High Recovery of Functional Islets Stored at Low and Ultralow Temperatures

BACKGROUND: Poor recovery of islets upon cryopreservation is the main hurdle in islet banking. Pancreatic islets have a poor antioxidative defense mechanism, and exposure of islets to low temperature leads to oxidative stress. AIM: We aimed to investigate whether known compounds such as metformin, γ aminobutyric acid (GABA), docosahexanoic acid (DHA), or eicosapentaenoic acid (EPA) alone or in combination are capable of reducing oxidative stress for better islet recovery upon storage at suboptimal temperatures. METHODS: Islets isolated from mouse pancreas were stored at low temperature (4°C) for 15 days and at ultralow temperature (-196°C) for 30 days with or without additives. After revival from cold storage, islets were assessed by using three methods: (1) specificity by dithizone (DTZ), (2) viability by fluorescein diacetate/propidium iodide (FDA/PI) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide (MTT) assay, and (3) functionality by glucose-stimulated insulin secretion (GSIS). The oxidative status of the islets stored at suboptimal temperatures was determined by both intracellular free radical release (fluorometric analysis) and lipid peroxidation (enzymatic determination). RESULTS: Supplementation with additives led to an improvement in islet survival upon storage at suboptimal temperatures, without depletion of insulin secretory activity, which was comparable to that of controls. The additives acted as cryoprotectants and antioxidants as revealed by high recovery of viable islets and reduction in total reactive oxygen species (ROS) and malonidealdehyde (MDA), respectively. CONCLUSIONS: Our results demonstrate for the first time that supplementation with EPA, DHA, and metformin may lead to higher islet recovery from -196°C storage, enabling proper islet banking.     

Freeze-all,for whom,when, and how

Background: The ‘freeze-all’ practice refers to the cryopreservation of all mature oocytes or viable embryos after ovarian stimulation. The development of the vitrification technique has been crucial to make this approach a reality, since it increases the post-thaw survival rates and permits comparable implantation rates with fresh embryos. Nonetheless, as implantation probabilities are comparable to fresh embryo transfer in normo-responder patients, the freeze- all strategy has demonstrated no benefits overall.Method: Narrative review in which we give an overview of this approach, discuss recent advances in the field, as well as for whom, when and how it is recommended to emply the freeze-all technique.Results: However, there is some clinical evidence that shows its feasibility. Thus, it has been demonstrated that elevation of progesterone at the end of ovarian stimulation decreases the implantation rates after the transfer of day 6 blastocysts in fresh and some uterine pathologies; freeze-all is also the preferred option for patients undergoing pre-implantation genetic testing, since there is an improvement of the results and it allows for inclusion of all blastocysts of the cohort. In high responders, the freeze-all strategy optimizes the response whilst also minimizing the risk of ovarian hyperstimulation syndrome.Conclusion: Due to the different cases that a reproductive expert might encounter, it is essential to highlight benefits and drawbacks of this practice.     

Belgian and European Experience with the European Homograft Bank (EHB) Cryopreserved Allograft Valves.-Assessment of a 20 Year Activity

European Homograft Bank (EHB) has been selecting, preparing, storing and distributing the cryopreserved allograft valves in Belgium and some other European Countries since 1989. It was established in 1988 by a pathologist and the cardiac and vascular surgeons from Belgian and other European centres as an inter-university, international nonprofit association. Due to its neutral behavior and very high quality criteria, European Homograft Bank became one of the prominent heart valve banks in Europe and wider. It collaborates with the transplant coordination in donor selection as well as with the huge network of the implanting surgeons in Belgium and other European Countries. The EHB responsible discusses with the implanting surgeon the allograft selection on basis of the indication and the patients state of emergency.

A total of 8.911 donor heart valves have been evaluated in EHB during the last 20 years. After selection, 5.258 allograft valves (1.996 aortic, 3.189 pulmonary and 73 mitral) were cryopreserved and stored in vapors of liquid nitrogen between 6 weeks and 5 years. A total of 4.516 allograft valves (1.391 aortic, 2.620 pulmonary and 48 mitral) were implanted in the left or right ventricular outflow tract for replacement of the diseased aortic or pulmonary valve and for mitral or tricuspid valve replacement or repair. In 1.380 cases the allograft valves were used for right ventricular outflow tract reconstruction as part of the Ross-procedure, whereas in 668 cases the allograft valve served for replacement of the aortic valve for endocarditis. The most important indications for use of cryopreserved allograft valves were: important cardiac and valve malformation in children, female patients of child-bearing age with diseased cardiac valves, cases with contra-indication for anti-coagulation and the patients with severe endocarditis with septal or annular abscesses. Although the number of the donation increased by year, the available allograft valves in stock are still insufficient to respond to all the surgeons’ request for different indications.     

Preservation of sperm of cancer patients: extent of use and pregnancy outcome in a tertiary infertility center

Sperm cryopreservation is the best modality to ensure future fertility for males diagnosed with cancer. The extent to which cryopreserved sperm is actually used for impregnation, the fertility treatment options that are available and the success rates of these treatments have not been investigated in depth. The medical records of 682 patients who cryopreserved sperm cells due to cancer treatment were analyzed. Seventy of these patients withdrew their frozen sperm for fertility treatments over a 20-year period (most within the first 4 years after cryopreservation). Sperm quality of different malignancies and outcomes of assisted reproduction treatment (ART) for pregnancy achievement in relation to the type of treatment and the type of malignancy were evaluated. The results showed that the rate of using cryo-thawed sperm from cancer patients for fertility treatments in our unit was 10.3%. Sperm quality indices differed between different types of malignancies, with the poorest quality measured in testicular cancer. Conception was achieved in 46 of the 184 ART cycles (25%), and resulted in 36 deliveries. The use of intracytoplasmic sperm injection (ICSI) methodology yielded a significantly higher pregnancy rate (37.4%) than intrauterine insemination (IUI; 11.5%) and was similar to other groups of infertile couples using these modalities. In vitro fertilization (IVF) failed to produce pregnancies. In conclusion, the rate of use of cryopresseved sperm in cancer patients is relatively low (10.3%). Achievement of pregnancies by ICSI presents the best option but when there are enough stored sperm samples and adequate quality, IUI can be employed. Cryopreservation is nevertheless the best option to preserve future fertility potential and hope for cancer patients.     

Successful Cryopreservation of Microvenous Allografts

Vein grafts are used in approximately 20% of microsurgical cases. Although autogenous veins currently form the major source, they are associated with increased operating time and donor site scars. Cryopreserved allograft veins may serve as an alternative source. To our knowledge, cryopreservation of veins (1 mm or less) has not been reported. In this article we have described the process of cryopreserving rat veins (less than 1 mm in diameter) and their preliminary use as interpositional vein grafts.     

Preliminary Results with an Apico-Aortic Valved Conduit (AAVC) Using Polyurethane and Aortic Allograft Valves

Stenosis of the left ventricular outflow tract is one of the most frequent congenital cardiac anomalies in dogs. The subvalvular stenosis is described as a ring of fibrous tissue located in the left ventricular outflow tract immediately below the aortic valve. The best method of surgical treatment in dogs seems to be relief of the aortic obstruction with a ventricular aortic prosthesis. A new trileaflet valve manufactured out of polyurethane has been developed. For the conduit a woven Dacron graft has been used. In 5 male dogs a valved conduit was implanted between the apex of the left ventricle and the aorta thoracica without using hypothermia and/or cardiopulmonary bypass. Four dogs survived the surgical procedure and showed no clinical symptoms postoperatively. Blood flow through the conduit was demonstrated postoperatively by angiocardiography for a maximum of 22 days, the longest surviving time being 8 weeks. Trials for the replacement of the artificial valve in the conduit by a canine aortic allograft valve are in progress. Studies were done with the cryopreservation of fresh aortic valves. Sterilization, cryoconservation, and storage methods for the allograft are described     

Predictors of sperm recovery after cryopreservation in testicular cancer

Our objective was to identify predictors of improved postthaw semen quality in men with testicular cancer banking sperm for fertility preservation. We reviewed 173 individual semen samples provided by 67 men with testicular germ cell tumor (TGCT) who cryopreserved sperm before gonadotoxic treatment between 1994 and 2010 at our tertiary university medical center. Our main outcomes measures were independent predictors for the greater postthaw total motile count (TMC) in men with TGCT. Men with NSGCT were more likely to be younger (P < 0.01) and had high cancer stage (II or III, P < 0.01) compared with men with seminoma. In our multiple regression model, NSGCT histology, use of density gradient purification, and fresh TMC > median fresh TMC each had increased odds of a postthaw TMC greater than median postthaw TMC. Interestingly, age, advanced cancer stage (II or III), rapid freezing protocol, and motility enhancer did not show increased odds of improved postthaw TMC in our models. In conclusion, men with TGCT or poor fresh TMC should consider preserving additional vials (at least 15 vials) before oncologic treatment. Density gradient purification should be routinely used to optimize postthaw TMC in men with TGCT. Larger, randomized studies evaluating cancer stage and various cryopreservation techniques are needed to assist in counseling men with TGCT regarding fertility preservation and optimizing cryosurvival.     

以细胞筛为载体的玻璃化冷冻和慢速程序化冷冻保存人卵巢组织的效果比较

目的比较以细胞筛为载体的玻璃化冷冻与慢速程序化冷冻用于人卵巢组织冷冻的效果。方法人卵巢组织取皮质切块后,随机分为3组:新鲜组织对照组(F组)、慢速程序化冷冻组(S组)及以细胞筛为载体玻璃化冷冻组(V组)。卵巢组织冷冻复苏后,固定切片后行苏木素-伊红染色,观察卵泡形态;使用TdT介导的dUTP缺口末端标记技术,观察卵泡凋亡情况;部分卵巢组织体外培养,隔日采集培养液后检测雌二醇(E2)浓度。比较三组卵泡正常形态率、卵泡凋亡率及E2浓度。结果与F组原始卵泡正常形态率(91.1%)相比,V组原始卵泡正常形态率(88.1%)无显著差异(P0.05),而S组原始卵泡正常形态率(79.6%)显著下降(P0.001);V组初级卵泡正常形态率(74.2%)与S组(73.6%)相似,但两组均低于F组(89.0%)(P0.05);凋亡检测中,3组凋亡率无显著差异(P0.05);体外培养2周,各组E2浓度持续上升,F组E2浓度显著高于S组、V组(P0.001),而S组与V组E2浓度无显著差异(P0.05)。结论以细胞筛为载体的玻璃化冷冻能较好地保存人卵巢组织,复苏后组织体外培养后,还可持续分泌E2。     

Spermatogonial survival after cryopreservation and short-term orthotopic immature human cryptorchid testicular tissue grafting to immunodeficient mice

BACKGROUND: Fertility preservation has become an urgent clinical requisite for prepubertal male cancer patients undergoing gonadotoxic treatment. As these patients do not yet produce spermatozoa for freezing, only immature tissue is available for storage. We studied the survival and proliferative activity of spermatogonia and Sertoli cells after cryopreservation of cryptorchid testicular tissue pieces followed by xenografting for 21 days. METHODS AND RESULTS: Single pieces of tissue from cryptorchid testes (2-9 mm(3)) of young boys (2-12 years) were cryopreserved, thawed and transplanted into the scrotum of mice. Quantitative morphometric and immunohistochemical techniques were used to evaluate the integrity of the tissue, as well as the survival and proliferative capacity of spermatogonia and Sertoli cells before and after freezing/thawing/grafting. Three weeks after grafting, cryopreserved tissue was removed and analysed. Most of the tubules (88.3%) were intact and there was no fibrosis or sclerosis, 14.5% of the initial spermatogonial population remained, as identified by the MAGE A4 antibody, and 32% of these cells showed proliferative activity evidenced by Ki67, compared to 17.8% before cryopreservation and grafting. The number of Sertoli cells was unchanged and 5.1% were Ki67-positive, compared to none at all before freezing and grafting. CONCLUSIONS: Through our orthotopic xenografting model, we have demonstrated the survival and proliferative activity of spermatogonia and Sertoli cells in cryopreserved immature human cryptorchid tissue. Testicular tissue banking may thus prove to be a promising technique for the preservation of fertility in prepubertal boys undergoing oncological treatments. As the stem cell niche is maintained, the cryopreserved tissue can potentially be used for future autotransplantation. In addition, whole tissue freezing does not exclude alternative clinical uses, including isolated cell transplantation after dissociation, selection and enrichment. However, as this work was done on cryptorchid tissue, studies on normal immature testicular tissue, involving longer grafting periods, are needed to demonstrate a differentiation capacity before clinical implementation. Ethical and safety issues should also be addressed.     

High levels of oxidation–reduction potential in frozen-thawed human semen are significantly correlated with poor post-thaw sperm quality

This study examined the relationship between oxidation–reduction potential (ORP) in frozen-thawed semen and the post-thaw sperm parameters. Levels of ORP were measured in 25 samples from men presenting for routine infertility work-up and were expressed as millivolt (mV)/10⁶ sperm/ml. Frozen-thawed samples were examined for post-thaw total motility (TM%), progressive motility (PM%), total sperm count (TSC) and ORP. The cryo-survival rate (CSR) was calculated as post-thaw TM/pre-freeze TM × 100. Data are provided as median and interquartile range (25th and 75th percentiles). The post-thaw TM% (10.0% [4.00%, 15.1%]), PM% (5.88% [2.97%, 9.33%]) and TSC (12.5 [10.0, 17.5] × 10⁶ sperm) were significantly lower than the pre-freeze TM% (45.9% [32.9%, 59.1%], PM% (31.5% [24.4%, 40.0%] and TSC (120 [90, 250] ×10⁶ sperm) (p < .001). Post-thaw ORP (2.62 [2.52, 3.13] mV/10⁶ sperm/ml) was significantly higher than pre-freeze ORP (0.73 [0.54, 1.21] mV/10⁶ sperm/ml; p < .001). The CSR was 21.7% (11.3%, 31.9%). The post-thaw seminal ORP was negatively correlated with post-thaw TM% (r = −.5; p = .02), PM% (r = −.41; p = .03), TSC (r = −.60; p = .03) and CSR (r = −.52; p = .01). Increased levels of ORP are significantly correlated with poor post-thaw sperm quality and CSR.