How effective is the RT-PCR assay for detecting SARS-CoV-2 in human semen?

The outbreak of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has highlighted concerns about semen samples for cryobanking.

SARS-CoV-2 is an angiotensin-converting enzyme 2 (ACE2) host receptor and TMPRSS2 invade target cells. Transient viremia during SARS-CoV-2 infection and expression of TMPRSS2 and ACE2 in testes and other accessory glands can lead to shedding of SARS-CoV-2 in the male reproductive tract.

Background

Although SARS-CoV-2 replication rarely occurs in the male reproductive system, certain specific male cells may serve as viral reservoirs after systemic infection with SARS-CoV-2.

Additionally, semen samples can be contaminated with SARS-CoV-2 during sperm collection. The potential for SARS-CoV-2 in her cryopreserved semen samples is a concern for patients undergoing treatment with assisted reproductive technology (ART) and fertility preservation.

Previously, four studies had shown the presence of the SARS-CoV-2 genome in semen samples. Three of these studies evaluated semen samples using a commercially available reverse transcription-polymerase chain reaction (RT-PCR) assay, but one did not describe the mechanism of virus detection.

However, these studies included small populations and did not include validated methods for detecting SARS-CoV-2 in semen samples. Additionally, most studies used the commercially available His RT-PCR assay, which was approved for respiratory samples only, and targeted only two or three viral genes.

Three studies used digital droplet PCR (dd-PCR) for good level detection of the SARS-CoV-2 genome. However, even in this process, most of the sample types were not semen. In such studies, the limit of detection (LOD) of semen samples should be confirmed.

Another study used the “SpermCOVID test” for SARS-CoV-2 genome detection in respiratory samples, but not in semen samples.

new research in Reproductive Biomedicine Online The journal aimed to develop a standardized protocol for SARS-CoV-2 RNA detection in semen fractions used for ART. This included high-performance RT-PCR assays using sperm specimens and cryopreserved semen for SARS-CoV-2 RNA detection. In addition, we also determined the effectiveness of this method according to different semen characteristics.

About research

In this study, we collected semen samples from patients undergoing routine semen analysis for infertility between July 2020 and March 2021, regardless of clinical criteria, body mass index, or age. None of the patients reported fever or other SARS symptoms. -CoV-2. They also reported that they had not been exposed to someone with SARS-CoV-2 symptoms. Conventional semen analysis, leukocyte detection, and evaluation or morphology were then performed, followed by sample preparation.

Normal sperm samples were thawed at 4°C and pooled for analytical testing. After extracting RNA from 160 µl of each sample, the SARS-CoV-2 genome was amplified and the SARS-CoV-2 RT-PCR assay was analytically validated.Influence of sperm quality based on efficacy of RT-PCR was analyzed. Finally, precision assay and LOD and limit of quantification (LOQ) of the SARS-CoV-2 RT-PCR assay were performed.

findings

As a result, we found that a highly accurate assay for semen types with high SARS-CoV-2 RNA concentrations, N and ORF1ab viral targets, and semen types with low SARS-CoV-2 RNA concentrations met the expected requirements. Reported. Furthermore, the S gene was reported to be the least sensitive viral target of the assay, whereas the N gene was the most sensitive target.

Cryoprotective media and semen characteristics were observed not to affect the efficacy of the detection method. The LOD was reported to be 0.33 SARS-CoV-2 genome copies/μl sample for semen and 0.23 SARSCoV-2 genome copies/μl for sperm. Finally, the LOQ was said to be 1 viral genome copy per µl of sample.

Therefore, this study was able to determine the presence of SARS-CoV-2 RNA in cryopreserved sperm and semen. The method was effective regardless of sperm characteristics and semen type. Therefore, this method can ensure the safety of semen donors and patients who benefit from fertility-preserving techniques during the SARS-CoV-2 pandemic.