Humans ; Vitrification ; Consensus ; Oocytes ; Fertilization in Vitro ; Cryopreservation

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

Cryopreservation of rare testicular-retrieved spermatozoa for intracytoplasmic sperm injection (ICSI) in patients with severe oligozoospermia and azoospermia remains a major challenge in clinical practice. This study evaluated the Cryopiece system as a potential technique to cryopreserve rare human spermatozoa for ICSI. Small numbers of ejaculated (24 patients) and testicular (13 patients) spermatozoa were cryopreserved using the Cryopiece system. The total number of recovered spermatozoa and motility were assessed after thawing. Thirty-seven couples underwent ICSI using spermatozoa cryopreserved by the Cryopiece system, and ICSI outcomes (rates of fertilization, embryo cleavage, and clinical pregnancy) were evaluated. The average sperm post-thaw retrieval rate was 79.1%, and motility was 29.7%. Ejaculated spermatozoa had a higher post-thaw motility (32.5%) than testicular spermatozoa (21.8%; P = 0.005). ICSI achieved a fertilization rate of 61.9%, embryo cleavage rate of 84.6%, and clinical pregnancy rate of 43.3%. The ICSI outcomes in the ejaculated and testicular frozen-thawed spermatozoa were similar. Assisted oocyte activation (AOA) after ICSI with motile (72.1%) or immotile (71.9%) spermatozoa resulted in a significantly higher fertilization rate than that when using motile spermatozoa without AOA (52.0%; P = 0.005). However, AOA did not enhance the clinical pregnancy rate (55.6% or 40.0% vs 35.3%; P = 0.703). The Cryopiece system is simple and useful for the cryopreservation of small numbers of ejaculated or testicular spermatozoa for ICSI in patients with severe oligozoospermia or nonobstructive azoospermia.
Azoospermia ; Cryopreservation ; Female ; Humans ; Male ; Oligospermia ; Pregnancy ; Pregnancy Rate ; Retrospective Studies ; Semen ; Sperm Injections, Intracytoplasmic ; Sperm Motility ; Spermatozoa ; Testis

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*

Cryopreservation ; Humans ; Male ; Methanol ; Nitrogen ; Papillomavirus Infections ; Pilot Projects ; Semen Preservation ; Spermatozoa

Objective To investigate the effects of self-made carriers on the cryopreservation of ovarian tissue of sheep. Methods Thirty-two ovaries were randomly assigned to fresh group,programmed freezing group,self-made carrier I vitrification group,and self-made carrier Ⅱ vitrification group.The morphology,proliferation,apoptosis,and estrogen level of the ovarian tissue in each group were observed. Results After cryopreservation,the morphology normal rate of the primordial follicles in programmed freezing group,self-made carrier I vitrification group,and self-made carrier Ⅱ vitrification group were 74.2%,72.8%,and 72.3%,respectively,lower than that(83.7%)in the fresh group(χ
Animals ; Cryopreservation ; Female ; Freezing ; Ovarian Follicle ; Ovary ; Sheep ; Vitrification

Oocyte cryopreservation is widely used for clinical and social reasons. Previous studies have demonstrated that conventional slow-freezing cryopreservation procedures, but not storage time, can alter the gene expression profiles of frozen oocytes. Whether vitrification procedures and the related frozen storage durations have any effects on the transcriptomes of human metaphase II oocytes remain unknown. Four women (30-32 years old) who had undergone IVF treatment were recruited for this study. RNA-Seq profiles of 3 fresh oocytes and 13 surviving vitrified-thawed oocytes (3, 3, 4, and 3 oocytes were cryostored for 1,2, 3, and 12 months) were analyzed at a single-cell resolution. A total of 1987 genes were differentially expressed in the 13 vitrified-thawed oocytes. However, no differentially expressed genes were found between any two groups among the 1-, 2-, 3-, and 12-month storage groups. Further analysis revealed that the aberrant genes in the vitrified oocytes were closely related to oogenesis and development. Our findings indicated that the effects of vitrification on the transcriptomes of mature human oocytes are induced by the procedure itself, suggesting that long-term cryostorage of human oocytes is safe.
Adult ; Cryopreservation ; Female ; Humans ; Metaphase ; Oocytes ; RNA-Seq ; Vitrification

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

Background: The increasing number of young survivors after cancer treatment and of patients with non-malignant conditions who are at risk for subfertility has resulted in a demand for fertility preservation services, including the Philippines. Objective: The aim of this paper is to provide an overview of the history, indications, and management principles of fertility preservation. Also, the available strategies in the Philippines in both pre-pubertal and post-pubertal men and women and future directions of the field in the country will be discussed. Materials and methods: Literature review, historical accounts Results and conclusions Fertility preservation should be a priority when treating children and adults of reproductive age with agents that have deleterious effects on the gonads. If harmful treatment will be used, the options of fertility preservation should be discussed, as early as possible by the primary physician in collaboration with the oncologist and the reproductive medicine specialist. Most of the known options for fertility preservation are available in the Philippines and are being implemented in the local IVF centers. Recent developments hint of a potentially faster progress in the field with the establishment of the Philippine Society for Fertility Preservation in collaboration with other professional societies and a linkage with the Department of Health with the signing into law of the National Integrated Cancer Control Act of 2019.
Fertility Preservation ; Cryopreservation ; Oocytes ; Ovary ; Fertility

BACKGROUND: It is currently unknown whether patients with a fever after controlled ovulation during egg retrieval could increase the risk of pelvic infection or not, and fever itself may affect endometrial receptivity or embryo quality with poor pregnancy outcomes. The aim of this study was to analyze the outcomes of patients with fever during oocyte retrieval after the first frozen-thawed embryo transfer (FET) cycle. METHODS: This was a 1:3 retrospective paired study matched for age. In this study, 58 infertility patients (Group 1) had a fever during the control ovulation, and the time of the oocyte retrieval was within 72 hours, they underwent ovum pick up and whole embryo freezing ("freeze-all" strategy). The control subjects (Group 2) are 174 patients matched for age who underwent whole embryo freezing for other reasons. The baseline characteristics, clinical data of ovarian stimulation, and outcomes, such as the clinical pregnancy rate, ongoing clinical pregnancy rate were compared between the two groups in the subsequent FET cycle. RESULTS: All patients had no pelvic inflammatory disease after oocyte retrieval. Anti-Mullerian hormone (AMH) levels (4.2 vs. 2.2, P <0.001) were higher in group 2, and the number of oocytes retrieved, and fertilization rate were lower in group 1 (P < 0.001), but the endometrial thickness, the number of embryo transfers, and the type of luteal support supplementation were similar between the two groups. Regarding pregnancy outcomes in the subsequent FET cycle, the implantation rate, clinical pregnancy rate, early spontaneous rate, ectopic pregnancy rate, and ongoing pregnancy rate were all not significantly different. Further regression analyses showed that the clinical pregnancy rate and ongoing pregnancy rate were also not significantly different. CONCLUSIONS Transvaginal ultrasound-guided follicular puncture for oocyte retrieval is a safe and minimally invasive method for patients with fever. Moreover, the fever had almost no effect on embryo quality.
Cryopreservation ; Female ; Fertilization in Vitro ; Freezing ; Humans ; Infertility ; Oocyte Retrieval ; Oocytes ; Ovulation Induction ; Pregnancy ; Pregnancy Outcome ; Pregnancy Rate ; Retrospective Studies