Descemet Membrane Endothelial Keratoplasty: NGS Metagenomics-Optimized Tissue Preparation and Surgical Technique. Doctoral Thesis

Title: Next-generation sequencing for the detection of microorganisms present in human donor corneal preservation medium. Aim: To detect the presence of microorganisms in the storage media of human donor corneas using next-generation sequencing method. Methods: Seven samples from organ culture (OC) group (Cornea Max, Eurobio, Les Ulis, France) with one control (sterile media without any cornea) and seven samples from hypothermic storage group (Cornea Cold, Eurobio) with one control were used for this study. The corneas were placed in the respective storage media for 14 days before collecting the samples. Storage media (2 mL) from each sample were collected in RNAase-free tubes and shipped for ribosomal RNA sequencing of 16S and 18S. Simultaneously, another 1 mL of media sample was used for conventional diagnostic method (CDM) using Bactec instruments. Results: In both, OC and hypothermic storage and control samples, the most abundant genera were Pseudomonas, Comamonas, Stenotrophomonas, Alcanivorax, Brevundimonas and Nitrobacter. Acidovorax, Acetobacter and Hydrogenophilus were detected mostly in the hypothermic storage group. The most abundant fungal pathogen detected belonged to the genus Malassezia, which was found in both the storage conditions. CDM was negative for microorganisms in all the samples. Conclusion: Metagenomics provides full taxonomic profiling of the detected genomic material of the organisms and thus has the potential to deliver a much wider microbiological diagnostic approach than CDM. The costs and turnaround time need to be reduced, and; the detection of viable organisms would help this technology to be introduced into routine clinical practice.

Descemet Membrane Endothelial Keratoplasty: NGS Metagenomics-Optimized Tissue Preparation and Surgical Technique. Summary of the Doctoral Thesis

Title: Next-generation sequencing for the detection of microorganisms present in human donor corneal preservation medium. Aim: To detect the presence of microorganisms in the storage media of human donor corneas using next-generation sequencing method. Methods: Seven samples from organ culture (OC) group (Cornea Max, Eurobio, Les Ulis, France) with one control (sterile media without any cornea) and seven samples from hypothermic storage group (Cornea Cold, Eurobio) with one control were used for this study. The corneas were placed in the respective storage media for 14 days before collecting the samples. Storage media (2 mL) from each sample were collected in RNAase-free tubes and shipped for ribosomal RNA sequencing of 16S and 18S. Simultaneously, another 1 mL of media sample was used for conventional diagnostic method (CDM) using Bactec instruments. Results: In both, OC and hypothermic storage and control samples, the most abundant genera were Pseudomonas, Comamonas, Stenotrophomonas, Alcanivorax, Brevundimonas and Nitrobacter. Acidovorax, Acetobacter and Hydrogenophilus were detected mostly in the hypothermic storage group. The most abundant fungal pathogen detected belonged to the genus Malassezia, which was found in both the storage conditions. CDM was negative for microorganisms in all the samples. Conclusion: Metagenomics provides full taxonomic profiling of the detected genomic material of the organisms and thus has the potential to deliver a much wider microbiological diagnostic approach than CDM. The costs and turnaround time need to be reduced, and; the detection of viable organisms would help this technology to be introduced into routine clinical practice.

Killing efficacy of a new hypothermic corneal storage medium against the micro-organisms frequently found in human donor cornea intended for transplantation

ObjectiveTo study the in vitro killing efficacy of Kerasave (AL.CHI.MI.A Srl), a medium provided with amphotericin B tablet for hypothermic storage of human donor corneas, against relevant contaminants associated with postkeratoplasty infections.Methods and AnalysisThe antimicrobial activity of Kerasave was determined after 0, 3 and 14 days of incubation at 2°C–8°C, inoculating Kerasave and the control medium with 105–106 colony forming units (CFU) of Candida albicans (CA), Fusarium solani (FS), Aspergillus brasiliensis (AB), Staphylococcus aureus (SA), Enterococcus faecalis (EF), Bacillus subtilis spizizenii (BS), Pseudomonas aeruginosa (PA), Enterobacter cloacae (EC) and Klebsiella pneumoniae (KP). Log10 reductions at different time intervals were determined by assessing the number of viable CFU using the serial dilution plating technique.ResultsAfter 3 days, Kerasave induced the highest log10 decrease in the concentrations of KP, PA, CA and EC (5.37, 4.15, 2.97 and 2.67, respectively; all p<0.001). The log10 decreases of SA and EF were 2.27 and 2.11, respectively (all p<0.001). The lowest log10 decrease was observed in BS, AB and FS concentrations (0.25, 0.30 and 0.67, respectively; p<0.001 for BS and AB and p=0.004 for FS). After 14 days, the microbial count of CA, FS, SA, EF, PA and EC further decreased (p=0.006 for FS; p<0.001 for the others).ConclusionKerasave effectively reduced or kept unchanged the microbial concentration of almost all tested strains after 3 days. Thus, this novel medium represents a valuable tool to control the microbial contamination of human donor corneas during hypothermic storage for up to 14 days before transplantation.

PSXV-14 Level of spontaneous oxidative modification of sperm plasma proteins in stallions of different ages

The objective of the study was to assess the level of spontaneous oxidative modification of sperm plasma proteins (OMP) in young and mature stallions as indicators of the severity of oxidative stress. Sperm from 12 stallions aged 14–18 years (average age 15.8±1.9 years) (Group I) and 12 stallions aged 3–5 years (average age 4.3±0.6 years; Group II) was obtained during the breeding season (February-May). Each ejaculate was evaluated by volume, concentration, total number of spermatozoa, as well as by the progressive motility and survival of spermatozoa in diluted sperm during its hypothermic storage at +4°C. For a quantitative assessment of the OMP level, a spectrophotometric analysis of 2,4-dinitrophenylhydrazones formed during the interaction of carbonyl derivatives of proteins with 2,4-dinitrophenylhydrazine was carried out (SF-2000, Russia). The level of aldehyde- and ketone-dinitrophenylhydrazones of neutral (ADNPHn, KDNPHn) and basic (ADNPGb, KDNPGb), and the total level of OMP (S OMP) was measured. Significance of differences was assessed by the Mann-Whitney test, the results were statistically significant at P ≤ 0.05. The total level of spontaneous OMP of sperm plasma in aged stallions is statistically significantly higher than in young stallions (531.7 and 384.3 Uod/g protein, respectively, P &lt; 0.05). This indicates a higher degree of oxidative stress in Group I, a more significant damage to the amino acid residues of sperm plasma proteins. In Group I a shift in the absorption spectrum towards neutral aldehyde derivatives (367.6 and 255.8 Uod/g protein, respectively, P &lt; 0.05) was noted. We assume that the pro-oxidant systems in a stallion body increasingly prevail over the protective ones with the age. This leads to an increase in the level of OMP in the sperm and a decrease in the survival of spermatozoa during the hypothermic storage of diluted sperm. Authors acknowledge financial support from Russian Science Foundation, Grant No: 20-16-00101.

Hypothermic storage in salt-free preservative solution alter motility duration in sterlet sperm

Together with concentration, motility is one of the most important characteristics of sturgeon sperm, determining its quality and suitability for insemination. After activation in water, the duration of progressive sperm motility is also important, and this time should not be less than that required for fertilization. Motility of spermatozoa depends on their physiological state, maturity, age and intracellular reserves of macroergic substances. During hypothermic storage, the percentage of spermatozoa that can be activated decreases progressively due to depletion of ATP supply or cell death. To improve the hypothermic storage of sterlet sperm, we have developed salt-free preservative solution ISGT-80 based on glucose and trehalose. During storage of sterlet sperm specimens from 20 males in ISGT-80 for 18 days, we observed, along with a progressive decrease in the percentage of motile spermatozoa, an alteration in the duration of their motility. On the 3rd to 6th day of storage, the time of half-loss of motility (τ50) increased significantly by approximately 1 min on average compared with fresh samples, then gradually decreased, however, not descending to the initial value. The reasons for this prolongation of motility are not clear, but we do not exclude the predominant death of spermatozoa with a short motility duration in the first days of storage and selection in favor of long-lived spermatozoa. Such gametic selection can lead to a shift in allele frequencies at heterozygous loci in the offspring. Thus, hypothermic storage of sperm could become an attractive subject for genetic research with the aim of developing new selection tools in sturgeon breeding.

Effect of ozonation on resistance of ovine and human erythrocytes to hypothermic storage

Long-term hypothermic storage of animal blood can lead to the loss of its quality and can cause complications in recipient animals after transfusion, so the search for new methods of increasing the preservation of erythrocytes after hypothermic storage continues. The article presents the data of the ozonation effect on the preservation rate of ovine and human erythrocytes during hypothermic storage with Alsever’s solution and mannitol medium. Hemolysis, osmotic fragility and distribution density of ovine and human erythrocytes by the sphericity index were determined at different stages of hypothermic storage. The ovine erythrocytes in the control had a lower osmotic resistance compared to human erythrocytes; however, their osmotic fragility did not change significantly after hypothermic storage for 8 weeks, and for human erythrocytes, it significantly increased. Storage of ovine and human erythrocytes longer than 8 weeks led to a sharp hemolysis, while the level of hemolysis of ovine erythrocytes was lower than that of human erythrocytes. Preservation of ozonated erythrocytes is higher than non-ozonated ones during prolonged hypothermic storage. The effect of ozonation on the preservation rate of red blood cells depended on the composition of the preservation media. Hypothermal storage of human erythrocytes in Alsever’s solution for up to 8 weeks led to a shift in the density of distribution according to the sphericity index towards spheroidization of cells; in a medium with mannitol, the number of flattened cell forms increased. After 8 weeks of hypothermic storage of ovine erythrocytes, most of the cells had high sphericity indices. Pretreatment of human and sheep erythrocytes with ozone allows the distribution of cells to be kept closer to the control during long-term storage, which is especially pronounced in mannitol medium. Ovine erythrocytes retained the ability to form rosettes with human T-lymphocytes after hypothermic storage for up to 12 weeks. Thus, ozone pretreatment and the use of mannitol as part of hypothermic storage medium could be an approach to improve the quality of preserved ovine erythrocytes.

PSV-35 Correlation of acute-phase blood proteins level and quality and cryostability of sperm in horses

The aim of this work was to study the relationship between acute phase indicators of a biochemical blood test with the quality and cryostability sperm in stallions. Semen from 59 stallions (aged from 5 to 15 years) were collected during the breeding season (February–May). Blood plasma samples were studied using a Vegasys automatic biochemical analyzer (Analyzer Medical System, Italy). The following parameters were determined in blood plasma: total protein, level of albumin, globulin, albumin/globulin ratio, C-reactive protein, fibrinogen. Each sperm sample was evaluated by volume, concentration, total sperm, as well as progressive motility and survival of sperm in chilled and thawed sperm during hypothermic storage at +40С. The normality of the distribution of quantitative traits was determined using the Shapiro-Wilk test. For each of the indicators, the Spearman coefficient was calculated. Significance of differences was assessed using the Mann-Whitney test, the results were statistically significant at P ≤0.05. The concentration of biochemical parameters in blood serum averaged: total protein - 67.4 ± 0.7 g/l, albumin - 39.4±0.6 g/l, globulins - 28.4±0.8 g/l, C reactive protein - 0.3±0.1 mg/l, fibrinogen - 3.1±0.1 g/l. A significant negative correlation between the plasma fibrinogen and sperm survival during hypothermic storage (at +4ºС) of chilled (r=0.48; P &lt; 0.001) sperm and progressive sperm motility after thawing (r =-0.29; &lt; 0.05) was found. We suggest that an increase in the level of fibrinogen in the blood may indicate the presence of latent inflammatory, immuno-mediated processes in the body, including those affecting the quality and cryostability of sperm in stallions. Authors acknowledge financial support from Russian Science Foundation, Grant No: 20-16-00101, development program of Bioresource collections “Cryobank of genetic recourses the All-Russian Research Institute for Horse Breeding.”

PSV-39 The relationship between structural integrity and sperm survival during hypothermic storage of stallion sperm

The aim of this work was to study the relationship between ultrastructural integrity and sperm survival during hypothermic storage of chilled and cryopreserved stallion sperm. Semen from 37 stallions, were collected during the breeding season. The structural integrity of sperm was studied using a Hitachi 700 electron microscope. Progressive motility was evaluated using an Olympus BX 41 phase contrast microscope. Sperm survival was defined as the ability of spermatozoa to maintain progressive motility above 5% during hypothermic (+4C) storage of sperm. Spearman nonparametric correlation analysis was used. Sperm survival at a temperature +4°C in chilled semen averaged 115.4±11.47 hours. After freezing sperm survival decreased on average to 48.9±5.56 hours (P &lt; 0.001). A reliable correlation between the number of sperm with intact heads and sperm survival at a temperature of + 4°C in diluted (r=0.60; P &lt; 0.001) and thawed (r=0.63; P &lt; 0.001) sperm was established. A significant negative correlation between the number of spermatozoa with degradation of the acrosome in native sperm and their survival (r=-0.48; P &lt; 0.01) under the hypothermic storage was found. For cryopreserved semen, a similar dependence has not been established. A significant correlation between the number of spermatozoa with the normal position of the acrosome and the sperm survival rate in native sperm was also established (r=0.52; P &lt; 0.01). A significant negative correlation between sperm with acrosome hypoplasia and sperm survival was found (r=-0.67; P &lt; 0.001). We assume that the structural integrity and ability of spermatozoa to maintain progressive motility for a long time under conditions of hypothermic storage depends on the quality of spermatogenesis and the resistance of cells to low and ultra-low temperatures. Authors acknowledge financial support from Russian Science Foundation, Grant No: 20-16-00101, development program of Bioresource collections “Cryobank of genetic recourses the All-Russian Research Institute for Horse Breeding.”