冷冻保存软骨移植的实验研究

本实验采用低温冷冻异体兔胎软骨移植，实验组和新鲜软骨移植对照组。每组各移植软骨１２０块，于植入后１５、３０、６０、９０、１２０ｄ取出软骨标本，进行肉眼观察，光镜和电镜检查，免疫组化检查，结果冻存软骨无吸收和再生，亦无坏死，排异反应轻，对照组有不同程度的增长，部分出现钙化，边缘灶性坏死，本实验对软骨的冻存在临床应用上有着重要的意义。     

Experience with the cryopreservation of human embryos using the mouse as a model to establish successful techniques

Mouse embryos at the one-, two-, and eight-cell stages have been used to optimize the conditions for cryopreservation of human oocytes and embryos. For storage in glass vials using 1.5 M dimethyl sulfoxide (DMSO) as a cryoprotectant and slow cooling (0.3°C/min), phosphate-buffered medium was superior to Hepes-buffered medium. Termination of slow cooling at –80°C before transfer to liquid nitrogen with subsequent slow thawing (–8°C/min) resulted in more embryos surviving than when cooling terminated at –40°C and rapid thawing (–500°C/min) was employed. Dilution of DMSO upon thawing with medium containing 0.5M sucrose gave higher embryo survival rates than a stepwise (0.25 M decrements) dilution. Using these techniques, three pregnancies were established upon the transfer of 11 frozenthawed embryos to seven patients. Rates of embryo survival using the simpler cryopreservation technique of ice-free vitrification in 0.25-ml straws have been disappointing.     

Comparative study on density gradients and swim-up preparation techniques utilizing neat and cryopreserved spermatozoa

AIM: To 1) compare post-wash and post-thaw parameters of sperm processed with PureSperm density gradient technique and swim-up method; and 2) test the efficacy of two commonly available density gradient media PureSperm and ISolate. METHODS: This prospective study used semen specimens from 22 patients. Specimens from nine patients were processed by both PureSperm density gradient and swim-up method. These specimens were then cryopreserved. Thirteen specimens were processed by both PureSperm (40 % and 80 %) and Isolate (50 % and 90 %) double density gradient techniques. The two fractions processed by both PureSperm and swim-up were analyzed for post-wash sperm characteristics. Post-thaw analysis was done after 24 hours. Sperm fractions obtained after processing with PureSperm and ISolate were compared for post-wash sperm characteristics and ROS levels. Results: Specimens prepared with PureSperm had significantly higher median total motile sperm counts (TMSC) (32.2 x 10(6) vs. 17.6 x 10(6)), recovery rates (69.2 % vs. 50.0 %), and longevity at 4 hours (83.0 % vs. 55.0 %) compared to specimen prepared by swim-up. Post-thaw specimens also had a higher recovery and longevity at 4 hours with PureSperm as compared to the swim-up. Semen specimens processed by PureSperm had significantly higher total sperm count, TMSC, and percentage recovery rates (30.0 % vs. 19.7 %) than ISolate. CONCLUSION: Semen quality is better preserved in fresh and cryopreserved semen prepared with PureSperm density gradient compared to swim-up. A significant enrichment of sperm is observed with PureSperm compared to ISolate. Higher recovery rates of mature motile sperm obtained after PureSperm sperm preparation may be beneficial for successful ART.     

Managing risk associated with cryopreservation

Patients who consent to the frozen storage of sperm or embryos quite rightly expect the storing centre to do everything reasonably possible to keep them in optimum conditions. Both the process of cryopreservation and the cryofacility are loaded with risk, from patient/sample processing, through to the eventual utilization or disposal of specimens. The risk management process should focus on minimizing losses, including staff injury, premature warming of cells and tissues, mistaken identity, and transmission of infection. Early warning and monitoring systems should be in place for quality assurance and to prevent incidents involving cryovessels turning critical. Centres must ensure that every reasonable practical measure that can be put in place is done so, and that resourcing of the service adequately reflects the liability it represents.     

Treatment of the blastic transformation of chronic granulocytic leukemia using high dose BCNU chemotherapy and cryopreserved autologous peripheral blood stem cells

Seven nonsplenectomized patients with blastic CGL have received high dose BCNU chemotherapy followed by cryopreserved peripheral blood stem cells (PBSC). The PBSC obtained at diagnosis were stored in the vapor phase of liquid nitrogen in 10% dimethyl sulfoxide for 11-46 months prior to use. Patients received 2.9 X 10(8) (1.9-7.8) thawed washed mononuclear cells/kg over 30 minutes with minimal morbidity. One patient was not rendered pancytopenic and died with blastic leukemia at 4 months. One patient, previously treated with daily busulfan, died of progressive hepatic failure 2 months after high dose BCNU. Restoration of the chronic phase of CGL was observed in the remaining five patients. Peripheral blood counts returned to normal ranges after a median of 19 days. Median survival for all patients is 11 months. Cytogenetic studies revealed elimination of acquired aneuploid cell lines in four of seven patients with persistence of Ph1. We conclude that: 1) frozen PBSC retain their viability for up to 4 years after cryopreservation and 2) the use of autologous PBSC following ablative chemotherapy may be associated with both symptomatic and karyotypic improvement in patients with blastic CGL.     

Fetal rat intestinal transplantation: cryopreservation and cyclosporin A

Successful preservation of small bowel by cryobiologic techniques would increase the feasibility of intestinal transplants. Immunosuppression by Cyclosporin A (CyA) has also increased interest in intestinal transplantation. We have investigated the effect of cryopreservation and immunosuppression in fetal rat intestinal transplantation. Segments of fetal bowel implanted isogeneically into the paravertebral gutter of young rats were found to grow in a high percentage of animals (53% to 100%). Segments frozen to -20 degrees C or -40 degrees C at two rates of cooling, grew isogeneically (50% to 89%), demonstrating the feasibility of cryopreservation. Histologic examination of this bowel showed preservation of structure. When these segments were cooled and implanted allogeneically, no immunosuppressive effect was found. Segments protected by daily CyA administration grew. No synergistic effect was seen by associating CyA and cryopreservation. These experiments suggest the possibility of creating fetal small bowel long-term banking.     

Potassium assay in blastomere of two-cell mouse embryo after equilibration and washing from cryoprotector

The need for effective methods of cryopreservation of early mammalian embryos necessitates the study of the mechanisms of blastomere adaptation to the equilibration procedure and subsequent washing from the cryoprotector. The osmotic effects during these procedures can cause electrolyte imbalance in embryonic cells. Intracellular potassium concentrations at the stage of two-blastomere mouse embryo were studied by electron probe microanalysis. Translated fromByulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 138, No. 7, pp. 48–49, July, 2004     

Improved in vitro fertilization embryo quality and pregnancy rates with intracytoplasmic sperm injection of sperm from fresh testicular biopsy samples vs. frozen biopsy samples

OBJECTIVE: To compare the outcomes of first-attempt IVF-intracytoplasmic sperm injection (ICSI) cycles when using fresh testicular biopsy samples vs. frozen biopsy samples. DESIGN: Retrospective chart review of 92 consecutive first-attempt IVF-ICSI cycles. SETTING: Two IVF programs. PATIENT(S): Forty consecutive first-attempt IVF-ICSI patients using sperm from fresh testicular biopsy samples and 52 consecutive first-attempt IVF-ICSI cycles using frozen testicular biopsy samples. INTERVENTION(S): Testicular biopsy, IVF-ICSI with fresh and frozen-thawed spermatozoa. MAIN OUTCOME MEASURE(S): Fertilization rates, embryo quality, pregnancy, delivery, and spontaneous abortion rates. RESULT(S): A significantly increased ICSI fertilization percentage was obtained with frozen testicular biopsy samples (76.5% +/- 3.1%) vs. fresh biopsy samples (68.3% +/- 2.6%). However, embryo quality, pregnancy, and delivery rates were higher in the fresh biopsy group. Mean embryo score was 4.54 +/- 0.31 and 3.62 +/- 0.2 in the fresh vs. frozen group, respectively. Chemical pregnancy rates (60% vs. 49.1%), clinical pregnancy rates (56.4% vs. 41.2%), and delivery rates (48.7% vs. 31.2%) were each higher in the fresh group vs. frozen group. Accordingly, the spontaneous abortion rate was lower in the fresh group (21.7%) vs. the frozen group (33.3%). CONCLUSION(S): Although the use of frozen biopsy samples has logistical advantages, we conclude it may be advantageous to use fresh testicular biopsy samples in IVF-ICSI cases whenever possible, as fresh specimens yielded significantly improved embryo quality, generally higher pregnancy rates, and lower spontaneous abortion rates.     

Fertility after cancer: a prospective review of assisted reproductive outcome with banked semen specimens

OBJECTIVE: To examine the outcome of assisted reproduction techniques (ART) using cryopreserved semen from patients with cancer. DESIGN: Prospective. SETTING: Therapeutic semen banking program at a tertiary healthcare center. PATIENT(S): Twenty-nine men with cancer who cryopreserved their sperm before treatment at our facility from 1982 to 2001 and withdrew their samples for assisted reproduction (IUI, IVF, or intracytoplasmic sperm injection [ICSI]). INTERVENTION(S): Sperm bank records were used to identify the patients. Information on fertility potential indices was obtained from medical records and through interviews. Of the 29 patients, 9 had testicular cancer, 12 had Hodgkin's disease, and 8 had other types of cancer. MAIN OUTCOME MEASURE(S): Pregnancy and live births. RESULT(S): A total of 87 ART cycles (42 IUI, 26 IVF, and 19 ICSI) was performed. Of those cycles, 18.3% resulted in pregnancy (7% IUI, 23% IVF, and 37% ICSI), and 75% of the pregnancies resulted in a live birth (100% IUI, 83% IVF, and 57% ICSI). There was no significant difference in the outcomes when the results were stratified by type of ART and malignancy. None of the 11 infants who were born had congenital anomalies. CONCLUSION(S): Our findings emphasize the need for physicians to discuss the issue of semen cryopreservation with all men of reproductive age who have cancer before antineoplastic therapy is started.     

Quantitative assessment of ischemic tissue damage in ovarian cortical tissue with or without antioxidant (ascorbic acid) treatment

OBJECTIVE: To estimate ischemic tissue damage in ovarian cortex and to evaluate the effectiveness of ascorbic acid, an antioxidant, to protect ovarian tissue from apoptosis caused by ischemia. DESIGN: In vitro laboratory experiments. SETTING: Academic research institute. INTERVENTION(S): Fresh and frozen/thawed cortical sections of bovine ovaries were incubated in MEM medium with or without ascorbic acid for a duration of 3, 24, and 48 hours at 37 degrees C. MAIN OUTCOME MEASURE(S): Oxygen consumption rates, lactate dehydrogenase concentrations, apoptosis rates determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining, and DNA fragmentation analysis. RESULT(S): The oxygen consumption rates were correlated inversely with the duration of incubation. When the rates of apoptosis in primordial follicles with or without ascorbic acid treatment were compared, there was no statistically significant difference between the two groups. However, the ascorbic acid treatment group showed significantly decreased apoptosis in ovarian cortex (stromal cells) with 24 hours of incubation. CONCLUSION(S): The correlation between ischemic tissue damage and the duration of ischemia was verified. Ovarian cortex could tolerate ischemia at least for 3 hours. Ascorbic acid treatment reduced apoptosis in ovarian cortex up to 24 hours of incubation in vitro. It appeared that stromal cells were more vulnerable to ischemia compared to primordial follicles.