Long-term Cryostorage Research Articles

O-298 Embryo long-term storage does not affect ART outcome: analysis of 58,001 vitrified blastocysts

Abstract Study question Does duration of storage affect clinical outcome in frozen embryo transfer (FET) cycles? Summary answer Long-term storage does not affect live birth rate (LBR) in elective and non-elective FET cycles. What is known already Despite the success rates provided by the embryo vitrification programs, some studies found an association between the duration of cryo-storage and decreased clinical outcome, raising concern about the potential negative effect that prolonged storage may have on embryos. Study design, size, duration This was a retrospective multicenter study including a total of 58001 vitrified/warmed day-5 blastocysts from two different populations according to the reason for FET: elective FET which involved patients undergoing freeze all cycles (N = 16,615 blastocysts and 16,615 patients); and non-elective FET including 41,386 embryos from 25,571 patients. FETs were performed in the period between January 2010 and December 2020. Participants/materials, setting, methods Main outcome was live birth rate (LBR) and secondary outcomes were embryo survival, miscarriage, clinical and ongoing pregnancy rates. The impact of storage time and the potential confounding variables was assessed by univariate analyses. Those covariables found to significantly modify the LBR were included in a multivariable logistic regression. GEE (generalized estimated equation) was performed to allow the analysis of repeated measurements. Odds ratios (OR) and 95% confidence intervals (CI) were provided. Main results and the role of chance The blastocysts sample was clustered into deciles which provided specific storage duration categories that ranked from ≤0.67 to ≥ 4.34 and ≤1.8 to ≥ 34.81 months in freeze-all and non-elective FET, respectively. In the freeze-all group, maximum storage time was 34.8 months (2.9 years), while it was 121.2 months (10.1 years) in the non-elective FET group. Embryo survival did not show statistical differences across the categories of storage time in freeze-all and non-elective FET groups. In the freeze-all group, statistical differences were found for LBR across some, but not all the subgroups of storage duration. Similarly, in the non-elective FET group, LBR among the deciles showed statistical differences in some comparisons but not in all. However, after adjusting for confounding variables, the multivariate analysis showed no association between storage time and LBR in both groups. Blastocyst quality, body mass index (BMI), number of retrieved oocytes, endometrial preparation, male factor and uterine factor were related to the drop in the LBR in the freeze-all group (p < 0.05). In the non-elective FET group, the variables that showed significant association with the LBR were age at retrieval and at FET, number of embryos transferred, number of retrieved oocytes, BMI, endometrial preparation, sperm origin and female factor. Limitations, reasons for caution The retrospective design of the study represents its main limitation. Wider implications of the findings Our results demonstrate the safety of long-term cryo-storage. This is reassuring for both IVF practitioners and patients especially considering the exponential increase in FET cycles observed in the last decades. Trial registration number Not applicable

P-375 Long-term cryostorage of donor semen does not affect egg donor cycles outcomes

Abstract Study question Does prolonged donor semen cryopreservation impact in success rates of egg donor (ED) IVF cycles? Summary answer Prolonged donor semen cryopreservation does not affect good quality embryos or pregnancy rates in ED-IVF cycles. What is known already Births from cryopreserved semen stored for over 20 years have been documented. In 2004, a case of an IVF birth with semen frozen for 21 years was reported, and in 2005 another case with an IUI birth using semen frozen for 28 years was published. Recently, in 2019, a child born through IVF with semen frozen for over 26 years was reported in UK. In 2019, a study found that prolonged storage of these samples did not affect the outcomes of IVF with own eggs or IUI. Study design, size, duration We analysed data from 969 ED-IVF cycles performed at our centre between 2007 and 2022. Initially, cycles were classified into two independent groups of study to eliminate the impact of differences in embryo culture time. Group 1 or short culture (until Day 3) included 511 cycles from 2007 to 2013, and Group 2 or long culture (until Day5/6) included 458 cycles from 2014 to 2022. Participants/materials, setting, methods Donor oocytes were inseminated by ICSI using donor sperm from our bank. We analysed the relationship between the years of semen cryopreservation and the number of good quality embryos in both groups using Pearson correlation coefficient. Afterwards, the groups were divided into three subgroups based on the years of cryopreservation: 0-<3, ≥3-5, ≥5. Pregnancy and term pregnancy rates were calculated, and their relationship with time of semen cryopreservation were analysed using the Chi-square statistic. Main results and the role of chance No relationship was found between the years of cryopreservation and the number of good quality embryos in either group (Group 1 r=-0.05 and Group 2 r = 0.04). Pregnancy rates for the three subgroups 0-<3, ≥3-5, ≥5 were as follows: 219/454(48.24%),17/ 39(43.59%) and 6/18(33.3%) respectively in Group 1 and 141/213 (66.2%), 62/104 (59.62%), and 85/141 (60.28%) in Group 2. No statistically significant relationship was found in either group (Group 1 p-value=0.41 and Group 2 p-value=0.38). Term pregnancy rates for the three subgroups 0-<3, ≥3-5, ≥5 were as follows: 139/219 (63.47%), 10/17 (58.82%) and 4/6 (66.67%) respectively in Group 1 and 89/141 (63.12%), 39/62 (63.12%) and 56/85 (65.88%) in Group 2. No statistically significant relationship was found in either group (Group 1 p-value=0.91 and Group 2 p-value=0.90). The findings of this study indicate that prolonged donor semen cryopreservation does not appear to have a negative impact on success rates in ED-IVF cycles, both in terms of embryo development, and pregnancy and term pregnancy rates. Limitations, reasons for caution In Group 1 there was a big difference in sample size between subgroups, but it was due to the nature of the study. Wider implications of the findings Despite it seems that prolonged cryopreservation of semen does not affect embryonic viability, there is a lack of many molecular studies to ensure that this does not impact in cellular mechanisms or epigenetics. Trial registration number Not Applicable

Proof of stability of an RSV Controlled Human Infection Model challenge agent

In 2018, SGS Belgium NV developed RSV-NICA (Respiratory Syncytial Virus-Nasobronchial Infective Challenge Agent), an RSV type A challenge agent for use in RSV Controlled Human Infection Model (CHIM) studies.It is widely recognized that the stability of RSV can be influenced by a variety of environmental parameters, such as temperature and pH. Consequently, our objective was to evaluate the stability of the viral titer of RSV-NICA following five years of controlled storage and to determine the uniformity of the viral titers across different vials of a GMP-qualified batch of RSV-NICA. In addition, we examined the capacity of RSV-NICA to infect human primary airway epithelial cells (MucilAir™), the principal target cells of RSV, and evaluated the influence of single and recurrent freeze–thaw cycles on the infectious viral titer of the challenge agent.The aliquoted RSV-NICA virus stock was subjected to standard virological and molecular methods to gather data on the titer and consistency of the viral titer contained within 24 representative vials of the stock. Our findings illustrate that over a span of five years of cryo-storage, the infectious viral titer in 75% of the tested vials exhibited a comparable average infectious viral titer (4.75 ± 0.06 vs 4.99 ± 0.11; p-value = 0.14). A considerable reduction down to an undetectable level of infectious virus was observed in the remaining vials. RSV-NICA demonstrated its capacity to effectively infect differentiated human airway epithelial cells, with active virus replication detected in these cells through increasing RSV genome copy number over time. Virus tropism for ciliated cells was suggested by the inhibition of cilia beating coupled with an increase in viral RNA titers. No discernable impact on membrane barrier function of the epithelial lung tissues nor cytotoxicity was detected. Pooling of vials with infectious titers > 4.0 log10 TCID50/ml and freeze-thawing of these combined vials showed no deterioration of the infectious titer. Furthermore, pooling and re-aliquoting of vials spanning the entire range of viral titers (including vials with undetectable infectious virus) along with subjecting the vials to three repeated freeze–thaw cycles did not result in a decrease of the infectious titers in the tested vials.Taken together, our findings indicate that long-term cryo-storage of vials containing RSV-NICA challenge agent may influence the infectious viral titer of the virus, leading to a decrease in the homogeneity of this titer throughout the challenge stock. However, our study also demonstrates that when heterogeneity of the infectious titer of an RSV stock is observed, rounds of pooling, re-aliquoting and subsequent re-titration serve as an effective method not only to restore the homogeneity of the infectious titer of an RSV-A stock, but also to optimize patient-safety, scientific and operational aspects of viral inoculation of study participants during at least the period of one RSV CHIM trial. RSV-NICA is a stable, suitable CHIM challenge agent that can be utilized in efficacy trials for RSV vaccines and antiviral entities.

Preservation of functionality, immunophenotype, and recovery of HIV RNA from PBMCs cryopreserved for more than 20 years.

Many research laboratories have long-term repositories of cryopreserved peripheral blood mononuclear cells (PBMC), which are costly to maintain but are of uncertain utility for immunological studies after decades in storage. This study investigated preservation of cell surface phenotypes and in-vitro functional capacity of PBMC from viraemic HIV+ patients and healthy seronegative control subjects, after more than 20 years of cryopreservation. PBMC were assessed by 18-colour flow cytometry for major lymphocyte subsets within T, B, NK, and dendritic cells and monocytes. Markers of T-cell differentiation and activation were compared with original immunophenotyping performed in 1995/1996 on fresh blood at the time of collection. Functionality of PBMC was assessed by culture with influenza antigen or polyclonal T-cell activation, to measure upregulation of activation-induced CD25 and CD134 (OX40) on CD4 T cells and cytokine production at day 2, and proliferative CD25+ CD4 blasts at day 7. RNA was extracted from cultures containing proliferating CD4+ blast cells, and intracellular HIV RNA was measured using short amplicons for both the Double R and pol region pi code assays, whereas long 4-kbp amplicons were sequenced. All major lymphocyte and T-cell subpopulations were conserved after long-term cryostorage, except for decreased proportions of activated CD38+HLA-DR+ CD4 and CD8 T cells in PBMC from HIV+ patients. Otherwise, differences in T-cell subpopulations between recent and long-term cryopreserved PBMC primarily reflected donor age-associated or HIV infection-associated effects on phenotypes. Proportions of naïve, memory, and effector subsets of T cells from thawed PBMC correlated with results from the original flow cytometric analysis of respective fresh blood samples. Antigen-specific and polyclonal T-cell responses were readily detected in cryopreserved PBMC from HIV+ patients and healthy control donors. Intracellular HIV RNA quantitation by pi code assay correlated with original plasma viral RNA load results. Full-length intracellular and supernatant-derived amplicons were generated from 5/12 donors, and sequences were ≥80% wild-type, consistent with replication competence. This unique study provides strong rationale and validity for using well-maintained biorepositories to support immunovirological research even decades after collection.

THE RESISTANCE OF OOCYTES AND IN VITRO PRODUCED CATTLE EMBRYOS TO CRYOPRESERVATION

In vitro fertilization and production of bovine embryos in vitro is method, which improves utilization of animal reproduction potential. In connection with cryopreservation methods and ability of long-term cryostorage of embryos offers suitable tool for positive influence of endangered species or breeds. In vitro embryo production with subsequent embryo transfer contributes to the improvement of reproduction rates and bypassing some reproduction failure in high productive animal breeding. The method is generally used for in vitro production of transferable bovine embryos. Worldwide about 411 000 of IVP bovine embryos are annually transferred to recipients (IETS, 2013). Most of them were transferred immediately after producing, which is difficult for organization and synchronization of animals. In contrast to this possibility the storage of embryos for later using brings undisputable advantages. In order to protect animal genetic resources and to preserve the gene pool of rare and endangered animals, it is necessary to establish gene banks containing stocks of gametes and embryos of these animals. Optimization of the methods of in vitro production of bovine embryos and increasing their resistance to cryopreservation process is an actual challenge to ensure long-term storage in the gene bank of animal resources. The main goal of this mini-review is to provide general insight into the task of oocyte and embryo resistance to cryopreservation.

Features of the leukocyte pool of umbilical cell blood cell concentrate prepared for long-term cryostorage

Allogeneic hematopoietic cell transplantation is the standard of care for patients with highrisk acute leukemia. Cord blood (UCB) is an alternative source of allogeneic hematopoietic stem cells for patients who need transplantation but do not have a related donor. The purpose of our study was to evaluate the characteristics of leukocyte pool (cellular composition, immunophenotype) of umbilical cord blood (UCB) cell concentrates, which were harvested for public long-term cryostorage for the needs of transplantology. A study of 1096 samples of umbilical cord blood of full-term newborns before and after processing [isolation of hematopoietic stem cells (HSC) concentrate] was carried out. The number of cells in the pooled concentrate of HSC at a volume of 25 mL, after long-term cryostorage was as follows: leukocytes, 17.41±0.36 × 108; HSCs with CD34+ immunophenotype, 5.25±3.6 × 106; natural killer cells (CD3-CD16+CD56+), 1.65+0.7 × 108. Analysis of the NKT cell population revealed 6.22±2.1% of cells with CD3+CD16/CD56+ immunophenotype. The contents of CD3+CD56+ cells was 2.69±2.4%, the relative amount of CD3+CD16+ was 2.53±1.2%. In the pooled NKT cell preparations, 5.15±2.1% of CD56+CD16 cells were detected, and two minor subpopulations were also identified, which differ in CD56 and CD16 antigen expression: the level of CD56-CD16+ was 2.91±1.2%, and the ratio of CD56+CD16+ cells was 0.69±0.3%. The obtained data on relative amounts and characteristics of cellular immunophenotype as well as the heterogeneity of the NK and NKT cell population in the HSC UCB concentrate, subjected to long-term cryostorage must be taken into account when selecting the HSC UCB concentrates for the purpose of transplantation in oncohematological diseases.

Storage time does not influence pregnancy and neonatal outcomes for first single vitrified high-quality blastocyst transfer cycle

Does blastocyst storage time have an impact on pregnancy and neonatal outcomes following the first single vitrified/warmed high-quality blastocyst transfer cycle for young women? Retrospective cohort study in a university-affiliated reproductive medical centre. A total of 2938 patients undergoing their first frozen embryo transfer (FET) cycle with a single high-quality blastocyst (Day 5: 3BB and above; Day 6: 4BB and above) transferred were divided into five groups: Group A with storage time ≤3 months (n = 1621), Group B with storage time of 4-6 months (n = 657), Group C with storage time of 7-12 months (n = 225), Group D with storage time of 13-24 months (n = 104), and Group E with storage time of 25-98 months (n = 331). After adjusting for confounding factors by multivariate logistic regression, there were no significant differences in live birth rate [Group A as reference; Group B: adjusted odds ratio (aOR) 0.954 (95% CI 0.791- 1.151); Group C: aOR 0.905 (95% CI 0.674-1.214); Group D: aOR 0.727 (95% CI 0.474-1.114); Group E: aOR 1.185 (955 CI 0.873-1.608)], β-human-chorionic-gonadotropin-positive rate, clinical pregnancy rate and miscarriage rate between Group A and the other groups. Among all singletons born after FET, there were no significant differences with regards to gestational age, preterm birth, birthweight, low birthweight, high birthweight and macrosomia. Long-term cryostorage of human vitrified high-quality blastocysts does not affect pregnancy or neonatal outcomes.

Banking of perinatal mesenchymal stem/stromal cells for stem cell-based personalized medicine over lifetime: Matters arising.

Mesenchymal stromal/stem cells (MSCs) are currently applied in regenerative medicine and tissue engineering. Numerous clinical studies have indicated that MSCs from different tissue sources can provide therapeutic benefits for patients. MSCs derived from either human adult or perinatal tissues have their own unique advantages in their medical practices. Usually, clinical studies are conducted by using of cultured MSCs after thawing or short-term cryopreserved-then-thawed MSCs prior to administration for the treatment of a wide range of diseases and medical disorders. Currently, cryogenically banking perinatal MSCs for potential personalized medicine for later use in lifetime has raised growing interest in China as well as in many other countries. Meanwhile, this has led to questions regarding the availability, stability, consistency, multipotency, and therapeutic efficiency of the potential perinatal MSC-derived therapeutic products after long-term cryostorage. This opinion review does not minimize any therapeutic benefit of perinatal MSCs in many diseases after short-term cryopreservation. This article mainly describes what is known about banking perinatal MSCs in China and, importantly, it is to recognize the limitation and uncertainty of the perinatal MSCs stored in cryobanks for stem cell medical treatments in whole life. This article also provides several recommendations for banking of perinatal MSCs for potentially future personalized medicine, albeit it is impossible to anticipate whether the donor will benefit from banked MSCs during her/his lifetime.

Prolong cryopreservation duration negatively affects pregnancy outcomes of vitrified-warmed blastocyst transfers using an open-device system: A retrospective cohort study

ObjectiveTo investigate the impact of cryopreservation (CP) duration on pregnancy outcomes of vitrified-warmed blastocysts transfers using an open-device liquid-nitrogen (LN2) system. MethodsThis retrospective cohort study was conducted on 6327 first vitrified-warmed single blastocyst transfer cycles with autologous oocytes from January 2015 to December 2020. The CP duration was initially divided into six groups: Group I: 0–3 months (n = 4309); Group II: 4–6 months (n = 1061); Group III: 7–12 months (n = 304); Group IV: 13–24 months (n = 113); Group V: 25–72 months (n = 466); Group VI: 73–120 months (n = 74). Multivariate logistic regression was performed to evaluate the independent effect of CP duration on pregnancy outcomes. To further examine the time limit of vitrification, propensity score matching (PSM) was applied to compare pregnancy outcome of patients with storage duration of 25–120 months to those of 0–24 months. After that, pregnancy outcomes were compared among the subgroups of Group I’: 0–24 months, Group II’: 25–48 months, Group III’: 49–72 months, Group IV’: 73–120 months. Stratification analysis based on embryo quality was also performed. Primary outcomes were clinical pregnancy rate and live birth rate. Secondary outcomes were implantation, biochemical pregnancy rate, ongoing pregnancy rate and early miscarriage rate. ResultsLogistic regression demonstrated that the odds of pregnancy outcomes were similar across Group I to IV. However, the implantation rate, chances of biochemical pregnancy, clinical pregnancy, ongoing pregnancy, and live birth significantly decreased as the storage duration increased up to 25 months, while miscarriage rate did not significantly differ between groups. Subgroup analysis confirmed a dramatical decrease of clinical pregnancy and live birth rate when cryopreserved for more than 24 months. After that, the slope was relatively steady between 25 and 72 months, then steeply decreased again as CP reached 73–120 months. In addition, there was a more remarkable decline of pregnancy outcomes in the average quality embryo transfers than in the high quality embryo transfers as cryopreservation storage increased. ConclusionProlonged cryopreservation of vitrified blastocysts in an open-device LN2 system up to 24 months might negatively affect pregnancy outcomes. This negative impact progresses as storage duration increases, especially when exceeds 72 months. Average quality embryo appears to be less sustainable with long-term cryo-storage.

Long-term cryostorage does not negatively affect the recovery of Caenorhabditis elegans

Cryostorage of Caenorhabditis elegans nematodes is important to maintain the many lines used for research. The standard method uses 15% of glycerol in M9-Buffer and a cooling rate of 1 °C/min; then worms can be stored in a −80 °C freezer or in liquid nitrogen. The recovery of C. elegans from stocks stored in liquid nitrogen is reported to be in the range of 35–45% and slightly decreases after years of storage. The storage at −80 °C is also considered safe, but the recovery is not as high as in liquid nitrogen. These observations have not been experimentally reported and therefore require verification. In this study, the standard methods were used in a set of experiments to compare the recovery of larvae and adult worms stored at −80 °C or in liquid nitrogen, after short- (a week) or long-term storage (3.5 years). No differences were observed in recovery, either for the time of storage or for the temperature of storage. Recovery of larvae was 32% at −80 °C and 36% in liquid nitrogen after 3.5 yr and that was not significantly different from the 7-d recovery rates. Adult worm recovery was below 5% for all treatments. These results suggest that both methods of storage can be used to successfully store C. elegans larvae for at least 3.5 years.

Do-it-Yourself Mock Community Standard for Multi-Step Assessment of Microbiome Protocols.

Microbiomes provide critical functions that support animals, plants, and ecosystems. High-throughput sequencing (HTS) has become an essential tool for the cultivation-independent study of microbiomes found in diverse environments, but requires effective and meaningful controls. One such critical control is a mock microbial community, which is used as a positive control for nucleic acid extraction, marker gene amplification, and sequencing. While mock community standards can be purchased, they can be costly and often include only medically relevant microbial strains that are not expected to be major players in non-human microbiomes. As an alternative, it is possible to design and construct a do-it-yourself (DIY) mock community, which can then be used as a positive control that is specifically customized to the protocol needs of a particular study system. In this article, we describe protocols to select appropriate microbial strains for the construction of a mock community. We first describe the steps to verify the identity of community members via Sanger sequencing. Then, we provide guidance on assembling and storing the DIY mock community as viable whole cells. This includes steps to create standard growth curves referenced to plate counts for each member, so that the community members can be quantified and later compared in terms of their "expected versus returned" relative contributions after sequencing. We also describe appropriate methods for the cryostorage of the fully assembled mock community as viable whole cells, so that they can be used as a unit in a microbiome analysis, from the lysis and nucleic acid extraction steps onwards. Finally, we provide an example of returned data and interpretation of DIY mock community sequences, discussing how to assess possible contamination and identify protocol biases for particular members. Overall, DIY mock communities serve to determine success and possible bias in a cultivation-independent microbiome analysis. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Strain identification and verification using Sanger sequencing Basic Protocol 2: Creation of glycerol stocks of each mock community strain for long-term cryostorage Basic Protocol 3: Assessment of strain freezer viability without cryoprotectant Basic Protocol 4: Creation of standard curve to determine CFU/ml of a liquid culture as a function of optical density Basic Protocol 5: Full mock community assembly using community concentration calculations and standard curves.

Novel Long-Term Cryo-Storage Using Vitrification and Laser Warming Techniques.

Vitrification and laser warming have gained popularity over the traditional convective warming techniques in cryopreservation. Laser warming is rapid with uniform effects, thus preventing ice crystal formation in samples. Contemporary laser warming studies have focused on proof-of-concept experiments. Yet, no protocols or techniques have been developed to address the problem of warming samples from long-term storage. Herein, a new approach to laser warming samples without exposing the samples to ambient temperature is introduced. The new device presented has a mean laser-hitting accuracy of 76% ± 16% and a rewarming rate of 59% ± 25% on samples with <1 μL in volume. Although these rates depend on the choice of vitrification solution and mastery of the technique, the approach described represents a successful first step toward laser warming samples from long-term cryo-storage.

PB2187: VIABILITY AND PROLIFERATIVE POTENTIAL OF AUTOGRAFT HEMATOPOIETIC STEM CELLS

Background: Autograft cellularity is a fundamental condition for the timing of hematopoiesis recovery in patients with multiple myeloma (MM) after autologous hematopoietic stem cell transplantation (AutoSCT). Taking the number of CD34+ cells in a volume of 2x10/6/kg of the patient’s weight as a suboptimal value that provides fast and reliable graft engraftment, it remains unclear whether this number will be sufficient for subsequent transplantation, if tandem AutoHSCT assumes an interval of 6 months, and repeated - not earlier 36 months. Aims: Determination of the preservation of colony-forming (CFU) and CD34+ cells in the autograft of MM patients depending on the terms of their cryopreservation. Methods: A retrospective analysis of the results of the study of 127 samples of the product of leukocytapheresis was performed. The prepared cell suspension was placed in disposable CryoMacs Freesing bags and mixed with dimethyl sulfoxide to its final concentration of 7.5%. Freezing was carried out in a Cryo 560-16 Planer RLC programmed freezer. Containers with cell suspension were stored in liquid nitrogen. To assess the effect of the duration of the cryopreservation period on CFU, the numerical values of each sample were converted into percentages: the primary CFU values from each sample were taken as 100%, and the CFU values after the cryopreservation period were compared with them. Results: Depending on the terms of cryostorage, 3 groups were formed. Group 1: 83 samples, shelf life from 1 to 5.9 months. Group 2: 34 samples, shelf life from 6 to 10 months. Group 3: 10 samples, shelf life over 10 months. A trend towards a decrease in the number of colonies was revealed as the duration of the cryopreservation period increased. Thus, the number of CFU-totals decreased from 66% recorded after 1–5.9 months of storage, to 60% after 6–10 months and to 44% of the original number when stored for more than 10 months; p=0.131. The decrease in the number of colony-forming cells of granulomonocytopoiesis was distinct in cell suspension samples stored for more than 10 months, but also did not reach significant values: 64%, 58% and 37%, respectively; p=0.451. On the contrary, the decrease in the number of burst-forming cells of erythrocytopoiesis (BFU-E) after 10 months of cryostorage was significantly lower than at earlier periods of cell suspension thawing. So if the content of BFU-E after 1–5.9 and 6–10 months of storage was 65% and 57% of the initial amount, respectively, then after 10 months it was only 26%; p=0.01. When analyzing the preservation of CD34+ cells, a tendency was also found to decrease in their percentage as the duration of cryopreservation increased, but the difference did not reach a significant value: 77% during thawing after 1–5.9 months of cryopreservation, 49% after 6–10 months and 39% in case of cell suspension thawing after 10 months of storage; p=0.465. Despite the fact that in some patients with the maximum terms of cell suspension cryopreservation, the need for a larger number of doses of donor erythrocytes was recorded and there were longer terms for platelet recovery than in patients in the other two groups, the median values of the analyzed parameters did not differ. Summary/Conclusion: When planning the first and subsequent, repeated, AutoHSCT in MM patients, it is advisable to prepare an autograft with a greater than suboptimal cellularity due to a possible decrease in the number of CFU during long-term cryostorage of the cell suspension.

The effect of extended cryo-storage following vitrification on embryo competence: a systematic review and meta-analysis.

Few studies explored whether prolonged cryo-storage after vitrification affects embryo competence and perinatal outcomes. This systematic review and meta-analysis aims at highlighting any putative impact of cryo-storage duration on cryo-survival, miscarriage, live birth and major malformations. A systematic review was performed using MEDLINE (PubMed), ISI Web of Knowledge, Scopus and Embase databases up to June 2021. Data were combined to obtain a pooled OR, and meta-analysis was conducted using a random effects model. Out of 1,389 screened abstracts, 22 papers were assessed for eligibility, and 5 studies were included (N = 18,047 embryos). Prolonged cryo-storage was defined as > 12months (N = 3389 embryos). Subgroup analysis was performed for untested vitrified cleavage stage embryos (N = 1739 embryos) and for untested and euploid vitrified blastocysts (N = 13,596 and 2712 embryos, respectively). Survival rate, miscarriage, live birth and major malformation rates were all similar in the two groups. These data further support the safety of long-term cryo-storage of human embryos beyond 12months. This is reassuring for good prognosis patients with surplus embryos, couples seeking a second child from supernumerary embryos and women postponing the transfer for clinical or personal reasons.

Cryopreserved Stem Cells Incur Damages Due To Terrestrial Cosmic Rays Impairing Their Integrity Upon Long-Term Storage

Stem cells have the capacity to ensure the renewal of tissues and organs. They could be used in the future for a wide range of therapeutic purposes and are preserved at liquid nitrogen temperature to prevent any chemical or biological activity up to several decades before their use. We show that the cryogenized cells accumulate damages coming from natural radiations, potentially inducing DNA double-strand breaks (DSBs). Such DNA damage in stem cells could lead to either mortality of the cells upon thawing or a mutation diminishing the therapeutic potential of the treatment. Many studies show how stem cells react to different levels of radiation; the effect of terrestrial cosmic rays being key, it is thus also important to investigate the effect of the natural radiation on the cryopreserved stem cell behavior over time. Our study showed that the cryostored stem cells totally shielded from cosmic rays had less DSBs upon long-term storage. This could have important implications on the long-term cryostorage strategy and quality control of different cell banks.

Freshly Thawed Cryobanked Human Neural Stem Cells Engraft within Endogenous Neurogenic Niches and Restore Cognitive Function after Chronic Traumatic Brain Injury.

Human neural stem cells (hNSCs) have potential as a cell therapy after traumatic brain injury (TBI). While various studies have demonstrated the efficacy of NSCs from ongoing culture, there is a significant gap in our understanding of freshly thawed cells from cryobanked stocks-a more clinically relevant source. To address these shortfalls, the therapeutic potential of our previously validated Shef-6.0 human embryonic stem cell (hESC)-derived hNSC line was tested after long-term cryostorage and thawing before transplant. Immunodeficient athymic nude rats received a moderate unilateral controlled cortical impact (CCI) injury. At four weeks post-injury, 6 × 105 freshly thawed hNSCs were transplanted into six injection sites (two ipsi- and four contra-lateral) with 53.4% of cells surviving three months post-transplant. Interestingly, most hNSCs were engrafted in the meninges and the lining of lateral ventricles, associated with high CXCR4 expression and a chemotactic response to SDF1alpha (CXCL12). While some expressed markers of neuron, astrocyte, and oligodendrocyte lineages, the majority remained progenitors, identified through doublecortin expression (78.1%). Importantly, transplantation resulted in improved spatial learning and memory in Morris water maze navigation and reduced risk taking in an elevated plus maze. Investigating potential mechanisms of action, we identified an increase in ipsilateral host hippocampus cornu ammonis (CA) neuron survival, contralateral dentate gyrus (DG) volume, and DG neural progenitor morphology as well as a reduction in neuroinflammation. Together, these findings validate the potential of hNSCs to improve function after TBI and demonstrate that long-term biobanking of cells and thawing aliquots before use may be suitable for clinical deployment.

P–783 Clinical, obstetric and perinatal outcomes after vitrified-warmed euploid blastocyst transfer are independent of cryo-storage duration

Abstract Study question Is cryo-storage duration associated with the outcomes after vitrified-warmed euploid single blastocyst transfer? Summary answer Lower live-birth-rates from blastocysts cryo-stored for periods longer than 3-months are mostly imputable to the worse quality of the embryos being warmed across sequential transfers. What is known already Blastocyst vitrification is crucial in modern IVF. Given its widespread application, a constant comprehensive monitoring of its effect on reproductive outcomes is pivotal. For instance, the effect of cryo-storage duration on embryo implantation potential, gestational and perinatal outcomes is object of a still ongoing investigation. The evidence in this regard are contrasting especially with regard to similar or decreased live birth rates among blastocysts subject to long-term cryo-storage. When investigating the neonatal outcomes, instead, no impact of blastocyst cryo-storage duration has ever been reported to date. Yet, data on euploid blastocysts and adjusted for quality and full-blastulation day are needed. Study design, size, duration Retrospective observational study. We included 2688 vitrified-warmed euploid single blastocyst transfers. The primary outcome was the live-birth-rates (LBR) according to cryo-storage duration clustered as ≤ 60, 61–90, 91–180, 181–360, 361–720, 721–1080 and &amp;gt;1080-days. The secondary outcomes were the miscarriage rate, the rates of gestational and perinatal issues among the deliveries, and the mean gestational age and birthweight among the babies born. All data were adjusted for confounders through linear or logistic regression analyses. Participants/materials, setting, methods: We included all vitrified-warmed transfers (range:1–8) conducted between May–2013 and March–2020 by 1884 patients (age:38±3yr) undergoing one blastocyst stage PGT-A cycle and obtaining ≥1 euploid embryo at our private clinic. Among putative confounders, only the number of sequential transfer from the same patient, blastocyst quality (Gardner’s scheme) and full-blastulation day (5–7) significantly associated with the LBR through univariate regressions. No association was reported for sperm factor, maternal age, incubator, and culture media. Main results and the role of chance The LBR of euploid blastocysts cryo-stored for ≤60-days was 49.4% (N = 319/646) versus 48.7% (N = 292/599; OR:0.98,95%CI:0.78–1.21,p=0.82) between 61–90-days, 42.9% (N = 291/679; OR:0.77,95%CI:0.62–0.96,p=0.02) between 91–180-days, 41.7% (N = 169/405; OR:0.73,95%CI:0.57–0.94,p=0.02) between 181–360-days, 34.7% (N = 50/144; OR:0.55,95%CI:0.37–0.79,p&amp;lt;0.01) between 361–720-days, 53.4% (N = 63/118; OR:1.17,95%CI:0.79–1.74,p=0.42) between 721–1080-days, and 50.5% (N = 49/97; OR:1.05,95%CI:0.68–1.60,p=0.83) for &amp;gt;1080-days. However, when these data were adjusted for blastocyst quality and full-blastulation day, all the multivariate-OR were not-significant. Indeed, the longer the cryo-storage period the worse the quality of the euploid blastocysts transferred (e.g. AA-blastocysts were 74% among embryos cryo-stored for ≤90-days, but always &amp;lt;70% for embryos cryo-stored for longer periods, p &amp;lt; 0.01; similarly, day5-blastocysts were ∼50% among embryos cryo-stored for ≤90-days, but always &amp;lt;50% for embryos cryo-stored for longer periods, p = 0.02). The miscarriage-rate (overall 14%, ranging 7–18%) was not associated with cryo-storage duration already from univariate regressions. Also the gestational (overall 6%, ranging 0–8%) and perinatal issues rates (overall 3%, ranging 0–5%) were not associated with cryo-storage duration already from the univariate regressions. Neither the gestational age nor the birthweight showed significant associations with cryo-storage duration, as confirmed by linear regressions. In fact the rate of newborns whose weight was normal-for-gestational-age was similar across all cryo-storage duration groups (overall 81%, ranging 80–83%). Limitations, reasons for caution The prevalence of first transfers decreases from ≥95% for procedures conducted ≤90-days from vitrification to 71%, 39%, 22% and 4% for procedures conducted between 91–180, 181–360, 361–720 and &amp;gt;720-days, respectively. However, also the sequential number of transfer was not associated with the LBR when adjusted for blastocyst-quality and full-blastulation day. Wider implications of the findings: Cryo-storage by vitrification is confirmed safe in the hands of experienced operators, and its duration does not impact any outcome. This information is valuable for freeze-all cycles, but also for women cryo-preserving surplus embryos for second pregnancies; in this regard, 6.8% of the patients in this study delivered ≥2 LBs. Trial registration number Not applicable

P-783 Clinical, obstetric and perinatal outcomes after vitrified-warmed euploid blastocyst transfer are independent of cryo-storage duration

Abstract Study question Is cryo-storage duration associated with the outcomes after vitrified-warmed euploid single blastocyst transfer? Summary answer Lower live-birth-rates from blastocysts cryo-stored for periods longer than 3-months are mostly imputable to the worse quality of the embryos being warmed across sequential transfers. What is known already Blastocyst vitrification is crucial in modern IVF. Given its widespread application, a constant comprehensive monitoring of its effect on reproductive outcomes is pivotal. For instance, the effect of cryo-storage duration on embryo implantation potential, gestational and perinatal outcomes is object of a still ongoing investigation. The evidence in this regard are contrasting especially with regard to similar or decreased live birth rates among blastocysts subject to long-term cryo-storage. When investigating the neonatal outcomes, instead, no impact of blastocyst cryo-storage duration has ever been reported to date. Yet, data on euploid blastocysts and adjusted for quality and full-blastulation day are needed. Study design, size, duration Retrospective observational study. We included 2688 vitrified-warmed euploid single blastocyst transfers. The primary outcome was the live-birth-rates (LBR) according to cryo-storage duration clustered as ≤ 60, 61-90, 91-180, 181-360, 361-720, 721-1080 and &amp;gt;1080-days. The secondary outcomes were the miscarriage rate, the rates of gestational and perinatal issues among the deliveries, and the mean gestational age and birthweight among the babies born. All data were adjusted for confounders through linear or logistic regression analyses. Participants/materials, setting, methods We included all vitrified-warmed transfers (range:1-8) conducted between May-2013 and March-2020 by 1884 patients (age:38±3yr) undergoing one blastocyst stage PGT-A cycle and obtaining ≥1 euploid embryo at our private clinic. Among putative confounders, only the number of sequential transfer from the same patient, blastocyst quality (Gardner’s scheme) and full-blastulation day (5-7) significantly associated with the LBR through univariate regressions. No association was reported for sperm factor, maternal age, incubator, and culture media. Main results and the role of chance The LBR of euploid blastocysts cryo-stored for ≤60-days was 49.4% (N = 319/646) versus 48.7% (N = 292/599; OR:0.98,95%CI:0.78-1.21,p = 0.82) between 61-90-days, 42.9% (N = 291/679; OR:0.77,95%CI:0.62-0.96,p = 0.02) between 91-180-days, 41.7% (N = 169/405; OR:0.73,95%CI:0.57-0.94,p = 0.02) between 181-360-days, 34.7% (N = 50/144; OR:0.55,95%CI:0.37-0.79,p &amp;lt; 0.01) between 361-720-days, 53.4% (N = 63/118; OR:1.17,95%CI:0.79-1.74,p = 0.42) between 721-1080-days, and 50.5% (N = 49/97; OR:1.05,95%CI:0.68-1.60,p = 0.83) for &amp;gt;1080-days. However, when these data were adjusted for blastocyst quality and full-blastulation day, all the multivariate-OR were not-significant. Indeed, the longer the cryo-storage period the worse the quality of the euploid blastocysts transferred (e.g. AA-blastocysts were 74% among embryos cryo-stored for ≤90-days, but always &amp;lt; 70% for embryos cryo-stored for longer periods, p &amp;lt; 0.01; similarly, day5-blastocysts were ∼50% among embryos cryo-stored for ≤90-days, but always &amp;lt; 50% for embryos cryo-stored for longer periods, p = 0.02). The miscarriage-rate (overall 14%, ranging 7-18%) was not associated with cryo-storage duration already from univariate regressions. Also the gestational (overall 6%, ranging 0-8%) and perinatal issues rates (overall 3%, ranging 0-5%) were not associated with cryo-storage duration already from the univariate regressions. Neither the gestational age nor the birthweight showed significant associations with cryo-storage duration, as confirmed by linear regressions. In fact the rate of newborns whose weight was normal-for-gestational-age was similar across all cryo-storage duration groups (overall 81%, ranging 80-83%). Limitations, reasons for caution The prevalence of first transfers decreases from ≥95% for procedures conducted ≤90-days from vitrification to 71%, 39%, 22% and 4% for procedures conducted between 91-180, 181-360, 361-720 and &amp;gt;720-days, respectively. However, also the sequential number of transfer was not associated with the LBR when adjusted for blastocyst-quality and full-blastulation day. Wider implications of the findings Cryo-storage by vitrification is confirmed safe in the hands of experienced operators, and its duration does not impact any outcome. This information is valuable for freeze-all cycles, but also for women cryo-preserving surplus embryos for second pregnancies; in this regard, 6.8% of the patients in this study delivered ≥2 LBs. Trial registration number not applicable

Long-Term Cryostorage of Mesenchymal Stem Cell-Containing Hybrid Hydrogel Scaffolds Based on Fibrin and Collagen.

The most difficult issue when using tissue engineering products is enabling the ability to store them without losing their restorative capacity. The numbers and viability of mesenchymal stem cells encapsulated in a hydrogel scaffold after cryostorage at −80 °C (by using, individually, two kinds of cryoprotectors—Bambanker and 10% DMSO (Dimethyl sulfoxide) solution) for 3, 6, 9, and 12 months were determined, with subsequent assessment of cell proliferation after 96 h. The analysis of the cellular component was performed using fluorescence microscopy and the two fluorochromes—Hoechst 3334 and NucGreenTM Dead 488. The experimental protocol ensured the preservation of cells in the scaffold structure, retaining both high viability and proliferative activity during storage for 3 months. Longer storage of scaffolds led to their significant changes. Therefore, after 6 months, the proliferative activity of cells decreased. Cryostorage of scaffolds for 9 months led to a decrease in cells’ viability and proliferative activity. As a result of cryostorage of scaffolds for 12 months, a decrease in viability and proliferative activity of cells was observed, as well as pronounced changes in the structure of the hydrogel. The described scaffold cryostorage protocol could become the basis for the development of storage protocols for such tissue engineering products, and for helping to extend the possibilities of their clinical use while accelerating their commercialization.

Does duration of cryostorage affect survival rate, pregnancy, and neonatal outcomes? Large-scale single-center study of slush nitrogen (SN2 ) vitrified-warmed blastocysts.

To evaluate the effects of the duration of cryostorage on clinical outcomes after embryo transfer of vitrified blastocysts stored in an open-device slush-nitrogen (SN2 ) system. A retrospective cohort study was carried out on 1632 autologous vitrified-warmed blastocyst transfer cycles between January 2013 and June 2014. Duration of cryostorage was divided into four groups: Group I: 0-6months (n=937); Group II: 7-12months (n=299); Group III: 13-24months (n=165); and Group IV: ≥25months (n=231). The effects of the duration of cryostorage on the survival rate (SR), clinical pregnancy rate (CPR), live birth rate (LBR), and neonatal outcomes of vitrified blastocysts stored in an open-device SN2 system were evaluated. There were no significant differences between groups in SR, CPR, LBR, and neonatal outcomes after autologous vitrified-warmed blastocyst transfer. Multivariate logistic regression analysis showed no effect on LBR from duration of cryostorage. Vitrification using SN2 and long-term cryostorage in an open-device system are safe and effective and do not significantly affect clinical outcomes after embryo transfer.