In the 1960s, sperm cryopreservation was developed as a method to preserve fertility. Currently, techniques for the cryopreservation of human spermatozoa have been widely used in assisted reproduction. However, although sperm cryobiology has made notable achievements, the optimal method for the recovery of viable spermatozoa after cryopreservation remains elusive. Postthawing sperm quality can be affected by cryoprotectants, ice formation, storage conditions, and osmotic stress during the freezing process. This review discusses recent advances in different cryopreservation techniques, cryoprotectants, and freezing and thawing methods during cryopreservation and new indications for the use of cryopreserved spermatozoa.
Humans ; Male ; Semen Preservation/methods* ; Sperm Motility ; Semen ; Cryopreservation/methods* ; Spermatozoa ; Cryoprotective Agents/pharmacology*

Ice easily recrystallizes during warming after vitrification, and antifreeze protein (AFP) can inhibit the re-crystallization. However, no study has evaluated the effect of AFP treatment only thereon during warming. This study sought to compare AFP treatment protocols: a conventional protocol with AFP treatment during vitrification and first-step warming and a new protocol with AFP treatment during the first-step warming only. According to the protocols, 10 mg/mL of LeIBP (a type of AFP) was used. Five-week-old B6D2F1 mouse ovaries were randomly divided into a vitrified-warmed control and two experimental groups, one treated with the conventional AFP treatment protocol (LeIBP-all) and the other with the new AFP treatment protocol (LeIBP-w). For evaluation, ratios of ovarian follicle integrity, apoptosis, and DNA double-strand (DDS) damage/repairing were analyzed. The LeIBP-treated groups showed significantly higher intact follicle ratios than the control, and the results were similar between the LeIBP-treated groups. Apoptotic follicle ratios were significantly lower in both LeIBP-treated groups than the control, and the results were not significantly different between the LeIBP-treated groups. With regard to DDS damage/repairing follicle ratio, significantly lower ratios were recorded in both LeIBP-treated groups, compared to the control, and the results were similar between the LeIBP-treated groups. This study demonstrated that both protocols with LeIBP had a beneficial effect on maintaining follicle integrity and preventing follicle apoptosis and DDS damage. Moreover, the new protocol showed similar results to the conventional protocol. This new protocol could optimize the mouse ovary vitrification-warming procedure using AFP, while minimizing the treatment steps.
Animals ; Antifreeze Proteins/*pharmacology ; Apoptosis/drug effects ; Cryopreservation ; Cryoprotective Agents/pharmacology ; Female ; Mice ; Ovarian Follicle/cytology/drug effects ; Ovary/cytology/drug effects/*physiology ; *Vitrification/drug effects

Organ transplantation is an effective approach for the treatment of end-stage organ failures. Currently, the donor organs used for clinical transplantation are all preserved at above-zero temperatures. These preservation methods are well-established and simple but the storage time lasts for only 4-12 h. Some researchers tried to extend the organ storage time by improving protectant and HLA matching to raise the use of stored organs and prolong the long-term survival of organs. These efforts still fall short of the clinical demand for organ transplantation. Moreover, a great many organs were wasted due to limited storage time, HLA mismatch, patients' conditions or distance involved. Therefore, preserving organs for several weeks or even months and establishing Organ Bank are the tough challenges and have become a shared goal of global scholars. This article reviews some issues involved in the cryopreservation of organs, such as use of cryoprotecting agents, freezing and thawing methods in the cryopreservation of hearts, kidneys and other organs.
Cryopreservation ; methods ; Cryoprotective Agents ; adverse effects ; pharmacology ; Humans ; Organ Preservation ; methods

BACKGROUNDL-proline is a natural, nontoxic cryoprotectant that helps cells and tissues to tolerate freezing in a variety of plants and animals. The use of L-proline in mammalian oocyte cryopreservation is rare. In this study, we explored the cryobiological characteristics of L-proline and evaluated its protective effect in mouse oocyte cryopreservation.

METHODSThe freezing property of L-proline was detected by Raman spectroscopy and osmometer. Mature oocytes obtained from 8-week-old B6D2F1 mice were vitrified in a solution consisting various concentration of L-proline with a reduced proportion of dimethyl sulfoxide (DMSO) and ethylene glycol (EG), comparing with the control group (15% DMSO and 15% EG without L-proline). The survival rate, 5-methylcytosine (5-mC) expression, fertilization rate, two-cell rate, and blastocyst rate in vitro were assessed by immunofluorescence and in vitro fertilization. Data were analyzed by Chi-square test.

RESULTSL-proline can penetrate the oocyte membrane within 1 min. The osmotic pressure of 2.00 mol/L L-proline mixture is similar to that of the control group. The survival rate of the postthawed oocyte in 2.00 mol/L L-proline combining 7.5% DMSO and 10% EG is significantly higher than that of the control group. There is no difference of 5-mC expression between the L-proline combination groups and control. The fertilization rate, two-cell rate, and blastocyst rate in vitro from oocyte vitrified in 2.00 mol/L L-proline combining 7.5% DMSO and 10% EG solution are similar to that of control.

CONCLUSIONSIt indicated that an appropriate concentration of L-proline can improve the cryopreservation efficiency of mouse oocytes with low concentrations of DMSO and EG, which may be applicable to human oocyte vitrification.

Animals ; Cryopreservation ; methods ; Cryoprotective Agents ; pharmacology ; Female ; Fertilization in Vitro ; Hydrogen-Ion Concentration ; Male ; Mice ; Oocytes ; drug effects ; Osmotic Pressure ; Proline ; pharmacology ; Spectrum Analysis, Raman ; Vitrification

OBJECTIVETo search for an optimal protocol and freezing conditions for the cryopreservation of microamount round spermatids of the mouse.

METHODSWe compared the survival rates of frozen-thawed microamount round spermatids of the mouse achieved by vitrification or standard slow freezing with different concentrations of glycerol (5, 7, or 9%) and different lengths of equilibrium time (0, 15, 30, 45, or 60 min).

RESULTSUnder the conditions of 7% glycerol and 30 min equilibrium, both vitrification and standard slow freezing achieved high survival rates of spermatids, and the former obtained an even higher rate than the latter ([72.9 ± 15.4]% vs [58.2 ± 17.7]%, P < 0.05).

CONCLUSIONA high rate of frozen-thawed microamount round spermatids of the mouse can be achieved by vitrification under the conditions of 7% glycerol and 30 min equilibrium.

Animals ; Cell Survival ; Cryopreservation ; methods ; Cryoprotective Agents ; administration & dosage ; pharmacology ; Glycerol ; administration & dosage ; pharmacology ; Male ; Mice ; Spermatids ; Time Factors ; Vitrification ; drug effects

OBJECTIVETo investigate the protective effect of lycopene against cryopreservation injury of post-thawing human sperm and its mechanism.

METHODSSemen samples were collected from 25 volunteers, each sample equally divided into four parts to be cryopreserved with cryoprotectant only (Ly0 control) or cryoprotectant + lycopene at the concentrations of 2 (Ly2), 5 (Ly5), and 10 µmol/L (Ly10), respectively. Before and after thawing, the semen samples were subjected to computer-assisted semen analysis ( CASA) for sperm kinematics, flow cytometry for sperm apoptosis, thiobarbituric acid assay for malondialdehyde (MDA) concentration, and JC-1 fluorescent staining for the sperm mitochondrial membrane potential (MMP).

RESULTSAfter cryopreservation, sperm motility was markedly decreased in all the groups (P < 0.01). The rate of sperm apoptosis was significantly lower in the Ly5 group than in the Ly0 control ([25.68 ± 4.36]% vs [33.26 ± 4.78]%, P < 0.05), while sperm MMP remarkably higher in the former than in the latter ([66.18 ± 14.23]% vs [55.24 ± 12.31]%, P < 0.05). The Ly2, Ly5 and Ly10 groups showed no statistically significance differences in the MDA level from the Ly0 control (P > 0.05).

CONCLUSIONAddition of lycopene at a proper concentration to cryoprotectant may reduce oxidative damage to sperm mitochondria in the freezing-thawing process, attenuate oxidative stress injury induced by reactive oxygen species to sperm plasma membrane, and improve the anti-apoptosis ability of sperm.

Apoptosis ; Carotenoids ; pharmacology ; Cryopreservation ; Cryoprotective Agents ; pharmacology ; Flow Cytometry ; Humans ; Male ; Malondialdehyde ; analysis ; Oxidative Stress ; Reactive Oxygen Species ; Semen Analysis ; Semen Preservation ; adverse effects ; methods ; Sperm Motility ; Spermatozoa ; drug effects ; physiology

PURPOSE: To investigate the effect of antifreeze protein (AFP) supplementation on ovarian vitrification and transplantation. MATERIALS AND METHODS: In this experimental study, we researched a total of 182 ovaries from 4-week-old ICR mice. The equilibration solution included 20% ethylene glycol (EG), and the vitrification solution included 40% EG, 18% Ficoll, and 0.3 M sucrose. Intact ovaries were first suspended in 1 mL of equilibration solution for 10 min, and then mixed with 0.5 mL of vitrification solution for 5 min. Ovaries were randomly assigned to 3 groups and 0, 5, or 20 mg/mL of type III AFP was added into the vitrification solution (control, AFP5, and AFP20 groups, respectively). The vitrified ovaries were evaluated after warming and 2 weeks after autotransplantation. The main outcome measurements are follicular morphology and apoptosis assessed by histology and the TUNEL assay. RESULTS: A significantly higher intact follicle ratio was shown in the AFP treated groups (control, 28.9%; AFP5, 42.3%; and AFP20, 44.7%). The rate of apoptotic follicles was significantly lower in the AFP treated groups (control, 26.6%; AFP5, 18.7%; and AFP20, 12.6%). After transplantation of the vitrified-warmed ovaries, a significantly higher intact follicle ratio was shown in the AFP20 group. The rate of apoptotic follicles was similar among the groups. CONCLUSION: The results of the present study suggest that supplementing AFP in the vitrification solution has beneficial effects on the survival of ovarian tissue during cryopreservation and transplantation.
Animals ; Antifreeze Proteins/*pharmacology ; Apoptosis/drug effects ; Cryopreservation/*methods ; Cryoprotective Agents/*pharmacology ; Female ; Fertility Preservation ; Humans ; Mice ; Mice, Inbred ICR ; Ovarian Follicle/drug effects ; Ovary/*drug effects/*transplantation ; *Vitrification

The effects of mouse oocyte vitrification on mitochondrial membrane potential and distribution were explored in this study. The collected mouse oocytes were randomly divided into vitrification and control groups. Ethylene glycol (EG) and dimethylsulphoxide (DMSO) were used as cryoprotectants in the vitrification group. The mitochondrial function and distribution in the oocytes were examined by using the fluorescent probes, JC-1 and Mito Tracker green. The results showed that the ratio of red to green fluorescence in mouse oocytes was significantly decreased after thawing in the vitrification group as compared with the control group (1.28 vs. 1.70, P<0.05). The percentage of polarized distribution of the mitochondria in oocytes was conspicuously reduced in the vitrification group when compared with the control group (31% vs. 63%, P<0.05). It was suggested that vitrification significantly affects the mitochondrial function and distribution in oocytes and reduces the potential of oocyte fertilization and embryo development.
Animals ; Cryopreservation ; methods ; Cryoprotective Agents ; pharmacology ; Dimethyl Sulfoxide ; pharmacology ; Ethylene Glycol ; pharmacology ; Female ; Fluorescent Dyes ; metabolism ; Membrane Potential, Mitochondrial ; drug effects ; physiology ; Mice ; Microscopy, Fluorescence ; Mitochondria ; drug effects ; metabolism ; Oocytes ; drug effects ; physiology ; Temperature ; Vitrification

Nano-cryopreservation may become a new way in the next generation of cryopreservation technology. However, research using nanoparticles in oocytes vitrification has not been reported in the literature. In this study, HA nanoparticles with different diameters were added into cryoprotectant and M II-stage porcine oocytes were vitrified by Cryotop. The results showed that nanoparticles improved the survival rate of cryopreserved M II-stage porcine oocytes, but the difference between nanoparticles with different diameters of was not significant. In order to study the mechanism of nano-cryopreservation, the cooling rate of cryoprotectant was measured by ultra-fast temperature measurement system and the melting enthalpy of cryoprotectant was measured by differential scanning calorimeter (DSC). The results showed that the adding of nanoparitcles could not increase the cooling rate of cryoprotectant, but could decreases the amount of ice crystals during freezing and warming. Therefore, the mechanical injury within and outside cells might be effectively reduced.
Animals ; Cell Survival ; physiology ; Cryopreservation ; methods ; veterinary ; Cryoprotective Agents ; pharmacology ; Durapatite ; pharmacology ; Female ; Fertilization in Vitro ; methods ; veterinary ; Metaphase ; Nanoparticles ; Oocytes ; cytology ; Swine ; Vitrification

This study was purposed to evaluate the effect of different lyophilizing protectants including human albumin, glucan, polyvinyl pyrrolidone and glycerine on lyophilized trehalose-loading red blood cells (RBC), then to screen the optimal lyophilizing protectant. The RBC were incubated in 800 mmol/L concentration of trehalose solution at 37°C for 7 hours, and washed 3 times with PBS solution to obtain the trehalose-loading RBC. The trehalose-loading RBC in control group were directly lyophilized without lyophilizing protectants, the trehalose-loading RBC in the experimental group were mixed with Lyophilizing protectants. The samples of 2 groups were kept at room temperature for 30 minutes, pre-frozen at -80°C for 24 hours, then lyophilized in freeze-dryer for 24 hours. Finally the samples were quickly rehydrated by 6% HES at 37°C. The recovery rate and hemolysis rate of hemoglobin were detected by using cyanohemoglobin detection kit. The water content of unhydrated samples were detected at the same time. The results showed that when the moisture content of sample was 3% - 5%, the recovery rate of hemoglobin in control group was 33.57 ± 2.89%, and that in experimental group was 51.15 ± 1.98%, there was statistically significant difference between the control and experimental group (P < 0.05). When the different concentration of dextran solution was chosen as protectants, the recovery rate of hemoglobin of lyophilized RBC was obviously lower. The higher concentration of dextran, the better the recovery rate. The recovery rate of hemoglobin was 22.15 ± 4.12% when the concentration of dextran was 36%, there were statistically significant difference between the two groups (P < 0.05). When the different concentration of polyvinyl pyrrolidone (PVP) solutions was chosen as protectants, especially the concentration below 40%, the recovery rate of hemoglobin of lyophilized RBC was significantly belower than the control group, there was statistically significant difference between the two groups (P < 0.05). When 10% glycerol was used as protectants, the recovery rate of hemoglobin was 3.93 ± 1.80%. There was also statistically significant difference between the two groups (P < 0.05). It is concluded that human serum albumin shows an important protective effect on the lyophilization of the trehalose-loading red blood cells. The dextran and PVP at the concentration lower than 40% can decrease the protective effect of trehalose in cells. Glycerol can not be chosen as protectant for lyophilized trehalose-loading red blood cells.
Blood Preservation ; methods ; Cryoprotective Agents ; pharmacology ; Erythrocytes ; drug effects ; Freeze Drying ; methods ; Humans ; Trehalose ; pharmacology