The effects of repeated freezing and thawing on bovine sperm morphometry and function.

Refreezing the remaining genetic resources after in vitro fertilization (IVF) can conserve genetic materials. However, the precise damage inflicted by repeated freezing and thawing on bovine sperm and its underlying mechanism remain largely unexplored. Thus, this study investigates the impact of repeated freeze-thaw cycles on sperm. Our findings indicate that such cycles significantly reduce sperm viability and motility. Furthermore, the integrity of the sperm plasma membrane and acrosome is compromised during this process, exacerbating the advanced apoptosis triggered by oxidative stress. Additionally, transmission electron microscopy exposed severe damage to the plasma membranes of both the sperm head and tail. Notably, the "9 + 2" structure of the tail was disrupted, along with a significant decrease in the level of the axonemal protein DNAH10, leading to reduced sperm motility. IVF outcomes revealed that repeated freeze-thaw cycles considerably impair sperm fertilization capability, ultimately reducing the blastocyst rate. In summary, our research demonstrates that repeated freeze-thaw cycles lead to a decline in sperm viability and motility, attributed to oxidative stress-induced apoptosis and DNAH10-related dynamic deficiency. As a result, the utility of semen is compromised after repeated freezing.

Evaluation of the regeneration process of the sciatic nerve of a mouse animal model after application of freezing, crushing or electrocoagulation damage / Ocena procesu regeneracji nerwu kulszowego mysiego modelu zwierzecego po zastosowaniu uszkodzenia metoda mrozenia, zmiazdzenia i elektrokoagulacji.

The problem of regeneration of damaged peripheral nerves is an ongoing topic and has long been the subject of intensive research worldwide. This study examined the morphological and functional evaluation of the regeneration process within the damaged sciatic nerve, a mouse animal model. The effect of impaired expression of the TSC-1 gene on the process of nerve regeneration was evaluated, depending on the mode of damage. The research object consisted of 48, 2-month-old male TSC lines. The test group consisted of animals that underwent damage to the sciatic nerve by crushing, freezing and electrocoagulation, while the control group includes mice whose sciatic nerve was not damaged. Behavioral tests were conducted to evaluate the functional return of the limb, after 3,5,7 and 14 days. The first changes in the regeneration process of the damaged neurite are observed as early as day 3 after the injury, while on day 14 after the injury the functional return of the damaged limb was noted.

Photosynthesis on the edge: photoinhibition, desiccation and freezing tolerance of Antarctic bryophytes.

In Antarctica, multiple stresses (low temperatures, drought and excessive irradiance) hamper photosynthesis even in summer. We hypothesize that controlled inactivation of PSII reaction centres, a mechanism widely studied by pioneer work of Fred Chow and co-workers, may effectively guarantee functional photosynthesis under these conditions. Thus, we analysed the energy partitioning through photosystems in response to temperature in 15 bryophyte species presenting different worldwide distributions but all growing in Livingston Island, under controlled and field conditions. We additionally tested their tolerance to desiccation and freezing and compared those with their capability for sexual reproduction in Antarctica (as a proxy to overall fitness). Under field conditions, when irradiance rules air temperature by the warming of shoots (up to 20 °C under sunny days), a predominance of sustained photoinhibition beyond dynamic heat dissipation was observed at low temperatures. Antarctic endemic and polar species showed the largest increases of photoinhibition at low temperatures. On the contrary, the variation of thermal dissipation with temperature was not linked to species distribution. Instead, maximum non-photochemical quenching at 20 °C was related (strongly and positively) with desiccation tolerance, which also correlated with fertility in Antarctica, but not with freezing tolerance. Although all the analysed species tolerated - 20 °C when dry, the tolerance to freezing in hydrated state ranged from the exceptional ability of Schistidium rivulare (that survived for 14 months at - 80 °C) to the susceptibility of Bryum pseudotriquetrum (that died after 1 day at - 20 °C unless being desiccated before freezing).

Influence of laboratory procedures on postthawing cell viability and hematopoietic engraftment after autologous peripheral blood stem cell transplantation.

BACKGROUND: The kinetics of hematopoietic recovery after autologous stem cell transplantation (ASCT) may be affected by laboratory procedures. The aim of this study was to evaluate the influence of characteristics of the cryopreserved units of peripheral blood stem cells (PBSC) on postthawing cell viability and engraftment outcomes after ASCT. STUDY DESIGN AND METHODS: This was a retrospective cohort study including individuals referred for ASCT. Cryopreservation was conducted at a single processing facility between 2014 and 2019, and patients received clinical care at six transplant centers. Covariates and outcome data were retrieved from participants' records. RESULTS: The study population comprised 619 patients (345 [55.7%] male). Median age was 53 years. Multiple myeloma was the most common diagnosis (62.7%). Higher preapheresis CD34+ cell count, lower nucleated cell (NC) concentration per cryobag, and composition of the cryoprotectant solution (5% dimethyl sulfoxide [DMSO] and 6% hydroxyethyl starch) were statistically significantly associated with higher postthawing cell viability. The linear regression model for time to neutrophil and platelet engraftment included the infused CD34+ cell dose and the composition of the cryoprotectant solution. Patients who had PBSC cryopreserved using 10% DMSO solution presented six times higher odds (odds ratio [OR] = 6.9; 95% confidence interval [CI]: 2.2-21.1; p = .001) of delayed neutrophil engraftment (>14 days) and two times higher odds (OR = 2.3, 95%CI: 1.4-3.7; p = .001) of prolonged hospitalization (>18 days). DISCUSSION: The study showed that mobilization efficacy, NC concentration, and the composition of the cryoprotectant solution significantly affected postthawing cell viability. In addition, the composition of the cryoprotectant solution significantly impacted engraftment outcomes and time of hospitalization after ASCT.

Superiority of Higher-Volume Fresh Feces Compared to Lower-Volume Frozen Feces in Fecal Microbiota Transplantation for Recurrent Clostridioides Difficile Colitis.

GOALS: To compare the clinical outcomes of different protocols for fecal microbiota transplantation (FMT) in two community hospitals with similar patient demographics. BACKGROUND: FMT is commonly performed for recurrent or refractory Clostridioides difficile infection (rCDI). The clinical efficacy of FMT for this indication has been well established. However, there has been no standardization or optimization of the amount of fecal material, method of feces preparation, or route of delivery for FMT. STUDY: In this retrospective study, patients with rCDI received FMT using commercially available frozen fecal preparation (22.7 g) at Center A and locally prepared fresh fecal filtrate (30-50 g) at Center B. The primary outcome was defined as complete resolution of clinical symptoms related to rCDI after at least 8 weeks of follow-up. RESULTS: Fifty patients from each center were included in the study. Clinical success after initial FMT with lower-volume frozen fecal preparation at Center A was 32/50 (64.0%) compared to 49/50 (98.0%) with higher-volume fresh fecal filtrate at Center B (p < 0.0001). Seventeen patients in Center A and 1 patient in Center B underwent at least one repeat FMT. Overall clinical success was achieved in 43/50 (86%) of patients in Center A and 50/50 (100%) in Center B (p = 0.012). CONCLUSIONS: Our results suggest superior clinical efficacy of a larger amount of fresh fecal filtrate over a smaller amount of commercially available frozen fecal preparation. Further studies are needed to examine the effect of varying amounts of feces and the optimal protocol for FMT in patients with rCDI.

The effect of temperature on the stability of PCSK-9 monoclonal antibody: an experimental study.

BACKGROUND: PCSK9 monoclonal antibody lowers plasma PCSK9 and LDL-cholesterol levels. The manufacturers recommend drug storage at 2-8 °C, and not above 25 °C. This study aimed to investigate drug stability at various temperatures that this drug could be exposed to during medication handling and transportation in tropical countries. METHODS: Alirocumab and evolocumab were tested in 3 study conditions: room temperature (RT), cooler device with cold pack, and freeze-thaw for 9 and 18 h. Heated drugs were used as negative control. Free plasma PCSK9 levels from 9 hyperlipidemia subjects were measured with ELISA. RESULTS: Average subject age was 49.2 ± 18.4 years. Percent PCSK9 inhibition significantly declined in heated drugs compared to baseline. Average RT during the study period was 30.4 ±2.6 °C. Change in percent PCSK9 inhibition of PCSK9 mAb at RT from baseline was - 5.8 ± 4.4% (P = 0.005) and - 11.0 ± 8.9% (P = 0.006) for alirocumab at 9 h and 18 h, and - 9.7 ± 11.8% (P = 0.04) and - 15.1 ± 14.3% (P = 0.01) for evolocumab at 9 and 18 h, respectively. In contrast, there were no significant changes in percent PCSK9 inhibition from baseline when PCSK9 mAb was stored in a cooler. In freeze-thaw condition, changes in percent PCSK9 inhibition from baseline to 9 and 18 h were - 5.2 ± 2.9% (P = 0.001) and - 2.6 ± 4.9% (P = 0.16) for alirocumab, and - 1.8 ± 4.2% (P = 0.24) and 0.4 ± 6.1% (P = 0.83) for evolocumab. CONCLUSION: Proper drug storage according to manufacturer's recommendation is essential. Drug storage at RT in tropical climate for longer than 9 h significantly decreased drug efficacy; however, storage in a cooler device with cold pack for up to 18 h is safe.

Fresh embryo transfer versus freeze-only after in vitro maturation with a pre-maturation step in women with high antral follicle count: a randomized controlled pilot study.

PURPOSE: In vitro maturation (IVM) is an alternative to in vitro fertilization (IVF) for women at high risk of developing ovarian hyperstimulation syndrome (OHSS). This study determined the effectiveness and safety of a freeze-only strategy versus fresh embryo transfer (ET) after IVM with a pre-maturation step (CAPA-IVM) in women with a high antral follicle count (AFC). METHODS: This randomized, controlled pilot study (NCT04297553) was conducted between March and November 2020. Forty women aged 18-37 years with a high AFC (≥24 follicles in both ovaries) undergoing one cycle of CAPA-IVM were randomized to a freeze-only strategy with subsequent frozen ET (n = 20) or to fresh ET (n = 20). The primary endpoint was ongoing pregnancy resulting in live birth after the first ET of the started treatment cycle. RESULTS: The ongoing pregnancy rate in the freeze-only group (65%) was significantly higher than that in the fresh ET group (25%; p = 0.03), as was the live birth rate (60% versus 20%; p = 0.02). Clinical pregnancy rate was numerically, but not significantly, higher after frozen versus fresh ET (70% versus 35%; p = 0.06), while the number of day 3 or good quality embryos, endometrial thickness on the day of oocyte pick-up, implantation rate, and positive pregnancy test rate did not differ significantly between groups. No cases of OHSS were observed, and miscarriage and multiple pregnancy rates were similar in the two groups. CONCLUSIONS: These findings suggest that the effectiveness of CAPA-IVM could be improved considerably by using a freeze-only strategy followed by frozen ET in subsequent cycles. TRIAL REGISTRATION NUMBER: NCT04297553 ( www.clinicaltrials.gov ).

Damage of Granusola and Stroma Cells Exposed to Mouse Ovarian Tissue Cryopreservation: Potential Mechanism of Ovarian Injury.

BACKGROUND: Several obstacles must be overcome before ovarian tissue cryopreservation can be used as a standard procedure. OBJECTIVE: To carry out a morphologic and functional study of the effect of cryopreservation on mouse follicles and stroma cells. MATERIALS AND METHODS: Female mice were divided into three groups (control, fresh graft and cryopreserved graft). Ultrastructural features of follicles and stroma cells were evaluated using transmission electron microscopy. After autologous transplantation, micro-vessel densities of grafts were examined. RESULTS: Vacuoles in granulosa cells and stromal cells are significantly greater than that of oocytes. The microvessel density of fresh grafts is significantly higher than that in frozen-thawed grafts. CONCLUSION: Granusola and stroma cells, rather than oocytes, are vulnerable to cryoinjury. Injuries to granulosa cells and stromal cells could be the critical part of ovarian damage caused by cryopreservation.

Impacts of Specific Cryoprotectants on Sperm Freezing and Relationships Between Cryodamage and Oxidation Stress Parameters in Awassi Ram Sperm.

BACKGROUND: The role of oxidative stress during cryoprotectant treatment has received little attention. OBJECTIVE: To assess the effects of different cryoprotectants and discover relationships between cryodamage and oxidative stress parameters on Awassi ram sperm. MATERIALS AND METHODS: The sperm samples diluted with Salamon's tris-citrate (TRIS) containing 20% centrifuged egg yolk and 0.5, 1.0 or 1.5 M Glycerol (Gly), methanol (M), 2-methoxyethanol (2-ME), dimethylacetamide (DMA) and 1.2 propanediol (PR). After 2 h of equilibration at +4 ºC, the sperm samples were frozen in liquid nitrogen vapour and stored. RESULTS: The best post-thaw motility (43.3%, 41.7%) of sperm was achieved when protected with 0.5 and 1.0 M glycerol. Arylesterase and ceruloplasmin parameters were significantly different after equilibration, whereas sulfhydryl groups were significantly different after freezing in their respective groups (P < 0.05). CONCLUSION: The increased use of glycerol caused greater loss of motility. The role of oxidative stress in freezing was also found to be limited.

Effect of Freeze-Thaw Cycles on the Oxidation of Protein and Fat and Its Relationship with the Formation of Heterocyclic Aromatic Amines and Advanced Glycation End Products in Raw Meat.

The aim of this research was to investigate the effect of the number of freeze-thaw cycles (0, 1, 3, 5, and 7) on porcine longissimus protein and lipid oxidation, as well as changes in heterocyclic aromatic amines (HAAs) and advanced glycation end products (AGEs) and their precursors. We analyzed the relationship among HAAs, AGEs, oxidation, and precursors and found the following results after seven freeze-thaw cycles. The HAAs, Norharman and Harman, were 20.33% and 16.67% higher, respectively. The AGEs, Nε-carboxyethyllysine (CEL) and Nε-carboxymethyllysine (CML), were 11.81% and 14.02% higher, respectively. Glucose, creatine, and creatinine were reduced by 33.92%, 5.93%, and 1.12%, respectively after seven freeze-thaw cycles. Norharman was significantly correlated with thiobarbituric acid reactive substances (TBARS; r2 = 0.910) and glucose (r2 = -0.914). Harman was significantly correlated to TBARS (r2 = 0.951), carbonyl (r2 = 0.990), and glucose (r2 = -0.920). CEL was correlated to TBARS (r2 = 0.992) and carbonyl (r2 = 0.933). These changes suggest that oxidation and the Maillard reaction during freeze-thaw cycles promote HAA and AGE production in raw pork.

Influence of Xenon on Survival of Sperm of Common Frog Rana temporaria during Slow Freezing.

We studied the effect of xenon on the survival rate of the spermatozoa of the common frog Rana temporaria during slow freezing with saturation of the suspension with xenon at a pressure of up to 1.2 bar. The cryoprotective properties of xenon were analyzed in comparison with nitrogen. No specific cryoprotective effect of xenon was revealed. Viability of spermatozoa pretreated with xenon at atmospheric pressure (0 bar) or under excess pressure of 0.6 bar and frozen in a cryoprotective medium with dimethylformamide, sucrose, and BSA did not differ significantly. The use of overpressure of xenon of 1.0 or 1.2 bar in the pretreatment and freezing process significantly impaired viability of the biomaterial.

Vitrification-induced activation of lysosomal cathepsin B perturbs spindle assembly checkpoint function in mouse oocytes.

As the age of child-bearing increases and correlates with infertility, cryopreservation of female gametes is becoming common-place in ART. However, the developmental competence of vitrified oocytes has remained low. The underlying mechanisms responsible for reduced oocyte quality post-vitrification are largely unknown. Mouse cumulus-oocyte complexes were vitrified using a cryoloop technique and a mixture of dimethylsulphoxide, ethylene glycol and trehalose as cryoprotectants. Fresh and vitrified/thawed oocytes were compared for chromosome alignment, spindle morphology, kinetochore-microtubule attachments, spindle assembly checkpoint (SAC) and aneuploidy. Although the majority of vitrified oocytes extruded the first polar body (PB), they had a significant increase of chromosome misalignment, abnormal spindle formation and aneuploidy at metaphase II. In contrast to controls, vitrified oocytes extruded the first PB in the presence of nocodazole and etoposide, which should induce metaphase I arrest in a SAC-dependent manner. The fluorescence intensity of mitotic arrest deficient 2 (MAD2), an essential SAC protein, at kinetochores was reduced in vitrified oocytes, indicating that the SAC is weakened after vitrification/thawing. Furthermore, we found that vitrification-associated stress disrupted lysosomal function and stimulated cathepsin B activity, with a subsequent activation of caspase 3. MAD2 localization and SAC function in vitrified oocytes were restored upon treatment with a cathepsin B or a caspase 3 inhibitor. This study was conducted using mouse oocytes, therefore confirming these results in human oocytes is a prerequisite before applying these findings in IVF clinics. Here, we uncovered underlying molecular pathways that contribute to an understanding of how vitrification compromises oocyte quality. Regulating these pathways will be a step toward improving oocyte quality post vitrification and potentially increasing the efficiency of the vitrification program.

Thermal stability of cytokines: A review.

BACKGROUND: The role of cytokines in various disease states is a burgeoning field of academic study and clinical application, however there are no consensus documents on how certain cytokines should be stored prior to quantification. This information is especially of interest to researchers assembling a biobank or clinicians who have to transport specimens to a different location in order to be tested. OBJECTIVE: To review the literature and synthesize prior findings on cytokine storage and freeze/thaw stability. DESIGN: We searched PubMed for articles related to cytokine storage stability. All articles were analyzed for cytokines studied, source of reported cytokine concentration (i.e., human whole blood or serum, concentrations from other species or bodily sources were excluded), and reported statistical results. RESULTS: We identified and synthesized results of 23 peer-reviewed articles which published data on the storage and freeze/thaw stability of 33 different cytokines and chemokines. CONCLUSION: There is a wide variety of reported cytokine storage and freeze/thaw stability. Interleukin-6 and tumor necrosis factor alpha are the most widely studied cytokines in regard to temperature stability. In a few cytokines, a clear consensus can be reached as to storage safety at particular temperatures, but in most, more research needs to be done and we advise the clinician or researcher to use caution in interpreting cytokine concentration results after a long period of storage or several freeze/thaw cycles.

Potency and Quality of Reconstituted Botulinum Neurotoxin Type A According to Storage Temperatures.

BACKGROUND: In clinical practice, one of the most important issues regarding the use of botulinum neurotoxin A (BoNT-A) is the proper storage conditions and the change in potency and quality over time after reconstitution. OBJECTIVE: This study aimed to investigate the change in potency and quality of reconstituted prabotulinumtoxin A (PraBoNT-A) over time when stored at different storage temperatures. MATERIALS AND METHODS: ICR/CD-1 mice and PraBoNT-A were used for the mouse intraperitoneal lethal dose 50% (LD50) test. A thorough quality evaluation of the product was performed. RESULTS: All of the reconstituted PraBoNT-A stored at different temperatures met the evaluation criteria for the suggested limits of estimated potency and for the quality assessment at every evaluated time point. When the stability of reconstituted PraBoNT-A was evaluated by regression analysis, the shelf life of reconstituted PraBoNT-A was found to be 99.24, 73.80, and 16.34 weeks in the case of PraBoNT-A stored at freezing, refrigeration, or room temperatures, respectively. CONCLUSION: Based on the results, the authors conclude that the efficacy and quality of the reconstituted PraBoNT-A product are not compromised at least for a certain period of time and that the shelf life of reconstituted PraBoNT-A is longest when stored at the freezing temperature.

The efficacy of antioxidants in sperm parameters and production of reactive oxygen species levels during the freeze-thaw process: A systematic review and meta-analysis.

To investigate the impact of antioxidants in sperm parameters and reduction in reactive oxygen species production during the freeze-thaw process. PubMed, Scopus, Web of Science, Embase and Cochrane central library were systematically searched. Of the 1583 articles, 23 studies were selected for data extraction. Our results show that antioxidants improved sperm progressive motility (standardised mean difference (SMD) = 1; 95% CI: 0.62, 1.38; p < .001) and viability (SMD = 1.20; 95% CI: 0.50, 1.91; p = .001) and reduced sperm DNA fragmentation (SDF) and hydrogen peroxide (H2 O2 ) production, but there was no significant improvement in total sperm motility after thawing. Acetyl-l-carnitine/l-carnitine, melatonin and catalase had a significant positive impact on progressive motility. The role of tempol and melatonin in improving viability was significant compared to other antioxidants. Moreover, a significant reduction in SDF was observed after addition of butylated hydroxytoluene, tempol and vitamin E. However, the prevention of H2 O2 production was significant only after the addition of tempol. Our overall results displayed the positive impact of antioxidants on progressive sperm motility, viability and reduction in SDF and H2 O2 production, but no significant impact of antioxidants on total sperm motility was seen during the freeze-thaw process.

Do patient factors influence embryologists' decisions to freeze borderline blastocysts?

RESEARCH QUESTION: To investigate whether patient factors influence the decision to freeze a blastocyst with low implantation potential. DESIGN: This experimental study assessed 170 practicing embryologists from a variety of countries who were recruited via an online survey. Participants were currently practicing embryologists, who grade blastocysts as part of this role. The survey presented decision-making 'vignettes' to participants. These included specific patient information, as well as an image of an expanded blastocyst that was of borderline quality for inner cell mass and trophectoderm, for which the embryologist selected whether or not to freeze. High/low maternal age, the presence/absence of other top quality blastocysts, and the presence/absence of previously unsuccessful IVF cycles were systematically varied within the patient information in a 2 × 2 × 2 design. Participants reported how likely they would be to freeze a particular blastocyst on a scale of 1 (Extremely Unlikely) to 7 (Extremely Likely), and whether or not they would ultimately freeze each blastocyst (Yes or No). RESULTS: Lower maternal age, no other high-quality blastocysts within the cohort, and multiple unsuccessful IVF cycles were associated with greater likelihood of recommending to freeze (P < .001). Furthermore, significant interactions among all three patient factors were noted. CONCLUSION: This study provides evidence suggesting that when faced with an uncertain blastocyst, factors pertaining to the patient (maternal age, the presence/absence of other top quality blastocysts, and the presence/absence of previously unsuccessful IVF cycles) influence the decision to freeze.

Water-transport and intracellular ice formation of human adipose-derived stem cells during freezing.

The key to optimizing the cryopreservation strategy of human adipose-derived stem cells (hADSCs) is to identify the biophysical characteristics during freezing. Systematic freezing experiments were conducted under a cryo-microscope system to investigate the cryoinjury mechanism for hADSCs at different cooling rates. By simultaneously fitting morphological change data to the water-transport equation at 5, 10 and 20 °C/min, the plasma membrane hydraulic conductivity, Lpg, and activation energy, ELp, were determined. Moreover, the optimal cooling rate was also predicted by using mathematical model methods. Additionally, the surface-catalyzed nucleation (SCN) parameters were calculated by fitting in numerical models, Ω0SCN and k0SCN were determined at cooling rates of 30, 45 and 60 °C/min. These results may provide potential application value for cryopreservation of hADSCs.