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PubMed1995

Cytogenetic effects of cryopreservation on human sperm: assessment using an improved method for analyzing human sperm chromosomes.

OBJECTIVE: To evaluate any cytogenetic effects of cryopreservation on human sperm by comparing the frequencies of sperm chromosome anomalies and sex ratios before and after freezing. METHODS: Using in vitro fertilization of zona-free hamster oocytes, analysis of sperm chromosomes was first performed on portions of fresh human semen samples. The residual semen was then analyzed for sperm chromosomes after cryopreservation for several weeks. Sperm donors were 5 healthy men aged 26-38 years. RESULTS: A total of 166 sperm karyotypes were analyzed, 94 before freezing and 72 after freezing. The results indicated no significant differences between fresh and frozen sperm in either frequencies of aneuploidy (fresh: 0%, frozen: 2.8%) or structural anomalies (fresh: 7.5%, frozen: 9.7%). The sex ratios did not differ from the expected 1:1 ratio under either condition. CONCLUSIONS: The results of these studies indicate that cryopreservation does not exert any cytogenetic mutagenicity on human spermatozoa or alter X/Y ratio of human sperm.

Adult↗
PubMed1997

Reactive oxygen species generation by seminal cells during cryopreservation.

OBJECTIVES: To test the hypothesis that conventionally used procedures for semen cryopreservation may cause an increase in the production of reactive oxygen species (ROS) by sperm or by seminal leukocytes, which may contribute to poor sperm function following cryopreservation. METHODS: Eighteen semen specimens with normal parameters from healthy male donors 22 to 40 years of age were each divided into two portions. The first portion was combined 1:1 with Test Yolk Buffer-Glycerol Freezing Medium and was frozen by gradual cooling into liquid nitrogen (-196 degrees C). The second portion was washed and the cells were resuspended in Sperm Washing Medium (SWM) and incubated at room temperature to serve as controls. After a period of treatment, frozen samples were thawed and semen cells were washed and resuspended in SWM. ROS generation by semen cells from each treatment group was measured on a luminometer. Sperm motility, sperm viability, and sperm membrane integrity were also measured in both control and freeze-thaw samples. To further assess ROS generation by semen cells during the cooling process, aliquots of washed semen cells and purified polymorphonuclear leukocytes (PMNs) were incubated separately at different temperature conditions (37 degrees C, 22 degrees C, 4 degrees C, and -20 degrees C). ROS activity in each treatment group was measured and compared with each other. RESULTS: In both semen cells and PMNs, ROS activity increased significantly during the cooling process. The highest ROS levels were recorded in both groups when cooled to 4 degrees C. The ROS levels were extremely low in samples cooled to -20 degrees C and in freeze-thaw samples, probably due to marked loss of cell viability. CONCLUSIONS: Gradual reduction of temperature during the process of semen cryopreservation can cause a significant ROS generation by semen cells. ROS is particularly elevated during cooling if the semen sample is contaminated by more than 0.5 x 10(6) leukocytes. Removal of leukocytes from semen samples or treatment with antioxidants prior to cryopreservation may improve sperm viability and function.

Adult↗
PubMed1996

The hypo-osmotic swelling test for selection of viable sperm for intracytoplasmic sperm injection in men with complete asthenozoospermia.

OBJECTIVE: To determine the ability of the hypo-osmotic swelling test to select viable sperm from nonmotile sperm samples for intracytoplasmic sperm injection (ICSI). DESIGN: Nonrandomized, sequential comparative study. PATIENTS: Thirteen couples enrolled in our ICSI program had 16 cycles in which sperm preparations with 0% motility were obtained. Five cycles used cryopreserved epididymal sperm with complete asthenozoospermia. INTERVENTIONS: In eight cycles, the semen samples were washed through a Percoll gradient and sperm were selected randomly for ICSI. In another eight cycles, the washed sperm were placed in a hypo-osmotic solution (75 mM fructose; 25 mM sodium citrate dihydrate) and the sperm with curled tails taken up with the microinjection needle, rinsed, and used ICSI. MAIN OUTCOME MEASURES: Fertilization rate per oocyte injected as determined by the presence of two pronuclei at 18 hours after retrieval and embryo cleavage rate per oocyte injected at 48 hours after retrieval. RESULTS: With random sperm injection, the fertilization and cleavage rates were 26% and 23%, respectively. In contrast, after injection of sperm selected using the hypo-osmotic swelling test, fertilization and cleavage rates were significantly greater (43% and 39%, respectively). There were three pregnancies in the eight cycles with the hypo-osmotic swelling test-selected sperm, including two from frozen epididymal sperm. CONCLUSION: Based on these preliminary observations, we believe that the hypo-osmotic swelling test will prove to be valuable for increasing fertilization and cleavage rates and pregnancy rates in ICSI cycles where no motile sperm are recovered.

Adult↗
PubMed1994

[The influence of different cryopreservation buffers on the fertilizing ability of human sperm "in vitro". II. Evaluation of the fertilizing potential of human sperm after cryopreservation].

We tried to define a potential ability of human spermatozoa for fertilization after 3 month preservation of samples in a liquid nitrogen. The functional test were applied: penetration of sperm to cervical mucus ("Penetrak") and sperm penetration to hamster zona-free oocytes. We would like to emphasize that presented way of cryo-preservation in 3 selected media with fast cooling of samples allow to maintain fertilizing ability of spermatozoa and subsequent insemination.

Adult↗
PubMed1994

Composition and behavior of head membrane lipids of fresh and cryopreserved boar sperm.

Head plasma membranes were isolated from fresh or cryopreserved ejaculated boar spermatozoa and the lipids were extracted for determination of lipid fluidity (n = 6 for fresh and cryopreserved) and for compositional analysis (n = 5 for fresh, 6 for cryopreserved). Composition of the egg yolk extender was also determined. For fluidity determination, the mixed lipids were allowed to form natural liposomes. Bilayer fluidity of these liposomes was analyzed in the presence or the absence of 1 mM Ca2+ with the probes tPNA, which preferentially locates into gel-phase areas, and cPNA, which enters fluid and gel-phase areas equally and thus assesses bulk lipids. Fluidity of liposomes declined significantly during controlled-rate cooling for all samples. Compared to lipids from fresh membranes, gel lipids from cryopreserved cells lost fluidity at a significantly more rapid rate, as did bulk lipids in the presence of Ca2+ (P < 0.001). Fluidity increased during subsequent rewarming (5 to 50 degrees C), again at a slower rate for lipids from fresh cells, with the cryopreservation effect being significant for all probe/Ca2+ combinations (P < or = 0.05). Calcium altered the fluidity characteristics of membrane lipids from fresh but not cryopreserved sperm when analyzed during cooling with cPNA (P < 0.01) and during rewarming with cPNA (P < 0.0001) and tPNA (P < 0.05). Lipids from cryopreserved cells contained significantly less sphingomyelin (14.6 +/- 1.1 vs 22.4 +/- 1.6 mol%) and more phosphatidylcholine (51.5 +/- 2.0 vs 40.5 +/- 2.4%). The octadecanoate (18:0) content in both phosphatidylserine and phosphatidylethanolamine decreased after cryopreservation (P < 0.05). The polyunsaturated fatty acids docosatetraenoate (22:4) and/or arachidonate (20:4) increased in these phospholipids and in sphingomyelin and phosphatidylinositol (P < 0.05). The alterations in the molecular interactions, composition, and Ca2+ sensitivity of membrane lipids may interfere with the normal membrane events of fertilization.

Animals↗
PubMed1992

Cryopreservation, survival and function of intrastriatal fetal mesencephalic grafts in a rat model of Parkinson's disease.

In the present study we quantitatively assessed to what extent freeze-storage at liquid nitrogen temperature influences the survival and function of fetal mesencephalic grafts in the dopamine-depleted rat striatum. Ventral mesencephalic (VM) tissue was dissected from rat fetuses and stored overnight in a preservative medium at 4 degrees C (hibernation). It was grafted intrastriatally either as a fresh cell suspension or was frozen as tissue fragments or as a cell suspension after stepwise incubation in ascending concentrations of dimethyl-sulphoxide. Following a cryopreservation interval of 80 days in liquid nitrogen, the frozen samples were rapidly thawed, rinsed, and grafted. Cellular viabilities of graft cell suspensions, as assessed by ethidium bromide/acridine orange staining, were decreased from 90% in fresh tissue to 38-35% in frozen and thawed tissue. Amphetamine-induced turning behavior at 6 weeks post-grafting was significantly attenuated in hosts that had received fresh grafts or grafts that were frozen as tissue fragments. Tyrosine hydroxylase-(TH-) immunocytochemistry of recipient brains revealed significant decreases in TH-positive graft cell numbers in rats grafted with cryopreserved tissue (38-42% of fresh tissue). Moreover, the dye exclusion viability of thawed VM tissue was found to accurately predict the subsequent graft survival. There was no difference with respect to graft cell numbers between the two freezing methods employed, though block storage seems to be more simple from a practical point of view.(ABSTRACT TRUNCATED AT 250 WORDS)

Amphetamine↗
PubMed2000

Intracytoplasmic sperm injection with cryopreserved testicular spermatozoa.

To assess if testicular sperm cryopreservation is a valid alternative to repetition of testicular sperm retrieval techniques, results of a cryopreservation technique in pills have been retrospectively analyzed. Enough motile spermatozoa for ICSI were obtained in 172 from 190 (90.5%) frozen-thawed testicular sperm samples. Overall, 249 couples underwent 390 ICSI cycles, 156 using fresh and 234 using cryopreserved testicular sperm. Mean two-pronuclear fertilization rates per cycle were not significantly different after ICSI with fresh (62.0%) or with cryopreserved (63.2%) spermatozoa. Mean embryo cleavage rate per cycle was higher in the fresh (90.6%) than in the cryopreserved (84.6%) group (P = 0.016). However, clinical pregnancy rates per cycle (28.2% with fresh vs 27.8% with cryopreserved), implantation rates (12.2% vs 13.1%) and ongoing pregnancy rates per cycle (22.4% vs 21.8%) were not significantly different. Cryopreservation of testicular spermatozoa is an effective technique that can be used both in obstructive and in non-obstructive azoospermia.

Adolescent↗
PubMed1991

Metabolic toxicity of fluorescent stains on thawed cryopreserved bovine sperm cells.

Several fluorescent probes, including derivatives of carboxyfluorescein, carbocyanine, ethidium, and rhodamine, have been used to assess sperm viability. However, the effects of these fluorescent dyes on the metabolic activity of sperm cells have not been systematically examined. This study was conducted to determine the effect of specific fluorescent stains on the metabolic processes of sperm. Cryopreserved bovine sperm cells were thawed, fluorescently stained, and examined using metabolic and flow cytometric techniques. Sperm were stained with either rhodamine 123 (Rhod-123), the aliphatic cell-tracking compound PKH2-GL, dihydro-ethidium (HED), the bisbenzimide stain Hoechst 33342 (Ho33342), or left unstained. The stained samples were compared for metabolic activity, cell staining pattern, and fluorescent intensity over a 180-min period. Samples stained with HED, Ho33342, and PKH2-GL had less oxygen uptake when compared with the unstained sperm samples (p greater than 0.05). Unstained samples and samples stained with Rhod-123 had similar oxygen consumption. The carbon dioxide produced during the 180 min was not different between controls and stained samples. Therefore, some fluorescent probes inhibit the oxygen metabolism of thawed, cryopreserved bovine sperm cells.

Animals↗
PubMed2004

Vitrification of goat, sheep, and cattle skin samples from whole ear extirpated after death and maintained at different storage times and temperatures.

Proper tissue preservation from a wide range of animals of different species is of paramount importance, as these tissue samples could be used to reintroduce lost genes back into the breeding pool by somatic cloning. We aim to study the temporal and thermal post-mortem limits, tested in rabbits and pigs, within which there will be guarantees of obtaining living skin cells in goat, sheep, and cattle. We also intend to study the effect of vitrification on the ability of ear skin cells, stored at different times and temperatures, to attach to the substratum and grow in vitro after warming. Ears were stored either at 4 degrees C for 12, 252, and 348 h post-mortem (hpm), or at room temperature (22-25 degrees C) for 60 and 96 hpm. In all cases, skin samples from these ears were sorted into two groups: one group was in vitro cultured immediately after storage, and the other group was vitrified after storage and further in vitro cultured. In goat and sheep, no differences in attachment (100%: goat; 90-100%: sheep) or subconfluence (75-100%: goat; 70-100%: sheep) rates were observed between experimental groups. However, in days of culture to reach subconfluence, significant differences between non-vitrified and vitrified groups were observed when ears were stored at 4 degrees C for 12 and 252 hpm. In cattle, with respect to attachment rate, vitrified samples from ears stored at 22-25 degrees C for 60 hpm were different from non-vitrified control group (60 vs. 100%, respectively; P < 0.05). Also, days of culture to reach subconfluence were analysed by a non-parametric Cox Survival Analysis. In general, results from ANOVA and Survival Analysis were similar, because the proportion of censored data was quite low (9%), so the bias when using ANOVA is not too high. In spite of all the above, the lowest survival rates (75%: goat; 70%: sheep; and 40%: cattle) were sufficiently high to enable collection of skin samples from the majority of dead animals and their cryopreservation.

Animals↗
PubMed1977

Detection of antibody to autologous human leukemia cells by immune adherence assays.

The sera of 21 adult patients with acute leukemia were studied for the presence of antibody reacting with surface antigens of autologous leukemia cells. Sequential serum samples were obtained from patients and were tested on cryopreserved leukemia cells in immune adherence assays. Three patients showed autologous serum reactivity and the serum of one of them was analyzed in detail. This antibody reacted with autologous acute lymphocytic leukemia cells but not with autologous cells obtained from peripheral blood, bone marrow, or spleen during clinical remission. In absorption tests, the antigen could not be detected on normal autologous or allogeneic blood lymphocytes, lymphoblastoid lines of T- or B-cell origin, or cells infected with simian sarcoma virus, baboon C-type virus, or Mason-Pfizer virus. Leukemia cells from two other patients with acute lymphocytic leukemia and one patient with acute nonlymphocytic leukemia absorbed specific reactivity. These studies indicate that certain acute leukemia cells express a common antigen that elicits a humoral immune response in the autologous host.

Acute Disease↗
PubMed2002

Target-selected inactivation of the zebrafish rag1 gene.

The zebrafish has become a favorite organism for genetic analysis of vertebrate development, but methods for generating mutants by reverse genetic approaches have been lacking. We report a method to obtain stable mutants of a gene based on knowledge of the gene sequence only. Parental fish were mutagenized with N-ethyl-N-nitrosourea; in 2679 F1 fish, the rag1 gene was analyzed for heterozygous mutations by resequencing. In total, we found 15 mutations: 9 resulted in amino acid substitutions and 1 resulted in a premature stop codon. This truncation mutant was found to be homozygous viable and defective in V(D)J joining. Although presumably immune deficient, these homozygous rag1 mutant fish are able to reach adulthood and are fertile. As sperm samples from all 2679 F1 fish were collected and cryopreserved, we have in principle generated a mutant library from which mutants of most zebrafish genes can be isolated.

Amino Acid Substitution↗
PubMed1990

Cryopreserved/thawed semen for in vitro fertilization: results from fertile donors and infertile patients.

We evaluated the in vitro fertilization (IVF) outcome in 54 cycles using cryopreserved/thawed semen from fertile donors. Controls were other IVF patients matched by time frame, female age, stimulation protocol, number of pre-embryos transferred, and absence of a male factor using freshly ejaculated normal semen samples. In the study group and controls, respectively, post-thaw swim-up motility was 83.1% and 89.5%; fertilization rate of preovulatory oocytes (91.8%, 95.7%) and ongoing pregnancy rate (PR) per transfer (21.1%, 25.0%) were similar. The excellent fertilization rate with frozen/thawed semen was achieved through high-concentration insemination (0.5 x 10(6) motile sperm/mL). With use of frozen/thawed samples from infertile men (normal and subfertile samples), PR was similar but fertilization rate was lower. Cryopreserved semen is a valuable option for infertile couples in IVF therapy.

Cryopreservation↗
PubMed1997

Percutaneous technique for aspiration of sperm from the epididymis and testicle.

Percutaneous sperm aspiration from the epididymis (PESA) or testicle (TESA) may be used as a diagnostic technique to confirm sperm production and as a method to harvest sperm for use in intracytoplasmic sperm injection (ICSI). We performed 43 PESA/TESA procedures in men considered to have nonreconstructible obstructive azoospermia. This short outpatient procedure was performed using a butterfly needle under intravenous i.v. sedation and local anesthesia. In 17 diagnostic aspirations, diagnoses of normal spermatogenesis, maturation arrest, and germ cell aplasia were established. In 26 therapeutic sperm aspirations for use with ICSI, adequate samples of sperm were obtained and residual sperm was cryopreserved. In the ICSI cycles, fertilization and pregnancy rates of 59% and 32%, respectively, were obtained. PESA/TESA is a minimally invasive sperm retrieval technique and appears to be an effective alternative to microsurgical epididymal sperm aspiration, which is more invasive, costly, and technically more difficult. There have been no acute or chronic complications in this patient population, including nine men who underwent a second successful procedure.

Anesthesia, Local↗
PubMed1997

[Development of specimen banks for research in molecular epidemiology].

In recent years, methodology has been implemented with the purpose to establish DNA diagnostic banking by means of transformed cell lines, direct extracted DNA, cryopreserved blood or other tissues and the study of samples embedded in paraffin. The objectives of the storage of individual DNA samples are for academic and research purposes, principally for gene mapping, and for diagnostic services to those who are at genetic risk and may be interested in family-based linkage testing, for establishing forensic DNA data bank, and for tissue transplantation. These developments implicate important legal and ethical issues, and institute rights and obligations of the donors and of the persons that conduct the banks.

Biological Specimen Banks↗
PubMed1994

[The influence of different cryoprotectant buffers on the fertilizing ability of human sperm "in vitro". I. The choice of cryoprotective buffers and methods of freezing].

We attempted to find the optimal way of cryopreservation of male gametes. We have been tested 9 cryoprotectants currently existing in the world literature. These fluids have been so far separately investigated in different centers of infertility treatment. In this study we compared the effectivity of these fluids in preservation of viable sperm. Basic seminological parameters and two variants of freezing were tested. There were noted significant differences between fast and slow cooling of sperm samples. Nevertheless after fast cooling we found a marked decrease of seminological parameters in preserved sperm when compared to fresh samples.

Cryopreservation↗
PubMed2003

Water status and thermal analysis of alginate beads used in cryopreservation of plant germplasm.

Encapsulation and dehydration techniques using alginate beads are used increasingly for the pre-treatment of various plant materials for cryopreservation to improve survival post-cryogenic storage. This study reports the effects of the water content of beads (formed with 3% (w/v) alginic acid in liquid S-RIB), polymerisation time (in 100 mM calcium chloride solution), osmotic dehydration (in 0.75 M sucrose solution), and evaporative air desiccation on the thermal properties of alginate beads used in cryopreservation protocols. Experimental beads were assayed using a differential scanning calorimeter (DSC) with a cooling programme to -150 degrees C, followed by re-warming. Resultant thermograms were evaluated with particular reference to the onset temperature and enthalpy of the melt endotherm from which the quantities of frozen and unfrozen water were calculated. Treatments were applied sequentially to samples of beads and their thermal features evaluated at each stage of the protocol. Using 'standard' beads (40-55 mg fresh weight), formed using plastic disposable pipettes, the degree of polymerisation (>10 min) proportionally reduced their dry weight and increased their water content. Thermal characteristics of the beads were unaffected by polymerisation times >10 min, but the maximum level (23%) of unfrozen (osmotically inactive) water was achieved after 15 min polymerisation. Osmotic dehydration using 0.75 M sucrose significantly lowered bead water content and mean dry weight approximately doubled with 20-24 h immersion time. Bead desiccation in still air reduced their water content by 83% of fresh weight, whilst dry weight remained constant. After 8 h desiccation in air between 27 and 37% of the water in the bead was osmotically inactive (unfrozen) in DSC scans. Desiccation >18 h reduced this fraction to zero. The melt onset temperature and the enthalpy of melting were directly related to bead water content. The unfrozen water fraction increased substantially with reduced water content of the beads (from 23 to 37% of total water content), concomitant with a reduction in the ratio of unfrozen to frozen water from 1:3 to 1:2. For successful vitrification and the production of a glass that did not destabilise on rewarming, a bead water content of ca. 26% of fresh weight (0.4 g waterg(-1) dry weight) was required, much of which was osmotically inactive water. These data are discussed in relation to optimal pre-treatments for alginate bead encapsulation techniques used in the cryopreservation of a range of plant germplasm. It is proposed that increased standardisation of alginate beads, in terms of volume, fresh weight, and water content, is required to reduce the variability in physical and thermal features, which in turn will improve survival of plant samples post-cryopreservation.

Alginates↗
PubMed2001

Effect of cryopreservation on human articular chondrocyte viability, proliferation, and collagen expression.

Autotransplantation of human chondrocytes is an alternative therapeutic treatment for focal lesions of cartilage. During the process of isolation and culture of chondrocytes some problems that render the implantation of the cells unsuitable can occur. For security, some cells must be stored using cryopreservation. The objective of this study was to analyze the effect of cryopreservation on cellular viability, proliferation, and collagen expression of human chondrocytes. Human osteoarthritic cartilage (n = 23) was obtained and transferred to a sterile flask containing Dulbecco's modified Eagle's medium (DMEM) and antibiotics. Chondrocytes were isolated, cultured for 3-4 weeks, and frozen in DMEM containing 10% human serum and 10% dimethyl sulfoxide by use of three different protocols. A cellular fraction was frozen directly to -80 degrees C (Protocol I). Another fraction was directly frozen to -80 degrees C and 24 h later introduced into liquid nitrogen (Protocol II). The last aliquot was frozen with controlled freezing using a freezing rate of -1 degrees C/min to a temperature of -40 degrees C, 2 degrees C/min to -60 degrees C, and 5 degrees C/min to -150 degrees C (Protocol III). Cells were cryopreserved for 2 weeks. Cells from each cryopreservation method were then cultured for 7 days and cellular proliferation was evaluated by the counting of the total cells in each flask. Cryopreservation had a negative effect on chondrocyte survival and proliferation. The survival after cryopreservation with the three protocols was 70-75%. There was no significative difference between the methods used to cryopreserve (P = 0.4117). However, there was a significant difference among the donors (P = 0.0111). Cellular proliferation of chondrocytes was reduced by cryopreservation (P = 0.024). The rate of proliferation of different groups was control samples 6.56, Protocol I 4.66, Protocol II 4.69, and Protocol III 5.58. Statistical analysis showed that the programmed protocol was the best method to preserve cellular functions. Chondrocytes were able to express collagen type II 1 week after cryopreservation. Cryopreservation modifies the survival and proliferation of chondrocytes. Of all protocols used to cryopreserve, the programmed protocol seems to be the best technique. Cryopreservation does not alter the collagen type II expression.

Cartilage, Articular↗
PubMed2000

Molecular strategies to define HLA haplotype loss in microdissected tumor cells.

Loss of heterozygosity (LOH) of chromosome 6p21 is an important mechanism that generates HLA haplotype loss in various human tumors. This mechanism produces non-reversible HLA-deficient tumor cells that can escape T cell immune responses in peptide-vaccinated cancer patients. However, the exact frequency of this mechanism is still unknown, because contaminating stroma in solid tumor tissues masks the tumor DNA obtained from solid samples. A microdissection technique was applied to 4-8 microm sections of cryopreserved tumor tissues from a group of colorectal and laryngeal carcinomas. Fifteen patients were analyzed for the presence of LOH associated with the beta(2)-microglobulin gene in chromosome 15, and five patients for LOH associated with HLA genes in chromosome 6. In two cases, autologous metastasis tissue samples were also available. The patients were selected for showing an altered HLA class I tumor phenotype as determined by immunohistological techniques. DNA was obtained from this microdissected material and amplified in order to detect the presence or absence of nine previously selected microsatellite markers. HLA sequence based typing (SBT) was also applied to these microdissected DNA samples to define the HLA genotype. Microdissection greatly improved the definition of LOH, with nearly 100% signal reduction in one of the alleles. In addition, this procedure allowed us to detect beta(2)-microglobulin LOH in tumors that expressed some HLA molecules. Our data indicate that this procedure can be successfully applied to microdissected samples from solid tumors, thus enhancing the power and sensitivity of LOH detection.

Colorectal Neoplasms↗