Seven days ex vivo perfusion of whole ewe ovaries with follicular maturation and oocyte retrieval: towards the development of an alternative fertility preservation method.

Fertility preservation methods for prepubertal women about to undergo gonadotoxic chemo and/or radiation therapy are limited. Therefore, the aim of this study was to investigate the feasibility to develop an alternative fertility preservation method based on an ex vivo perfusion platform for whole ewe ovaries. Thirteen ewe ovaries were divided into two groups (group 1 and 2) that were perfused in a bioreactor for up to 7days. Group 1 (n =3) were stimulated with human menopausal gonadotropin (hMG) administered in single daily dose, while group 2 (n =10) were stimulated continuously for 24h. The perfused ovaries in group 1 showed no significant differences in follicular density, sub-follicular morphology and oocyte quality after ischaemia and after ex vivo perfusion compared with non-perfused control ovaries. The perfused ovaries in group 2 showed a significant decrease in the follicular reserve and oocyte quality compared with the control group. In total, 16 GV-MI oocytes were retrieved from both groups. This study describes for the first time the ex vivo maintenance of viable follicles of ewe ovaries with oocyte integrity and the retrieval of oocytes after ex vivo hormonal perfusion with two different protocols for up to 7days.

Luminal polyethylene glycol solution delays the onset of preservation injury in the human intestine.

The organ damage incurred during the cold storage (CS) of intestinal grafts has short and long-term consequences. Animal studies suggest that additional luminal preservation (LP) with polyethylene glycol (PEG) may alleviate this damage. This study aims to validate these findings using human intestines. Ileal segments, perfused intravascularly with IGL-1 solution, were procured from 32 multiorgan donors and divided into two parts: one containing a PEG 3350-based solution introduced luminally (LP group) and another one without luminal treatment (control). Sampling was performed after 4 h, 8 h, 14 h, and 24 h of CS. Histology was assessed using the Chiu/Park score. Tight junctions (TJ), several inflammatory markers, and transcription factors were examined by immunofluorescence, ddPCR, and western blot. Tissue water content (edema) was also measured. Apoptotic activity was assessed with caspase -2, -3, and -9 assays. LP significantly lowered mucosal injury at all time points. Redistribution of TJ proteins occurred earlier and more severely in the control group. After 24 h of CS, LP intestines showed an emerging unfolding protein response. Increased caspase-3 and -9 activity was found in the control group. The current results indicate that luminal PEG is safe and effective in reducing damage to the intestinal epithelium during CS.

The development of an extended normothermic ex vivo reperfusion model of the sheep uterus to evaluate organ quality after cold ischemia in relation to uterus transplantation.

INTRODUCTION: Uterus transplantation has recently proved that infertility in women with uterine factor infertility can be cured. It is still an experimental procedure with numerous critical details remaining to be established, including tolerance to warm and cold ischemic insults. In preparation for human uterus transplantation trials, most teams use the sheep as a model system for research and team training, since the vasculature and the uterus is of similar size as in the human. We, therefore, aimed to develop an ex vivo sheep uterus reperfusion platform that mimics the reperfusion situation so that initial assessments and comparisons can be performed without the need for costly and labor-intensive in vivo transplantation experiments. MATERIAL AND METHODS: Isolated sheep uteri were perfused with the preservation solution IGL-1 and were then exposed to cold ischemia for either 4 (n = 6) or 48 hours (n = 7). Uteri were then reperfused for 48 hours under normothermic conditions with an oxygenated recirculating perfusate containing growth factors and synthetic oxygen carriers. Histological and biochemical analysis of the perfusate was conducted to assess reperfusion injury. RESULTS: Quantification of cell density indicated no significant edema in the myometrium or in the endometrium of uteri exposed to 4 hours cold ischemia and then a normothermic ex vivo reperfusion for 48 hours. Only the outer serosa layer and the inner columnar luminal epithelial cells were affected by the reperfusion. However, a much faster and severe reperfusion damage of all uterine layers were evident during the reperfusion experiment following 48 hours of cold ischemia. This was indicated by major accumulation of extracellular fluid, presence of apoptotic-labeled glandular epithelial layer and vascular endothelium. A significant accumulation of lactate was measured in the perfusate with a subsequent decrease in pH. CONCLUSIONS: We developed a novel ex vivo sheep uterus model for prolonged perfusion. This model proved to be able to distinguish reperfusion injury-related differences associated to organ preservation. The experimental setup is a platform that can be used to conduct further studies on uterine ischemia- and reperfusion injury that may lead to improved human uterus transplantation protocols.

Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 Gene Editing Technique in Xenotransplantation.

Genetically modified pigs have been considered favorable resources in xenotransplantation. Microinjection of randomly integrating transgenes into zygotes, somatic cell nuclear transfer, homologous recombination, zinc finger nucleases, transcription activator-like effector nucleases, and most recently, clustered regularly interspaced short palindromic repeats-cas9 (CRISPR/Cas9) are the techniques that have been used to generate these animals. Here, we provide an overview of the CRISPR approaches that have been used to modify genes which are vital in improving xenograft survival rate, including cytidine monophosphate-N-acetylneuraminic acid hydroxylase, B1,4N-acetylgalactosaminyltransferase, isoglobotrihexosylceramide synthase, class I MHC, von Willebrand factor, C3, and porcine endogenous retroviruses. In addition, we will mention the importance of potential candidate genes which could be targeted using CRISPR/Cas9.

Significantly Accelerated Wound Healing of Full-Thickness Skin Using a Novel Composite Gel of Porcine Acellular Dermal Matrix and Human Peripheral Blood Cells.

Here we report the fabrication of a novel composite gel from decellularized gal-gal-knockout porcine skin and human peripheral blood mononuclear cells (hPBMCs) for full-thickness skin wound healing. Decellularized skin extracellular matrix (ECM) powder was prepared via chemical treatment, freeze drying, and homogenization. The powder was mixed with culture medium containing hyaluronic acid to generate a pig skin gel (PSG). The effect of the gel in regeneration of full-thickness wounds was studied in nude mice. We found significantly accelerated wound closure already on day 15 in animals treated with PSG only or PSG + hPBMCs compared to untreated and hyaluronic acid-treated controls (p < 0.05). Addition of the hPBMCs to the gel resulted in marked increase of host blood vessels as well as the presence of human blood vessels. At day 25, histologically, the wounds in animals treated with PSG only or PSG + hPBMCs were completely closed compared to those of controls. Thus, the gel facilitated generation of new skin with well-arranged epidermal cells and restored bilayer structure of the epidermis and dermis. These results suggest that porcine skin ECM gel together with human cells may be a novel and promising biomaterial for medical applications especially for patients with acute and chronic skin wounds.