Ultra-rapid cooling and rewarming rate is a critical technical approach to achieve ice-free cells during the freezing and melting process. A set of ultra-rapid solid surface freeze-thaw visualization system was developed based on a sapphire flim, and experiments on droplet freeze-thaw were carried out under different cryoprotectant components, volumes and laser energies. The results showed that the cooling rate of 1 μL mixed cryoprotectant [1.5 mol/L propylene glycol (PG) + 1.5 mol/L ethylene glycol (EG) + 0.5 mol/L trehalose (TRE)] could be 9.2×10 ³ °C/min. The volume range of 1-8 μL droplets could be vitrified. After comparing the proportions of multiple cryoprotectants, the combination of equal proportion mixed permeability protectant and trehalose had the best vitrification freezing effect and more uniform crystallization characteristics. During the rewarming operation, the heating curve of glassy droplets containing gold nanoparticles was measured for the first time under the action of 400-1 200 W laser power, and the rewarming rate was up to the order of 10 ⁶ °C/min. According to the droplet images of different power rewarming processes, the laser power range for ice-free rewarming with micron-level resolution was clarified to be 1 400-1 600 W. The work of this paper simultaneously realizes the ultra-high-speed temperature ramp-up, transient visual observation and temperature measurement of droplets, providing technical means for judging the ice free droplets during the freeze-thaw process. It is conducive to promoting the development of ultra-rapid freeze-thaw technology for biological cells and tissues.
Freezing ; Vitrification ; Cryopreservation/methods* ; Trehalose ; Gold ; Rewarming ; Metal Nanoparticles ; Cryoprotective Agents ; Lasers

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*

OBJECTIVES: To evaluate the effects of different pretreatment methods and preservation time on RNA quality of peripheral blood samples, and to optimize the preservation method of peripheral blood samples. METHODS: Eight pretreatment methods were used to preprocess the peripheral blood from 3 healthy unrelated individuals and the treated samples were stored at -80 ℃. Total RNA of samples was extracted using Quick-RNA^TM Miniprep Plus kit. DNA/RNA Shield^TM was added to peripheral blood and total RNA was extracted after preservation at -80 ℃ for 0, 5, 10, 15, 30 and 60 days, respectively. The concentration, purity and integrity of RNA were determined. Statistical analyses were performed by SPSS 22.0 software to compare the differences in RNA yield, purity and integrity among the eight pretreatment methods. RESULTS: In terms of purity, leukocyte pretreated with RNAlater^TM and directly cryopreservation peripheral blood showed the worst purity. The other six methods showed better purity. In terms of yield, blood cells with DNA/RNA Shield^TM came out with the highest yield, followed by peripheral blood with DNA/RNA Shield^TM. In terms of integrity, peripheral blood preserved in PAXgene Blood RNA tube method had the best integrity. Except for peripheral blood pretreated with DNA/RNA Shield^TM and blood cells pretreated with DNA/RNA shield^TM, the other five methods had statistical differences when compared to the method by keeping peripheral blood in PAXgene Blood RNA tube. The purity of RNA stored at six-time gradients ranged from 1.815 to 1.952. With the increase of storage time, RNA yield decreased from 4.516 ng to 1.039 ng, and RNA integrity decreased from 8.533 to 7.150. CONCLUSIONS According to the results of total RNA's yield, purity and integrity, peripheral blood pretreated with DNA/RNA Shield^TM was the best pretreatment method. After the pretreatment, samples can be preserved for up to 60 days in low temperature.
Blood Specimen Collection/methods* ; Cryopreservation ; DNA/analysis* ; Humans ; RNA

Oxidative stress and apoptosis are the key factors that limit the hypothermic preservation time of donor hearts to within 4-6 h. The aim of this study was to investigate whether the histone deacetylase 3 (HDAC3) inhibitor RGFP966 could protect against cardiac injury induced by prolonged hypothermic preservation. Rat hearts were hypothermically preserved in Celsior solution with or without RGFP966 for 12 h followed by 60 min of reperfusion. Hemodynamic parameters during reperfusion were evaluated. The expression and phosphorylation levels of mammalian STE20-like kinase-1 (Mst1) and Yes-associated protein (YAP) were determined by western blotting. Cell apoptosis was measured by the terminal deoxynucleotidyl-transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) method. Addition of RGFP966 in Celsior solution significantly inhibited cardiac dysfunction induced by hypothermic preservation. RGFP966 inhibited the hypothermic preservation-induced increase of the phosphorylated (p)-Mst1/Mst1 and p-YAP/YAP ratios, prevented a reduction in total YAP protein expression, and increased the nuclear YAP protein level. Verteporfin (VP), a small molecular inhibitor of YAP-transcriptional enhanced associate domain (TEAD) interaction, partially abolished the protective effect of RGFP966 on cardiac function, and reduced lactate dehydrogenase activity and malondialdehyde content. RGFP966 increased superoxide dismutase, catalase, and glutathione peroxidase gene and protein expression, which was abolished by VP. RGFP966 inhibited hypothermic preservation-induced overexpression of B-cell lymphoma protein 2 (Bcl-2)-associated X (Bax) and cleaved caspase-3, increased Bcl-2 mRNA and protein expression, and reduced cardiomyocyte apoptosis. The antioxidant and anti-apoptotic effects of RGFP966 were cancelled by VP. The results suggest that supplementation of Celsior solution with RGFP966 attenuated prolonged hypothermic preservation-induced cardiac dysfunction. The mechanism may involve inhibition of oxidative stress and apoptosis via inactivation of the YAP pathway.
Acrylamides/pharmacology* ; Animals ; Apoptosis/drug effects* ; Cryopreservation ; Disaccharides/pharmacology* ; Electrolytes/pharmacology* ; Glutamates/pharmacology* ; Glutathione/pharmacology* ; Heart/physiology* ; Heart Transplantation/methods* ; Hepatocyte Growth Factor/antagonists & inhibitors* ; Histidine/pharmacology* ; Histone Deacetylase Inhibitors/pharmacology* ; Intracellular Signaling Peptides and Proteins/antagonists & inhibitors* ; Male ; Mannitol/pharmacology* ; Oxidative Stress/drug effects* ; Phenylenediamines/pharmacology* ; Proto-Oncogene Proteins/antagonists & inhibitors* ; Rats ; Rats, Sprague-Dawley ; Signal Transduction/drug effects* ; YAP-Signaling Proteins

PURPOSE: Sperm cryopreservation before cancer treatment is the most effective method to preserve the fertility of male patients. We present our 21 years experience with sperm cryopreservation for cancer patients, including an examination of semen quality, the current status of cryopreserved sperm, and the rate of sperm use for assisted reproductive technology (ART). MATERIALS AND METHODS: A total of 721 cancer patients at Fertility Center of CHA Gangnam Medical Center successfully performed sperm cryopreservation for fertility preservation from January 1996 to December 2016. Medical chart review was used to analyze patient age, marital status, cancer type, semen volume, sperm counts and motility, length of storage, and current banking status. RESULTS: The major cancers of the 721 patients were leukemia (28.4%), lymphoma (18.3%), testis cancer (10.0%). The mean age at cryopreservation was 27.0 years, and 111 patients (15.4%) performed sperm cryopreservation during or after cancer treatment. The mean sperm concentration was 66.7±66.3 ×106/mL and the mean sperm motility was 33.8%±16.3%. During median follow-up duration of 75 months (range, 1–226 months), 44 patients (6.1%) used their banked sperm at our fertility center for ART and 9 patients (1.2%) transferred their banked sperm to another center. The median duration from cryopreservation to use was 51 months (range, 1–158 months). CONCLUSIONS: Sperm cryopreservation before gonadotoxic treatment is the most reliable method to preserve the fertility of male cancer patients. Sperm cryopreservation should be offered as a standard of care for all men planning cancer therapy.
Cryopreservation ; Fertility Preservation ; Fertility ; Follow-Up Studies ; Humans ; Leukemia ; Lymphoma ; Male ; Marital Status ; Methods ; Reproductive Techniques, Assisted ; Semen ; Semen Analysis ; Semen Preservation ; Sperm Count ; Sperm Motility ; Spermatozoa ; Standard of Care ; Testicular Neoplasms

The survival rates of boys and men with cancer have increased due to advances in cancer treatments; however, maintenance of quality of life, including fertility preservation, remains a major issue. Fertile male patients who receive radiation and/or chemotherapy face temporary, long-term, or permanent gonadal damage, particularly with exposure to alkylating agents and whole-body irradiation, which sometimes induce critical germ cell damage. These cytotoxic treatments have a significant impact on a patient's ability to have their own biological offspring, which is of particular concern to cancer patients of reproductive age. Therefore, various strategies are needed in order to preserve male fertility. Sperm cryopreservation is an effective method for preserving spermatozoa. Advances have also been achieved in pre-pubertal germ cell storage and research to generate differentiated male germ cells from various types of stem cells, including embryonic stem cells, induced pluripotent stem cells, and spermatogonial stem cells. These approaches offer hope to many patients in whom germ cell loss is associated with sterility, but are still experimental and preliminary. This review examines the current understanding of the effects of chemotherapy and radiation on male fertility.
Alkylating Agents ; Cryopreservation ; Drug Therapy ; Embryonic Stem Cells ; Fertility ; Fertility Preservation ; Germ Cells ; Gonads ; Hope ; Humans ; Induced Pluripotent Stem Cells ; Infertility ; Infertility, Male ; Male ; Methods ; Quality of Life ; Radiotherapy ; Spermatogenesis ; Spermatozoa ; Stem Cells ; Survival Rate ; Whole-Body Irradiation

Primordial follicle activation is a process in which individual primordial follicles leave their dormant state and enter a growth phase. While existing hormone stimulation strategies targeted the growing follicles, the remaining dormant primordial follicles were ruled out from clinical use. Recently, in vitro activation (IVA), which is a method for controlling primordial follicle activation, has provided an innovative technology for primary ovarian insufficiency (POI) patients. IVA was developed based on Hippo signaling and phosphatase and tensin homolog (PTEN)/phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT)/forkhead box O3 (FOXO3) signaling modulation. With this method, dormant primordial follicles are activated to enter growth phase and developed into competent oocytes. IVA has been successfully applied in POI patients who only have a few remaining remnant primordial follicles in the ovary, and healthy pregnancies and deliveries have been reported. IVA may also provide a promising option for fertility preservation in cancer patients and prepubertal girls whose fertility preservation choices are limited to tissue cryopreservation. Here, we review the basic mechanisms, translational studies, and current clinical results for IVA. Limitations and further study requirements that could potentially optimize IVA for future use will also be discussed.
Cryopreservation ; Female ; Fertility Preservation ; Humans ; In Vitro Techniques ; Methods ; Oocytes ; Ovarian Follicle ; Ovary ; Phosphotransferases ; Pregnancy ; Primary Ovarian Insufficiency

INTRODUCTION: This study investigated the differences in clinical pregnancy rate (CPR), live birth rate (LBR) and multiple pregnancy rate (MPR) between double cleavage-stage embryo transfers compared to single and double blastocysts stage embryo transfers in a single academic medical centre. MATERIALS AND METHODS: This was a retrospective cohort study performed at the KK Women's and Children's Hospital In Vitro Fertilisation (KKIVF) Centre of all women who underwent fresh-cycle in vitro fertilisation/intracytoplasmic sperm injection (IVF/ICSI) cycles over a 5-year period. The outcome measures were CPR, LBR and MPR. The study included 5294 cycles, of which 539 patients underwent single embryo transfer (SET); 4533 patients underwent double embryo transfer (DET); 84 patients underwent double blastocyst embryo transfer (DBT); and 65 patients underwent single blastocyst embryo transfer (SBT). RESULTS: The mean age of patients undergoing single blastocysts stage embryo transfer was lower than the other 2 groups. The DET, single and double blastocysts stage embryo transfer groups achieved similar LBR (33.9%, 38.7%, 35.4%, >0.05) and CPR (42.4%, 46.2%, 46.9%). CONCLUSION We found that single blastocysts stage embryo transfer is associated with similar LBR and CPR compared to double blastocysts stage embryo transfer and DET, with lower MPRs, and should be offered as standard practice, where possible.
Adult ; Cohort Studies ; Cryopreservation ; methods ; statistics & numerical data ; Embryo Transfer ; adverse effects ; methods ; statistics & numerical data ; Female ; Fertilization in Vitro ; statistics & numerical data ; Humans ; Live Birth ; epidemiology ; Pregnancy ; Pregnancy Outcome ; epidemiology ; Pregnancy Rate ; Pregnancy, Multiple ; statistics & numerical data ; Retrospective Studies ; Singapore ; Single Embryo Transfer ; adverse effects ; methods ; statistics & numerical data ; Sperm Injections, Intracytoplasmic ; statistics & numerical data

BACKGROUND AND OBJECTIVES: The human Amniotic epithelial cells (AME) derived from amniotic membrane of placenta have been considered as the potential fetal stem cell source with minimal or no ethical concerns and are important therapeutic tool for anti-fibrotic and regenerative therapies. METHODS AND RESULTS: Here, we evaluated the isolation, media screening, scale-up and characterization of AME cells. The isolation, expansion of AMEs were performed by sequential passaging and growth kinetics studies. The AMEs were characterized using immunocytochemistry, immunophenotyping, In-vitro differentiation, and anti-fibrotic assays. The growth kinetics study revealed that the AME cultured in Ultraculture (UC) and DMEM knockout (DMEM-KO) have prominently higher growth rate compared to others. Overall, the AMEs cultured from 5 different media retained basic morphological characteristics and the functional characteristics. CONCLUSIONS: Our result suggests that the AMEs can be successfully cultured in UC based complete media without losing its epithelial cell characteristics even after passaging for passage 2 (P2). However, a careful and methodical pre-clinical and clinical translation studies need to be conducted to show its safety and efficacy.
Amnion ; Cell- and Tissue-Based Therapy ; Cryopreservation ; Epithelial Cells ; Fetal Stem Cells ; Humans ; Immunohistochemistry ; Immunophenotyping ; Kinetics ; Mass Screening ; Methods ; Placenta ; Tissue Engineering

ObjectiveTo investigate the effects of resveratrol in the cryopreservation medium on the quality and function of post-thaw sperm.

METHODSSemen samples were obtained from 50 normozoospermic and 50 oligoasthenozoospermic men, liquefied and then cryopreserved in the glycerol-egg yolk-citrate (GEYC) medium with or without 30 μmol/L resveratrol. Sperm motility, viability and acrosome reaction (AR) were examined before and after thawing. Sperm lipid peroxidation and the level of reactive oxygen species (ROS) were measured using commercial malondialdehyde (MDA) and the ROS assay kit. Sperm mitochondrial membrane potential (MMP) and DNA damage were determined by Rhodamine 123 staining and TUNEL.

RESULTSThe percentage of progressively motile sperm (PMS), total sperm motility, sperm viability, MMP and AR were significantly decreased (P <0.05) while the levels of sperm ROS, MDA and DNA fragmentation index (DFI) remarkably increased in both the normozoospermia and oligoasthenozoospermia groups after cryopreservation as compared with those in the fresh ejaculate (P <0.05). In comparison with the non-resveratrol control, the post-thaw sperm cryopreserved with 30 μmol/L resveratrol showed markedly higher PMS (［32.7 ± 4.8］ vs ［43.1 ± 6.3］ %, P <0.05), total motility (［44.8 ± 6.9］ vs ［56.9 ± 7.4］ %, P <0.05), viability (［52.3 ± 6.1］ vs ［67.5 ± 5.6］ %, P <0.05), MMP (［56.5 ± 7.0］ vs ［63.4 ± 7.5］ %, P <0.05) and AR (［16.6 ± 3.8］ vs ［26.3 ± 4.7］ %, P <0.05) but lower ROS, MDA and DFI (all P <0.05) in the normozoospermia group, and so did the post-thaw sperm in the oligoasthenozoospermia group, with a particularly lower DFI (［28.5 ± 4.8］ vs ［36.3 ± 5.7］%, P <0.01).

CONCLUSIONSResveratrol in the cryopreservation medium can improve the quality and function of post-thaw human sperm by reducing cryopreservation-induced sperm injury and the level of ROS.

Acrosome ; drug effects ; Animals ; Antioxidants ; pharmacology ; Cryopreservation ; methods ; DNA Fragmentation ; Humans ; Lipid Peroxidation ; Male ; Malondialdehyde ; Membrane Potential, Mitochondrial ; Reactive Oxygen Species ; analysis ; Resveratrol ; pharmacology ; Semen Analysis ; Semen Preservation ; adverse effects ; Sperm Motility ; drug effects ; Spermatozoa ; drug effects ; physiology