In vitro evidence against productive SARS-CoV-2 infection of human testicular cells: Bystander effects of infection mediate testicular injury.

The hallmark of severe COVID-19 involves systemic cytokine storm and multi-organ injury including testicular inflammation, reduced testosterone, and germ cell depletion. The ACE2 receptor is also expressed in the resident testicular cells, however, SARS-CoV-2 infection and mechanisms of testicular injury are not fully understood. The testicular injury could be initiated by direct virus infection or exposure to systemic inflammatory mediators or viral antigens. We characterized SARS-CoV-2 infection in different human testicular 2D and 3D culture systems including primary Sertoli cells, Leydig cells, mixed seminiferous tubule cells (STC), and 3D human testicular organoids (HTO). Data shows that SARS-CoV-2 does not productively infect any testicular cell type. However, exposure of STC and HTO to inflammatory supernatant from infected airway epithelial cells and COVID-19 plasma decreased cell viability and resulted in the death of undifferentiated spermatogonia. Further, exposure to only SARS-CoV-2 Envelope protein caused inflammatory response and cytopathic effects dependent on TLR2, while Spike 1 or Nucleocapsid proteins did not. A similar trend was observed in the K18-hACE2 transgenic mice which demonstrated a disrupted tissue architecture with no evidence of virus replication in the testis that correlated with peak lung inflammation. Virus antigens including Spike 1 and Envelope proteins were also detected in the serum during the acute stage of the disease. Collectively, these data strongly suggest that testicular injury associated with SARS-CoV-2 infection is likely an indirect effect of exposure to systemic inflammation and/or SARS-CoV-2 antigens. Data also provide novel insights into the mechanism of testicular injury and could explain the clinical manifestation of testicular symptoms associated with severe COVID-19.

In vitro evidence against productive SARS-CoV-2 infection of human testicular cells: Bystander effects of infection mediate testicular injury.

The hallmark of severe COVID-19 involves systemic cytokine storm and multi-organ failure including testicular injury and germ cell depletion. The ACE2 receptor is also expressed in the resident testicular cells however, SARS-CoV-2 infection and mechanisms of testicular injury are not fully understood. The testicular injury can likely result either from direct virus infection of resident cells or by exposure to systemic inflammatory mediators or virus antigens. We here characterized SARS-CoV-2 infection in different human testicular 2D and 3D models including primary Sertoli cells, Leydig cells, mixed seminiferous tubule cells (STC), and 3D human testicular organoids (HTO). Data shows that SARS-CoV-2 does not establish a productive infection in any testicular cell types. However, exposure of STC and HTO to inflammatory supernatant from infected airway epithelial cells and COVID-19 plasma depicted a significant decrease in cell viability and death of undifferentiated spermatogonia. Further, exposure to only SARS-CoV-2 envelope protein, but not Spike or nucleocapsid proteins led to cytopathic effects on testicular cells that was dependent on the TLR2 receptor. A similar trend was observed in the K18h-ACE2 mouse model which revealed gross pathology in the absence of virus replication in the testis. Collectively, data strongly indicates that the testicular injury is not due to direct infection of SARS-CoV-2 but more likely an indirect effect of exposure to systemic inflammation or SARS-CoV-2 antigens. Data also provide novel insights into the mechanism of testicular injury and could explain the clinical manifestation of testicular symptoms associated with severe COVID-19.

Morphometric and immunohistochemical analysis as a method to identify undifferentiated spermatogonial cells in adult subjects with Klinefelter syndrome: a cohort study.

OBJECTIVE: To study the prevalence of spermatogonia in adult subjects with Klinefelter syndrome (KS) using MAGE-A4 and UCHL1 (PGP9.5) immunohistochemistry as markers for undifferentiated spermatogonial cells. We aimed to compare this method to the gold standard of hematoxylin and eosin (H & E) staining with histologic analysis in the largest reported cohort of adult subjects with KS. DESIGN: A retrospective cohort study. SETTING: Infertility Clinic and Institute for Regenerative Medicine. PATIENT(S): This study consisted of 79 adult subjects with KS and 12 adult control subjects. INTERVENTION(S): The subjects with KS (n = 79) underwent bilateral testicular biopsy in an initial effort to recover spermatozoa for in vitro fertilization and intracytoplasmic sperm injection. The institutional review board approved the use of a portion of the archived diagnostic pathology paraffin blocks for the study. The samples were superimposed onto microscopic slides and labeled with the PGP9.5 and MAGE-A4 antibodies. Subjects (n = 12) who had previously consented to be organ donors via the National Disease Research Interchange were selected as controls. Dedicated genitourinary pathologists examined the H & E-, PGP9.5-, and MAGE-A4-stained tissue for presence of undifferentiated spermatogonia and spermatozoa with the use of a virtual microscopy software. MAIN OUTCOME MEASURE(S): The primary outcome was the presence of MAGE-A4-positive or UCHL1-positive tubules that indicate undifferentiated spermatogonia. Supportive outcomes include assessing the biopsy specimen for the following: total surface area; total seminiferous tubule surface area; total interstitium surface area; the total number of seminiferous tubules; and MAGE-A4- negative or UCHL1-negative tubules. Additionally, clinical information, such as age, karyotype, height, weight, mean testicle size, and hormonal panel (luteinizing hormone, follicle-stimulating hormone, and testosterone), was obtained and used in a single and multivariable analysis with linear regression to determine predictive factors for the number of UCHL1-positive tubules. RESULT(S): The mean age of the subjects in the KS group was 32.9 ± 0.7 years (range, 16-48). UCHL1 (PGP9.5) and MAGE-A4 staining showed that 74.7% (n = 59) and 40.5% (n = 32) of the subjects with KS, respectively, were positive for undifferentiated spermatogonia compared with 100% (n = 12) of the control subjects who were positive for both the markers. Hematoxylin and eosin with microscopic analysis showed that only 10.1% (n = 8) of the subjects were positive for spermatogonia. The mean number of positive tubules per subject with KS was 11.8 ± 1.8 for UCHL1 and 3.7 ± 1.0 for MAGE-A4. Secondary analysis showed 7 (8.9%) adult subjects with KS as positive for spermatozoa on biopsy. The population having negative testicular sperm extraction results (n = 72) showed a spermatogonia-positive rate of 1.4%, (n = 1), 72.2% (n = 52), and 34.7% (n = 25) using H & E, UCHL1, and MAGE-A4, respectively. Further analysis showed that 54 (75.0%) subjects were either positive for UCHL1 or MAGE-A4. Twenty (27.8%) subjects were positive for both UCHL1 and MAGE-A4. Multivariate analysis with linear regression showed no significant correlation between clinical variables and the number of UCHL1-positive tubules found on biopsy specimens. CONCLUSION(S): We report a cohort of adult subjects with KS undergoing analysis for the presence of undifferentiated spermatogonia. UCHL1 and MAGE-A4 immunostaining appear to be an effective way of identifying undifferentiated spermatogonia in testicular biopsy specimens of subjects with KS. Despite observing deterioration in the testicular architecture, many patients remain positive for undifferentiated spermatogonia, which could be harvested and potentially used for infertility therapy in a patient with KS who is azoospermic and has negative testicular sperm extraction results.

Fertility preservation for pediatric male cancer patients: illustrating contemporary and future options; a case report.

The main aim of current pediatric male fertility preservation programs is storing spermatogonia stem cell (SSC) prior to starting cancer treatment. From July 1st, 2014 to May 1st, 2020; 170 patients have been recruited in Wake Forest Testicular Tissue Banking Program. The existence of multiple testis biopsies in different time points and detailed histological analyses of a unique cancer patient, provided an educational opportunity to investigate testis condition in different phases of cancer management. A pediatric male cancer patient with B-cell acute lymphoblastic leukemia (ALL) had multiple testicular leukemia recurrences and went through several testicular biopsies, to identify leukemic infiltration as well as considering fertility preservation. Infiltration of leukemia cells into both testes was identified. Neither elongated spermatid nor sperm were detected, but germ cells including SSC, spermatocyte and round spermatid could be identified in the stored tissue even after initial cancer treatment. Different germ cells were identified by hematoxylin and eosin (H&E) staining and specific immunohistochemical (IHC) markers including PGP9.5/UCHL1 or MAGE-A4 (spermatogonia), SYCP3 (spermatocyte) and PRM1 (round spermatid). This emphasizes the importance of offering testicular biopsy to pediatric cancer patients at risk of infertility regardless to the stage of cancer treatment, although earlier biopsy is preferred. Promising research on in vitro spermatogenesis and auto-transplantation support the practice of SSC preservation. In addition, finding and storing round spermatids isolated from testicular biopsy provides a currently available option of round spermatid injection (ROSI). Given the complexity of managing cancer while considering fertility preservation, a multidisciplinary collaboration is important to achieve optimal overall outcomes.

Purging of malignant cell contamination prior to spermatogonia stem cell autotransplantation to preserve fertility: progress & prospects.

PURPOSE OF REVIEW: This systematic review evaluates the state of the art in terms of strategies used to detect and remove contaminated malignant cells from testicular biopsy prior to spermatogonia stem cells (SSCs) autotransplantation to restore fertility. RECENT FINDINGS: Several trials have been done in past two decades to determine the reliable methods of detecting and purging cancer cells prior to SSCs autotransplantation. SUMMARY: The success in treating childhood cancer has dramatically increased over the past few decades. This leads to increasing demand for a method of fertility preservation for patients with pediatric cancer, as many cancer therapies can be gonadotoxic. Storing the SSCs prior to chemo- or radiation therapies and transplanting them back has been tested as a method of restoring fertility in rodents and nonhuman primate models. This has promise for restoring fertility in childhood cancer survivors. One of the major concerns is the possibility of malignant cell presence in testicular tissue biopsies that could re-introduce cancer to the patient after SSCs autotransplantation. Non-solid cancers - especially hematologic malignancies - have the risk of being transplanted back into patients after SSCs cryopreservation even if they were only present in small number in the stored testicular tissue biopsy.