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Histopathology and Ultrastructural Findings of Fatal COVID-19 Infections on Testis
Justin K. Achua, Kevin Y. Chu, Emad Ibrahim, Kajal Khodamoradi, Katiana S. Delma, Oleksii A. Iakymenko, Oleksandr N. Kryvenko, Himanshu Arora, Ranjith Ramasamy
Purpose: To evaluate the presence and analyze the pathological changes within the testes of patients who died or recovered from severe acute respiratory syndrome coronavirus 2 (COVID-19) complications.
Materials and Methods: Testis tissue was collected from autopsies of COVID-19 positive (n=6) and negative men (n=3). Formalin-fixed paraffin-embedded tissues were stained with hematoxylin and eosin (H&E) and subjected to immunofluorescence for angiotensin-converting enzyme 2 (ACE-2) expression. Fluorescent-labeled tissue slides were imaged on a quantitative pathology scope with various zoom levels allowing for qualitative and quantitative interpretation. Tissue from four COVID-19 positive autopsy cases and a live seroconverted patient was imaged with transmission electron microscopy (TEM).
Results: H&E histomorphology showed three of the six COVID-19 biopsies had normal spermatogenesis while the remaining three had impaired spermatogenesis. TEM showed the COVID-19 virus in testis tissue of one COVID-19 positive autopsy case and the live biopsy, H&E stain on the same autopsy case demonstrated interstitial macrophage and leukocyte infiltration. Immunofluorescent stained slides from six COVID-19 positive men demonstrated a direct association between increased quantitative ACE-2 levels and impairment of spermatogenesis.
Conclusions: The novel COVID-19 has an affinity for ACE-2 receptors. Since ACE-2 receptor expression is high in the testes, we hypothesized that COVID-19 is prevalent in testes tissue of infected patients. This study suggests the male reproductive tract, specifically the testes, may be targets of COVID-19 infection. We found an inverse association between ACE-2 receptor levels and spermatogenesis, suggesting a possible mechanism of how COVID-19 can cause infertility.
INTRODUCTION
The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, COVID-19) first appeared in Wuhan, China in December 2019, and has since become a global pandemic [1]. This novel coronavirus has resulted in over 6 million confirmed cases and more than 370,000 deaths within the first 6 months of spread, confirmed cases as of this writing (August 23, 2020) has reached 23,057,288 globally (https://covid19.who. int/). COVID-19 initially appeared exclusively as a respiratory tract infection. As our understanding of the virus grew, it became apparent that the virus additionally affects other organs of the human body, such as the liver, kidneys, and gastrointestinal tract. There is a male preponderance for the virus and early studies showed worse disease severity and duration in men compared to women. This preponderance has resulted in an increased incidence of the disease and morbidity rate in men that is double that of women [2]. The 2005 SARS-CoV virus, a respiratory virus part of the same family as the SARS-CoV-2 virus, was also investigated regarding its effects on testes tissue. Xu et al [3] found that all six patients who died of SARS-CoV displayed widespread germ cell destruction with few to no spermatozoon, thickened seminiferous tubule basement membranes, as well as lymphocyte and macrophage infiltration. They suggested orchitis is a complication of SARS-CoV. Previous studies have found that deceased COVID-19 men displayed testicular tissue injury in association with inflammatory infiltrates on light microscopy. COVID-19 was detected via reverse transcriptase-polymerase chain reaction (RT-PCR); however, transmission electron microscopy (TEM) failed to identify the virus [4]. Other groups have detected COVID-19 in autopsy testes tissue via RT-PCR, TEM, and immunohistochemistry [5]. Studies have managed to find COVID-19 in the ejaculate of acutely infected men via RT-PCR while others have failed to detect the virus via similar methods [6,7]. Although much has been discovered about the novel coronavirus, there remain mixed findings regarding the pathogenesis of the COVID-19 virus in the male reproductive system that need to be researched.
Pathological studies have shown that the primary target organ of COVID-19 is the lungs. It is believed that this is due to an increased expression of angiotensin-converting enzyme 2 (ACE-2) receptors in lung tissue, of which COVID-19 has a high affinity of binding and subsequent entry [8-10]. Studies have shown the potential risk of COVID-19 impacting and damaging other organs that express ACE-2 receptors, including the heart, kidneys, bladder, oral cavity, esophagus, and ileum [9,11,12]. Interestingly, the ACE-2 receptor is widely expressed in the testes [13]. It has been found that prior to viral entry via ACE-2 the SARS-CoV-2 viral spike proteins must be primed via the transmembrane protease, serine 2 (TMPRSS2). Androgens via the androgen receptor are the only known transcription promoters for the TMPRSS2 gene [14,15]. Since both ACE-2, as well as TMPRSS2, have been shown to be expressed in testis tissue, via single-cell and single nucleus RNA-seq studies, we believe the high androgen environment of the testes will allow for viral entry [16]. In addition, multiple studies have reported that the use of renin-angiotensin system inhibitors has neither been shown to confer any protective effects, nor impact testing positive rates or mortality [17-19]. Additionally, it has been shown that viruses, such as human immunodeficiency virus, hepatitis B virus, and mumps, can cross the blood-testis barrier and cause viral orchitis resulting in infertility and cancer [20]. In this study, we hypothesized that the SARS-CoV-2 virus can be present in the testis and impact spermatogenesis. We also evaluated the association between ACE-2 receptor levels and the impact on spermatogenesis.
CONCLUSIONS
The presence of SARS-CoV-2 viral particles in the testicular tissue fills a fundamental gap in knowledge of the affected organs and possible sequelae of COVID-19 in men. The findings of this study could be the first step in discovering impacts on fertility or the possibility of sexual transmission of the virus. On the basis of these preliminary findings, we believe that COVID-19 can penetrate the blood-testis barrier and enter the testis in some men. The presence of the virus can still be identified in the testis after patients have seroconverted. ACE-2 receptor density in testis tissue may be a factor influencing the extent of damage to cells responsible for spermatogenesis, with higher ACE-2 expression possibly leading to poorer spermatogenesis. However, further experiments are needed to validate this association. The relationship between possible visual viral particles on TEM and leukocyte infiltration suggests the COVID-19 virus may enter the testis and potentially cause orchitis. Further studies need to be undertaken to better understand the effects of this virus on reproductive organs.
Justin K. Achua, Kevin Y. Chu, Emad Ibrahim, Kajal Khodamoradi, Katiana S. Delma, Oleksii A. Iakymenko, Oleksandr N. Kryvenko, Himanshu Arora, Ranjith Ramasamy
Purpose: To evaluate the presence and analyze the pathological changes within the testes of patients who died or recovered from severe acute respiratory syndrome coronavirus 2 (COVID-19) complications.
Materials and Methods: Testis tissue was collected from autopsies of COVID-19 positive (n=6) and negative men (n=3). Formalin-fixed paraffin-embedded tissues were stained with hematoxylin and eosin (H&E) and subjected to immunofluorescence for angiotensin-converting enzyme 2 (ACE-2) expression. Fluorescent-labeled tissue slides were imaged on a quantitative pathology scope with various zoom levels allowing for qualitative and quantitative interpretation. Tissue from four COVID-19 positive autopsy cases and a live seroconverted patient was imaged with transmission electron microscopy (TEM).
Results: H&E histomorphology showed three of the six COVID-19 biopsies had normal spermatogenesis while the remaining three had impaired spermatogenesis. TEM showed the COVID-19 virus in testis tissue of one COVID-19 positive autopsy case and the live biopsy, H&E stain on the same autopsy case demonstrated interstitial macrophage and leukocyte infiltration. Immunofluorescent stained slides from six COVID-19 positive men demonstrated a direct association between increased quantitative ACE-2 levels and impairment of spermatogenesis.
Conclusions: The novel COVID-19 has an affinity for ACE-2 receptors. Since ACE-2 receptor expression is high in the testes, we hypothesized that COVID-19 is prevalent in testes tissue of infected patients. This study suggests the male reproductive tract, specifically the testes, may be targets of COVID-19 infection. We found an inverse association between ACE-2 receptor levels and spermatogenesis, suggesting a possible mechanism of how COVID-19 can cause infertility.
INTRODUCTION
The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, COVID-19) first appeared in Wuhan, China in December 2019, and has since become a global pandemic [1]. This novel coronavirus has resulted in over 6 million confirmed cases and more than 370,000 deaths within the first 6 months of spread, confirmed cases as of this writing (August 23, 2020) has reached 23,057,288 globally (https://covid19.who. int/). COVID-19 initially appeared exclusively as a respiratory tract infection. As our understanding of the virus grew, it became apparent that the virus additionally affects other organs of the human body, such as the liver, kidneys, and gastrointestinal tract. There is a male preponderance for the virus and early studies showed worse disease severity and duration in men compared to women. This preponderance has resulted in an increased incidence of the disease and morbidity rate in men that is double that of women [2]. The 2005 SARS-CoV virus, a respiratory virus part of the same family as the SARS-CoV-2 virus, was also investigated regarding its effects on testes tissue. Xu et al [3] found that all six patients who died of SARS-CoV displayed widespread germ cell destruction with few to no spermatozoon, thickened seminiferous tubule basement membranes, as well as lymphocyte and macrophage infiltration. They suggested orchitis is a complication of SARS-CoV. Previous studies have found that deceased COVID-19 men displayed testicular tissue injury in association with inflammatory infiltrates on light microscopy. COVID-19 was detected via reverse transcriptase-polymerase chain reaction (RT-PCR); however, transmission electron microscopy (TEM) failed to identify the virus [4]. Other groups have detected COVID-19 in autopsy testes tissue via RT-PCR, TEM, and immunohistochemistry [5]. Studies have managed to find COVID-19 in the ejaculate of acutely infected men via RT-PCR while others have failed to detect the virus via similar methods [6,7]. Although much has been discovered about the novel coronavirus, there remain mixed findings regarding the pathogenesis of the COVID-19 virus in the male reproductive system that need to be researched.
Pathological studies have shown that the primary target organ of COVID-19 is the lungs. It is believed that this is due to an increased expression of angiotensin-converting enzyme 2 (ACE-2) receptors in lung tissue, of which COVID-19 has a high affinity of binding and subsequent entry [8-10]. Studies have shown the potential risk of COVID-19 impacting and damaging other organs that express ACE-2 receptors, including the heart, kidneys, bladder, oral cavity, esophagus, and ileum [9,11,12]. Interestingly, the ACE-2 receptor is widely expressed in the testes [13]. It has been found that prior to viral entry via ACE-2 the SARS-CoV-2 viral spike proteins must be primed via the transmembrane protease, serine 2 (TMPRSS2). Androgens via the androgen receptor are the only known transcription promoters for the TMPRSS2 gene [14,15]. Since both ACE-2, as well as TMPRSS2, have been shown to be expressed in testis tissue, via single-cell and single nucleus RNA-seq studies, we believe the high androgen environment of the testes will allow for viral entry [16]. In addition, multiple studies have reported that the use of renin-angiotensin system inhibitors has neither been shown to confer any protective effects, nor impact testing positive rates or mortality [17-19]. Additionally, it has been shown that viruses, such as human immunodeficiency virus, hepatitis B virus, and mumps, can cross the blood-testis barrier and cause viral orchitis resulting in infertility and cancer [20]. In this study, we hypothesized that the SARS-CoV-2 virus can be present in the testis and impact spermatogenesis. We also evaluated the association between ACE-2 receptor levels and the impact on spermatogenesis.
CONCLUSIONS
The presence of SARS-CoV-2 viral particles in the testicular tissue fills a fundamental gap in knowledge of the affected organs and possible sequelae of COVID-19 in men. The findings of this study could be the first step in discovering impacts on fertility or the possibility of sexual transmission of the virus. On the basis of these preliminary findings, we believe that COVID-19 can penetrate the blood-testis barrier and enter the testis in some men. The presence of the virus can still be identified in the testis after patients have seroconverted. ACE-2 receptor density in testis tissue may be a factor influencing the extent of damage to cells responsible for spermatogenesis, with higher ACE-2 expression possibly leading to poorer spermatogenesis. However, further experiments are needed to validate this association. The relationship between possible visual viral particles on TEM and leukocyte infiltration suggests the COVID-19 virus may enter the testis and potentially cause orchitis. Further studies need to be undertaken to better understand the effects of this virus on reproductive organs.
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