The samples were collected at the laboratory of Cryos International, Orlando, FL, between March 10, 2020, and October 29, 2020, and approved by the Western Institutional Review Board (20200888). Eligibility criteria were men between the ages of 18 and 60, with an intact reproductive tract, and who tested positive for SARS-CoV-2 RNA 2 by nasopharyngeal swabs using rt-PCR, regardless of symptomatology.
Exclusion criteria included men who had a vasectomy, men with a urologic condition that inhibits ejaculation, taking testosterone or hormonal treatment that can impact semen production, undergoing chemotherapy, on immunosuppressant medication, and with a concurrent acute or chronic infection.
Study participants were instructed to provide a semen specimen free of contamination by cleaning the penis and hands as well as avoiding saliva during collection. Specimens were collected in a sterile collection cup and allowed to liquefy before aliquoting into cryovials. The specimens were frozen using a controlled rate freezer and stored under liquid nitrogen at −196 °C. Specimens were transported to Florida Gulf Coast University, Fort Myers, FL, using a dry shipper to maintain −196 °C.
Upon arrival, samples were stored at −80 °C until processed. When processed, samples were thawed and 200 μL aliquoted from the original cryovial. Aliquot was centrifuged at 3500 rpm for 5 min in a table-top centrifuge (Grant PCV-2400 Combined Centrifuge/Vortex Mixer, Grant Instruments, Cambridge, UK) to spin-out any cellular material or debris. Then 140 μL supernatant from centrifuged samples was transferred into a new tube. Any remaining, unused sample was frozen back and stored at −80 °C. Viral RNA was extracted from the 140 μL collected supernatant following the protocol outlined in the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany). A water-only control (RNA Blank) was included with each round of extraction to check for RNA contamination. Sample viral RNA extractions were eluted in 60 μL nuclease-free water and stored at 4 °C (24 h maximum) until ready for qRT-PCR application.
Reactions for real-time RT-PCR were assembled using GoTaq® Probe 1-Step RT-qPCR System (Promega, Madison, WI). Twenty-microliter reactions were prepared, using 5 μL of RNA extract template per reaction. Each sample extracted, including controls, was tested in triplicate. Additional water-only, no template control (NTC), was added to check for contamination of master-mix and/or individual samples during loading. The US Centers for Disease Control and Prevention (CDC, USA) panel of 2019-Novel Coronavirus (2019-nCoV) primers and probes, specifically for SARS-CoV-2 targets N1 and N2, were utilized in recommended concentrations from IDT-DNA, 2019-nCoV Research-Use-Only Kit (Integrated DNA Technologies, Coralville, IA). *A link to CDC panel of primers and probes sequences is listed at the end of “Materials and methods” section. A dilution series of Sars-CoV-2_USA_WA1 N-gene transcript RNA (shared by Dr. Nathan Grubaugh at Yale University, New Haven, CT) served as a positive control and to create duplicate standards for assessing genome copy number.
Reactions were run on CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA) under the following conditions: Reverse transcription at 45 °C for 15 min followed by single cycle of 95 °C for 2 min, then 45 cycles of PCR with denaturation at 95 °C for 10 s and combined annealing/extension at 55 °C for 45 s.
Limit of detection was determined to be 20 genome copies of positive transcript RNA. Control RNA stock was serially diluted tenfold and then verified by qRT-PCR with both N1 and N2 primer sets.
Samples’ amplification curves and Cp values were compared to those of positive control standards in order to determine whether positive or negative for presence of SARS-CoV-2 RNA.
To assess the potential for qRT-PCR inhibitors from the samples, positive control reactions with N-gene transcript RNA were spiked with sample RNA extracts.
*Link to CDC panel of 2019-Novel Coronavirus (2019-nCoV) primers and probes: