Histopathology and Ultrastructural Findings of Fatal COVID-19 Infections on Testis

Abstract

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.

Keywords: Autopsy; COVID-19; Coronavirus; Infertility; Testis.

Fig. 1. Ultrastructure features of testes from live seroconverted severe acute respiratory syndrome coronavirus 2 (COVID-19) patient and patient who died due to COVID-19 pneumonia. (A) Coronavirus-like spiked viral particles (blue arrows) in the seminiferous tubules of a live patient who had previously contracted the COVID-19 virus and subsequently seroconverted. (B) Coronavirus-like spiked viral particles (green dotted arrows) in the seminiferous tubules postmortems of a patient how had been acutely infected with the COVID-19 virus.

Fig. 2. Histological and ultrastructural features of testes under 40× magnification from postmortems of patient demonstrating inflammation and the severe acute respiratory syndrome coronavirus 2 (COVID-19) viral particle. (A) H&E stained sections showing hyalinization and thickening of the basement membrane of the seminiferous tubules with lymphocyte infiltration. (B) Coronavirus-like particles (green dotted arrow) with distinctive spikes seen in the cytoplasm of the interstitial cells of the testes, magnified image in lower left corner.

Fig. 3. Expression of angiotensin-converting enzyme 2 (ACE-2) in human testicular cells using ACE-2 Rabbit antibody. Scatter plot quantitating the number of cells expressing ACE-2 (x-axis). ^*It denotes a significant difference (p≤0.05) between cases of normal and abnormal spermatogenesis.

Fig. 4. Immunofluorescence of human testicular cells showing expression of angiotensin-converting enzyme 2 (ACE-2) and DAPI (4′,6-diamidino-2-phenylindole), grouped according to normal vs abnormal spermatogenesis. (A) Severe acute respiratory syndrome coronavirus 2 (COVID-19) positive cases with normal spermatogenesis on histomorphology had weaker ACE-2 staining of testes tissue. (B) COVID-19 positive cases with abnormal spermatogenesis on histomorphology had stronger ACE-2 staining of testes tissue.

Fig. 5. Spectrum of pathologic abnormalities in spermatogenesis of testes from postmortem patients with severe acute respiratory syndrome coronavirus 2 (COVID-19) and the associated angiotensin-converting enzyme 2 (ACE-2) immunofluorescence. (A, B) H&E stained slide (20× magnification) showing normal spermatogenesis and decreased immunofluorescent expression of ACE-2. (C, D) H&E stained slide (20× magnification) showing hypospermatogenesis and occasional sclerosis with high immunofluorescent expression of ACE-2.