Impaired spermatogenesis in COVID-19 patients

2.1. Study design

The current observational study has a pair of two case-control investigations. The first part of the study is a case series in which autopsy specimens of testis and epididymis were obtained from patients who died of COVID-19 (n=6), and an equal number of surgical specimens from male patients other than of COVID-19 were taken as controls (n=6), the specimens included in the investigation for the control group were of the patients with prostate cancer and had no other history of medical illness (as per the documented medical history records). These specimens were obtained from the Department of Forensic Medicine, Tongji Medical College, Huazhong University of Science and Technology.

In the second part of the study (cross-sectional cohort), we recruited male COVID-19 inpatients and age-matching male healthy controls. A total of 31 eligible male COVID-19 inpatients showed interest in participating in this research between 26th February 2020 and 12th March 2020, of these, 23 patients provided their semen samples; the remaining eight patients declined to provide samples on the day of collection (primarily because of weakness and some did not explain the reasons) and thus were excluded from the study. The patients provided the semen samples were classified as mild cases (n=9) or ordinary cases (n=14) according to the “Guidance for COVID-19” (the sixth edition) from the National Health Commission of the PRC [23]. We also enrolled in age-matched healthy male controls (n=22), who visited the Center of Reproductive Medicine, Tongji Medical College, for counseling for fathering the second child or infertility due to his partner (wife) during August to October 2019 (before the COVID-19 pandemic). For the control group, a search was conducted in the hospitals’ research database using age range and reproductive health status. An independent investigator screened out the list of all healthy male volunteers and performed matching with COVID-19 group participants based on the age range of these participants. All controls provided written consent, allowing us to store their semen samples in liquid nitrogen for further research purposes. The COVID-19 inpatients were provided with a sterile 50-ml tube and asked to produce a semen sample by masturbation. The semen was sealed in the tube with a cap and allowed to liquefy 30 min at room temperature before transferring it to the laboratory for SARS-CoV-2 RNA detection.

The eligibility criteria for enrollment of male inpatients for COVID-19 group includes; (a) age above 18 years, (b) positive for SARS-CoV-2 RNA in throat swab tests within the recent seven days, (c) received no steroidal drug treatments while receiving anti-COVID-19 treatment, (d) have offspring through natural pregnancy or without offspring due to identified problem of his wife, (e) never received treatment for infertility, including drugs treatment or assisted reproductive techniques, (f) no history of diseases may influence spermatogenesis (obesity, diabetes, cryptorchidism, varicocele, testicular torsion, mumps, genital tract infection, exposure to environmental chemicals, etc.). While the eligibility criteria for the control group includes: (a) age above 18 years, (b) not receiving any steroidal drug treatments for any disease, (c) have offspring through natural pregnancy or without offspring due to identified problem of his wife, (d) never received treatment for infertility, including drugs treatment or assisted reproductive techniques, (e) no history of diseases may influence spermatogenesis. The participants were excluded from the study if they do not comprehend the defined criteria.

Both parts of the current study were reviewed and approved by the Ethics Committee of Center of Reproductive Medicine, Tongji Medical College, Huazhong University of Science and Technology (No. 2020001). The participants provided written, informed consent for their participation in this study. The Study complies with STROBE guidelines for reporting study.

2.2. Histopathology

Tissues were fixed in 10% neutral-buffered formalin and then embedded in paraffin. Continuous 4-µm sections of the testis and epididymis were cut on a microtome (KD-3390, Huanxi), and used for hematoxylin and eosin staining, TUNEL assay, and immunohistochemistry. TUNEL assay was performed with an in-situ cell death detection kit (fluorescein, Roche) according to the manufacturer's instruction. Apoptotic cells were marked by green fluorescence, and DAPI was used to mark the nuclei. Photographs were taken under a fluorescence microscope at 200 × total magnification for cell counting. At least 5000 cells were counted for each sample. When the cell number reached 5000, other cells in this photograph were counted.

2.3. Immunohistochemistry

Immunohistochemistry was used to determine numbers of T lymphocytes (CD3+) and macrophages (CD68+) in specimens, and IgG distribution in testicular tissues, as well as the expression of the receptor for SARS-CoV-2, ACE2 in testicular and epididymal cells. The primary antibodies used included monoclonal mouse anti-human CD3 (clone F7.2.38, Dako), monoclonal mouse anti-human CD68 (clone KP1, Dako), monoclonal mouse anti-human ACE2 (MM0073-11A31, Abcam). Monoclonal rabbit anti-human DDX4 (EPR21789, Abcam) was used to marker germ cells in the testis. Antigen retrieval was performed in EDTA antigen retrieval solution (G1206, Servicer Bio.) by microwave oven heating, and then sections were incubated with a blocking solution. After blocking, sections were incubated overnight with the primary antibody at 4 °C. Biotinylated secondary antibodies and DAB (AR1022, Boster) were used for CD3, CD68. Alexa Flour 594 donkey anti-mouse IgG (Ref21203, Life) and Alexa Flour 488 donkey anti-rabbit IgG (Ref21206, Life) was used for the detection of ACE2 and DDX4, respectively. For IgG detection in the testis, sections were incubated with biotinylated rabbit anti-human IgG (SA1024, Boster) and stained with AEC (SA1020, Boster).

In order to count the percentage of T lymphocytes and macrophages in the interstitium of testicular tissue, at least 200 cells in the interstitium with round nucleus were counted for each specimen. Cells were counted under a light microscope at 200 × total magnification. When the cell number reached 200, other cells in this vision were counted.

2.4. Detection SARS-CoV-2 RNA in semen

There is no commercial kit specific for viral RNA isolation from semen. The Trizol LS reagent was recommended for reliable RNA isolation for viral RNA detection in semen by different groups [24,25]. Seminal plasma contains some agents that inhibit PCR. RNA from seminal plasma was isolated by Trizol LS (Invitrogen) as previously described [26] and subjected to SARS-CoV-2 viral RNA detection with RT-qPCR. For each sample, a total of 200 μl seminal plasma was used. The RNA pellet was dissolved in 30 µl RNase-free water. Moreover, 20 μl RNA was added to 20 µl RT-qPCR mix (Maccura, Chengdu, China), which determine ORF1ab, E gene, and N gene of SARS-CoV-2 within in a multiplex PCR reaction (primers are provided in Supplemental Table 1). RT-qPCR was performed on a PCR cycler (7500, ABI) with conditions: a cycle of reverse transcription at 55 °C for 15 min; a cycle for initial denaturation at 95 °C for 2 min, followed by 40 amplification cycles with denaturation at 95 °C for 15 s, annealing, extension, and signal detection at 58 °C for 35 s. SARS-CoV-2 positive culture medium from the throat swabs was spiked in the semen as the positive control.

2.5. Sperm concentration and leucocytes

The concentrations of sperm and leucocytes in semen were determined under a microscope according to the manual of WHO (Fifth Edition) [27]. Sperm motility was not determined because semen was transferred and stored in cold temperatures.

2.6. IL-6, TNF-α, MCP-1 determination in semen

Immune factors were determined in semen with ELISA kits following the manufacturer's protocol (Cusabio, Wuhan, China) to determine levels interleukin-6 (IL-6, CSB-E04638h), tumor necrosis factor-α (TNF-α, CSB-E04740h), monocyte chemoattractant protein-1 (MCP-1, CSB-E04655h) in semen.

2.7. Statistics

The statistical analysis was performed using SPSS (version: 20.0). Since the percentages of cells, levels of seminal immune factors, and sperm concentrations were positively skewed, these data were transformed to natural logarithms before analysis and expressed as geometric mean with its 95% CI. The difference between groups was analyzed using t-test, and mean ratios with their 95%CIs were calculated. The level of significance was determined as a p-value of less than 0.05.

2.8. Role of funding source

These funding bodies are public institutions, and they had no role in study conception, design, interpretation of results, and manuscript preparation.

3.1. Study characteristics

Autopsied testicular and epididymal specimens of six deceased COVID-19 male cases (case no: A1, A4, A6, A7, A8, A10) were studied. The control specimen included in the study were of the patients with prostate cancers (PC1769, PC3894, PC2947, PC1074, PC9819, PC8664). The age of the COVID-19 and control patients ranges from 51 to 83 (69.3±11.5) and 56 to 85 years (69.0±9.4), respectively. The clinical characteristics of cases of both groups are shown in Supplemental Table 2.

In the second part of this study, a total of 23 Male COVID-19 inpatients and 22 age-matched healthy participants as controls were eligible for enrollment in this study according to our eligibility criteria described in the Methods and provided semen samples. The age of COVID-19 patients and controls ranges from 27 to 55 years old (40.8±8.5) and 30 to 54 years (40.1±5.8), respectively. All study participants of both groups had no history of infertility or ever had received any fertility treatment. Among these participants, a total of 21 COVID-19 inpatients and 16 control males have offspring through natural pregnancy, while remaining two COVID-19 inpatients and six control males yet have not attained fatherhood due to known infertile state of their partners (wife). That is, they were fertile or supposed to be fertile. No steroid treatment was applied in these COVID-19 inpatients. The clinical characteristics of both cohort groups are shown in Supplemental Table 3.

3.2. Histopathology

Histopathological assessment of testes and epididymides from deceased COVID-19 patient compared to age-matched control showed noticeable changes; it included the presence of interstitial edema and congestion both in testes and epididymides in all of the cases. Obvious red cell exudation was observed both in testes and epididymides of cases A7, A8, and in epididymides of case A10. Thinning of seminiferous epithelium (decreased cell layers) was observed (Fig. 1A). Also, the seminiferous tubules were seen to have higher spermatogenic epithelial shedding (Fig. 1A). This finding demonstrated impaired spermatogenesis in these deceased COVID-19 patients.

The proportion of apoptotic testicular cells was determined using the TUNEL assay. Most apoptotic cells were located within seminiferous tubules. The proportion of apoptotic cells in the testes of COVID-19 deceased patients was significantly higher with 2.95 folds (95%CI 1.26–6.90, p=0.018) compared to the average of apoptotic cells in the testes of control patients (Fig. 1B).

3.3. Immunological mediators

Observation of T-lymphocyte (CD3+) and macrophage (CD68+) infiltration in testicular tissues showed an increased concentration of T-lymphocyte and macrophage in the interstitium of testicular tissue of the COVID-19 patients compared to control patients testes, with mean ratios of 2.35 (95%CI 1.41–3.91, p=0.004) and 1.44 (95% CI 1.13–1.85, p=0.008), respectively (Fig. 2A). Additionally, we also found the obvious infiltration of T-lymphocytes around the blood vessels of the testes and epididymis tissue and noticed occasional T-lymphocytes presence around the epididymal duct of the COVID-19 patients (Fig. 2B).

We further investigated the distribution of IgG in COVID-19 patients' testis, of the six patients, we identified the IgG precipitation in seminiferous tubules of four patients (cases A1, A6, A8, A10) (Fig. 2C), while, no IgG precipitation within seminiferous tubules was observed in control cases.

3.4. ACE2 protein expression profiling in testicular cells

Immunochemistry with fluorescence was performed to detect the ACE2 protein expression in testicular cells using marker DDX4 expression by the spermatogenic cells. Consistent with the previous transcript expression results [13,19], we observed that the expression of ACE2 was highly expressed in Leydig cells (Supplemental Fig. 1). However, ACE2 is not strong in the spermatogonia, in which the ACE2 mRNA is abundant [13,19]. No obvious difference in the ACE2 expression between COVID-19 patients and the control was observed.

3.5. SARS-CoV-2 RNA detection in semen

Totally 23 COVID-19 inpatients provided semen samples. The days from the diagnosis to providing semen samples ranges from 4 to 42 days (averaged 25.8 days). All patients have positive SARS-CoV-2 RNA results in throat swab tests within seven days before the semen collection. All semen samples of inpatients showed negative results of SARS-CoV-2 RNA in semen, while the control semen sample with spiked SARS-CoV-2 positive culture medium showed a positive result.

3.6. Sperm concentrations and immune factors in semen

Upon examining semen specimens, nine out of the 23 COVID-19 inpatients (39.1%) showed sperm concentration less than 15 × 10⁶/ml, which relates to the recommended criteria of WHO for diagnosing oligozoospermia [27]. Furthermore, all these nine patients who showed oligozoospermia had offspring without any history of the use of assisted reproductive technology or treatment for infertility (Supplemental Table 3). Moreover, the averaged sperm concentration of COVID-19 inpatients was significantly decreased when compared with the age-matched control males, with a mean ratio of 0.29(95%CI 0.12–0.71, p=0.008, Table 1).

An increase in seminal leucocytes of more than 1 × 10⁶/ml, which is the recommended criteria of WHO for leucocytospermia [27] was observed in 14 patients (60.9%). The percentage of cases with abnormal seminal leucocytes of COVID-19 inpatients was significantly higher than the control males (p=0.006, Table 1). Levels of proinflammatory cytokines and chemokines, including IL-6, TNF-α, and MCP-1, which play a significant role in the immunopathology of SARS-COV-2 [28] were determined in semen samples. Compared to controls, we observed a significant increase of seminal IL-6 (1.72 folds, 95%CI 1.02–2.89, p=0.042), TNF-α (1.60 folds, 95%CI 1.15–2.23, p=0.006), and MCP-1 (1.88 folds, 95%CI 1.12–3.16, p=0.018) levels were observed in COVID-19 patients (Fig. 3).

We further compared sperm concentration and seminal leucocytes among different subgroups of COVID-19 inpatients. However, no obvious difference of these parameters was observed between the mild cases and ordinary cases, or between the cases with or without a history of high fever (≥39 °C), maybe due to the limited size of patients (Table 1).