key: cord-0801404-vzlo8h2e
authors: Rajput, Sandeep K; Khan, Shaihla A; Goheen, Benjamin B; Engelhorn, Heidi J; Logsdon, Deirdre M; Grimm, Courtney K; Kile, Rebecca A; West, Rachel C; Yuan, Ye; Schoolcraft, William B; McCormick, Sue; Krisher, Rebecca L; Swain, Jason E
title: Absence of SARS-CoV-2 (COVID-19) virus within an active IVF laboratory using strict patient screening and safety criteria
date: 2021-03-07
journal: Reprod Biomed Online
DOI: 10.1016/j.rbmo.2021.03.005
sha: 39fdfea6bbb2146251183e3586a993acd70d24dd
doc_id: 801404
cord_uid: vzlo8h2e

Research Question: In the early stages of the COVID-19 pandemic, IVF clinics stopped the majority of patient treatment cycles to minimize the risk of disease transmission. The risk of SARS-CoV-2 viral exposure and potential cross contamination within the IVF lab remains largely unclear. To that end, the objective of this study was to examine follicular fluid (FF), culture media (M) and vitrification solution (VS) for SARS-CoV-2 in an IVF lab. Design: Prospective clinical study. All females undergoing transvaginal oocyte retrieval (TVOR) were required to have a negative SARS-CoV-2 RNA test 3-4 days prior to the procedure. Male partners were not tested. All cases utilized intracytoplasmic sperm injection (ICSI). The first tube of FF aspirated during oocyte retrieval, M drops following removal of embryos on day 5, and VS after blastocyst cryopreservation were analyzed for SARS-CoV-2 RNA. Results: In total, M from 61 patients, VS from 200 patients, and FF from 300 patients were analyzed. All samples were negative for SARS-CoV-2 viral RNA. Conclusion(s): With stringent safety protocols in place, including female patient testing and symptom-based screening of men, the presence of SARS-CoV-2 RNA was not detected in FF, M or VS. This work demonstrates the possibility of implementing a rapid laboratory screening assay for SARS-CoV-2 and has implications for safe laboratory operations, including cryostorage recommendations.

Recently, ASRM, ESHRE, and IFFS released a joint statement affirming that reproduction is an essential human right and that infertility is a time sensitive although treatable disease affecting 10-12% of couples of reproductive age, the treatment of which should be considered essential care (Veiga et al., 2020) . Following the nearly global cessation of infertility treatment at the beginning of the COVID19 pandemic, restarting the practice of ART during the ongoing pandemic environment is largely an uncharted proposition operating under a "proceed-withcaution" approach. Clinicians and embryologists have implemented best practices as recommended by guidelines from their professional societies. However, hard scientific data is scarce because of the novel nature of SARS-CoV-2, and adjustments to safe practices based on new knowledge and updated recommendations must often be made in real time. Some of the strategies adopted by clinics worldwide include patient and staff questionnaires and testing, adoption of tele-health appointments, use of personal protective equipment (PPE), and elevated cleaning and disinfection of facilities. Unfortunately, in the embryology laboratory, the risk to gametes and embryos is relatively unknown and management strategies are based upon previous guidelines for known viral pathogens (Simopoulou et al., 2020) .

Recent evidence of the presence of the receptor and protease necessary for SARS-CoV-2 infection (ACE2 and TMPRSS2) in human oocytes and embryos (Essahib et al., 2020 , Rajput et al., 2020 highlights the potential for infection or cross-contamination in the IVF lab and the imperative to implement all possible precautions to keep gametes, embryos, staff, and patients safe. To minimize risk of virus exposure in the clinic and laboratory, both ASRM and ESHRE recommend the use of questionnaire based triage to identify potentially infected patients and staff, and the use of established infection control procedures. However, the two societies diverge in their recommendations for SARS-CoV-2 testing to mitigate risk in the setting of assisted reproduction. These recommendations continue to change over time. Regardless, use of these tests without understanding their inherent sensitivity, specificity, and temporal limitations may lead to inadvertent infection of patients, staff, physicians, gametes and/or embryos (La Marca and Nelson, 2020) .

Although infertility treatment has now resumed, there is still much unknown about the novel coronavirus in the context of the IVF laboratory. We do not know if infection of follicular cells, gametes, or embryos occurs in vivo or in vitro, and if so if the virus may be infectious within the laboratory resulting in cross contamination of biological samples and/or staff. There remains concern in the embryology community about how the virus may impact laboratory outcomes as well as staff safety (Anifandis et al., 2020 , Arav, 2020 . Some suggested precautions for the IVF laboratory specifically including UV disinfection of liquid nitrogen used for vitrification and warming gametes and embryos, extensive washing, closed vitrification systems, reducing the use of embryo micromanipulation techniques that breach the zona pellucida, and eliminating the transfer of any embryos without an intact zona pellucida until more information becomes available . However, it is difficult to ascertain if these precautions are indeed necessary without further scientific knowledge.

Because the risk of SARS-CoV-2 viral exposure and potential cross contamination within the IVF laboratory remains largely unclear, the objective of this study was to assess the true risk of exposure to SARS-CoV-2 in an active IVF laboratory when strict patient screening procedures are in place.

This study included SARS-CoV-2 screening of 300 follicular fluid (FF), 200 vitrification solution (VS) and 61 embryo culture medium (M) samples. Samples were collected during the IVF cycle of patients undergoing fertility treatment at the Colorado Center for Reproductive Medicine (CCRM). . Because transvaginal oocyte retrievals were performed under sedation and under the guidance of our anesthesia group, each female patient undergoing transvaginal oocyte retrieval (TVOR) was required to have a negative SARS-CoV-2 PCR test 3-5 days prior to the retrieval procedure. This timing ensured adequate time for results to be reviewed prior to triggering the patient to avoid having to cancel the retrieval. Due to lack of anesthesia use, as well as for cost, male partners were examined using a symptom-based screening approach the day of collection. All cases examined in this study utilized standard semen preparation of a double layer gradient followed by sperm swim-up for use during Intracytoplasmic Sperm Injection (ICSI). The first tube of FF aspirated during TVOR, which may have involved puncture of more than 1 follicle, was collected for analysis. Collection entailed pipetting up to 20ul of the follicular fluid aspirate from the dish using an RNase/DNAase free tip into a PCR tube. Embryos were cultured in a sequential media system, and M samples were collected on day 5 (following change from cleavage media to blastocyst media on day 3). Care was used to avoid drawing up mineral oil overlay. Vitrification solution (VS) samples were collected after blastocyst exposure to serve as the final dilution step before embryos are placed into storage tanks. Immediately after collection, self-inactivating replication incompetent lentivirus (LV) particles (SHC003V# Sigma) containing the single stranded viral RNA genome were inoculated into each sample as a positive control for viral RNA stability.

The outline of sample processing before RNA isolation is provided in Figure 1 . Briefly, a total of 100 LV particles were inoculated into ~2 ml of follicular fluid, 250-450 µl of VS, and ~50 µl of M samples collected from patients. After mixing LV, samples were stored at 4°C and processed for RNA isolation within 5 days of collection. FF samples were centrifuged at 250 x g for 5 min and the obtained supernatant passed through a 0.22 micron filter (Sigma #SLGV033RS) to remove cells and cellular debris. To concentrate the VS (if >200 µl) and filtered FF (~2 ml), samples were loaded into amicon filter column (Sigma # UFC810024) and centrifuged at 4°C in a fixed angle rotor at 7500 x g until 200 µl of sample was left in the column. Since concentrated FF samples were highly viscous due to the high amount of protein present, a protein removal step was performed before RNA isolation. A total of 800 µl QIAzol was added to each sample, incubated for 5 min, and mixed with 200 µl of chloroform. After centrifugation at 12000 x g for 10 min at 4°C, the supernatant (deproteinized FF) was used for RNA isolation. A total of ~50 µl of M and ~200 µl of concentrated VS and deproteinized FF samples were subjected to RNA isolation using QIAamp Viral RNA Mini Kit (Qiagen # 52906) following the manufacturers protocol with some modifications. Briefly, 2 µg of carrier RNA/sample was used for each isolation and RNA was eluted into 16 µl of nuclease-free (NF) water. After determining the purity and yield on Nanodrop, a total of 14 µl of RNA was used for cDNA synthesis using SuperScript™ IV VILO™ Master Mix (Thermo # 11756050).

A TaqMan-based multiplex qPCR assay was developed to detect N1, N2, and ORF1ab loci of the SARS-CoV-2 genome as well as the lentivirus genome (external control) in a single tube reaction. N1 and N2 probes with FAM, ORF1ab with Cy5, and LV probe with SUN labeling were used with their respective primers to prepare the TaqMan assay (TMA) for each loci. Each TMA was tested individually using their specific cDNA template equivalent to 100 copies of LV RNA and SARS-CoV-2 synthetic control RNA in qPCR. Reaction mixture (20 µl) containing 2X TaqMan™ fast advanced master mix (ABI # 4444557), 2 µl of cDNA (100 copies), and 1 µl of TMA was amplified as follows; Uracil-N glycosylase (UNG) incubation at 50°C for 2 minutes, initial denaturation at 95°C for 2 min and 40 cycles at 95°C for 1 s, 60°C for 20 s. Amplified PCR products were measured based on the fluorescence resulting from TaqMan probe hydrolysis after every cycle. After confirming the specificity for their template, each TMA ((N1, N2, ORF 1ab and LV) was mixed in equal concentration to develop multiplex TMA.

To further test the sensitivity and cross reactivity of the multiplex assay, cDNA equivalent to 100, 20, and 4 copies of the lentivirus and SARS-CoV-2 genome was used to perform the TaqMan-based qPCR as described above. A mixture of SARS-CoV-2 and lentivirus cDNA (equivalent to 100 copies of each) with multiplex TMA was also tested by qPCR to confirm amplification of all the target loci (N1, N2, ORF1ab, and LV) in a single tube reaction. All primers and probes used in this assay are provided in Table 1 . The developed multiplex qPCR assay was used to test for the presence of SARS-CoV-2 in all the diagnostic samples. A total of 2 µl of diluted (1:2) cDNA of each diagnostic sample was used to perform the qPCR along with the positive control (mixture of SARS-CoV-2 and lentivirus cDNA) and negative control (nuclease free water) reactions. Since lentivirus particles were mixed in each diagnostic sample as an external positive control, samples with no amplification of the lentivirus genome in qPCR were considered false negative and removed from the analysis. Samples with higher than 37 Ct value in qPCR amplification in any channel were also considered negative.

Multiplexed RT-qPCR assay targeting LV genome (external control) and N1, N2 and ORF1ab loci of SARS-CoV2 genome was developed and tested with cDNA synthesized from 100, 20 and 4 copies of lentivirus RNA and SARS-CoV2 synthetic RNA. Results demonstrated specific amplification of SUN labeled LV TMA (Red) with all three concentrations of lentivirus cDNA.

In addition, lentivirus cDNA did not show any cross amplification with SARS-CoV-2 TMA (blue and orange) present in the reaction (Figure 2A ). We observed similar results with SARS-CoV-2 cDNA. Specific amplification of FAM labeled N1/N2 (blue) and Cy5 labeled ORF1ab

(Orange) was detected in qPCR with all three concentrations of SARS-CoV-2 cDNA, but no cross reactivity was observed with LV TMA (Red) present in the reaction ( Figure 2B ). To test if this assay could successfully amplify all the target loci present in the reaction, a mixture of 100 copies of lentivirus and SARS-CoV2 cDNA was used as a template in a qPCR reaction. We observed a clear amplification of N1N2, ORF1ab, and LV loci ( Figure 2C ), demonstrating that our multiplex assay can detect both SARS-CoV-2 and lentivirus genomes if present in diagnostic samples. No amplification curve was observed in negative controls that contained all sets of primers and probes with no LV and Sars-CoV-2 genome ( Figure 2D) , thereby, strengthening our validation for primer specificity for the corresponding locus of the genome of interest. Our results indicate that we can successfully multiplex N1, N2, ORF1ab and LV primers and respective probes in a TaqMan-based RT-qPCR assay.

Following validation, the TaqMan-based RT-qPCR assay was used to screen SARS-CoV-2 in 300 FF, 61 M, and 200 VS samples. These fluids were selected based on the following: FF was examined to determine if the virus might be exposed to the oocytes in the lab, either directly from follicular fluid or via mixture with trace blood amounts encountered during the normal retrieval process. Media (M) samples were examined because this is where resulting embryos spend the majority of their time and to determine if the exposure to processed sperm from untested males during ICSI or if the dilution through embryo rinsing/culture might impact presence/absence of the virus when in the extended presence of the developing embryo. Finally, VS was examined because it is the final step in the dilution process before embryos are placed into LN2 tanks and may provide insight into possible risk of cross contamination between frozen samples.

Our results showed no amplification of SARS-CoV-2 genome with N1, N2 and ORF1ab loci specific TMA in any of the samples analyzed. As expected, lentiviral amplification signal was observed in all patient samples that were analyzed and included in the diagnostic study.

Amplification curves for N1, N2, ORF1ab and LV genomes were observed in the positive control (mixture of lentivirus and SARS-CoV2 cDNA) included in each RT-qPCR experiment ( Figure 3 ). The negative controls in each experiment did not amplify.

The emergence of the respiratory virus, SARS-CoV-2, with possible aerosol transmission, has had tremendous impacts on fertility treatments. Among the various concerns raised from this global pandemic was the possibility of lab staff infection from contaminated patient samples, cross contamination of samples while inside the laboratory, or even reinfection of patients if subsequently using previously infected samples. An abundance of opinions and commentaries on suggested safety precautions and procedural modifications for the IVF laboratory flooded the literature (Alviggi et al., 2020 , Anifandis et al., 2020 , Arav, 2020 , Corona et al., 2020 , Maggiulli et al., 2020 , Perry et al., 2020 , Simopoulou et al., 2020 ) (De Santis et al. 2020 ). With a paucity of data available, these publications were often extremely cautious in their recommendations to avoid unknown or unforeseen issues. This approach was and is prudent to alleviate concerns and permit a safe reopening for essential fertility treatments. However, as new data emerges, an evidenced-based approach to combat infection or cross contamination risks is feasible and patient counseling and laboratory procedures can be adjusted accordingly.

Until now, the presence and unknown impact of SARS-CoV-2 in the IVF laboratory has been a concern, but most studies have not examined the laboratory environment for the presence of the virus. Initially viewed as a respiratory virus, as opposed to a sexually transmitted disease, emerging data made it clear that the virus could be detected in various tissues or bodily fluids .

Receptors involved in SARS-CoV-2 signaling were identified in reproductive tissues and cells (Rajput et al., 2020 , Stanley et al., 2020 . However, whether active virus can be present or whether the virus could act directly upon gametes or embryos to impact development or function is still unknown. Since the existing SARS-CoV-2 PCR based protocol has not been optimized for complex samples like follicular fluid, culture media and vitrification solutions, we first developed a reliable multiplex RT-qPCR protocol for these samples and utilized it to screen culture medium from 61 patients, vitrification solution from 200 patients, and follicular fluid from 300 patients. Our data demonstrating the absence of viral particles in these samples demonstrates that the SARS-CoV-2 virus can successfully be excluded from the IVF laboratory, providing reassurance that cross-contamination of the virus between gametes and embryos, as well as exposure of embryologists, is minimal. Importantly, all female patients tested negative with a PCR-based nasal swab test 3-5 days prior to oocyte retrieval and were asymptomatic the day of the procedure. It is unknown if any of the women had prior SARS-CoV-2 infections. That being said, follicular fluid is routinely contaminated with varying amounts of blood. An initial study from China indicated that SARS-CoV-2 was present in <1% of blood samples from infected patients . However, whether infection is possible via blood is unknown. At least one publication describes a platelet transfusion from a SARS-CoV-2 infected individual to an uninfected person with no subsequent infection as a result (Cho et al., 2020) . Additionally, because no ovarian follicle aspirates in this study contained SARS-CoV-2 virus, it is unclear if the SARS-CoV-2 virus may be introduced into the oocyte, especially considering the zona pellucida barrier. Importantly, we were not able to quantify the varying amounts of blood present in the various follicular aspirates. Interestingly, one recent study examined oocytes from two SARS-CoV-2 infected women. Women were diagnosed the day of their oocyte retrieval as COVID infected via PCR assay, which may have implications on the incubation period and presence in blood, follicular fluid or other tissues. However, none of the oocytes examined displayed any viral RNA for the SARS-CoV-2 gene (Barragan et al., 2020) .

Our data is the first known report examining embryo culture media within the clinical IVF laboratory for the presence of SARS-CoV-2. No virus was detected in samples obtained from embryo culture media microdroplets. Importantly, several embryo culture media samples examined included those from patients who did not have prior follicular fluid samples tested.

Additionally, all cycles in this study utilized ICSI. Sperm preparation for ICSI entailed the routine use of a 45/90 gradient, sperm washing and a final swim-up step prior to the normal dilutions within polyvinyl propylene (PVP) and other media encountered during routine ICSI procedures. Interestingly, there are mixed reports on the presence or absence of SARS-CoV-2 in semen. One of the first studies out of Wuhan, China did not detect virus in the semen from 34 SARS-CoV-2 sero-positive men when tested 8-75 days after diagnosis (Pan et al., 2020) . A second study of 38 patients indicated that 6 of the semen samples had virus detected 6-16 days after symptom onset, though whether active virus was present is unknown . A third report of 12 SARS-CoV-2 infected men in the recovery phase from China demonstrated no virus in semen . While no symptomatic male patients were knowingly involved in IVF treatments in this study, it is unknown if any of the male patients were asymptomatic and actively infected with SARS-CoV-2. This demonstrates that with basic, symptom based screening of men and testing of females with normal lab procedures using ICSI, that SARS-CoV-2 was not present in culture media. This has implications for concerns and recommendations regarding zona breaching, future embryo transfer or modes of possible transmission/cross contamination in the lab . Importantly, it has been suggested that ICSI may carry the potential to directly place a sperm with SARS-CoV-2 virus directly into the oocyte, which could impact the developing embryo (Perry et al., 2020) . It is unknown if this is the case and may be a possibility if using semen with active virus. This is also the first known report examining embryo vitrification solutions for the presence of SARS-CoV-2. Similar to embryo culture media, no virus was detected in vitrification solution samples. This is not surprising given the lack of viral detection in follicular fluid and embryo culture media as well as the additional dilution that occurs during solution exposure. It does serve to further reinforce that with thorough testing and symptom-based screening approaches, as well as the extensive dilution experienced in the IVF laboratory, presence of the SARS-CoV-2 virus can be avoided. This may have implications for subsequent recommendations regarding cross-contamination and cryo-storage procedure modifications for oocytes and preimplantation embryos, such as the use of closed cryo-devices, sterilization of liquid nitrogen, use of separate tanks, or other measures (Arav, 2020 , Yakass and Woodward, 2020 . These recommendations may differ from those used for cryopreserving or storing semen (Corona et al., 2020) .

Importantly, the multiplex-RT-qPCR assay developed in this study targets three loci of the SARS-CoV2 genome to provide better specificity as well as implementing lentivirus as an external control, which is more similar in size and nature to SARS-CoV-2 compared to the phage RNA used in most of the commercially available kits. The protocol used in this study is sensitive enough to detect 50 viral particles in 2 ml of samples and 4 copies of viral genome/qPCR reaction. The multiplex nature of the assay makes it not only cost effective but four times less time-consuming compared to uniplex PCR. Unlike commercially available kits, we have provided complete details of reagents and protocols for RNA isolation and cDNA synthesis as well as optimized primers and probes sequences used in the multiplex assay to aid in direct implementation for SARS-CoV-2 screening in any laboratory. The relatively simple nature and rapid turn-around time of this assay means it could be incorporated into the IVF laboratory during routine embryo culture. If virus is not identified around day 3 of embryo culture, the time when many labs change media over in a sequential system, or the day when embryo handling can easily occur for things like zona breaking or embryo grading, then there should be confidence that no virus is present in later stages of culture or after further dilution during cryopreservation.

While virus could be below the detectable limit of the assay, or perhaps inside the cells and not present in media, this is unlikely.

As we continue to learn about the SAR-CoV-2 virus and the impact it may or may not have on gametes and embryos as well as any resulting pregnancy and offspring, the field of ART must react accordingly. These data demonstrate that with proper patient screening, testing, and taking appropriate safety precautions, the virus can be effectively avoided within media in the IVF lab. It is important to note this does not mean that SARS-CoV-2 cannot be present inside the lab, particularly in the absence of patient testing, as our study presumably did not include actively infected women or symptomatic men. However, our results, as well as other emerging studies, do highlight the possibility of continuing to conduct IVF in a safe environment without dramatic changes to existing laboratory protocols for embryos, patients, and laboratory staff. Jason E. Swain, PhD, HCLD is the Corporate Laboratory Director of the CCRM IVF Laboratory Network. His primary research interests include pursuit of methods to improve in vitro embryo culture conditions through reduction of environmental stressors via modification of both the physical and chemical culture environment.

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