Cryopreservation Method Research Articles

P-129 Transferring blastocysts derived from frozen-thawed cleavage embryos versus frozen-thawed blastocysts: A comparative analysis of pregnancy outcomes

Abstract Study question Is there an enhancement in pregnancy outcomes when cleavage-stage embryos are thawed, cultured for transfer as blastocysts, as opposed to transferring thawed blastocysts? Summary answer The strategy of culturing frozen-thawed cleavage embryos for 2 days and transferring them as blastocysts is not inferior to the transfer of frozen-thawed blastocysts. What is known already Embryo cryopreservation in IVF has many advantages, allowing couples to postpone pregnancy for personal reasons and reducing the risk of ovarian hyperstimulation syndrome. In the past, the common practice involved freezing and transferring cleavage embryos. However, a current trend favors single-embryo transfers at the blastocyst stage, promoting potential self-selection during extended culture. Several studies have raised concerns about potential damage during blastocyst vitrification, prompting investigation into whether cryopreserving cleavage-stage embryos for subsequent culturing to blastocysts yields superior outcomes compared to direct transfer of thawed blastocysts. Study design, size, duration This observational study was conducted at Hung Vuong Hospital from January 2022 to December 2023. We included 509 patients who underwent IVF followed by FET cycles. The D3-5 group (n = 167) comprised embryos that were frozen and thawed at the cleavage stage, subsequently extended two more days of culturing to the blastocyst stage for transfer. The D5 group (n = 342) involved frozen and thawed blastocysts. We excluded donor cycles, surrogacy, and cycles with preimplantation genetics testing. Participants/materials, setting, methods We used vitrification method for embryo cryopreservation. The freezing and thawing process were performed using either Kitazato or Cryotec medium kit, following manufacture’s instruction. Regarding the D3-5 group, thawed cleavage embryos were cultured for two more days until blastulation and transfer. Regarding the D5 group, blastocysts transfer was performed at least 2 hours after warming. The embryo transfer procedure was performed under abdominal ultrasonographical guidance. Pregnancy results were recorded and compared between two groups. Main results and the role of chance Regarding the D3-5 group, a noteworthy proportion of 65.3% cycles were unfortunately cancelled. The primary reason for cycle cancellations was the absence of developed blastocysts for transfer (85.3%). The remaining 14.7% of cancellations were attributed to other reasons. Fertilization rates were comparable between the two groups, with 80.8% in the D3-5 group and 81.5% in the D5 group (p = 0.464). However, the blastulation rate in the D3-5 group (40.1%) was lower than in the D5 group (46.4%), (p = 0.016). Patients in the D5 group demonstrated slightly better pregnancy outcomes compared to the D3-5 group: implantation rate (59.9% versus 51.7%, p = 0.302), clinical pregnancy rate (52.3% versus 44.8%, p = 0.36), ongoing pregnancy rate (37.7% versus 34.5%, p = 0746), live birth rate (33.9% versus 31%, p = 0.78), and miscarriage rate (26.3% versus 20.7%, p = 0.46). However, these differences were not statistical significance. Limitations, reasons for caution This is an observational study, we observed that thawing the cleavage-stage embryos then subsequently culture to the blastocyst stage may lead to the lower blastulation rate or even absence of developed blastocysts for transfer. This approach also increases the workload for embryologists. Wider implications of the findings Culturing frozen-thawed cleavage embryos for 2 days and transferring them as blastocysts increases workload for embryologists and poses a risk of cycle cancellation. We propose that the use of frozen-thawed blastocysts may be a more efficient and patient-friendly option. Trial registration number not applicable

P-325 Comparing slow-freezing and vitrification endometrial biopsy cryopreservation protocols for tissue cryodamage and functional organoid formation

Abstract Study question Are slow-freezing and vitrification suitable methods for cryopreserving endometrial biopsies intended for downstream research applications and organoid formation? Summary answer Both slow-frozen and vitrified endometrial biopsies resulted in successful organoid formation thus can be considered similar to fresh endometrial biopsies in preserving the cellular viability. What is known already Passive slow freezing (PSF) protocols have demonstrated efficacy for cryopreserving endometrial biopsies with subsequently viable isolated epithelial and stromal cells. Additionally, single-cell transcriptomic analyses and successful generation of endometrial organoids suggest a little impact of cryopreservation. By avoiding ice crystal formation, vitrification (VT) could potentially prevent mechanical injury and improve tissue integrity compared to slow freezing. Study design, size, duration Experimental study using endometrial biopsies collected using a Pipelle from eight healthy volunteers. Samples were retrieved at three cycle phases: proliferative (n = 2), mid-secretory (7 days post-LH surge, LH + 7, n = 2) and late-secretory phase (LH + 11, n = 4). Each biopsy was divided into fresh, slow-freezing and vitrification sub-samples for further comparison of outcomes according to microscopic histological analysis, transmission electron microscopy (TEM), gene expression profiling evaluation and generation of endometrial epithelial organoids (from 2 slow-frozen and 2 vitrified samples). Participants/materials, setting, methods PSF was performed using Mr Frosty with samples embedded in 1x DMEM, 30% FBS and 7.5% DMSO at -80ºC. Vitrification was performed using 40% ethylene glycol (EG); 30% Ficoll; 0.5 M Sucrose and 10 mg/ml HSA, and preserved in liquid nitrogen. Tissue morphology was evaluated by traditional histology, TEM and gene expression profiling. The viability of endometrial cells and the ability to form epithelial organoids were evaluated before and after two different freezing protocols. Main results and the role of chance Our analysis showed that both PSF and vitrification techniques are appropriate for cryopreservation and storage of endometrial biopsies aimed at organoid generation, as no significant differences were found between fresh and cryopreserved tissue samples. However, ultrastructural changes were observed by TEM in PSF samples indicating an impact on mitochondria of epithelial and stromal cells with increased swelling. Changes were also observed in epithelial cells in vitrified samples, with decreased nuclear granularity, clarity, and open karyoplasm. On TEM analyses changes in the chromatin status were more evident in vitrified samples compared to fresh and PSF samples. PSF treated samples showed similarly high quality of epithelial and stromal nuclear staining when compared to the fresh samples in haematoxylin and eosin-stained slides, while VT of samples resulted in a significant decrease of both epithelial and stromal nuclear clarity. However, despite these changes observed in the tissue morphology, all three groups displayed similar transcriptional profiles and endometrial organoids were generated from both slow-frozen and vitrified samples, indicating very little or no impact of the freezing method used for post-thaw tissue functionality. Limitations, reasons for caution Although our study demonstrated relatively mild damages in both freezing protocols, we cannot exclude the absence of additional changes not evaluated in this study, such as those concerning the epigenetic constitution of the cells. Therefore, more extended research with larger sample size is needed for further validation of the results. Wider implications of the findings Our findings indicate that both slow-freezing and vitrification methods applied to endometrial biopsies are effective in maintaining tissue morphology and functionality. Further optimization of cryopreservation methods of endometrium could contribute to future experimental and clinical use of cryopreserved endometrial tissue. Trial registration number not applicable

Optimization of cryopreservation methods for somatic cells of the Tobet dog breed

The Tobet dog breed, a national heritage of Kazakhstan, is threatened with extinction and conservation measures are urgently needed. This study presents a pioneering approach to preserve the genetic diversity and survival of this endangered breed through cryopreservation and cryobanking. We have optimised methods for cryopreservation of somatic cells (fibroblasts) isolated from skin explants of Tobet dogs and investigated the effects of in vitro cultivation on cell viability. Our results emphasise the importance of selecting appropriate culture media and cryoprotectants. Dulbecco's Modified Eagle Medium (DMEM) and ethylene glycol (EG) were found to be the most effective agents for increasing the growth rate and viability of fibroblasts after thawing. Through careful experimentation, including the evaluation of equilibrium and non-equilibrium cryopreservation techniques and the application of various cryoprotectants, we have established the first Tobet cryobank for somatic cells. The establishment of a cryobank represents a significant step towards the conservation of the Tobet dog breed and provides a model for the conservation of other endangered species. This study not only contributes to the preservation of Kazakhstan's cultural and biological heritage, but also opens up new avenues for the application of biotechnological approaches to wildlife conservation.

Plunge-Freezing Cryopreservation of Tendons.

Allograft transplantation is an important method for tendon reconstruction after injury, and its clinical success highly relies on the storage and transportation of the grafts. Cryopreservation is a promising strategy for tendon storage. In this study, we report a novel cryopreservation agent (CPA) formulation with a high biocompatibility for tendon cryopreservation. Mainly composed of natural zwitterionic betaine and the biocompatible polymer poly(vinylpyrrolidone) (PVP), it exhibited ideal abilities to depress the freezing point and inhibit ice growth and recrystallization. Notably, after cryopreservation via plunge-freezing for 1 month, Young's modulus (144 MPa, 98% of fresh tendons) and ultimate stress (46.7 MPa, 99% of fresh tendons) remained stable, and the cross-linking of collagen microfibers, protein structures, and glycosaminoglycan (GAG) contents changed slightly. These results indicate that the formulation (5 wt % betaine and 5 wt % PVP in phosphate-buffered saline, PBS solution) effectively maintains the biomechanical properties and tissue structure. This work offers a novel cryopreservation method for tendons and may also provide insights into the long-term preservation of various other tissues.

Temporal analysis of cryopreservation effects on human sperm phospholipase C zeta expression profile: Laboratory preliminary findings

Objectives: The objectives of this study were to compare the sperm parameters and sperm phospholipase C zeta (PLC ζ) expression profile before and after vitrification. Materials and Methods: Pre-vitrification and post-vitrification analysis of semen samples of 14 infertile men was carried out for sperm parameters such as motility, vitality, and morphology by standard semen analysis procedures, whereas the proportion of sperms exhibiting PLC ζ and its localization pattern was assessed by indirect immunofluorescence. The temporal analysis was done thrice: over 1 week, 1 month, and 3 months of cryostorage. Statistical Analysis: Statistical analyses were performed using GraphPad Prism version 8.0.1 (San Diego, California, USA). Data were expressed as mean ± standard deviation. Parametric or non-parametric tests were employed for comparisons according to the normality of data distribution and the available data set. P value of 0.05 was considered statistically significant. Results: Vitrification was found to be associated with a decrease in the percentage of sperm motility (P ≤ 0.0001), vitality (P ≤ 0.0001), spermatozoa exhibiting normal morphology (P > 0.05), and PLC ζ protein (P > 0.05), however, the latter two, only, insignificantly. There was increased dominance in the post-acrosomal localization of PLC ζ after vitrification (P ≤ 0.001). Conclusions: The post-acrosomal localization of PLC ζ has been reported to have the highest positive correlation with oocyte fertilization and the present study showed the predominant pattern of the same. The implications for quality maintenance for long storage periods can be suggested as better sperm quality was observed at 3 months of storage during this study. This raises the hypothesis that the vitrification method of sperm cryopreservation may be the method of choice for routine clinical use in the assisted reproductive technology settings.

Novel cryopreservation medium for enhanced stability of T cells at −80°C

The increasing demand for immune cell applications, both in clinical settings and in research laboratories, has highlighted the critical need for cryopreservation (banking) methods for T cells. While conventional techniques such as freezing with liquid nitrogen remain prevalent, they pose significant challenges including high equipment costs, safety considerations, and logistical hurdles in transportation. Our cryopreservation medium, C80EZ®, represents a novel approach, leveraging biocompatible polysaccharides as cryoprotectants to enable safe storage at −80°C. This paper presents a comprehensive series of tests assessing the effectiveness of C80EZ® in shielding T cells from the detrimental effects of cryopreservation. Importantly, our findings demonstrate that C80EZ® not only ensures the survival of T cells, with a particular emphasis on preserving the CD8+ subsets, but also maintains their critical function in targeting and eliminating cancer cells.

A quick and effective modification method to improve the patency and endothelialization of cryopreserved allogenic blood vessels

Cryopreserved allogeneic blood vessels have received considerable attention for vascular reconstruction owing to their easy availability and excellent anti-infective property. However, current cryopreservation methods cause significant endothelium loss alongside directly exposing smooth muscle cells and extracellular matrix components to the circulating blood, which can induce thrombosis and/or restenosis. We covalently coupled PEGylated phospholipids (DMPE-PEG) with anti-coagulant bivalirudin (abbreviated as DPB) and endothelial progenitor cell (EPC)-capturing TPSLEQRTVYAK (TPS) (abbreviated as DPT), respectively. Both DPT and DPB were co-modified onto the lumen surface of cryopreserved allogeneic vessels through hydrophobic interaction between DMPE-PEG and the phospholipid bilayer of surviving cells. Cy7-labled DMPE-PEG was used to investigate the saturation concentration and incubation time on the lumen surface of cryopreserved vessels. DPB and DPT co-modified cryopreserved vessels were characterized through different assays, including tissue viability and gene expression, hemocompatibility, and EPC capture in vitro and an artery implantation model in vivo. Co-modification of DPB and DPT attained luminal saturation as earlier as 10 min while preserving the viability of the residual cells on the cryopreserved vessels. The optimally co-modified cryopreserved allogeneic vessels showed that the DPB can improve the hemocompatibility by reducing fibrinogen adsorption and platelets adhesion alongside protecting the EPCs-capturing function of the DPT via inhibiting the masking by blood components, which altogether promoted the patency and endothelialization. This work provided a quick, biocompatible and an effective approach to functionalize tissue-engineered constructs containing alive cells.

Effect of rapid cooling, frozen storage, and thawing on the passive viscoelastic properties and structure of the rat aorta

Biological tissues decay over time after harvesting, which alters their biomechanical properties. This poses logistical challenges for studies investigating passive arterial biomechanics as tissues need to be characterized shortly after excision. Freezing and cryopreservation methods can help alleviate the need for biomechanical testing of fresh tissue in human ex vivo studies. However, these methods tend to eliminate or reduce arterial cell functionality and affect passive biomechanics. Furthermore, their impact on dynamic arterial biomechanics remains unknown despite arterial viscoelastic properties being an integral component contributing to arterial stiffness under in vivo loading conditions. The present study aims to investigate the impact of rapid cooling and subsequent storage at −80 °C on the passive viscoelastic properties of arterial tissue and aid in ascertaining whether this is a suitable method to delay tissue analysis for studies investigating passive arterial biomechanics. Control and frozen abdominal rat aorta segments were quasi-statically and dynamically tested using a biaxial testing set-up. The results were modeled using a constituent-based quasi-linear viscoelastic modeling framework, yielding directional stiffness parameters, individual constituent biomechanical contributions, and a quantification of viscoelastic stiffening under dynamic pressurization conditions. Frozen samples displayed significantly decreased wall thickness, viscoelastic dissipation, viscoelastic stiffening, and significantly decreased circumferential deformation with changes in luminal pressure. Furthermore, frozen samples displayed significantly increased circumferential stiffness, pulse wave velocity, and collagen load bearing. Consequently, these changes should be considered when utilizing this tissue preservation method to delay biomechanical characterization of rat aortic tissue.

New Approach to the Cryopreservation of GV Oocytes and Cumulus Cells through the Lens of Preserving the Intercellular Gap Junctions Based on the Bovine Model.

Differences in structural and functional properties between oocytes and cumulus cells (CCs) may cause low vitrification efficiency for cumulus-oocyte complexes (COCs). We have suggested that the disconnection of CCs and oocytes in order to further cryopreservation in various ways will positively affect the viability after thawing, while further co-culture in vitro will contribute to the restoration of lost intercellular gap junctions. This study aimed to determine the optimal method of cryopreservation of the suspension of CCs to mature GV oocytes in vitro and to determine the level of mRNA expression of the genes (GJA1, GJA4; BCL2, BAX) and gene-specific epigenetic marks (DNMT3A) after cryopreservation and in vitro maturation (IVM) in various culture systems. We have shown that the slow freezing of CCs in microstraws preserved the largest number of viable cells with intact DNA compared with the methods of vitrification and slow freezing in microdroplets. Cryopreservation caused the upregulation of the genes Cx37 and Cx43 in the oocytes to restore gap junctions between cells. In conclusion, the presence of CCs in the co-culture system during IVM of oocytes played an important role in the regulation of the expression of the intercellular proteins Cx37 and Cx43, apoptotic changes, and oocyte methylation. Slow freezing in microstraws was considered to be an optimal method for cryopreservation of CCs.

Short- and long term preservation of cardiac tissue-engineered constructs

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): This research was financially supported by the Gravitation Program ‘‘Materials Driven Regeneration,’’ funded by the Netherlands Organization for Scientific Research (024.003.013) and the EU-funded project BRAV∃ (H2020, ID:874827). Background Cardiac tissue engineering (cTE) has already advanced towards the first clinical trials, investigating safety and feasibility of cTE construct transplantation in failing hearts. However, the lack of well-established preservation methods poses a hindrance to further scalability, commercialization, and transportation, thereby reducing their clinical implementation. Purpose In this study, hypothermic (short-term) preservation (4°C), and slow and fast freezing methods for cryopreservation, at -196°C and -150°C respectively, were investigated as potential solutions to extend the cTE construct implantation window and increase their clinical applicability. Material and Methods The cTE model used consisted of induced-pluripotent stem cell-derived cardiomyocytes and human cardiac fibroblasts embedded in a natural-derived hydrogel and supported by a polymeric melt electrowritten hexagonal scaffold. Constructs, composed of cardiomyocytes of different maturity, were preserved for three days, using several commercially available preservation protocols and solutions. Their viability, function (BPM and calcium handling), and metabolic activity were investigated after rewarming. Results Our observations suggest that cardiomyocyte maturation did not influence post-rewarming viability, however, aging positively influenced construct function pre- and post-storage. Hypothermic preservation in Hypothermosol ensured retained construct viability and function. Furthermore, fast-freezing outperformed slow-freezing, but both viability and function were severely reduced after rewarming. Conclusion In conclusion, whereas long-term preservation remains a challenge, hypothermic preservation in Hypothermosol represents a promising solution for cTE construct short-term preservation and potential transportation, aiding in off-the-shelf availability, ultimately increasing their clinical applicability.Graphical abstract

Cryopreservation of date palm tissue cultures by vitrification and assessment of genetic stability using ISSR analysis

In this work, a successful cryopreservation method of date palm tissue cultures by vitrification was established. Callus and somatic embryos of three date palm cultivars were exposed to dimethylsulfoxide (DMSO) and Plant Vitrification Solution 2 (PVS2) for different durations before plunging into liquid nitrogen for cryoprotection. After freezing phase, the cryopreserved cultures were thawed and cultured on recovery media. Among different times of exposure to DMSO (10%), 60 min gave best results of survival and regrowth of the date palm tissue cultures. Regarding recovery, ‘Zaghlool’ cultivar gave the highest values of callus fresh mass (0.5 g) and growth value (1.00). Also, the highest differentiation percentages (75%) and number of proliferated shootlets (8.50) were recorded with ‘Zaghlool’ cultivar. Concerning PVS2, survival and regrowth gradually increased as exposure time increasing till 30 min and then decreased. Unlike of callus cultures, the survival and regrowth rates of somatic embryos were relatively higher at all PVS2 exposure time. With regard to recovery, the genotype effect took same trend in cryopreservation of callus cultures, since ‘Zaghlool’ was superior in growth parameters in comparison to the other two cultivars. On the other hand, genetic stability of the cryopreserved date palm tissue cultures was assessed using ISSR markers. Among four screened primers, three gave monomorphic bands; all of these banding profiles were similar to those of non-cryopreserved cultures. Generally, the results of ISSR showed high genetic similarity suggesting that cryopreservation using vitrification does not affect genetic stability of date palm tissue cultures. The results of this study demonstrated PVS2 was more effective in cryoprotection of date palm tissue cultures compared with DMSO and cryopreservation by vitrification is a suitable tool for conservation of genetic stable date palm germplasm.

Vitrification of human blastocysts for couples undergoing assisted reproduction: an updated review.

Over the past 40years there has been a worldwide critical change in the field of assisted reproduction technology (ART), leading to the increased application of single blastocyst transfer, which is extremely important to avoid the risks of multiple pregnancy and associated complications for both mother and babies. Indeed, advancements in ART over the last few decades have been obtained thanks to several improvements, including ovarian stimulation, embryo culture conditions and, of course, progress in cryopreservation methods, especially with the application of vitrification. The ability to cryopreserve human embryos has improved significantly with vitrification compared to the initially adopted slow-freezing procedures. Since the introduction of vitrification, it has become the gold standard method to effectively cryopreserve human blastocysts. However, some new protocols are now being explored, such as the short warming procedure and even shorter exposure to the equilibration solution before vitrification, which seem to provide optimal results. Therefore, the main aim of the current narrative review, will be to illustrate the benefit of vitrification as an effective method to cryopreserve the human blastocyst and to illustrate new protocols and variations which in future may increase the performance of vitrification protocols.

Effect of resveratrol supplementation in conventional slow and ultra-rapid freezing media on the quality and fertility of bull sperm

The study investigated the impact of resveratrol (RES) on bull sperm cryopreservation employing conventional slow (CS) and ultra-rapid (UR) freezing methods on sperm quality and in vitro fertility. Twenty-four ejaculates from four bulls were divided into four groups based on the cryopreservation method and RES addition: CS-RES (n = 80), CS-Co (n = 80), UR-RES (n = 24), and UR-Co (n = 24). The CS freezing involved exposing sperm straws with 5% glycerol to liquid nitrogen (LN2) vapors, while UR freezing submerged sperm drops with 100 mM sucrose directly into LN2. Overall, sperm kinematic parameters and integrity of plasma and acrosome membranes significantly decreased (P < 0.001) after cryopreservation. Post-thaw values of motilities (total [TM] and progressive [PSM]), velocities (curvilinear and straight-line), beat cross frequency (BCF), and sperm with intact plasma membrane/intact acrosome (PI-/PNA-) were higher (P < 0.05) with CS-RES and CS-Co treatments compared to UR-RES and UR-Co treatments. CS-RES treatment resulted in greater percentages (P < 0.05) of TM, PSM, PI-/PNA-, and fertility (blastocyst rate) than their control, CS-Co; while UR-RES showed higher BCF values (P < 0.05) than its control, UR-Co. Additionally, UR-RES treatment exhibited lower oxidative stress percentages than UR-Co (P < 0.05). This study presents the following conclusions: (1) the CS freezing resulted in better cryosurvival of bull sperm than UR freezing; (2) the RES supplementation to CS freezing medium improved sperm motility, membrane integrity, and fertility; and (3) despite low cryosurvival sperm and fertility, the RES addition to ultra-rapid freezing medium reduced oxidative stress.

Evaluation of microRNA expression profiles in human sperm frozen using permeable cryoprotectant-free droplet vitrification and conventional methods.

For sperm cryopreservation, the conventional method, which requires glycerol, has been used for a long time. In addition, the permeable cryoprotectant-free vitrification method has been continuously studied. Although the differences of cryopreservation effects between the two methods have being studied, differences in microRNA (miRNA) profiles between them remain unclear. In this study, we investigated the differences in miRNA expression profiles among conventional freezing sperm, droplet vitrification freezing sperm and fresh human sperm. We also analyzed the differences between these methods in terms of differentially expressed miRNAs (DEmiRs) related to early embryonic development and paternal epigenetics. Our results showed no significant differences between the cryopreservation methods in terms of sperm motility ratio, plasma membrane integrity, DNA integrity, mitochondrial membrane potential, acrosome integrity, and ultrastructural damage. However, sperm miRNA-sequencing showed differences between the two methods in terms of the numbers of DEmiRs (28 and 19 with vitrification using a nonpermeable cryoprotectant and the conventional method, respectively) in postthaw and fresh sperm specimens. DEmiRs related to early embryonic development and paternal epigenetics mainly included common DEmiRs between the groups. Our results showed that the differences between conventional freezing and droplet vitrification were minimal in terms of miRNA expression related to embryonic development and epigenetics. Changes in sperm miRNA expression due to freezing are not always detrimental to embryonic development. This study compared differences in miRNA expression profiles before and after cryopreservation between cryopreservation by conventional and vitrification methods. It offers a new perspective to evaluate various methods of sperm cryopreservation.

Abstract PO5-01-09: Exploring cellular heterogeneity of localised breast cancers

Abstract Breast cancer is a heterogeneous disease at multiple levels, ranging from subtype differences between patients (inter-patient heterogeneity) to the diverse composition of malignant cells, heterogeneity of hormone receptor (HR) expression and cellular makeup within single breast cancer samples (intra-tumour heterogeneity). Despite an increase in the number of effective therapies available, many patients will experience an incomplete treatment response and subsequent relapse. These adverse treatment outcomes may be attributed to the often overlooked but critical factor of cellular heterogeneity. In order to gain deeper insights into intra-tumour heterogeneity, we have applied single-cell technologies on a cohort comprising 250 primary, untreated breast cancers. We have optimized methods for tissue cryopreservation, eliminating the need for fresh sample processing, and multiplex tissue profiling. Together, these are cost-efficient processes that allow for improved handling of small tissue sizes, such as biopsies, and reduce batch effect. To ensure accurate and reliable data processing, we have developed a scalable computational workflow that includes benchmarked methods for sample SNP-demultiplexing, doublet detection, high-resolution cell annotation and cellular integration. Finally, we are extending our existing methods1 to study the cellular heterogeneity of breast cancers. Our method, scSubtyper, explores the phenotypic differences between malignant cells within tumours, by comparing each single cell to distinct features associated with different molecular subtypes and assigning each cell to one of these subtypes. Our previous study1 and preliminary results of this project revealed that over 90% of the samples exhibit a mix of malignant cells of different subtypes, and 50% of samples contain cells that have characteristics of all subtypes, demonstrating cellular heterogeneity exists not only exists between malignant cells, but also within malignant cells of a tumour. Our second approach, known as ecotyping, assesses patterns of cell type frequencies across samples and groups them based on similarity of these co-occurences. Our preliminary results have revealed the existence of 5 ecotypes that lack significant associations with samples clinical subtypes. Applying the same approach exclusively within the HR-positive samples identified 4 ecotypes characterized by distinct abundances of immune and stromal cells. This analysis revealed that ecotypes are not a simple surrogate for clinical and molecular subtypes, but their presence could drive different response to treatment. Together, our high-throughput tissue processing and computational approaches to studying intra-tumour heterogeneity are now being applied to our large, well annotated, clinical cohort. Supported by the preliminary results, we hypothesize that this study will play a vital role in optimizing breast cancer patient stratification to improve treatment management and outcome. 1. Wu, Sunny Z., et al. "A single-cell and spatially resolved atlas of human breast cancers." Nature genetics 53.9 (2021): 1334-1347. Citation Format: Beata Kiedik, Daniel Roden, Kate Harvey, Ghamdan Al-Eryani, Sunny Wu, Mun Hui, Sandra O'Toole, Elgene Lim, Charles Perou, Alex Swarbrick. Exploring cellular heterogeneity of localised breast cancers [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-01-09.

Kriopreservasi Sperma Manusia: Systematic Review

This study aims to identify the types of methods and effects of human sperm cryopreservation. This review was written using a literature review method from international databases such as Scopus and PubMed. The results of the research show that by choosing a cryopreservation method, choosing a cryoprotectant agent such as TAT-PRDX2, canthaxanthin, or Holotheria parva coleomic extract, can increase sperm motility, viability and integrity after cryopreservation, the right cryoprotectant agent can defend cells from oxidative stress and fragmentation DNA. Keywords: Cryopreservation, Human, Sperm

Ultrastructural examination of cryodamage in Paracentrotus lividus eggs during cryopreservation

This study examinates the challenges of cryopreserving sea urchin (Paracentrotus lividus) eggs, a task hindered by factors like low membrane permeability and high sensitivity to cryoprotective agents (CPAs). While successful cryopreservation has been achieved for some marine invertebrates, eggs remain problematic due to their unique characteristics. The study explores the impact of various CPAs and cryopreservation techniques on sea urchin eggs, employing scanning and transmission electron microscopy to analyze cellular damage. The findings reveal that exposure to low CPA concentrations (0.5 M) did not induce significant damage to eggs. However, high concentrations (3 M) proved highly detrimental. Every cryopreservation approach investigated in this study resulted in irreversible damage to the sea urchin eggs, rendering them nonviable for future use. The research sheds light on the importance of understanding the structural alterations induced by CPAs and cryopreservation methods. This knowledge is essential for refining cryopreservation methods, potentially paving the way for successful preservation of these challenging cells.

Improving vitrification efficiency of human in vitro matured oocytes by the addition of LEA proteins.

Can the addition of late embryogenesis-abundant (LEA) proteins as a cryoprotective agent during the vitrification cryopreservation of in vitro matured oocytes enhance their developmental potential after fertilization? LEA proteins improve the developmental potential of human in vitro matured oocytes following cryopreservation, mostly by downregulating FOS genes, reducing oxidative stress, and inhibiting the formation of ice crystals. Various factors in the vitrification process, including cryoprotectant toxicity, osmotic stress, and ice crystal formation during rewarming, can cause fatal damage to oocytes, thereby affecting the oocytes developmental potential and subsequent clinical outcomes. Recent studies have shown that LEA proteins possess high hydrophilicity and inherent stress tolerance, and can reduce low-temperature damage, although the molecular mechanism it exerts protective effects is still unclear. Two LEA proteins extracted and purified by us were added to solutions for vitrification-warming of oocytes at concentrations of 10, 100, and 200 µg/mL, to determine the optimal protective concentration for each protein. Individual oocyte samples were collected for transcriptomic analysis, with each group consisting of three sample replicates. Immature oocytes were collected from patients who were undergoing combined in vitro fertilization (IVF) treatment and who had met the designated inclusion and exclusion criteria. These oocytes underwent in vitro maturation (IVM) culture for experimental research. A fluorescence microscope was used to detect the levels of mitochondrial membrane potential (MMP), reactive oxygen species (ROS), and calcium in the mitochondria of vitrified-warmed human oocytes treated with different concentrations of LEA proteins, and the protective effect of the protein on mitochondrial function was assessed. The levels of intracellular ice recrystallization inhibition (IRI) in human oocytes after vitrification-warming were characterized by the cryomicroscope, to determine the LEA proteins inhibitory effect on recrystallization. By analyzing transcriptome sequencing data to investigate the potential mechanism through which LEA proteins exert their cryoprotective effects. The secondary structures of AfrLEA2 and AfrLEA3m proteins were shown to consist of a large number of α-helices and the proteins were shown to be highly hydrophilic, in agreement with previous reports. Confocal microscopy results showed that the immunofluorescence of AfrLEA2-FITC and AfrLEA3m-FITC-labeled proteins appeared to be extracellular and did not penetrate the cell membrane compared with the fluorescein isothiocyanate (FITC) control group, indicating that both AfrLEA2 and AfrLEA3m proteins were extracellular. The group treated with 100 µg/mL AfrLEA2 or AfrLEA3mprotein had more uniform cytoplasmic particles and fewer vacuoles compared to the 10 and 200 µg/mL groups and were closest to the fresh group. In the 100 µg/mL groups, MMPs were significantly higher while ROS and calcium levels were significantly lower than those in the control group and were closer to the levels observed in fresh oocytes. Meanwhile, 100 µg/mL of AfrLEA2 or AfrLEA3m protein caused smaller ice crystal formation in the IRI assay compared to the control group treated with dimethylsulphoxide (DMSO) and ethylene glycol (EG); thus, the recrystallization inhibition was superior to that with the conventional cryoprotectants DMSO and EG. Further results revealed that the proteins improved the developmental potential of human oocytes following cryopreservation, likely by downregulating FOS genes and reducing oxidative stress. The in vitro-matured metaphase II (IVM-MII) oocytes used in the study, due to ethical constraints, may not accurately reflect the condition of MII oocytes in general. The AfrLEA2 and AfrLEA3m proteins are recombinant proteins and their synthetic stability needs to be further explored. LEA proteins, as a non-toxic and effective cryoprotectant, can reduce the cryoinjury of oocytes during cryopreservation. It provides a new promising method for cryopreservation of various cell types. This work was supported by the National Key Research and Development Program of China (2022YFC2703000) and the National Natural Science Foundation of China (52206064). The authors declare no competing interest. N/A.

Ultra-Rapid Freezing and Rapid Freezing Methods in Clinical ICSI Program: Effects on Sperm Biological Characteristics, DNA Methylation Stability, DNA Methyltransferase Activity, and Embryo Morphokinetics

The purpose of this study was to evaluate the differences in sperm function and embryo morphokinetics following sperm cryopreservation by ultra-rapid freezing or rapid freezing methods compared to fresh spermatozoa. Thirty samples of normozoospermia were divided equally into fresh, ultra-rapid freezing, and rapid freezing groups. In the rapid freezing, sperm suspension was placed horizontally on nitrogen vapor. In the ultra-rapid freezing, sperm suspension with a straw-in-straw system was directly immersed in liquid nitrogen. Sperm function was assessed in terms of motility, morphology, viability, mitochondrial membrane potential, sperm DNA fragmentation, and acrosome reaction status. Also, the effects of two cryopreservation methods were assessed on global DNA methylation and DNA methyltransferase activity. Moreover, 730 embryos in three groups were cultured using time-lapse imaging until day 6 for embryo morphokinetics. Progressive motility (38.80 ± 4.21 vs. 34.86 ± 4.19; p &lt;0.001) and viability (64.30 ± 6.24 vs. 58.10 ± 8.69; p&lt;0.01) in ultra-rapid freezing were significantly higher than rapid group. DNA fragmentation and acrosome reaction were significantly increased in both cryopreserved groups (p &lt;0.001). However, DNA fragmentation (16.30 ± 1.14 vs. 14.33 ± 2.94; p&lt;0.01) was significantly higher in the rapid than the ultra-rapid freezing group. No significant differences were noted in global DNA methylation (p&gt;0.05) and DNA methyltransferases activity (p&gt;0.05) in fresh compared to cryopreservation groups. The kinetic times, including tPB2, tPNa, tPNf, t2, t3, t4, t5, t6, t7, t8, and tM, showed a significant delay in cell divisions in both cryopreservation groups. Furthermore, tPNa, tPNf, and t8 occurred with a significantly higher delay in embryos fertilized by sperm from the rapid freezing compared to the ultra-rapid freezing group. In addition, blastocysts formation was similar in both cryopreservation groups. Ultra-rapid freezing preserved the sperm biological integrity and lead to better embryo morphokinetics compared to the rapid freezing method. However, both methods of sperm cryopreservation were epigenetically safe.

The outcome of tissue cryopreservation on the cellular, molecular and epigenetic characteristics of endometrial tissue and stromal cells

Research questionWhat impact does the cryopreservation of endometrial tissue have on cell characteristics and molecular and epigenetic profile changes in endometrial tissue and stromal cells? DesignCellular properties, such as proliferation efficiency, surface marker expression and the differentiation potency of endometrial stromal cells (ESC) isolated from fresh (Native) and cryopreserved (Cryo) tissue were compared. Moreover, changes in the expression of genes associated with pluripotency, endometrial function and epigenetic regulation and microRNA (miRNA, miR) were assessed, as were levels of DNA methylation and histone modifications. ResultsNative and Cryo cells exhibit very similar profiles including cell surface marker expression, differentiation potency and histone modifications, except for a decrease in proliferative potency and cell surface marker SUSD2 expression in Cryo cells. It was demonstrated that endometrial tissue cryopreservation led to an up-regulated expression of genes associated with pluripotency (NANOG, OCT4 [also known as POU5F1]). This confirms that despite being recovered from cryopreserved differentiated tissue, cells retained their stemness properties. In addition, alterations in DNA methyltransferase (DNMT1, DNMT3A, DNMT3B) gene regulation were observed, along with a down-regulation of hsa-miR145-5p in Cryo ESC. ConclusionsThese findings contribute to a deeper understanding of the complex effects of endometrial tissue cryopreservation, providing insights for both medical and basic research applications. Since different tissues possess unique characteristics, it is essential to select the most suitable cryopreservation method for each tissue individually. Furthermore, the study findings indicate the potential utility of slow-cooling cryopreservation for both normal and pathological endometrial tissue samples, with the purpose of isolating stromal cell cultures.