key: cord-0894624-wwrzceef
authors: Adiga, Satish Kumar; Tholeti, Prathima; Uppangala, Shubhashree; Kalthur, Guruprasad; Gualtieri, Roberto; Talevi, Riccardo
title: Fertility preservation during COVID-19 pandemic: mitigating the viral contamination risk to reproductive cells in cryostorage
date: 2020-09-15
journal: Reprod Biomed Online
DOI: 10.1016/j.rbmo.2020.09.013
sha: d23e94d04437fd619798164cbd304fd9cd84c4e0
doc_id: 894624
cord_uid: wwrzceef

Reopening fertility care services across the world in the midst of a pandemic brings with it numerous concerns that need immediate addressal, such as the impact of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) on the male and female reproductive cells and the plausible risk of cross-contamination and transmission. There exists little literature on confirmatory reports of the association of SARS-CoV-2 with reproductive tissues, gametes, and embryos due to the novelty of the disease. Cryobanking, an essential service in fertility preservation, carries the risk of cross-contamination through cryogenic medium and thus calls for risk-mitigation strategies. This review aims to address the available literature on the presence of SARS-CoV-2 on tissues, gametes, and embryos with a special reference to the possible sources of cross-contamination through liquid nitrogen. Strategies for risk-mitigation have been extrapolated from reports dealing with other viruses to the current global crisis, for safety in fertility treatment services in general and oncofertility in specific.

The current pandemic of Coronavirus Disease 2019 caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has focused the attention of healthcare service providers across the globe away from all other non-emergency health problems including fertility (ESHRE, 2020a; Tesarik, 2020) . ASRM's recent guidelines state that the window of opportunity for infertile couples is finite and postponing for too long could reduce the chances of pregnancy (ASRM, 2020a) . Importantly, individuals needing fertility preservation due to cancer or other conditions which requires gonadotoxic treatment, need urgent procedures to store their reproductive cells. With fertility services slowly resuming globally (ESHRE 2020b) , the aim of this review is to formulate a better understanding of the impact of SARS-CoV-2 on male and female gametes and embryos and determine the risks involved with cross-contamination of viral pathogens such as SARS-CoV-2 during cryostorage in liquid nitrogen (LN2). This review also highlights strategies to mitigate the risks through safety and precautionary measures.

Due to the novelty of COVID-19 causing virus, SARS-CoV-2, few reports are available documenting the detection of this virus on male and female reproductive cells or tissues (Corona et al, 2020; Jing et al, 2020) . SARS-CoV-2 belongs to the Coronavirus family and as its genome sequence is 82% identical to SARS-CoV, (Chan et al, 2020) , the findings from this family subtype (SARS-CoV) could help in understanding the SARS-CoV-2 pathophysiology on reproductive cells (Segars et al, 2020) . Important structural commonalities between SARS-CoV-2 and SARS-CoV have been recently demonstrated with respect to receptor binding domains and mechanism of cell entry (Hoffmann et al, 2020) . Both viruses utilize the spike glycoprotein for entry into target cells. Entry into the host cells depends on binding of the virus to the angiotensin-converting enzyme 2 (ACE2) and occurs after priming of the spike glycoprotein by the host cell serine protease TMPRSS2 (Hoffmann et al, 2020) . Since human germ cells and early embryos express high levels of ACE2, there is potential risk of SARS-CoV-2 being associated with reproductive cells. In the males, ACE2 has been shown to be expressed in spermatogonia, Leydig and Sertoli cells.

Gene Set Enrichment Analysis showed that gene ontology categories associated with viral replication and transmission are markedly expressed in these cells lending support to the hypothesis that the human testicle could represent a potential target for SARS-CoV-2 infection (Wang and Xu, 2020) . Currently, about 27 viruses over a wide range of families have been found in the human semen and in case of Zika, Ebola and Marburg, replicating viruses have been demonstrated in the semen and can be sexually transmitted (Salam and Horby, 2017 ). An indication that SARS-CoV, closely related to SARS-CoV-2, may affect the male reproductive system by causing orchitis was demonstrated in six individuals, however, in situ hybridization did not detect SARS-CoV genomic sequences in the testes suggesting that the pathology may represent a complication of SARS-CoV infection (Xu et al. 2006) .

As SARS-CoV-2 has been found in blood of 1% of symptomatic patients , the possibility of male reproductive tract infections, especially in patients with systemic or local inflammation compromising the blood-testes/deferens/epididymis barriers, remains a concern. Till now the presence of SARS-CoV-2 in human semen is controversial as the few studies performed reported conflicting data. Li et al. (2020) reported the presence of the virus in the semen of 26.7% patients in the acute phase of infection and 8.7% of recovering patients. However, the study is scarcely detailed about procedures adopted to avoid contamination of semen samples and proof about the real infectivity of the virus detected in the semen is lacking. On the contrary, Pan et al. (2020) (2020) failed to detect the presence of the virus in urine and semen of one patient 8 days after SARS-CoV-2 PCR detection but one day before a further negative pharyngeal swab. Other recent studies Guo et al, 2020) also did not detect SARS-CoV-2 RNA in semen samples in patients with recent infection or recovering or in a testicular sample of one patient who died of COVID-19. However, Holtmann et al.,(2020) have reported impairment of semen parameters in patients with moderate SARS-CoV-2 infection though the virus was not detected in the semen, but whether this impairment was due to the infection itself or the associated treatment was not determined. Overall, due to the limited number of subjects analysed, the stage of infection at the time of semen sampling and conflicting evidence, the possibility that SARS-CoV-2 presence in the semen of the infected patients cannot be completely ruled out, especially in asymptomatic cases (Kashi, 2020) .

From the available literature it appears that the female reproductive tract is less impacted by SARS-CoV-2 than the males (Segars et al, 2020) as SARS-CoV was not demonstrated in the ovaries and uterine tissues through immunohistochemistry and in situ hybridization studies (Ding et al,2004) although ACE2 is widely expressed in the ovary (Jing et al., 2020), uterus and vagina (Vaz-Silva et al, 2009) . The presence of ACE-2 indicates the female reproductive organs as potential targets for SARS-CoV-2 infection, however no evidences of infection or sexual transmission were reported till now (Cui et al, 2020) .

Gametes obtained from patients with other viral illnesses, such as human immunodeficiency virus and hepatitis, must be treated with special precautions to reduce exposure of the noninfected partner and cross-contamination of reproductive tissue within the laboratory (ASRM, 6 2013). Whether these precautions are to be recommended currently for SARS-CoV-2, given the lack of insufficient evidence for transmission through blood or sexual contact Cui et al, 2020 ) is yet to be seen. While there is no universal recommendation for screening oocyte or semen donors for SARS-CoV-2, ASRM recommends screening questions in asymptomatic donors, to avoid potential infections (ASRM, 2020b). These are areas in which further investigation is necessary to assure the safety of stored gametes and the safety of patients undergoing assisted reproduction.

With the emerging global and regional success of oncofertility (Ataman et al, 2016; Rashedi et al, 2020) , and the American Society of Clinical Oncology recommending guidelines to offer fertility preservation services to oncological patients (Oktay et al, 2018) , many young and adult cancer patients preserve their spermatozoa, testicular tissues, ovarian tissues, oocytes or embryos for a fertile future. Multiple cryopreservation protocols using various cryoprotectants have been developed for each of the reproductive material to ensure the longterm safe storage and effective recovery while also sustaining the fertility potential Although, elective procedures using ART are being preferably cancelled or postponed during this pandemic, fertility preservation is an emergency requirement even in limited resource settings, as cancer treatment cannot be delayed (Salama et al, 2020a) . However, there are concerns about risks of cross-contamination during cryo-storage and reintroduction of virus when patients need fertility restoration. 7

Cryostorage, while being beneficial also carries multiple concerns pertaining to contamination of LN2 leading to transmission of infectious diseases between samples (Tomlinson, 2005) or transmission of infectious disease to patient itself years later during fertility restoration. Although the presence of SARS-CoV-2 in semen and female reproductive fluids is still under debate, a relevant risk in cryopreservation is the potential transmission of pathogens during preparation procedures and cryostorage. Noticeably, the transmission of pathogens in samples stored in fertility cryobanks has never been reported (Yakass and Woodward, 2020) , but concerns of cross-contamination among cryopreserved samples arose after the report of human hepatitis virus transmission from bone marrow transplants stored in the same LN2 tank (Tedder et al, 1995) . One of the reasons for cryoresistance of viruses could be the use of specific cryoprotectants used to protect germplasm during the process of freeze-thawing, which could also confer protection to the enveloped viruses thereby leading to transmission of the pathogen to the other stored biomaterials (Hubalek, 2003; Bielanski et al 2000; Bielanski et al 2009; Alikani, 2018) . Reports of viruses such as HIV, hepatitis, influenza, and papillomavirus retaining their infectivity after cryopreservation has brought upon the realization that LN2 exposed to the virus can be a biohazard leading to cross-contamination (Schaefer et al, 1976; Tedder et al, 1995; Byers, 1999; Bielanski, 2012; Merrill et al, 2018) . Studies on virus cross-contamination in ART cryobanks are controversial. Bielanski et al. (2000) reported absence of transmission of bovine viral diarrhoea virus (BVDV) and bovine herpesvirus-1 (BHV-1) from infected semen and embryos straws to non-infected samples stored in the same LN2 tanks, but crosscontamination to frozen embryos was seen during experimental contamination of LN2 with the same viruses . On the other hand, Cobo et al. (2012) , failed to detect the presence of viral RNA or DNA sequences in LN2 used for oocyte or embryo vitrification in patients 8 seropositive for human immunodeficiency virus (HIV), hepatitis C virus (HCV), and hepatitis B virus (HBV) undergoing ART cycles. Nevertheless, while repeated washings could effectively eliminate the viral pathogens (ASRM 2020), the possibility that viruses could cross the zona pellucida (ZP) is a major concern. The ZP is a glycoprotein envelope that acts as a physical barrier against microorganisms and might protect oocytes and embryos by viral representing an additional concern considering that human preimplantation embryos highly express ACE2, that is required by SARS-CoV-2 for cell entry (Yan et al., 2013) .

From the clinical perspective, patients can be reassured that their gametes are not infected by SARS-CoV-2 and therefore cryopreserved, however, SARS-CoV-2 testing should be made mandatory for all fertility preservation patients (Dellino et al., 2020) . Apart from contaminated clinical samples, there is a substantial risk of iatrogenic infection of the samples in the laboratory from the operators. It should be noted that the particles of SARS-CoV-2 could be present during cryopreservation and may survive post-thaw as viruses are usually resistant to the freeze-thaw process (ASRM 2020a). This warrants the need for guidelines in safe handling and cryopreservation of biomaterials during fertility preservation.

9 Several reproductive societies and clinical groups provided recommendations and guidelines on the management of ART patients during the current SARS-CoV-2 global pandemic (ESHRE 2020b; Dellino et al, 2020; Vaiarelli et al, 2020; SART, 2020; Alteri et al, 2020; Huyser, 2014) . ESHRE and ASRM recommend testing of both the partners for SARS-Cov-2 before initiating ART treatment (ASRM, 2020a; ESHRE, 2020b). Esteves et al. (2020) , have proposed remedies that include identifying oncological patients who can neither delay the treatment nor experience infertility post gonadotoxic therapy and give them priority for fertility preservation after testing for SARS-CoV-2 (Esteves et al, 2020) .

Cross contamination during cryostorage possibly arises due to the type of cryopreservation device such as open or closed cryosystems used. In a closed device, such as sealed straws, the reproductive material is not directly exposed to LN2 therefore reducing the hazard of crosscontamination (Shapiro et al, 2020) . However, use of screw capped plastic vials can lead to contact with surrounding LN2 by vacuum creation in the vial due to condensation of the atmosphere at such low temperature thereby drawing in the liquid nitrogen (Woods and Thirumala, 2011) . Several closed carrier systems have been employed for cryopreservation, such as microvolume air cooling (MVAC) device (Punyawai et al, 2015) , high-security vitrification straw (HSV) or Cryotip, some of which have been used for human embryo cryopreservation thereby hermetically isolating the reproductive cells (Arav, 2020; Abdel-Hafez et al, 2011; Kuwayama et al, 2005) . Hence, ESHRE in its latest guidelines recommends the use of high-security straws and vapor-phase storage for cryopreservation of reproductive samples from COVID-19 positive patients (ESHRE, 2020b) . To avoid contamination while using open carrier system during vitrification, Arav et al, (2016) have devised a bench-top device to produce cooled clean liquid air (ClAir) that has similar temperature to that of LN2. It may also be safe to use a secondary enclosure for cryodevices or to store samples of patients who are suspected/infected in separate LN2 cryotanks to avoid disease transmission through contaminated LN2 (Bielanski et al, 2009) . Sharing of LN2 between patient samples during cryopreservation is not advisable (Pomeroy and Schiewe, 2020) . LN2 has been used as an efficient cryogenic medium but considering the risk of cross-contamination, vapour phase nitrogen has been shown to be a safer alternative (Abdel-Hafez et al, 2011; ASRM 2020c) , although the risk cannot be negated (Grout and Morris, 2009 ). Vapour phase nitrogen storage appears to be practical especially in cases of unwashed semen samples or those awaiting viral test results (Schiewe et al, 2018) . Whilst taking these precautions it is also important to ensure the sterility of LN2 (ASRM 2020; Bielanski et al, 2009; Larman et al, 2014) , using certain devices (http://www.freepatentsonline.com/5737926.html), especially with regard to viral pathogens whose route of transmission is different than those of blood borne viral pathogens. Small volumes of LN2 required for vitrification can be filtered using 0.2μm filters (McBurnie & Bardo, 2002) , however its efficacy on eliminating virus such as SARS-COV-2 remains to be proved. Periodic disinfection of the cryotanks and tools used during cryopreservation, by UV exposure or chemical disinfectants, is recommended to reduce cross-contamination (Bielanski et al, 2009; Larman et al, 2014) . Sterilization of LN2 has been reported by Vajta et al. (1998) who performed cooling in liquid nitrogen that has been filtered through 0.2μm pore size disposable filters to eliminate contaminants, and by Parmegiani et al. (2009) through emitting a minimum UV radiation dose in a temperature controlled manner in a short interval for effective sterilization. Care should be taken however, during cleaning so as not to put the stored reproductive samples at risk during removal from cryotanks (Tao et al, 2020) . Though no studies have been reported so far on the presence of SARS-Cov-2 in commercially produced medical-purpose LN2, contamination risks should be considered.

Permegiani et al. (2012) , have reported a safe 3-step washing procedure with sterile LN2 to eliminate pathogens from human cryopreserved specimens to minimize the risk of contamination, but as it was only studied on bacteria and fungi, the effectiveness on viral

pathogens is yet to be seen. Nevertheless, it may be advisable to follow this procedure in the current scenario to eliminate the viral pathogens (ASRM, 2020a). Also, repeated and efficient washing of gametes and embryos before cryopreservation and after thawing can reduce the infectivity of the disease by high dilution of viral particles (ASRM 2020a; Bielanski, 2009; ) .

Such washing-dilution procedures dilute the infective agents far below the threshold level required for causing a clinical infection, to a probability of <0.0002% even with open vitrification systems (Vajta et al, 2015) , however, measures should be taken to prevent such low probability as well. Sperm-washing procedures such as double density gradient followed by swim-up has been shown to separate motile sperm free of viral particles in males infected with HIV or HCV and such sperm wash procedures could be used for other viral infection (ASRM, 2013) .

Based on the above discussion, implementing following cross-contamination risk mitigation strategies may ensure safety to patients, personnel and laboratories offering cryo-facilities:

-Periodic disinfection of cryocontainers or ensuring sterility of factory derived LN2 (Bielanski, and Vajta, 2009 ).

-Storage of suspected/infected samples in separate cryo-containers away from other healthy patient's samples (Bielanski, and Vajta, 2009 ).

-Use of closed carrier devices during cryopreservation to prevent direct contact with LN2 or "double bagging" of the cryodevices (Bielanski, and Vajta, 2009 ).

-Storage of samples in vapour phase nitrogen instead of LN2 to minimize risk is practical in cases of unwashed semen samples or those awaiting viral test results (Schiewe et al., 2018) 12 -Repeated washing of gametes/embryos before cryopreservation and after thawing to dilute the viral infectivity (ASRM, 2020a).

While the above stated precautions can minimize the risk of cross-contamination thereby ensuring safety to the patient, the safety of the personnel working in the laboratory is also equally important (Vajta et al, 2013) . In many embryology laboratories across the world, standard precautions are not regularly followed while handling LN2 (Vajta et al, 2013) , proving to be a health hazard for the personnel. It has been demonstrated that aerosol mist particles of 1-5µm sizes are generated near the liquid nitrogen surface, up to a distance of 10-20cm, which could be due to evaporation and floating of particles (Lee, 2020) . This could pose a risk to personnel while handling contaminated LN2 containers. Hence, it is important for the personnel in cryo units to use cryo-accessories such as safety goggles or face visors and protective clothing (Vajta et al, 2013) while handling cryo-samples and containers, to ensure safety and efficiency.

With fertility services resuming across the world, it is important for healthcare providers to be aware of the impact of SARS-CoV-2 on male and female reproductive cells and tissues and the risk of cross-contamination and transmission through cryobanking services. While there is controversial evidence on presence of SARS-CoV-2 in seminal plasma of COVID-19

infected or recovering patients, so far, there are no reports demonstrating its presence on the female reproductive cells and tissues. The current scenario of COVID-19 is expected to last for several months to a year at least, with rebounds expected to occur (ASRM, 2020a), hence new strategies have to be adopted when offering fertility services, especially pertaining to cryopreservation, to combat the COVID-19.

Cryobanking using liquid nitrogen carries the risk of cross-contamination by viral pathogens leading to disease transmission, therefore it is important to understand and mitigate such risks through safety and precautionary measures. Some of the measures include testing of both the partners for SARS-CoV-2 before initiating treatment, use of closed-carrier cryodevices, sanitary cryostorage protocols and efficient washing of gametes or embryos during cryopreservation, which would help in reducing the risk of disease transmission. However, the feasibility of adopting these strategies especially in developing countries, with limited resources and already existing challenges to support oncofertility programs, would require a strong global network that enables sharing resources, methodologies and experiences for building competency (Salama et al, 2020b) .

The authors declare no conflict of interest. 

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Professor Adiga is heading the ART laboratory and Centre for Fertility Preservation at Manipal, India. His research interests are fertility preservation, preimplantation biology, non-invasive gamete and embryo selection

This review aims to address the available literature on the presence of SARS-CoV-2 on tissues, gametes, and embryos with a special reference to the possible sources of crosscontamination through liquid nitrogen. Strategies for risk-mitigation have been extrapolated from reports dealing with other viruses to the current global crisis, for safety in fertility treatment services in general and oncofertility in specific.