3†Emergency salvage of time expired clinical corneas during the COVID-19 pandemic.

INTRODUCTION: Corneas for clinical use can be stored for a maximum of 28 days in organ culture medium after death. At the beginning of the COVID-19 pandemic in 2020 it became apparent that; a rare situation was arising in that clinical operations were being cancelled and that there would be a surplus of "clinical grade" corneas. Consequently, when the corneas reached the end of the storage period, if the tissue had appropriate consent, they were transferred to the Research Tissue Bank (RTB). However, University research had also stopped due to the pandemic and there was a situation where the RTB had good quality tissue without any users. Rather than discarding the tissue, a decision was made to store the tissue for future use by cryopreservation. MATERIALS AND METHODS: An established protocol for cryopreserving heart valves was adapted. Individual corneas were placed into wax histology cassettes then inside a Hemofreeze heart valve cryopreservation bag with 100 ml cryopreservation medium (10% Dimethyl sulphoxide)). They were frozen in a controlled rate freezer (Planer, UK) to below -150oC and stored in vapour phase over liquid nitrogen (VPLN) below -190oC. To assess morphology, six corneas were cut in half, one half was processed for histology whilst the other half was cryopreserved, stored for 1 week then thawed and processed for histology. The stains used were Haematoxylin and Eosin (H&E) and Miller's with Elastic Van Gieson (EVG). RESULTS: Comparative histological examination indicated that there were no visible, major, detrimental changes in morphology in the cryopreserved group as compared to the controls. Subsequently, a further, 144 corneas were cryopreserved. Samples were assessed for handling properties by eye bank technicians and ophthalmologists. The eye bank technicians felt that the corneas may be suitable for training purposes such a DSAEK or DMEK. The ophthalmologists said that they had no preference between the fresh or cryopreserved corneas, and both would be equally suitable for training purposes. CONCLUSION: Time expired, organ-cultured corneas, can be successfully cryopreserved using an established protocol by adapting the storage container and conditions. These corneas are suitable for training purposes and may prevent discard of corneas in future.

A French single-center experience on allogeneic stem cell transplant cryopreservation during severe acute respiratory syndrome coronavirus 2 pandemic.

BACKGROUND AIMS: Allogeneic hematopoietic stem cell transplantation (allo-SCT) is a curative treatment for chemo-resistant hematological malignancies. Because of transport restriction imposed by the coronavirus disease 2019 pandemic, regulatory bodies and societies recommended graft cryopreservation before recipient conditioning. However, the freezing and thawing processes, including washing steps, might impair CD34+ cell recovery and viability, thereby impacting the recipient engraftment. Over 1 year (between March 2020 and May 2021), we aimed to analyze the results of frozen/thawed peripheral blood stem cell allografts in terms of stem cell quality and clinical outcomes. METHODS: Transplant quality was evaluated by comparing total nucleated cells (TNCs), CD34+ cells and colony-forming unit-granulocyte/macrophage (CFU-GM)/kg numbers as well as TNC and CD34+ cell viabilities before and after thawing. Intrinsic biological parameters such as granulocyte, platelet and CD34+ cell concentrations were analyzed, as they might be responsible for a quality loss. The impact of the CD34+ cell richness of the graft on TNC and CD34 yields was evaluated by designing three groups of transplants based on their CD34 /kg value at collection: >8 × 10 6/kg, between 6 and 8 × 106/kg and <6 × 106/kg. The consequences of cryopreservation were compared in the fresh and thawed group by evaluating the main transplant outcomes. RESULTS: Over 1 year, 76 recipients were included in the study; 57 patients received a thawed and 19 patients a fresh allo-SCT. None received allo-SCT from a severe acute respiratory syndrome coronavirus 2-positive donor. The freezing of 57 transplants led to the storage of 309 bags, for a mean storage time (between freezing and thawing) of 14 days. For the fresh transplant group, only 41 bags were stored for potential future donor lymphocyte infusions. Regarding the graft characteristics at collection, median number of cryopreserved TNC and CD34+ cells/kg were greater than those for fresh infusions. After thawing, median yields were 74.0%, 69.0% and 48.0% for TNC, CD34+ cells and CFU-GM, respectively. The median TNC dose/kg obtained after thawing was 5.8 × 108, with a median viability of 76%. The median CD34+ cells/kg was 5 × 106, with a median viability of 87%. In the fresh transplant group, the median TNC/kg was 5.9 × 108/kg, and the median CD34+ cells/kg and CFU-GM/kg were 6 × 106/kg and 276.5 × 104/kg, respectively. Sixty-one percent of the thawed transplants were out of specifications regarding the CD34+ cells/ kg requested cell dose (6 × 106/kg) and 85% of them would have had this dose if their hematopoietic stem cell transplant had been infused fresh. Regarding fresh grafts, 15.8% contained less than 6 × 106 CD34+ cells /kg and came from peripheral blood stem cells that did not reach 6 × 106 CD34+ cells /kg at collection. Regarding the factor that impaired CD34 and TNC yield after thawing, no significant impact of the granulocyte count, the platelet count or the CD34+ cells concentration/µL was observed. However, grafts containing more than 8 × 10 6/kg at collection showed a significantly lower TNC and CD34 yield. CONCLUSIONS: Transplant outcomes (engraftment, graft-versus-host disease, infections, relapse or death) were not significantly different between the two groups.

Effects of extended transport on cryopreserved allogeneic hematopoietic progenitor cell (HPC) product quality and optimal methods to assess HPC stability.

BACKGROUND: Since the beginning of the COVID-19 pandemic, cryopreservation of hematopoietic progenitor cell (HPC) products has been increasingly used to ensure allogeneic donor graft availability prior to recipient conditioning for transplantation. However, in addition to variables such as graft transport duration and storage conditions, the cryopreservation process itself may adversely affect graft quality. Furthermore, the optimal methods to assess graft quality have not yet been determined. STUDY DESIGN AND METHODS: A retrospective review was performed on all cryopreserved HPCs processed and thawed at our facility from 2007 to 2020, including both those collected onsite and by the National Marrow Donor Program (NMDP). HPC viability studies were also performed on fresh products, retention vials, and corresponding final thawed products by staining for 7-AAD (flow cytometry), AO/PI (Cellometer), and trypan blue (manual microscopy). Comparisons were made using the Mann-Whitney test. RESULTS: For HPC products collected by apheresis (HPC(A)), pre-cryopreservation and post-thaw viabilities, as well as total nucleated cell recoveries were lower for products collected by the NMDP compared to those collected onsite. However, there were no differences seen in CD34+ cell recoveries. Greater variation in viability testing was observed using image-based assays compared to flow-based assays, and on cryo-thawed versus fresh samples. No significant differences were observed between viability measurements obtained on retention vials versus corresponding final thawed product bags. DISCUSSION: Our studies suggest extended transport may contribute to lower post-thaw viabilities, but without affecting CD34+ cell recoveries. To assess HPC viability prior to thaw, testing of retention vials offers predictive utility, particularly when automated analyzers are used.

Cryopreservation of Human Spermatozoa: Functional, Molecular and Clinical Aspects.

Cryopreservation is an expanding strategy to allow not only fertility preservation for individuals who need such procedures because of gonadotoxic treatments, active duty in dangerous occupations or social reasons and gamete donation for couples where conception is denied, but also for animal breeding and preservation of endangered animal species. Despite the improvement in semen cryopreservation techniques and the worldwide expansion of semen banks, damage to spermatozoa and the consequent impairment of its functions still remain unsolved problems, conditioning the choice of the technique in assisted reproduction procedures. Although many studies have attempted to find solutions to limit sperm damage following cryopreservation and identify possible markers of damage susceptibility, active research in this field is still required in order to optimize the process. Here, we review the available evidence regarding structural, molecular and functional damage occurring in cryopreserved human spermatozoa and the possible strategies to prevent it and optimize the procedures. Finally, we review the results on assisted reproduction technique (ARTs) outcomes following the use of cryopreserved spermatozoa.

Neither COVID-19, nor cryopreservation, prevented allogeneic product infusion: A report from the National Marrow Donor Program.

Background: The Coronavirus Disease 2019 (COVID-19) pandemic in early 2020 has resulted in an unprecedented level of uncertainty and challenge for the stem cell donor registries. To address these challenges, rapid strategies were implemented by the National Marrow Donor Registry (NMDP) and its network partners. Herein, we aim to report the impact of the COVID-19 pandemic on the collection, utilization of grafts, and short-term outcomes of patients who received stem cell products from COVID-19-positive donors. Methods: NMDP data during the early phase (1 March 2020 through 1 May 2020) of the pandemic were compared to the later phase (1 March 2021 through 1 May 2021). Odds ratios were calculated to determine the impact of the pandemic on graft sources requested by transplant centers (TCs). The Kruskal-Wallis test was used to test the effect of the pandemic on the disease indication, volume of searches, and number of products not infused. Results: Although there was an initial decline in overall donor searches during the early phase of the pandemic, these numbers increased reaching pre-pandemic levels during the later phase. Urgent malignant diseases remained the most common indication for transplant in 2021. The pandemic necessitated cryopreservation of stem cell products due to transportation restrictions as well as clinical uncertainties in managing the virus. Cryopreserved grafts remained the most common requested grafts throughout the pandemic. In the later phase of the pandemic, the total numbers of requests for fresh grafts increased, mostly due to the increase in requests for fresh bone marrow (BM) grafts. As the pandemic continued, TCs became more accepting of cryopreservation, resulting in a reduction in the number of products not infused. Lastly, no short-term deleterious outcomes were noted among the patients who had stem cell products infused from a SARS-CoV-2-positive donor. Conclusion: Throughout the pandemic, the NMDP and TCs worked tirelessly to ensure that patients would receive lifesaving grafts when needed. The data reported here, although limited by small numbers, illustrate that transplantation from donors with COVID-19 is feasible and safe.

Pfizer SARS-CoV-2 BNT162b2 mRNA vaccination (BNT162b2) has no adverse effect on elective oocyte cryopreservation outcomes.

RESEARCH QUESTION: Do elective oocyte cryopreservation outcomes in women 1-13 months after SARS-CoV-2 vaccination alter compared with unvaccinated women and do different time intervals between vaccination and ovarian stimulation impact these outcomes? DESIGN: This retrospective cohort study, conducted in a university-affiliated IVF centre, included 232 elective oocyte cryopreservation cycles of vaccinated and unvaccinated patients, without previous infection with the SARS-CoV-2 virus, between December 2020 and January 2022. Two control groups - pre-pandemic (January 2019 to February 2020) and intra-pandemic (December 2020 to January 2022) unvaccinated groups - were compared with the vaccinated group, further divided into four subgroups (under 3, 3-6, 6-9 and 9-13 months). The primary outcome was the elective oocyte cryopreservation cycle outcomes - number of retrieved and number of mature oocytes. RESULTS: The vaccinated group demonstrated comparable outcomes with regards to number of retrieved and mature oocytes compared with the pre-pandemic and intra-pandemic unvaccinated groups (12.6 ± 8.0 versus 13.0 ± 8.2 and 12.5 ± 7.4 retrieved and 10.1 ± 6.9 versus 9.5 ± 6.4 and 10.1 ± 6.3 mature oocytes, respectively; not significant for both). Similar results were noted in a comparison between the intra-pandemic unvaccinated group and the four vaccinated subgroups. No correlation was found between the parameter of days from vaccination and cycle outcomes. Similarly, analysis of covariance showed no association between vaccination status and timing and number of mature oocytes. CONCLUSIONS: The SARS-CoV-2 vaccination does not alter the outcomes of elective oocyte cryopreservation procedures. This is true even in a relatively long time interval of 9 to 13 months from vaccination.

Measurement of Mitochondrial Respiration in Cryopreserved Human Peripheral Blood Mononuclear Cells (PBMCs).

Inflammatory diseases caused by infectious agents such as the SARS-CoV-2 virus can lead to impaired reductive-oxidative (REDOX) balance and disrupted mitochondrial function. Peripheral blood mononuclear cells (PBMCs) provide a useful model for studying the effects of inflammatory diseases on mitochondrial function but can be limited by the need to store these cells by cryopreservation prior to assay. Here, we describe a method for improving and determining PBMC viability with normalization of values to number of living cells. The approach can be applied not only to PBMC samples derived from patients with diseases marked by an altered inflammatory response such as viral infections.

Can We Cryopreserve the Sperm of COVID-19 Patients During the Pandemic?

An extreme strain has been placed on healthcare facilities in the COVID-19 era. Initial stage of the pandemic, national and international societies for reproductive medicine suggested the suspension of new IVF treatments and non-essential cryopreservation of gametes. Accordingly, the demands of cryopreservation of semen with COVID-19 patients also was suspended by some of cryobanks to protect staff and patients from unnecessary viral exposure at the acute stage. However, the pandemic may stay with us longer than expected. In addition, there will be some male COVID-19 patients with cancer or critically illness who needs to cryopreserve their semen before medical treatments, otherwise they might loss the chance of getting their own offspring. In this document, we summarize available evidence to deepen and expand awareness of feasibility of sperm cryopreservation and propose some suggestions to help cryobanks carry out sperm preservation procedure for COVID-19 male patients.

The impact of past COVID-19 infection on pregnancy rates in frozen embryo transfer cycles.

PURPOSE: To study the effect of SARS-CoV-2 infection on pregnancy rates in frozen embryo transfer (FET) cycles. METHODS: A retrospective cohort study including women under the age of 42 with documented SARS-CoV-2 infection up to 1 year prior to treatment, undergoing FET cycles in the first half of 2021, with transfer of embryos generated prior to the infection. Controls were SARS-CoV-2 non-diagnosed, non-vaccinated women matched by age, number, and day of embryo transfer. Demographic and cycle characteristics and outcomes were compared. RESULTS: Forty-one recovered women and 41 controls were included. Pregnancy rates were 29% and 49% respectively (p = 0.070). Stratification by time from SARS-CoV-2 infection to transfer into ≤ 60 and > 60 days revealed a difference in pregnancy rates, with women in the COVID group having lower pregnancy rates if infected in proximity to the transfer (21% vs. 55%; p = 0.006). In a logistic regression model, infection was a significant variable (p = 0.05, OR 0.325, 95% CI 0.106-0.998). Logistic regression applied on the subgroup of women infected in proximity to the transfer further strengthened the univariate results, with COVID-19 remaining a significant parameter (p = 0.005, OR 0.072, 95% CI 0.012-0.450). CONCLUSIONS: In FET cycles of patients with past SARS-CoV-2 infection, in which oocytes were retrieved prior to infection, decreased pregnancy rates were observed, specifically in patients who recovered less than 60 days prior to embryo transfer. Pending further studies, in cases of FET cycles with limited number of embryos, postponing embryo transfer for at least 60 days following recovery from COVID-19 might be considered when feasible.

Quantitative and Functional Assessment of the Influence of Routinely Used Cryopreservation Media on Mononuclear Leukocytes for Medical Research.

Quantitative and functional analysis of mononuclear leukocyte populations is an invaluable tool to understand the role of the immune system in the pathogenesis of a disease. Cryopreservation of mononuclear cells (MNCs) is routinely used to guarantee similar experimental conditions. Immune cells react differently to cryopreservation, and populations and functions of immune cells change during the process of freeze-thawing. To allow for a setup that preserves cell number and function optimally, we tested four different cryopreservation media. MNCs from 15 human individuals were analyzed. Before freezing and after thawing, the distribution of leukocytes was quantified by flow cytometry. Cultured cells were stimulated using lipopolysaccharide, and their immune response was quantified by flow cytometry, quantitative polymerase chain reaction (qPCR), and enzyme-linked immunosorbent assay (ELISA). Ultimately, the performance of the cryopreservation media was ranked. Cell recovery and viability were different between the media. Cryopreservation led to changes in the relative number of monocytes, T cells, B cells, and their subsets. The inflammatory response of MNCs was altered by cryopreservation, enhancing the basal production of inflammatory cytokines. Different cryopreservation media induce biases, which needs to be considered when designing a study relying on cryopreservation. Here, we provide an overview of four different cryopreservation media for choosing the optimal medium for a specific task.

Meeting the COVID challenge: Optimizing vCD34+ in cryopreserved HPC samples for implementation of an external QA Program.

BACKGROUND: The COVID-19 pandemic has forced a fundamental change in the global procurement of allogeneic hematopoietic progenitor cells (HPCs) for transplantation. To better meet the emergent challenges of transporting cryopreserved allogeneic HPC during pandemics, there is an urgent need for External Quality Assurance (EQA) programs to evaluate reproducibility and harmonization of viable CD34+ cell (vCD34+) HPC enumeration, as the current EQA programs are unsuitable for analysis of vCD34+. The cost-effective distribution of HPC cryopreserved reference samples (CRSs) with acceptable reproducibility and specificity is key to the success of a vCD34+ EQA program. METHODS: Cryopreserved HPC samples (n = 11) were either stored on dry ice for 1 to 4 days or for 1 day followed by liquid nitrogen (LN) storage for 1 to 3 days to assess optimal conditions for vCD34+ EQA. Flow cytometric enumeration of vCD34+ HPCs was performed using a single platform assay combined with 7-AAD viability dye exclusion. The optimum transportation condition was validated in pilot and multicenter national studies (n = 12). RESULTS: A combination of 1 day on dry ice followed by LN storage stabilized viability compared with continuous storage on dry ice. This study demonstrates that dispatch of CRSs on dry ice to recipient centers across a distance of ≤4000 km within 26 h, followed by LN storage, resulted in reproducible intercenter vCD34+ enumeration. The estimated cost of safer and more convenient dry ice delivery is >20-fold lower than that of LN. CONCLUSION: This approach can form the basis for economically and scientifically acceptable distribution of CRSs for external vCD34+ EQA.

High cryo-resistance of SARS-CoV-2 virus: Increased risk of re-contamination at transplantation of cryopreserved ovarian tissue after COVID-19 pandemic.

Cryopreservation and re-transplantation of ovarian tissue after anticancer treatment is important medical technology. Today, during a pandemic, the risk of contamination of transplanted cells with SARS-CoV-2 virus is extremely high. Data about cryo-resistance (virulence and/or infectivity) of SARS-CoV-2 are limited. Analysis and systematization of literature data allow us to draw the following conclusions: 1) The cytoplasmic membrane of somatic cell, like envelope of corona viruses, consists of lipid bilayer and this membrane, like envelope of corona virus, contains membrane proteins. Thus, we can consider the cytoplasmic membrane of an ordinary somatic cell as a model of the envelope membrane of SARS-CoV-2. It is expected that the response of the virus to cryopreservation is similar to that of a somatic cell. SARS-CoV-2 is more poor-water and more protein-rich than somatic cell, and this virus is much more cryo-resistant. 2) The exposure of somatic cells at low positive temperatures increases a viability of these cells. The safety of the virus is also in direct proportion to the decrease in temperature: the positive effect of low temperatures on SARS-CoV-2 virus has been experimentally proven. 3) Resistance of SARS-CoV-2 to cryoprotectant-free cryopreservation is extremely high. The high viability rate of SARS-CoV-2 after freezing-drying confirms its high cryo-resistance. 4) The risk of SARS-CoV-2 infection after transplantation of cryopreserved ovarian tissues that have been contaminated with this virus, increases significantly. Our own experimental data on the increase in the viability of cancer cells after cryopreservation allow us to formulate a hypothesis about increasing of viability (virulence and/or infectivity) of SARS-CoV-2 virus after cryopreservation.

Development of Optimized Vitrification Procedures Using Closed Carrier System to Improve the Survival and Developmental Competence of Vitrified Mouse Oocytes.

The open carrier system (OC) is used for vitrification due to its high efficiency in preserving female fertility, but concerns remain that it bears possible risks of cross-contamination. Closed carrier systems (CC) could be an alternative to the OC to increase safety. However, the viability and developmental competence of vitrified/warmed (VW) oocytes using the CC were significantly lower than with OC. We aimed to improve the efficiency of the CC. Metaphase II oocytes were collected from mice after superovulation and subjected to in vitro fertilization after vitrification/warming. Increasing the cooling/warming rate and exposure time to cryoprotectants as key parameters for the CC effectively improved the survival rate and developmental competence of VW oocytes. When all the conditions that improved the outcomes were applied to the conventional CC, hereafter named the modified vitrification/warming procedure using CC (mVW-CC), the viability and developmental competence of VW oocytes were significantly improved as compared to those of VW oocytes in the CC. Furthermore, mVW-CC increased the spindle normality of VW oocytes, as well as the cell number of blastocysts developed from VW oocytes. Collectively, our mVW-CC optimized for mouse oocytes can be utilized for humans without concerns regarding possible cross-contamination during vitrification in the future.

Optimizing Flow Cytometric Analysis of Immune Cells in Samples Requiring Cryopreservation from Tumor-Bearing Mice.

Most shared resource flow cytometry facilities do not permit analysis of radioactive samples. We are investigating low-dose molecular targeted radionuclide therapy (MTRT) as an immunomodulator in combination with in situ tumor vaccines and need to analyze radioactive samples from MTRT-treated mice using flow cytometry. Further, the sudden shutdown of core facilities in response to the COVID-19 pandemic has created an unprecedented work stoppage. In these and other research settings, a robust and reliable means of cryopreservation of immune samples is required. We evaluated different fixation and cryopreservation protocols of disaggregated tumor cells with the aim of identifying a protocol for subsequent flow cytometry of the thawed sample, which most accurately reflects the flow cytometric analysis of the tumor immune microenvironment of a freshly disaggregated and analyzed sample. Cohorts of C57BL/6 mice bearing B78 melanoma tumors were evaluated using dual lymphoid and myeloid immunophenotyping panels involving fixation and cryopreservation at three distinct points during the workflow. Results demonstrate that freezing samples after all staining and fixation are completed most accurately matches the results from noncryopreserved equivalent samples. We observed that cryopreservation of living, unfixed cells introduces a nonuniform alteration to PD1 expression. We confirm the utility of our cryopreservation protocol by comparing tumors treated with in situ tumor vaccines, analyzing both fresh and cryopreserved tumor samples with similar results. Last, we use this cryopreservation protocol with radioactive specimens to demonstrate potentially beneficial effector cell changes to the tumor immune microenvironment following administration of a novel MTRT in a dose- and time-dependent manner.

Easy and quick (EQ) sperm freezing method for urgent preservation of mouse strains.

Cryopreservation of mouse spermatozoa is widely used for the efficient preservation and safe transport of valuable mouse strains. However, the current cryopreservation method requires special containers (plastic straws), undefined chemicals (e.g., skim milk), liquid nitrogen, and expertise when handling sperm suspensions. Here, we report an easy and quick (EQ) sperm freezing method. The main procedure consists of only one step: dissecting a single cauda epididymis in a microtube containing 20% raffinose solution, which is then stored in a -80 °C freezer. The frozen-thawed spermatozoa retain practical fertilization rates after 1 (51%) or even 3 months (25%) with the C57BL/6 J strain, the most sensitive strain for sperm freezing. More than half of the embryos thus obtained developed into offspring after embryo transfer. Importantly, spermatozoa stored at -80 °C can be transferred into liquid nitrogen for indefinite storage. As far as we know, our EQ method is the easiest and quickest method for mouse sperm freezing and should be applicable in all laboratories without expertise in sperm cryopreservation. This technique can help avoid the loss of irreplaceable strains because of closure of animal rooms in emergency situations such as unexpected microbiological contamination or social emergencies such as the COVID-19 threat.

Changes in Hematopoietic Cell Transplantation Practices in Response to COVID-19: A Survey from the Worldwide Network for Blood & Marrow Transplantation.

SARS-CoV-2 has spread rapidly worldwide, but the full impact of the COVID-19 pandemic on the field of hematopoietic cell transplantation (HCT) remains unknown. To understand this better, an 18-item online survey was disseminated by the Worldwide Network for Blood & Marrow Transplantation with questions exploring SARS-CoV-2 testing algorithms, mobilization, and cryopreservation strategies and COVID-19 infections in allogeneic related and autologous hematopoietic progenitor cell (HPC) donors. The aim of this survey was to assess the impact of the outbreak on policies relating to HPC mobilization, collection, and processing with respect to changes in daily routine. A total of 91 individual responses from distinct centers in 6 continents were available for analysis. In these centers, the majority (72%) of allogeneic related and autologous donors are routinely tested for SARS-CoV-2 before HPC collection, and 80% of centers implement cryopreservation of allogeneic HPC grafts before commencing conditioning regimens in patients. Five related and 14 autologous donors who tested positive for COVID-19 did not experience any unexpected adverse events or reactions during growth factor administration (eg, hyperinflammatory syndrome). These data are limited by the small number of survey respondents but nonetheless suggest that centers are following the recommendations of appropriate scientific organizations and provide some preliminary data to suggest areas of further study.

Effect of different human tissue processing techniques on SARS-CoV-2 inactivation-review.

The safety of the tissue transplant recipient is a top priority for tissue banks, and the emergence of the new coronavirus SARS-CoV-2 has raised significant concerns about the risks of releasing tissue for clinical use. In the present study, we conducted a literature review about the potential infectivity of SARS-CoV-2 in different biological tissues and the influence of various tissue processing and sterilization procedures on viral inactivation. The search revealed that SARS-CoV-2 binds to the human angiotensin-converting enzyme receptor to penetrate human cells. These receptors are present in skin cells, musculoskeletal tissue, amniotic membranes, cardiovascular tissue and ocular tissues, including the cornea. In general, we found that coronaviruses are stable at low temperatures, and inactivated upon exposure to extreme heat and pH. Notably, gamma irradiation, which has already been employed to inactivate SARS and MERS, could be useful for sterilizing skin, amnion and musculoskeletal tissues against SARS-CoV-2. We conclude that due to the limited information about the effects of physical and chemical tissue processing methods on viral neutralization, rigorous donor screening is still essential for tissue transplant recipient safety.

Cryopreservation in reproductive medicine during the COVID-19 pandemic: rethinking policies and European safety regulations.

Cryopreservation of reproductive cells and tissues represents an essential aspect of ART practices that might be particularly strategic and helpful during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emergency. However, recommendations on how and when to preserve reproductive tissues and cells during a novel severe pandemic are scanty. This article uses a SWOT (strengths, weaknesses, opportunities and threats) analysis to identify favourable and unfavourable factors and to recognize challenges and obstacles related to the use of cryopreservation procedures during the spreading of a new virus. One of the strengths associated with the cryopreservation is represented by the availability of robust European guidelines on storage safety to prevent sample contamination or cross-contamination by pathogens. These recommendations should be deep-rooted in all ART laboratories. Weaknesses include uncertainties regarding the management of COVID-19 affected asymptomatic patients, the suboptimal accuracy of diagnostic tests for the disease, the nebulous prospective regarding the duration of the pandemic and the additional costs. The application of the strategy represents an opportunity to postpone pregnancy in order to avoid a severe infectious disease during gestation while concomitantly counteracting the possible detrimental effect of time. Critical threats, at present still undefined, are represented by potential adverse events for the mother and offspring due to infected gametes or embryos after thawing and, subsequently, the re-spreading of the virus.

High-security closed devices are efficient and safe to protect human oocytes from potential risk of viral contamination during vitrification and storage especially in the COVID-19 pandemic.

PURPOSE: The main purpose and research question of the study are to compare the efficacy of high-security closed versus open devices for human oocytes' vitrification. METHODS: A prospective randomized study was conducted. A total of 737 patients attending the Infertility and IVF Unit at S.Orsola University Hospital (Italy) between October 2015 and April 2020 were randomly assigned to two groups. A total of 368 patients were assigned to group 1 (High-Security Vitrification™ - HSV) and 369 to group 2 (Cryotop® open system). Oocyte survival, fertilization, cleavage, pregnancy, implantation, and miscarriage rate were compared between the two groups. RESULTS: No statistically significant differences were observed on survival rate (70.3% vs. 73.3%), fertilization rate (70.8% vs. 74.9%), cleavage rate (90.6% vs. 90.3%), pregnancy/transfer ratio (32.0% vs. 31.8%), implantation rate (19.7% vs. 19.9%), nor miscarriage rates (22.1% vs. 21.5%) between the two groups. Women's mean age in group 1 (36.18 ± 3.92) and group 2 (35.88 ± 3.88) was not significantly different (P = .297). A total of 4029 oocytes were vitrified (1980 and 2049 in groups 1 and 2 respectively). A total of 2564 were warmed (1469 and 1095 in groups 1 and 2 respectively). A total of 1386 morphologically eligible oocytes were inseminated by intracytoplasmic sperm injection (792 and 594 respectively, P = .304). CONCLUSIONS: The present study shows that the replacement of the open vitrification system by a closed one has no impact on in vitro and in vivo survival, development, pregnancy and implantation rate. Furthermore, to ensure safety, especially during the current COVID-19 pandemic, the use of the closed device eliminates the potential samples' contamination during vitrification and storage.