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[The comparison of the process of five different vitrification devices].

Over the last decade, several methods have been designed to improve the survival rate of vitrificated embryos. Although some teams have succeeded, the main remaining drawback of these methods is that they do not provide a leak proof environment for cryopreserved biological samples. To respond to that demand in respect with the European reglementation, the Cryo Bio System company (CBS) designed the HSV High Security Vitrification Kit (HSV). This system is composed of three distinct parts, a High Security thermal-autogenic sealed clear straw, a capillary with its extremity in form of a gutter, and an introducer that can be mounted on the manipulation rod before introduction into the straw. In this study, we confirmed that the CBS vitrification kit is a suitable method for vitrification in association with a small amount of cryoprotector enriched viscous media such as 25 microM Ficool 400, 750 mM Sucrose, 1% Bovine albumin, 20% Dimethyl Sulfoxide and 20% Ethylene glycol in a Phosphate buffered saline solution. We also evaluated the speed of the temperature decrease during vitrification in comparison with four other commercially available non-aseptic methods and showed the protective role of the CBS system during transfer. These physical data have now been confirmed biologically by P. Vanderzwalmen who obtained easily reproductible good results with human embryo using our method. Today, the HSV represents the unique aseptic alternative device (EC and FDA approved) for embryos, oocytes, and biological samples vitrification.

Cryopreservation↗

[Analysis of hemopoietic progenitor cells in bone marrow and peripheral blood samples which have undergone different periods of cryopreservation].

Bone marrow (BM) and peripheral blood stem cell (PBSC) samples from patients undergoing autologous transplant were tested to evaluate the effects of cryopreservation. Cell viability was assessed as well as the proliferative capability of CFU-GM and BFU-E (myeloid and erythroid progenitors respectively). Moreover, long term culture (LTC) of stromal cells was used to test their functionality. A total of 23 samples were studied: 5 from AML patients, 7 MM, 6 NHL, 3 ALL and 2 HL. Nine patients received autologous bone marrow transplant (ABMT) and the remaining 14 PBSC. The cells were frozen during 24 to 33 days before infusion and 16 to 40 months before culture. Forty percent of AML and MM samples gave rise to colonies in vitro while the other hematology diseases tested showed colony growth in almost 100% of the cases. Samples from patients with lymphoid diseases exhibited a good correlation between the percentage of CD34+ cells and the number of colonies developed in culture. Nevertheless, there was no correlation when ALL and MM were tested suggesting that the underlying disease may have affected the growth in culture. The stromal layer was fully developed on BM samples, but on PBSC samples it only generated macrophages and fibroblasts. Our results suggest that the efficacy of cryopreservation of hematopoietic cells can be measured by means of CFU-GM and BFU-E culture and that the period the samples remained frozen did not affect the growth capability of the cells.

Adult↗

Analysis of outcomes of cryopreserved surgically retrieved sperm for IVF/ICSI.

We evaluated our experience to date with in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) after either cryopreserved sperm or sperm produced on the date of IVF/ICSI was used. We performed a retrospective statistical analysis of data derived from 188 women undergoing IVF/ICSI cycles using surgically retrieved sperm. A total of 318 IVF/ICSI treatment cycles with 3280 ova were performed using testicular sperm extraction (TESE, 304 cycles) or microsurgical epididymal sperm aspiration (MESA, 14 cycles). Sperm obtained at time of IVF/ICSI (fresh) or thawed cryopreserved sperm samples were used in 38 and 280 of the ICSI cycles, respectively. For IVF/ICSI cycles using both TESE and MESA sperm, the fertilization rate was 59.9% for cryopreserved sperm, and 53.6% when fresh sperm was used (chi2 P-alpha < .02, Cramer's 0.04). The fertilization rate for the TESE group alone was 60.0% for cryopreserved sperm and 55.1% for fresh sperm (chi2P-alpha = .075). Cohen effect size was computed at 0.03; yielding for P-beta = .8, 6597 ova would be required to demonstrate similarity between fresh and cryopreserved sperm in the TESE group. To demonstrate superiority of cryopreserved sperm in this group at a P-alpha significance level of .05, 7524 ova would be necessary. The pregnancy rate for the TESE group was 27.3% for cryopreserved sperm and 27% for fresh sperm. Further analysis of the pregnancy data in this group, using the methods described, yielded a chi2 P-alpha and power of 0.971 (effect size calculated at 0.002). While our fertilization rates for cryopreserved sperm are greater in analyses of surgically derived sperm, based on the 7 years required to obtain data on 3280 ova, full numerical resolution of the issue of whether cryopreserved sperm is superior or similar will not be available until approximately 2010. However, we believe these results, along with the similarity shown in pregnancy rates achieved with both types of sperm, clearly indicate that cryopreserved sperm is not inferior to fresh sperm.

Cryopreservation↗

Microbial screening of UC blood units by an automated culture system: effect of delayed testing on bacterial detection.

BACKGROUND: Microbial screening is a mandatory test for banked UC blood (UCB) to comply with the code of good manufacturing practice (GMP). Cord blood banks (CBBs) are not always closely located to a GMP-licensed microbiology laboratory, resulting in time delays for transport of specimens prior to microbiological testing. This study investigated the influence of >/=24 h delays in initiating automated microbial screening on the detection of bacteria in UCB, by analysis of specimens deliberately spiked with bacteria and the recovery of bacteria from cryopreserved spiked-UCB. MATERIALS AND METHODS: UCB was processed according to standard CBB procedures and spiked with Staphylococcus epidermidis or Escherichia coli [2-2000 colony forming units (CFU)/mL]. Spiked-UCB (0.5 mL) was (1) held at room temperature (RT) and inoculated into pediatric BacT/Alert bottles (bioMérieux) at Days 1, 4 and 7 (delayed inoculation); and (2) inoculated directly (Day 0) into replicate BacT/Alert bottles and held at RT for 1, 4 or 7 days before loading onto the BacT/ALERT system (delayed loading). Spiked-UCB samples were cryopreserved. Bacterial counts were quantitated on horse blood agar plates. RESULTS: Bacterial growth in UCB spiked with a single bacterium was capable of detection by the BacT/ALERT system. S. epidermidis grew in all conditions of delayed testing (ie. delayed inoculation and delayed loading). E. coli failed to grow under conditions of delayed inoculation but grew at all time points of delayed loading. S. epidermidis and E. coli were recovered from cryopreserved spiked-UCB. DISCUSSION: Inoculation of culture bottles as soon as possible after sample preparation is preferable. Bacteria can maintain viability in BacT/ALERT bottles inoculated and held at RT for up to 7 days prior to automated culture testing. Bacteria can be successfully recovered from cryopreserved UCB.

Bacteria↗

Cryopreservation of spermatozoa from wild-born Namibian cheetahs (Acinonyx jubatus) and influence of glycerol on cryosurvival.

Sperm cryopreservation is a valuable tool for the genetic management of ex situ populations. This study was conducted to assess: (1) semen characteristics of wild-born cheetahs; and (2) the impact of three types of glycerol influence (duration of exposure, temperature, and method of addition) on sperm cryosensitivity. To evaluate the impact of duration of glycerol exposure, spermatozoa were incubated in Test Yolk Buffer (TYB) with 4% glycerol at ambient temperature (approximately 22 degrees C) for 15 vs. 60 min before cryopreservation. To evaluate the influence of temperature and method of glycerol addition, spermatozoa were resuspended at ambient temperature either in TYB with 0% glycerol followed by addition of 8% glycerol (1:1 v/v; at ambient temperature vs. 5 degrees C) or directly in TYB with 4% glycerol. All samples were cryopreserved in straws over liquid nitrogen vapor and evaluated for sperm motility and acrosomal integrity after thawing. Semen samples (n = 23; n = 13 males) contained a high proportion (78%) of pleiomorphic spermatozoa. Ejaculates also contained a high proportion of acrosome-intact (86%) and motile spermatozoa (78%). Immediately after thawing, a significant proportion of spermatozoa retained intact acrosomes (range, 48-67%) and motility (range, 40-49%). After thawing, incubation in glycerol for 60 min at ambient temperature before freezing decreased (p < 0.05) sperm motility and acrosomal integrity at one time-point each (pre-centrifugation and post-centrifugation, respectively). However, method or temperature of glycerol addition had no (p > 0.05) impact on sperm cryosurvival. In summary, (1) wild-born cheetahs produce high proportions of pleiomorphic spermatozoa but with a high proportion of intact acrosomes; and (2) resuspension in 4% glycerol, followed by exposure for up to 60 min at ambient temperature, had minimal effect on sperm motility and acrosomal integrity after cryopreservation. Results indicate the feasibility of cryopreserving cheetah spermatozoa under field conditions, providing a user-friendly method to capture and store gametes to enhance genetic management.

Acinonyx↗

Immunomagnetic removal of cryo-damaged human spermatozoa.

AIM: To estimate the dissipation of mitochondrial transmembrane potential (mTMP, DeltaPsim) and activation of sperm caspases (aCP) as signs of apoptosis in human spermatozoa during cryopreservation and to evaluate the efficiency of immunomagnetic cell separation (MACS) of these spermatozoa via annexin V-binding. METHODS: The mTMP and aCP in fresh and cryopreserved spermatozoa were detected by fluorescence microscopy and by Western blots. The sperm suspensions were divided into two sperm fractions (with intact and deteriorated membranes) by magnetic cell separation (MiniMACS, Miltenyi Biotec, Bergisch Gladbach, Germany) in dependence on their binding to superparamagnetic annexin V-microbeads (AN-MB). RESULTS: The cryopreservation decreased the portion of spermatozoa with intact mTMP from 80.1 % +/- 7.2 % to 53.5 % +/- 13.1 % and increased the spermatozoa with activated pan-caspases (aCP) from 21.8 % +/- 2.6 % to 47.7 % +/- 5.8 % (n=10; mean +/- SEM; P <0.01). The activation of caspases 1, 3, 8, and 9 in the cryopreserved spermatozoa was confirmed by Western blots (n=22). MACS reduced significantly the percentage of cryopreserved spermatozoa with dissipated mTMP to 8.1 +/- 3.9 (P <0.01) and also those with aCP to 9.3 % +/- 2.2 %. Western blot analyses confirmed the increase of the activated caspase3, 9, and 8 in the AN-MB-positive fraction (P <0.05) compared with the AN-MB-negative fraction. The MACS separation effect was confirmed by anti-annexin V-antibodies. There was no significant influence of the separation column and the magnetic field on the sperm functions. CONCLUSION: The cryopreservation impaired the mTMP and enhanced the activation status of caspases in human spermatozoa. The immunomagnetic sperm separation via binding of AN-MB could deplete low quality spermatozoa from cryopreserved semen samples.

Apoptosis↗

Effects of cryopreservation on the mononuclear cells of patients with lung cancer and normal controls.

Studies were carried out to determine the effects of cryopreservation on the mitogen-induced proliferative and immunoglobulin-producing abilities of peripheral blood mononuclear cells (MNC) of patients with lung cancer and normal controls. One-half of each sample of cells was tested fresh, while the other half was frozen, thawed immediately, and cultured at the same time. The responses of each sample of cryopreserved cells were compared to the responses of fresh cells from the same individual in simultaneous assays. The cryopreserved mononuclear cells of most of the lung cancer patients showed significantly enhanced plaque-forming cell (PFC) responses after stimulation with pokeweed mitogen (PWM). No such significant differences were observed between the proliferative responses of cryopreserved and fresh cells against phytohemagglutinin (PHA) or PWM stimulation. The cryopreserved MNC of some of the normal controls also showed a similar increase in the PFC responses, although to a lesser extent. Coculture of cryopreserved B cell-enriched populations of cells with fresh T cell-enriched fractions obtained from these patients also resulted in the generation of a higher number of PFCs as compared to the number of PFCs observed after coculture of fresh B and T cell-enriched populations. The results indicate that the suppressor activities of monocytes/macrophages and other non-T cells (NK cells) are sensitive to cryopreservation. The results also show that the MNC of patients with lung cancer can be cryopreserved and used for subsequent B and T cell assays.

Adult↗

Effects of bovine serum albumin on function of cryopreserved stallion spermatozoa during medium culture and uterine tube epithelial cell coculture.

OBJECTIVE: To compare function of cultured cryopreserved stallion spermatozoa in a modified Tyrode's medium (TM), with or without bovine serum albumin (BSA), or in uterine tube (oviduct) epithelial cell (OEC) coculture in TM, with or without BSA. SAMPLE POPULATION: Cryopreserved spermatozoa from 6 proven stallions and OEC from bovine reproductive tracts in follicular phase. PROCEDURE: Thawed spermatozoa were cultured in TM, with or without BSA, or cocultured with OEC monolayers in TM, with or without BSA. Percentages of capacitated and acrosome-reacted spermatozoa were measured at 5 hours for TM cultures. Spermatozoal survival and motility characteristics were observed over time for all culture methods. Number of spermatozoa attaching to OEC were compared for cocultures. RESULTS: Use of TM without BSA altered spermatozoal function in cell-free medium culture and OEC coculture. A higher percentage of spermatozoa were acrosome reacted in TM with BSA, although percentages of capacitated spermatozoa did not differ. Spermatozoa survived longer and maintained superior motion in TM culture without BSA and in OEC cocultures. More spermatozoa were able to attach to OEC in TM without BSA. CONCLUSIONS: Incubation of cryopreserved spermatozoa in media with BSA resulted in rapid decrease in percentage of intact, motile spermatozoa and limited their ability to interact with OEC. CLINICAL RELEVANCE: Current culture media used for assisted reproduction techniques in horses do not provide functionally capacitated spermatozoa. Removal of BSA from such media improves spermatozoal quality and survival.

Animals↗

Effect of cryoprotective additives and cryopreservation protocol on sperm membrane lipid peroxidation and recovery of motile human sperm.

Sperm membrane damage during cryopreservation reduces the recovery of motile sperm. The present study investigates changes in sperm motility and membrane lipid peroxidation (LPO) in response to two changes in the standard sperm cryopreservation/thawing methodology: 1) the addition of platelet-activating factor (PAF) and pentoxifylline (PTX) as cryoprotective additives, and 2) the alteration of sample thawing time. PAF (1 microM) and PTX (3 mM) were added to fresh sperm samples prior to cryopreservation. After 2 weeks the samples were thawed either quickly (5 minutes at 37 degrees C) or slowly (30 minutes at 4 degrees C) and evaluated for sperm motility and LPO. Thawing time influenced both post-thaw motility and LPO. Samples thawed quickly exhibited a 31% increase in motility recovery (35.2 +/- 4.3% in quick-thaw samples; 24.3 +/- 3.9% in slow-thaw samples) and a 23% lower LPO level (23.3 +/- 3.4% in quick-thaw samples; 30.09 +/- 4.4% in slow-thaw samples) compared to samples thawed slowly. Results also demonstrated that PAF (49 +/- 1.7%) or PTX (42.6 +/- 1.5%) enhance post-thaw motility in comparison to control (35.8 +/- 1.2%), whereas neither PAF nor PTX affect post-thaw LPO (19.1 +/- 2.2% in controls; 20.2 +/- 1.7% in PAF samples; 20.5 +/- 1.4% in PTX samples). These results support observations that there is a negative correlation between sperm motility and LPO in cryopreserved samples. The results also discount the hypothesis that LPO protection is a result of the cryoprotective action of PAF or PTX.

Cell Membrane↗

[Evaluation of the interest of semen culture before cryoconservation].

OBJECTIVES: French guidelines recommend screening all patients for virus infection prior to cryopreservation of their semen. In case of viral risk, the use of specific high secure CBS straws is recommended. The objective of this work was to evaluate the microbiological risk by testing all semen samples before cryopreservation. PATIENTS AND METHODS: Fifty one patients underwent a semen culture before cryopreservation. RESULTS: The fifty one patients were classed into 3 groups following semen culture results: group I: negative culture (39/51, 76.47%), group II: positive culture with microbiological contamination (7/51, 13.73%) and group III: positive culture with pathogen (5/51, 9.8%). For 3 patients of the latter group, we tested a three-layer density gradient to eliminate bacteria before Assisted Reproductive Techniques. DISCUSSION AND CONCLUSIONS: This paper discusses the risks related to microbiological contamination and the options available in this case.

Bacterial Infections↗

Autologous peripheral blood progenitor cells are a potential source of parvovirus B19 infection.

BACKGROUND: Parvovirus B19 is a cause of delayed red blood cell (RBC) engraftment after marrow transplantation (BMT). The diagnosis of parvovirus infection requires serologic and DNA testing in the context of clinical disease and characteristic marrow morphologic findings; however, the source of infection is often difficult to determine. STUDY DESIGN AND METHODS: Investigation of a case of delayed RBC engraftment and pure RBC aplasia (PRCA) occurring 3 months after autologous peripheral blood progenitor cell (PBPC) transplantation in a patient with high-risk diffuse large B-cell lymphoma. DNA testing of serum and of a sample of cryopreserved PBPCs was performed. RESULTS: Marrow morphology showed a maturational arrest of erythroid cells with giant proerythroblasts. Polymerase chain reaction and nucleic acid hybridization confirmed the presence of parvovirus DNA in the serum and in a sample of sequestered PBPCs saved at the time of PBPC harvest. PRCA resolved after the administration of intravenous immune globulin. CONCLUSION: Autologous PBPCs are a potential source of parvovirus infection, which may cause significant disease after autologous BMT.

DNA, Viral↗

[Requests for utilization of a semen bank among oncological patients. Semen cryopreservation prior to chemotherapy, radiotherapy and surgery].

OBJECTIVES: Oncological therapies, either surgery, radiotherapy or chemotherapy, may cause irreversible subfertiliy/infertility. Both chemotherapy and radiotherapy have cytotoxic effects on gametogenesis and there are not preventive alternatives currently available. The objective of this article is to review current criteria for semen cryopreservation and its usefulness as a method for preservation of fertility in patients with cancer. METHODS/RESULTS: We reviewed a large group of recent original articles and systematic reviews on the issue with a common feature: evaluation of fertility status after oncological therapy. CONCLUSIONS: Every male patient in fertile age who could wish future fatherhood should be offered the option to storage cryopreserved semen samples before starting oncological therapies, with the exception of patients with azoospermia at the time of diagnosis.

Cryopreservation↗

Vitrification and rapid freezing of rabbit fetal tissues and skin samples from rabbits and pigs.

Vitrification (3.58 M EG and 2.82 M DMSO in PBS with 20% FCS) and rapid-freezing (0.25 M sucrose, 2.25 M EG, and 2.25 M DMSO in PBS with 20% FCS) procedures were assayed to cryopreserve rabbit tissue samples from 12-day fetuses, and skin samples from live born pups and adult rabbits. These methods were also assayed to cryopreserve pig skin samples obtained from abattoir animals. The ability of rabbit tissue samples to attach and colonize the substratum by cell proliferation was not affected by the assayed cryopreservation procedures, regardless of specimen age. In porcines, sample attachment and cell proliferation capability of primary cultures were not affected by applied cryopreservation procedures. Almost all primary cultures from cryopreserved skin samples reached confluency (from 92 to 100%). Results reported here allow us to establish in both species, rabbit and pig, a cryobank of skin samples from adult specimens classified as outliers for longevity (in rabbits) and prolificacy (in pigs).

Animals↗

Low temperature-induced dimerization of the bovine sperm serine protease, BSp66.

BSp120 and BSp66 are trypsin-like serine proteases from bovine spermatozoa. The former is active in cryopreserved sperm samples while the latter shows proteolytic activity in recently obtained fresh sperm. Both proteases are immunologically related and co-localize in the apical portion of the sperm head. In Western blots with specific antibodies, sperm samples incubated with reducing agents showed a decrease in the amount of BSp120, while BSp66 was detected with both anti-BSp120 and anti-BSp66 antibodies. BSp120 was evident in frozen intact spermatozoa after 60 days of semen cryopreservation and the kinetic of appearance of this protein was coincident with the decrease in the amount of BSp66. Identical results were obtained by freezing sperm extracts from fresh semen at -20 degrees C. Our results suggest that BSp120 results from disulfide bond-dimerization of BSp66 and that this process may be induced by temperatures below zero in both intact spermatozoa and in sperm extracts.

Animals↗

Cryopreservation of sperm from adolescents and adults with malignancies.

Although cryopreservation of sperm is performed routinely in adults, only a small amount of information is available on its feasibility in adolescent patients with malignancies. Of 936 patients who were candidates for sperm cryopreservation, 851 (111 adolescents and 740 adults) were eligible for this retrospective analysis after excluding patients with relapses of the original or secondary cancers, known bitesticular lesions, or an unknown diagnosis. In general, patients were seen before initiation of treatment for malignancies. However, unilateral ablation of the testis was performed in 61% of patients with testicular cancer before cryopreservation of samples. Patients were grouped according to primary diagnosis and age. Measurements included testicular volume, semen analysis, and serum hormones (luteinizing hormone [LH], follicle-stimulating hormone [FSH], and testosterone). The youngest patient with an ejaculate containing sperm was 13.5 years old. No significant differences in any investigated parameter could be detected for any diagnosis among the 111 adolescents (age, <20 years). In contrast, adult patients with testicular cancer showed higher FSH values and lower sperm concentrations than adult patients with lymphomas, leukemias, and bone cancers. Patients younger than 16 years had lower ejaculate volumes than men older than 25 years, and testosterone levels were higher in patients aged 20-29 years than in the youngest patient group. Cryopreservation of sperm can be performed in adolescent patients with overall success rates (defined as the observation of at least a single motile sperm after the thawing procedure) similar to those observed in adults and should be recommended even to oncological patients younger than 15 years, provided that these patients can produce a semen sample.

Adolescent↗

Importance of explant size and origin and of preconditioning treatments for cryopreservation of garlic shoot apices by vitrification.

This paper investigates the effect of the origin and size of the explants employed and of the preconditioning (cold acclimation, preculture) and loading treatments on survival and regeneration of cryopreserved garlic shoot apices using vitrification with the PVS3 vitrification solution. Both the origin and size of explants had a significant effect on regeneration of cryopreserved apices. Higher regeneration was generally observed with apices excised from bulbs and bulbils, followed by cloves, and those originated from larger propagules regrew more rapidly. Smaller apices (1.5 or 3.0 mm in diameter) displayed higher regeneration than large ones (4.5 mm in diameter). Cold acclimation at 5 degree C of apices before freezing had no positive effect on regeneration after cryopreservation. Preculture of apices at 10 or 23 degree C for more than 3 days had a detrimental effect on regeneration. The optimal sucrose concentration in the preculture medium was 0.3-0.5 M. Loading apices for 30 or 60 min at 23 degree C in medium containing 2 M glycerol + 0.4 M sucrose or 1 M glycerol + 0.8 M sucrose had no effect on regeneration after cryopreservation, in comparison with apices cryopreserved without loading treatment. Under optimal conditions, regeneration of cryopreserved apices sampled from large cloves was above 90 percent.

Acclimatization↗

Pregnancy following gamete intra-fallopian transfer (GIFT) with cryopreserved semen from infertile men following therapy to lymphomas or testicular tumour: report of three cases.

Cryopreserved spermatozoa from men having treatment for lymphoma or testicular tumour have been reported to function sub-optimally. We report on three successful pregnancies following gamete intra-Fallopian transfer (GIFT) using such cryopreserved sperm samples. In one case, ovum donation was required since the woman had premature ovarian failure.

Adult↗

The effects of follicular fluid and platelet-activating factor on motion characteristics of poor-quality cryopreserved human sperm.

PURPOSE: To evaluate the effects of follicular fluid and platelet-activating factor on sperm motion characteristics of cryopreserved oligospermic and normospermic samples. METHODS: Sperm motion characteristics were evaluated prior to cryopreservation, immediately after thawing and following incubation in human tubal fluid, follicular fluid, or 1-microM platelet-activating factor cultures. Sixteen oligospermic samples and 20 normospermic samples were examined. Sperm motion characteristics were analyzed manually according to WHO criteria (1999) and also with an automated videomicrography system. RESULT(S): Incubation in follicular fluid increased overall motility and the percentage of sperm with fast progressive motility in normospermic but not oligospermic samples. Incubation with platelet-activating factor increased overall motility and the percentage of sperm showing nonprogressive motility in both oligospermic and normospermic samples. CONCLUSION(S): The stimulatory effects of culture in follicular fluid as seen in normospermic samples do not show a significant benefit in oligospermic cryopreserved samples. Platelet-activating factor and follicular fluid increase motility via different mechanisms. Incubation of oligospermic cryopreserved sperm with PAF increases the number of motile sperm, thereby enabling easier identification of viable sperm for intracytoplasmic sperm injection in samples with severe asthenozoospermia.

Cryopreservation↗