Effects of different freezing techniques on the rejection of allogeneic vascular transplantation / 中国组织工程研究

BACKGROUND:Cryopreservation can better ensure the integrity of the vascular structure.How to reduce its immunogenicity to improve rejection after transplantation has attracted more and more attention. OBJECTIVE:To review the research progress of freezing treatment to reduce vascular immunogenicity after allogeneic vascular transplantation in order to provide a reference for clinical research. METHODS:A systematic search of Chinese and English databases CNKI,WanFang and PubMed,as well as online websites Baidu and Google Scholar since the establishment of the database has published literature on reducing vascular immunogenicity after allogeneic vascular transplantation.Keywords were"cardiovascular disease,endothelial cells,cryopreservation,blood vessel transplantation or vascular graft,immunogenicity,immune rejection,allograft or allogeneic transplantation or allograft transplantation and cryoprotectant".A total of 68 articles were included according to the inclusion and exclusion criteria. RESULTS AND CONCLUSION:(1)The review found that the rejection of allogeneic vascular transplantation can be reduced by improving the existing freezing technology,which mainly involves the selection of freezing and thawing methods and cryoprotectants.(2)The existing research suggests that the freeze-drying method is superior to the low-temperature cryopreservation,but due to the limited conditions,it is still dominated by low-temperature cryopreservation.Among them,vitrification cryopreservation,slow rewarming and the use of stainless steel and even silver-containing materials are better than programmed cryopreservation and rapid rewarming.(3)The combined selection of permeable and non-permeable cryoprotectants can further reduce the occurrence of rejection while reducing their toxicity.

Serum-free cell cryopreservation based on self-assembled peptides / 中国生物制品学杂志

@#Objective To achieve serum-free and low-DMSO efficient cell cryopreservation based on self-assembled peptides(Peptide).Methods Hepa1-6 cells were cryopreserved by combining three cryoprotectants(CPAs),FBS,DMSO and Peptide. Confocal microscopy,flow cytometry and cell counting were used to measure the survival rate,recovery rate and proliferation ability of the cells after freezing,and the ice inhibition of CPAs was tested by differential scanning calorimetry(DSC). Finally,a CPA with high efficiency for cell cryopreservation was determined.Results Peptide formed a fibrous network hydrogel in DMSO solution to encapsulate cells and supported them in a three-dimensional(3D)stereological distribution. Under serum-free conditions,the use of 0. 1 wt% Peptide combined with 5% DMSO as a novel composite CPA for cryopreservation of Hepa1-6 cells significantly improved the survival rate and post-freezing recovery rate compared with the5% DMSO and 0. 1 wt% Peptide groups,and there was no significant difference with the 10% DMSO + 20% FBS group(t =0. 983 and 2. 164,respectively,each P > 0. 05). In addition,compared with the 10% DMSO + 20% FBS group,0. 1 wt%Peptide + 5% DMSO group showed no significant difference in cell proliferation ability after freezing-thawing(t = 3. 513,P > 0. 05). In the end,DSC tests showed that Peptide-based CPA had good ice inhibition performance.Conclusion The use of 0. 1 wt% Peptide combined with 5% DMSO as a novel composite CPA has a good effect on cell cryopreservation.

Discussion on Ethical Value and Application Principle of Cryopreservation and Transplantation of Human Ovarian Tissue / 中国医学伦理学

In recent years, with the continuous progress of medical technology, the survival rate of cancer patients after treatment has been continuously improved, and more and more young cancer patients begin to pay attention to the fertility problem after survival. For prepubertal or adolescent cancer patients who require urgent chemoradiotherapy, and for reproductive female patients, ovarian tissue cryopreservation (OTC) follows by transplantation is the only option to preserve their fertility at present. Although the OTC technology has been carried out as a routine clinical project in a few medical institutions in China, it is still in the stage of clinical trial research in majority medical institutions. There are still many technical and ethical challenges in clinical practice of OTC technology. Therefore, this paper discussed the ethical principles that should be followed in clinical practice of human OTC and transplantation, and briefly analyzed the corresponding ethical issues. When implementing this technology, the indications should be followed strictly, the wishes of patients should be respected and true and full informed consent should be obtained while ensuring that the cancer treatment of patients is not delayed. Besides, it is significants to accumulate enough experience for minor patients to fully protect their rights and interests and promote the construction of relevant national laws and regulations.

Evaluation of survival and functionality in frozen platelets

ABSTRACT Objective: Prolong platelet survival and functionality up to 28 days. Methods: A sample of apheresis platelets was evaluated, distributed in 3 groups according to the cryopreservative solution used: DMS05%+2%albumin; DMSO5%+NaC10,9% and DMS05%+Dextrose2%. They were then frozen at -80 °C and thawed at 7, 14 and 28 days. The in vitro survival and viability were assessed by the post-thaw platelet count and the CD41, CD61 and CD42a staining percentages by flow cytometry. The functionality was determined with the percentage of post-stimulation aggregation with INm-thrombin using the Chromo-Log490 aggregometer. The control group (CG) consisted of fresh platelets under constant agitation at 22 °C. Results: A total of 72 platelet aliquots was analyzed. The CG presented a platelet-count of 1934 ± 0.5 × 109/L and a 100% viability. The percentages of CD41, CD61 and CD42a labeling were 99, 98.5 and 96.5%, respectively. The percentage of aggregation was 99%. On day 7 of the post-freezing, the platelet count for groups 1, 2 and 3 was 1,844 ± 102, 1,856 ± 76 and 1,752 ± 226, with the viability of 98, 96 and 95%, respectively. On day 14, the counts were 1,722 ± 238, 1,649 ± 215 and 1,578 ± 223 with the viability of 96, 95 and 94% and, on day 28, they were 1,602 ± 374, 1,438.6 ± 429 and 1,406.6 ± 436, with the viability of 96, 94 and 93%, respectively. Groupl presented a higher expression of membrane antigens. Aggregation percentages were 90, 98 and 89% at day 7, 88%, 98 and 87% at day 14 and 84%, 95 and 82% at day of the 28 post-freezing, respectively, with group2 presenting the best results. Conclusion: The results support cryopreservation as a reasonable method to prolong platelet survival up to 28 days, maintaining its functionality and viability greater than 50%.

Timing of semen cryopreservation: before or after processing?

Abstract Objective: Seminal cryopreservation causes significant damage to the sperm; therefore, different methods of cryopreservation have been studied. The aim of the study was to compare the effects of density gradient processing and washing/centrifugation with seminal plasma removal for cryopreservation in semen parameters. Methods: Seminal samples of 26 normozoospermic patients were divided into 3 parts: with seminal plasma; after washing/centrifugation; and after selection through density gradient. The samples were cryopreserved for at least two weeks. Motility, sperm count, morphology and viability were evaluated before cryopreservation and after thawing. Results: Density gradient processing selected motile and viable sperm with normal morphology in fresh samples (p<0.05). Cryopreservation negatively affected all sperm parameters regardless of the processing performed, and even if the sperm recovery was lower in the density gradient after the thawing, progressive motility, total motility, viability and morphology remained higher (p<0.05). Conclusion: Cryopreservation significantly compromises sperm parameters (motility, morphology, viability). In normozoospermic patients, the density gradients select better quality spermatozoa compared to other processing methods; this benefit was kept after thawing.

Social egg freezing: a gateway to fertility insurance

Background: There is a decline in fertility due to delayed childbearing and hence, elective egg freezing (oocyte cryopreservation) offers a solution. Two primary techniques are used for human oocyte cryopreservation: slow freezing and vitrification. Vitrification is highlighted as a promising method. The present study was conducted to evaluate the pregnancy outcomes from oocytes that were frozen for social reasons and oocytes were frozen by method of vitrification.Methods: This retrospective study was conducted at RISAA IVF International Fertility Centre, New Delhi, collecting the data available over 6 years (2017-2023). It focused on freezing of oocytes and collected data on frozen, thawed, and fertilized oocytes, analysing thawing, survival, and fertilization rates by patient age group. Data was processed using Microsoft Excel and SPSS, with quantitative data presented as mean and standard deviation, and qualitative data as frequencies and proportions.Results: In this study of 25 patients who froze their oocytes, the average age at cryopreservation was 33.38 years. The mean age at implantation was 36.48 years. On average, patients had 10 retrieved oocytes. Most patients with thawed oocytes were in the 35-37 age group, although the highest survival and fertilization rates (86.9%) occurred in the above-40 age group. Established pregnancies were more common in the 35-37 age group, with 5 out of 10 pregnancies regardless of the day of embryo transfer.Conclusions: Elective egg freezing (oocyte cryopreservation) has emerged as a valuable solution to preserve fertility.

Estudio exploratorio de las motivaciones para la criopreservación de ovocitos en forma electiva / An exploratory study of motivations for elective oocyte cryopreservation

INTRODUCCIÓN: La vitrificación de óvulos es un avance científico que ha cambiado la perspectiva reproductiva de la sociedad humana. Este procedimiento se ha ofrecido como alternativa a la postergación voluntaria del embarazo, confiriéndole a la mujer una nueva perspectiva en su autonomía reproductiva. El número de mujeres que consultan y luego optan por congelar ovocitos ha aumentado en forma casi exponencial en Chile y en todo el mundo. En nuestro país, hay poco conocimiento acerca de la motivación, experiencia y resultados de la criopreservación electiva de ovocitos en Chile. El objetivo fue conocer la motivación, experiencia y el deseo reproductivo futuro de este grupo de mujeres sometidas a esta técnica. MÉTODOS: Estudio descriptivo transversal, basado en un cuestionario enviado por correo electrónico en el que participaron mujeres que se habían sometido previamente a criopreservación electiva de ovocitos entre enero de 2011 y diciembre de 2019 en Clínica Alemana, Santiago de Chile. RESULTADOS: De 342 mujeres que habían completado un ciclo de criopreservación electiva de ovocitos, 193 aceptaron participar y de estas, 98 (51%) de las mujeres contestaron la encuesta en forma satisfactoria. Se establecieron criterios de exclusión a aquellas mujeres que se habían sometido a este procedimiento por indicación médica como la endometriosis, el cáncer y la baja reserva ovárica. El motivo más frecuente para realizarse el procedimiento fue la edad (44%). En relación al procedimiento; el 94% no se arrepiente de haberlo realizado y 74% de las mujeres cree que utilizará sus ovocitos en algún momento de su vida. Por último, desde que se realizaron la criopreservación de ovocitos a la fecha, el 11% de las mujeres encuestadas ha usado sus ovocitos vitrificados y 27% ha logrado embarazarse con estos. CONCLUSIÓN: Las mujeres que se someten a criopreservación electiva de ovocitos por razones sociales, son principalmente mujeres sin pareja que tiene como motivación principal su edad reproductiva y la gran mayoría de ellas no se arrepienten de haberlo realizado.

INTRODUCTON: Oocyte vitrification is a scientific advance that has changed the reproductive perspective of human society. This procedure has been offered as an alternative to the voluntary postponement of pregnancy, giving women a new perspective on their reproductive autonomy. The number of women who consult and then choose to freeze oocytes has increased almost exponentially in Chile and throughout the world. There is little knowledge about the motivation, experience, and results of elective oocyte cryopreservation in Chile. The objective was to know the motivation, experience, and future reproductive desire of the women who underwent this technique. METHODS: Cross-sectional descriptive study based on a questionnaire sent by e-mail in which females who had previously undergone elective oocyte cryopreservation between January 2011 and December 2019 at Clínica Alemana, Santiago, Chile, participated. RESULTS: Of 342 women who had completed a cycle of elective oocyte cryopreservation, 193 agreed to participate, and of these, 98 (51%) answered the survey satisfactorily. Women who underwent this procedure for medical indication, including endometriosis, cancer, and low ovarian reserve, were excluded. The most frequent reason for the procedure was age (44%). Concerning the procedure: 94% do not regret having it done, and 74% of the women believe that they will use their oocytes at some point in their lives. Finally, from the time of oocyte cryopreservation to date, 11% of the surveyed women have used their vitrified oocytes, and 27% have become pregnant. CONCLUSIONS: Women who undergo elective oocyte cryopreservation for social reasons are mainly women without a partner whose main motivation is their reproductive age. The vast majority do not regret doing so.

CRYOPRESERVED SEMEN OF Piaractus orinoquensis (Serrasalmidae): POST-THAW STORAGE TIMES AND ACTIVATING SOLUTIONS / Semen congelado de Piaractus orinoquensis (Serrasalmidae): Almacenamiento posdescongelación y soluciones activadoras

ABSTRACT Piaractus orinoquensis is a native species from the Colombian Orinoquia and several studies have contributed to the standardization of protocols for seminal cryopreservation of this species. The main goal of this study was to evaluate the sperm motility, duration of sperm motility (DM), pH, sperm membrane integrity (SMI) and fertility of semen cryopreserved for seven years and subjected to different post-thaw storage times (PST). Semen from five males (3.04 ± 0.3 kg) was diluted 1:4 (semen:diluent) with 10 % dimethylsulfoxide, 5.5 % glucose and 12 % egg yolk in 4 mL macrotubes and frozen in liquid nitrogen. Four PST were evaluated: 0, 15, 45 and 60 min. The sperm cells were activated with 1 % NaHCO3 (SB) and 0.9 % NaCl (SC) to determine sperm motility, DM, fertility rate, SMI and pH through PST. Significative motility reduction (p < .05) was observed through the PST and between the PST of 0 and 60 min for DM, both for treatments activated with SB and with SC. The SMI had a significant reduction (p < .05) after 60 min of post-thaw storage and the pH did not vary during PST. The fertility rate decreased significantly (p < .05) between time 0 and 60 min of PST. PST affects the seminal quality and fertility of P. orinoquensis, the best fertilization results were obtained by activating post-thawing sperm motility with 1 % SB. The long-term cryopreserved semen (seven years) of the species maintains its fertilization capacity with values like those obtained with fresh sperm.

RESUMEN Piaractus orinoquensis es una especie de la Orinoquía Colombiana en la cual se han realizado numerosos estudios para estandarizar protocolos de crioconservación seminal. El objeto de este estudio fue evaluar la motilidad espermática, duración de la motilidad espermática (DM), pH, integridad de membrana espermática (SMI) y fertilidad de semen crioconservado por siete años, sometido a diferentes tiempos de almacenamiento posdescongelación (PST). Semen de 5 machos (3,4 ± 0,3 kg) fue diluido 1:4 (semen: diluyente) con dimetilsulfóxido 10 %, glucosa 5,5 %, yema de huevo 12 %, en macrotubos de 4 mL y congelado en nitrógeno líquido. Cuatro PST fueron evaluados: 0, 15, 45 y 60 min. Las células espermáticas fueron activadas con NaHCO3 1% (SB) y NaCl 0,9 % (SC) para determinar motilidad espermática, DM, tasa de fertilización, SMI y pH a través de PST. Se observó reducción significativa de motilidad (p < ,05) a través del PST, también en DM entre tiempo 0 y tiempo 60 min. de PST en semen activado con SB y SC. SMI tuvo una reducción significativa (p < ,05) luego de 60 min. de almacenamiento posdescongelación y el pH no varió durante el PST. La tasa de fertilización decayó significativamente (p < ,05) entre 0 y 60 minutos de PST. La calidad seminal y fertilidad en P. orinoquensis fue afectada por PST. Los mejores resultados de fertilización se obtuvieron activando la motilidad con SB. El semen crioconservado a largo plazo (siete años), conservó su capacidad fertilizante con valores similares a los obtenidos con semen fresco.

Cryopreservation as a strategy for prevention of ovarian hyperstimulation syndrome in a public assisted reproduction service in São Paulo - Brazil

SUMMARY OBJECTIVE: This study aimed to evaluate the prevalence of ovarian hyperstimulation syndrome (OHSS) and associated risk factors in patients undergoing fertilization cycles at risk of OHSS (≥15 antral follicles or ≥15 oocytes aspirated) and submitted to cryopreservation of all embryos in the Human Reproduction Service of the Pérola Byington Hospital (Referral Center for Women's Health) in São Paulo, SP, Brazil. METHODS: This cross-sectional, institutional, descriptive study of secondary data from patients' charts enrolled in the Assisted Reproduction Service of the Pérola Byington Hospital at risk of OHSS after controlled ovarian stimulation and submitted to cryopreservation of all embryos was conducted between January 2015 and September 2017. RESULTS: OHSS occurred in 47.5% of cycles, all with mild severity, and there were no moderate or severe cases of OHSS. CONCLUSION: The cryopreservation of all embryos is associated with a reduction in moderate and severe forms of OHSS. Risk factors for OHSS should be evaluated prior to initiation of treatment, with less intense stimulation protocols accordingly.

Comparison of the Effects of Single Sperm Cryopreservation and Conventional Cryopreservation on Embryo Culture and Pregnancy in Patients Undergoing Micro-TESE / 中山大学学报(医学科学版)

ObjectiveTo compare the effects of single sperm cryopreservation and conventional cryopreservation on embryo culture and pregnancy in patients undergoing microdissection testicular sperm extraction （micro-TESE）. MethodsA retrospective analysis was done on the patients who underwent micro-TESE at the Reproductive Medicine Center in the Sixth Affiliated Hospital of Sun Yat-Sen University between January 2018 and December 2021. The single sperm cryopreservation group included 39 patients undergoing single sperm cryopreservation and 307 MII oocytes. The conventional cryopreservation group included 39 patients undergoing conventional cryopreservation and 410 MII oocytes. Propensity score matching （PSM） was performed to balance the selection bias. The fertility rate， embryo culture and pregnancy of these two groups were compared. ResultsThere was no statistical difference in age， body mass index （BMI）， years of infertility， basal FSH， basal LH， basal E2， AMH， AFC， number of oocytes retrieved and number of transferred embryos between the two groups （P>0.05）. No significant difference was found in fertilization rate （65.8% vs. 60.49%）， available embryo rate （67.82% vs. 58.87%） and high-quality embryo rate （70.80% vs. 75.34%）. The single sperm cryopreservation group had significantly lower clinical pregnancy rate than conventional cryopreservation group （45.45% vs. 70.0%， P=0.049）. ConclusionIf the patients undergoing micro-TESE have very few sperms， single sperm cryopreservation could be an effective choice to increase the utilization of each sperm and ensure the subsequent sperm retrieval after thawing， but the clinical pregnancy rate is decreased. Due to the limited number of cases， we need a large additional number of cases to observe and analyze.

Effect of follicular size on clinical outcomes of frozen-thawed embryo transfer induced by hCG on ovulation in natural cycles / 中华妇产科杂志

Objective:To investigate the effect of follicular size on the clinical outcomes of frozen-thawed embryo transfer induced by human chorionic gonadotropin (hCG) of natural cycles on ovulation.Methods:Clinical data of 427 cycles of frozen-thawed single blastocyst transfer in Nanjing Drum Tower Hospital from January 2016 to December 2019 were retrospectively analyzed. The patients were divided into 15-16 mm group (15≤diameter≤16 mm, n=66), 16-17 mm group (16<diameter≤17 mm, n=101), 17-18 mm group (17<diameter≤18 mm, n=125), 18-20 mm group (18<diameter≤20 mm, n=109),>20 mm group (diameter>20 mm, n=26), according to the maximum follicle diameter on the induction day of hCG ovulation induction. The estradiol and luteinizing hormone (LH) levels, and clinical pregnancy rate, abortion rate and live birth rate were compared in five groups. Results:There were statistically significant differences in estradiol and LH levels among the five groups on the day of hCG induction (all P<0.05). Estradiol levels in 15-16 mm group to >20 mm group gradually increased on the day of hCG induction, and estradiol level in 15-16 mm group was significantly lower than those in 17-18 mm group, 18-20 mm group and >20 mm group (median: 1 002.3 vs 1 103.3 vs 1 171.2 vs 1 539.0 pmol/L), with statistical significances ( P=0.034, P<0.001, P=0.002). On the day of hCG induction, LH levels in 15-16 mm group to >20 mm group showed a decreasing trend, and LH level in 15-16 mm group was significantly higher than those in 17-18 mm group and >20 mm group (median: 37.73 vs 28.24 vs 24.11 U/L), with statistically significant differences ( P=0.007, P=0.006). There were no significant differences in clinical pregnancy rate, abortion rate and live birth rate in 15-16 mm group to >20 mm group (all P>0.05). Conclusion:In the natural cycle protocol of hCG induced ovulation, the small follicle group could achieve similar clinical outcomes compared with normal sized follicles in the single blastocyst transfer of frozen-thawed embryos.

Droplet freeze-thawing system based on solid surface vitrification and laser rewarming / 生物医学工程学杂志

Ultra-rapid cooling and rewarming rate is a critical technical approach to achieve ice-free cells during the freezing and melting process. A set of ultra-rapid solid surface freeze-thaw visualization system was developed based on a sapphire flim, and experiments on droplet freeze-thaw were carried out under different cryoprotectant components, volumes and laser energies. The results showed that the cooling rate of 1 μL mixed cryoprotectant [1.5 mol/L propylene glycol (PG) + 1.5 mol/L ethylene glycol (EG) + 0.5 mol/L trehalose (TRE)] could be 9.2×10 3 °C/min. The volume range of 1-8 μL droplets could be vitrified. After comparing the proportions of multiple cryoprotectants, the combination of equal proportion mixed permeability protectant and trehalose had the best vitrification freezing effect and more uniform crystallization characteristics. During the rewarming operation, the heating curve of glassy droplets containing gold nanoparticles was measured for the first time under the action of 400-1 200 W laser power, and the rewarming rate was up to the order of 10 6 °C/min. According to the droplet images of different power rewarming processes, the laser power range for ice-free rewarming with micron-level resolution was clarified to be 1 400-1 600 W. The work of this paper simultaneously realizes the ultra-high-speed temperature ramp-up, transient visual observation and temperature measurement of droplets, providing technical means for judging the ice free droplets during the freeze-thaw process. It is conducive to promoting the development of ultra-rapid freeze-thaw technology for biological cells and tissues.

A model for preservation of thymocyte-depleted thymus

DiGeorge syndrome is a disorder caused by a microdeletion on the long arm of chromosome 22. Approximately 1% of patients diagnosed with DiGeorge syndrome may have an absence of a functional thymus, which characterizes the complete form of the syndrome. These patients require urgent treatment to reconstitute T cell immunity. Thymus transplantation is a promising investigational procedure for reconstitution of thymic function in infants with congenital athymia. Here, we demonstrate a possible optimization of the preparation of thymus slices for transplantation through prior depletion of thymocytes and leukocyte cell lineages followed by cryopreservation with cryoprotective media (5% dextran FP 40, 5% Me2SO, and 5% FBS) while preserving tissue architecture. Thymus fragments were stored in liquid nitrogen at -196°C for 30 days or one year. The tissue architecture of the fragments was preserved, including the distinction between medullary thymic epithelial cells (TECs), cortical TECs, and Hassall bodies. Moreover, depleted thymus fragments cryopreserved for one year were recolonized by intrathymic injections of 3×106 thymocytes per mL, demonstrating the capability of these fragments to support T cell development. Thus, this technique opens up the possibility of freezing and storing large volumes of thymus tissue for immediate transplantation into patients with DiGeorge syndrome or atypical (Omenn-like) phenotype.

Opinions of female academicians on oocyte freezing: a qualitative study

SUMMARY OBJECTIVE: The aim of this study was to evaluate the opinions of female academicians about oocyte freezing. METHODS: This qualitative study included 12 single academic women who had not yet entered menopause, did not have children, and were continuing their doctoral education in Istanbul, Turkey, between August and September 2022. Data were collected with semi-structured interviews and evaluated by content analysis. RESULTS: Three main themes were "Difficulty of fertility in academics," "Advantages of oocyte freezing," and "Concerns about oocyte freezing." Participants mostly had positive attitudes about the advantages of oocyte cryopreservation, but they had concerns about pregnancies obtained with frozen oocytes. CONCLUSION: The academic women attributed fertility as an obstacle to their career and experienced anxiety about fertility. They were aware of the advantages of oocyte cryopreservation; however, they defined the pregnancy with oocyte freezing as artificial.

Structural Integrity and Cellular Viability of Cryopreserved Allograft Heart Valves in Right Ventricular Outflow Tract Reconstruction: Correlation of Histopathological Changes with Donor Characteristics and Preservation Times

Abstract Introduction: Cryopreserved allograft heart valves (CAHV) show longer event-free survival compared to other types of protheses. However, all patients develop early and/or late allograft failure. Negative predictors are clinical, and there is a lack of evidence whether they correspond with the microscopic structure of CAHV. We assessed histopathological signs of structural degeneration, degree of cellular preservation, and presence of antigen-presenting cells (APC) in CAHV and correlated the changes with donor clinical characteristics, cryopreservation times, and CAHV types and diameters. Methods: Fifty-seven CAHV (48 pulmonary, nine aortic) used for transplantation between November/2017 and May/2019 were included. Donor variables were age, gender, blood group, height, weight, and body surface area (BSA). Types and diameters of CAHV, cold ischemia time, period from decontamination to cryopreservation, and cryopreservation time were recorded. During surgery, arterial wall (n=56) and valvar cusp (n=20) samples were obtained from the CAHV and subjected to microscopy. Microscopic structure was assessed using basic staining methods and immunohistochemistry (IHC). Results: Most of the samples showed signs of degeneration, usually of mild degree, and markedly reduced cellular preservation, more pronounced in aortic CAHV, correlating with arterial APC counts in both basic staining and IHC. There was also a correlation between the degree of degeneration of arterial samples and age, height, weight, and BSA of the donors. These findings were independent of preservation times. Conclusion: CAHV show markedly reduced cellular preservation negatively correlating with the numbers of APC. More preserved CAHV may be therefore prone to stronger immune rejection.

Collection, cryopreservation and thawing of stem cells for children weighing less than 25 Kg with high-risk neuroblastoma: A single center results in Morocco

ABSTRACT Introduction: An important component of the advances made in neuroblastoma treatment has been the use of peripheral blood stem cells to support high-dose chemotherapy. In this study, we report our experience on a series of small children who have undergone standard and large volume leukaphersis (LVL) procedures, provide an update on a single institution's experience with cryopreservation of autologous peripheral blood stem cells (PBSCs), using 10% dimethyl sulfoxide (DMSO) and applying post-thaw DMSO depletion and analyze a number of variables that may affect viability. Methods: A total of 36 aphereses were performed on 29 children weighing less than 25 kg between July 2016 and October 2019 at the Ibn Sina university hospital. Results: Seven females and twenty-two males, median bodyweight 14 kg (9 - 22). A single apheresis was sufficient to obtain at least 3 × 106/kg body weight (BW) of CD34+ cells in 82.8% of the cases. The LVL was performed in 22 aphereses. A median number of 5.9 × 106/ kg CD34 cells were collected per apheresis. A total of 60 PBSC samples were cryopreserved and 46 samples were infused. The mean cell viability percentage decreased from 94.75 ± 1.14% before freezing to 70.84 ± 8.6% after thawing (p < 0.001). No correlation was found between post-thaw viability and storage time (r = -0.233; p = 0.234) or number of total nucleated cells (r = 0.344; p = 0.073). Conclusion: Leukapheresis is safe and feasible in small pediatric patients if the appropriate measures are used. Cryopreservation poses numerous challenges, especially a decrease in cell viability after thawing.

Association of bull semen protein estimates and SDS-PAGE profiles on semen freezability

It is vital to identify the ejaculate with good freezability by determining the biochemical makeup of the ejaculate at the pre-freeze stage. The present study targeted to assess the use of the protein estimates and profiles at the pre-freeze stage as markers of freezability in Frieswal populations. Storing the proteins for proteomic studies is always tricky in the case of animal studies, where accessibility to liquid nitrogen is limited. Hence alternative storing approaches need to be optimized. The second part of this study examined the protein concentration and protein profiles of RNALater and frozen stored sperm cells to assess the use of RNALater preservation in sperm proteomic studies. Sperm and seminal plasma protein concentrations were quantified using Bradford assay, and total protein quantities were derived. The seminal plasma and sperm protein profiles were generated with SDS-PAGE. The protein estimates and SDS-PAGE profiles of good and poor freeze-groups were similar. Also, sperm and seminal plasma protein concentration were not correlated with the semen volume and sperm count. Even though the yield was comparatively less, the protein profiles of sperm preserved by RNALater were similar to that of frozen sperms. The present study results indicate that the protein estimates and qualitative profiles of sperm and seminal plasma proteins may not be sufficient to reveal the differences in the proteome of good and poor freezable bulls at the macro level. Hence, the protein estimates and profiles of neat semen may not be helpful for the prediction of freezability at the pre-freeze stage. Secondly, this study indicates that RNALater preservation helps store sperms for proteome analysis studies.

Processamento e conservação de enxertos de pele: uma abordagem com foco nos métodos de criopreservação, liofilização e glicerolização / Skin grafts processing and conservation: an approach focusing on cryopreservation, freeze-drying and glycerol conservation methods

Introdução: o uso de substitutos cutâneos para o tratamento de diversas feridas graves é uma forma eficiente de prevenir infecções e favorecer o processo de reepitelização. No entanto, tecidos biológicos estão suscetíveis a degradação e contaminação. Por isso, devem ser submetidos a rigorosos protocolos de processamento e testes que comprovem suas contribuições benéficas e segurança de aplicação. Objetivo: trazer uma abordagem sobre as principais características dos métodos de criopreservação, glicerolização e liofilização e sua consequencia nos aspectos imunológicos, microbiológicos e de viabilidade tecidual de enxertos de pele humana. Metodologia: foi realizada uma busca online utilizando as palavras chaves "criopreservação", "liofilização", "glicerolização", "enxertos", "processamento tecidual" e "engenharia dos tecidos" em múltiplas combinações nos bancos de dados PubMed, LILACS e ScienceDirect. Resultados: 200 artigos científicos foram obtidos, 26 excluídos por duplicidade, 92 selecionados para leitura integral a partir da leitura de seus resumos e 27 utilizados na construção desta revisão. A liofilização e a glicerolização são métodos semelhantes considerando a viabilidade tecidual. O uso de glicerol traz como principal desvantagem sua citotoxicidade quando comparado aos outros métodos. A criopreservação mantém os tecidos viáveis. Contudo, pode ser mais cara e trazer riscos de transmissão de microorganismos patogênicos. De modo geral, não é bem estabelecido quais os melhores métodos de conservação para uma adequada conservação da viabilidade dos enxertos de pele. Considerações Finais: os 3 métodos, liofilização, glicerolização e criopreservação, possuem aplicabilidade na conservação de enxertos. A falta de padronização na aplicação de enxertos apesar de sua frequente aplicação e a escassez de estudos recentes sobre o tema justificam o presente estudo.

Introduction: the use of skin substitutes for treatment of several wounds is an efficient way to prevent infections and allow the re-epithelialization process. However, biological tissues are susceptible to degradation and contamination. Therefore, they must undergo rigorous processing and testing protocols that prove their beneficial contributions and application security. Objective:to bring an approach on the main characteristics of cryopreservation, freeze-drying and glycerol conservation methods and their implications on immunological, microbiological and tissue viability aspects when applied to human skin grafts. Methodology:a mostly online search was performed using the keywords "cryopreservation", "freeze-drying", "glycerol conservation", "grafts", "tissue processing" and "tissue engineering" in multiple combinations in PubMed, LILACS and ScienceDirect databases. Results: 200 scientific articles were rescued, 26 excluded by duplicity, 92 selected for full reading from the reading of their abstracts and 27 used in the construction of this review. Freeze-drying and glycerol conservation are similar methods, with glycerol conservation having greater economic advantage. The use of glycerol presents cytotoxicity when compared to the other methods. Cryopreservation keeps tissues viable, however, is more expensive and carry risks of transmission of pathogenic microorganisms. Overall, there is a lack of clarity about the importance of viability in the performance of skin grafts. Final considerations: the 3 methods have applicability in graft conservation. The lack of standardization in graft application despite its frequent application and the scarcity of recent studies on the subject justify the present study.

Clinical outcomes after twice frozen-thawed embryo transfer / 中国医师杂志

Objective:To investigate the effect of repeated freezing and thawing on the clinical outcome of embryo transfer.Methods:A total of 48 cycles of twice frozen-thawed embryo (blastocyst) transfer in the reproductive medicine department, Shenzhen Luohu People′s Hospital from 2015 to 2020 were collected as the observation group, and 98 cycles of one frozen-thawed blastocyst transfer in the same period were randomly selected as the control group. The patient′s age, endometrial thickness, average number of transferred embryos, average number of high-quality embryos transferred, embryo recovery rate, implantation rate, clinical pregnancy rate, abortion rate, ectopic pregnancy rate, and live birth rate were compared.Results:There was no significant difference in age, endometrial thickness, number of embryos transferred and high-quality embryos transferred between the two groups (all P>0.05). There was no significant difference in embryo recovery rate, ectopic pregnancy rate, abortion rate and live birth rate between the two groups (all P>0.05). The embryo implantation rate and clinical pregnancy rate in the observation group were significantly lower than those in the control group (32.47% vs 55.70%, 39.58% vs 66.33%, all P<0.05). Conclusions:The clinical pregnancy rate was significantly lower than that of the control group after repeated vitrification of frozen-thawed embryo transfer, which may affect the subsequent developmental potential of embryos.

Establishment and clinical application of Kidd, Diego, Duffy blood group system, Shaanxi Province / 中国输血杂志

【Objective】 To screen individuals with rare blood type of Kidd, Diego, Duffy blood group system among the voluntary blood donor in Shaanxi province and to establish on-line and physical database of rare blood type. 【Methods】 Jk(a-b-)phenotype donors were screened by 2 mol/L urea hemolysis test. Blood donors with Di(a+ b-) phenotype were screened by genotyping; Fy(a-) and D-- phenotype donors were screened by modified antiglobulin assay. 【Results】 Three cases of Jk(a-b-) phenotype were detected out of 158 484 voluntary blood donors. The distribution frequency of Jk(a-b-) phenotype was 0.019‰. Di(a+ b-) phenotype was detected in 2(0.436‰) cases out of 4 586 voluntary blood donor. Fy(a-) phenotype was detected in 8(4.034‰) cases out of 1 983 voluntary blood donors. D-- phenotype was not detected in 29 430 voluntary blood donors. 【Conclusion】 The on-line database of Kidd, Diego, Duffy blood group system had been established by large-size screening of blood donor samples, which can conclude the region′s population distribution and genetic characteristics of RBC blood group. And physical database could further be established using the technology of red blood cells cryopreservation when the conditionspermit, so as to provide the most compatible blood for the clinical effectively improve blood transfusion safety, and provide data support for blood early warning.