key: cord-0727012-5q7fmrqo
authors: Duarte‐Neto, Amaro N.; Teixeira, Thiago A.; Caldini, Elia G.; Kanamura, Cristina T.; Gomes‐Gouvêa, Michele S.; dos Santos, Angela B. G.; Monteiro, Renata A. A.; Pinho, João R. R.; Mauad, Thais; da Silva, Luiz F. F.; Saldiva, Paulo H. N.; Dolhnikoff, Marisa; Leite, Katia R. M.; Hallak, Jorge
title: Testicular pathology in fatal COVID‐19: A descriptive autopsy study
date: 2021-07-16
journal: Andrology
DOI: 10.1111/andr.13073
sha: 8d4a90f5b75e816740a2d30b1b56e4fe4aac98ad
doc_id: 727012
cord_uid: 5q7fmrqo

BACKGROUND: Multi‐organ damage is a common feature of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection, going beyond the initially observed severe pneumonia. Evidence that the testis is also compromised is growing. OBJECTIVE: To describe the pathological findings in testes from fatal cases of COVID‐19, including the detection of viral particles and antigens, and inflammatory cell subsets. MATERIALS AND METHODS: Postmortem testicular samples were obtained by percutaneous puncture from 11 deceased men and examined by reverse‐transcription polymerase chain reaction (RT‐PCR) for RNA detection and by light and electron microscopy (EM) for SARS‐CoV‐2. Immunohistochemistry (IHC) for the SARS‐CoV‐2 N‐protein and lymphocytic and histiocytic markers was also performed. RESULTS: Eight patients had mild interstitial orchitis, composed mainly of CD68+ and TCD8+ cells. Fibrin thrombi were detected in five cases. All cases presented congestion, interstitial edema, thickening of the tubular basal membrane, decreased Leydig and Sertoli cells with reduced spermatogenesis, and strong expression of vascular cell adhesion molecule (VCAM) in vessels. IHC detected SARS‐Cov‐2 antigen in Leydig cells, Sertoli cells, spermatogonia, and fibroblasts in all cases. EM detected viral particles in the cytoplasm of fibroblasts, endothelium, Sertoli and Leydig cells, spermatids, and epithelial cells of the rete testis in four cases, while RT‐PCR detected SARS‐CoV‐2 RNA in three cases. DISCUSSION AND CONCLUSION: The COVID‐19‐associated testicular lesion revealed a combination of orchitis, vascular changes, basal membrane thickening, Leydig and Sertoli cell scarcity, and reduced spermatogenesis associated with SARS‐CoV‐2 local infection that may impair hormonal function and fertility in men.

The severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is a recently-emerged coronavirus that was first reported in late 2019 at the Chinese city of Wuhan and is the causative agent of the new and mostly unknown pandemic disease, COVID-19, with a wide variety of implications in human health. 1, 2 Compared to the previously described beta coronavirus, this new entity spreads strategically in silence for more extended periods before patients become clinically symptomatic and the virus is eventually identified. 3 By April 15th, 2021, the pandemic had affected almost 140 million cases and caused close to three million deaths, with the USA, India, Brazil, France, and Russia, as the five countries with the highest number of cases. 4 COVID-19 manifests mainly by respiratory symptoms. 1, 2 However, as the pandemic progresses, this paradigm has changed as cumulative evidence has shown that COVID-19 is a systemic condition. 5 Therefore, understanding multiple organ involvement is crucial to fully comprehend COVID-19 pathophysiology. 3, 6 It is believed that systemic involvement occurs because of the presence of angiotensin-converting enzyme 2 (ACE2), the cell surface receptor for SARS-CoV-2, and transmembrane serine protease 2 (TMPRSS2), responsible for priming the viral S protein to facilitate viral entry, in several organs and tissues. Recognizing the expression of these proteins throughout the human body is essential to understand the clinical manifestations and predict potential lesions in organs that are often overlooked. 7 SARS-CoV-2 infection of the male reproductive tract (MRT) has gained interest as the male gender has the highest number of cases and the highest risk of severe COVID-19, including hospitalization rates in intensive care units and a high proportion of cases with unfavorable outcomes. 8, 9 The testes have the highest ACE2 expression in the human body, according to data from the Genotype-Tissue Expression Project, 10 and this receptor is primarily found in spermatogonia, Leydig, and Sertoli cells. 11 Theoretically, this fact renders testicles highly susceptible to SARS-CoV-2 infection and damage, and, therefore, male COVID-19 patients are prone to develop testicular morphofunctional changes. 12 It remains unclear whether these potential alterations can translate into prolonged or even permanent damage, negatively impacting the fertility status or establishing an early-onset hormonal imbalance. [12] [13] [14] [15] The COVID-19 pandemic in Brazil began on February 26th, 2020. By April 15th, 2021, more than 13.5 million confirmed cases had been registered, with more than 350 thousand deaths. We have previously described the autopsy findings on our first ten fatal cases of COVID-19 (including five male patients), in which we found signs of orchitis in the two testicle samples obtained through a percutaneous puncture. 16 The present study aims to detail the histological description of SARS-CoV-2-related testicular damage and investigate viral particles and antigens in the lesions by immunohistochemistry (IHC), reverse-transcription polymerase chain reaction (RT-PCR), and electron microscopy (EM), in a greater number of testicular samples from fatal cases of COVID-19. The autopsy was performed after written consent from the nextof-kin as previously described. 16 Ultrasound-guided minimally invasive autopsy (MIA-US) was employed to examine and guide tissue sampling, using Tru-Cut semi-automatic coaxial needles (14G 

The 11 patients had the following characteristics (Table 1) Thrombi in pulmonary vessels were seen in eight cases (73%). Secondary pneumonia with associated suppurative infiltrate was seen in six cases.

Eight cases (73%) presented orchitis, characterized by a mild-tomoderate interstitial and mononuclear inflammatory infiltrate, composed mainly of CD68+ cells and a few TCD8+ cells. Few TCD4+ and CD57+ cells were found, and CD20+ cells were rare. No inflammatory cells were observed within the tubules, or vasculitis (Table 1 and Figures 1A and B, Figure 4 ). Interstitial edema was pronounced in all cases. Vascular changes (congestion, endothelial tumefaction, fibrin thrombi, or organized thrombi) were also present in all cases (Table 1 and Figure 1C -E). Leydig cells were reduced and disarranged in all samples ( Figure 1A and B). Focal tubular atrophy was present in seven cases and partial tubular thickening in three ( Figure 1A and B, Figure 2 ). Sertoli cells showed detachment from the basement membrane, and spermatogonia were sloughed in the tubular lumen ( Figure 1B) . A decrease in spermatogenesis was observed in six cases. Interstitial apoptotic cells were not found by H&E and EM analysis.

The SARS-CoV-2 N-capsid protein was detected in all cases in Leydig cells, Sertoli cells, spermatogonia, and fibroblasts (Figure 2A locations, col: collagen fibrils. Part of the nucleus (nu) is occupied by the mesh-like structure of decondensed chromatin fibers (asterisk). The arrowheads highlight that these chromatin fibers are continuous with those of the heterochromatin. Inset corresponds to a higher magnification of a coronavirus particle showing nucleocapsids (white arrowhead) and surface projections resembling spikes (black arrow). Note that the SARS-CoV-2 particle is located within a membrane compartment (black arrowhead) typically attached to the inner membrane surface. (F) Rete testis epithelial cells showing part of the nucleus (nu) and the cytoplasm containing coronavirus particles (arrows) inside vesicular compartments. Inset corresponds to a higher magnification of a viral-like particle attached to the vesicular membrane (black arrowhead). Note nucleocapsids (white arrowhead) and spikes at virus surface (black arrow). (G) EM of a virus-infected cell (n: nucleus) inside the lumen of a capillary of the testis interstitium. The inset image corresponds to a higher magnification of the area surrounded by dotted lines showing some viral particles within a membrane-bounded vacuole (white arrow). The cytoplasm presents fragmented areas and it is possible to observe a virus particle-free (arrow) in the capillary lumen. The endothelial cell (en) is detached from the basal membrane and some areas of vascular are wall ruptured (arrowheads). (H) Low magnification EM of a representative blood capillary found in the testis interstitium. The vascular lumen (lu) is filled by cellular debris, bundles of fibrin fibers (asterisks), and an organized microthrombus (th). (I) Low magnification EM of an enlarged capillary of the testis interstitium. The lumen (lu) was full of aggregated erythrocytes (er). The endothelial layer (en) is damaged or absent, while fibrin deposits (arrows) are found adjacent to the basement membrane. These electron microscopy results corroborate the immunohistochemical findings (SARS-CoV-2 N-antigen detection) in the testicular samples. 

We previously reported orchitis in two fatal cases of COVID-19 at the beginning of the epidemic in São Paulo state, Brazil. 16 As the epidemic progressed and the number of autopsies increased, the testicular findings in fatal cases of COVID-19 remained frequent, and we now present the results of a more significant number of cases.

The present study shows that there are testicular lesions in patients with fatal COVID-19, which can be attributed to inflammatory changes associated with SARS-CoV-2 in the testicular parenchyma and to varying degrees of vascular damage with secondary ischemia.

SARS-CoV acts as an archetype for the COVID-19 testicular pathophysiological hypothesis because it shares exactly the same cell invasion mechanism. 19 During the SARS-2005 epidemic in China, Xu et al.

described testicular lesions in six deceased men: orchitis, germ cell damage, presence of scarce or no spermatozoa in the seminiferous tubules, basement membrane thickening, peritubular fibrosis, interstitial vascular congestion, leukocyte infiltration, and decrease in Leydig cells were the main findings. 20 We found similar testicular lesions in our cases, which other authors have also observed. [21] [22] [23] [24] Orchitis was found in 73% of our cases, but this rate may be higher since the inflammation is focal and may have been underestimated in the samples obtained percutaneously, given their small size. Interstitial orchitis was mild, composed mainly of macrophages, with lower participation of other cell types. This profile was also found by other authors, [21] [22] [23] [24] but the presence of CD8 + cells in ten of our cases suggests that cytotoxicity plays a relevant role in SARS-CoV-2 orchitis.

SARS-Cov-2 was detected in testicular tissue by IHC, EM, and through detection of viral RNA by rRT-PCR. IHC was positive in Leydig cells, Sertoli cells, spermatogonia, and fibroblasts in all cases, even in those with more than 30 days of disease (see Table 1 and Figure The mumps virus (MuV) is the prototype of an agent that causes testicular damage. The MuV-related orchitis presents congestion, interstitial edema, lymphocytic infiltration, seminiferous tubule congestion, tubular hyalinization, germinal epithelium atrophy, and fibrosis. 27, 28 In the MuV orchitis, germ cell apoptosis can be induced by the C-X-C motif chemokine ligand 10 produced by infected Sertoli cells, and spermatogenesis impairment is observed due to paracrine dysregulation by macrophages, Sertoli, and Leydig cells. 29 Based on autopsy studies, HIV-1 causes a myriad of testicular damages, including germ cell loss, perivasculitis, lymphocytic infiltration, interstitial fibrosis, basement membrane thickening, testicular atrophy, and the "Sertoli-cell-only" syndrome, the most common testicular change found in this infection (43%). 30 The testis is a primary target of the Zika virus (ZIKV), acting as an anatomic reservoir with high viral loads, even over prolonged periods. 31 20 These studies also reported the loss of spermatogonia through apoptosis [21] [22] [23] , the presence of SARS-CoV-2 antigen in interstitial and intratubular cells, 22 and viral particles by EM. 21, 22 In the present study, we also demonstrate testes' vascular changes, suggesting ischemia as an alternative mechanism of tissue damage, and the presence of antigens and viral particles in different cell types in the testicular parenchyma, including infection of fibroblasts, which can be associated with tubular fibrosis.

Our study has some limitations. First, we did not present data on patients' semen since they were all in severe clinical condition from the first day of hospitalization. Second, we did not compare our results with a control group. Our study is descriptive, but our findings agree with those of other authors. Our conclusions need to be validated with a more significant number of cases and compared with matched controls. Prospective studies evaluating the function of the MRT using noninvasive methods (e.g., semen analysis, hormonal status, and Dopplercolor ultrasound), with histological correspondence in cases of COVID-19 with different clinical outcomes, may provide helpful answers.

In conclusion, we present testicular histological changes in 11 COVID- 

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The authors declare that they have no conflict of interest