. Andrea Kauffmann-Zeh
  9. Lucila G. G. Pacifico
  10. Alice F. Versiani
  11. Maísa M. Antunes
  12. Fernanda R. Souza
  13. Geovanni D. Cassali
  14. André L. Caldeira-Brant
  15. Hélio Chiarini-Garcia
  16. Fernanda G. de Souza
  17. Vivian V. Costa
  18. Flavio G. da Fonseca
  19. Maurício L. Nogueira
  20. Guilherme R. F. Campos
  21. Lucas M. Kangussu
  22. Estefânia M. N. Martins
  23. Loudiana M. Antonio
  24. Cintia Bittar
  25. Paula Rahal
  26. Renato S. Aguiar
  27. Bárbara P. Mendes
  28. Marcela S. Procópio
  29. Thiago P. Furtado
  30. Yuri L. Guimaraes
  31. Gustavo B. Menezes
  32. Ana Martinez-Marchal
  33. Kyle E. Orwig
  34. Miguel Brieño-Enríquez
  35. Marcelo H. Furtado

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Abstract

Background

Cellular entry of SARS-CoV-2 has been shown to rely on angiotensin-converting enzyme 2 (ACE2) receptors, whose expression in the testis is among the highest in the body. Additionally, the risk of mortality seems higher among male COVID-19 patients, and though much has been published since the first cases of COVID-19, there remain unanswered questions regarding SARS-CoV-2 impact on testes and potential consequences for reproductive health. We investigated testicular alterations in non-vaccinated deceased COVID-19-patients, the precise location of the virus, its replicative activity, and the immune, vascular, and molecular fluctuations involved in the pathogenesis.

Results

We found that SARS-CoV-2 testicular tropism is higher than previously thought and that reliable viral detection in the testis requires sensitive nanosensors or RT-qPCR using a specific methodology. Through an in vitro experiment exposing VERO cells to testicular macerates, we observed viral content in all samples, and the subgenomic RNA’s presence reinforced the replicative activity of SARS-CoV-2 in testes of the severe COVID-19 patients. The cellular structures and viral particles, observed by transmission electron microscopy, indicated that macrophages and spermatogonial cells are the main SARS-CoV-2 lodging sites, where new virions form inside the endoplasmic reticulum Golgi intermediate complex. Moreover, we showed infiltrative infected monocytes migrating into the testicular parenchyma. SARS-CoV-2 maintains its replicative and infective abilities long after the patient’s infection. Further, we demonstrated high levels of angiotensin II and activated immune cells in the testes of deceased patients. The infected testes show thickening of the tunica propria, germ cell apoptosis, Sertoli cell barrier loss, evident hemorrhage, angiogenesis, Leydig cell inhibition, inflammation, and fibrosis.

Conclusions

Our findings indicate that high angiotensin II levels and activation of mast cells and macrophages may be critical for testicular pathogenesis. Importantly, our findings suggest that patients who become critically ill may exhibit severe alterations and harbor the active virus in the testes.

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  1. Version published to 10.1186/s12915-022-01497-8
  2. PREreview

    This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/6207581.

    Main Claims & Relevance:

    This preprint investigates the impact of SARS-CoV-2 infection on the testes of males who died of the infection. Of the 11 non vaccinated male patients who were included in the study, RT-qPCR revealed the presence of the virus in 10 patients' testicles. Notably, COVID was detected in the testes up to 26 days after symptoms began, which suggests that the testes may act as a viral sanctuary harboring the virus for some time after patients may test negative by nasal swab. Upon further investigation, several virus infected macrophages were detected in blood vessels, parenchyma, and the seminiferous tubules. Additionally, spermatogonial cells displayed intense spike protein labeling. The authors claim that this suggests a "trojan horse" like mechanism where macrophages can bring the virus into the immune privileged area of the testicles and infect the tissue within. Longer duration of severe disease was correlated with a loss of germ cells, as well as fibrosis of the parenchyma and tubules. The authors claim that this may suggest that severe COVID may be associated with an increased risk of infertility.

     

    Are the findings strong, reliable, potentially informative, not informative, or misleading?

    The findings are reliable, although certain claims are not substantiated by the evidence presented in the preprint. The histological findings are well powered and controlled against a group of healthy individuals. However, the sample size is not adequate to support the claim that germ cell loss and the progression of the pathogeny is weakly or not correlated with age. The authors also comment that the claims that COVID may cause infertility in these patients are not substantiated, as it is possible that recovery may occur. In order to adequately assess this claim, further studies must be made.

     

    How might these ideas presented by the main claims further knowledge of the COVID-19 Pandemic?

    The findings of this preprint are not novel, as previous literature has revealed similar findings (1, 2). Despite this, the ideas presented by this preprint contribute to the body of knowledge of SARS-CoV-2 and thus are valuable to use inform future care of patients. As well, this preprint highlights the need for a study to be performed to illuminate the possible infertility resulting from COVID infection.

     

    References

    1. Ma, X., Guan, C., Chen, R. et al. Pathological and molecular examinations of postmortem testis biopsies reveal SARS-CoV-2 infection in the testis and spermatogenesis damage in COVID-19 patients. Cell Mol Immunol 18, 487–489 (2021). https://doi.org/10.1038/s41423-020-00604-5
    2. Duarte-Neto, AN, Teixeira, TA, Caldini, EG, et al. Testicular pathology in fatal COVID-19: A descriptive autopsy study. Andrology. 2022; 10: 13– 23. https://doi.org/10.1111/andr.13073

     

  3. ScreenIT

    SciScore for 10.1101/2022.02.05.22270327: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    EthicsIRB: The Research Ethics Committee of the Mater Dei Hospital and the National Research Ethics Committee (CONEP) approved this investigation under the number CAAE: 30999320.1.0000.5128.
    Consent: Postmortem collection of both testicles was performed after a legally responsible family member signed an informed consent document.
    Sex as a biological variableCOVID-19 PATIENTS: In 2021, we enrolled 11 non-vaccinated male patients deceased from COVID-19 complications, confirmed by SARS-CoV-2 RT-qPCR performed during their hospital stay, initially admitted in hospitals of Belo Horizonte, Brazil.
    RandomizationSeminiferous tubule measurements: Seminiferous tubules were analyzed using computer-assisted image analysis of 30 randomly chosen seminiferous tubules cross-sections per donor.
    BlindingAfter this blinded analysis, to understand the evolution of testicular pathogeny, we examined the clinical data of COVID-19 patients.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    They were covalently functionalized with the polyclonal antibody anti-Spike protein (Rhea Biotech, Brazil) and a polyclonal antibody anti-Nucleocapsid protein (CTVacinas, Brazil) through a carbodiimide-activated amidation reaction.
    anti-Spike protein (Rhea Biotech, Brazil)
    suggested: None
    anti-Nucleocapsid protein (CTVacinas, Brazil)
    suggested: None
    The binding between the gold surface and the antibodies was mediated by adding a capping layer formed by α-lipoic acid.
    α-lipoic acid.
    suggested: None
    To determine the concentration of antibodies in the LSPR-nanosensor, an antibody curve was carried out ranging from 0.25µg to 10µg for anti-S and anti-N proteins, respectively (Supplemental Fig.
    anti-N
    suggested: None
    Immunofluorescence against Spike protein: Immunofluorescence was performed using a validated primary anti-S protein antibody(12) (Rhea Biotech; IM-0828) to detect and corroborate the viral presence in the testicular parenchyma (Supplemental Table 2).
    anti-S protein antibody(12)
    suggested: None
    Immunohistochemistry in Vero cells: To confirm viral isolation from the patients’ testes, we performed an immunohistochemistry assay using the anti-S commercial antibody (Rhea Biotech, Brazil).
    anti-S commercial antibody (Rhea Biotech, Brazil).
    suggested: None
    Then, the cells were incubated overnight with the anti-S antibody (dilution 1:500) at RT.
    anti-S
    suggested: (LSBio (LifeSpan Cat# LS-C91688-500, RRID:AB_10635347)
    The cells were also incubated with an anti-rabbit IgG antibody conjugated to horseradish peroxidase (Promega, USA), diluted at 1:2500 at RT for 60 min.
    anti-rabbit IgG
    suggested: None
    Reactions were visualized using biotin-conjugated secondary antibodies (anti-goat: 1:100 dilution, Abcam, ab6740; anti-mouse: 1:200 dilution, Imuny, IC1M02; anti-rabbit: 1:200 dilution, Abcam, ab6720) combined with Elite ABC Kit (Vector Laboratories, USA).
    anti-goat
    suggested: (Abcam Cat# ab6720, RRID:AB_954902)
    anti-mouse
    suggested: None
    IC1M02
    suggested: None
    anti-rabbit
    suggested: (Abcam Cat# ab6720, RRID:AB_954902)
    Experimental Models: Cell Lines
    SentencesResources
    Vero CCL-81 cells were seeded in a T25 flask until complete confluence was achieved.
    CCL-81
    suggested: None
    Viral detection in Vero cell culture: To confirm the isolation of SARS-CoV-2, we extracted the total RNA from the supernatant using a TRIzol extraction protocol.
    Vero
    suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)
    Software and Algorithms
    SentencesResources
    A 2mM α-lipoic acid solution (LA; Sigma Aldrich, USA) in ethanol was added to the GNR suspension (0.039mg/mL).
    LA; Sigma Aldrich
    suggested: None
    Spectra analyses were performed using OriginPro version 9.0.
    OriginPro
    suggested: None
    The histomorphometric analyses were performed using the CaseViewer software (3DHISTECH, Hungary) and the Image J v.
    CaseViewer
    suggested: (CaseViewer, RRID:SCR_017654)
    Images of the two techniques were captured in a Spot Insight Color digital camera adapted for Olympus BX-40, using the Spot software version 3.4.5.
    Spot
    suggested: (Spot, RRID:SCR_018915)
    Images were analyzed with Image J v.
    Image J
    suggested: (ImageJ, RRID:SCR_003070)
    Signal detection was obtained via peroxidase substrate 3,39-diaminobenzidine (DAB; Sigma Aldrich, USA) reaction and counterstaining with Mayer’s hematoxylin (Merck, USA).
    DAB; Sigma Aldrich
    suggested: None
    Graphs and statistical analyses were conducted using GraphPad PRISM v6.0 (GraphPad Software, Inc).
    GraphPad PRISM
    suggested: (GraphPad Prism, RRID:SCR_002798)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    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.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 33, 35 and 39. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  4. Version published to 10.1101/2022.02.05.22270327v1 on medRxiv