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PubMed2005

In vitro fertilization-intracytoplasmic sperm injection success rates with cryopreserved sperm from patients with malignant disease.

OBJECTIVE: To describe the success rate of intracytoplasmic sperm injection (ICSI) using thawed cryopreserved sperm in male cancer patients. DESIGN: Retrospective consecutive study. SETTING: IVF unit in a tertiary university hospital. PATIENT(S): Twenty-one couples treated by ICSI using the frozen-thawed sperm of husbands treated for cancer. INTERVENTION(S): Follow-up of all treatments by ICSI. MAIN OUTCOME MEASURE(S): Pregnancy rate, sperm count, and fertilization rate. RESULT(S): Male patients being treated for cancer froze between one and 29 sperm samples. Post-thaw sperm count ranged from 1 x 10(5) to 106 x 10(6). The mean fertilization rate by ICSI was 60% (range, 33%-100%), and the pregnancy rate was 42%. Twenty-six pregnancies were obtained resulting in 23 children (13 singleton and 10 twins) and eight spontaneous abortions (31%). Delivery was obtained in 12 of the 21 treated couples (57%). The lowest total motile sperm count that resulted in a pregnancy was 1 x 10(5). CONCLUSION(S): Male cancer patients should be encouraged to freeze numerous sperm samples even when sperm count and motility are poor. In these cases, ICSI is a powerful technique compared with intrauterine injection since thawed sperm samples with poor parameters can produce relatively high fertilization rates resulting in normal pregnancies and deliveries. The possibility to repeat treatments even in the face of a limited number of sperm samples appears to be of importance.

Adult↗
PubMed2006

Dilution of spermatozoa results in improved viability following a 24 h storage period but decreased acrosome integrity following cryopreservation.

This study was designed to investigate the physiological factors affecting the reduced viability of cryopreserved spermatozoa following dilution. Ninety-six ejaculates were collected from 13 bulls and diluted to 10 x 10(6) and 60 x 10(6) sperm/ml in a commercial long term extender (Eqcellsire; IMV) and in an egg yolk extender. Samples diluted in the egg yolk extender were frozen in 0.25 ml straws. Samples diluted in the Eqcellsire were stored at room temperature for 24 h and assessed for sperm cell viability using SYBR14 and PI (Molecular Probes) and osmotic resistance. Frozen samples were thawed and assessed for viability, osmotic resistance and acrosome intergrity. Acrosome integrity was measured using Mitotracker, PI and PNA-FITC (Molecular Probes). Spermatozoa diluted to 10 x 10(6) sperm/ml and stored at ambient temperature had a higher proportion of viable spermatozoa (P < 0.01) and were less susceptible to osmotic stress (P < 0.01) than sperm diluted to 60 x 10(6) sperm/ml. Following cryopreservation there was no concentration-related difference in the proportion of viable spermatozoa and their relative susceptibility to osmotic stress. Spermatozoa diluted to lower cell concentrations had a higher proportion of viable cells that were acrosome reacted (P < 0.001). It is suggested that the higher proportion of acrosome reacted cells may result from an increased proportion of cells in a capacitated-like state in the spermatozoa diluted to lower concentrations. A Spearmans ranked correlation demonstrates a relationship between individual bull spermatozoa following dilution or cryopreservation for viability (r2 = 0.98; P < 0.001) or osmotic resistance (r2 = 0.87; P < 0.001) suggesting a variation in these characteristics between bulls.

Acrosome↗
PubMed1993

Comparative cryopreservation and capacitation of spermatozoa from epididymides and vasa deferentia of the domestic cat.

Methods of cryopreservation for spermatozoa from domestic cat epididymides and vasa deferentia were compared as models for posthumous gamete salvage from non-domestic felids. Spermatozoa were collected either immediately after castration (Fresh, n = 37) or after being cooled (5 degrees C) in tissue overnight (Cool, n = 37) and released into one of three extenders containing 20% egg yolk and 3% glycerol for cryopreservation: (1) TE: Tris buffer, citric acid and fructose; (2) TC: Tris buffer, citric acid and glucose, or (3) CP: lactose, and frozen over lipid nitrogen. Before and after freezing, each sperm cell sample was evaluated for motility and percentage morphologically normal cells. Samples were also evaluated for their ability to initiate fertilization using a zona attachment assay. Neither percentage morphologically normal spermatozoa nor percentage motility differed among the three diluents for prefreeze and post-thaw samples, regardless of the collection treatment. However, CP tended to provide lower post-thaw status than did the TE and TC cryoextenders. Before freezing, there was no difference in percentage motility between the Fresh and Cool groups (mean: 76 versus 72%, respectively); however, progressive status and normal morphology were lower (P < 0.05) in Cool (3.0 and 57%) than in Fresh (3.4 and 64%) samples. After thawing there was a greater decline (P < 0.05) in percentage motility in the Cool than in the Fresh group (34 versus 24%) and the number of intact acrosomes dropped from prefreeze values of 66.7 +/- 6.3 and 56.4 +/- 4.8% to 17.8 +/- 3.9 and 20.9 +/- 4.6% after thawing in the Fresh and Cool groups, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1999

A laparoscopic technique for obtaining ovarian cortical biopsy specimens for fertility conservation in patients with cancer.

OBJECTIVE: To evaluate the efficacy of a newly designed round biopter as a practical and safe method for collecting ovarian tissue for cryopreservation in young women with cancer before chemotherapy. DESIGN: Prospective study of young women volunteering for research (Leeds, United Kingdom) and patients with cancer (Jerusalem, Israel and Leeds, United Kingdom) undergoing laparoscopic ovarian cortical tissue biopsy and cryopreservation before administration of high-dose radiochemotherapy. SETTING: Two university-based tertiary referral centers of oncology and gynecology (Hadassah Medical Center, Israel; Leeds General Infirmary, United Kingdom). PATIENT(S): Twenty female volunteers undergoing routine laparoscopic gynecologic procedures (age, 25-34 years) and 20 young women (age, 11-30 years) with advanced cancer requiring potentially sterilizing radiochemotherapy. INTERVENTION(S): Cortical ovarian tissue biopsies performed under laparoscopy with use of the round biopter. RESULT(S): The laparoscopic sampling procedure was uncomplicated in all cases. In treated patients, five to six samples were obtained (5 mm in diameter; 2-3 mm in depth) using the round biopter, and radiochemotherapy was administered without delay. In volunteers, no adhesions were noted at repeat laparoscopy (9 patients). All biopsy specimens were cryopreserved, and histologic examination confirmed the presence of many primordial follicles. CONCLUSION(S): Laparoscopic ovarian biopsy performed with the round biopter is a safe and efficient method for collecting ovarian tissue for cryopreservation in patients with cancer.

Adolescent↗
PubMed2001

Increased numbers of circulating donor-specific T helper lymphocytes after human heart valve transplantation.

Implantation of cryopreserved human donor heart valves for either congenital or acquired cardiac disease has been performed since the last three decades. Although the clinical outcome is good, long-term valve degeneration resulting in dysfunction has been observed. A specific immunological response of the recipient against the allograft has been proposed as one of the factors involved in this process. Helper T lymphocytes play an important intermediate role in cellular and humoral immune response. Increasing numbers of circulating donor-specific helper T lymphocytes precursors (HTLp) correlate with graft rejection after organ transplantation. To investigate whether cryopreserved human donor heart valves are able to induce a donor-specific T helper response, we monitored the HTLp frequencies (HTLpf) in peripheral blood samples of 13 patients after valve allograft transplantation by use of a limiting dilution assay followed by an interleukin-2 bioassay. Prior to transplantation, HTLpf specific for donor and third-party antigens showed individual baseline levels. After allografting, the antidonor frequencies significantly increased in 11 of the 13 patients (P = 0.02). This was not found for stimulation with third-party spleen cells (P = 0.68), which indicates a donor-specific response. Maximal donor-specific HTLpf were already found at 1--2 months after operation. Valve allograft transplantation induces an increase in the numbers of donor-specific HTLp in peripheral blood of the patients. Analogous to organ transplantation, these HTLp may play a crucial role in events that lead to valve damage. Therefore, monitoring of HTLp in peripheral blood samples might be informative for donor valve degeneration (rejection) and subsequently valve allograft failure.

Adult↗
PubMed2002

CD56 and PGP expression in acute myeloid leukemia: impact on clinical outcome.

BACKGROUND AND OBJECTIVES: Overexpression of P-glycoprotein (PGP), a multidrug-related (MDR) protein, is one of the most important factors responsible for reduced drug sensitivity in acute myeloid leukemia (AML). Recently, we demonstrated that the presence of CD56 antigen, an isoform of the neural adhesion molecule, in AML cells is a negative independent prognostic factor for the achievement of complete remission (CR) and correlates with shorter survival. Since in our previous report we observed a more frequent PGP expression in CD56+ patients, we hypothesized that the reduced response to chemotherapy in this group of patients was due to increased PGP-mediated drug efflux. To confirm this hypothesis in this study PGP and CD56 expression on AML cells was correlated with other clinical and biological features and treatment response. DESIGN AND METHODS: Immunophenotypic analysis, including evaluation of CD56 and PGP expression, was performed using multiparameter flow cytometry on fresh and/or cryopreserved blast cells, obtained after informed consent, from bone marrow and/or peripheral blood of 143 consecutive newly diagnosed AML cases at the time of diagnosis. Samples expressing CD56 in at least 15% or more cells were considered as positive (CD56+). PGP expression was expressed as a mean fluorescence index (MFI) i.e. as the ratio of sample mean fluorescence channel and the isotypic control mean fluorescence channel. RESULTS: Overall results showed that 67/143 cases were PGP-/CD56-, 23/143 were PGP+ /CD56+, 40/143 were PGP+/CD56- and the remaining 13/143 were PGP-/CD56+. CD56+ and PGP+ on AML cells significantly reduced the CR rate (83% in the PGP-/CD56- group vs 60% in the PGP-/CD56+ group, 46% in the PGP+/CD56- group and 58% in the PGP+/CD56+ group, p = 0.002). In addition we observed a significantly higher proportion of total failures in patients expressing PGP or CD56 compared to in the group not expressing either (73% vs 27%, respectively; p = 0.0001). CD56 and PGP overexpression influenced the overall survival: in fact, the median survival of CD56+ and PGP+ patients ranged from 10 to 23 months, while the actuarial survival of CD56-/PGP- patients at 5 years is 52% (p = 0.023). INTERPRETATION AND CONCLUSIONS: Our data underline the independent negative prognostic role of PGP and CD56 expression in acute myeloid leukemia. Since the mechanism by which CD56 reduces drug sensitivity is still unknown, further investigations are required.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1979

Dilution techniques for optimum recovery of cryopreserved bone marrow cells.

Bone marrow granulocyte progenitor cells (CFU-C) were assayed in methyl-cellulose prior to cryopreservation in Dimethylsulfoxide (DMSO) and after thawing and diluting the DMSO. The time of dilution from 10% to 1% DMSO and the temperature of the sample and diluting media were studied. Compared with samples diluted at 0 degrees-4 degrees C, samples which were diluted at 24 degrees C were more viable by Trypan Blue exclusion (p less than .01) and had greater CFU-C growth in vitro (p less than .01). There was no advantage to prolonging dilution time from 10 minutes at a constant rate to 40 minutes using stepwise technique. Recovery of CFU-C at 24 degrees C ranged from 40% to 114% with a mean +/- S.D. of 67% +/- 19.5%. There was evidence that clonogenic cells were selectively preserved under the conditions described.

Bone Marrow Cells↗
PubMed1999

Differential effects of IL-2 incubation on hematopoietic potential of autologous bone marrow and mobilized PBSC from patients with hematologic malignancies.

Culturing of hematopoietic progenitor cells for 24 h with IL-2 generates cytotoxic effector cells that mediate in vitro and possibly in vivo antitumor activity. We examined the effect of IL-2 incubation on progenitor cells from 24 patients with hematologic malignancies using paired autologous bone marrow (ABM) and PBSC to determine differences in hematopoietic potential. Cells were cryopreserved and stored in liquid nitrogen until conditioning therapy was completed. After thawing, cells were incubated with IL-2 for 24 h at 37 degrees C. Paired samples of ABM and PBSC from the same patient were analyzed for nucleated and mononuclear cell number, CD34 antigen expression, and colony-forming unit (CFU) activity before and after IL-2 incubation. There was a significant decrease in the average number of mononuclear cells (MNC) (x10(8)/kg) (<0.001) and CD34+ cells (x10(6)/kg) (0.006) from both ABM and PBSC after 24 h IL-2 culture (ABM MNC: 0.6+/-0.1 vs. 0.4+/-0.0, p = <0.001; PBSC MNC: 4.4+/-0.5 vs. 3.7+/-0.4, p = 0.03; ABM CD34+: 2.4+/-0.5 vs. 1.3+/-0.3, p = <0.001; PBSC CD34+: 6.6+/-1.8 vs. 5.0+/-1.2, p = 0.05). However, whereas ABM CFU/10(5) MNC plated (269.3+/-47.2 vs. 385.6+/-70.6) were significantly increased (p = 0.005), there was no change in PBSC CFU (271.0+/-47.2 vs. 257.3+/-48.5). The mean plating efficiency (%) of ABM CD34+ cells was markedly increased after IL-2 incubation (10.1+/-3.3 vs. 19.0+/-7.2, p = 0.04), although it was lower than that of PBSC CD34+ cells, which did not change significantly in culture (29.4+/-5.5 vs. 36.0+/-6.5). Additional work is in progress to determine the cause and significance of the enhanced plating efficiency of the ABM progenitor cells.

Adult↗
PubMed2005

Respiratory burst and NAD(P)H oxidase activity are involved in capacitation of cryopreserved bovine spermatozoa.

Heparin (a glycosaminoglycan) and quercetin (a calcium-ATPase plasma membrane specific inhibitor) induce bovine sperm capacitation. Mitochondria from frozen semen are capable of generating oxidative energy. The aim of the study was to determine oxygen uptake variation and the participation of diphenileneiodonium (DPI)-sensitive oxidases from spermatozoa capacitated with heparin or quercetin. Oxygen uptake was measured polarographically and 2 microM diphenileneiodonium (DPI) was used as a specific inhibitor of NAD(P)H-oxidases. Sperm capacitation was determined by the chlorotetracycline technique. Heparin produced a respiratory burst (17.0+/-3.2 microL O2/h/10(8) spermatozoa; mean+/-S.D.) versus control (11.3+/-0.9 microL O2/h/10(8) spermatozoa; P<0.05). Oxygen uptake and sperm hypermotility were inhibited by cyanide. Treatment with DPI blocked heparin capacitation and oxygen uptake (cyanide-sensitive) decreased to control levels. Respiration of quercetin-treated samples (cyanide-sensitive; 9.7+/-0.7 microL O2/h/10(8) spermatozoa) was not significantly different from the controls; oxygen uptake was not modified by DPI, but quercetin capacitation was inhibited (P<0.05). The effect of DPI with heparin confirmed that oxidases participate in capacitation induction. The addition of superoxide dismutase and/or catalase to heparin- or quercetin-treated samples, failed to modify oxygen uptake and blocked capacitation (P<0.05), suggesting that the superoxide anion (O2*-) participates in the capacitation induction. High mitochondrial activity from heparin-treated samples indicated that energy requirements, especially for hypermotility, were supported by the respiratory chain. Although a respiratory burst was not produced by quercetin, DPI-sensitive-oxidases (O2*- source) were necessary for capacitation. In cryopreserved bovine spermatozoa, heparin- or quercetin-induced capacitation required different levels of mitochondrial energy and DPI-sensitive oxidase activity.

Animals↗
PubMed2005

Cryopreservation of European eel (Anguilla anguilla) sperm using different extenders and cryoprotectants. Short communication.

Experiments were carried out on the sperm cryopreservation of artificially induced eels. The effects of several extenders and two cryoprotectants on the motility of spermatozoa were investigated. The highest post-thaw motility was observed with the combination of Tanaka's extender and DMSO as cryoprotectant. Further dilution after thawing resulted in complete loss of motility in samples frozen in presence of DMSO while sperm frozen with methanol as cryoprotectant retained its motility after further dilution.

Anguilla↗
PubMed2006

Animal genetic resources conservation in the Netherlands and Europe: poultry perspective.

Increased global use of highly productive breeds of farm animals has been coupled to loss of genetic diversity in most species. In European countries, various governmental, non-governmental, and private organizations try to preserve genetic diversity of livestock in situ (e.g., by stimulating the use of indigenous, rare breeds by farmers; in nature reserves; or in noncommercial farms). In the case of poultry, maintaining in situ populations of the noncommercial (fancy) breeds largely relies on hobby farmers. In addition to in situ conservation, gene banks are being established for ex situ conservation. In at least 2 countries, France and The Netherlands, there are limited collections of frozen semen of rare poultry breeds. Since 2003, the CGN has started with a more systematic effort to collect, freeze, and store semen of indigenous Dutch poultry breeds. At present, the CGN gene bank contains semen of 11 Dutch rare poultry breeds. Also, CGN has performed research on the methodology for cryopreservation of fowl semen. This recent work was focused on finding a suitable replacement for glycerol, which is contraceptive in the hen, as a cryoprotectant. For reasons of hygiene and sample identification, we favored straw freezing, as opposed to the highly effective pellet freezing method. A significant interaction was found between cooling rate and cryoprotectant concentration. Best post-thaw sperm quality was obtained when combining 0.6 mol of dimethylacetamide/L with a cooling rate of +/- 200 degrees C/min. Inseminations twice per week with 0.3 billion sperm per insemination resulted in 97 and 88% fertilized eggs with fresh and frozen semen, respectively. In 2005, CGN has used this straw freezing method to extend the collection of poultry semen in the Dutch gene bank.

Animals↗
PubMed1998

Cryopreservation of immature human oocytes and ovarian tissue: an emerging technology?

OBJECTIVE: To review the potential for cryopreserving immature follicles either in situ or after isolation from ovarian stroma and to consider the options for fertility by transplantation or in vitro follicle growth. DESIGN: The problems of storing embryos and mature (metaphase II) oocytes were considered in light of the needs of patients to protect fertility before undergoing potentially sterilizing therapy for cancer. The evidence from the experimental biology literature showing that immature oocytes (prophase I) in primordial follicles can be cryopreserved successfully and transplanted to produce fertile eggs was reviewed. The review, which was compiled from MEDLINE and other bibliographic databases, is intended to emphasize the practical opportunities for this technology and the need for future research rather than to be a comprehensive treatment of the subject. CONCLUSION(S): The disappointing results obtained with the cryopreservation of oocytes at metaphase II and ethical concerns about embryo storage are giving impetus to the banking of ovarian tissue for patients who require conservation of fertility. The numbers of needy patients are growing as long-term survivorship after high-dose chemotherapy and bone marrow transplantation rises. More speculatively, if ovarian tissue banking becomes a proven effective method, young, healthy women may request storage of ovarian biopsy samples to keep their reproductive options open in midlife when oocyte fertility is declining. Although the cryotechnology is not yet perfected, the major question now is how to use the tissue most effectively after thawing. For the present, ovarian tissue cryopreservation is still at the experimental stage, but it holds the promise of valuable applications.

Cellular Senescence↗
PubMedDate not supplied

Pressure shift freezing of pork muscle: effect on color, drip loss, texture, and protein stability.

Cylindrical specimens (50 mm diameter and 160 mm length) of fresh pork muscle (boneless rib portions) packed in plastic bags were frozen by pressure shift freezing (PSF) at 100, 150, and 200 MPa, air blast freezing (ABF), and liquid immersion freezing (LIF). Temperature and phase transformations of the muscle tissue were monitored during the freezing process at three locations: center, midway between the center and the surface, and near the surface. Pork muscle quality changes [color, drip loss (both thawing and cooking), texture (shear force), and protein stability (DSC thermal profiles)] were evaluated after thawing the frozen samples at room temperature (20 degrees C). Employing pressures above 150 MPa caused very significant (P < 0.01) color changes in pork muscle during the PSF process. The PSF process reduced thawing drip loss of pork muscle but did not cause obvious changes in total drip loss following thawing and subsequent cooking. PSF at 150 and 200 MPa resulted in considerable denaturation of myofibrillar proteins of pork muscle. The PSF process also caused an increase in the pork muscle toughness as compared with that of unfrozen, ABF, and LIF samples.

Animals↗
PubMed1996

Detection of p53 gene abnormality by sequence analysis of archival paraffin tissue. A comparison with fresh-frozen specimens.

This parallel study was designed to compare the sensitivity and specificity of detection of point mutations in fresh-frozen and formalin-fixed, paraffin-embedded breast cancer tissue. Sequence analysis of exon 5 of p53 gene was performed on polymerase chain reaction-amplified DNA from 25 infiltrating ductal carcinomas of the breast. Four tumor showed mutations with identical base substitutions in their respective codons of both frozen and paraffin-embedded specimens. We conclude that subtle genetic alterations can be detected in archival paraffin tissue with an accuracy comparable to that of fresh-frozen histologic samples.

Breast Neoplasms↗
PubMed2005

Cryopreservation of cultured periosteum: effect of different cryoprotectants and pre-incubation protocols on cell viability and osteogenic potential.

Evidence has accumulated that periosteal cells have a great potential to regenerate bone. We have demonstrated that cultured periosteum (CP) in membrane form is an effective device to regenerate alveolar bone. To increase the availability of CP in a clinical environment, an effective cryopreservation protocol for CP has been developed. In this study, three different cryoprotectants (Me(2)SO, glycerol, and ethylene glycol) were used. The effect on cell viability of pre-incubation temperature, pre-incubation time, and agitation during incubation was investigated. Samples were stored at -196 degrees C for 10 days. Cell viability was assessed by a colorimetric cell viability assay using a tetrazolium salt, and the assay results were confirmed by confocal laser scanning microscopy after staining with a combination of calcein AM and ethidium homodimer-1. The activity of the cells after thawing was assessed by alkaline phosphatase assay. To assess the osteogenic potential of cryopreserved CP, the CP was grafted to calvarial defects in athymic rats. The greatest cell viability was obtained in the group equilibrated at 37 degrees C for 30 min with Me(2)SO, under agitation, showing 63.3 +/- 10.5% recovery. After cryopreservation, the cell growth of surviving cells was identical when Me(2)SO was used as a cryoprotectant. Alkaline phosphatase (ALP) activity was maintained in the groups cryopreserved with Me(2)SO and glycerol. The transplantation experiment showed that the calvarial defects were completely closed by grafting cryopreserved CP, which demonstrates that the osteogenic property of CP was well maintained. An efficient cryopreservation protocol for CP has been developed and this will provide a convenient and effective treatment option for bone regeneration in clinics.

Alkaline Phosphatase↗