Freezing Protocol Research Articles

Cryopreservation of Noble Scallop Mimachlamys nobilis sperm: Optimization study of cooling rate and thawing temperature

In marine biology reproduction research, the cryopreservation of sperm is a crucial area, especially for economically valuable species. Effective freezing techniques help maintain genetic diversity and support sustainable development in aquaculture. This study examined the effects of freezing protocols and thawing methods on the cryopreservation of Mimachlamys nobilis sperm. Experimental results indicated that a cooling rate of 5 °C/min and an equilibration time of 5 minutes were the most suitable conditions for maintaining the structural and functional integrity of the sperm. Comparisons of different thawing temperatures showed that 38 °C provided the best results, minimizing ice recrystallization and cellular damage. Additionally, it was found that a 10 % concentration of DMSO effectively balanced protection and cytotoxicity, maintaining sperm viability and motility. Transmission electron microscopy further revealed the impact of freezing conditions on sperm, including damage to the plasma membrane, dissolution, blurring and swelling of the acrosome and mitochondria, and cristae fragmentation and loss. This study emphasizes the importance of optimizing freezing and thawing protocols and accurately controlling cryoprotectant concentration, providing key insights for the effective long-term preservation of M.nobilis sperm. This is significant for the conservation of marine organisms and aquaculture.

Biopsy vitrification: New tool for endometrial tissue cryopreservation for research applications

Patient-derived endometrial biopsies serve as a crucial source for molecular studies, highlighting the necessity for tissue cryopreservation methods that preserve cell viability and tissue morphology with minimal to no impact. The passive slow freezing (PSF) protocol has demonstrated efficacy for cryopreserving endometrial biopsies, allowing for the subsequent isolation of viable epithelial and stromal cells. Vitrification (VT) enables the avoidance of ice crystal formation and could therefore potentially prevent mechanical injury to tissues. In this study, PSF and VT techniques were applied to endometrial biopsies, and the effects of cryopreservation on tissue samples were evaluated using traditional histology. In addition, transmission electron microscopy (TEM), gene expression profiling analyses, the viability of endometrial cells, and the ability to form epithelial organoids were compared between PSF and VT endometrial biopsies in a subset of samples. The histology and TEM studies demonstrated relatively mild cellular and sub-cellular damage in both cryopreservation protocols which did not affect tissue functionality and the formation of the organoids. Additionally, the cryopreservation methodology did not affect the gene expression profile of the 68 endometrial-receptivity associated genes studied. In conclusion, our findings indicate that although current cryopreservation methodologies need further improvements, they still allow us to achieve acceptable cell viability and functionality, showing promising potential for facilitating the utilization of cryopreserved endometrial tissue samples for research purposes.

Optimized partial freezing protocol enables 10-day storage of rat livers

Preserving organs at subzero temperatures with halted metabolic activity holds the potential to prolong preservation and expand the donor organ pool for transplant. Our group recently introduced partial freezing, a novel approach in high-subzero storage at -15 °C, enabling 5-day storage of rodent livers through precise control over ice nucleation and unfrozen fraction. However, increased vascular resistance and tissue edema suggested a need for improvements to extend viable preservation. Here, we describe an optimized partial freezing protocol with key optimizations, including an increased concentration of polyethylene glycol (PEG) to enhance membrane stability while minimizing shear stress during cryoprotectant unloading with an acclimation period and a maintained osmotic balance through an increase in bovine serum albumin (BSA). These approaches ensured the viability during preservation and recovery processes, promoting liver function and ensuring optimal preservation. This was evidenced by increased oxygen consumption, decreased vascular resistance, and edema. Ultimately, we show that using the optimized protocol, livers can be stored for 10 days with comparable vascular resistance and lactate levels to 5 days, outperforming the viability of time-matched static cold stored (SCS) livers as the current gold standard. This study represents a significant advancement in expanding organ availability through prolonged preservation, thereby revolutionizing transplant medicine.

Production and Evaluation of Frozen Semen of Sahiwal Bull Using Two Different Diluters and Protocols

The experiment was designed to evaluate the effects of diluters and freezing protocols on frozen semen production of Sahiwal breeding bulls. Imported 100% Sahiwal breeding bulls were then reared in the American dairy limited (ADL). Semen samples are processed and frozen once a week in the ADL. Spermatozoa with various motility patterns were collected; these included post-thawing, progressive, fast, slow, circular, and local. Computer Assisted Semen Analyzer (CASA) was used to measure the straight-line velocity (VSL), curvilinear velocity (VCL) and linearity (LIN) parameters. Two diluters (tris egg-yolk and tris egg-yolk aqua) and freezing protocols (slow freezing and fast freezing) were used for frozen semen production of these bulls. The significantly highest (P<0.05) number of post-thawing total, progressive and progressive fast motility (78.42±1.88, 73.50±1.90 and 59.68±2.10%), VCL (115.31±3.82µm/s) and lowest slow, circular and local motility (13.44±1.40, 0.57±0.08 and 4.92±0.41%), LIN (0.39±0.01%) was found in diluter 1 and freezing protocol 2. On the other hand, highest number of circular motility (0.77±0.18%) and VSL (44.83±1.17µm/s) was found in diluter 1 and freezing protocol 1 whereas highest number of slow and local motility (18.84±1.23 and 7.46±0.57%) and LIN (0.61±0.20%) and lowest progressive fast motility (48.73±2.28%), VCL (98.90±3.42µm/s), VSL (39.76±1.33µm/s) was found in diluter 2 freezing protocol 2 and also lowest post-thawing and progressive motility (73.44±1.94 and 66.51±2.02%) was observed in diluter 2 freezing protocol 1. The study reveals that diluter 1(one step egg yolk based) and freezing protocol 1 (5 steps) and 2 (2 steps) seems to be an efficient method for frozen semen production.

Liposome-based Freezing Medium Improves the Outcome of Mouse Prepubertal Testicular Tissue Cryopreservation

AbstractCryopreservation of testicular tissue holds an important role in the field of fertility preservation, particularly for prepubertal boys diagnosed with cancer. However, prepubertal testicular tissue cryopreservation is still considered to be in the experimental stage necessitating the refinement of cryopreservation protocol. Considering the fact that loss of membrane lipids is the primary cause of freeze–thaw-induced loss of testicular cell functions, in this study, we explored the beneficial properties of exogenous supplementation of membrane lipids in the form of liposomes in enhancing the cryosurvival of prepubertal testicular tissue. The freezing medium supplemented with liposomes (prepared from soy lecithin, phosphatidylethanolamine, phosphatidylserine, and cholesterol) was used for the experiments. Prepubertal testicular tissues from Swiss albino mice were cryopreserved in a liposome-containing freezing medium (LFM) composed of 0.25 mg/mL liposomes, 5% DMSO, and 30% FCS in the DMEM/F12 medium using a slow freezing protocol. The tissues were thawed and assessed for various testicular cell functions. Freezing in LFM mitigated the loss of viability, decreased malondialdehyde level (p < 0.05), and reduced apoptosis (p < 0.05) in the testicular cells compared to the testicular tissue cryopreserved in the control freezing medium (CFM). Further, DMSO (5%) appears to be the ideal penetrating cryoprotectant for prepubertal testicular tissue cryopreservation with liposome-based freezing medium. Similar enhancement in cryosurvival of cells was observed in adult human testicular tissue frozen with LFM. These findings highlight the translational value of liposome-based freezing medium in the cryopreservation of testicular tissue of prepubertal boys undergoing chemotherapy.

The characteristics of frozen-thawed rooster sperm using various intracellular cryoprotectants

Cryopreservation of rooster semen is essential for conserving genetic resources, genetic improvement, and increasing productivity. However, the nature of avian sperm presents a global issue in ensuring superior frozen semen for artificial insemination. Thus, the present study aimed to evaluate the impact of using dimethylacetamide (DMA), dimethyl sulfoxide (DMSO), and ethylene glycol (EG) as cryoprotectants on post-thawed sperm motility, quality, antioxidant indicators, and fertilizing capacity. Twice a week, fresh semen ejaculates were collected from 15 adult roosters and immediately evaluated to constitute a pool from clean and qualified samples. The pooled semen was further diluted at a ratio of 1:2 (v/v) with an extender and then subjected to a freezing protocol in a liquid nitrogen vapor after adding a cryoprotectant solution containing 6% of either DMA, DMSO, or EG, respectively. After thawing, characteristics of sperm motion, quality, antioxidants, and fertilizing ability were evaluated and compared to fresh and cooled semen as controls. The results demonstrated that semen cooling negatively affected some parameters of sperm motility, quality, antioxidant biomarkers, and fertility. In comparison to the DMSO and EG groups, employing DMA considerably (P < 0.05) raised the percentages of sperm progressive motility, viability, plasma membrane intactness, and DNA integrity. The DMA group showed a significant increase in the catalase and glutathione reduced antioxidant enzyme activity and a reduction in nitric oxide and lipid peroxidation. After artificial insemination, the DMA and DMSO groups exhibited considerably (P < 0.05) better rates of hatchability and fertility than the EG group. It is concluded that freezing extenders containing 6% DMA is better than DMSO or EG to improve the post thaw semen quality and fertility in chickens.

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

O-312 Home semen freezing for fertility patients at the time of global travel restrictions due to COVID-19 pandemic

Abstract Study question Can an alternative process for freezing semen be utilized for patients in need of fertility care during the coronavirus disease 2019 (COVID-19) pandemic travel restrictions? Summary answer At-home sperm freezing was developed during COVID-19 to offer fertility care for travel restricted patients, which provided similar IVF outcomes compared to laboratory frozen sperm. What is known already Cases of COVID-19 were first detected in December 2019 spreading rapidly across the world leading to declaration of a pandemic on March 11, 2020 and resulting in international border closures and global travel restrictions lasting into 2023. Sperm freezing by trained laboratory personnel is well established and safe clinical practice. While home semen collection kits existed to extend sperm survival for hours allowing sufficient time for the sample to reach facility to be frozen in the laboratory, there were no home semen freezing methods for longer time constraints available for our patients. Study design, size, duration A specialized home semen freezing process has been developed and validated in-house on research samples. Retrospective analysis was performed to assess home sperm freezing efficiency in patients undergoing fertility treatment. IVF outcomes were compared between home sperm freezing to the samples frozen in our laboratory (control) from 2020 to 2023. Participants/materials, setting, methods In total 508 semen samples frozen at home have been received at the clinic, with 495 (98%) of samples arriving within set time of 18 days in optimal frozen condition. Delivery of 12 samples were delayed causing sample thawing during transportation that required a new at home freezing. Only IVF with ova donors were included to compare outcomes of 448 IVF with semen frozen at home to 421 IVF with semen frozen in the laboratory. Main results and the role of chance Only 17 (3%) of home semen samples arrived from patients within Canada, while 491 (97%) were from patients of 27 different countries of several regions: Europe 346 (68%), Australia 73 (15%), Middle East 36 (7%), USA 26 (5%), Asia 7 (1.5%), and Latin America 3 (0.5%). Men from the study group were similar to control in terms of age (36 ± 5.2 versus 36.8 ± 5.8). Post thaw test was performed on all home freezing samples as a quality control to ensure that sufficient sample was available for IVF: mean sperm concentration was 25.3 M/ml ± 14.8 and motility of 21% ± 10. IVF outcomes between samples frozen at home or in the lab were compared; fertilization (80.3% ± 14.6 v. 79.6 ± 19.3), blastulation (66.5% ± 20.7 v. 66.6% ± 19.3%), clinical pregnancy (39.1% v. 38.8%), miscarriage (14.6% v.6.7%) or life birth (30.9% v. 31.6%), respectively, were not statistically different. PGT-A was performed on 7224 blastocysts with results controlled by the age of ova donors and were not statistically different for embryos derived from sperm frozen at home and in the laboratory with euploidy (65.3% v. 64.6%), mosaic (14.8% v.13.8%) and aneuploidy (19.9% v. 21.6%), respectively. Limitations, reasons for caution Pre-freezing semen analysis was only available for laboratory frozen samples. Post-thaw results were not compared between home and laboratory frozen sample as different freezing protocols with two dilution factor (1:1 v. 3:1) and different sample volume per frozen unit (0.1-1.2 ml in vial v. 0.5 ml in straw) were used. Wider implications of the findings In cases where it is logistically difficult or impossible for sperm providers to travel to the clinic for IVF treatment, home semen freezing performed by the patient is a viable and safe method. Trial registration number not applicable

P-161 Mechanical phenotyping of mammalian oocytes using Atomic Force Microscopy (AFM) to evaluate their developmental potential

Abstract Study question Can we measure the mechanical properties of mammalian oocytes with AFM and use them as a biomarker to identify oocytes with the best development potential? Summary answer We characterized mammalian oocytes’ mechanical properties using AFM, correlated them with oocyte cortex organization, and explored these parameters in contexts critical for oocyte quality. What is known already Oocyte production is crucial for reproduction but prone to errors, yielding a significant number of poor-quality oocytes detrimental to fertility and offspring development. Indeed, oocyte quality determines embryo health post-fertilization. Current selection methods for oocytes rely on subjective morphological analyses. Remarkably, human and mouse oocyte quality correlates with mechanical properties, particularly cortical tension (CT). CT defects in oocytes are rather frequent and impair early embryonic development after fertilization. However, they do not induce any visible oocyte morphological alteration. Mechanical properties could, therefore, be used as a biomarker of oocyte quality. Study design, size, duration We designed an AFM protocol for the measurement and analysis of mammalian oocytes’ mechanical properties, including a new elasto-capillary model describing oocyte mechanics. Concomitantly, we analyzed oocyte cortex organization using spinning disk microscopy in live. We measured mouse oocytes at various stages of meiotic maturation (prophaseI, meiosisI, meiosisII), mutant mouse oocytes with known cortical tension defects, oocytes coming from young and aged mice, and human oocytes at various stages of meiotic maturation (prophaseI, meiosisI, meiosisII). Participants/materials, setting, methods Mouse oocytes were collected from 11-week-old and 44-56-week-old OF1 female mice. Human oocytes were collected from patients who underwent ovarian stimulation for Assisted Reproduction Technologies (ART) as part of their protocol. Only oocytes that were in prophaseI or meiosisI after ovarian punction and thus not suitable for the ART procedure were used in this study. All patients gave informed consent (agreement CNIL number 22_5725). Main results and the role of chance Our AFM approach allows measuring various mechanical parameters, including oocyte cortical tension, elasticity, and capillary indentation rate, quantifying whether the oocyte behaves predominantly as a droplet with surface tension or as an elastic material. We find that these parameters correlate to specific oocyte cortex organization, showing that a thin actin cortex enriched in myosin II is associated with high tension, high elastic modulus, and low capillary indentation rate. In contrast, a thick actin cortex with few myosin II is associated with low cortical tension, low elasticity, and high capillary indentation rate. We go further and show that these parameters can predict cortex organization. Indeed, oocytes from aged mice display altered mechanical parameters, reflecting specific changes in their cortex organization. Finally, we transferred our approach to human oocytes. We show that the mechanical parameters of mouse and human oocytes are in the same range. Still, in humans, they evolve differently from those of mice during meiotic maturation, again reflecting specific changes in the organization of their cortex. Thus, mechanical parameters correlate with oocytes quality, and can predict cortex organization in mouse and human. Limitations, reasons for caution AFM is hardly usable for clinical application, we are thus working in parallel on developing non-invasive devices to perform high-throughput measurements of oocyte mechanical properties in a non-invasive manner, to serve as a mechanical biomarker to identify oocytes with the best developmental potential for ART. Wider implications of the findings Ultimately, this project could improve ART procedures by increasing their success rate while decreasing treatment cycles and risks for the patient and newborn, and optimize protocols for oocyte freezing for fertility preservation. Trial registration number not applicable

Optimized Partial Freezing Protocol Enables 10-Day Storage of Rat Livers.

Preserving organs at subzero temperatures with halted metabolic activity holds the potential to prolong preservation and expand the donor organ pool for transplant. Our group recently introduced partial freezing, a novel approach in high-subzero storage at -15°C, enabling 5 days storage of rodent livers through precise control over ice nucleation and unfrozen fraction. However, increased vascular resistance and tissue edema suggested a need for improvements to extend viable preservation. Here, we describe an optimized partial freezing protocol with key optimizations including increased concentration of propylene glycol to reduce ice recrystallization and maintained osmotic balance through an increase in bovine serum albumin, all while minimizing sheer stress during cryoprotectant unloading with an acclimation period. These approaches ensured the viability during preservation and recovery processes, promoting liver function and ensuring optimal preservation. This was evidenced by increased oxygen consumption, decreased vascular resistance and edema. Ultimately, we show that using the optimized protocol, livers can be stored for 10 days with comparable vascular resistance and lactate levels to 5 days, outperforming the viability of time-matched cold stored livers as the current gold standard. This study represents a significant advancement in expanding organ availability through prolonged preservation and thereby revolutionizing transplant medicine.

Effect of interleukin 6 (IL-6) on sperm quality, kinematic parameters, acrosome integrity, apoptosis, ultrastructure, and molecular docking in cryopreserved ram spermatozoa

Sperm cryopreservation can lead to subfertility due to potential damage to sperm DNA, membranes, and overall motility caused by the freeze-thaw process. Interleukin-6 (IL-6) is a versatile cytokine with various roles in reproductive processes. However, the impacts of IL-6 supplementation on cryopreserved ram sperm have not been thoroughly investigated. Therefore, this study aims to assess the influence of IL-6 on the sperm quality of cryopreserved ram sperm. Ram semen was collected, pooled, and extended with tris-citrate soybean lecithin extender supplemented with 0, 50, 100, and 200 ng/mL of IL-6. The samples experienced a standard freezing protocol, and sperm quality, kinematic parameters, ultrastructure, and molecular docking of cryopreserved ram spermatozoa were evaluated. The results showed that sperm kinematics, viability, progressive motility, and membrane integrity were significantly enhanced by the addition of 100 or 200 ng of IL-6/mL (p < 0.05). Semen supplemented with 100 or 200 ng/mL of IL-6 also exhibited higher percentages of sperm kinematics, including DAP, DCL, DSL, VSL, VAP, VCL, and ALH, compared to other groups (p < 0.05). IL-6 supplementation enhanced acrosome integrity, and reduced caspase-3 activity in post-thawed ram spermatozoa (p < 0.05) compared to untreated group. Supplementation with IL-6 (200 ng/mL) significantly decreased oxidative biomarkers (NO, MDA, and H2O2) (p < 0.001) and improved total antioxidant capacity (p < 0.05). The percentage of sperm damage (tail, head, and midpiece) was significantly reduced by IL-6 supplementation (p < 0.05). Electron micrographs showed that supplementation with 100 or 200 ng/mL IL-6 protected acrosome stability, plasma membrane integrity, and sustained the ultrastructure integrity of cryopreserved ram spermatozoa. The docking exploration indicates a higher binding affinity with sperm function biomarkers, including caspase 3, BCL2, and PSMA6, with binding energies of − 52.30 kcal/mol, − 56.04 kcal/mol, and − 57.06 kcal/mol, respectively. In conclusion, the addition of IL-6 to the freezing extender can enhance the post-thaw quality of cryopreserved ram spermatozoa.

Long-term follow-up of pulmonary vein cryoballoon isolation using a double 120s vs single 240s standard freeze protocol

Abstract Background Recently, pulmonary vein isolation (PVI) using a double 120s freeze protocol has demonstrated to be non-inferior to a single 240s protocol despite a potentially increased procedural safety. Main advantages of a double 120s protocol are a reduction of phrenic nerve injury and an increase of the mean minimal esophageal temperature during the procedure. Methods We performed a long-term follow-up comparison between groups of patients with history of symptomatic atrial fibrillation undergoing PVI with second generation cryoballoon (CB) between 2019 and 2020. Our usual energy dosing protocol was modified including 2 times 120 seconds applications instead of a single 240 seconds application. Of A total of 160 consecutive patients, 80 were treated with double 120s (CB120) and 80 with single 240s (CB240) freeze protocol. 107 patients were, successfully, followed-up. The primary end-point was freedom from atrial tachyarrhythmia. Results Before PVI a total of 11/52 patients (21%) had persistent AF and 41/52 (79%) had paroxysmal AF in the CB120 group; a total of 20/55 patients (36%) had persistent AF and 35/55 (64%) had paroxysmal AF in the CB240 group. On a mean follow-up time of 17 months, freedom from atrial tachyarrhythmia was 63% in those patients undergoing CB240 and 74% with CB120 (log-rank P=0.255). Conclusions The double 120s freeze protocol is not-inferior as compared to the standard 240s in terms of freedom from recurrences on a long term follow-up, but with a more favorable procedural safety profile.

Sperm melatonin receptors, seminal plasma melatonin and semen freezability in goats

Goat bucks are seasonal breeders that show variation in sperm quality, endogenous melatonin (MLT), and presumably in the expression of MLT receptors on the sperm throughout the year, which may modify sperm freezability. The aim of this study was to determine whether sperm freezability is associated with (i) endogenous melatonin levels in seminal plasma and (ii) the expression of sperm plasma membrane melatonin receptors (MT1, MT2). To evaluate this, spermatozoa from seven Saanen goat bucks were cryopreserved throughout the year in Mexico using a standard freezing protocol. Seminal plasma MLT concentrations were determined by ELISA and the expression and localization of MT1 and MT2 were detected by immunocytochemistry and confirmed by western blotting. The recovery rate of progressive motility after thawing was higher in spring than autumn and winter; in contrast, the F pattern (CTC assay) was higher in winter than in the other seasons. A proportional increase in the AR pattern (CTC assay) was smaller in winter than in the other seasons and the proportion of sperm showing high plasma membrane fluidity was higher in spring than in summer and autumn. The seminal plasma MLT concentrations showed no significant interseasonal differences. The MT1 receptor was immunolocalised at the apical region of the sperm head, while MT2 was mainly localised in the neck. The relative expression of MLT receptors showed significant differences between summer and winter for all bands, except at 75 kDa of MT2. In conclusion, there was an association between the relative expression of MT1 and MT2 receptors throughout the year and sperm freezability in goat bucks in México. Post-thaw sperm quality is enhanced in semen samples collected during breeding season.

Characterization of Freezing Processes in Drug Substance Bottles by Ice Core Sampling

Freezing of biological drug substance (DS) is a critical unit operation that may impact product quality, potentially leading to protein aggregation and sub-visible particle formation. Cryo-concentration has been identified as a critical parameter to impact protein stability during freezing and should therefore be minimized. The macroscopic cryo-concentration, in the following only referred to as cryo-concentration, is majorly influenced by the freezing rate, which is in turn impacted by product independent process parameters such as the DS container, its size and fill level, and the freezing equipment. (At-scale) process characterization studies are crucial to understand and optimize freezing processes. However, evaluating cryo-concentration requires sampling of the frozen bulk, which is typically performed by cutting the ice block into pieces for subsequent analysis. Also, the large amount of product requirement for these studies is a major limitation. In this study, we report the development of a simple methodology for experimental characterization of frozen DS in bottles at relevant scale using a surrogate solution. The novel ice core sampling technique identifies the axial ice core in the center to be indicative for cryo-concentration, which was measured by osmolality, and concentrations of histidine and polysorbate 80 (PS80), whereas osmolality revealed to be a sensitive read-out. Finally, we exemplify the suitability of the method to study cryo-concentration in DS bottles by comparing cryo-concentrations from different freezing protocols (-80°C vs -40°C). Prolonged stress times during freezing correlated to a higher extent of cryo-concentration quantified by osmolality in the axial center of a 2 L DS bottle.Graphical

First report on successful delivery after retransplantation of vitrified, rapid warmed ovarian tissue in Europe

Research questionCryopreservation of ovarian tissue is one feasible option to preserve female fertility prior to cancer treatment. The slow freezing protocol represents the current standard approach, while vitrification has been suggested as a promising alternative. This paper reports the follow-up and first successful delivery after retransplantation of vitrified, rapid warmed ovarian tissue in Europe. DesignAfter the patient received a diagnosis of breast cancer, ovarian tissue was removed laparoscopically and sent via overnight transportation to University Hospital Bonn for vitrification on site. The patient was treated with chemotherapy, leading to ovarian failure. After 2 years, retransplantation of the vitrified, rapid warmed tissue was conducted on site. ResultsTwo months after grafting, the patient reported regular menstrual cycles. After 1 further month a clinical pregnancy occurred, which ended in a spontaneous abortion at the 8th week of pregnancy. Six months after grafting, another naturally conceived pregnancy was determined, resulting in the birth of a healthy boy 14 months after retransplantation of the ovarian tissue. ConclusionsComplementing the successful deliveries reported by the groups of Suzuki (Japan) and Silber (USA) regarding vitrified tissue, the current results confirm the high potential of this cryopreservation method in a clinical routine setting as an alternative approach to the widespread slow freezing method.

Effects of different cryopreservation parameters on the differences between trypan blue and fluorescent SYTO 13/GelRed assays

Post-thaw cell viability assessment is very important in cryopreservation because it is the main assessment method used to optimize cryopreservation protocols for each cell type; hence, having standardized accurate, quick, and reliable assays for post-thaw cell viability measurements is of utmost importance. The trypan blue exclusion assay and nucleic-acid-binding fluorescence-based assays are two different methods for cell viability assessment. Both assays identify cells with damaged membranes by whether they let a compound enter the cell. In this study, these two assays are compared in the context of cryopreservation and the impacts of important cryopreservation parameters on the differences in measurements are investigated. H9c2 myoblasts were cryopreserved with different freezing protocols. Cell membrane integrities were measured immediately after thaw as well as after cryoprotectant removal by a hemocytometer-based trypan blue dye exclusion assay and a dual fluorometric SYTO 13/GelRed assay; and the results were compared. This study quantifies how (i) the absence or presence of different cryoprotectants, (ii) different cell–cryoprotectant incubation conditions, and (iii) the presence or removal of cryoprotectants after thaw affect the differences between these two viability assays.

Evaluation of a New Cystoscopic Cryocatheter and Method for the In Situ Destruction of Bladder Cancer: Preliminary In Vivo Study.

Purpose: To assess the ability to deliver full-thickness bladder wall cryoablation through a cystoscopic approach using a new closed-loop 6F cryocatheter and thermal dose-controlled protocol. Materials and Methods: Evaluations were conducted using a chronic porcine model wherein 10 lesions/animal were created throughout the bladder (bladder wall, trigone region, ureteral orifice, and distal ureter). A 6F cryocatheter was passed through the working channel of a flexible cystoscope. Single 1- and 1.5-minute freeze protocols in a saline environment were evaluated and resultant lesion size was determined. A laparoscopic approach was utilized to observe the transmural extension of the ice propagation. Results: Studies demonstrated the generation of transmural lesions characterized by full-thickness histologic necrosis after freezing for 1.5 minutes regardless of tissue thickness (range 2-12 mm). All animals were found to have good overall health (maintained weight, appetite, mobility, and energy levels) throughout the recovery period. No significant deviations were noted in complete blood count and serum chemistry bloodwork except for elevated creatine kinase levels. Importantly, no fistulas or perforations were noted. Conclusions: The cryocatheter was able to rapidly and effectively freeze the bladder wall through a cystoscopic approach. The results showed the ability to consistently ablate an ∼1 cm diameter and up to 1.2 cm deep using a single 1.5-minute freeze protocol. Analysis of the ablation efficacy revealed ∼80% destruction within the frozen mass. Although further testing and refinement are needed, these studies demonstrate the potential of this new approach to provide a next-generation strategy for the treatment of bladder cancer.

The Assessment of a Novel Endoscopic Ultrasound-Compatible Cryocatheter to Ablate Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease that may be treated utilizing thermal therapies. Cryoablation is an effective, minimally invasive therapy that has been utilized for the treatment of various cancers, offering patients a quicker recovery and reduced side effects. Cryoablation has been utilized on a limited basis for the treatment of PDAC. With the recent reports on the success of cryoablation, there is a growing interest in the use of cryoablation as a standalone, minimally invasive procedure to treat PDAC. While offering a promising path, the application of cryoablation to PDAC is limited by current technologies. As such, there is a need for the development of new devices to support advanced treatment strategies for PDAC. To this end, this study investigated the performance of a new endoscopic ultrasound-compatible cryoablation catheter technology, FrostBite. We hypothesized that FrostBite would enable the rapid, effective, minimally invasive delivery of ultra-cold temperatures to target tissues, resulting in effective ablation via an endoscopic approach. Thermal properties and ablative efficacy were evaluated using a heat-loaded gel model, tissue-engineered models (TEMs), and an initial in vivo porcine study. Freeze protocols evaluated included single and repeat 3 and 5 min applications. Isotherm assessment revealed the generation of a 2.2 cm diameter frozen mass with the -20 °C isotherm reaching a diameter of 1.5 cm following a single 5 min freeze. TEM studies revealed the achievement of temperatures ≤ -20 °C at a diameter of 1.9 cm after a 5 min freeze. Fluorescent imaging conducted 24 h post-thaw demonstrated a uniformly shaped ellipsoidal ablative zone with a midline diameter of 2.5 cm, resulting in a total ablative volume of 6.9 cm3 after a single 5 min freeze. In vivo findings consistently demonstrated the generation of ablative areas measuring 2.03 cm × 3.2 cm. These studies demonstrate the potential of the FrostBite cryocatheter as an endoscopic ultrasound-based treatment option. The data suggest that FrostBite may provide for the rapid, effective, controllable freezing of cancerous pancreatic and liver tissues. This ablative power also offers the potential of improved safety margins via the minimally invasive nature of an endoscopic ultrasound-based approach or natural orifice transluminal endoscopic surgery (NOTES)-based approach. The results of this pre-clinical feasibility study show promise, affirming the need for further investigation into the potential of the FrostBite cryocatheter as an advanced, minimally invasive cryoablative technology.

Characterization and seminal cryopreservation of three species of birds of prey.

Assisted reproduction techniques in birds have contributed to many species' conservation and sustainable use. One of these techniques is semen cryopreservation, which is possible following the discovery of suitable cryoprotectants. This study aimed to characterize the fresh and post-thaw ejaculates of different species of birds of prey. The following species were included in the study: red-tailed hawk (Buteo jamaicensis) n=3, golden eagle (Aquila chrysaetos) n=3, and Harris's hawk (Parabuteo unicinctus) n=3. Twenty-five ejaculates were obtained for each species. The percentage of spermatozoa motility, viability, and morphology were evaluated. Evident differences were observed among the ejaculates of the three species, particularly in sperm length and between the fresh and post-thaw parameters of the same species in which the motility reduced to approximately 40% after thawing. It was demonstrated that sperm cryopreservation of the studied species was possible using the same freezing protocol. This study showed that sperm characteristics could influence the parameters obtained during their in vitro conservation, both in the fresh and post-thaw states.

High-Pressure Freezing and Low-Temperature Processing of Seeds for Electron Microscopy and Electron Tomography.

High-pressure freezing/freeze substitution has been used to preserve biological samples for ultrastructure study instead of chemical fixation. For most plant samples, the water content is too high and cannot be properly preserved during cryofixation. Additionally, the cell wall is a barrier that prevents the substitution of water with the resin. In this chapter, we will discuss modified high-pressure freezing and subsequent processing protocols based on our routinely used methodology for examining Arabidopsis seeds in transmission electron microscopy and electron tomography.