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PubMed2006

Comparison of protocols for cryopreservation of rhesus monkey spermatozoa by post-thaw motility recovery and hyperactivation.

Cryopreservation of spermatozoa is useful for gene banking and for in vitro fertilisation (IVF). This study compared several published cryopreservation techniques to find the most efficient for rhesus macaques. Effectiveness was assessed by sperm longevity (post-thaw motility % and duration) and ability to hyperactivate in response to chemical activators (caffeine, dibutyryl cyclic AMP). Each ejaculate from three males was treated with four published cryopreservation protocols (Seier et al. 1993; Sanchez-Partida et al. 2000; Si et al. 2000; Isachenko et al. 2005). Upon thawing, each sub-sample was incubated either at 37 degrees C in 5% CO2 in air with or without activators or at approximately 22 degrees C in atmospheric air without activators for 0-24 h. Samples cryopreserved using one method showed zero motility and were not included in the 2 ;2 G-test statistical analysis. The other methods all demonstrated good immediate post-thaw motility rates (68%, 73% and 62% respectively) and underwent capacitation after exposure to activators. Sperm motility in each treatment decreased over time at both temperatures but overall, incubation at 22 degrees C preserved motility better in all three methods. In summary, cryopreservation of rhesus spermatozoa using the method published by Sanchez-Partida et al. or Seier et al. appeared best, potentially supporting gene banking as well as allowing for multiple IVF uses from the same sample.

Animals↗
PubMed1993

Effects of genotype and cryopreservation of avian semen on fertility and number of perivitelline spermatozoa.

1. The fertility of freshly diluted and cryopreserved samples of semen obtained from a population of chickens selected for duration of fertility of cryopreserved spermatozoa (FS line) and its unselected control (FC line) were compared over a range of spermatozoa concentrations (10, 40, 80, and 160 x 10(6) sperm/50 microliters insemination). 2. The spermatozoa of the FS line had greater fertility than spermatozoa of the FC line, whether freshly diluted or cryopreserved. Cryopreservation resulted in a reduction in fertility, regardless of line. There were no significant line by genotype interactions. 3. There were fewer spermatozoa from the FC line than the FS line found in the perivitelline membrane (perivitelline spermatozoa). The increase in number of perivitelline spermatozoa with increasing sperm concentration was greater in the FS than FC line. However, the slope of the increase in sperm number in the perivitelline membrane with increasing concentrations of cryopreserved spermatozoa was zero. 4. A minimum of 10(3) perivitelline spermatozoa must be found on day 2 post-insemination for duration of fertility to exceed three days. The ability to produce spermatozoa capable of reaching the forming perivitelline membrane appears to be a quantitative, rather than a qualitative, trait and may be subject to genetic manipulation.

Animals↗
PubMed2004

Bacterial risk and sperm cryopreservation.

Prior to sperm cryopreservation, French guidelines only recommend viral screening for serological status towards human immunodeficiency virus, hepatitis B and C viruses and Treponema palidum. The probability of semen infection by other bacterial pathogens is not taken into consideration by the current recommendations. The objective of the present study was to evaluate this risk and a strategy to reduce it prospectively. Ninety-six patients consulting for sperm cryopreservation underwent a semen culture simultaneously to cryopreservation. The patients were classified into three groups following semen culture results: negative culture (group 1, 77/96, 80.2%), positive culture with saprophytic agents (group 2, 9/96, 9.4%) and positive culture with pathogen agents (group 3, 10/96, 10.4%). For six patients of the latter group showing a genital infection with Ureaplasma urealyticum, a discontinuous gradient selection performed on the cryopreserved sample was efficient to discard bacteria. These data emphasize the usefulness to cultivate semen simultaneously to cryopreservation and demonstrate the ability to remove some microbial agents from semen before its use in assisted reproductive techniques.

Anti-Bacterial Agents↗
PubMed1996

Sperm quality improvement in cryopreserved human semen.

PURPOSE: We determined if separation of spermatozoa (washed) on a discontinuous colloidal suspension of silica (Percoll) density gradient before cryopreservation improves post-thaw motility compared to an unprocessed (raw) cryopreserved sample. MATERIALS AND METHODS: Ten normal healthy volunteers recruited into the andrology laboratory donor program were studied. Raw and washed cryopreserved spermatozoa were compared for loss of motility with time, motion characteristics, viability and membrane integrity after incubation for 1, 6 and 24 hours. Within group comparisons were made to baseline measurements (0 hours before incubation). RESULTS: Raw and washed cryopreserved spermatozoa showed statistically significant decreases in motility and other motion characteristics after thawing. There were significant decreases in motility and other motion characteristics after incubation periods of 1, 6 and 24 hours, and significant decreases in viability and membrane integrity at 6 and 24 hours in the unprocessed spermatozoa. Although, motility and motion characteristics of washed samples decreased significantly with longer incubation periods, loss of motility with time (longevity) was greater in raw samples. Washed samples retained greater sperm motility for up to 24 hours (p < 0.03). CONCLUSIONS: Specimens prepared by Percoll separation techniques before freezing offer the possibility of selecting spermatozoa that retain motility for up to 24 hours. This finding can be of benefit for couples undergoing intrauterine insemination to achieve pregnancy.

Cell Survival↗
PubMed1989

Human fetal liver as a valuable source of haemopoietic stem cells for allogeneic bone marrow transplantation.

The CFU-GM and T cell contents of human fetal livers were studied at various times between 6-14 weeks of gestation. The number of CFU-GM increased parallel to gestational age, especially after week 10. Cells bearing mature T cell markers, however, were found only in one case out of 35 fetal liver samples. Cryopreservation of fetal liver cells hardly affected the viability and proliferative capacity of CFU-GM in the sample. According to these findings fetal liver is, at least up to the 14th gestational week, practically free of mature T cells but it does contain a considerable amount of CFU-GM (an accepted indicator of pluripotent stem cell content), consequently fetal liver can be considered as a valuable source of haemopoietic stem cells for allogeneic bone marrow transplantation for children.

Antigens, Differentiation, T-Lymphocyte↗
PubMed2000

The effects of prolonged cryopreservation on the biomechanical properties of bone allografts: a microbiological, histological and mechanical study.

Bone allografting is the most common form of allotransplantation in modern medicine. Bone banking is usually the major part of most tissue banks throughout the world. Several years ago, many standards of bone banking were set empirically, and have never been evaluated. One particular parameter or standard was outdating graft materials after 5 years of storage. This study was conducted to evaluate the effect of prolonged cryopreservation on the biomechanical properties of bone allografts and establish whether graft materials become contaminated during long-term storage.Proximal humeral bone allografts were obtained from the bone bank after 1, 3 and 5 years of -80 degrees C cryopreservation. Samples of each humeral head, i.e., cartilage, subchondral bone and spongy bone were histologically examined for inter- and intra-cellular changes. A three-point mechanical bending test was used on identical pieces of cortical bone to compare fresh and cryopreserved materials. Fresh-retrieved cortical bone using identically-sized segments, served as a control. Cultures were taken from each respective sample to determine contamination or sterility.Results of both the histological and mechanical testing showed that there were no significant, qualitative histological, or quantitative mechanical differences among the samples. All the cultures were negative. Therefore, based on this study's parameters, bone allografts can safely be used after a cryopreservation period of over 5 years and should not be discarded.

Journal Article↗
PubMed1996

Comparison of seminal quality in Holstein bulls as yearlings and as mature sires.

Semen quality was compared in 5 Holstein bulls from samples collected as young sires (yearlings) and again as mature bulls after a mean interval of 1,265 d. At both sampling periods, the semen was examined for ejaculate volume, sperm numbers, post-thaw progressive motility and sperm viability. Sperm viability was assessed on cryopreserved samples with fluorescent SYBR-14 to stain living spermatozoa and propidium iodide (PI) to identify dead spermatozoa. The fluorescent populations of stained spermatozoa were quantified by flow cytometry. The percentages of living spermatozoa for the individual bulls, as determined by green fluorescence of SYBR-14, ranged from 44 +/- 3.1 to 54 +/- 0.3 for yearlings, and from 38 +/- 1.5 to 55 +/- 1.0 for mature sires. No differences in sperm viability were found between samples taken from yearling bulls and those of mature bulls. The percentage of spermatozoa stained with SYBR-14 was negatively correlated (r = -0.97; P = 0.0001) with the percentage of dead spermatozoa as indicated by PI staining. Comparisons of identical samples run on 2 different flow cytometers indicated that either flow instrument could be used to assess sperm viability. Although the individual bulls differed (P < 0.05) in ejaculate volume and sperm numbers as yearlings, they did not differ in these parameters as mature bulls. The average number of spermatozoa per ejaculate changed as a result of maturation, increasing from 6.2 +/- 1.0 to 10.7 +/- 1.1 x 10(9). Aging was significantly correlated with ejaculate volume (r = 0.76; P = 0.01) but not with the total number of spermatozoa per ejaculate (r = 0.51; P = 0.13). The maturational changes that occurred in the 5 bulls were minimal with the exception of the increased volume of the ejaculate and the number of spermatozoa per ejaculate.

Journal Article↗
PubMed2004

[Assessment on effect of short-term cryopreservation of cord blood hematopoietic cells].

To study the effects of short-term cryopreservation of cord blood hematopoietic cells in liquid nitrogen, the viability and function of cord blood hematopoietic cells were investigated by using each of 8 samples cryopreserved for six months, one and two years after thawing respectively. Nucleated cells (NC) were detected by blood cell analyzer. CD34+ cells were analyzed by flow cytometry, CFU-GM were cultured and detected in vitro, the survival rate was determined by trypan blue staining. The results showed that the differences of recovery rate of NC, CD34+, CFU-GM were nonsignificant at three different cryopreserved times. In conclusion, the short-term storage in liquid nitrogen showed a good effect on cord blood hematopoietic cell without any significant change of activities and number of the cryopreserved hematopoietic cells.

Cell Survival↗
PubMed2003

CD4+ and CD8+ cells in cryopreserved human PBMC maintain full functionality in cytokine ELISPOT assays.

The frequency and the cytokine signature of antigen-specific T cells in the blood reflect the magnitude and the quality of T cell immunity in vivo. Recently, cytokine enzyme-linked immunospot (ELISPOT) assays performed on freshly isolated peripheral blood mononuclear cells (PBMC) emerged as a promising tool for monitoring these key parameters, providing direct feedback information on the efficacy of vaccinations and immune therapies. However, performing ELISPOT assays with freshly isolated cells is not readily feasible in the context of clinical trials. The ability to obtain valid ELISPOT data on cryopreserved samples would greatly enhance ex vivo immune monitoring capabilities. We have therefore systematically studied antigen-specific T cell responses in freshly isolated PBMC and after cryopreservation. Four healthy donors were selected that displayed T cell responses to six recall antigens. The antigen reactive T cells were defined as CD4 or CD8 cells, and their cytokine effector class was established measuring interferon (IFN)-gamma, interleukin (IL)-2, IL-4 and IL-5. The donors were bled at three different time points, and their PBMC were tested fresh and after freeze-thawing. The results showed that the frequencies and type 1/type 2 cytokine signatures of recall antigen-specific CD4 and CD8 cells are unaffected after cryopreservation. In contrast to these data obtained on human PBMC, cryopreservation of murine spleen cells causes a decrease in cytokine secretion.

Animals↗
PubMed2006

Cryopreservation and sperm DNA integrity.

Cryopreservation of sperm is an extremely important issue in the field of male infertility as freezing can have detrimental effects on a variety of sperm functions, some of them not accessible to the traditional semen quality analysis. In this study, chromatin structure variations in human spermatozoa in semen were studied with the sperm chromatin structure assay (SCSA), both before and after cryopreservation. Samples were divided into two aliquots: the first was analysed without further treatment, while the second was stored in liquid nitrogen at -196 degrees C using standard cryopreservation techniques. The fresh and thawed aliquots were also assessed by light and fluorescence microscopy (after Acridine Orange staining, AO), and computer-assisted semen analysis (CASA) of motility. Overall sperm quality was found to deteriorate after cryopreservation. When thawed spermatozoa were subjected to an extra swim-up round, a general improvement in nuclear maturity was seen in post-rise spermatozoa.

Acridine Orange↗
PubMedDate not supplied

Evidence that membrane stress contributes more than lipid peroxidation to sublethal cryodamage in cryopreserved human sperm: glycerol and other polyols as sole cryoprotectant.

One effect of cryopreservation on human sperm is sublethal cryodamage, in which cell viability post-thaw is lost more rapidly at later times than in fresh cells. We hypothesized two modes of sublethal cryodamage: one is peroxidation-related involving plasma membrane damage due to lipid peroxidation; the other is membrane stress-related involving membrane embrittlement during phase transitions occurring during freeze-thaw. If the peroxidation-related mode contributed substantially to sublethal cryodamage, the hypothesis predicts that lipid peroxidation inhibitors should reduce this damage. To test this prediction, we examined the effect of the lipid peroxidation inhibitors, hypotaurine, bovine serum albumin (BSA), and alpha-tocopherol (Vit. E) on the time to loss of motility (TLM), taken as a measure of cell viability over time, for sperm samples cryopreserved in glycerol plus egg yolk medium. These agents had no effect on TLM of these samples, indicating that this mode contributes little to sublethal cryodamage. If the membrane stress-related mode contributed, the hypothesis predicts rapid recovery of motility in the presence of egg yolk plus glycerol, but slow recovery in the presence of glycerol alone. It also predicts that an appropriate polyol may be both necessary and sufficient for cryopreservation. In the presence of egg yolk plus glycerol, motility recovery was complete within 5 minutes, but the percent motile cells then decreased linearly with time. With glycerol alone in the range 3-12%, at 5 minutes post-thaw the percent motile cells was 5-10%, but by 40 minutes post-thaw had risen to 60-80%, approaching that in the fresh sample, and was maintained up to 4 hours. In the absence of glycerol, the percentage of motile cells post-thaw was nil and remained nil up to 4 hours. The polyols, erythritol, ribitol, and sorbitol had similar effects to that of glycerol, but the recovery of motility was not as complete. These results indicate that the membrane stress-related mode contributes substantially to sublethal cryodamage. They also indicate that glycerol and other polyols can function alone as cryoprotectants, but that recovery of motility is slow in these systems.

Adult↗
PubMed1997

[Pregnancy obtained by intracytoplasmic oocyte injection of spermatozoa retrieved from a cryopreserved testicular biopsy].

We have obtained a clinical pregnancy in a 26 year old patient by means of in vitro oocyte fertilization by intracytoplasmic injection of spermatozoa obtained from a cryopreserved testicular biopsy. In a first attempt performed with fresh biopsy material, the woman became pregnant, but the pregnancy ended in a spontaneous abortion. In a second cycle, the spermatozoa were retrieved from a cryopreserved sample saved from the first attempt. Twelve metaphase II oocytes were collected and injected; from these, nine became fertilized, three preembryos were transferred at the eighth-cell stage and the other six were cryopreserved. An ongoing clinical pregnancy was obtained with two gestational sacs.

Adult↗
PubMed2003

Trehalose ameliorates the cryopreservation of cord blood in a preclinical system and increases the recovery of CFUs, long-term culture-initiating cells, and nonobese diabetic-SCID repopulating cells.

BACKGROUND: The cryopreservation of HPCs in DMSO has been practiced by cord blood (CB) banks worldwide. Inevitably, some detriment to biologic function occurs as the result of freezing injuries and DMSO toxicity. Trehalose, a disaccharide, is a natural cryoprotectant in organisms capable of surviving extreme dehydration and cold. The objective of this study was to establish the cryopreservation of CB under preclinical conditions using trehalose as a supplement to DMSO. STUDY DESIGN AND METHODS: In a preclinical protocol, the effects of 5-percent trehalose with 10-percent DMSO or 5-percent DMSO on the cryopreservation of CB MNCs or nucleated cells (NCs) were further evaluated. The read-out system consisted of a panel of HPCs: early progenitors (CFU-GEMM, long-term culture-initiating cells [LTC-IC]) and committed progenitors (CFU-GM, CFU/BFU-E, CFU-megakaryocyte [CFU-MK]). The homing and engraftment capacity of these cells were assessed in nonobese diabetic (NOD)-SCID mice. RESULTS: Trehalose increased the recoveries of CFU-GM, CFU/BFU-E, CFU-GEMM, and LTC-IC by over 7.25 percent (mean), 11.9 percent, 19.2 percent, and 12.9 percent, respectively, when compared with those in paired CB samples cryopreserved in 10-percent DMSO. Freezing and thawing reduced the yields of CFU-MK by 35.5 percent (mean) and 28.4 percent in MNC and NC samples, respectively, and the inclusion of 5-percent trehalose significantly retrieved these progenitor cells to over 90 percent of fresh samples. The improved recovery of functional HPLs was reflected by their multilineage engraftment in NOD-SCID mice. CONCLUSION: Trehalose at 5 percent significantly ameliorates the cryopreservation of CB progenitor cells at a preclinical protocol. The increased recoveries of these cells might potentially improve the engraftment outcomes of CB transplants.

Animals↗
PubMed2004

Alternative cerebrospinal fluid tests to diagnose neurosyphilis in HIV-infected individuals.

OBJECTIVE: To identify alternatives to the CSF-Venereal Disease Research Laboratory (VDRL) test for the diagnosis of neurosyphilis in HIV-infected individuals. METHODS: CSF fluorescent treponemal antibody (FTA) reactivity and % CSF lymphocytes that were B cells in fresh and frozen samples were determined for 47 HIV-infected cases with syphilis and 26 HIV-infected controls. As for serum, CSF fluorescent treponemal antibody reactivity > or =2+ was considered positive. Based on the results in controls and cases with normal CSF measures, cut-offs for elevated CSF B cells were proposed to be > or =9% in fresh and > or =20% in frozen samples. Neurosyphilis was defined as a reactive CSF-VDRL. RESULTS: CSF-FTA-ABS (absorbed) and CSF-FTA (unabsorbed and undiluted) were 100% sensitive for the diagnosis of neurosyphilis. Elevated % CSF B cells in fresh and cryopreserved samples was specific (100%) but not sensitive (40 and 43%) in post hoc analyses. The results of CSF-FTA and assessment of % CSF B cells together allowed 16% of cases with pleocytosis but nonreactive CSF-VDRL to be diagnosed with neurosyphilis and 26% to be diagnosed as not having neurosyphilis. CONCLUSION: When the CSF-VDRL is nonreactive, CSF-FTA and % CSF B cells may help exclude or establish the diagnosis of neurosyphilis.

Absorption↗
PubMed2000

Cryodamage to plasma membrane integrity in head and tail regions of human sperm.

AIM: To investigate the effect of cryopreservation on the plasma membrane integrity in the head and tail regions of individual sperm, and the relationship between intact cryopreserved sperm and its motility and zona-free hamster oocyte penetration rate. METHODS: The eosin Y exclusion and the hypoosmotic swelling tests were combined to form a single test (HOS-EY test) to identify the spermatozoa with four types of membrane integrity. RESULTS: After cryopreservation, there was a marked decline in the percentage of spermatozoa with Type IV membrane integrity (head membrane intact/tail membrane intact), and a significant increase in those with Type I (head membrane damaged/tail membrane damaged) and Type III (head membrane damaged/tail membrane intact) membrane integrity (n = 50, P < 0.01). The value of Type II integrity had a wide range of variability, whereas Type II (head membrane intact/tail membrane damaged) was uncommon after thawing. A high correlation was observed between the percentage of Type IV integrity and sperm motility ( n = 50, r = 0.74, P < 0.01). However, the values of Type IV integrity were usually lower than those of post-thaw motility in most cryopreserved samples. The value of Type IV integrity did not correlate with the sperm penetration rate (n = 25, r = 0.22, P > 0.05). CONCLUSION: (1) The HOS-EY test has the advantage of showing four patterns of membrane integrity in individual spermatozoon; (2) Cryopreservation causes a significant membrane rupture in the head and tail regions of spermatozoa; Type III is the main transitional state of membrane cryodamage; (3) Cryodamage to head and tail membrane may occur independently; the presence of an intact tail membrane does not necessarily indicate the intactness of head membrane. (4) Intact membranes are closely related to post-thaw motility, but do not reflect the fertilizing potential.

Adult↗
PubMed1996

Suitability of the hypo-osmotic swelling test for assessing the viability of cryopreserved sperm.

OBJECTIVE: To determine whether the hypo-osmotic swelling test can assess viability of frozen spermatozoa. DESIGN: Fresh semen specimens were divided into two aliquots: one was not processed and the other was processed by swim-up. Both aliquots were then cryopreserved by the liquid nitrogen vapor method. SETTING: Andrology laboratory at a tertiary care facility. PATIENT(S): Eleven volunteers with proven fertility. INTERVENTION(S): The hypo-osmotic swelling test and the sperm viability assessment by Hoechst 33258 (Sigma Chemical Co., St. Louis, MO) and eosin-nigrosin strains were performed in each aliquot. MAIN OUTCOME MEASURE(S): The results of hypo-osmotic swelling test as indicated by swelling of sperm tails were compared with the viability results obtained by Hoechst 33258 stain before freezing and by Hoechst-33258 and eosin-nigrosin stains after freezing. RESULT(S): The hypo-osmotic swelling test and Hoechst 33258 viability results were correlated highly in unprocessed and processed fresh specimens (r = 0.95). After cryopreservation, there was no correlation between hypo-osmotic swelling test and Hoechst 33258 or between hypo-osmotic swelling test and eosin-nigrosin. Prefreeze sperm processing did not influence the post-thaw results. Viability assessed by Hoechst 33258 and eosin-nigrosin was well correlated (r = 0.72). CONCLUSION(S): The hypo-osmotic swelling test can accurately evaluate viability in fresh human spermatozoa but not in cryopreserved ones. Thus, this test cannot be used to select viable cells in cryopreserved samples for assisted reproductive procedures, such as intracytoplasmic sperm injection.

Aniline Compounds↗
PubMedDate not supplied

Chondrocyte recovery in cryopreserved porcine articular cartilage after bone carrier alteration.

In order to investigate the consequences on the distribution of cell recovery through a cross-section of articular cartilage, the pathway for ice nucleation and diffusion of water and solutes in porcine osteochondral tissue was altered by drilling a 2mm diameter hole through the subchondral bone to the base of the cartilage. Samples equilibrated with 1M dimethyl sulfoxide were cooled at 1 C/min to -30 degrees C then stored in liquid nitrogen. A significant increase in chondrocyte recovery was documented when compared to samples cryopreserved without holes (48.3 percent vs 28.6 percent, P=0.003). The most significant change due to bone base modification was an increase in recovery in the middle section of the cartilage. These results provide insight into mechanisms of cryoinjury in tissue systems.

Animals↗
PubMed1994

Effects of cryopreservation on the intracellular calcium concentration of human spermatozoa and its response to progesterone.

The intracellular free calcium concentration [Ca2+]i of sperm from 23 ejaculates was measured before and after cryopreservation using the fluorescent probe Fura-2. Spermatozoa were treated with 3.18 microM progesterone so that the regulation of [Ca2+]i in a dynamic situation could be studied. [Ca2+]i (nM) was 290 +/- 13 in fresh spermatozoa vs. 550 +/- 26 in cryopreserved samples (mean +/- S.E.M. P < 0.0001 paired t-test). Progesterone at a dose of 3.18 microM stimulated a large and rapid increase in [Ca2+]i to a peak value > 1 microM after 10-20 seconds. [Ca2+]i then declined to a slightly raised basal level over the next 30-40 seconds. This phenomenon occurred in all the fresh samples, but about half the frozen thawed samples failed to respond. The peak [Ca2+] attained by frozen samples which did respond after the addition of progesterone was similar to that observed with fresh sperm. The calcium channel blocker verapamil (200 microM) completely inhibited the transient rise in [Ca2+]i produced by progesterone, but 100 microM verapamil had only a partial effect. We conclude that (1) cryopreservation causes a substantial elevation of the [Ca2+]i in human spermatozoa and (2) damage to the plasma membrane during cryopreservation may result in the loss of the progesterone receptor. Both factors may contribute to the loss of fertility after cryopreservation.

Calcium↗