Pre-freeze Samples Research Articles

Egg Yolk-Supplemented Tris-Citric Acid Extender Improves the Prefreezing and Post-Thaw Sperm Quality Indices of Guinea Pig (Cavia porcellus) Epididymal Spermatozoa.

This study aimed to assess the suitability of egg yolk (EY) supplementation to a tris-citric acid-based extender on cryosurvival of guinea pig (Cavia porcellus) epididymal spermatozoa. Two synthetic-based extenders, tris-citric acid-glucose plus 20% EY (TCG-EY) and tris-citric acid-fructose (TCF) both with 5% glycerol, were compared. Thirty-two epididymides were recovered from 16 adult guinea pig males by gonadectomy, and then the sperm samples were retrieved by retrograde flushing using TCG-EY and TCF extenders for left or right epididymis, respectively. TCG-EY and TCF sperm samples were frozen in static liquid nitrogen vapors through a two-step cooling procedure. Before freezing, the percentage of progressive sperm motility and sperm with intact plasma and acrosome membranes from TCG-EY sperm samples were higher (p < 0.05) than those diluted with TCF. Post-thaw sperm kinematic variables and membrane integrity were drastically reduced (p < 0.001) compared with prefreezing samples, regardless of extender type. The post-thaw plasmatic and acrosome membrane integrity from TCG-EY sperm samples was higher (p < 0.05) than those from TCF samples. Except for the length, the morphometric head dimensions of sperm diluted with TCG-EY or TCF did not vary (p > 0.05) after the freezing-thawing process compared with the prefreezing samples. In conclusion, despite greater cell cryoinjury with both extenders, the EY supplementation exerted greater cell membrane protection before and after the freezing-thawing process. This research shows an in-depth analysis of guinea pig sperm cryopreservation; however, more studies are recommended.

Cryopreservation alters the immune characteristics of platelets.

Cryopreserved platelets are under clinical evaluation as they offer improvements in shelf-life and potentially hemostatic effectiveness. However, the effect of cryopreservation on characteristics related to the immune function of platelets has not been examined. Buffy coat derived platelets were cryopreserved at -80°C using 5%-6% dimethylsulfoxide (DMSO, n=8). Paired testing was conducted pre-freeze (PF), post-thaw (PT0), and after 24 h of post-thaw storage at room temperature (PT24). The concentration of biological response modifiers (BRMs) in the supernatant was measured using commercial ELISAs and surface receptor abundance was assessed by flow cytometry. Cryopreservation resulted in increased RANTES, PF4, and C3a but decreased IL-1β, OX40L, IL-13, IL-27, CD40L, and C5a concentrations in the supernatant, compared to PF samples. C4a, endocan, and HMGB1 concentrations were similar between the PF and PT0 groups. The abundance of surface-expressed P-selectin, siglec-7, TLR3, TLR7, and TLR9 was increased PT0; while CD40, CLEC2, ICAM-2, and MHC-I were decreased, compared to PF. The surface abundance of CD40L, B7-2, DC-SIGN, HCAM, TLR1, TLR2, TLR4, and TLR6 was unchanged by cryopreservation. Following 24 h of post-thaw storage, all immune associated receptors and TLRs increased to levels higher than observed on PF and PT0 platelets. Cryopreservation alters the immune phenotype of platelets. Understanding the clinical implications of the observed changes in BRM release and receptor abundance are essential, as they may influence the likelihood of adverse events.

Salvaging urospermic ejaculates from brown bear (Ursus arctos)

The objective of this study was to reverse the osmotic stress of sperm in urine contaminated bear ejaculates that were obtained by electroejaculation using pre-freezing washing or density gradient centrifugation isolation. In Experiment 1, ejaculates were divided into six aliquots, five were diluted in each washing extender: 200, 300, 400, 500 and 700mOsm/kg (prepared from a Tes–Tris–Fructose base, adding water or fructose as corresponds), at a 1:2 ratio (raw semen: washing solution, v/v); and the other aliquot was handled without washing (Control group). Samples were centrifuged at 600×g for 6min prior to freezing. In Experiment 2, ejaculates were divided into two aliquots: one was diluted 1:1 with TCG (Tris–Citric acid–Glucose) and centrifuged at 600×g for 6min (Centrifugation Control; C-Control); the other was treated with PureSperm® density gradient column. After treatments, samples were cryopreserved. Sperm motility, viability (SYBR-14/propidium iodide (PI)) and acrosomal status (peanut agglutinin-fluorescein isothiocyanate (PNA-FITC)/PI) were analyzed before and after freezing. Ejaculates with an initial osmolality of less than 120mOsm/kg treated with pre-freezing washing, and the Control sample had greater pre-freezing sperm motility than the raw ejaculate, but sperm viability was not different among these groups. The samples washed with 700mOsm/kg solutions had the least pre-freezing viability. In the post-thawing evaluation, pre-freezing washing treatments did not provide any improvement in comparison with the Control sample, and treatment with 700mOsm/kg extender had deleterious effects in all urospermic samples. PureSperm® density gradient centrifugation applied to urospermic raw semen was suitable for improving sperm motility and viability of pre-freezing samples and the selected spermatozoa had greater freezing capacity.

Effect of testicle postmortem storage on goat frozen-thawed epididymal sperm quality as a tool to improve genebanking in local breeds

The interest to develop assisted reproductive technologies and cryobanking for farm animal genetic resource conservation has recently increased. However, cryopreservation for ex-situ management of genetic diversity sometimes is not routinely feasible, owing to the lack of facilities (AI centres, laboratories) and expertise near the local breed farming area. In these cases, epididymal sperm obtained from slaughtered or castrated animals, associated with the possibility of managing rather long periods between animal death, sperm recovery and freezing, would increase the opportunities to create semen storages. This investigation addresses the pre-freeze/post-thaw quality of goat epididymal sperm as a function of testicle storage temperature (environment or +5°C) and time elapsed between animal’s death and sperm recovery (0, 24, 48, 72 h) to establish the optimal protocols for the recovery and cryopreservation of epididymal sperm in this species. Testicles of 50 mature bucks collected at the abattoir were divided in two groups: half of the testicles (n=50) were transported to the laboratory at environment temperature (E), whereas the remaining half (n=50) at a refrigeration temperature (R) of +5°C. In the two groups (E) and (R), one testicle from each pair was processed after slaughter forming the time 0 groups (0E and 0R). The contralateral testicle was processed after 24, 48 or 72 h of storage, at the corresponding temperature. Sperm motility and kinetic parameters, viability and morphology were assessed in pre-freeze and post-thaw samples. Until 48 h postmortem, both E and R temperatures are able to maintain good pre-freeze epididymal sperm quality. After 48 h postmortem, R temperature is fundamental to reduce epididymal sperm quality decay in pre-freeze samples. Moreover, testicle refrigeration also has a positive impact on post-thaw samples, allowing a lower decline through time considering total motility, kinetics parameters, sperm viability and sperm abnormalities. Therefore, when sperm cryopreservation is not immediately practicable, goat testicles should be transported and stored at 5°C up to a maximum of 48 h postmortem to ensure an acceptable sperm quality.

Effects of cryopreservation on microbial‐contaminated cord blood

Cord blood units (CBUs) are associated with significant risk of exposure to microbial contamination during collection and processing; however, the survival of bacteria within a CBU is poorly understood. This study aimed to determine whether contaminating organisms in CBU survive the cryopreservation, frozen storage, and subsequent thawing conditions before infusion. A total of 134 CBUs rejected from banking due to known contamination were thawed and rescreened using blood culture bottles (BacT/ALERT, bioMérieux). An additional 61 fresh CBUs were deliberately spiked with a range of microbial organisms and evaluated both before freeze and after thaw. Microbial contaminants were detected after thaw in 63% of stored contaminated CBUs and 85% of spiked CBUs. Postthaw organism detection in spiked cord blood (CB) was higher in adult culture bottles (80%) than pediatric culture bottles (61%). Twenty percent of spiked organisms, particularly Bacillus subtilis, Escherichia coli, Clostridium sporogenes, and Propionibacterium acnes, were not detected in prefreeze samples but were detectable after thaw. This study demonstrates that the majority of contaminating organisms isolated in a prefreeze sample of CB have the ability to survive cryopreservation, frozen storage, and thawing. Further, CBUs reported as microbial free may contain microbial contamination, which could result in transplantation of contaminated CB and be potentially deleterious to a patient.

A Bayesian Adjustment for Multiplicative Measurement Errors for a Calibration Problem with Application to a Stem Cell Study

We develop a Bayesian approach to a calibration problem with one interested covariate subject to multiplicative measurement errors. Our work is motivated by a stem cell study with the objective of establishing the recommended minimum doses for stem cell engraftment after a blood transplant. When determining a safe stem cell dose based on the prefreeze samples, the postcryopreservation recovery rate enters in the model as a multiplicative measurement error term, as shown in the model. We examine the impact of ignoring measurement errors in terms of asymptotic bias in the regression coefficient. According to the general structure of data available in practice, we propose a two-stage Bayesian method to perform model estimation via R2WinBUGS (Sturtz, Ligges, and Gelman, 2005, Journal of Statistical Software 12, 1-16). We illustrate this method by the aforementioned motivating example. The results of this study allow routine peripheral blood stem cell processing laboratories to establish recommended minimum stem cell doses for transplant and develop a systematic approach for further deciding whether the postthaw analysis is warranted.

Cryopreservation of buffy-coat-derived platelet concentrates in dimethyl sulfoxide and platelet additive solution

Platelets prepared in plasma can be frozen in 6% dimethyl sulfoxide (Me 2SO) and stored for extended periods at −80 °C. The aim of this study was to reduce the plasma present in the cryopreserved product, by substituting plasma with platelet additive solution (PAS; SSP+), whilst maintaining in vitro platelet quality. Buffy coat-derived pooled leukoreduced platelet concentrates were frozen in a mixture of SSP+, plasma and 6% Me 2SO. The platelets were concentrated, to avoid post-thaw washing, and frozen at −80 °C. The cryopreserved platelet units ( n = 9) were rapidly thawed at 37 °C, reconstituted in 50% SSP+/plasma and stored at 22 °C. Platelet recovery and quality were examined 1 and 24 h post-thaw and compared to the pre-freeze samples. Upon thawing, platelet recovery ranged from 60% to 80%. However, there were differences between frozen and liquid-stored platelets, including a reduction in aggregation in response to ADP and collagen; increased CD62P expression; decreased viability; increased apoptosis and some loss of mitochondrial membrane integrity. Some recovery of these parameters was detected at 24 h post-thaw, indicating an extended shelf-life may be possible. The data suggests that freezing platelets in 6% Me 2SO and additive solution produces acceptable in vitro platelet quality.

Aldehyde Dehydrogenase (ALDH) Vs CD34 for Predicting Engraftment After Autologous Hematopoietic Progenitor Cell (autoHPC) Transplant

AbstractAbstract 4441 Introduction:When the cell surface marker CD34 became available for enumerating HPC there was controversy as to whether it was useful for predicting time to engraftment after autologous peripheral blood HPC transplant (autoHPCT). It was demonstrated that a threshold dose of 2.0–2.5 × 106 CD34+ cells/kg in the product, measured before cryopreservation, predicted reliable engraftment within a reasonable period of time. CD34 is expressed on the surface of HPC ranging from primitive to committed progenitor cells. ALDH is highly expressed in primitive cells such as HPC, endothelial progenitor cells and mesenchymal stromal cells. ALDHbr cells are by definition viable, while measurement of viable CD34+ cells requires an additional assay. We tested the correlation of CD34 and ALDH activity in fresh HPC products and then examined the usefulness of these two assays for predicting time to WBC and platelet engraftment after autoHPCT. Materials and Methods:We identified 42 consecutive HPC apheresis products used for autoHPCT for which data on CD34+ numbers and viability as well as ALDHbr cell numbers were available. Data from 5 of these product were not used for predicting engraftment due to lack of engraftment data. Viability was assesed by flow cytometry on pre-cryopreservation (fresh) and post-thaw samples by looking at propidium iodide or 7-AAD uptake within the CD34+ population identified using the ISHAGE method. ALDH activity was measured by flow cytometry on pre-freeze samples using the manufacturers prescribed method. HPC were cryopreserved using a controlled rate freezer and 10% DMSO, then stored in liquid nitrogen. Data on time to WBC and platelet engraftment were determined by chart review. The patents received a median of 3.78 × 106 CD34+ cells/kg (range 2.43–8.59 × 106 CD34+ cells/kg) for autoHPCT. Regression analyses were performed to see if viable CD34 in the fresh product, and in the thawed product, correlated with ALDH and whether any of these measures correlate with time to WBC (ANC >500/mm3) or platelet engraftment (>20,000/mm3). Results:The correlation coefficients for ALDHbr vs viable CD34+ cells pre-cryopreservation (r=0.97; n=42) and for ALDHbr cells vs post-thaw CD34+ cells (r=0.96; n=42) were both significant. The percent of variation for ALDHbr cells to WBC engraftment (r2 = 2.2%; n=37) and for CD34+ cells both pre-freezing and post-thaw to WBC engraftment (r2 =1.9%; n=37) were not significant. The r2 for ALDHbr cells and platelet engraftment was 0.9%, the r2 for CD34+ cells and platelet engraftment was 1.1% pre-freeze (n=37) and was 0.9% post-thaw (n=37) were also not significant. Conclusion:Enumeration of HPC by ALDH correlates well with CD34, suggesting that the two assays are equivalent when analyzing autologous peripheral blood. Neither the number of ALDHbr cells or CD34+ cells in fresh products, nor the number of viable CD34+ cells in the thawed products, correlates with time to WBC and platelet engraftment. Since our patients received at least 2.4 × 106 CD34+ cells/kg, our observations support previous data showing no correlation between cell dose and time to engraftment if more than the HPC threshold dose for engraftment is given for transplant. Disclosures:No relevant conflicts of interest to declare.

Assessment of cord blood hematopoietic cell parameters before and after cryopreservation

The testing of cord blood (CB) progenitor and stem cell units for transplantation suitability involves enumeration of total nucleated cells before freezing. CD34+ cell counts may also be a means of determining suitability. Studies have been conducted to evaluate how specific storage conditions influence cell counts. CB units were processed by hydroxyethyl starch volume reduction. Cryopreserved-thawed samples were diluted 1:3 without washing. CD34+ cells were measured with three commercially available assay methods. In specific studies, apoptosis-indicating reagents were included. CB units were analyzed for nucleated cells, aldehyde dehydrogenase-containing cells, and progenitor colonies. CD34+ cell levels and nucleated cells were retained during storage in test tubes at 1 to 6 degrees C for 3 days. Cryopreserved-thawed samples showed a reduction in CD34+ cells relative to prefreeze levels with the largest decrease with the Stem-Kit (Beckman Coulter) restricted gating procedure. Prefreeze samples contained minimal numbers of presumed apoptotic cells detected with 7-aminoactinomycin D or SYTO16, but after cryopreservation-thawing there was an increase. Nucleated cell levels determined with a hematology analyzer or flow cytometry were reduced after thawing. Cryopreservation-thawing reduced the percentage of CD34+ cells positive for the presence of aldehyde dehydrogenase and the number of progenitor colonies. These differences were significant. These studies indicate that CD34+ cell counts were maintained when CB samples were stored at 1 to 6 degrees C in test tubes for 3 days. Cryopreservation-thawing resulted in changes in a number of parameters including the percentage of CD34+ cells that were aldehyde dehydrogenase(+) and the number of 7-aminoactinomycin D(+) cells and SYTO16(low) cells.

Effect of Pentoxifylline Containing Human Sperm Cryopreservation Medium on Post-Thaw Motility of Human Spermatozoa and Lipid Peroxidation Status of Human Semen

The process of freeze-thawing impairs sperm motility and membrane integrity and results in an overall loss of sperm fertilizing ability. The damage occurs mostly by the production of reactive oxygen species. Pentoxifylline acts as a potent motility activator by inhibiting the phosphodiesterase activity as well as an antioxidant. The present study was carried out to assess the effect of pentoxifylline containing human sperm cryopreservation medium {egg-yolk, glucose, fructose, glycine containing and glycerol based medium where the effective concentration of glycerol in medium-semen mixture is 7.5% (V/V)} on human sperm motility, forward progression and lipid peroxidation status of frozen thawed semen. Prospective experimental study. A final concentration of 3.6 mM pentoxifylline/L of medium-semen mixture was selected for this study. A comparative study between both the media (the Human Sperm Cryopreservation Medium[HSPM] alone and the HSPM containing 7.2 mM pentoxifylline/L of medium) was carried out in 26 normozoospermic human semen samples from patients attending the University clinic. After preliminary analysis, each semen sample was divided into 2 aliquots, each of 300 μL and one was mixed with the medium (M) alone and the other with the pentoxifylline containing medium (Mp), both in 1: 1 dilution and preserved at -196°C in liquid nitrogen for 1 week. The motility characteristics were assessed and lipid peroxidation status was estimated by malonaldehyde (MDA) formation in both the media preserved spermatozoa on thawing and it was compared with that of pre-freeze ones. The mean percentage (± SD) motility was 66.9 ± 15.2 % in pre-freeze samples; it was 31.3 ± 17.0% (p < 0.005) and 46.2 ± 19.4% (p > 0.07) after thawing in spermatozoa preserved in medium M and Mp respectively. The mean percentage (± SD) of 5% forward progression was 56.6 ± 16.4 % in pre-freeze samples; it declined to 21.8 ± 16.9% (p < 0.005) and 37.3 ± 20.9% (p > 0.01) on thawing in sperm preserved in medium M and Mp, respectively. The lipid peroxidation of whole semen was 22.6 ± 11.9 nM in pre-freeze condition and increased to 43.0 ±11.3 (p < 0.01) in samples preserved in medium M and 30.6 ± 15.8 (p > 0.3) nM of MDA/ml of semen in samples preserved in Mp. The addition of pentoxifylline with sperm cryopreservation medium not only provides a comparatively higher yield of spermatozoa with % motility and forward progression on thawing but also minimizes the lipid peroxidation of sperm membrane on cryopreservation. Hence, pentoxifylline could be used as a supplement in human sperm cryopreservation medium.

192 PROBLEMS USING JC-1 TO ASSESS MITOCHONDRIAL STATUS IN BROWN BEAR (URSUS ARCTOS) SEMEN

Brown bear is a highly endangered species in Spain and could benefit from biological resource banking. Currently, we are studying several reproductive aspects in order to aquire the knowledge for establishment of a germplasm bank for this species. One of our objectives is to develop adequate protocols for the evaluation of bear sperm before and after cryopreservation. We have used the fluorescent probe JC-1 protocol, which differentially stains mitochondria, according to its activity (Garner DL et al. 1997 Biol. Reprod. 57, 1401–1406). Here we describe one problem that arose using this staining for evaluation of extended bear semen. We electroejaculated 13 adult brown bears (Ursus arctos) (206–311 kg) housed in a half-freedom regime in the Cabarceno Park (Cantabria, Spain). Anesthesia was performed with tiletamine + zolazepan (Zoletil 100®, 7 mg/kg; Virbac, Spain), and ketamine (Imalgene 1000®, 2 mg/kg; Mericl, Sain). We used an electroejaculator (PT Electronics®; Boring, OR, USA) with a 3-electrode transrectal probe (26 mm in diameter, 320 mm long). Ejaculation occurred at 10 V/250 mA. Samples were extended (prepared in our laboratory, Anel L et al. 2003 Theriogenology 60, 1293–1308; M3 modified) and cooled to 5°C for 70 min (pre-freezing protocol). We analyzed individual (MI) and progressive (MP) motility by means of an automated motility analyzer (Hamilton Thorne Biosciences, Inc., Beverly, MA, USA), using a phase contrast microscope (Nikon, ×10). Mitochondrial status was analyzed after diluting the sample 1:100 with buffered medium (20 mM HEPES, 153 mM NaCl, 2.5 mM KOH, 10 mM glucose; Sigma, Madrid, Spain) and adding JC-1 (6.8 μM final; Molecular Probes, The Netherlands). After 30 min at 37°C, 100 cells were counted with an epifluorescence microscope (Nikon, ×400), determining the percentage of sperm with orange-stained (active) mitochondria. We analyzed a total of 55 samples in three different models: fresh, pre-freezing, and thawed. We divided the samples into successful JC-1 staining (valids: V) or failed JC-1 staining (not valid: NV) (depending on the aspect of the stained cells). In not-valid samples we observed a greenish background, with almost no fluorescent spermatozoa. These observations were consistent in a given sample, giving the same V or NV result when we repeated the staining. In fresh and thawed groups there were no NV samples, but in the pre-freezing group there were 40 NV samples (73%). We calculated Pearson correlations (SAS; SAS Institute, Inc., Cary, NC, USA) between percent JC-1 orange population and MI and MP in fresh (r = 0.40 and 0.33; P &lt; 0.001), thawed (r = 0.61 and 0.43; P &lt; 0.001) and pre-freezing samples (r = −0.11 and −0.24; P &gt; 0.05), all respectively. When pre-freezing samples were split between V and NV, the former had good correlations (r = 0.74 and 0.49; P &lt; 0.05), and NV still did not (r = −0.17, −0.27; P &gt; 0.05). We conclude that JC-1 staining is not reliable for the pre-freezing analysis of bear sperm, at least under the conditions described here. This could be due to the interaction of the extender or the refrigeration treatment with the sperm. However, this problem did not occur in the analysis of fresh and thawed samples. Nevertheless, it may be advisable to test other mitochondrial probes for analyzing this kind of samples.

The effect of temperature and the duration of cryopreservation on human sperm chromatin

The present study was carried out to assess the effect of different freezing temperatures and duration of freezing on human sperm chromatin integrity. Prospective study. Semen samples were obtained from 93 randomly selected men attending the University Clinic, specimens from 5 proven fertile volunteers served as the controls. After preliminary semen analysis, each sample was divided in five aliquots and mixed with commercially available cryopreservation medium (Tardigrade, IMV, France). One aliquot (A) was used for the pre-freeze study of chromatin integrity, two other aliquots (B1, B2) were frozen in a mechanical freezer at -80°C and the last two aliquots (C1, C2) were frozen in liquid nitrogen (-196°C). The duration of cryopreservation for aliquots B1 and C1 was 1 week, and 3 months for aliquots B2 and C2. Chromatin cryo-injury was examined under fluorescent microscope using acridine orange. The mean percentage of spermatozoa with intact chromatin in pre-freeze samples (aliquot A) was 90.1 ± 5.9%, it showed no difference from that of whole semen without a cryopreservative. After 1 week of freezing, chromatin integrity reduced to 76.5 ± 7.7% and 81.6 ± 8.1% in aliquots B1sC1, respectively. It dropped to 69.6 ± 8.6% and 76.3 ± 7.1% for aliquots B2sC2, respectively, upon prolonged freezing for 3 months. The process of freeze-thawing has an adverse effect on human sperm chromatin. The loss of chromatin integrity is more prominent in case of mechanical freezing at -80°C than with liquid nitrogen, both at 1 week as well as after prolonged preservation for three months. Therefore, cryopreservation in liquid nitrogen should be recommended for freezing of semen specimens used for assisted reproduction.

High-efficiency recovery of functional hematopoietic progenitor and stem cells from human cord blood cryopreserved for 15 years.

Transplanted cord blood (CB) hematopoietic stem cells (HSC) and progenitor cells (HPC) can treat malignant and nonmalignant disorders. Because long-term cryopreservation is critical for CB banking and transplantation, we assessed the efficiency of recovery of viable HSCHPC from individual CBs stored frozen for 15 yr. Average recoveries (+/- 1 SD) of defrosted nucleated cells, colony-forming unit-granulocyte, -macrophage (CFU-GM), burst-forming unit-erythroid (BFU-E), and colony-forming unit-granulocyte, -erythrocyte, -monocyte, and -megakaryocyte (CFU-GEMM) were, respectively, 83 +/- 12, 95 +/- 16, 84 +/- 25, and 85 +/- 25 using the same culture conditions as for prefreeze samples. Proliferative capacities of CFU-GM, BFU-E, and CFU-GEMM were intact as colonies generated respectively contained up to 22,500, 182,500, and 292,500 cells. Self-renewal of CFU-GEMM was also retained as replating efficiency of single CFU-GEMM colonies into 2 degrees dishes was >96% and yielded 2 degrees colonies of CFU-GM, BFU-E, and CFU-GEMM. Moreover, CD34(+)CD38(-) cells isolated by FACS after thawing yielded >250-fold ex vivo expansion of HPC. To assess HSC capability, defrosts from single collections were bead-separated into CD34(+) cells and infused into sublethally irradiated nonobese diabetic (NOD)severe combined immunodeficient (SCID) mice. CD45(+) human cell engraftment with multilineage phenotypes was detected in mice after 11-13 wk; engrafting levels were comparable to that reported with fresh CB. Thus, immature human CB cells with high proliferative, replating, ex vivo expansion and mouse NODSCID engrafting ability can be stored frozen for >15 yr, can be efficiently retrieved, and most likely remain effective for clinical transplantation.

Cryopreservation of fractionated, highly motile human spermatozoa: effect on membrane phosphatidylserine externalization and lipid peroxidation.

This study investigated lipid peroxidation (LPO) and membrane integrity following cryopreservation-thawing. Infertile men (study group) and donors (control group) were examined. Purified populations of highly motile spermatozoa were cryopreserved using TEST-yolk buffer and glycerol (TYB-G) followed by quick thaw. LPO was measured by a spectrophotometric assay, with and without a ferrous ion promoter. Annexin V binding was used to assess membrane translocation of phosphatidylserine (PS). Pre-freeze LPO was significantly higher in the study than in the control group (P = 0.03). In both groups, LPO measurements after thawing were significantly higher than the pre-freeze samples not exposed to TYB-G (P = 0.002 and P = 0.001 respectively). However, when the pre-freeze samples with TYB-G were compared with the post-thaw samples (all exposed to TYB-G), these differences were not significant. There was a significant increase in PS externalization following cryopreservation in both groups (P = 0.02 and P = 0.003 respectively). In donors, pre-freeze LPO concentrations had a significant positive correlation with thawed spermatozoa depicting PS externalization (r = 0.77, P = 0.04). Although patients had higher basal LPO than donors, LPO did not differ between fresh and cryopreserved-thawed fractionated motile spermatozoa. Freezing-thawing was associated with translocation of PS to the external membrane leaflet.

Cryopreservation-thawing of fractionated human spermatozoa and plasma membrane translocation of phosphatidylserine

Objective: [1] To evaluate sperm membrane damage during cryopreservation-thawing by the assessment of phosphatidylserine (PS) translocation and [2] to examine the relationship between reactive oxygen species (ROS) and cryopreservation-related alterations. Design: Prospective cohort study. Setting: University-based center. Patient(s): Men consulting for infertility and fertile donors (controls). Intervention(s): Semen processing was performed by density gradient separation followed by cryopreservation and thawing. Main Outcome Measure(s): Membrane PS translocation was evaluated with annexin V binding, generation of ROS was detected by chemiluminescence, and motion parameters were assessed by computer analysis. Result(s): Annexin V binding was detected in the prefreeze fractions with high and low sperm motility. In the patient group, there were significantly higher postthaw levels of annexin V binding in both fractions when compared with prefreezing values. However, such induction of PS translocation was significantly higher in the fractions with high sperm motility. Significantly higher ROS levels were detected in prefreeze samples of the fractions with low sperm motility. Conclusion(s): In the population of men studied, [1] cryopreservation-thawing was associated with induction of membrane PS translocation; [2] postthaw ROS levels were lower than before freezing; and [3] neither annexin V binding results nor the generation of ROS were able to accurately predict sperm cryosurvival rates.

Plasma Membrane Translocation of Phosphatidylserine and Determination of Reactive Oxygen Species: A Study of Their Association With the Cryopreservation-Thawing Survival of Fractionated Human Spermatozoa

Objectives: (1) To evaluate sub-lethal plasma membrane damage of sperm during cryopreservation-thawing by the assessment of phosphatidylserine (PS) translocation, (2) to examine the relationship between generation of reactive oxygen species (ROS) and cryopreservation-related membrane alterations, and (3) to study the value of membrane PS translocation for prediction of the ability to survive cryopreservation.Design: Prospective cohort study.Materials and Methods: Sub-fertile semen samples (n=10) were studied. Membrane PS translocation was evaluated with annexin V binding and generation of ROS was measured by chemiluminescence using luminol. Motility parameters were assessed by computer analysis. All measurements were performed in purified semen fractions with high and low sperm motility (Percoll density gradient separation) independently, before and after cryopreservation-thawing using TEST-Yolk buffer and glycerol.Results: Freezing-thawing induced significant levels of PS externalization in both sperm fractions. However, the induction of PS translocation in the fractions with high sperm motility was significantly higher than that of the fractions with low sperm motility. Significantly higher ROS levels were detected in pre-freeze samples of the fractions with low sperm motility compared to the fractions with superior motility. After freezing-thawing, ROS levels were significantly reduced in both fractions. There was a significant and negative correlation between annexin V staining before freezing and the ability to survive cryopreservation in the fractions with high sperm motility (r=−0.45, p=0.006). Furthermore, levels of annexin V staining <10% in a given fraction with high sperm motility predicted a >45% cryosurvival rate, with a sensitivity of 83% and a specificity of 80%.Conclusions: (1) Cryopreservation-thawing induced membrane PS translocation; (2) the effects of cryopreservation on sperm plasma membrane integrity were not associated with an increase in ROS generation as detected by chemiluminescence; and (3) the degree of annexin V binding before freezing may be used to predict the ability to survive cryopreservation-thawing. Objectives: (1) To evaluate sub-lethal plasma membrane damage of sperm during cryopreservation-thawing by the assessment of phosphatidylserine (PS) translocation, (2) to examine the relationship between generation of reactive oxygen species (ROS) and cryopreservation-related membrane alterations, and (3) to study the value of membrane PS translocation for prediction of the ability to survive cryopreservation. Design: Prospective cohort study. Materials and Methods: Sub-fertile semen samples (n=10) were studied. Membrane PS translocation was evaluated with annexin V binding and generation of ROS was measured by chemiluminescence using luminol. Motility parameters were assessed by computer analysis. All measurements were performed in purified semen fractions with high and low sperm motility (Percoll density gradient separation) independently, before and after cryopreservation-thawing using TEST-Yolk buffer and glycerol. Results: Freezing-thawing induced significant levels of PS externalization in both sperm fractions. However, the induction of PS translocation in the fractions with high sperm motility was significantly higher than that of the fractions with low sperm motility. Significantly higher ROS levels were detected in pre-freeze samples of the fractions with low sperm motility compared to the fractions with superior motility. After freezing-thawing, ROS levels were significantly reduced in both fractions. There was a significant and negative correlation between annexin V staining before freezing and the ability to survive cryopreservation in the fractions with high sperm motility (r=−0.45, p=0.006). Furthermore, levels of annexin V staining <10% in a given fraction with high sperm motility predicted a >45% cryosurvival rate, with a sensitivity of 83% and a specificity of 80%. Conclusions: (1) Cryopreservation-thawing induced membrane PS translocation; (2) the effects of cryopreservation on sperm plasma membrane integrity were not associated with an increase in ROS generation as detected by chemiluminescence; and (3) the degree of annexin V binding before freezing may be used to predict the ability to survive cryopreservation-thawing.

Effects of cryopreservation on bull sperm head morphometry.

Artificial insemination using cryopreserved semen is a common management tool of the contemporary livestock producer. However, cryopreservation is detrimental to sperm function and fertility, killing some 50% of the spermatozoa during the process. Prediction of cryopreservation damage from prefreeze samples remains elusive. Computer-automated sperm head morphometry was used in this study to determine the effects of cryopreservation on bovine sperm head morphometry. Semen was collected from 18 bulls and was divided. One portion was extended to 200 x 10(6) sperm/ml and a microscope slide was prepared, while the remaining portion was cryopreserved in a Triscitrate-yolk extender. After thawing, the cryopreserved samples were prepared on microscope slides. All slides were air dried and were stained with hematoxylin and rose bengal. The morphometric dimensions for length, width, width/length, area, and perimeter for a minimum of 200 sperm heads were analyzed from each slide by computer-aided sperm head morphometry analysis, and the mean measurements were recorded. Bull sperm heads were significantly (P < 0.01) smaller in cryopreserved spermatozoa than in the companion extended samples for length (8.56+/-0.07 vs. 8.63+/-0.08 microm), width (4.39+/-0.05 vs. 4.48+/-0.05 microm), area (28.42+/-0.07 vs. 29.14+/-0.08 microm), and perimeter (23.33+/-0.21 vs. 23.70+/-0.23 microm) for all bulls. Width/length was also different (0.513 vs. 0.519). In addition, differences (P < 0.05) were found within 14 of 18 bulls for at least four of the morphometric parameters. The percent change in measures after cryopreservation were correlated (P < or = 0.05) to the variability of the extended sample. Variations in sperm head measurements were lower (P < or = 0.05) in extended samples of the four bulls in which no changes occurred than in extended samples of the remaining 14 bulls. These data suggest that the variability in sperm head measurements of individual bulls, or ejaculates, may be an indicator of sperm cryosurvivability.