Assessment of DNA integrity and morphology of ejaculated spermatozoa from fertile and infertile men before and after cryopreservation

Cryopreservation of human spermatozoa is extensively used in artificial insemination and IVF programmes. Despite various advances in cryopreservation methodology, the recovery rate of functional post-thaw spermatozoa remains mediocre, with sperm motility being significantly decreased after freezing. This aim of this study was to investigate the effects of cryopreservation on both DNA integrity and morphology of spermatozoa from fertile and infertile men. Semen samples were obtained from 17 fertile and 40 infertile men. All samples were prepared by discontinuous Percoll density centrifugation (95.0:47.5). Samples were divided into aliquots to allow direct comparison of fresh and frozen spermatozoa from the same ejaculate. Aliquots for cryopreservation were mixed with a commercial cryoprotectant and frozen by static phase vapour cooling before plunging into liquid nitrogen. Thawing was carried out slowly at room temperature. Sperm DNA integrity was determined using a modified alkaline single cell gel electrophoresis (comet) assay and sperm morphology analysed using the Tygerberg criteria. DNA of semen and prepared spermatozoa from fertile men was found to be unaffected by cryopreservation. In marked contrast, spermatozoa from infertile men were significantly damaged by freeze-thawing. Cryopreservation had a detrimental effect on morphology of semen and prepared samples from fertile and infertile men.     

Cryoloop vitrification yields superior survival of Rhesus monkey blastocysts

BACKGROUND: Vitrification using the cryoloop procedure was evaluated for preservation of non-human primate blastocysts by comparing survival results from two different cryoprotectant mixtures with prior results from controlled rate cooling. METHODS: Rhesus monkey blastocysts were produced by intracytoplasmic sperm injection of mature oocytes from cycling females stimulated with recombinant human hormones. Morphologically well-formed blastocysts were divided between Procedure A (2.8 mol/l dimethylsulphoxide and 3.6 mol/l ethylene glycol with 0.65 mol/l sucrose and 25 micromol/l Ficoll in TALP-HEPES with 20% fetal bovine serum (TH20)) and Procedure B (3.4 mol/l glycerol and 4.5 mol/l ethylene glycol in TH20). After >48 h in liquid nitrogen, the removal of cryoprotectants was accomplished in the presence of a 3-step series of decreasing sucrose concentrations in TH20. Surviving embryos were co-cultured on buffalo rat liver cells. RESULTS: Of 16 blastocysts vitrified via Procedure A, 38% survived with minimal lysis and only one hatched in culture; in contrast, of 33 blastocysts vitrified by Procedure B, 85% survived and 71% hatched. Of 22 blastocysts cryopreserved by conventional slow cooling, 36% survived and 6% hatched. Transfer into three recipients, each with two embryos vitrified with Procedure B, resulted in a successful twin-term pregnancy. CONCLUSION: Modified cryoloop vitrification with a final solution of 3.4 mol/l glycerol and 4.5 mol/l ethylene glycol is a promising procedure for preserving Rhesus monkey blastocysts that is simple, rapid, and inexpensive.     

Feasibility of vitrification as a storage method for tissue-engineered blood vessels

It is well established that, in multicellular systems, conventional cryopreservation results in damaging ice formation, both in the cells and in the surrounding extracellular matrix. As an alternative to conventional cryopreservation, we performed a feasibility study using vitrification (ice-free cryopreservation) to cryopreserve tissue-engineered blood vessels. Fresh, frozen, and vitrified tissue-engineered blood vessels were compared using histological methods, cellular viability, and mechanical properties. Cryosubstitution methods were used to determine the location of ice in conventionally cryopreserved engineered vessels. Ice formation was negligible (0.0 +/- 0.0% of vessel area) in the vitrified specimens, and extensive (68.3 +/- 4.5% of vessel area) in the extracellular matrix of frozen specimens. The metabolic assay and TUNEL staining results indicated that vitrified tissue had similar viability to fresh controls. The contractility results for vitrified samples were >82.7% of fresh controls and, in marked contrast, the results for frozen samples were only 10.7% of fresh controls (p < 0.001). Passive mechanical testing revealed enhanced tissue strength after both freezing and vitrification. Vitrification is a feasible storage method for tissue-engineered blood vessel constructs, and their successful storage brings these constructs one step closer to clinical utility.     

五种玻璃化冻存试剂单独或组合应用对髓核细胞的毒性分析

BACKGROUND: Transplantation of allogeneic intervertebral disc can be facilitated by the cryopreservation of the intervertebral disc. But the traditional cryopreservation methods always lead to the appearing of ice crystals inside and outside the cells which can cause cellular injury. The vitrification method that can avoid the formation of ice crystals have been widely applied in the cryopreservation field. However, only a few reports have assessed the vitrified cryopreservation of the intervertebral disc, and the toxicity of cryoprotectants to the nucleus pulposus cells have not been fully explored. OBJECTIVE: To determine the order of toxicity of five commonly used cryoprotectants that are used alone or in combination to rabbit nucleus pulposus cells, and to select the optimal cryoprotectant for the vitrification of the intervertebral disc. METHODS: We chose five most commonly used cryoprotectants including dimethyl sulphoxide, formamide, ethylene glycol, propylene glycol and glycerol. Then, 5 single commonly used cryoprotectants, 10 mixed agents containing any 2 commonly used cryoprotectants, and 10 mixed agents containing any 3 commonly used cryoprotectants were formulated. Cell viability of nucleus pulposus cells was determined using cell counting kit-8 and fluorescein diacetate/propidium iodide method. All data obtained were analyzed statistically to choose the appropriate combining scheme with less toxicity. RESULTS AND CONCLUSION: The order of the toxicity of these five commonly used cryoprotectants from low to high was ethylene glycol, glycerol, formamide, dimethyl sulphoxide, and propylene glycol. The toxicity of the combined agents containing two or three commonly used cryoprotectants was lower than that of any commonly used cryoprotectants that were used to formulate them. The toxicity of the mixed agents that contained ethylene glycol or glycerol was lower than that of any other mixed agents. So we can choose the mixed cryoprotectants that contain ethylene glycol and (or) glycerol for the vitrification of the intervertebral disc.       

Preferential vitrification of water in small alginate microcapsules significantly augments cell cryopreservation by vitrification

The morphological changes of small (~100 μm) alginate microcapsules and the biophysical alterations of water in the microcapsules during cryopreservation were studied using cryomicroscopy and scanning calorimetry. It was found that water in the small microcapsules can be preferentially vitrified over water in the bulk solution in the presence of 10% (v/v) or more dimethylsulfoxide (DMSO, a cryoprotectant), which resulted in an intact morphology of the microcapsules post cryopreservation with a cooling rate of 100°C/min. A small amount of Ca²⁺ (up to 0.15 M) was also found to help maintain the microcapsule integrity during cryopreservation, which is attributed to the enhancement of the alginate matrix strength by Ca²⁺ rather than promoting vitrification of water in the microcapsules. The preferential vitrification of water in small microcapsules was further found to significantly augment cell cryopreservation by vitrification at a low concentration of cryoprotectants (i.e., 10% (v/v)) using a small quartz microcapillary (400 μm in diameter). Therefore, the small alginate microcapsule could be a great system for protecting living cells that are highly sensitive to stresses due to freezing (i.e., ice formation) and high concentration of cryoprotectants from injury during cryopreservation.     

Open pulled straws for vitrification of mature mouse oocytes preserve patterns of meiotic spindles and chromosomes better than conventional straws

Vitrification of oocytes has been applied recently for humans, but remains elusive. The microtubules of oocytes are vulnerable to cryoprotectants and thermal changes. Using mouse oocytes, the effects of vitrification in open pulled straws (OPS) were investigated on survival, the meiotic spindle, and chromosomes and compared with conventional straws. Mature oocytes were allocated to four groups for exposure to cryoprotectants, vitrification in conventional straws, or vitrification in OPS. They were diluted in stepwise sucrose solutions. Oocytes without treatments were used as controls. The surviving oocytes were stained for meiotic spindles and chromosomes. After dilution, all of the oocytes exposed to cryoprotectants survived. Vitrification sometimes resulted in lysis so that survival using OPS (62%) was significantly (P < 0.05) smaller than that using conventional straws (81%). Oocytes exposed to cryoprotectants or vitrified exhibited serious disturbances of microtubules immediately post-dilution. After 1 h incubation, the microtubules could repolymerize so that the OPS group had significantly (P < 0.05) more normal spindles (78%) than did the conventional straw group (21%). The former also tended to have more compact chromosomes (87%) than did the latter (78%). OPS for vitrification of oocytes achieve more rapid cooling, warming, and dilution and so reduce spindle injury. However, the lower survival rate in OPS needs improvement.     

Cryopreservation of alginate-fibrin beads involving bone marrow derived mesenchymal stromal cells by vitrification

Application of cell--biomaterial systems in regenerative medicine can be facilitated by their successful low temperature preservation. Vitrification, which avoids ice crystal formation by amorphous solidification, is an emerging approach to cryopreservation. Developing vitrification strategy, effective cryopreservation of alginate-fibrin beads with porcine mesenchymal stromal cells has been achieved in this study. The cell-biomaterial constructs were pre-cultured for 20 days before cryopreservation, allowing for cell proliferation and construct stabilization. Ethylene glycol (EG) was employed as the basic cryoprotectant for two equilibration solutions. Successful cryopreservation of the constructs was achieved using vitrification solution composed of penetrating (EG MW 62 Da) and non-penetrating (sucrose MW 342 Da) cryoprotectants. Stepwise procedure of introduction to and removal of cryoprotectants was brief; direct plunging into liquid nitrogen was applied. Cell viability, evaluated by combining live/death staining and confocal laser microscopy, was similar for both control and vitrified cells in the beads. No detectable damage of microstructure of cryopreserved beads was found as shown by scanning electron microscopy. Both osteogenically induced control and vitrified cells in the constructs were equally capable of mineral production and deposition. There was no statistically significant difference in metabolic activity and proliferation between both groups during the entire culture period. Our study leads to the conclusion that the developed cryopreservation protocol allowed to maintain the integrity of the beads while preserving the ability of the pig bone marrow derived mesenchymal stromal cells to proliferate and subsequently differentiate; demonstrating that vitrification is a promising approach for cryopreservation of "ready-to-use" cell-biomaterial constructs.     

Developmental rate and ultrastructure of vitrified human pronuclear oocytes after step-wise versus direct rehydration

BACKGROUND: This study compared the viability of human pronuclear oocytes subjected to vitrification followed by post-thaw step-wise removal of cryoprotectants versus direct rehydration, in terms of their subsequent in vitro survival and ultrastructural features. METHODS: A total of 115 three-pronuclei stage oocytes were cryopreserved in super-open-pulled straws by vitrification in 40% ethylene glycol + 0.75 mol/l sucrose for either 1 min or 10 s at 38 degrees C, followed by direct plunging into liquid nitrogen. After thawing, oocytes vitrified for 1 min (group 1) or 10 s (group 2) were expelled into a graded series of sucrose solutions (1.0, 0.75, 0.5, 0.25 and 0.12 mol/l) for removal of the cryoprotectant in five 2.5 min steps. A second batch of oocytes vitrified for either 1 min (group 3) or 10 s (group 4) were directly expelled into culture medium at 38 degrees C after thawing. Finally, the ultrastructural changes occurring in oocytes in each of the treatment groups were evaluated. RESULTS: Oocyte development (division to two-blastomere stage) rates after in vitro culture were 82, 83, 0 and 0% for groups 1, 2, 3 and 4, respectively. The harsh osmotic process involved in direct rehydration provoked ultrastructural changes, including the disruption of cytoplasmic and pronuclear membranes as well as intracellular organelles. CONCLUSION: The direct post-thaw rehydration of human pronuclear oocytes has lethal osmotic effects, such that protocols for vitrifying human pronuclear oocytes should include the step-wise removal of the cryoprotectant.     

Comparison of the effects of controlled-rate cryopreservation and vitrification on 2-cell mouse embryos and their subsequent development.

Effects of two cryopreservation procedures (conventional slow controlled-rate freezing using a programmable freezer and vitrification by direct plunging into liquid nitrogen) were compared on 2-cell embryos and their subsequent development to blastocysts, fresh or cryopreserved 2-cell mouse embryos were developed into blastocysts in vitro. The percentage of vitrified embryos which developed into blastocysts was significantly lower than that of fresh and slow controlled-rate frozen embryos. Although blastocysts from each cryopreservation procedure appeared morphologically normal and neither number of cells in the blastocysts nor in-vitro trophoblast spreading differed significantly, there were significant differences in their functional viability. First, the glucose incorporation activity in terms of [(3)H]2-deoxyglucose (2-DG) uptake in vitrified and thawed 2-cell embryos significantly decreased compared with fresh or slow controlled-rate frozen and thawed 2-cell embryos. Second, 2-DG uptake by blastocysts developed in vitro from fresh 2-cell embryos and from slow controlled-rate frozen or vitrified 2-cell embryos was 105 +/- 75, 43.0 +/- 28.3 and 22.0 +/- 11.4 fmol/embryo/h respectively. Third, the implantation rate of blastocysts developed in vitro from vitrified 2-cell embryos (10.2%) was significantly lower than that from fresh 2-cell embryos (30.8%) or slow controlled-rate frozen 2-cell embryos (22.1%). Since these data suggest that cryopreservation may have ulterior consequences on the functional development of embryos and that vitrification may exert a more harmful effect than slow controlled-rate freezing, more attention should be paid to its safety before vitrification is used routinely in a clinical programme.     

Novel direct cover vitrification for cryopreservation of ovarian tissues increases follicle viability and pregnancy capability in mice

BACKGROUND: Cryopreservation of ovarian tissue is valuable for fertility preservation. We develop an innovative vitrification method using less concentrated cryoprotectants and direct application of liquid nitrogen to the ovarian tissue (direct cover vitrification, DCV) to improve its efficiency. METHODS: Ovaries of 5- to 6-week-old C57BL/6J mice were randomly allocated to four groups: DCV, conventional vitrification, slow-freezing and non-frozen controls. Experiment 1: observing the follicle morphology. Experiment 2: assessing viability. Experiment 3: investigating the ultrastructure. Experiment 4: examining the follicle number after grafting. Experiment 5: ascertaining pregnancy potential by allogeneic orthotopic transplantation. RESULTS: The percentages of morphologically normal or viable follicles from DCV were significantly greater than those achieved from conventional vitrification and slow freezing (P < 0.01). The ultrastructure of primordial follicles from DCV appeared better than that achieved from conventional vitrification and slow freezing. After grafting, the follicle number from DCV was greater than conventional vitrification (P = 0.001) and slow freezing (P = 0.021). The pregnancy rate of DCV was higher than conventional vitrification (P < 0.01). The litter size from DCV was comparable with that from non-frozen graft and was significantly greater than that achieved from conventional vitrification and slow freezing (P < 0.01). CONCLUSIONS: DCV is highly efficient for cryopreservation of ovarian tissue. Using less concentrated cryoprotectants appears to reduce toxicity. Direct cover by liquid nitrogen maximizes cooling that could facilitate vitrification and prevent ice crystal injury.     

冷冻载体对扩张期囊胚解冻结局的影响

背景：囊胚的冷冻复苏对人类辅助生殖技术的发展具有重要意义,合适的冷冻载体可以降低冷冻毒性提高冷冻效率,是改善囊胚冷冻复苏结局的研究热点之一。 目的：观察不同冷冻载体对昆明小鼠扩张期囊胚玻璃化冷冻解冻结局的影响。 方法：以新鲜昆明小鼠扩张期囊胚作为对照组,随机选取同期小鼠胚进行玻璃化冷冻解冻,冷冻前使用激光仪行人工皱缩,使用玻璃化法进行冷冻和解冻,按实验载体不同分为冷冻环组、闭合拉细麦管组和自制麦管叶片组;冷冻环组和自制麦管叶片组投入液氮前与冷冻保护剂接触时间 <40 s,40-60 s和60-90 s;解冻后囊胚使用胚胎囊期培养基添加10%血清替代品进行微滴培养。比较解冻后组间囊胚丢失率、囊胚复苏率和培养24 h囊胚孵出率。 结果与结论：相比闭合拉细麦管组,冷冻环组和自制麦管叶片组的囊胚丢失率降低,胚胎复苏率增高(P < 0.05),各冷冻组复苏囊胚培养24 h孵出率均低于对照组(P < 0.05)。使用冷冻环载体和自制麦管叶片载体冷冻解冻囊胚,投入液氮前与冷冻保护剂接触时间90 s内不同时间胚胎复苏率和孵出率的比较差异无显著性意义。结果说明冷冻环载体和自制麦管叶片载体用于昆明小鼠扩张期囊胚玻璃化解冻复苏的效果较好。     

Cryopreservation of human embryos using ethylene glycol in controlled slow freezing

BACKGROUND: Ethylene glycol (EG) has been successfully used as a cryoprotectant for vitrification of mammalian formula embryos (including human embryos) due to its low formula weight and high permeation into cells compared with other cryoprotectants, including propylene glycol (PROH). This study was carried out to evaluate the permeation and toxicity of EG and to investigate the effects of its use in a slow-freezing protocol on post-thaw development of mouse embryos and on pregnancy outcome of frozen human embryos. METHODS: Spare human embryos after embryo transfer were cryopreserved using 1.5 mol/l EG or PROH using a slow-freezing protocol which had been tested previously in mouse experiments. RESULTS: The post-thaw survival rate of human embryos in the EG group (80.6%) was significantly higher than that in the PROH group (65.2%, P < 0.05). The implantation and clinical pregnancy rates of human embryos in the EG group (20.3 and 46.9%) were significantly higher than those in the PROH group (7.5 and 24.6%, P < 0.05). CONCLUSIONS: Ethylene glycol may be a good substitute for PROH to cryopreserve human embryos using a slow-freezing protocol.     

Survivability of rabbit amniotic fluid-derived mesenchymal stem cells post slow-freezing or vitrification

This work aimed to evaluate the effect of two distinct cryopreservation procedures - conventional slow-freezing and vitrification, on survivability and mesenchymal marker expression stability of rabbit amniotic fluid-derived mesenchymal stem cells (rAF-MSCs). Cells at passage 2 were slowly frozen, using 10% of dimethylsulfoxide, or vitrified, using 40% of ethylene glycol, 0.5 M sucrose and 18% Ficoll 70. After three months storage in liquid nitrogen, viability, chromosomal stability, ultrastructure, surface and intracellular marker expression and differentiation potential of cells were evaluated immediately post-thawing/warming and after additional culture for 48–72 h. Our results showed decreased (P ≤ 0.05) viability of cells post-thawing/warming. However, after additional culture, the viability was similar to those in fresh counterparts in both cryopreserved groups. Increase (P ≤ 0.05) in the population doubling time of vitrified cells was observed, while doubling time of slow-frozen cells remained similar to non-cryopreserved cells. No changes in karyotype (chromosomal numbers) were observed in frozen/vitrified AF-MSCs, and histological staining confirmed similar differentiation potential of fresh and frozen/vitrified cells. Analysis of mesenchymal marker expression by qPCR showed that both cryopreservation approaches significantly affected expression of CD73 and CD90 surface markers. These changes were not detected using flow cytometry. In summary, the conventional slow-freezing and vitrification are reliable and effective approaches for the cryopreservation of rabbit AF-MSCs. Nevertheless, our study confirmed affected expression of some mesenchymal markers following cryopreservation.     

Ultra-rapid freezing of very low numbers of sperm using cryoloops

BACKGROUND: With the availability of ICSI, men with severe oligozoospermia (<5x10(6)/ml) are able to reproduce. Current methods for cryopreservation of severe oligozoospermic samples are labour intensive and costly. The objective of this study was to evaluate whether freezing small numbers of motile sperm (approximately 100) was feasible using cryoloops. METHODS: Initial tests assessed the effect of various dilutions of cryoprotectants on pre-freezing sperm motility. Several solutions were further evaluated for their ability to cryoprotect sperm during ultra-rapid freezing. Sperm were placed on cryoloops and held in liquid nitrogen vapour for 5 min prior to freezing (ultra-rapid freezing) or directly submerged into liquid nitrogen. Using the optimal cryoprotectant and technique from these experiments, ultra-rapid and standard slow-rate freezing protocols were compared. RESULTS: Optimal sperm survival was seen when sperm in cryoloops were placed in liquid nitrogen vapour in test yolk buffer with 12% v/v glycerol versus other cryoprotectants. Using this cryoprotectant, post-thaw sperm motility is comparable between ultra-rapid and slow-rate freezing methods. CONCLUSION: Ultra-rapid freezing of very low numbers of sperm is feasible using cryoloops suspended in liquid nitrogen vapour for 5 min.     

Blastocoele collapse by micropipetting prior to vitrification gives excellent survival and pregnancy outcomes for human day 5 and 6 expanded blastocysts

BACKGROUND: Manual puncture of the trophectoderm of human blastocysts with a needle before vitrification increases their survival rate, but the embryos take a long time to re-expand. This study examined whether causing human blastocysts to collapse by manual pipetting before vitrification would allow more rapid re-expansion and improve pregnancy rates. METHODS: After embryo transfer in IVF cycles, surplus embryos that developed to the expanded blastocyst stage were placed in cryoprotectant and then artificially shrunk by mechanical pipetting with a fine hand-drawn glass pipette slightly smaller in diameter than the blastocyst. The shrunken embryos were placed in a small volume of vitrification solution and plunged into liquid nitrogen on a cryotop. The blastocysts were thawed by warming and then dilution in 1 mol/l sucrose. RESULTS: Of 49 expanded vitrified blastocysts, 48 (98%) re-expanded within 3 h after warming. Following transfer (48 blastocysts in 28 cycles), 14 women (50%) became clinically pregnant, and the implantation rate was 33% (16/48). Eight healthy babies have been born in six deliveries, and the other eight pregnancies are ongoing. To date, there have been no spontaneous abortions. CONCLUSIONS: The results suggest that artificial shrinkage with pipetting is a simple and effective technique to assist successful cryopreservation of expanded blastocysts by vitrification.     

An improved protocol for dilution of cryoprotectants from vitrified human blastocysts

BACKGROUND: The purposes of this study were to compare the survival rate of human blastocysts thawed by two different methods after vitrification using electron microscopic (EM) grids, and to report the successful pregnancies and live births resulting from the transfer of blastocysts that had survived the established thawing method. METHODS: The blastocysts produced from three pronuclei zygotes in IVF cycles were vitrified on an EM grid. After dilution of cryoprotectant by either a 6- or 2-step method, the survival rate of the blastocysts was compared. RESULTS: The survival rate of blastocysts thawed using the 6-step method (82.6%, 100/121) was significantly higher than that of the 2-step method (50.6%, 87/172; P < 0.01). Therefore, we applied the 6-step method in our blastocyst cryopreservation programme. Overall, 34.1% (14/41) of clinical pregnancies and 11 live births were achieved. CONCLUSIONS: The present results indicated that a 6-step thawing method was more effective than a 2-step thawing method for thawing vitrified human blastocysts.     

Development of a novel electrophoretic system for the isolation of human spermatozoa

BACKGROUND: Optimization of assisted conception outcomes involves the development of rapid, safe, effective techniques for the isolation of functional human spermatozoa free from significant DNA damage. In this study we describe a novel electrophoretic sperm isolation technique that achieves these objectives. METHODS: The separation system consisted of a cassette comprising two 400 mul chambers separated by a polycarbonate filter containing 5 micromol/l pores and bounded by a 15 kDa polyacrylamide membrane to allow the free circulation of buffer. Semen was introduced into one chamber, current applied (75 mA at variable voltage) and within seconds a purified suspension of spermatozoa could be collected from the adjacent chamber. These cells were assessed for their count, viability, motility, morphology and DNA integrity. RESULTS: The suspensions generated by the electrophoretic separation technique contained motile, viable, morphologically normal spermatozoa and exhibited low levels of DNA damage. Moreover, these cell suspensions were free from contaminating cells, including leukocytes. The technique was comparable to discontinuous gradient centrifugation except that it took a fraction of the time and generated cells with significantly less DNA damage. CONCLUSION: Electrophoretic separation represents a highly effective, novel approach for the isolation of spermatozoa for assisted conception purposes.     

Expression of Hsp60 and Grp78 in the human endometrium and oviduct, and their effect on sperm functions

BACKGROUND: Within the female genital tract, spermatozoa undergo a series of membranous and intracellular transformations to become competent at fertilizing the oocyte. In the bovine, previous studies have shown that two oviductal proteins, heat shock protein 60 (Hsp60) and glucose regulated protein 78 (Grp78), bind to spermatozoa and may be involved in this acquisition of fertilizing competence. METHODS: Immunohistochemical studies were performed on human endometrial and oviduct tissues to localize these two chaperones in the female genital tract. Human spermatozoa were incubated under capacitating conditions in the presence or absence of recombinant Hsp60 or Grp78. Following a 4-h incubation, the effects of these proteins were evaluated on sperm acrosomal integrity, motility, protein phosphotyrosine content and free intracellular calcium concentrations. RESULTS: Both chaperones were present in the uterus and oviduct epithelial cells and were shown to bind to human spermatozoa. Incubation with either exogenous Hsp60 or Grp78 did not affect sperm viability, motility or acrosomal integrity. Hsp60 partially prevented the increase in p81 phosphotyrosine content induced by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine and both chaperones significantly increased the sperm intracellular calcium concentration. Moreover, the progesterone-induced increase in intracellular calcium was higher when sperm were pre-treated with either Hsp60 or Grp78. CONCLUSIONS: Our study suggests that these two proteins may affect human sperm intracellular signalling pathways and capacitation.     

Thermal Expansion of Blood Vessels and Muscle Specimens Permeated with DMSO,DP6, and VS55 at Cryogenic Temperatures

As part of an ongoing effort to characterize the continuum mechanics during cryopreservation via vitrification (vitreous in Latin means glass), the current study focuses on measuring the thermal expansion of cryoprotective agents in the presence and absence of biological samples of bovine muscle and goat arteries. The cryoprotectants under investigation are 7.05M DMSO, DP6, and VS55, with permeation time of up to 48 h. This study focuses on the upper part of the cryogenic temperature range, where the cryoprotectant behaves like a liquid in all practical applications. The specific lower boundary of this range is unique to the cryoprotectant composition and to the applied cooling rate. The measurement device used in the current study is a modification of a prototype presented recently, in which modifications are made to accommodate biological samples. Based on thermal strain observations, it is concluded that the presence of tissue samples promotes crystallization (the tissue samples serve as potential nucleation sites). It is shown in this study that the thermal strain of tissue samples permeated with 7.05M DMSO and DP6 is no greater than the thermal expansion of the same cryoprotectants in the absence of tissue samples, and in some cases it is significantly less. For a given cryoprotectant in comparable thermal conditions, experiments on bovine muscle samples show significantly higher scattered data than experiments on artery samples.     

Artificial shrinkage of blastocoeles using either a micro-needle or a laser pulse prior to the cooling steps of vitrification improves survival rate and pregnancy outcome of vitrified human blastocysts

BACKGROUND: Since we reported the first successful birth from a blastocyst vitrified using a cryoloop technique, our results showed that the survival rate of vitrified blastocysts was negatively correlated with the expansion of the blastocoele. We speculated that a large blastocoele may disturb the efficacy of vitrification. Therefore, we evaluated the effectiveness of artificial shrinkage (AS) of blastocoeles before vitrification, on increasing the survival rate of vitrified blastocysts. METHODS: Supernumerary expanded blastocysts on day 5 were vitrified after AS, which was performed by puncturing the blastocoele with a micro-needle, or by making a hole in the blastocoele with a laser pulse. After warming, viable blastocysts (confirmed by re-expansion of the blastocoele) were transferred to patients with hormone replacement cycle. We compared these data with those of our previous report where AS was not carried out. RESULTS: The survival rate was significantly higher (97.2%, 488/502) in this study than that of the previous report (86%). After 266 transferable cycles, 160 patients became pregnant (60.2%), which was significantly higher than our previous results (34.1%, 29/85). The implantation rate was 46.7% (209/448). CONCLUSIONS: Our results revealed that the survival rate and the pregnancy rate of vitrified expanded and hatching blastocysts can be improved by using AS to collapse the blastocele before vitrification.