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PubMed2000

Is a review of standard procedures for cryopreservation needed?: safe and effective cryopreservation-should sperm banks and fertility centres move toward storage in nitrogen vapour?

A recent consultation documentation by the Human Fertilisation and Embryology Authority (HFEA) which focused on the safe cryopreservation of gametes and embryos highlighted the need for a review of the way that fertility clinics in the UK store potentially infective material. The main points for consideration were to: (i) ensure containers used for cryopreservation are guaranteed by manufacturers to withstand low temperatures; (ii) use secondary containers, i.e. 'double bagging' of samples if stored in the liquid phase; and (iii) store in nitrogen vapour as a 'safer' alternative. In this article we examine a number of issues related to vapour storage which need careful consideration, including safety, cost and the effectiveness of various storage techniques in maintaining gamete and embryo viability. We also discuss the effectiveness of vapour storage in comparison with current liquid nitrogen storage techniques. In conclusion, we propose that fertility clinics should be compelled to review their cryopreservation procedures, not just because of new legislation or indeed fear of litigation but by a moral obligation.

Bacterial Infections↗
PubMed2000

Bone marrow transplantation depleted of T cells followed by repletion with incremental doses of donor lymphocytes for relapsing patients with chronic myeloid leukemia: a therapeutic strategy.

BACKGROUND: For patients with chronic myeloid leukemia (CML), long-term survival after stem cell transplantation requires adequate control of graft-versus-host disease (GVHD) and disease recurrence. Relapsing patients respond to donor lymphocyte infusion (DLI) but develop life-threatening complications. METHODS: Patients with CML in first chronic phase received bone marrow (n = 14) or peripheral blood progenitor cell transplants (n = 4) from HLA-identical siblings. GVHD prophylaxis was by ex vivo T-cell depletion with CAMPATH 1G. If disease recurred, donors' mononuclear cells were collected by apheresis, the CD3 samples commencing at 10(6)/kg were aliquoted at half-log increment intervals, cryopreserved, and infused until disease clearance. RESULTS: Eighteen patients (median age: 32.5 years) received transplants. All engrafted without procedure-related mortality. Fourteen patients relapsed, and 13 entered the DLI program. Two developed extensive GVHD after single schedule infusions ranging from 89x10(6) to 670x10(6) mononuclear cells/kg, and one survives in complete remission (CR). The rest, treated with incremental dose DLI, experienced no acute toxicities. One, who had developed grade III steroid-responsive GVHD, died in CR2 from opportunistic infections. Steroids reversed limited cutaneous GVHD and elevated liver enzymes in five patients. Three others developed pancytopenia, and two restored blood counts only after donor peripheral blood progenitor cell infusions. Molecular CR2 was established in 12/13 patients, occurring in 10/11 (91%) on the incremental program at a median accumulation of 67 (range: 5-166) x10(6) CD3 cells/kg. Sixteen of 18 (89%) survive at median of 854.5 days from bone marrow transplantation, 4 in CR1 and 10 in CR2 at a median disease-free survival (for remission 2) duration of 341 days. The median combined disease-free survival of the 14 patients in CR 1+2 is 660 days, with 99% average performance status. CONCLUSIONS: Escalating DLI leads to safe new molecular CR in most CML relapse patients. These results raise the possibility of using "safe" transplantation programs of T-cell depletion, that include graded DLI as prevention against disease recurrence.

Adolescent↗
PubMed1992

[Cryopreservation of unfertilized rat oocytes by ultrarapid freezing].

Unfertilized rat oocytes were placed in a highly concentrated solution of cryoprotectant (DAP 224:2M dimethylsulphoxide, 2M acetamide, 4M propylene glycol in PB1) in 0.5 ml sampling tubes and then immediately immersed into liquid nitrogen; thawing was conducted in a 37 degrees C waterbath. After thawing, 630 out of 968 oocytes (65.1%) were morphologically normal. After insemination in vitro of cryopreserved oocytes, the proportion of pronuclear oocytes with spermatail (s), male (s) and female pronuclei (8-10 h post insemination), and 2-cell embryos with two identical blastomeres (28-30 h post insemination) was 60.8% (152/250) and 29.8% (39/131), respectively. One hundred and fifty oocytes that were judged as pronuclear oocytes under the inverted microscope 8-10 h after insemination were transferred to the oviducts of pseudopregnant recipients; 18.7% (28/150) of the oocytes developed to normal young.

Animals↗
PubMed2001

Effects of three cryopreservation methods and two semen extenders on the quality of dog semen after thawing.

The aim of this study was to evaluate the influence of two diluents, one with and the other without Orvus ES paste, and three methods of cryopreservation on the quality of dog semen after thawing. The investigation was carried out on 126 ejaculates collected from 37 dogs. In Expt 1, sperm-rich fractions of ejaculates were frozen in 0.25 ml minitubes and pellets. In Expt 2, each sperm-rich fraction of ejaculate was divided and extended in two diluents, one with and the other without Orvus ES paste. Samples of semen were frozen in pellets and 0.5 ml French straws. Motility, percentage of spermatozoa with normal acrosomes and longevity of spermatozoa were significantly higher (P < 0.05) in semen samples frozen in pellets than in samples frozen in 0.25 ml minitubes. Aspartate aminotransferase activity in extracellular fluid was significantly (P < 0.05) lower in semen frozen in pellets. There were no significant differences in quality after thawing between semen samples frozen in pellets and 0.5 ml French straws. Longevity, unlike acrosome status and aspartate aminotransferase activity, was greater in samples extended in Tris-buffered diluent with addition of Orvus ES paste, regardless of the method of cryopreservation.

Animals↗
PubMed2006

Within- and between-subject variation in semen parameters in infertile men and normal semen donors.

OBJECTIVE: To determine within- and between-subject variation of semen parameters in infertile and normal men. DESIGN: Retrospective analysis of 74 infertile men and 65 normal men producing five or more ejaculates each. SETTING: Clinical andrology laboratory. SUBJECT(S): Men presenting to an infertility clinic for semen analysis and normal men providing donor semen for cryopreservation. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Within-subject coefficients of variation (CVw) and between-subject coefficients of variation (CVb), calculated for sperm count, motility, and velocity, semen volume, and motility and velocity after cryopreservation. RESULT(S): The number of ejaculates ranged 5-20 in patients (479 total) and 5-136 in donors (2,043 total). Sperm counts, motility, and velocity were statistically greater in donors, whereas semen volume was significantly greater in patients. The CVw in patients and donors was greatest in sperm counts (54.2% and 45.8%, respectively) and lowest in velocity (20.1% and 19.9%, respectively). The CVb in patients and donors was markedly higher than CVw for all parameters in both patients and donors. The CVw was lower, and the CVb was higher, in donors compared with patients. The CVw was not affected by age of the individual or abstinence period. Cryopreservation resulted in a marked increase in variability. Intraclass correlations indicated that a minimum of three semen samples were required to achieve reliability. CONCLUSION(S): These data indicate large within- and between-subject variation in sperm parameters, especially sperm count, in both patients and healthy donors, and further substantiate the need for measurement of multiple ejaculates before characterizing a man as normal or infertile.

Adult↗
PubMed2001

High degree of interlaboratory reproducibility of human immunodeficiency virus type 1 protease and reverse transcriptase sequencing of plasma samples from heavily treated patients.

We assessed the reproducibility of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and protease sequencing using cryopreserved plasma aliquots obtained from 46 heavily treated HIV-1-infected individuals in two laboratories using dideoxynucleotide sequencing. The rates of complete sequence concordance between the two laboratories were 99.1% for the protease sequence and 99.0% for the RT sequence. Approximately 90% of the discordances were partial, defined as one laboratory detecting a mixture and the second laboratory detecting only one of the mixture's components. Only 0.1% of the nucleotides were completely discordant between the two laboratories, and these were significantly more likely to occur in plasma samples with lower plasma HIV-1 RNA levels. Nucleotide mixtures were detected at approximately 1% of the nucleotide positions, and in every case in which one laboratory detected a mixture, the second laboratory either detected the same mixture or detected one of the mixture's components. The high rate of concordance in detecting mixtures and the fact that most discordances between the two laboratories were partial suggest that most discordances were caused by variation in sampling of the HIV-1 quasispecies by PCR rather than by technical errors in the sequencing process itself.

Amino Acid Sequence↗
PubMed2001

Laboratory studies of cryopreservation of sperm and trochophore larvae of the eastern oyster.

The eastern oyster, Crassostrea virginica, is the most important cultured oyster species of the Atlantic and Gulf coasts of the United States. Cryopreservation of gametes and larvae of aquatic organisms has increased in importance in recent years. However, studies on the cryopreservation of sperm and larvae of mollusks have focused on the Pacific oyster, Crassostrea gigas. The present study was conducted to improve cryopreservation of sperm and trochophore larvae and to assess fertilizing ability and male-to-male variation of thawed sperm of the eastern oyster. Sperm were diluted in 12 cryoprotectant solutions composed of Hanks' balanced salt solution without calcium and 0, 5, 10, 15, 20, and 25% (v/v) propylene glycol with or without 0.25 M sucrose. Trochophore larvae were suspended in artificial seawater and 10 or 15% propylene glycol (v/v). Sperm or trochophore larvae were placed in 5-mL macrotubes and allowed to equilibrate for 15 min. The macrotubes were cooled in a controlled-rate freezer at a rate of 2.5 degrees C per min until reaching a final temperature of -30 degrees C and were plunged into liquid nitrogen. After storage for 2 weeks, the samples were thawed in a water bath at 70 degrees C for 15 s. Overall, for cryopreservation of sperm and larvae, best results were obtained using 10 or 15% propylene glycol. Thawed sperm presented significant male-to-male variation in fertilizing ability. Survival of thawed larvae decreased as the concentration of larvae per macrotube increased. The procedures developed in this study for sperm and larvae are suitable for production of seedstock in commercial oyster hatcheries.

Animals↗
PubMed2006

Comparing ethylene glycol with glycerol for cryopreservation of canine semen in egg-yolk TRIS extenders.

The objective of this work was to evaluate the possibility of substituting glycerol (G) for ethylene glycol (EG) when cryopreserving dog semen. A total of 15 ejaculates from 13 dogs was pooled into five samples and frozen in egg-yolk Tris extenders with variable ethylene glycol and glycerol concentrations, with or without Equex STM Paste. Two widely used glycerol extenders (Uppsala Equex II and Norwegian) were utilized as controls. Semen quality parameters assessed after thawing were total subjective motility (TSM), computer assisted sperm analysis (CASA), eosin-nigrosin staining, and flow cytometry (FC) after staining with the PI/Fitc-PSA (fluorescein isotiocianate conjugated with the agglutinin of Pisum sativum, PSA) fluorochromes. No advantages were seen in using EG to replace G when freezing dog semen or combining EG and G in the freezing medium. The Uppsala Equex II provided the best overall post-thaw parameters, followed by the egg-yolk Tris experimental extender with 5% EG and Equex STM Paste. The extender with 4% EG produced similar results to the Norwegian extender. High correlations (r>0.98) were obtained between eosin-nigrosin staining and FC, as well as between subjective and computerized motility assessment (r>0.90).

Acrosome↗
PubMed2006

Cryopreservation of Mexican fruit flies by vitrification: stage selection and avoidance of thermal stress.

This report presents details of a vitrification methodology for the cryopreservation of embryos of the Mexican fruit fly, Anastrepha ludens. The overall summary of the data indicates that selecting the correct developmental stage for cryopreservation is the most important criterion. The key aspect in selection of the correct stage is to balance depletion of the gut yolk content against development of the embryonic cuticle. Embryogenesis was divided into four stages between 90 and 120 h after incubation at 21.7 degrees C. The classification was based on the intestinal yolk content and the initial development of mandibular-maxillary complex. Stages having low mid-gut yolk content and the appearance of mouth hooks were found to be the most suitable for cryopreservation. Embryos developing at 30 degrees C had premature cuticle formation relative to gut development and significantly lower hatching after cryopreservation. Vitrification of embryos by direct quenching in liquid nitrogen was less effective than quenching after annealing the samples in liquid nitrogen vapor. Quenched samples of vitrification solutions containing 1,2-ethanediol as the major component exhibited fractures. Fracturing occurred less frequently when the solutions were annealed and when containing polyethylene glycol. Hatching of vitrified embryos stored in liquid nitrogen for over 12 months was not statistically different from those held for only 15 min. Our protocol yielded normalized hatching rates that ranged as high as 61%. Selecting the exact stage for cryopreservation from a population of embryos obtained by collection from ovipositing females during a span of just 30 min resulted in nearly 80% of the embryos hatching into larvae.

Animals↗
PubMed1997

An intrauterine insemination-ready cryopreservation method compared with sperm recovery after conventional freezing and post-thaw processing.

OBJECTIVE: To test a sucrose-glycerol cryoprotectant for IUI-ready sperm preparation. DESIGN: Semen aliquots from normozoospermic donors either were subjected to conventional semen freezing (TES and Tris yolk buffer in 7.4% final glycerol) with post-thaw processing or were preprocessed and frozen in HEPES-buffered human tubal fluid with 1% human serum albumin, 4% sucrose, and 6% glycerol. All aliquots were cooled to 4 degrees C, exposed to liquid nitrogen vapors, and stored in liquid nitrogen. Aliquots from each were processed by centrifugation resuspension or by centrifugation in Percoll (Pharmacia, Alameda, CA) before sperm parameters were analyzed. SETTING: University-based andrology laboratory. MAIN OUTCOME MEASURE(S): Recovery of motile sperm. RESULT(S): Percoll processing produced preparations with higher percentages of motile cells; however, cryopreserved sperm had a lower recovery of motile sperm compared with Percoll-processed fresh semen or centrifugation/resuspension-processed fresh or frozen samples. The percentages of sperm with normal morphologies were significantly increased in the IUI-ready samples compared with samples frozen conventionally. The IUI-ready Percoll-processed sample produced the best results, with a final mean motility of 36% and an overall yield of motile sperm of 17.4%. CONCLUSION(S): The sucrose-glycerol-based cryoprotectant produced an IUI-ready preparation with motile sperm recovery comparable to that of conventional semen cryopreservation but with improved percent morphology.

Centrifugation, Density Gradient↗
PubMed2000

Comparison of protective media and freezing techniques for cryopreservation of human semen.

OBJECTIVE: To evaluate the influence of cryopreservation medium and freezing-thawing techniques on human sperm motility and morphology. STUDY DESIGN: 63 semen samples were obtained from 39 donors to the artificial insemination programme. Possible effects of the sperm dilution with cryomedium on the motility were examined 10 min after exposure of 24 high initial quality semen samples to TEST-yolk ¿zwitterion-citrate-egg yolk extender containing TES [N-Tris (hydroxymethyl) methylaminoethane sulfonic acid] and Tris [(hydroxymethyl) aminomethane]¿ and human sperm preservation medium (HSPM). Post-thaw sperm motility from 24 frozen semen samples was examined comparing the cryoprotective efficacy of TEST-yolk and HSPM following different freezing techniques (vapour freezing, fast programmable freezing and slow programmable freezing). The relationship of sperm morphology to the effects of freezing was investigated on 39 semen samples following different freezing techniques. Post-thaw sperm motility from 39 frozen semen samples was compared among three groups divided according to the percentage of morphologically normal cells (<40, 40-50 and >50%) in fresh semen. RESULTS: Exposure of spermatozoa to cryomedia for 10 min at room temperature significantly reduced motility in TEST-yolk treatment group for 9% and in HSPM treatment group for 18% (P<0.01). The recovery of motile sperms (mean+/-standard deviation) was 49+/-15.7, 43+/-15.2 and 52+/-16.8% when TEST-yolk was used and 34+/-17.8, 32+/-18.2 and 50+/-13.6% when HSPM was used as a cryopreservative following vapour freezing, and fast and slow programmable freezing, respectively. Following vapour freezing and also following fast programmable freezing, the recovery of motile sperm was significantly higher (P<0.05) after addition of TEST-yolk medium than after addition of HSPM. Post-thaw motility of the sperm cryopreserved in HSPM showed significant differences (P<0.05) after three different freezing techniques. The recovery of motile sperms was 57+/-26.4, 38+/-8.6 and 38+/-17.3% in groups with >50, 40-50 and <40% morphologically normal cells, respectively. The percentage of morphologically normal spermatozoa was reduced 8% after vapour freezing and 6 and 3% after fast and slow programmable freezing, respectively. The results were statistically analysed using SAS/STAT software. CONCLUSIONS: Slow programmable freezing was superior to vapour freezing and fast programmable freezing as a method for sperm cryopreservation. However, none of these methods of freezing had discernible effects on sperm morphology. Motility of spermatozoa decreased due to the exposure of semen to cryomedium. TEST-yolk was a superior cryomedium to HSPM. Fresh semen with more than 50% of morphologically normal cells showed the best recovery of motile cells after freezing and thawing.

Buffers↗
PubMed1988

Expression of immunological markers on leukemic cells before and after cryopreservation and thawing.

We have studied the effects of cryopreservation on the viability and on the expression of surface antigens of acute leukemia cells. Marrow samples were obtained at initial diagnosis from 89 patients with acute myeloid leukemia (AML), acute undifferentiated leukemia (AUL), and acute lymphoid leukemia (ALL). In AML, the mean viability was greater than 90% in the types M1, M4, and M5 of the French-American-British classification, 79% in M2, and 3% in M3 types. The viability was 74% in AUL. In ALL, the viability was 95% for pre-B leukemias, but only 2% in T-cell leukemias. The expression of myeloid antigens was studied before and after freezing and thawing using three monoclonal antibodies (NHL30.5, against poorly differentiated granulocytic leukemias, VIMC6 against differentiated granulocytic leukemias and granulocytes; and UCHM1 or CRIS-6, against monocytic leukemias and monocytes). The percentage of cells stained by NHL30.5 and UCHM1 or CRIS-6 was very similar before and after cryopreservation. For VIMC6, the mean staining after cryopreservation was 60% of the initial one. In pre-B ALL, the stainings by anti common ALL antigen before and after cryopreservation were also very similar. We conclude that leukemic cryopreserved cells are suitable for immunologic studies. The recovery is, however, very low in promyelocytic AML and T-cell ALL.

Acute Disease↗
PubMed1996

Activity of aspartate aminotransferase and acid phosphatase in cryopreserved trout sperm.

Milt of brown, rainbow and brook trout was cryopreserved. Activity of aspartate aminotransferase (AspAT) and acid phosphatase was assayed both in supernatants and in spermatozoa obtained from thawed sperm samples; additionally, post-thaw motility was evaluated. Enzyme activities differed according to fish species and were strongly affected by the type of cryoprotectant used. The activity in supernatants was usually higher than that in spermatozoa because of protein leakage from injured cells. AspAT activity in cryopreserved spermatozoa correlated positively with fertilization success in all three species. There was a negative correlation between activity of extracellular (supernatant) AspAT and fertilization rates in variants with dimethyl sulfoxide and dimethylacetamide-based extenders. The motility of thawed sperm, determined microscopically, provided some information on the cryopreservation efficiency of trout milt.

Acid Phosphatase↗
PubMed2003

Comparison of cryopreserved sperm in vaporous and liquid nitrogen.

OBJECTIVE: To evaluate the feasibility of using the vaporous phase of liquid nitrogen as a storage medium for human semen samples. STUDY DESIGN: Human semen samples were used in a 2-armed study. In the first arm, semen samples were rapidly frozen and randomly assigned to storage in either liquid nitrogen (-196 degrees C) or vapor-phase nitrogen (-189 degrees C). In the second arm, sperm specimens were frozen in a controlled, stepwise process and randomly assigned to storage in either the vaporous or liquid phase of liquid nitrogen. The samples were subsequently thawed and analyzed for sperm viability. RESULTS: The rapid freezing technique demonstrated no differences in the examined viability parameters between semen specimens stored in either liquid or vaporous nitrogen. Similarly, semen samples prepared by the slower, controlled freezing process also showed no differences in viability parameters after storage in either the liquid or vaporous phase of liquid nitrogen. The rapid freezing technique was equal to the slow, controlled freezing method in terms of preserving the integrity of the sperm when the specimens were subsequently stored in either liquid or vaporous nitrogen. CONCLUSION: Cryopreservation of human semen in the vapor phase of nitrogen is a viable alternative to storage in liquid-phase nitrogen.

Cryopreservation↗
PubMed2006

Cryopreservation of preantral ovarian follicles in situ from domestic cats (Felis catus) using different cryoprotective agents.

The objective of this investigation was to verify the structural characteristics of preantral follicles (PAF) of cat ovarian tissue after cryopreservation in 1.5 M glycerol or ethylene glycol, using a slow-freezing procedure. Ovaries (n = 10) from domestic cats were divided into fragments. One fragment was immediately preserved for classical histology (fresh control), and additional fragments were immersed in minimum essential medium plus 10% bovine fetal serum (MEM+BFS), or MEM+BFS supplemented with 1.5 M glycerol or ethylene glycol. The samples were frozen and plunged into liquid nitrogen. After 1 wk, the samples were thawed. A total of 600 PAF were evaluated. In the fresh control, there were 71.3% normal PAF. After thawing, the rates of normal PAF were 26.0, 39.3 and 58.0% for samples without cryoprotectant or with glycerol or ethylene glycol, respectively. We concluded that ethylene glycol was useful for the cryopreservation of feline PAF in situ.

Animals↗
PubMed1994

Improvement in quality of cryopreserved human spermatozoa by swim-up before freezing.

Selecting a population of spermatozoa by the swim-up technique yields, after freezing and thawing, a population of cells that contains proportionally more spermatozoa which are morphologically normal, fewer spermatozoa with damaged tail membranes, and a greater percentage of progressively motile spermatozoa with greater velocities and amplitudes of head displacement than those obtained after freezing and thawing the same semen samples in the normal way. This pattern was found for the semen from 10 patients and five volunteers. However, the cells selected by swim-up were as susceptible to the stresses caused by freezing and thawing as unselected spermatozoa in the original semen sample, and the improvement came from the better quality of the initial sample.

Adult↗
PubMedDate not supplied

Cryopreservation-Thawing of fractionated human spermatozoa is associated with membrane phosphatidylserine externalization and not DNA fragmentation.

The objective of these studies was to evaluate the effect of cryopreservation-thawing of human spermatozoa on DNA fragmentation and membrane integrity. This was a prospective, controlled cohort study, performed at a university-based infertility center. Ejaculates were examined from 5 donors and 16 men undergoing infertility evaluation. Purified sperm populations were prepared by gradient centrifugation, cryopreserved using a manual method and TEST-yolk buffer and glycerol (TYB-G), followed by quick-thaw. Annexin V binding was used for assessing membrane translocation of phosphatidylserine, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) was utilized for the evaluation of DNA fragmentation. The results were as follows: the percentage of live cells with intact membranes (annexin V-, live) was significantly reduced after cryopreservation-thawing. On the other hand, the percentages of live cells with phosphatidylserine translocation (annexin V-, live) and of necrotic (dead) cells increased significantly after thawing. TUNEL revealed percentages of cells with DNA fragmentation in the prefreeze and postthaw samples that were not significantly different. In a further attempt to examine differences in response to various cryoprotection protocols, experiments were carried out using no cryoprotection, glycerol alone, or TYB-G. Samples frozen with TYB-G demonstrated significantly higher percentages of live cells without phosphatidylserine translocation than the other conditions. We concluded that cryopreservation-thawing of human sperm from patients and donors was associated with membrane change, as revealed by membrane translocation of phosphatidylserine, while having no major impact on DNA fragmentation.

Annexin A5↗