Post-thaw Survival Research Articles

Different responses of larval fatty acid profiles to cryopreservation in two commercially important bivalves.

Larval cryopreservation techniques have been established in Pacific oysters and Mediterranean mussels. Although initial cryopreservation protocols for both species differed slightly in Ficoll PM 70 (FIC) concentration, better post-thaw larval survivability was produced in mussels than oysters. Furthermore, the post-thaw survivability in the latter could be improved significantly by the addition of lipids and antioxidants to the cryoprotectant agent. These findings have generated a unique opportunity to investigate the cryo-functions of both endogenous and exogenous fatty acids (FAs) in bivalves as mammalian studies indicated that lipid compositions could contribute the interspecific difference in gamete and embryo cryopreservation. Our study showed that the higher survivability of post-thaw larvae in mussels (65% vs. 34% in oysters) could be attributed to their higher proportion of total polyunsaturated fatty acids (PUFAs), especially C22:6, and higher resistance of FA profiles to cryopreservation. In oysters, on the other hand, their FA profiles were very sensitive to cryopreservation, with saturated FAs and PUFAs being significantly increased (from 37 to 41%) and decreased (from 35% to ~ 32%), respectively. Although exogenous lipids could significantly improve the survivability of post-thaw oyster larvae from 34 to 51%, their supplementation did not alter the response pattern of endogenous FA profiles to cryopreservation.

Current State and Challenges of Tissue and Organ Cryopreservation in Biobanking

Recent years have witnessed significant advancements in the cryopreservation of various tissues and cells, yet several challenges persist. This review evaluates the current state of cryopreservation, focusing on contemporary methods, notable achievements, and ongoing difficulties. Techniques such as slow freezing and vitrification have enabled the successful preservation of diverse biological materials, including embryos and ovarian tissue, marking substantial progress in reproductive medicine and regenerative therapies. These achievements highlight improved post-thaw survival and functionality of cryopreserved samples. However, there are remaining challenges such as ice crystal formation, which can lead to cell damage, and the cryopreservation of larger, more complex tissues and organs. This review also explores the role of cryoprotectants and the importance of optimizing both cooling and warming rates to enhance preservation outcomes. Future research priorities include developing new cryoprotective agents, elucidating the mechanisms of cryoinjury, and refining protocols for preserving complex tissues and organs. This comprehensive overview underscores the transformative potential of cryopreservation in biomedicine, while emphasizing the necessity for ongoing innovation to address existing challenges.

Effect of seminal plasma cholesterol and triacylglycerides concentrations and sperm morphology on semen freezability in domestic cats (Felis silvestris catus)

There is scarce information about the effect of sperm morphology and seminal plasma composition on cat semen freezability. Thus, this study aims to assess the effect of cat sperm morphology and seminal plasma cholesterol (CHOL) and triacylglyceride (TAG) concentrations on sperm post-thaw survival. Ejaculates (n = 49) were evaluated, and seminal plasma was separated and frozen until CHOL and TAG concentrations were measured. The sperm pellet was diluted in a tris-based egg yolk extender, frozen (n = 38), or processed for sperm ultrastructure study (n = 11). Abnormalities recorded were abnormal head shape and size, detached heads, knobbed or ruffled acrosomes, eccentric mid-piece insertion, proximal and distal cytoplasmic droplets, folded and coiled tails, and Dag defect. Ultramicroscopic evaluation detected several sperm abnormalities in fresh semen and some sperm damage in frozen semen. Seminal plasma lipids components were positively correlated with post-thaw motility and acrosome integrity. Higher freezability indices for motility and acrosome integrity were observed in frozen-thawed semen with high seminal plasma CHOL and TAG concentrations. No freezability differences were observed between teratozoospermic and normozoospermic ejaculates. Our results showed that even when seminal plasma was removed before cryopreservation, sperm survival after thawing was significantly higher in samples with high seminal plasma CHOL and TAG concentrations, indicating a rapid adherence to these compounds to the sperm plasma membrane, protecting sperm cells from temperature changes. Nevertheless, there were no differences in sperm freezability by sperm morphology.

Quercetin in semen extender curtails reactive oxygen and nitrogen species and improves functional attributes of cryopreserved buck semen

Cryopreservation of goat spermatozoa is challenging due to several factors, including one of the most essential, i.e., oxidative stress. It is particularly essential in goat semen due to its scanty ejaculate volume and high sperm concentration. This leaves a narrow sperm-to-seminal plasma ratio owing to marginal antioxidant support; moreover, semen extension further dilutes the antioxidant level, leading to an imbalance of oxidant-antioxidant equilibrium. The present study aimed to evaluate the effect of quercetin on curtailing oxidative stress and its reflection on the post-thaw survivability and membrane integrity of goat spermatozoa. For this study, six bucks were selected. Six ejaculates from each buck totaling 36 ejaculates were collected, which were then split into five parts; furthermore, each part was added with a semen extender having a particular concentration of additive. Group C without quercetin and T1 containing Vitamin E at 3 mmol/mL were considered the control and positive control respectively, whereas T2, T3, and T4 contain 10, 20, and 30 μmol/mL of Quercetin respectively. The final sperm concentration of each group was kept at 200 × 106 spermatozoa/mL. All groups were subjected to equilibration at 4 °C for 4 h, then filled in French mini (0.25 mL) straws, followed by sealing and cryopreservation. Samples after 72 h of cryopreservation were subjected to evaluation of plasma membrane integrity and viability through staining, acrosomal integrity, and mitochondrial membrane activity through flow cytometry. Evaluation of sperm kinematics as well as the oxidant-antioxidant status of sperm (ROS and nitric oxide) and seminal plasma (SOD, CAT, GPx, FRAP, and lipid peroxidation through MDA estimation) were also carried out. Quercetin, when supplemented at 20 μmol/mL in buck semen extender, significantly (p < 0.01) improved cryopreserved sperm functions in terms of plasma membrane integrity, viability, acrosomal integrity, mitochondrial membrane activity, and sperm kinematics of buck semen. Similarly, Quercetin supplementation at 20 μmol/mL significantly reduced reactive oxygen and nitrogen species (RONS) in sperm and improved the antioxidant status of seminal plasma, which was indicated by reduced oxidative damage and improved the antioxidant status of buck semen. In conclusion, Quercetin at 20 μmol/mL reduced oxidative stress, improved semen antioxidant status, and improved sperm membranes integrity and kinematics.

P-239 Comparative study between two commercial vitrification kits on survival and clinical outcome for blastocyst frozen embryo transfer

Abstract Study question To compare the post thaw survival and clinical outcome of blastocyst (D5/D6) transfer between two different commercially available kits for freezing and thawing. Summary answer There is significant difference on survival of post thaw blastocyst and clinical outcomes between Kitazato (Japan) and Irvine (USA) vitrification media. What is known already Vitrification is widely used in embryo preservation for its reliable outcome and the ease of use. Different brands of commercial freezing and thawing kits are available. The main difference between the two commercial vitrification kits used in this study are the different extracellur cryoprotectant used in which Kitazato contain trehalose supplemented with hydroxypropyl cellulose while Irvine contain sucrose with dextran serum supplement. Recent study suggested that cell apoptosis in specimen vitrified in trehalose media is lower in comparison to sucrose. Study design, size, duration Retrospective study was carried out from 2022-2023. Patient with uterine abnormalities, history of recurrent implantation failure (RIF), patient that underwent Preimplantation Genetic Testing (PGT) and those transferring D2/3 embryo were excluded. Survivability of post thaw blastocyst and clinical outcome of frozen blastocyst transfer were compared between Kitazato (N = 374) and Irvine (N = 253) vitrification kit. The data was analyzed using t-test and Anova. Participants/materials, setting, methods Blastocysts that were vitrified and warmed with vitrification kit from the same manufacturer were grouped together and compared. Patient in each vitrification arms were divided into two main age groups which are &amp;lt;37 years old and ≥37 years old. The survivability and clinical outcome of the two respective vitrification kits was compared between the age group, blastocyst grading prior to freezing (Good, Fair, Poor) and the day of blastocyst freezing (D5 or D6). Main results and the role of chance The overall mean post thawed survival was higher in Kitazato (95.8%) compare to Irvine (91.8%) (p &amp;lt; 0.001). In patient age &amp;lt;37 years old, Kitazato group performed better when compare to Irvine group. Post thawed survival using Kitazato warming was significantly better (p &amp;lt; 0.001) on good (AA,AB,BA) and fair graded (BB) blastocyst with mean survival of 97.1% vs 93.4% and 94.9% vs 87.6% respectively. Similarly, post thawed survival of D5 and D6 blastocysts were significantly higher in Kitazato group when compared to Irvine group with mean survival of 96.7% vs 92.8% and 94.8% vs 90.3% respectively. However no significant difference was observed in post thawed survival outcome for patient aged ≥ 37 years old between both groups in the same study parameters. In terms of clinical outcomes, there is also no difference observed between Kitazato group and Irvine group on beta hCG positive rates. Interestingly, the Irvine group shown to have significantly higher clinical pregnancy compare to Kitazato group (53% vs 60% respectively) but no significant difference was observed in sub group for patient aged &amp;lt; 37 years old and ≥ 37 years old. Furthermore, the implantation rate (IR) in Irvine group group (55.7%) is also significantly higher compare to the Kitazato group (50.5%). Limitations, reasons for caution Potential confounder factors such as stimulation protocol and the embryologist experience were not factored in may have affected the outcome of the study. Sample size can be increased to improve confidence in this study. Wider implications of the findings This study demonstrates that the post thaw survivability is better in the Kitazato group while the clinical outcome is better in the Irvine group. Thus this can give a better understanding of the respective commercial vitrification kits and provide insight for laboratories to choose for their clinical practices. Trial registration number Not Applicable

P-031 Viral crossroads: revisiting sperm processing in the context of Human Immunodeficiency Virus (HIV) infection

Abstract Study question Is extensive sperm washing, followed by viral load testing, necessary to ensure safety of assisted reproductive techniques in HIV-positive men? Summary answer In context of viral suppression, there is no added value of extensive sperm washing followed by viral load testing for HIV-positive men seeking fertility treatment. What is known already Sperm washing was initially introduced as a safe reproductive strategy for serodiscordant couples in which the male partner is seropositive. This technique uses a combination of density gradient centrifugation (DGC) and swim-up (SU) to separate the HIV-free spermatozoa from the seminal plasma containing most of the viral particles. Nowadays, due to the increased availability and efficacy of antiretroviral drugs, more patients are adequately suppressed and have no risk of transmitting HIV, which is reinforced by the ‘Undetectable Equals Untransmittable’ principle. This questions the need for extensive sperm washing followed by quantitative polymerase chain reaction (qPCR) testing in the IVF laboratory. Study design, size, duration This single center retrospective study investigates the effectiveness of our existing sperm washing procedures by evaluating the HIV viral load (VL) both in the raw and processed semen fractions, while considering the patient’s viral suppression status. All sperm wash procedures performed on fresh ejaculates between 2012 and 2023 were included. Additionally, clinical outcomes of the subsequent intracytoplasmic sperm injection (ICSI)-cycles were collected. Participants/materials, setting, methods Type 1 (wash-DGC-SU), type 2 (wash-DGC) or type 3 (double wash) sperm processing was executed based on the concentration and motility of the raw sample. The processed fraction was cryopreserved, and the VL was determined in both the raw and processed semen fraction using qPCR. A semen VL below 20 copies/mL was considered negative. Only processed fractions with a negative VL were suitable for further therapeutic use. Threshold for viral suppression was 50 copies/mL. Main results and the role of chance A number of 82 sperm processing procedures were performed for 68 seropositive men, with a mean age at donation of 41.9 ± 7.0 years. All patients were confirmed to be HIV-1 positive, with the exception of one individual presenting a co-infection with both HIV-1 and HIV-2. Incidences for type 1, type 2 and type 3 processing were 23.2%, 62.2% and 14.6%, with mean initial concentration of 91.3(±63.6), 42.1(±47.9) and 0.12(±0.15) million/mL, and mean progressive motility of 54(±12.7)%, 33(±16.8)% and 4(±3.6)% in the respective groups. In 91.5% (n = 75/82) of cases, the patient was suppressed at the time of sperm processing. In case of viral suppression, only one raw sample (n = 1/75, 1.3%) tested positive for HIV (94 copies/mL), likely due to the phenomenon of “viral shedding”. After processing, none of the fractions contained a residual VL. Post-thaw progressive motile sperm survival rate was on average 0.40 ± 0.26. Overall, 61 couples underwent a total of 120 ICSI-cycles, resulting in cumulative clinical pregnancy and cumulative live birth rates per couple of 57.4% (n = 35/61) and 42.6% (n = 26/61), respectively. Limitations, reasons for caution Given that HIV-positive individuals represent a minority within our patient population, the limited prevalence of sperm wash procedures performed over the past decade has led to a relatively small sample size for this study. Wider implications of the findings These findings have the potential to shape future guidelines concerning the use of sperm samples from HIV-positive men. This may optimize the efficiency of sperm processing protocols, while maintaining both the safety and efficacy of assisted reproductive techniques for this specific patient population. Trial registration number Not applicable

P-073 Microfluidic sperm selection yields competent spermatozoa with higher genomic integrity and cryostress tolerance

Abstract Study question Can microfluidic sperm sperm selection (MFSS) contribute to isolating spermatozoa with higher genomic integrity and cryostress resistance, while retaining embryo developmental competence? Summary answer In comparison to conventional density gradient (DG), MFSS yielded a higher proportion of motile spermatozoa coupled with enhanced genomic integrity and resistance to cryostress. What is known already The cryopreservation of human spermatozoa has long been a cornerstone in reproductive treatments, serving as a pivotal method for preserving male fertility for several decades. Despite the unlimited storage time of cryopreserved semen specimens, the thawing process inevitably diminishes the number of viable spermatozoa. The implementation of a technique capable of enhancing the motility of spermatozoa cryopreserved would yield a higher proportion of gametes with higher kinetics and genomic integrity at thawing. Study design, size, duration Since 2017, 183 men underwent cryopreservation utilizing MFSS. Post-processing semen parameters, including Sperm Chromatin Fragmentation(SCF), and post-thaw motility were compared with a DG control(n = 1979). Additionally, post-thaw parameters for 30 men who had previously employed DG and subsequently adopted MFSS for semen cryopreservation were included. In 12 specimens, a paired analysis was carried out on a same ejaculate. Moreover, we explored pregnancy outcomes with MFSS-selected specimens in some men that underwent ART. Participants/materials, setting, methods Semen parameters were analyzed, and DG was performed according to WHO 2021 guidelines. MFSS was carried out following manufacture’s protocol (ZyMōt® Multi 850µL). Cryoprotectant (Test Yolk Buffer) was added to the final sample before being immersed in liquid nitrogen. A minute aliquot of the processed sample, cryopreserved separately, was thawed for post-thaw motility. TUNEL assay was used to measure SCF (≤ 15% normal threshold). Main results and the role of chance In this study, 1979 men (40.0±7y) underwent DG prior to cryopreservation, resulting in a 44.3±25x106/mL concentration, 88.2±4% motility, and 2.9±1% normal morphology, serving as control. The remaining 183 men of a similar age underwent MFSS prior to cryopreservation. Despite a lower concentration at 26.1±15x106/mL(P &amp;lt; 0.001), MFSS resulted in a higher post-processing motility(97.8±2%, P &amp;lt; 0.001) and normal morphology(3.4±1%, P &amp;lt; 0.001). Post-thaw analysis revealed a remarkably higher post-thaw motility in MFSS (51.2±7% vs 43.6%±4%,P &amp;lt; 0.001). SCF averaged 17%(raw), 11%(DG) and 2%(MFSS,P&amp;lt;0.001). To confirm research hypothesis, a pilot study was done in 12 men (37.3±3y,3.0±2d abstinence) with their semen specimen equally aliquoted for DG or MFSS and cryopreserved. The post-thaw motility increased from 45.4±8%(DG) to 61.3±13%(MFSS,P&amp;lt;0.001). Subsequently, in men(n = 30, 40.8±6y, 3.0±2d abstinence) who underwent semen cryopreservation previously by DG and subsequent by MFSS, a similar trend was observed with a higher post-thaw motility (53.2±6% vs 43.2%±6%,P &amp;lt; 0.001). Among the 36 patients who utilized DG cryopreserved specimen for reproductive purposes, 11/19 (57.9%) patients achieved pregnancy in ICSI cycles. while 5/17 (29.4%) achieved pregnancy in an IUI cycle, Among the 18 couples who used MFSS cryopreserved specimen for ICSI, a pregnancy rate was reported (8/18,44.4%). A IUI pregnancy rate was also reported (4/18,22.2%) using MFSS cryopreserved specimen. Limitations, reasons for caution While MFSS has proven to increase post-thaw survival rate and maintain a lower SCF, it did not necessarily trend in a higher pregnancy in IUI cycles, possibly due to the borderline elevated SCF. This finding needs to be confirmed in a larger population. Wider implications of the findings In reproductive medicine, we strive to identify new techniques aimed at providing more competent gametes. MFSS was capable of isolating the most motile potion of spermatozoa with a higher genomic integrity. The utilization of MFSS for specimens to be cryopreserved appears promising in generating higher post-thaw recovery of motile spermatozoa. Trial registration number not applicable

Asian elephant (Elephas maximus) seminal plasma: establishing the proteome and effect on spermatozoa when added to cryomedium.

Context The removal or supplementation of ejaculates with seminal plasma (SP) can affect cryotolerance and post-thaw survival of spermatozoa in many species. In the Asian elephant (Elephas maximus ), elucidation of the SP proteome and investigation of how it affects spermatozoa may enable improvement of cryopreservation protocols. Aims Herein, we characterise the Asian elephant SP proteome and investigate the impacts of SP on sperm cryotolerance in the presence of conspecific or heterospecific SP. Methods Proteomic analysis of Asian elephant SP was performed using mass spectrometry on nine samples from three individuals. In a separate study, SP was removed from six ejaculates and spermatozoa were resuspended in Tris extender supplemented with: no seminal plasma (NOSP), conspecific SP from ejaculates exhibiting 'good' (GSP, >60%) or mixed sperm total motility (MSP), or horse SP (HSP). Samples underwent cryopreservation, and sperm parameters were compared prior to cryopreservation and after thawing (0 and 2h). Key results Mass spectrometry identified 155 proteins from an array of families. Significant differences were observed in post-thaw sperm quality between SP treatments: high concentrations of MSP (25%, v/v) displayed greater average path and straight-line velocity immediately after thawing (P P P Conclusions and implications These preliminary findings suggest the potential of SP to enhance the cryosurvival of Asian elephant spermatozoa, with HSP showing particularly promising results compared to conspecific SP (GSP). Further research into the specific effects of Asian elephant SP proteins is warranted.

Human recombinant interleukin-6 improves the morphological quality of cryopreserved in vitro produced bovine blastocysts

This work explored whether a well-characterized recombinant human interleukin-6 (hIL6) protein will influence in vitro produced (IVP) bovine embryo development and survival after cryopreservation. Cumulus oocyte complexes were collected from abattoir derived ovaries, matured for 24 h, and fertilized using pooled semen from Holstein bulls. Embryos were treated with 0, 25, 50, or 100 ng/mL hIL6 on day 5 post-fertilization. An increase in ICM cell numbers was observed in each hIL6 treatment, with the lowest hIL6 treatment having the same magnitude of response as the middle and highest hIL6 concentration. No effects on TE cell numbers were observed. The second study involved cryopreserving (via slow freezing) of hIL6-treated blastocysts, then examining post-thaw blastocyst survival by incubating for 24 h in the absence of hIL6 treatments. Blastocyst re-expansion and hatching rates were unaffected by any of the IL6 treatments, however, increases in both ICM and TE cell numbers were detected at 24 h post-thawing in blastocysts exposed to 100 ng/mL hIL6 but not lower concentrations before freezing. A reduction in the percentage of TUNEL-positive TE cells was observed after thawing in blastocysts exposed to 25, 50 and 100 ng/mL hIL6 before cryopreservation. No treatment-dependent changes in TUNEL-positive ICM cells were observed. In summary, hIL6 supplementation improves ICM cell numbers in bovine blastocysts to a degree that is commensurate with what has been observed when using bovine recombinant IL6. This positive effect of hIL6 on ICM cell numbers is maintained after freezing and thawing, and a novel improvement in post-thaw TE cell numbers occur in hIL6 treated embryos. This positive effect on TE cell numbers is attributed, at least in part, to an hIL6-dependent reduction in TE cell apoptosis.

Embryonic Parameters Associated With Outcomes of Single Blastocyst Frozen Transfer [ID 2683440

INTRODUCTION: It is unclear how various in vitro fertilization cycle characteristics may ultimately influence the fertility outcomes of patients utilizing assisted reproductive technology. This study investigates embryo characteristics to determine which features are most associated with clinical intrauterine pregnancy after frozen embryo transfer. METHODS: This retrospective study evaluates 535 single blastocyst frozen embryo transfers (FETs) in 2022 and received IRB approval from the University of Kansas. Logistic regression analyses were used to evaluate embryo characteristics and likelihood of clinical intrauterine pregnancy (IUP), defined as positive fetal cardiac activity on ultrasound after 12 weeks of gestation. Blastocyst survival (percentage of live cells), blastocoele re-expansion, and morphological grade were evaluated prior to embryo transfer. Significant embryonic parameters included blastocyst developmental stage and blastocyst grade before cryopreservation, usage of preimplantation genetic testing for aneuploidy (PGT-A), post-warm blastocyst survival (percentage of live cells), degree of blastocoele re-expansion, blastocyst grading before transfer, and presence of zona pellucida. RESULTS: Among 535 FET cycles, 257 (48.0%) resulted in clinical IUP. Multivariate logistic regression analysis showed a significant independent influence of blastocyst development time (day 5/6) (odds ratio [OR] 0.571; P=.003), morphological grade before cryopreservation (day 5/6) (OR 0.648; P=.013), usage of PGT-A (OR 1.704; P=.010), and percentage of post-thaw live cells (OR 0.322; P=.001) on embryo transfer success rates. While fully expanded blastocysts showed a significantly higher rate of clinical IUP, the degree of blastocoele re-expansion did not appear to be independently related to ongoing pregnancy when multivariate logistic regression analysis was employed (OR 2.886; 95% CI: 0.824–10.105; P=.097). CONCLUSION: In addition to blastocyst quality prior to cryopreservation, post-thaw blastocyst survival is an independent predictor of embryo recovery and further development potential. Additional understanding of the potential contributors to failed FETs can inform strategies for improvement. Further investigation is needed to determine how to minimize embryonic cell damage during the vitrification and warming process.

Cryopreservation of European flat oyster (Ostrea edulis) larvae using a controlled-rate freezer

Larvae of the European flat oyster (Ostrea edulis) were cryopreserved using two protocols originally developed for other bivalvian species with several modifications. Culture of the European flat oyster went through several setbacks due to parasitic diseases, thus, assisted reproductive technologies such as larval cryopreservation can aid in prompt restoration of lost populations and thus facilitate farming of the species. Veliger larvae were diluted in two basic extenders (0.4 M trehalose or 0.4 M sucrose) supplemented with ethylene glycol (EG), dimethyl-sulfoxide (DMSO) or propylene glycol (PG) in concentrations of 10–20% with or without the addition of polyvinylpyrrolidone (average MW-40,000; PVP-40). Larvae were loaded into 0.25-ml or 0.5-ml straws and cryopreserved in a controlled-rate freezer using two slow cooling profiles. Assessment of larval survival 20 min or 24 h post-thaw revealed that the cooling profiles and the type of extender used did not have a significant effect on survival rates. On the other hand, the type of cryoprotectant, straw volume and the time of assessment had a significant main effect on larval survival percentages (p < 0.001 in all three cases). Survival percentages of 70–80% were observed 20 min post-thaw which decreased to 40–60% at 24 h. Generally, EG and DMSO resulted in higher post-thaw larval survival than did PG when larvae were cryopreserved in 0.25-ml straws, whereas, these differences were not detected when 0.5-ml straws were employed. Results of these experiments will allow later studies on long-term survival of cryopreserved European flat oyster larvae.

Abstract 5229: Enhancing solid tumor treatment with engineered blood-derived Vδ1 platform: Safe and effective targeting of mesothelin-expressing tumors with Vδ1 γδT cells

Abstract Mesothelin (MSLN) is a solid tumor-associated antigen that has been thoroughly studied and is expressed in several tumor indications that have significant unmet therapeutic needs. Vδ1-enriched γδT cells differentiate themselves from other MSLN-targeted modalities by mitigating the challenges of targeting tumors with heterogeneous MSLN expression through their inherent capability to detect malignant cells while sparing normal cells. The ability to effectively combat cancer cells that express mesothelin is significantly increased by the expression of a chimeric antigen receptor (CAR). Novel armoring is being evaluated to improve engineered Vδ1 persistence in the solid tumor microenvironment. Cytokine armoring, through novel engineering, enhances sensitivity to cytokines and recovers cell fitness that is impaired by 'traditional' cytokine armoring options. Extensive in vitro and in vivo safety and efficacy studies were carried out to validate the product's potency. These studies aimed to determine cytotoxicity, expansion, and persistence. MSLN CAR cytokine-armored Vδ1 cells exhibited high post-thaw viability and survival. Furthermore, cytokine-armored Vδ1 cells displayed elevated innate receptor expression and gene-engineered cells maintained their baseline innate phenotype, indicating that they can maintain their ‘innate’ effectiveness over time. In vitro studies demonstrated broad anti-tumor activity against multiple tumor cell lines expressing various levels of mesothelin. Additionally, in long-term cytotoxicity assays, cytokine-armored cells demonstrated improved tumor-clearance capabilities. The effector cells also exhibited an enhanced innate cytotoxic phenotype that equips the cells with the capability to persist and proliferate in response to soluble cytokines and upon cytokine trans-presentation by adjacent cells. In vitro studies further revealed that there is a distinction in the killing activity between tumor and normal cells, which is important for ensuring that the product is safe for use in patients. An orthotopic lung tumor-bearing mouse model was developed to confirm their anti-tumor effectiveness in vivo. The model's outcomes demonstrated that MSLN CAR cytokine-armored Vδ1 cells exhibit higher in vivo efficacy in comparison to their unarmored counterparts at low effector-to-target ratios. With no significant healthy tissue damage found, the histopathology report confirmed an excellent safety profile. Terminal biodistribution studies demonstrated that armored drug candidates preferentially home to and persist in tumor-bearing tissues. Overall, these findings imply that the product might be highly effective in treating tumors that express mesothelin. This strategy could greatly advance our knowledge of how Vδ1-enriched γδT cells function as a platform against solid tumors. Citation Format: Nandhu Mohan Sobhana, Andre Simoes, Sarah Edwards, Sweta Mishra, Gary Gu, George Papadopoulos, Stefan Kiesgen, Rebecca Alade, njing Li, Andrea Venuso, Jyothi Kumaran, Kristy Waddington, Tabassum Huseni, Rajeev Karattil, Lilly Reynolds, Maria Sharif, David Wotherspoon, Corina Tudor, Istvan Kovacs, Mei Rosa Ng, Yana Wang, Lan Cao. Enhancing solid tumor treatment with engineered blood-derived Vδ1 platform: Safe and effective targeting of mesothelin-expressing tumors with Vδ1 γδT cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5229.

A droplet vitrification cryopreservation protocol for conservation of hops (Humulus lupulus) genetic resources

Hops (Humulus lupulus L.) is essentially used in the brewing industry as it contributes to flavor, and aroma of beer. However, the genetic diversity of hops is increasingly threatened by diseases, environmental changes, and urbanization. Cryopreservation has emerged as a pivotal strategy for safeguarding and maintaining the genetic diversity of hops. The present work presents a comprehensive study on the cryopreservation of hops, focusing on the development and optimization of a droplet vitrification based cryopreservation protocol. Shoot tips excised from one month old in vitro cultures were precultured on 0.3 M sucrose, dehydrated in a loading solution followed by treatment with PVS2 solution for different durations. Significant effect of PVS2 dehydration was observed on post-thaw survival and regeneration after cryoconservation with maximum 50% post-thaw regeneration observed in shoot tips dehydrated in PVS2 for 30 min. Genetic fidelity of the regenerated plants was confirmed using 30 ISSR markers. Reproducibility of the developed protocol was tested on seven other accessions and post thaw regeneration ranging from 43 to 70% was observed across the accessions. The present study reports a highly efficient protocol for conservation of hops germplasm. The results indicate that droplet vitrification can be used as a reliable and sustainable approach for hop genetic preservation, with high survival rates and minimal genetic alterations observed in cryopreserved samples. To the best of our knowledge, this is the first report on DV based cryopreservation of hops germplasm.

Influences of Supplementing Selective Members of the Interleukin-6 Cytokine Family on Bovine Oocyte Competency.

This work explored whether supplementing selective members of the interleukin-6 (IL6) cytokine family during in vitro bovine oocyte maturation affects maturation success, cumulus-oocyte complex (COC) gene expression, fertilization success, and embryo development potential. Human recombinant proteins for IL6, IL11, and leukemia inhibitory factor (LIF) were supplemented to COCs during the maturation period, then fertilization and embryo culture commenced without further cytokine supplementation. The first study determined that none of these cytokines influenced the rate that oocytes achieved arrest at meiosis II. The second study identified that LIF and IL11 supplementation increases AREG transcript abundance. Supplementation with IL6 supplementation did not affect AREG abundance but reduced HAS2 transcript abundance. Several other transcriptional markers of oocyte competency were not affected by any of the cytokines. The third study determined that supplementing these cytokines during maturation did not influence fertilization success, but either LIF or IL11 supplementation increased blastocyst development. No effect of IL6 supplementation on subsequent blastocyst development was detected. The fourth experiment explored whether each cytokine treatment affects the post-thaw survivability of cryopreserved IVP blastocysts. None of the cytokines supplemented during oocyte maturation produced any positive effects on post-thaw blastocyst re-expansion and hatching. In conclusion, these outcomes implicate IL11 and LIF as potentially useful supplements for improving bovine oocyte competency.

#83 : Human Sperm Cryo-Survival Rate

Background and Aims: Sperm cryopreservation plays an important role in the suite of services available to fertility patients. The ability to preserve the motility and viability of human spermatozoa is a key component in delivering successful assisted reproductive technologies (ART) outcomes. Cryopreservation methods expose sperm to stress, resulting in cellular damage which consequently leads to a reduction in their survival rate. The aim of this study was to evaluate the average cryo-survival rate of semen samples frozen in a clinical setting, to help improve and manage patient expectations prior to accessing this service. Method: Pre and post sperm freeze data from patients who presented at the Genea Sydney clinic for sperm cryopreservation between 2017 and 2022 was analysed. The post-thaw survival rate for 1467 specimens was assessed by averaging the total post-thaw motility with total pre-freeze motility. Only ejaculated samples, frozen on-site, who had a raw motility calculated as well as a post-thaw motility analysis able to be performed were included in the study. Results: The range of post-thaw survival was 57% to 67% per annum over the six years of examined data, with an overall cryo-survival rate of 62%. Conclusions: The data shows a consistent recovery rate of post-thaw sperm motility over the time period analysed. Literature indicates average sperm cryo-survival rates range from 30% to 50%, and previous advice to patients has been to expect around 50% sperm survival. With this new data, Genea can revise its guidance to match the current average figure of around 60%. In addition, future internal freeze optimisation studies can use this figure as a benchmark to improve upon techniques and processes.

When Should We Recommend Egg Freezing and When Is It Too Late?

Social oocyte freezing (SOF), a fast-growing field in ART is the elective cryopreservation of one’s own oocytes at a younger age when oocytes are of higher quality with the goal of prolonging a woman’s childbearing opportunity without any urgent medical reasons. The optimum age for SOF is said to be below the age of 35 years ( ASRM , 2013 ; ESHRE , 2012 ). SOF in women above 35 years of age is associated with lower post-thaw survival rates, a lower live birth rate per thawed oocyte, and an increased overall cost per livebirth as more cycles of oocyte freezing and thawing may be required. Beyond 42 years of age, SOF is unlikely to result in a livebirth due to suboptimal response to stimulation and a higher cycle cancellation rate ( ASRM , 2013 ; Doyle, et al. , 2016 ; Wennberg, et al. , 2019 ). The number of oocytes needed to freeze varies widely between studies but should be individualized according to the woman’s ovarian reserve as well as the IVF center’s own success rate data ( Teo, et al. , 2021 ). The utilization rate of frozen oocyte is low, ranging from 6 to 15%. The main reasons cited are a) women not wanting to start a family without a partner; b) preference for natural conception and, c) refusal to use a sperm donor ( Hammarberg, et al. , 2017 ). With the use of relatively safe IVF techniques and success rates similar to that of fresh oocytes, SOF has become an acceptable reproductive option for women today to extend their reproductive ability to achieve parenthood using their own eggs. It is, however, expensive and more than one cycle of oocyte freezing may be required to improve its efficacy. Thorough counselling must be done to advise women regarding the lack of robust evidence on its efficacy, long-term risks, and costs.

Controlled hydrogel-based encapsulation of macrophages determines cell survival and functionality upon cryopreservation

The development of novel cell-based therapies has increased the necessity to improve the long-term storage of cells. The current method of cryopreservation is far from optimal, causing ice-associated mechanical and osmotic damage to sensitive cells. Cell encapsulation is emerging as a new strategy to overcome those current limitations; however, few data are applicable to slow freezing, with conflicting results and multiple experimental conditions. The objective of this research work was to evaluate the impact of capsule size and encapsulation method on cell survival and functionality after a conventional freezing protocol. To this end, cells were encapsulated in alginate beads of different sizes, spanning the range of 200–2000 µm thanks to multiple extrusion techniques and conditions, and further cryopreserved using a slow cooling rate (−1°C/min) and 10 % DMSO as cryoprotectant. Our data show that there is a strong correlation between bead size and cell survival after a slow cooling cryopreservation process, with cell viabilities ranging from 7 to 70 % depending on the capsule size, with the smallest capsules (230 µm) achieving the highest level of survival. The obtained results indicate that the beads’ diameter, rather than their morphology or the technique used, plays a significant role in the post-thawing cell survival and functionality. These results show that a fine control of cell encapsulation in alginate hydrogels is required when it comes to overcoming the current limitations of long-term preservation techniques by slow cooling.

Successful cryopreservation of common carp (Cyprinus carpio) embryos: efficiency of methanol-based vitrification solutions with honey on post-thaw survival rate

Successful cryopreservation of common carp (Cyprinus carpio) embryos: efficiency of methanol-based vitrification solutions with honey on post-thaw survival rate

Strip Pulled Straw: Cost Effective Alternative Cryodevice for the Vitrification of Oocytes

BACKGROUND: Increased cooling and warming rates by using a suitable cryodevice allows the use of lower cryoprotectant concentration and reduces cryoinjuries as a result of the rapid passage through the 'dangerous' temperature zone. OBJECTIVE: To evaluate the effectiveness of newly customized strip pulled straw (SPS) with respect to post warming quality, viability, and in vitro maturation for immature oocytes post-vitrification of. MATERIALS AND METHODS: SPS was prepared using conventional French mini straw to combine the merits of OPS and the Cryotop system. Immature sheep oocytes were treated in 15% EG + 15% DMSO, loaded on SPS and plunged into liquid nitrogen (LN). Post thaw quality, viability, and maturation rates of oocytes were determined after 1 week storage in LN. RESULTS: SPS achieved a post-thaw morphological survival of 90.9% with 9.0% morphological defects, 96.4% viability and 51% in vitro maturation. In comparison to OPS, SPS had higher post thaw survival (86.5% vs 67.9%) and maturation rate (57.7% vs 50.5%) with lower morphological defects (13.5% vs 32.1%). Cumulus cell loss was the highest among morphological abnormalities of post warm oocytes in SPS (40.9%) and OPS (44.1%). The data showed acceptable post thaw survival, viability and in vitro maturation rate of immature ovine oocytes using SPS as compared to traditional OPS. CONCLUSION: SPS can be used as a cost effective alternative device for oocyte vitrification.

P-138 Protein concentration of 5 mg/mL in recovery medium for thawed blastocysts is equivalent to 10 mg/mL in post-thaw survival and pregnancy rates

Abstract Study question Are comparable survival and pregnancy rates obtained when recovery media are supplemented with 5mg/mL protein versus customary 10mg/mL protein in Frozen Embryo Transfer cycles? Summary answer Recovery medium containing 5mg/mL protein compared to 10mg/mL yielded similar outcomes in blastocyst survival and pregnancy rates. What is known already Accepted practice has been to culture thawed blastocysts in medium with the same protein concentration as the vitrification media (20% protein) to minimize osmotic stress and optimize survival and implantation rates. The potential to acquire similarly positive outcomes with supplementation of only 5 mg protein/ml has been explored and demonstrated, challenging the requirement for high protein levels in recovery media. Study design, size, duration From July through August 2022, a prospective study was conducted in a university laboratory to compare the survival and implantation rates in Frozen Embryo Transfer cycles using vitrified blastocysts recovered in Continuous Single Culture–NX Complete (CSCM-NXC) (5mg/mL Human Serum Albumin (HSA)) or global HP[LW1] (8.8mg/mL HSA and 1.2mg/mL α-and ß-globulins). 50 blastocysts were warmed per condition. Comparative analyses included survival and implantation rates, beta HCG levels, and clinical pregnancy rates by fetal cardiac activity. Participants/materials, setting, methods To evaluate the effect of protein level in recovery medium for vitrified blastocysts, CSCM-NXC and global HP, with 5mg/mL and 10mg/mL total protein, respectively, were compared. Blastocysts were vitrified in Irvine Scientific Vit Kit – Freeze and were warmed in Irvine Scientific Vit Kit – Warm. Minimum recovery time was the same for all blastocysts (30 minutes), and is defined as the time from completion of the warming procedure to the time of embryo transfer. Main results and the role of chance In both study arms, one blastocyst was removed from the data analysis due to embryo transfer cancellation that was not related to embryo survival or suitability for transfer, resulting in N = 49 for each group. Chi-square analysis was performed to assess statistical significance of all compared parameters, and a p value &amp;lt; 0.05 was considered significant. Average patient age (years) for CSCM-NXC was 36.1 and for global HP, 35.0. There was no difference between blastocysts warmed in CSCM-NXC and global HP in average recovery time, i.e., 3.8 and 3.9 hours, respectively, or survival rates, 98.4% and 98.1% respectively. Comparison of patients with blastocysts warmed in CSCM-NXC reflected a trend towards higher outcomes compared to those in global HP with initial positive beta HCG rates of 64.6% vs 56.3% and ongoing clinical pregnancy rates of 52.1% vs 45.8%, respectively. Implantation rates were 56.1% and 57.7% in CSCM-NXC and global HP, respectively. Incidence of monozygotic twinning of 0% in CSM-NXC and 3.3% in Global HP was observed. Limitations, reasons for caution As this is a challenge to accepted formulation of recovery media for vitrified blastocysts, specifically with respect to protein supplementation - a strategy for minimizing osmotic shock and potential damage to warming embryos - more expansive and robust studies must be undertaken to increase the statistical power of this study. Wider implications of the findings As frozen embryo transfers are utilized more routinely in assisted reproductive treatment, the clinical finding that a commercial recovery medium containing of 5mg/mL protein, the standard for routine embryo culture, can support the recovery of vitrified embryos and lead to successful pregnancy could increase the efficiency in the laboratory. Trial registration number na