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PubMed2005

Effect of cryopreservation on mitochondrial DNA of zebrafish (Danio rerio) blastomere cells.

Cryopreservation has been extensively used in human reproductive medicine, aquaculture and conservation programmes for endangered species. However, despite the growing successes of cryopreservation, post-thaw recovery of reproductive and embryonic cells very often remains poor. Many studies have been devoted to the mechanisms of cryodamage. It is known that cryopreservation causes extensive damage to membranes; reduce the metabolic activity of cells; and disturbs the mitochondrial bioenergetical processes of cells. But few investigations on the genetic stability of cells during cryopreservation have been performed, and the role of any genetic impact cryopreservation needs to be determined. Some indirect data in the literature suggests that progress in this field might come from investigating freezing damage to mitochondrial DNA (mtDNA), nuclear DNA and other genome-related structures. In this study, zebrafish (Danio rerio) blastomeres were treated in three different ways: control suspension of blastomere cells in phosphate buffered saline; equilibration of blastomeres with 2M dimethyl sulfoxide (Me2SO) for 1h at room temperature and cryopreservation using Me2SO as a cryoprotectant. Mitochondrial DNA was analysed in fresh cells and after the different treatments. Two different loci of mtDNA were amplified with the help of PCR and sequenced. The sequences were analysed and nuclear base substitutions were counted for both control and treated samples. The results showed that cryopreservation significantly increased the frequency of mutations (0.78+/-0.27% in comparison to 0.16+/-0.25% of control), whilst 2M Me2SO treatment did not bring a significant increase in frequency of mutations (0.24+/-0.28%). The distributions of the mutation locations were analysed. More investigations are needed to determine whether optimisation of cryopreservation protocol is possible to reduce these adverse effects; whether such mutations interfere with overall function of the cells; whether similar changes also occur in the nuclear DNA and whether such mutations happen in other species. Meanwhile, it is important to be cautious in making judgements of the effect of cryopreservation technique in assisted reproduction. This is the first report on the effect of cryopreservation on mtDNA.

Animals↗

PubMed2003

Masked MLL gene rearrangement was disclosed in the clinical course and sequential development of chromosome abnormality in a patient with therapy related acute myelogenous leukemia.

Therapy related acute myelogenous leukemia in a 55-year-old Japanese woman is described. She had been treated for a diagnosis of non-Hodgkin's lymphoma 2 years before the onset of the secondary leukemia. She was diagnosed as AML (FAB: M2) with monosomy 7, and successfully treated by an intensive combination chemotherapy followed by an autologous peripheral blood stem cell transplantation. The disease relapsed shortly after the treatment, and the karyotype analysis revealed a complex abnormality accompanied with t(9;11)(p22;q23), however, monosomy 7 was absent. Southern blotting analysis was performed, and MLL rearrangement was evident in both the bone marrow samples obtained at that time and the cryopreserved marrow cells obtained at the onset of the disease. The bone marrow sample stored in a Carnoy solution at the onset was further analyzed, and three karyotype panels showing 45,XX, -7, t(9;11)(p22;q23) were found. Like this situation, a masked MLL rearrangement may have existed in some cases with hematopoietic malignancies, and appear to be disclosed in the clinical course.

Cell Transformation, Neoplastic↗

PubMed1998

Quantitative immunophenotypic characterization, cryopreservation, and enrichment of second- and third-trimester human fetal cord blood hematopoietic stem cells (progenitor cells).

OBJECTIVE: The aims of this study were (1) to assess the hematopoietic stem cell (progenitor cell) contents of umbilical cord blood samples from second-trimester and early-third-trimester fetuses versus term fetuses and (2) to determine the feasibility of cryopreservation and enrichment of cord blood from fetuses of different gestational ages. STUDY DESIGN: Cord blood between 13 and 42 weeks' gestation (n = 31) was sampled after delivery or fetal expulsion. Fluorescence-activated cell sorting was used to measure CD34(+) and CD34(+)CD38(-) cell numbers. Samples were cryopreserved with 10% dimethylsulfoxide, and CD34(+) enrichment was performed by magnetically activated cell sorting with the MiniMACS system (Miltenyi Biotech, Bergisch Gladbach, Germany). Kruskal-Wallis analysis of variance and the Mann-Whitney U test were used for analysis of data. RESULTS: CD34(+) and CD34(+)CD38(-) cell contents were significantly higher in second- and early third-trimester fetuses than in term fetuses (CD34(+) 2.57% +/- 0.38%, 1.48% +/- 0. 31%, and 0.7% +/- 0.13%, respectively, P =.0067; CD34(+)CD38(-) 0. 72% +/- 0.26%, 0.18% +/- 0.05%, and 0.06% +/- 0.02%, respectively, P =.0132). Mononuclear cell recovery, viability, and CD34(+) cell purity after cryopreservation and enrichment were similar among different gestational ages. CONCLUSION: Cord blood stem cell content decreases significantly from the second trimester to term. Cryopreservation and enrichment of these cells from earlier gestational ages is feasible. This might be especially useful for allogeneic stem cell transplantation and for in utero gene therapy.

ADP-ribosyl Cyclase↗

PubMedDate not supplied

Initial experiences of a testicular sperm extraction programme for assisted reproduction in Ireland.

BACKGROUND: The technique aspirating spermatozoa from the testis is a new development in male infertility treatment. It is appropriate for infertile couples where the male has azoospermia but is still producing live motile spermatozoa in the testes. AIM: To describe the initial experiences of a testicular sperm extraction programme (TESE) coupled with intracytoplasmic sperm injection (ICSI) in 18 men during 1998. METHODS: Spermatozoa were obtained by direct aspiration from the testes using a 16 gauge needle with cannula and negative suction under local anaesthetic. All samples obtained were to be cryopreserved for use in a subsequent ICSI cycle. RESULTS: All five men with congenital bilateral absence of the vas deferens to be carriers of cystic fibrosis gene mutations. No gene deletions were found in their wives. No other cyto or molecular genetic abnormalities were otherwise found. Twenty one procedures were carried out. The mean number of aspirations was 1.72. Eleven samples from 10 men had sperm suitable for ICSI post-freeze. Post-procedure pain was the universal side-effect. Eight couples had a single attempt at ICSI, two couples two each. Fertilisation rate was 71.4%. Two pregnancies were achieved. CONCLUSION: TESE may give hope in selected cases of azoospermia of fathering a child without the involvement of a third party.

Adult↗

PubMed1996

Effects of phosphatidylserine and cholesterol liposomes on the viability, motility, and acrosomal integrity of stallion spermatozoa prior to and after cryopreservation.

Computer-assisted motion analyses (CASA) and flow cytometry were used to evaluate stallion spermatozoa prior to and after cryopreservation. Spermatozoa were pretreated with: (1) Hepes-buffered medium (SHB); (2) phosphatidylserine (PS) liposomes; or (3) liposomes composed of both PS and cholesterol (PSCH) prior to dilution in either SHB or skim milk-egg yolk extender (SMEY). After cooling to 5 degrees C in SHB, PS and PSCH pretreatment (23%). Spermatozoal motion parameters were higher for spermatozoa diluted in SMEY than dilution in SHB. In Experiment 2, motion parameters were compared for spermatozoa pretreated with PSCH liposomes and cryopreserved in either SMEY or a high salt-skim milk-egg yolk extender (CO). Spermatozoal motion characteristics were similar for all spermatozoal treatments after cooling at 5 degrees C. After cryopreservation, PSCH liposome-treated samples had higher percentages of motile spermatozoa than untreated samples regardless of freezing extender. Samples frozen in CO medium had higher percentages of motile spermatozoa than samples frozen in SMEY (P < 0.05; 63% in CO + PSCH and 54% in CO vs 55% in SMEY + PSCH and 48% in SMEY, respectively). In Experiment 3, spermatozoa were treated with dilauroylphosphatidylcholine (PC12) to induce the acrosome reaction. The percentages of viable cells and viable acrosome-reacted spermatozoa were higher for fresh spermatozoa than for cryopreserved spermatozoa (P < 0.05), but were not affected by PSCH liposome treatment (P > 0.05). Addition of PSCH liposomes improved recovery of motile spermatozoa after cryopreservation but did not affect the ability of spermatozoa to undergo a PC12-induced acrosome reaction.

Acrosome↗

PubMed2001

Morphological and functional changes of stallion spermatozoa after cryopreservation during breeding and non-breeding season.

The study compared quality and freezability of stallion semen during breeding and non-breeding seasons. Ejaculates were collected twice per week from four stallions during May (n = 24) and December (n = 24). The semen was mixed with skim milk extender, centrifuged and resuspended in fresh extender. Aliquots of this sperm suspension were separated from extender and diluted in TALP medium for sperm evaluation or with cryoextender (type "Gent" or a combination of Triladyl and skim milk). Samples of 0.5ml were cryopreserved in straws using a programmed freezer. Parameters of sperm quality were evaluated before and after freezing/thawing. These included percentages of motile spermatozoa and of morphological intact sperm. Typical injuries were demonstrated by scanning electron microscopy (S.E.M.). The acrosomal status was visualised using FITC-conjugated peanut agglutinin, and the acrosome reaction was induced by calcium ionophore A 23187. The chromatin stability was estimated by acridine orange test. In winter, the average percentages of motile and morphologically normal sperm (67 and 74.3%, respectively) were higher than during the breeding season in May (59 and 65.9%; P < 0.05). After freezing/thawing the proportions of vital and intact sperm decreased significantly. The number of motile sperm declined to 15 and 18% in May and December (range 5-40%), and of morphologically intact sperm to 51% in both seasons. Results of S.E.M. showed typical membrane ruptures in the acrosomal region and some sperm with abnormal necks. The proportion of frozen sperm with spontaneous acrosome reaction was higher during winter (86.5 versus 77.0%), suggesting a higher degree of membrane reactivity. Percentages of spermatozoa with denaturated chromatin were minimal and showed minimal differences between fresh and frozen state, stallions or seasons. An additional decondensation treatment with papain and DTE revealed a slightly enhanced number of spermatozoa with denaturable DNA after cryopreservation, especially in December (5.4 +/- 1.3%). The influence of cryoextenders was not significant for most sperm parameters, but there was a high variability between the stallions. Altogether, the influence of factors on the quality of spermatozoa has the following rank order: cryopreservation > stallion > season. Different cellular structures seem to have different susceptibilities to physicochemical stress. The cryopreservation of sperm during December results in survival rates similar to those measured during the breeding season, even more important for successful preservation is the selection of suitable semen donors.

Acrosome↗

PubMed2005

Comparison of in vitro laboratory analyses with the fertility of cryopreserved stallion spermatozoa.

Assessing the fertilizing potential of a semen sample is important for effective stallion management and for rapid progress in evaluating new cryopreservation technologies. Unfortunately, sperm motility does not estimate fertility well. These experiments established assays to measure cell viability, acrosomal integrity and mitochondrial function for cryopreserved stallion spermatozoa, using flow cytometry, and determined the variability associated with these assays. Correlations between results for these laboratory assays and stallion fertility were also determined. The inter-assay variability for visual motility, computer assisted motility, and sperm velocity, sperm viability, percent viable-acrosome intact cells and mitochondrial function of cells were all similar, however, intra-assay variability was lower for flow cytometric assays than for motility assays. The reliability of all assays were >0.72, except for sperm velocity (0.32). Although visual motility and the straightness of sperm motility conducted 90 min after thawing were correlated with seasonal fertility (0.56 and 0.55, respectively), data from no single assay were correlated with first-cycle fertility rates (P > 0.05). Best models using data from multiple assays explained 66 to 73, 76 to 89 and 79 to 94% of the variability in fertilizing potential, when two, three and four variables were included, respectively. Caution is required in interpreting these data, as only a few stallions were evaluated and relatively few mares were bred to each stallion, however, they do indicate that using a few rapid and inexpensive sperm assays, we can begin to understand factors important in stallion sperm fertilizing capacity, and we can use these assays to more effectively evaluate new methods for cryopreserving stallion spermatozoa.

Acrosome↗

PubMed1992

Solid-phase anti-CD3 antibody activation and cryopreservation of human tumor-infiltrating lymphocytes derived from epithelial ovarian cancer.

The effect of solid-phase anti-CD3 antibody activation and cryopreservation was evaluated on thirteen samples of tumor-infiltrating lymphocytes (TILs) derived from epithelial ovarian cancer. Seven preparations of TILs were cultured with or without solid-phase anti-CD3 antibody in addition to 100 units/ml of recombinant interleukin-2 (rIL-2). The proliferation rate of all of the seven TIL preparations stimulated by anti-CD3 antibody on the fourth or fifth day of culture was 3.4 to 9.8 times greater than that of lymphocytes cultured with rIL-2 alone. Furthermore, in an experiment with five TIL samples activated with anti-CD3 antibody, three of them showed augmented cytotoxic activity against autologous fresh tumor cells. The population of CD3+/CD8+ TILs was increased after 4-5 weeks of cultivation and CD8+ lymphocytes amounted to over 70% in all of seven preparations tested, whereas two of seven preparations not activated by anti-CD3 antibody were CD3+/CD4(+)-dominant. In addition, nine preparations of TILs cultured with rIL-2 were cryopreserved for several weeks; after recovery from cryopreservation, no major change was observed in cell surface markers, in growth rate or in cytotoxic activity. These results suggest that cryopreserved and/or anti-CD3 antibody-activated lymphocytes could conveniently be employed in a clinical trial of adoptive immunotherapy employing TIL.

Antibodies↗

PubMed2000

What is the benefit of preoperative sperm preservation for patients who undergo restorative proctocolectomy for benign diseases?

PURPOSE: In patients with benign colorectal diseases undergoing a restorative proctocolectomy with an ileal pouch-anal anastomosis, semen cryopreservation seems rational to enable the possibility of procreation in case surgery leads to sexual disorders or impotence. The aim of this study was to determine the preoperative and postoperative semen quality in patients undergoing ileal pouch-anal anastomosis. In addition, the study sought to determine the incidence of surgery-induced sexual dysfunction to evaluate the economic efficiency of semen cryopreservation as compared with alternatives such as microsurgical epididymal sperm aspiration. METHODS: Preoperative and postoperative semen analyses were offered to 97 patients with ileal pouch-anal anastomosis with benign colorectal diseases since 1989. The direct costs of the semen cryopreservation program were determined and compared with those of alternatives. RESULTS: In 34 of 40 consecutive patients with ileal pouch-anal anastomosis who made use of preoperative semen preservation, normal sperm concentrations, motility, and morphology were found. Mean semen characteristics of all 23 patients who returned for postoperative analysis were not different from preoperative values, but they were for total sperm number. Two patients developed temporary retrograde ejaculation postoperatively. None of the preserved semen samples was used, thus semen cryopreservation benefited none of these patients. The total costs of semen cryopreservation are between 2.2 and 5 times higher than the costs for one microsurgical epididymal sperm aspiration procedure. CONCLUSIONS: Preoperative semen cryopreservation in patients undergoing ileal pouch-anal anastomosis because of benign colorectal diseases is quite feasible. However, most likely because of improved surgical techniques and the increasing number of effective alternatives, preoperative semen cryopreservation in patients with ileal pouch-anal anastomosis is no longer cost effective.

Adult↗

PubMed2004

Retention of cellular properties of PBPCs following liquid storage and cryopreservation.

BACKGROUND: G-CSF-mobilized PBPCs are routinely cryopreserved within 24 hours of collection. The ability to hold PBPCs for extended time would offer increased flexibility for patients and hospitals. Retention of PBPC properties following overnight shipping, extended liquid storage at 1 to 6 degrees C, and cryopreservation was evaluated. STUDY DESIGN AND METHODS: PBPCs were stored in liquid at 1 to 6 degrees C up to 3 days, with and without shipping, and then cryopreserved in HES (6%), DMSO (5%), and HSA (4%). Thawed samples were assayed after two procedures, on dilution and after dilution and washing. Nucleated cells, viability, CD34+ cell number, committed progenitor colonies, and long-term culture-initiating cells were measured. RESULTS: CD34+ cell number, committed colony-forming cells, and long-term culture-initiating cells were essentially maintained when samples were stored in liquid for 1, 2, or 3 days before cryopreservation or after thawing and dilution. Nevertheless, significant (p < 0.05, paired t test) losses in total nucleated cell numbers were observed if thawed PBPC samples were washed before assay. CONCLUSION: PBPCs can be maintained at 1 to 6 degrees C for up to 3 days and can be cryopreserved after extended storage with properties minimally altered. Dilution alone, without centrifugation and washing, of thawed PBPC samples is a satisfactory procedure for preparing samples for in vitro assays.

Antigens, CD34↗

PubMed1999

Effect of freeze-thawing procedure on chromatin stability, morphological alteration and membrane integrity of human spermatozoa in fertile and subfertile men.

Cryopreservation is known to impair sperm motility and decrease the fertilization rate by detrimental effects on acrosomal structure and acrosin activity. However, the consequences of cryopreservation on the integrity of the sperm nucleus, chromatin stability and centrosome are less clear. The present study was designed to determine the effect of the freeze-thawing procedure on chromatin condensation (aniline blue staining) and the morphology (strict criteria) and membrane integrity of human spermatozoa. The structural and functional characteristics of the sperm plasma membrane were measured by the eosin-test and hypo-osmotic swelling test which were done separately. Sperm cryopreservation was performed on semen samples from two groups of men classified as fertile (n = 20) and subfertile (n = 72), based on their reproductive history and semen analysis according to WHO guidelines. The mean percentage of condensed chromatin, morphologically normal spermatozoa and membrane integrity in all semen samples investigated (n = 92) decreased significantly (p = 0.0001) after freeze-thawing, in comparison to the value observed prior to freezing. By comparing the semen samples between fertile and subfertile patients, significantly (p = 0.0009) greater damage was demonstrated in the subfertile than in the fertile group. Furthermore, no significant difference was observed between the two groups with regard to the morphological alteration and structural as well as functional damage of the sperm membrane. In conclusion, the freeze-thawing procedure significantly affects chromatin structure and sperm morphology, especially in the head and the tail regions, and this may explain the lower fertilization rate and IVF/ICSI outcome when frozen-thawed spermatozoa are used. In addition, this study demonstrates that chromatin condensation is a sensitive parameter for the evaluation of cryodamage of semen samples from fertile and subfertile patients, though subfertile patients with very poor semen characteristics have yet to be studied. It is therefore recommended that chromatin condensation be used as an additional parameter for the assessment of sperm quality after freeze-thawing.

Cell Membrane↗

PubMed2003

Assessment of fresh and frozen-thawed boar semen using an Annexin-V assay: a new method of evaluating sperm membrane integrity.

Detection of early changes in the sperm plasma membrane during cryopreservation is of utmost importance when designing freezing protocols. The present study evaluated the ability of an Annexin-V binding assay to detect early changes in sperm membrane integrity using flow cytometry (FC) in two different portions of the boar ejaculate, in cryopreserved semen. Using a split sample design, sperm motility was evaluated in fresh (controls) and frozen-thawed (FT) samples, both subjectively and by means of a computer-assisted motility assessment (CASA) system, while membrane integrity was assessed using Annexin-V (A) and propidium iodide (PI) staining in spermatozoa derived from the first sperm-rich fraction (Portion I) or the remaining ejaculate (Portion II). The A/PI technique revealed four sperm subpopulations, two PI negative (either A- (alive) or A+ (apoptotic)); and two PI positive (dead cells), either A+ (dead, late apoptotic or early necrotic cells) or A- (dead, late necrotic cells). Significant differences were found between the two portions of the ejaculate in the fresh (control) and FT samples. In the fresh controls, significantly more live, nonapoptotic spermatozoa (A-/PI-) were present in Portion I than in Portion II (P<0.001). Although apoptotic spermatozoa were detected in both semen portions, the frequency of live, early apoptotic (A+/PI-) cells was significantly lower in Portion I than in Portion II (P<0.001). Irrespective of the ejaculate portion considered, freezing and thawing significantly decreased the mean percentages of live spermatozoa (P<0.01), and dramatically increased the percentages of apoptotic or early necrotic cells (P<0.01), but not of early apoptotic cells (N.S.). The latter finding might suggest that apoptotic changes due to cryopreservation using the procedures applied in this trial are transient and lead to cell death. In conclusion, the Annexin-V binding assay was able to detect deleterious changes in the sperm plasma membrane at an earlier point than PI staining, thus representing a novel approach to investigating membrane integrity in this species. The finding that fewer spermatozoa in Portion I of the ejaculate showed early apoptosis post-freezing, suggests boar spermatozoa in this portion of the seminal plasma are less sensitive to the stress induced by cryopreservation.

Animals↗

PubMed2005

Maximizing the retention of antigen specific lymphocyte function after cryopreservation.

The ability to cryopreserve lymphocytes in peripheral blood mononuclear cells (PBMC) to retain their function after thawing is critical to the analysis of cancer immunotherapy studies. We evaluated a variety of cryopreservation strategies with the aim of developing an optimized protocol for freezing and thawing PBMC to retain viability and function. We determined several factors which do not affect cell viability after cryopreservation such as shipping frozen samples on dry ice, the length of time and speed at which samples are washed and centrifuged after thawing, and the number of cells frozen per container. Different media additives, however, did impact the viability of the cells after thawing. There was a significant reduction in the viability of the cells after freezing when using human AB serum compared to all other additives tested (p<0.000). A second critical parameter was the temperature of the media used to wash the cells after removal from the cryotubes. When the media was cooled to 4 degrees C prior to washing, the mean viability was 69.7+/-12.5%, at 25 degrees C 92.55+/-3.1%, and at 37 degrees C 95.11+/-2.5%. Finally, we used an optimized cryopreservation protocol with different media additives to determine if functional T cell responses to tetanus toxoid could be preserved. There was a statistically significant correlation between the tetanus specific stimulation index (S.I.) of the non-cryopreserved PBMC and SI obtained from cells frozen with media containing human serum albumin as compared to other additives such as dextran or fetal bovine serum.

Antigens↗

PubMed1997

An intralaboratory quality control program for quantitation of CD34+ cells by flow cytometry.

This study was undertaken to develop a quality control protocol to monitor instrument, operator, and CD34 assay performance. A dual level control system was established by cryopreserving aliquots of cells from peripheral blood progenitor cell (PBPC) collections exhibiting different percentages of CD34+ cells. Twenty-five samples from each control specimen were assayed to establish a control range (mean +/- 2 SD). Levey-Jennings graphs were prepared for each control specimen to plot multiple measurements of CD34%. No significant differences were observed between fresh or cryopreserved PBPC aliquots in terms of light scatter properties or CD34 antigen density within the gated cell population. Cryopreserved PBPC samples are ideal for serving as a positive methodology control for daily CD34 analysis. Furthermore, such a system can help identify problems with assay reagents, sample preparation technique, or incorrect data analysis.

Antigens, CD34↗

PubMed2001

Human UC-blood banking: impact of blood volume, cell separation and cryopreservation on leukocyte and CD34(+) cell recovery.

BACKGROUND: UC blood represents an increasingly useful source of hematopoietic stem cells for BMT, although, currently, low cell numbers generally limit its use to pediatric patients. We have determined parameters that influence the recovery of viable cells during processing and cryopreservation, in an effort to set guidelines for determining whether a sample will yield sufficient cells to be of use in the transplant setting. METHODS: UC blood was collected from donors from January 1996 to December 1999. Volume was reduced using Ficoll, followed by cryopreservation under liquid nitrogen. Total leukocyte and CD34(+)-cell counts were determined prior to processing and a subset of samples was also assessed post-processing and post-cryopreservation. RESULTS: Outcomes for 3816 samples were analyzed to determine the correlation between cell number, cell type, volume, and time between collection and processing. A positive relationship was observed between volume and cell number for both leukocytes and the CD34(+) cells. This correlation allowed us to determine the number of leukocytes and CD34(+) cells expected from a sample based on volume, and to set guidelines for determining the practicality of storing any given sample prior to processing and cryopreservation. DISCUSSION: Measuring blood volume gives a very useful indication of the total leukocyte and CD34(+) cell number. The majority (75%) of cord-blood samples contain sufficient leukocytes for a pediatric transplant, and the number of cells available can be determined prior to processing by measuring blood volume.

Antigens, CD34↗

PubMed1993

Detection of altered acrosomal physiology of cryopreserved human spermatozoa after sperm residence in the female reproductive tract.

At least some of the spermatozoa that remain motile following cryopreservation have sustained sublethal damage that reduces their functional capacity in vivo. Although it is believed that acrosomal damage is partly responsible for impaired sperm function in vivo, direct evidence for this hypothesis is lacking because spermatozoa have not been collected from the female reproductive tract for evaluation. In the study reported here, cervical mucus was collected from women 24 h after artificial insemination by cervical cup. For both cryopreserved and nonfrozen inseminates, spermatozoa within the cervical mucus and spermatozoa that migrated out of mucus into culture medium (t = 1 h) were viable and had intact acrosomes. However, although nonfrozen spermatozoa did not initially respond to induction of the acrosome reaction with follicular fluid, a significant proportion of cryopreserved spermatozoa did respond. These results demonstrate that cryopreservation increases the acrosomal lability of spermatozoa residing in the female reproductive tract. An in vitro test was developed to detect this form of cryodamage. Sperm-free mucus was collected before insemination and spermatozoa from the inseminate were allowed to swim into this column of mucus in vitro. Spermatozoa recovered from this mucus sample were compared with spermatozoa from the paired sample collected from the cervix 24 h later. This in vitro test could detect acrosomal lability in cryopreserved semen samples, and this approach may prove valuable for studying sublethal cryodamage to the acrosome.

Acrosome↗

PubMed2005

Assessment of the cryopreservation of equine spermatozoa in the presence of enzyme scavengers and antioxidants.

OBJECTIVE: To evaluate the effect of the addition of enzyme scavengers and antioxidants to the cryopreservation extender on characteristics of equine spermatozoa after freezing and thawing. SAMPLE POPULATION: 2 ejaculates collected from each of 5 stallions. PROCEDURE: Equine spermatozoa were cryopreserved in freezing extender alone (control samples) or with the addition of catalase (200 U/mL), superoxide dismutase (200 U/mL), reduced glutathione (10 mM), ascorbic acid (10 mM), alpha-tocopherol (25, 50, 100, or 500 microM or 1 mM), or the vehicle for alpha-tocopherol (0.5% ethanol). After thawing, spermatozoal motility was assessed via computer-assisted analysis and DNA fragmentation was assessed via the comet assay. Spermatozoal mitochondrial membrane potential, acrosomal integrity, and viability were determined by use of various specific staining techniques and flow cytometry. RESULTS: The addition of enzyme scavengers or antioxidants to cryopreservation extender did not improve spermatozoal motility, DNA fragmentation, acrosomal integrity, viability, or mitochondrial membrane potential after thawing. Superoxide dismutase increased DNA fragmentation, likely because of the additional oxidative stress caused by the generation of hydrogen peroxide by this enzyme. Interestingly, the addition of the vehicle for alpha-tocopherol resulted in a significant decrease in live acrosome-intact spermatozoa. CONCLUSIONS AND CLINICAL RELEVANCE: The addition of antioxidants to the cryopreservation extender did not improve the quality of equine spermatozoa after thawing, which suggests that the role of oxidative stress in cryopreservation-induced damage of equine spermatozoa requires further investigation. Our data suggest that solubilizing alpha-tocopherol in ethanol may affect spermatozoal viability; consequently, water-soluble analogues of alpha-tocopherol may be preferred for future investigations.

Animals↗

PubMed1996

Cryopreservation of semen from adolescent patients with malignancies.

In adult oncological patients semen cryopreservation offers the possibility of preserving fertility prior to aggressive therapy that may lead to infertility. The cryopreserved semen can later be used to induce pregnancies in the partner by techniques of assisted fertilization. In adolescent boys the question of fertility is often beyond consideration when the young patient's life is threatened acutely. However, improved survival rates increasingly prompt the question of quality of life after therapy, including fertility. Semen quality is known to be impaired in patients with malignancies and may be further impaired by the process of cryopreservation. Since normal values for semen in adolescents are not known and spermatogenesis may be impaired by the malignant disease, it was unclear whether semen samples from adolescents with malignancies warrant cryopreservation at all. In order to demonstrate the feasibility of semen cryopreservation in adolescent males, we compared the results from 12 pubertal boys aged 14-17 years with those from 17 young adults aged 18-20 years who had similar malignancies and, additionally, to 210 adults with malignancies (> 20 years). Luteinizing hormone serum values were significantly lower in adolescents than in adult patients. Follicle stimulating hormone showed a significant increase with age. Testosterone serum levels and testicular volumes showed similar distribution patterns in adolescent and adult men. Sperm concentrations, sperm motility, and normal sperm morphology in the adolescent patients did not show significant differences compared with adults. Thus cryopreservation of semen should be considered as an option to young male patients whose cancer therapy will include potentially gonadotoxic treatment.

Adolescent↗