Cryoprotectants-Free Vitrification and Conventional Freezing of Human Spermatozoa: A Comparative Transcript Profiling. International journal of molecular sciences INTRODUCTION:Spermatozoa cryopreservation is an important technique to preserve fertility for males. This study aimed at exploring the stability of epigenetics information in human spermatozoa, manipulated by two different technologies, freezing and vitrification. METHODS:Spermatozoa samples were distributed into three groups: 1. Fresh spermatozoa (control group), 2. Frozen spermatozoa, 3. Vitrified spermatozoa. Epigenetic differences of fresh and cryopreserved spermatozoa were evaluated using high-throughput RNA sequencing. RESULTS:Differentially expressed genes (DEGs) in frozen (1103 genes) and vitrified (333 genes) spermatozoa were evaluated. The bioinformatical analysis identified 8 and 15 significant pathways in groups of frozen and vitrified spermatozoa, respectively. The majority of these pathways are most relevant to immune and infectious diseases. The DEGs of the fertilization process are not detected during vitrification. The freezing process induces more down-regulation of genes and is relevant to apoptosis changes and immune response. CONCLUSION:Cryopreservation of human spermatozoa is an epigenetically safe method for male fertility preservation. Cryoprotectant-free vitrification can induce more minor biological changes in human spermatozoa, in comparison with conventional freezing. 10.3390/ijms23063047

A simple method of human sperm vitrification. Shah Dupesh,Rasappan ,Shila ,Gunasekaran Karthik MethodsX Human sperm vitrification is a novel method of sperm freezing which achieves cryopreservation due to ultra-rapid cooling rates that prevent ice-crystal formation. However, sperm vitrification protocols are still largely not standardized for routine clinical use and seldom achieve a post warm sperm survival of 25-35%. The study aim was to validate and optimize a simple method of sperm vitrification that yields a high survival rate of spermatozoa for clinical use. Semen samples from 10 normozoospermic patients were subject to a simple swim-up into pre-warmed gamete handling media. Swim-up specimens were mixed in a 1:1 ratio with 0.5 M sucrose. Swim up specimens were then directly dropped in liquid N2. After a week of storage samples where warmed at 42 degree Celsius and sperm motility and viability was estimated. The mean sperm total motility of the fresh sample after the swim up preparation was 94.3 ± 3.06 %. Upon, vitrification followed by warming the mean percentage of total motile sperm fraction recovered was 74.70 ± 5.60 %. The mean sperm progressive motility of vitrified-warmed spermatozoa was 68 ± 8.47 %. The overall mean percentage of motile sperm recovery was 70.05% of the fresh swim up sample in this study. The overall mean sperm viability as assessed using the HOST vitality test was 77.21 ± 7.52%. •This study presents a simple protocol on the 'droplet method' of sperm vitrification.•Sperm cells vitrified using our modified method show a >70% motility and viability rates compared to the routine 25% to 35% of reported survival with the original sperm vitrification/freezing methodologies. This survival is attributed to a crucial change in the warming step.•This method has the advantage of using no toxic cell permeating cryoprotectant or expensive programmable freezing devices. 10.1016/j.mex.2019.09.022