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PubMedDate not supplied

Influence of cryopreservation on the microelectrophoretic motility (EPM) of human spermatozoa.

The net surface charge of spermatozoa represents a characteristic of the plasmamembrane. It can be evaluated indirectly, by the measurement of the sperm electrophoretic motility (EPM). The EPM of washed human spermatozoa was determined in the microelectrophoresis to investigate three problems: (a) intra- and interindividual variation coefficients of EPM of cryopreserved spermatozoa, (b) effects of cryopreservation medium, temperature shock and cryoprotection on the EPM, (c) relationship between the EPM and the cryotolerance of the spermatozoa. The inter- and intrain-dividual variation coefficients of the sperm EPM of 24 cryopreserved semen samples of 6 fertile semen donors amounted to 7% and to 6%, respectively. The cryopreservation did not cause a significant change of sperm EPM in contrast to the temperature shock in liquid nitrogen. Temporary incubation of spermatozoa with egg yolk decreased their EPM. A significant correlation between EPM and the cryotolerance ascertained by the alteration of sperm motile efficiency could be found. After contact with egg yolk spermatozoa achieved a behaviour that corresponded to a better cryotolerance.

Cell Membrane↗
PubMed1993

Platelet activating factor and pentoxifylline as human sperm cryoprotectants.

OBJECTIVE: To explore whether the addition of platelet-activating factor (PAF) or pentoxifylline before cryopreservation improves the recovery of motile viable sperm and what role cyclic adenosine monophosphate (cAMP) plays in this recovery. DESIGN: Washed sperm was cryopreserved in the absence of and in the presence of PAF and pentoxifylline. After 2 weeks these samples were quick-thawed and evaluated before and after washing for sperm motility and other motion characteristics. Sperm viability and cAMP concentration were determined to compare the effects of these cryoprotectants. RESULTS: When sperm samples were cryopreserved in the presence of PAF or pentoxifylline, an improvement in the recovery of motile sperm in unwashed and washed post-thaw samples was observed. There were 38% more motile sperm recovered with PAF and 15% more with pentoxifylline when compared with untreated samples. In comparison with the unwashed samples, sperm motility in post-thaw samples was lowered by the washing procedure. When PAF was used as a cryoprotectant, a significant improvement in the linearity and straight line velocity of the post-thaw sperm was observed. When pentoxifylline was used as a cryoprotectant, lateral head displacement was significantly improved in the post-thaw samples than in the control group. Both PAF-and pentoxifylline-treated samples contained a greater number of viable sperm than the control. The cAMP concentrations in post-thaw samples were 12-fold higher in pentoxifylline-treated samples and 4-fold higher in PAF-treated samples when compared with the untreated control. A 4-fold decrease in cAMP concentration was observed in post-thaw control samples compared with fresh-washed sperm. CONCLUSIONS: The results of this study suggest that both PAF and pentoxifylline are useful cryoprotectants for the increased recovery of motile, viable sperm. Although increased recovery of motile sperm in pentoxifylline-treated samples is related to higher cAMP levels, the cryoprotective effect of PAF does not appear to be due to increased cAMP.

Cell Survival↗
PubMed1999

Nuclear chromatin variations in human spermatozoa undergoing swim-up and cryopreservation evaluated by the flow cytometric sperm chromatin structure assay.

The sperm chromatin structure assay (SCSA) is a flow cytometric (FCM) technique which exploits the metachromatic properties of Acridine Orange to monitor the susceptibility of sperm chromatin DNA to in-situ acid denaturation. SCSA was used to study the chromatin structure variations of human spermatozoa in semen, both before and after swim-up and after cryopreservation. Semen samples were provided by 19 healthy normozoospermic subjects attending pre-marriage checks. Each sample was divided into three aliquots: the first aliquot was evaluated without further treatment, the second underwent swim-up, and the third was stored according to standard cryopreservation techniques in liquid nitrogen at -196 degrees C. Samples were also analysed by light and fluorescence microscopy (after Acridine Orange staining to evaluate the number of green fluorescent sperm heads), and by computer-assisted semen analysis. The results showed that post-rise spermatozoa represent a subpopulation characterized by a general improvement of the morphological (reduction of the percentage of abnormal forms and heads, increase of the green head sperm percentage) and kinetic parameters. This subpopulation also exhibited improved chromatin structure properties, confirming that these cells have the best structural and functional characteristics, indicative of optimal fertilizing ability. On the other hand, overall sperm quality deteriorates after cryopreservation. When thawed spermatozoa underwent an additional swim-up round, a general improvement of nuclear maturity was seen in the post-rise spermatozoa.

Adult↗
PubMed1982

Positive correlation between high aryl hydrocarbon hydroxylase activity and primary lung cancer as analyzed in cryopreserved lymphocytes.

Blood samples from closely monitored patients at the Veterans Administration Hospital in Houston, Texas, were collected, coded, and sent to Microbiological Associates over an 8-month period. Lymphocytes were isolated and cryopreserved at -190 degrees. Lymphocyte samples were simultaneously thawed, phytohemagglutinin activated, and analyzed for benz(a)anthracene-induced aryl hydrocarbon hydroxylase (AHH) levels, [3H]thymidine incorporation, and reduced nicotinamide adenine dinucleotide-dependent cytochrome b5 (cytochrome c) reductase activity. Determinations were made at both 96 and 120 hr in culture, and peak activities were compared among a total of 51 individuals who expressed such lesions as squamous cell carcinomas (22%), adenocarcinomas (14%), oat cell carcinomas (6%), chronic obstructive pulmonary disease (22%), and other nonmalignant diseases. Of the 14 highest AHH/cytochrome c activities observed, all were found in patients with primary lung cancer. Mean AHH/cytochrome c activities were 0.89 for lung cancer patients (a total of 21) and 0.47 for noncancer patients (a total of 30) (p less than 0.001). No relationship was observed between AHH/cytochrome c activity and age of patient, numbers of cigarettes smoked, family history of cancer, location or histological type of tumor, or level of phytohemagglutinin blastogenesis ([3H]thymidine cpm/cytochrome c). Whether the higher AHH levels are the cause or the result of the primary lung cancer remains to be determined.

Adult↗
PubMed2005

Study design for genetic analysis in the Jackson Heart Study.

OBJECTIVE: This paper describes the preparation of genetic materials and the recruitment and initial characterization of a nested Family Study within the Jackson Heart Study (JHS) METHODS: Genomic DNA was prepared from all consenting JHS participants. In addition, family members of a subset of JHS participants were recruited to the JHS Family Study to allow heritability and linkage analyses and family-based association studies. Family Study participants completed the same questionnaires, measures, and procedures as all other JHS participants and provided blood samples for lymphocyte cryopreservation and transformation. RESULTS: DNA samples were obtained from 4726 JHS participants, including 1499 members of 291 families. In the family cohort, estimated heritabilities of body mass index, selected lipid levels, and blood pressure are highly significant, supporting the validity of the sample. DISCUSSION: The JHS data and genetic materials (DNA and cryopreserved cells) offer valuable opportunities to identify susceptibility alleles for common complex diseases by positional and candidate gene approaches.

Adult↗
PubMed1995

Disappearance of Langerhans cells and melanocytes after cryopreservation of skin.

Cryopreserved skin allografts have been extensively reported to remain viable for longer periods after grafting, both in the laboratory and in the clinic, than skin stored by other methods. We investigated the immunocytochemical and electron microscopic properties of samples of cryopreserved human skin (-196 degrees C) for comparison with fresh samples. In an immunocytochemical study of fresh skin, reagents S-100 and CDIa indicated numerous mesenchymal origin cells in the squamous cell layer, basal layer and dermis; 2B7 identified these cells in the basal layer and PC-10 identified them in the basal and squamous cell layers. In cryopreserved skin, however, few cells reacted to these reagents. An electron microscopic study of the cryopreserved skin showed Langerhans cells (LC); however, these had suffered degeneration, with partial defects of the cell membrane and vacuolation in the cytoplasm. We speculate these effects are responsible for the virtually complete abolition of LC membrane and cytoplasm markers. In summary, we detected few mesenchymal origin cells, melanocytes, Langerhans cells, or S-phase cells, in cryopreserved skin by immunocytochemical methods. Langerhans cells existed but had degenerated. These results indicate that cryopreservation at -196 degrees C causes degeneration of Langerhans cells, and that is the reason for the prolonged viability of cryopreserved allograft.

Adolescent↗
PubMed1998

Plasma membrane integrity of cryopreserved human sperm: an investigation of the results of the hypoosmotic swelling test, the water test, and eosin-Y staining.

OBJECTIVE: [1] To examine the relationship between sperm membrane integrity and motion parameters before and after cryopreservation; [2] to determine the capacity of the membrane integrity tests to predict the outcome of cryopreservation in fertile and infertile men; and [3] to examine the degree of agreement between tail and head membrane integrity of testicular and ejaculated immotile sperm cryopreserved for intracytoplasmic sperm injection. DESIGN: Prospective study. SETTING: Academic tertiary care institution. PATIENT(S): Fertile donors and normozoospermic oligozoospermic, and asthenozoospermic subfertile men. INTERVENTION(S): Semen samples were cryopreserved and thawed for analysis. MAIN OUTCOME MEASURE(S): Sperm membrane integrity and computer-assisted motion parameters. RESULT(S): The hypoosmotic swelling test and water test had a significant and positive correlation in the fresh and cryopreserved ejaculates of all groups. The results of the hypoosmotic swelling test correlated positively with the percent motility in the fresh ejaculates of fertile and subfertile men. None of the membrane integrity tests correlated with the cryosurvival rate in any group. In the ejaculated and testicular samples with no postcryopreservation motility, the simultaneous assessment of hypoosmotic swelling test and eosin showed that of 33% sperm exhibiting coiling with the hypoosmotic swelling test, only 9% were eosin negative, whereas 24% were eosin positive. CONCLUSION(S): [1] The water test may be a simpler replacement for the hypoosmotic swelling test; [2] none of the membrane integrity tests predicted sperm motility after cryopreservation; and [3] there was a high degree of disagreement between the hypoosmotic swelling test and eosin in the samples with no postcryopreservation motility.

Cell Membrane↗
PubMed1999

Salivary mutans streptococci and lactobacilli in 9- and 13-year-old Italian schoolchildren and the relation to oral health.

The prevalence and levels of mutans streptococci (MS) and lactobacilli (LB) in saliva and its possible correlation with dental caries and periodontal conditions was investigated in 473 Italian schoolchildren, 9 and 13 years of age. A clinical examination and sampling of stimulated whole saliva was carried out in the school and oral health was assessed as DMFT and CPITN using the WHO criteria. The saliva samples were frozen in liquid nitrogen and after thawing, cultivated on selective media. To test the influence of cryopreservation, fresh samples from 20 subjects were cultivated. Thirty-five percent of the children were caries-free with a mean DMFT of 1.9 at the age of 13. The majority exhibited healthy periodontal conditions. Salivary MS and LB were identified in 52% and 21% of the children, respectively. The prevalence of MS was higher among the 13-year-olds than the 9-year-olds while no such difference was found regarding LB. There was a statistically positive relationship (P < 0.01) between the levels of MS and LB and both were significantly correlated to caries (P < 0.01). The correlation coefficient of microbial recovery between frozen and unfrozen samples was 0.99. In conclusion, the data provided cross-sectional information of a clear positive relationship between selected micro-organisms in saliva and caries in 9- and 13-year-old children in spite of a relatively low prevalence of the disease. The findings are discussed in a risk selection perspective.

Adolescent↗
PubMed2004

Vitality of oligozoospermic semen samples is improved by both swim-up and density gradient centrifugation before cryopreservation.

PURPOSE: To ascertain whether washing sperm from oligozoospermic and normozoospermic samples before cryopreservation improves post-thaw vitality. METHODS: Normozoospermic (n = 18) and oligozoospermic (n = 16) samples were divided into three aliquots. The first aliquot remained untreated and the second and third aliquots were subjected to the swim-up and discontinuous density gradient sperm washing techniques respectively. Vitality staining was performed, samples mixed with cryopreservation media and frozen. Spermatozoa were thawed, stained, and vitality quantified and expressed as the percentage of live spermatozoa present. RESULTS: Post-thaw vitality in untreated aliquots from normozoospermic samples (24.9% +/- 2.3; mean +/- SEM) was significantly higher (unpaired t-tests; P < 0.01) than untreated oligozoospermic samples (11.9% +/- 2.3). Post-thaw vitality was significantly higher after swim-up in normozoospermic samples (35.6% +/- 2.1; P < 0.001; one-way ANOVA) and oligozoospermic samples (27.7% +/- 1.7; P < 0.01). Density gradient centrifugation significantly improved post-thaw vitality in oligozoospermic (22.4% +/- 1.0; P < 0.01) but not normozoospermic (30.8% +/- 1.8) samples. CONCLUSIONS: Sperm vitality in cryopreserved oligozoospermic samples was improved by both the swim-up and density gradient centrifugation washing techniques prior to freezing.

Cell Separation↗
PubMed2006

A new technique to evaluate the ability of cryoprotectors to prevent premature acrosome reaction in human spermatozoa.

Acrosome reaction (AR) induced by low temperature has been used to evaluate sperm function; it correlates adequately with the fertilization percentages in vitro. In this study, the technique of AR induction by low temperature was used to evaluate the effect in the protection of the acrosome by cryopreservatives normally used in human semen cryopreservation. Donor sperm selected by use of the migration sedimentation technique was incubated in human tubal fluid medium, added to dimethyl sulphoxide 1 m, ethylene glycol 0.75 m, glycerol 1 m, incubated at 4 degrees C and 20 degrees C (as a control) for 18 h, and then for 3 h at 37 degrees C in a cell incubator. The AR was evaluated by triple stain in 100 viable spermatozoa. The effect of cryopreservatives on acrosome preservation in samples incubated for 18 h at 4 degrees C was as follows: 78% intact acrosome for glycerol, 77.8% intact acrosome for dimethyl sulphoxide and 96.2% intact acrosome for ethylene glycol (P < 0.0025 compared with glycerol and dimethyl-sulphoxide). The sperm samples incubated with cryopreservatives for 18 h at 20 degrees C did not show an increase in the percentage of AR in samples incubated with glycerol and ethylene glycol, while a significant variation was observed in the sample incubated with dimethyl sulphoxide (P < 0.001). Additional incubation for 3 h at 37 degrees C significantly increased the AR only in the sample incubated with glycerol (P < 0.001). Acrosome preservation is essential in the fertilization process and the evaluation of acrosome reaction induction by low temperature test was satisfactory. This test proves that ethylene glycol presents a greater protective effect on the acrosome preservation of human spermatozoa.

Acrosome Reaction↗
PubMedDate not supplied

Ultrastructural parameters of fertile cryopreserved human sperm.

Two hundred and forty-four cryopreserved semen samples were used for artificial insemination by donor (AID). All samples were examined for motility and concentration. Thirty of the samples resulted in pregnancies. These samples were further examined ultrastructurally. There was no difference in sperm motility or concentration between the samples that did or did not result in a pregnancy. The ultrastructural characteristics of the samples that resulted in pregnancies revealed that only 15% of sperm (SD = 7.67) possessed normal morphology and had undamaged acrosomes after cryopreservation.

Acrosome↗
PubMed1981

Clonogenic cell survival in cryopreserved human tumour cells.

Cells from 3 human tumours have been grown in soft agar contained in Millipore diffusion chambers and implanted i.p. in mice. Clonal growth was obtained from fresh biopsy samples, from cryopreserved tissue, and from xenografts of the tissues in immune-suppressed mice. The radiosensitivities of a melanoma and an ovarian carcinoma were evaluated by in vitro irradiation before assay for colony formation. Xenografting did not modify the radiosensitivity of the melanoma. Cells from another tumour were exposed to Adriamycin or cyclophosphamide whilst contained within i.p. diffusion chambers; the sensitivity was similar for cryopreserved and xenografted cells. The results encourage further attempts to quantify the sensitivity of human tumour cells by these methods.

Animals↗
PubMed1997

The effects of a simplified method for cryopreservation and thawing procedures on peripheral blood stem cells.

A simplified method for cryopreservation at -80 degrees C of peripheral blood stem cells (PBSC) has been increasingly used for autologous PBSC transplantation in Japan. Although this method, using 6% hydroxyethyl starch (HES) and 5% dimethyl sulfoxide (DMSO) as a cryoprotectant without rate-controlled freezing, has several advantages over the conventional method using 10% DMSO with rate-controlled freezing, little is known about effects of long-term cryopreservation for years and thawing process on hematopoietic progenitors. We examined the recovery rates of BFU-E and CFU-GM in sample tubes cryopreserved by the simplified method under various conditions as follows: (1) long-term storage for 1-5 years; (2) DMSO exposure for 1 h after rapid thawing; and (3) thawing at a lower temperature other than 37 degrees C. In our study, we found that the recovery rates of BFU-E and CFU-GM were not affected by the length of cryopreservation period; they remained at more than 70% on average for 16-61 months. In our hands, a 1-h exposure to DMSO after rapid thawing was not toxic for hematopoietic progenitors. Furthermore, there was no significant difference in the recovery rates of BFU-E and CFU-GM between thawing at 37 degrees C and 20 degrees C. These observations indicate that PBSC cryopreserved for at least 5 years by the simplified method can be used clinically without losing hematopoietic activity, and suggest that hematopoietic activity of the thawed PBSC may be unaffected when PBSC are infused slowly within 60 min or even when PBSC are thawed gradually at room temperature.

Blood Preservation↗
PubMed1996

Quality assurance of CD34+ cell estimation in leucapheresis products.

In order to examine the feasibility of an external quality assurance (QA) scheme for CD34+ cell enumeration and to identify causes of the differences between laboratories for CD34 counts, we carried out a pilot QA exercise in two parts. There were eight participating laboratories in the initial study and each performed CD34 counts using their in-house method. A series of 12 samples of cryopreserved peripheral blood progenitor cells (PBPC) were analysed by each of the eight laboratories. A very wide range of values for all the samples was found for the different in-house methods. For the second part of the study, 12 laboratories analysed a different set of 12 PBPC samples and each used the same anti-CD34 antibody (HPCA-2 PE), anti-CD45 antibodies to identify leucocytes, and counted a minimum of 50,000 events. These measures have reduced the interlaboratory variation in results but this variation is still too high to allow us to realistically compare values between centres. Overall, most centres performed comparably, but there was one centre in part one of the study which gave results that were significantly different from the other centres.

Antigens, CD34↗
PubMed2000

Reproducibility of human immunodeficiency virus type 1 (HIV-1) protease and reverse transcriptase sequencing of plasma samples from heavily treated HIV-1-infected individuals.

The reproducibility of population-based human immunodeficiency virus type 1 (HIV-1) protease and reverse transcriptase (RT) sequencing was assessed using replicate aliquots of cryopreserved plasma samples obtained from seven heavily treated HIV-1-infected individuals. The sequence of each sample replicate was compared with the consensus sequence for that sample and 99.4% of 35128 amino acids were found to be concordant with the sample consensus. Partial discordances were present at 0.5% of positions and complete discordances were present at <0.1% of positions. To assess the reproducibility at detecting mutations (defined here as differences from the subtype B consensus sequence), the proportion of sequences having a mutation when at least two sequences from that sample had the same mutation were examined. There was a median of 13 protease and 18 RT mutations per sample for a total of 3126 mutations; 95% of these mutations were detected. However, sequencing of multiple clones from two samples demonstrated that those mutations present in a minority of clones were often not detected by population-based sequencing. These results suggest that HIV-1 protease and RT sequencing of circulating plasma virus is highly reproducible but that the sensitivity at detecting mutations may be low if those mutations are present as minor variants.

Anti-HIV Agents↗
PubMed2005

[Cryopreserved human sperm deposits: usability after decades of storage].

To determine the effect of long-term storage on cryopreserved human sperm, 238 individual samples from 34 cryopreserved sperm deposits that had been collected between 1976 and 1989 were evaluated at regular intervals. The material was from 34 patients, 18 of whom suffered from underlying malignant disease. Every 3 years a sample from each deposit was thawed for microscopic analysis. During the course of the storage period limited to a maximum of 21 years, all ejaculate parameters had deteriorated in comparison to fresh semen. Quantitative motility was the most sensitive parameter with a decrease of ca. 80%. Sperm density decreased hardly at all. The parameters of the criteria investigated did not decrease linearly or proportionally with the storage duration, but rather more directly after the freezing process or within the first 3 years. These changes became increasingly smaller as the storage time lengthened. Almost all of the deposits exhibited adequate quality at all times during storage. Consequently, 21-year permanent frozen storage in liquid nitrogen is safe regarding aspects of spermatology.

Cryopreservation↗
PubMed2002

Cryopreservation of peripheral blood mononuclear cells does not significantly affect the levels of spontaneous apoptosis after 24-h culture.

Studies performed with malaria patients living in endemic areas are frequently conducted in laboratories located hundreds of kilometer away from research centers, due to the difficulties in performing the assays in field conditions. Thus, we considered the potential indication of cryopreservation of peripheral blood mononuclear cells (PBMC), in most fieldwork, and decided to evaluate the effect of cryopreservation of PBMC on spontaneous apoptosis. The membrane integrity of PBMC was tested using three previously described protocols of cryopreservation. Cell samples were obtained from 19 healthy volunteers. Percentage of apoptotic nuclei in short-term PBMC cultures was determined by a sensitive method using 7-aminoactinomycin D followed by flow cytometry. Our results indicate that although cryopreservation can to some extent affect lymphocyte membrane integrity rates, flow cytometry analysis showed that frequencies of spontaneous apoptosis in cryopreserved cells were not significantly modified after 24-h culture. It is concluded that cryopreserved PBMC could be used for measuring spontaneous apoptosis and therefore, could be employed for the study of populations living in areas distant from research centers, allowing the comparative evaluation of samples obtained at different time.

Apoptosis↗