Review of human oocyte cryopreservation in ART programs: Current challenges and opportunities.

Oocyte cryopreservation has notably increased in recent times, to become an essential part of clinical infertility treatment. Since the 1980s, many improvements in oocyte cryopreservation (OC) have been adopted, including the great advance with the application of vitrification. The commonly used vitrification protocol applies different cryoprotectants (Ethylene glycol and/or DMSO and/or PROH and sucrose and/or Trehalose) and two different steps: firstly, exposure in equilibration solution for 5-15 min, followed by a vitrification solution for 60-90 s at room temperature. The warming method includes a first step for 1 min at 37 °C and 3 subsequent steps at room temperature to remove the cryoprotectant for a total of 9-12 min. In addition, biosafety is a critical aspect to mention, and it is related to devices used during the vitrification, mainly in terms of whether the biological vitrified material comes in direct contact with liquid nitrogen (open vitrification) or not (closed vitrification), where LN2 may contain potentially contaminating viruses or pathogens. Furthermore, during early development major waves of epigenetic reprogramming take place. Recent literature suggests that epigenetic and transcriptomic profiles are sensitive to the stress induced by vitrification, including osmotic shock, temperature, rapid changes of pH and toxicity of cryoprotectants. It is, therefore, important to better understand the potential perturbations of epigenetic modifications that may be associated with the globally used vitrification methods. Therefore, we here discuss the benefits and efficiency of human oocyte vitrification; we also review the evidence surrounding oocyte cryopreservation-related epigenetic modifications and potential epigenetic dysregulations, together with long-term consequences for offspring health.

Cryopreservation of Mammalian Oocytes: Slow Cooling and Vitrification as Successful Methods for Cryogenic Storage.

Two basic methods for the laboratory-focused cryopreservation of mammalian oocytes are described, based on work with murine oocytes. One method uses a relatively low concentration of the cryoprotectant propanediol plus sucrose and requires controlled rate cooling equipment to achieve a slow cooling rate. This method has also produced live births from cryopreserved human oocytes. The second method, which is described here, employs a high concentration of the cryoprotectant dimethyl sulfoxide plus a low concentration of polyethylene glycol. This is a vitrification method, which involves ultra-rapid cooling by plunging standard straws into liquid nitrogen vapor, hence avoiding the need for specialized equipment, but requires technical ability to manipulate the oocytes quickly in the highly concentrated cryoprotectant solutions. Murine oocytes that have been vitrified using this technique have resulted in live births. Vitrification using other cryoprotectant mixtures is now a popular clinically accepted method for cryobanking of human oocytes.

Reliability and Accuracy of Clinical Assessment and Digital Image Analysis for Steatosis Evaluation in Discarded Human Livers.

BACKGROUND: Accurate assessment of steatosis in procured livers is crucial to reduce the poor outcome associated with high-grade steatosis and to optimize the utilization of donor grafts. Clinical examination and digital image analysis (DIA) have been used for steatosis evaluation, but the validity of these methods is debated. This study aimed to compare these methods with standard histology for assessment of steatosis severity in human livers and to evaluate a revised classification system for automated fat measurement. METHODS: Clinical assessment of liver steatosis at time of retrieval and automated measurement were compared with standard histology in paraffinized and hematoxylin and eosin-stained slides, using a 4-grade scale for ordinal data and percentages for numerical values. RESULTS: Analysis of 42 human livers that were retrieved but not transplanted showed that clinical examination was not reliable for assigning steatosis grades (κw, 0.12; 95% CI, -0.06 to 0.30), overestimated steatosis severity, and had an accuracy of 67% for discriminating low- and high-grade steatosis. Digital image analysis had a substantial agreement on absolute fat percentage (intraclass correlation coefficient, 0.76; 95% CI, 0.63-0.84) and steatosis grades (κw, 0.70; 95% CI, 0.57-0.82), with 88% accuracy using the revised classification system. CONCLUSIONS: Clinical assessment of steatosis is inaccurate, and relying on this method alone could result in unnecessary discard of livers. Digital image analysis is feasible with higher accuracy and reliability, but further clinical studies are required to evaluate its clinical validity.

Urinary Neutrophil Gelatinase Associated Lipocalins (NGALs) predict acute kidney injury post liver transplant.

BACKGROUND: Acute Kidney Injury, a common complication of liver transplant, is associated with a significant increase in the risk of morbidity, mortality and graft loss. Current diagnostic criteria leaves a delay in diagnosis allowing further potential irreversible damage. Early biomarkers of renal injury are of clinical importance and Neutrophil Gelatinase Associated Lipocalins (NGALs) and Syndecan-1 were investigated. METHODS: AKI was defined according to the Acute Kidney Injury Network criteria. Urine and blood samples were collected pre-operatively, immediately post-op and 24 h post reperfusion to allow measurement of NGAL and Syndecan-1 levels. RESULTS: 13 of 27 patients developed an AKI. Patients who developed AKI had significantly higher peak transaminases. Urinary NGAL, plasma NGAL and Syndecan-1 levels were significantly elevated in all patients post reperfusion. Urinary NGAL levels immediately post-op were significantly higher in patients who developed an AKI than those that didn't [1319 ng/ml vs 46.56 ng/ml, p ≤ 0.001]. ROC curves were performed and urinary NGAL levels immediately post-op were an excellent biomarker for AKI with an area under the curve of 0.948 (0.847-1.00). CONCLUSIONS: Urinary NGAL levels measured immediately post-op accurately predict the development of AKI and their incorporation into clinical practise could allow early protocols to be developed to treat post transplant AKI.

Neutrophil Gelatinase-Associated Lipocalin (NGAL) in predicting acute kidney injury following orthotopic liver transplantation: A systematic review.

BACKGROUND: Acute kidney injury (AKI) is common after orthotopic liver transplantation (OLT) usually occurring early post-transplant. Multiple causes include graft preservation injury, blood loss, hypotension but also severity of recipient liver disease. Early intervention in AKI has both short and long term patient benefits. Unfortunately there are no current clinical biomarkers of early AKI. AIM: To assess the value of NGAL in predicting AKI following OLT. METHODS: Ovid MEDLINE and EMBASE were searched between the years of 2000 and 2017 for studies using keywords: Neutrophil Gelatinase Associated Lipocalin or NGAL variants combined with synonyms for liver transplantation. RESULTS: 96 studies were identified. 11 studies including 563 patients were considered suitable for analysis. Both urinary (uNGAL) and plasma NGAL (pNGAL) measurement were found to predict AKI after liver transplantation. Optimal reported area under the receiver-operator characteristics curve (AUROC) values of 0.5-0.83 and 0.54-0.86 respectively. CONCLUSIONS: NGAL is a good predictor of early AKI post OLT although there is considerable variation in the published results. Further studies with prospectively defined cut-off values, standardized definitions of AKI and rigorous data reporting should be conducted to establish its clinical usefulness and limitations.

Anaemia in patients with chronic liver disease and its association with morbidity and mortality following liver transplantation.

Pre-operative anaemia and the need for intra-operative transfusion have been associated with increased morbidity and mortality following cardiac and major non-cardiac surgery. Anaemia is highly prevalent in patients with severe chronic liver disease. Whether this correlates with an altered morbidity and mortality following liver transplant has not been established. METHODS: Prospectively collected data was analysed for the period 1998-2012. Donor and recipient characteristics, blood profiles and complications were recorded. Graft and patient survival was calculated. All patients were followed up for 1 year or until death. Pre-operative haemoglobin levels were correlated with patient demographics and outcome using a binary logistic regression analysis. RESULTS: Pre-operative anaemia, according to WHO criteria, occurred in 73% of patients. Anaemia was more common with advanced liver disease (higher MELD score). As MELD score increased, Haemoglobin levels dropped. Anaemic patients were more commonly transfused (p < 0.001), spent longer ventilated (7 day vs 5 days, p = 0.005) and required longer ITU stays (8 days vs 6 days, p = 0.015). Pre-operative anaemia did not correlate with patient morbidity or mortality. CONCLUSIONS: Reduced haemoglobin levels reflect the severity of chronic liver disease but are not an independent risk factor for a poor outcome following liver transplantation.

An Evaluation of Ischaemic Preconditioning as a Method of Reducing Ischaemia Reperfusion Injury in Liver Surgery and Transplantation.

Liver Ischaemia Reperfusion (IR) injury is a major cause of post-operative liver dysfunction, morbidity and mortality following liver resection surgery and transplantation. There are no proven therapies for IR injury in clinical practice and new approaches are required. Ischaemic Preconditioning (IPC) can be applied in both a direct and remote fashion and has been shown to ameliorate IR injury in small animal models. Its translation into clinical practice has been difficult, primarily by a lack of knowledge regarding the dominant protective mechanisms that it employs. A review of all current studies would suggest that IPC/RIPC relies on creating a small tissue injury resulting in the release of adenosine and l-arginine which act through the Adenosine receptors and the haem-oxygenase and endothelial nitric oxide synthase systems to reduce hepatocyte necrosis and improve the hepatic microcirculation post reperfusion. The next key step is to determine how long the stimulus requires to precondition humans to allow sufficient injury to occur to release the potential mediators. This would open the door to a new therapeutic chapter in this field.

Remote ischaemic preconditioning in orthotopic liver transplantation (RIPCOLT trial): a pilot randomized controlled feasibility study.

BACKGROUND: Ischaemia Reperfusion (IR) injury is a major cause of morbidity, mortality and graft loss following Orthotopic Liver Transplantation (OLT). Utilising marginal grafts, which are more susceptible to IR injury, makes this a key research goal. Remote Ischaemic Preconditioning (RIPC) has been shown to ameliorate hepatic IR injury in experimental models. Whether RIPC can reduce IR injury in human liver transplant recipients is unknown. METHODS: Forty patients undergoing liver transplantation were randomized to RIPC or a sham. RIPC was induced through three 5 min cycles of alternate ischaemia and reperfusion of the left leg prior to surgery. Data on clinical outcomes was collected prospectively. Per-operative cytokine levels were measured. RESULTS: Fourty five of 51 patients approached (88%) were willing to enroll in the study. Five patients were excluded and 40 randomized, of which 20 underwent RIPC which was successfully completed in all patients. There were no complications following RIPC. Median day 3 AST levels were slightly higher in the RIPC group (221 IU vs 149 IU, p = 1.00). CONCLUSIONS: RIPC is acceptable and safe in liver transplant recipients. This study has not demonstrated evidence of a reduction in short-term measures of IR injury. Longer follow up will be required and consideration of an altered protocol.

Cryoprotectants: A review of the actions and applications of cryoprotective solutes that modulate cell recovery from ultra-low temperatures.

Cryopreservation has become a central technology in many areas of clinical medicine, biotechnology, and species conservation within both plant and animal biology. Cryoprotective agents (CPAs) invariably play key roles in allowing cells to be processed for storage at deep cryogenic temperatures and to be recovered with high levels of appropriate functionality. As such, these CPA solutes possess a wide range of metabolic and biophysical effects that are both necessary for their modes of action, and potentially complicating for cell biological function. Early successes with cryopreservation were achieved by empirical methodology for choosing and applying CPAs. In recent decades, it has been possible to assemble objective information about CPA modes of action and to optimize their application to living systems, but there still remain significant gaps in our understanding. This review sets out the current status on the biological and chemical knowledge surrounding CPAs, and the conflicting effects of protection versus toxicity resulting from the use of these solutes, which are often required in molar concentrations, far exceeding levels found in normal metabolism. The biophysical properties of CPAs that allow them to facilitate different approaches to cryogenic storage, including vitrification, are highlighted. The topics are discussed with reference to the historical background of applying CPAs, and the relevance of cryoprotective solutes in natural freeze tolerant organisms. Improved cryopreservation success will be an essential step in many future areas such as regenerative medicine, seed banking, or stem cell technology. To achieve this, we will need to further improve our understanding of cryobiology, where better and safer CPAs will be key requirements.

The Grand Challenges of Organ Banking: Proceedings from the first global summit on complex tissue cryopreservation.

The first Organ Banking Summit was convened from Feb. 27 - March 1, 2015 in Palo Alto, CA, with events at Stanford University, NASA Research Park, and Lawrence Berkeley National Labs. Experts at the summit outlined the potential public health impact of organ banking, discussed the major remaining scientific challenges that need to be overcome in order to bank organs, and identified key opportunities to accelerate progress toward this goal. Many areas of public health could be revolutionized by the banking of organs and other complex tissues, including transplantation, oncofertility, tissue engineering, trauma medicine and emergency preparedness, basic biomedical research and drug discovery - and even space travel. Key remaining scientific sub-challenges were discussed including ice nucleation and growth, cryoprotectant and osmotic toxicities, chilling injury, thermo-mechanical stress, the need for rapid and uniform rewarming, and ischemia/reperfusion injury. A variety of opportunities to overcome these challenge areas were discussed, i.e. preconditioning for enhanced stress tolerance, nanoparticle rewarming, cyroprotectant screening strategies, and the use of cryoprotectant cocktails including ice binding agents.

Cryopreservation of mammalian oocytes.

Two methods for the laboratory-focused cryopreservation of mammalian oocytes are described, based on work with murine oocytes. One method uses a relatively low concentration of the cryoprotectant propanediol plus sucrose and requires controlled rate cooling equipment to achieve a slow cooling rate. Such a method has also produced live births from cryopreserved human oocytes. The second method described employs a high concentration of the cryoprotectant dimethyl sulfoxide plus a low concentration of polyethylene glycol. This is a vitrification method which involves ultrarapid cooling by plunging standard straws into liquid nitrogen vapor, hence avoiding the need for specialized equipment, but requires technical ability to manipulate the oocytes quickly in the highly concentrated cryoprotectant solutions. Murine oocytes vitrified using this technique has resulted in live births.

Endothelial nitric oxide synthase and heme oxygenase-1 act independently in liver ischemic preconditioning.

BACKGROUND: ischemic preconditioning (IPC) protects against liver ischemia-reperfusion (IR) injury. The mechanism involves nitric oxide metabolism but the importance of endothelial nitric oxide synthase (eNOS) has not been established. Heme oxygenase-1 (HO-1) protects against liver IR but it is unclear if this depends on nitric oxide synthase. MATERIALS AND METHODS: A mouse model of IPC with liver IR using wild-type (WT) and eNOS transgenic knockout (eNOS-/-) mice was developed to study the role of eNOS and its relationship to HO-1. Serum alanine aminotransferase level, liver histopathologic injury scores, and liver microcirculatory blood flow were measured. Western blots measured liver HO-1/2, eNOS, phosphorylated eNOS, inducible nitric oxide synthase, and reverse transcription-polymerase chain reaction (HO-1). A set of 24-h recovery experiments was undertaken on WT mice with measurement of serum alanine aminotransferase level, histologic injury score, and HO-1 by Western blot. RESULTS: In WT animals, IPC preceding IR resulted in a reduction in hepatocellular and histologic injury, and improvement in parenchymal perfusion. In contrast, IPC in the eNOS-/- model did not protect the animals from IR injury. There was no difference between the eNOS and phosphorylated eNOS expression in all the WT groups. HO-1 protein was not detected in the nonrecovery groups but HO-1 messenger RNA was detected in all groups. In WT recovery experiments, IPC was protective against IR injury. HO-1 protein was detected in the IPC + IR and IR only groups but not in the sham group. CONCLUSIONS: This study developed and used an eNOS-/- model to demonstrate that eNOS mediates protection against liver IR injury by IPC. The eNOS expression and activity and HO-1 expression are increased independently in liver IPC and IR, with HO-1 expression increased in the later stages of IPC and IR.

Molecular mechanisms of liver ischemia reperfusion injury: insights from transgenic knockout models.

Ischemia reperfusion injury is a major obstacle in liver resection and liver transplantation surgery. Understanding the mechanisms of liver ischemia reperfusion injury (IRI) and developing strategies to counteract this injury will therefore reduce acute complications in hepatic resection and transplantation, as well as expanding the potential pool of usable donor grafts. The initial liver injury is initiated by reactive oxygen species which cause direct cellular injury and also activate a cascade of molecular mediators leading to microvascular changes, increased apoptosis and acute inflammatory changes with increased hepatocyte necrosis. Some adaptive pathways are activated during reperfusion that reduce the reperfusion injury. IRI involves a complex interplay between neutrophils, natural killer T-cells cells, CD4+ T cell subtypes, cytokines, nitric oxide synthases, haem oxygenase-1, survival kinases such as the signal transducer and activator of transcription, Phosphatidylinositol 3-kinases/Akt and nuclear factor κß pathways. Transgenic animals, particularly genetic knockout models, have become a powerful tool at elucidating mechanisms of liver ischaemia reperfusion injury and are complementary to pharmacological studies. Targeted disruption of the protein at the genetic level is more specific and maintained than pharmacological inhibitors or stimulants of the same protein. This article reviews the evidence from knockout models of liver IRI about the cellular and molecular mechanisms underlying liver IRI.

Cryopreservation of alginate encapsulated mesenchymal stromal cells.

Human mesenchymal stromal cells (MSCs) can differentiate into various cell types, which makes them attractive for regenerative medicine and tissue engineering. Encapsulation of MSCs in alginate microspheres (AMS) is a novel and promising approach of tissue engineering. Application and research of such cell-hydrogel systems require selection of adequate cryopreservation protocols. In this study we investigated the response of MSCs encapsulated in AMS to different cryopreservation protocols. Bone marrow MSCs either encapsulated in AMS and or as cells in suspension, were cryopreserved with 5% and 10% of dimethyl sulfoxide (ME2SO) using conventional 2-step slow cooling (protocol 1). The viability and metabolism of MSCs in AMS following cryopreservation with 5% Me2SO were lower than in the group cryopreserved with 10% Me2SO. MSCs in suspension were more resistant to cryopreservation than cells in AMS when cryopreserved with 5% Me2SO, although when using a concentration of 10% Me2SO, no differences were detected. Comparisons of the viability and metabolic activity of MSC cryopreserved either in AMS or as cell suspensions with 10% ME2SO using protocol 1 (2-step cooling), protocol 2 (3-step slow cooling with induced ice nucleation) or protocol 3 (rapid 1-step freezing), showed that the highest viabilities and metabolic rates were obtained following cryopreservation of MSCs in AMS by protocol 2 (with controlled ice nucleation). Cryopreservation with protocol 3 resulted in critical damage of the encapsulated MSCs. After cryopreservation by protocol 2, AMS encapsulated MSCs were capable of achieving multilineage differentiation directed towards osteogenic, adipogenic and chondrogenic lineages. The data obtained indicate that cryo-banking of AMS encapsulated MSCs is feasible for future regenerative medicine projects.

Organ Preservation: Current Concepts and New Strategies for the Next Decade.

SUMMARY: Organ transplantation has developed over the past 50 years to reach the sophisticated and integrated clinical service of today through several advances in science. One of the most important of these has been the ability to apply organ preservation protocols to deliver donor organs of high quality, via a network of organ exchange to match the most suitable recipient patient to the best available organ, capable of rapid resumption of life-sustaining function in the recipient patient. This has only been possible by amassing a good understanding of the potential effects of hypoxic injury on donated organs, and how to prevent these by applying organ preservation. This review sets out the history of organ preservation, how applications of hypothermia have become central to the process, and what the current status is for the range of solid organs commonly transplanted. The science of organ preservation is constantly being updated with new knowledge and ideas, and the review also discusses what innovations are coming close to clinical reality to meet the growing demands for high quality organs in transplantation over the next few years.

In vivo study of a model tissue-engineered small-diameter vascular bypass graft.

Conventionally used vascular grafts such as polyester (Dacron) or expanded polytetrafluoroethylene perform inadequately as small-diameter vascular bypass grafts (SDBGs). SDBGs, which can maintain long-term patency and those that could potentially evolve with the somatic growth, are highly desirable in vascular surgery and thus research into tissue-engineered blood vessels (TEBVs) is of keen interest. A TEBV was developed by seeding endothelial cells onto a collagen matrix that was cross-linked and contracted by smooth muscle cells (SMCs). A polyester graft served as a scaffold. Recovery studies (12 TEBVs and seven controls) were carried out to assess in vivo endothelialization and long-term patency of TEBVs. Hemodynamic observations indicated para-anastomotic turbulences and high shear stress at anastomosis. Recovery studies demonstrated confluent endothelialization, thrombus-free surfaces, and patent TEBVs in all cases. Graft incorporation and neovascularization of the scaffold occurred in both hybrid and control grafts. However, thickened neointima formation occurred in TEBV grafts, which was most likely caused by the rigidity of polyester scaffold. Significant perigraft inflammatory changes could be observed in both TEBVs and control grafts at 1, 4, and 8 weeks. In conclusion, the TEBVs demonstrated satisfactory performance as an infra-renal-aortic graft in a porcine model. The TEBV serves as a promising model and facilitates the development of a TEBV in a clinical setting, potentially with human stem cells and with more biocompatible, biodegradable scaffolds that are mechanically more compliant with natural vessels.

Effect of remote ischemic preconditioning on liver ischemia/reperfusion injury using a new mouse model.

Ischemic preconditioning of remote organs (RIPC) reduces liver ischemia/reperfusion (IR) injury in the rabbit and rat. Mice are the only species available with a large number of transgenic strains. This study describes development and validation of a mouse model of hindlimb RIPC that attenuates liver IR injury. Mice were allocated to 4 groups: (1) Sham surgery; (2) RIPC: 6 cycles of 4 × 4 minutes ischemia/reperfusion of hindlimb; (3) IR: 40 minutes lobar (70%) hepatic ischemia and 2 hours reperfusion; (4) RIPC+IR: RIPC followed by IR group procedures. Plasma liver aminotransferases and hepatic histopathological and transmission electron microscopy studies were performed at the end of the experiment. Hepatic microcirculatory blood flow was measured throughout the experiment. Postoperative complications and animal survival were evaluated. Hindlimb RIPC using a tourniquet resulted in limb paralysis. Hindlimb RIPC using direct clamping of the femoral vessels showed no side effects. Compared to liver IR alone, RIPC+IR reduced plasma aminotransferases (P < 0.05) and histopathological and ultrastructural features of injury. Hepatic microcirculatory blood flow was preserved in the RIPC+IR compared to IR group (P < 0.05). There was no mortality in any of the groups. By demonstrating a consistent improvement in these features of liver IR injury with antecedent hindlimb RIPC and by minimizing experimental confounding variables, we validated this mouse model. In conclusion, we describe a validated mouse model of hindlimb RIPC that reduces liver IR injury. With the availability of transgenic mice strains, this model should prove useful in unraveling the mechanisms of protection of hindlimb RIPC.

Access to organs and tissues for research from the organ donor pool within the UK.

The donor organ transplant scenario offers one potential route to access high-quality human organs and tissues for research. There are well-established networks for co-ordinating organ donation events across many countries, including the UK, which include robust mechanisms for obtaining consent for ethically-approved research. Within the UK, the challenge for the next few years is to facilitate this research donation with respect to regulatory pathways directed by the Human Tissue Act, which covers all aspects of access to human tissues.