Nuno Costa-Borges, Ph.D.1, Mònica Acacio, M.Sc.1, Ivette Vanrell, M.Sc.1, Gloria Calderón, Ph.D.1
1Embryotools, Parc Cientific de Barcelona
Since the state of alarm was decreed in Spain due to the COVID-19 pandemic, we have gone through an initial skepticism phase that was then followed by the temporary cessation of all activities in the IVF laboratories, similarly to what happened in most European countries. During this period, the different national and international Scientific Societies have prepared guidelines with instructions for reproductive centers, which have been prepared on the basis of the available knowledge about the virus (1-3). These recommendations have been updated regularly and contributed to the evaluation of the possible risks of transmission of SARS-CoV-2 to reproductive cells and have helped IVF centers to implement preventive measures. Similarly, governmental agencies such as FDA in the US that regulate use of donor gametes or embryos provided additional guidance for cases where heterologous material is used (4). However, recommendations of additional screening and testing of donors has been quite minimal. Now that many centers are returning to normal activities, there are still doubts in regard to specific laboratory procedures. In particular, one of the most unsettled questions is the potential risk of cross-contamination with SARS-CoV-2 during cryopreservation procedures or the storage of the reproductive cells in liquid or vapor phase nitrogen tanks. In this regard, we consider that it is important to evaluate carefully the pros and cons of each of the measures proposed to avoid unnecessary risks.
Risks of cross contamination
The possible transmission of viruses or other pathogens among cryopreserved samples has always been a matter of concern in our field (5), since hepatitis B virus (HBV) transmission was reported between a hematopoietic cell line and stem cells stored in liquid nitrogen tanks (6). These concerns increased when other studies confirmed that bacteria and environmental fungi can survive at very low temperatures and were detected in liquid nitrogen and vapor phase tanks (7). In attempts to prevent cross-contamination, some groups proposed the use of sterile liquid nitrogen for the cryopreservation of human gametes or embryos (8).
However, the installation and use of liquid nitrogen sterilizers is a practice that is still not widespread among IVF laboratories possible due to disbelief in contamination risks or technology insufficiencies.
In the meantime, other investigations have evaluated the existence of cross-contamination in an IVF scenario. In a study performed in the mouse model, embryos were exposed to mouse hepatitis virus (MHV) and Pasteurella pneumotropica and were subsequently cryopreserved for up to 12 months together with embryos that had not been in contact with these pathogens. Once thawed and transferred, the mice resulting from non-inoculated embryos did not show signs of the presence of pathogens and thus no evidence of cross-contamination was concluded (9). More recently, the presence of virus has not been detected either in samples of culture medium or liquid nitrogen that had been in direct contact with oocytes and embryos from IVF patients seropositive for HIV, HCV or HBV (10). At the moment there are no cases reported of cross-contamination during the cryopreservation or storage of human oocytes or embryos. Therefore, current published general guidelines from the different Scientific Societies propose that precautions and procedures currently implemented in IVF laboratories for seropositive patients should be sufficient to avoid COVID-19 contamination as well (1-3).
Taking into account the exceptional moments that we are living and that the SARS-CoV-2 virus is highly contagious, as a preventive measure, many embryologists questioned whether more precautions should be taken, e.g., using quarantine tanks for the storage of samples from asymptomatic or confirmed COVID-19 positive patients, nitrogen vapor phase tanks to store samples cryopreserved in open devices; or the use of vitrification protocols based on closed devices with high security straws, rather than open systems.
The use of quarantine tanks
The use of nitrogen quarantine tanks, either in liquid or vapor phase, is employed in most IVF centers to store biological samples from seropositive patients since several years. In the current scenario, the aim would be to avoid storing samples from patients positive for SARS-CoV-2 in the same tank as other samples from healthy patients. Considering that cross contamination can theoretically occur, the use of quarantine tanks could represent an additional measure to reduce disease transmission. We should question though whether this measure is arriving on time, as it has been reported that the first cases of SARS-CoV-2 infected patients in Europe might have occurred in December 2019 or even before (November). At this time, as in the following months until WHO declared the pandemic state, IVF centers were working in full swing, so there is a chance that few asymptomatic patients infected with SARS-CoV-19 may have been treated before the cessation of activities. Consequently, at this moment, it is likely that there might be gametes or embryos collected from COVID-19 positive patients that may already be cryopreserved and are currently stored in the cryo-tanks of daily use together with samples from COVID-19 free patients. However, the fact that there may be a few patients who were asymptomatic and had their samples cryopreserved should not prevent the use of a quarantine tank for patients who are known to be COVID-19 positive before the treatment. How to test patients for the virus before the IVF treatment has also been in debate, as it should help to clarify the potential infectivity of gametes or embryos and thus the utility of using quarantine tanks. However, testing for the virus may not be worthwhile, if only a single test is offered prior to gamete retrieval. Therefore, a serial testing regime may offer more utility when deciding the use of quarantine tanks. Also, even if gametes or embryos from virus positive patients are cryopreserved, useful guidance to risks associated with their use should be explored further, as the presence of eventual viral particles in reproductive cells or in their surroundings is being questioned daily. According to recent publications, ACE2 and CD147 receptors for SARS-Cov-19 have been identified in human embryos (11). However, the zona pellucida could constitute an efficient barrier to prevent the entry of the virus into the embryonic cells, as shown for other pathogens of similar size (12). Furthermore, the washes carried out between the different in vitro culture procedures could also contribute to dilute the number of viral particles surrounding the embryos, reducing their chance of presence at the time of cryopreservation. This dilution could be even more significant during the storage of the cryopreserved samples, considering the large volume of liquid nitrogen tanks usually used in routine would reduce the number of viral particles circulating within the tank. Obviously, this hypothesis would apply only in the case of the so-called open vitrification systems, as in the closed devices there is no direct contact of the samples with liquid nitrogen. In addition, it is yet to demonstrate whether at such low temperatures there is any possibility of Brownian movement of virus particles circulating in the tanks, as at least the biological samples and the surrounding vitrification solution are likely to be in a solid glassy state.
Use of nitrogen vapor phase tanks
The use of nitrogen vapor phase tanks to store samples cryopreserved with open systems has been a common practice since several years in some IVF laboratories (13). This strategy allows a greater storage capacity of samples, at the same time as it reduces the circulation of pathogens that may be present in liquid nitrogen, reducing the eventual risk of cross contamination in open vitrification systems (13). The recommendation on the use of vapor phase tanks has been proposed in forums or webinars organized by different organizations. However, it should be noted that not all centers have the chance of acquiring vapor phase tanks, either because of their higher cost compared to conventional liquid nitrogen tanks or due to the lack of available space in the cryopreservation rooms. In addition, the utilization of these systems is not trivial, as the way samples are handled in nitrogen vapors requires appropriate training and caution. An incorrect manipulation could expose samples to unexpected temperature fluctuations, putting at risk the survival of oocytes or embryos. The use of nitrogen vapor phase tanks is associated with increased sensitivity to liquid nitrogen loss and thus subject to increased risk of temperature loss. The implementation of nitrogen vapor phase tanks may thus affect the daily routine of laboratories and requires proper training of the staff as well as the implementations of measures to prevent unnecessary risks associated with the handling of samples within vapor phase tanks.
Use of open or closed vitrification systems
The current vitrification protocols, either using open or closed cryo-devices, are usually associated with high survival rates. However, as these protocols are usually performed manually (in most laboratories), the consistency of results can vary greatly depending on skills level and practice of each operator. Considering that laboratories have stopped their activities for several weeks and any modification to protocols must be performed gradually and with a continuous evaluation of the results, we question whether the current moment would be the ideal for introducing alterations in such a delicate procedure, as recommended by some organizations on supporting the use of closed vitrification to avoid COVID-19 contamination.
It is expected that sub-optimal training or the incorrect application of a new protocol can result into a significant reduction on the efficiencies of the vitrification process. This would represent, undoubtedly, a higher risk than the theoretical likelihood of cross contamination with COVID-19 as a result of the use of an open system. In the absence of scientific evidence about cross contamination with SARS-Cov-19 virus, cautiousness should be the rule of thumb for IVF laboratories. However, each laboratory should perform a risk assessment analysis of facts considering their own particularities and conditions and taking into account that, in some cases, to avoid a problem, the implementation of precipitate measures (the cure) may end up being worse than the disease (potential cross-contamination risk).
- ASEBIR y SEF. 2020. Recomendaciones para la seguridad de riesgos ante la infección por coronavirus (SARS-Cov-2) en las unidades de reproducción asistida. Available online in: https://asebir.com/wp content/uploads/2020/04/Documento-Consenso-ASEBIR-SEF-COVID19-V2.pdf.
- ESHRE COVID-19 Working Group. ESHRE guidance on recommencing ART treatments. Available online in: https://www.eshre.eu/Press-Room/ESHRE-News.
- ASRM Patient Management and Clinical Recommendations During the Coronavirus (COVID-19) Pandemic. Available online in: https://www.asrm.org/globalassets/asrm/asrm-content/news-andpublications/covid-19/covidtaskforce.pdf.
- FDA: Important Information for Human Cell, Tissue, or Cellular or Tissue-based Product (HCT/P) Establishments Regarding the 2019 Novel Coronavirus Outbreak. February 14, 2020. Available online in: https://www.fda.gov/vaccines-blood-biologics/safety-availability-biologics/important-information-human-cell-tissue-or-cellular-or-tissue-based-product-hctp-establishments.
- Practice Committee of American Society for Reproductive Medicine (ASRM). Recommendations for reducing the risk of viral transmission during fertility treatment with the use of autologous gametes: a committee opinion. Fertil Steril. 2013 Feb;99(2):340-6.
- Tedder RS, Zuckerman MA, Goldstone AH, Hawkins AE, Fielding A, Briggs EM, Irwin D, Blair S, Gorman AM, Patterson KG, et al. Hepatitis B transmission from contaminated cryopreservation tank. Lancet. 1995 Jul 15;346(8968):137-40.
- Fountain D, Ralston M, Higgins N, Gorlin JB, Uhl L, Wheeler C, Antin JH, Churchill WH, Benjamin RJ. Liquid nitrogen freezers: a potential source of microbial contamination of hematopoietic stem cell components. Transfusion. 1997 Jun;37(6):585-91.
- Parmegiani L, Accorsi A, Bernardi S, Arnone A, Cognigni GE, Filicori M. A reliable procedure for decontamination before thawing of human specimens cryostored in liquid nitrogen: three washes with sterile liquid nitrogen (SLN2). Fertil Steril. 2012 Oct;98(4):870-5.
- Kyuwa S, Nishikawa T, Kaneko T, Nakashima T, Kawano K, Nakamura N, Noguchi K, Urano T, Itoh T, Nakagata N. Experimental evaluation of cross-contamination between cryotubes containing mouse 2-cell embryos and murine pathogens in liquid nitrogen tanks. Exp Anim. 2003; 52(1):67-70.
- Cobo A et al. Viral screening of spent culture media and liquid nitrogen samples of oocytes and embryos from hepatitis B, hepatitis C, and human immunodeficiency virus chronically infected women undergoing in vitro fertilization cycles. Fertil Steril. 2012; 97 (1): 74-78.
- Colaco S, Chhabria K, et al. Expression of SARS-CoV-2 receptor ACE2 and the spike protein processing enzymes in developing human embryos. Pre-print April 2020.
- Van Soom A, Wrathall AE, Herrler A, Nauwynck HJ. Is the zona pellucida an efficient barrier to viral infection? Reprod Fertil Dev. 2010;22(1):21-31.
- Cobo A., Storage of human oocytes in the vapor phase of nitrogen. Fertility & Sterility, 2010. Fertil Steril. 2010 Oct;94(5):1903-7.