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PubMed1999

DNA strand breaks in human sperm cells: a comparison between men with normal and oligozoospermic sperm samples.

BACKGROUND: The aim of this report was to compare the degree of DNA strand breaks in known normal fertile men to those with oligozoospermia, and evaluate the presence of DNA strand breaks in normal raw sperm, after Percoll and swim-up and following conventional cryopreservation, as all these preparation methods might differ in selection of healthy sperm cells. METHODS: Sperm samples from proven fertile sperm donors (n=20) and infertile men with oligozoospermia (n=33) were tested for the presence of DNA strand breaks in the spermatozoa, by direct immunoperoxidase detection of digoxigenin-labeled genomic DNA. A correlation to other sperm parameters, sperm counts, motility and Krüger's strict criteria was performed. RESULTS: DNA strand breaks were found significantly more often in sperm samples from men with oligozoospermia compared to sperm samples of normal fertile men. The degree of spermatozoa with DNA strand breaks was correlated proportional with the degree of morphological pathological sperm cells judged by Krüger's strict criteria. The percentage of spermatozoa with DNA strand breaks in the samples was not influenced by procedures such as the swim-up technique, Percoll gradients or cryopreservation and thawing. CONCLUSION: DNA strand breaks were found significantly more often in men with oligozoospermia compared to normospermic men. The DNA strand breaks might play an important role for the maturation process of the spermatozoa in the same way as apoptosis is controlling the number of early meiotic germ cells in the testis, and hereby play an important role in advanced fertility treatments (ICSI).

Apoptosis↗
PubMed2003

Anaplastic thyroid cancer evolved from papillary carcinoma: demonstration of anaplastic transformation by means of the inter-simple sequence repeat polymerase chain reaction.

BACKGROUND: In thyroid tumors, the coexistence of well- and poorly differentiated tumor types has led to the hypothesis that poorly differentiated thyroid tumors develop from well-differentiated thyroid tumors. By evaluating the genomic instability of histologically distinct but coexisting tumor foci, this study aimed to develop an improved understanding of thyroid tumorigenesis and tumor evolution. DESIGN: Laser capture microdissection (LCM) was carried out on archival formalin-fixed, paraffin-embedded sections from a tumor containing foci of classic papillary thyroid cancer and anaplastic thyroid cancer. DNA was extracted from each microdissected tumor focus. In addition, cryopreserved bulk normal and neoplastic thyroid tissue underwent DNA extraction. All DNA samples were subsequently evaluated for genomic instability by means of inter-simple sequence repeat polymerase chain reaction. RESULTS: The LCM DNA from each archival paraffin-embedded tumor focus demonstrated unique patterns of banding as compared with the cryopreserved tumor and normal tissue DNA. Thus, intratumoral variability in genomic instability was observed. Comparison of inter-simple sequence repeat polymerase chain reaction patterns of LCM DNA from adjacent foci of papillary and anaplastic tumors showed conserved genome alterations. CONCLUSIONS: At the genome level, thyroid tumors may be highly heterogeneous. The intratumoral histologic heterogeneity observed in thyroid neoplasms reflects genetically heterogeneous underlying tumor cell populations that are demonstrated by the observed differences in their rates and extents of genomic instability. The conserved genomic alterations in the microdissected papillary and anaplastic foci suggest intratumoral evolution, with transformation of a preexisting papillary tumor to anaplastic carcinoma.

Aged↗
PubMed1987

Baseline and mutagen-induced sister-chromatid exchanges in cultures of human whole blood and purified fresh or frozen lymphocytes.

Baseline and mutagen-induced levels of sister-chromatid exchanges were evaluated in 10 normal individuals. Cultures with whole blood or purified lymphocytes, either freshly isolated or after 1 or 6 months of cryopreservation, were analyzed to determine whether frozen lymphocytes are suitable for SCE studies. Whole blood and freshly isolated lymphocytes were cultured from samples taken at the beginning of the study (Time 0) and 6 months later (Time 6). Cryopreserved lymphocytes were recovered after 1 month (Time 1) and 6 months (Time 6) of cryopreservation and then challenged with mutagens in culture. The mutagens used were mitomycin C, 4-nitroquinoline-1-oxide, and N-methyl-N'-nitro-N-nitrosoguanidine. Purified lymphocytes had consistently and significantly higher baseline SCE frequencies than cells from whole blood cultures and were more sensitive to N-methyl-N'-nitro-N-nitrosoguanidine and 4-nitroquinoline-1-oxide. The response to mitomycin C was similar in all culture types. There was, overall, no consistent effect of freezing on baseline or induced sister-chromatid exchange frequencies in the purified lymphocytes. This suggests that purification and cryopreservation of human lymphocytes does not alter the baseline or mutagen-induced sister-chromatid exchange response and in certain epidemiological, occupational and monitoring situations may have logistical and technical advantages over the use of fresh whole blood.

4-Nitroquinoline-1-oxide↗
PubMed1999

The cryopreservation protocol optimal for progenitor recovery is not optimal for preservation of marrow repopulating ability.

The efficiency of five different cryopreservation protocols (our original controlled-rate and noncontrolled-rate protocols) was evaluated on the basis of the recovery after thawing of very primitive pluripotent hemopoietic stem cells (MRA(CFU-GM), pluripotent progenitors (CFU-Sd12) and committed granulocyte-monocyte progenitors (CFU-GM) in mouse bone marrow. Although the nucleated cell recovery and viability determined immediately after the thawing and washing of the cells were found to be similar, whether controlled-rate or noncontrolled-rate cryopreservation protocols were used, the recovery of MRA(CFU-GM), CFU-Sd12 and CFU-GM varied depending on the type of protocol and the cryoprotector (DMSO) concentrations used. It was shown that the controlled-rate protocol was more efficient, enabling better MRA(CFU-GM), CFU-Sd12 and CFU-GM recovery from frozen samples. The most efficient was the controlled-rate protocol of cryopreservation designed to compensate for the release of fusion heat, which enabled a better survival of CFU-Sd12 and CFU-GM when combined with a lower (5%) DMSO concentration. On the contrary, a satisfactory survival rate of very primitive stem cells (MRA(CFU-GM)) was achieved only when 10% DMSO was included with a five-step protocol of cryopreservation. These results point to adequately used controlled-rate freezing as essential for a highly efficient cryopreservation of some of the categories of hematopoietic stem and progenitor cells. At the same time, it was obvious that a higher DMSO concentration was necessary for the cryopreservation of very primitive stem cells, but not, however, for more mature progenitor cells (CFU-S, CFU-GM). These results imply the existence of a mechanism that decreases the intracellular concentration of DMSO in primitive MRA cells, which is not the case for less primitive progenitors.

Animals↗
PubMed1984

Improved recovery of post-thaw motility and vitality of human spermatozoa cryopreserved in the presence of dithiothreitol.

Semen was collected in the laboratory from nine healthy donors. The concentrations and the percentages of live and motile spermatozoa in all semen samples were within the normal range. Each sample was diluted with citrate-egg yolk-glycerol medium with and without 5 mM dithiothreitol (DTT). Samples were frozen in liquid nitrogen vapor (-70 degrees C) for 7 min and subsequently stored in liquid nitrogen. The effect of DTT in cryopreservation of sperm was determined by comparing percentage of motile and live spermatozoa between controls and DTT-treated post-thaw samples. Percentage of motile spermatozoa was determined by two techniques, laser Doppler velocimetry (LDV) and light microscopy. The percentage of live spermatozoa was measured by microscopic evaluation after staining with eosin-nigrosin. It was shown that the addition of DTT to the freezing medium significantly improved the recovery of motile and live spermatozoa in the post-thaw samples. The mean motility recovery, as measured by LDV, was 44.9% in the controls as compared to 73.9% in the DTT-treated samples. Similarly the mean recovery of live spermatozoa in the controls and DTT-treated samples was 66.5 and 86.6%, respectively. Based on these results, a new hypothesis implicating lipid peroxidation in cryoinjury is proposed. It is also suggested that the use of DTT in the freezing medium may offer an advantage over the commonly used techniques of human sperm cryopreservation.

Cell Survival↗
PubMed1999

Semen cryopreservation after orchidectomy in men with testicular cancer.

OBJECTIVE: To assess the feasibility of semen cryopreservation after orchidectomy in patients with testicular tumour. PATIENTS AND METHODS: The quality of semen samples was investigated in 36 men with testicular tumour (mean age 31.7 years, range 20-49) who were referred to our infertility centre for semen cryopreservation. For each patient, the number of straws, semen volume, number of spermatozoa, and sperm motility before and after freezing were evaluated. RESULTS: Fifteen patients (42%) banked sperm before and 21 (58%) after orchidectomy; the delay was >7 days in 19 patients (53%). The mean age, histological diagnosis and tumour stage did not differ significantly whatever the time of cryopreservation. Semen quality did not differ significantly in patients who cryopreserved sperm before or after orchidectomy and there were no significant differences in sperm values whatever the delay before preservation. Semen quality was the same in patients with seminoma or nonseminoma tumour. CONCLUSION: These findings indicate that spermatogenesis of the contralateral testis is sufficient for successful semen cryopreservation after orchidectomy. Urologists should be encouraged to increase the awareness among oncology teams and patients about the new developments in preserving fertility for patients with cancer.

Adult↗
PubMed1986

Cryopreservation of fetal rat liver tissue--a morphological investigation.

The present study was undertaken to define optimal conditions for cryopreservation of fetal rat liver tissue fragments. First, various cooling rates (1, 4, 10, 20 and 30 degrees C/min) and preserving temperatures (-20, -80 and -196 degrees C) were examined. Next, various concentrations of Me2SO (5, 10, 20 and 30 per cent vol/vol) were examined by freezing at a rate of 4 degrees C/min to -80 degrees C before transfer to -196 degrees C. All samples were preserved for at least one week. After recovery from cryopreservation, the fragments were transplanted into the spleens of syngeneic rats. Histological assessment of the grafts was made one month after transplantation. Consequently, the optimal cryopreserving conditions for fetal rat liver fragments were defined as follows: cooling rate was 1 to 10 degrees C/min, preserving temperature was at -196 degrees C, concentration of Me2SO was 20 per cent (final concentration: 10 per cent). In the long term observation, the stored liver fragments could be differentiated into adult hepatocytes and the surviving hepatocytes showed little difference from the nonfrozen controls, either histochemically or ultrastructurally. The surviving hepatocytes in the stored transplants were less numerous than in the nonfrozen ones and hepatic cell plates and sinusoids were nil.

Animals↗
PubMed2000

Freezing a few testicular spermatozoa retrieved by TESA.

Men with azoospermia can now be treated using testicular sperm aspiration (TESA). New aspirations, in subsequent cycles, may be avoided using cryopreservation. Conventional sperm freezing techniques are not suitable for TESA samples with a small number of spermatozoa. Testicular spermatozoa were obtained from 10 azoospermic men undergoing TESA for a diagnostic objective. Two different freezing protocols were performed according to the number of spermatozoa found in the final suspension: between 100-2000, we used TEST yolk buffer with glycerol, adding it to testicular sperm (Method I); for less than 100, we injected them into cell-free human zona pellucida before adding a freezing medium (Method II). Sperm and motility recovery rates were 1% and 32.3%, and 88.2% and 26.6% for methods I and II respectively. The fertilisation rate was 13.3% and 23% for methods I and II respectively. This study represents our preliminary experience in freezing testicular spermatozoa collected by TESA. Preliminary observations show that it is possible to freeze a few testicular spermatozoa inside evacuated zona pellucida.

Cleavage Stage, Ovum↗
PubMed2006

Comparative generation and characterization of pluripotent unrestricted somatic stem cells with mesenchymal stem cells from human cord blood.

OBJECTIVE: Generation and expansion of pluripotent unrestricted somatic stem cells (USSCs) and mesenchymal cells (MSCs) from human cord blood as well their functional characterization were assessed. METHODS: USSC generation was initiated from fresh and cryopreserved cord blood (CB). Culture conditions and functional assays distinguishing between USSCs and MSCs (e.g., endodermal differentiation) are described. RESULTS: USSC cultures were initiated from 573 placental CB samples with a total generation frequency of 35.4% (n = 203), whereas successful generation of USSCs from cryopreserved products was scare. Medium, dexamethasone, and the fetal calf serum source are the major parameters to generate and expand the cells in a pluripotent state (osteogeneic, neural, and endodermal differentiation). CONCLUSIONS: USSC or MSC cultures can be easily obtained from fresh CB samples, good manufacturing practice production of such cells is feasible. These USSCs or MSCs from CB, when matched for HLA antigens, may serve as an allogeneic stem cell source for the development of cellular therapy for tissue repair.

Cell Culture Techniques↗
PubMed2006

Effect of cryopreservation on sperm apoptotic deoxyribonucleic acid fragmentation in patients with oligozoospermia.

OBJECTIVE: To verify if the increase in sperm apoptotic DNA fragmentation during cryopreservation is greater in oligozoospermic patients than in normozoospermic controls. DESIGN: Controlled prospective study. SETTING: Patients in an academic research environment. PATIENT(S): Forty-seven patients with oligozoospermia (concentration <10 x 10(6) sperm/mL) and 30 normozoospermic men. INTERVENTION(S): Sperm cryopreservation using a standard protocol with a test-yolk buffer, glycerol as the cryoprotectant. MAIN OUTCOME MEASURE(S): Rate of apoptotic sperm DNA fragmentation as assessed by the terminal deoxynucleotidyl-mediated deoxyuridine triphosphate nickend labeling (TUNEL) assay, graded in apoptotic or nonapoptotic, before and after cryopreservation. RESULT(S): An increase in apoptotic DNA fragmentation was observed in all the groups, regardless of sperm concentration. Normozoospermic men presented a smaller rate of apoptotic DNA fragmentation than oligozoospermic patients, both in pre- and postcryopreservation samples. The increase in DNA fragmentation was similar in both groups. CONCLUSION(S): Cryopreservation induces apoptotic sperm DNA fragmentation in men, regardless of sperm concentration. Men with oligozoospermia present with higher precryopreservation and postcryopreservation apoptotic sperm DNA fragmentation. The increase in DNA fragmentation is similar in both groups.

Adult↗
PubMed2004

Preserving the reproductive potential of men and boys with cancer: current concepts and future prospects.

The introduction of ICSI has totally changed the reproductive prospects for boys and men who are treated for cancer. With post-pubertal boys and adult men, semen cryopreservation should be offered to every patient undergoing a cancer treatment since preservation of fertility cannot be guaranteed for an individual patient and treatment may shift to a more sterilizing regimen. In the ICSI era, all semen samples, even those containing only a few motile sperm, should be accepted for cryopreservation. Patients who are azoospermic at the time cancer is diagnosed may be offered testicular sperm extraction and cryopreservation of testicular tissue. With pre-pubertal boys, no prevention of sterility by sperm banking is possible since no active spermatogenesis is present. However, in the next decade, prevention of sterility in childhood cancer survivors will become a major challenge for reproductive medicine. In theory, testicular stem cell banking is the only way of preserving the future fertility of boys undergoing a sterilizing chemotherapy. In animal models, testicular stem cell transplantation has proved to be effective; however, it remains to be shown that this technique is clinically efficient as well, especially when frozen-thawed cells are to be transplanted. Malignancy recurrence prevention is an important prerequisite for any clinical application of testicular stem cell transplantation. Although still at the experimental stage, cryobanking of testicular tissue from pre-pubertal boys may now be considered an acceptable strategy.

Adolescent↗
PubMed2005

Thermal expansion of blood vessels and muscle specimens permeated with DMSO, DP6, and VS55 at cryogenic temperatures.

As part of an ongoing effort to characterize the continuum mechanics during cryopreservation via vitrification (vitreous in Latin means glass), the current study focuses on measuring the thermal expansion of cryoprotective agents in the presence and absence of biological samples of bovine muscle and goat arteries. The cryoprotectants under investigation are 7.05 M DMSO, DP6, and VS55, with permeation time of up to 48 h. This study focuses on the upper part of the cryogenic temperature range, where the cryoprotectant behaves like a liquid in all practical applications. The specific lower boundary of this range is unique to the cryoprotectant composition and to the applied cooling rate. The measurement device used in the current study is a modification of a prototype presented recently, in which modifications are made to accommodate biological samples. Based on thermal strain observations, it is concluded that the presence of tissue samples promotes crystallization (the tissue samples serve as potential nucleation sites). It is shown in this study that the thermal strain of tissue samples permeated with 7.05 M DMSO and DP6 is no greater than the thermal expansion of the same cryoprotectants in the absence of tissue samples, and in some cases it is significantly less. For a given cryoprotectant in comparable thermal conditions, experiments on bovine muscle samples show significantly higher scattered data than experiments on artery samples.

Animals↗
PubMed1989

[Autologous bone marrow transplantation--a new approach in the treatment of neoplastic hematologic diseases. I. Scientific principles and methodology of the treatment].

This first part of the review deals with fundamental knowledge, rationale and methods for the use of autologous bone marrow transplantation in the treatment of neoplastic diseases. Use of high-dose chemo- and radiotherapy in the treatment of neoplastic diseases is limited by side effects on haemopoietic tissues. Bone marrow transplantation offers a possibility to escalate the dose of cytotoxic therapy, but this possibility is limited by two main factors: need for matched allogeneic donor, and patient age below 45 years. This has led to application of autologous BMT for the treatment of older patients and those without compatible marrow donors. Samples of bone marrow collected before intensive myeloablative treatment are stored by means of cryopreservation. Viability and clonogenicity of stored bone marrow stem cells prior to reinfusion into the patient are tested by in vitro bone marrow culture (usually CFU-GM). Treatment of marrow samples in vitro by monoclonal antibodies and/or cytotoxic drugs are used in order to cleane ("purge") the marrow of residual neoplastic cells.

Bone Marrow Transplantation↗
PubMed2006

Determination of active TGF-beta2 in aqueous humor prior to and following cryopreservation.

Transforming growth factor-beta2 (TGF-beta2) is one of the most important immunosuppressive cytokines in the anterior chamber of the eye. It is secreted as a complex with latency-associated peptide as an inactive precursor. Only the activated form of TGF-beta2 can bind to its receptor and induce signaling. To date, the concentration of active TGF-beta2 in aqueous humor was exclusively determined using samples that had been preserved at -80 degrees C. Quantitative measurements of the activated form directly after sampling have not yet been taken. The aim of this study was to investigate the effect of cryopreservation on the concentration of active TGF-beta2 in the aqueous humor. Samples of aqueous humor were drawn from patients with either cataract or a corneal disorder for determination of TGF-beta2 using a Sandwich-ELISA. In group I (n=30, patients with corneal disorders or cataract), one part of each sample was tested for active TGF-beta2 directly after sampling, whereas the remaining material was stored at -80 degrees C for later analysis. Group II consisted of patients undergoing a simple cataract extraction (n=38), and active TGF-beta2 levels were determined within 3 h after sampling. In Group III (n=34, patients with corneal disorders or cataract), active TGF-beta2 was determined within 3 h after puncture, as were total TGF-beta2 levels after acidic activation for each sample. The average level of active TGF-beta2 in the aqueous humor of group I analyzed directly after sampling was 35+/-31 (median 37) pg/ml. In contrast, frozen samples from the same patients showed an average concentration of 155+/-103 (median 152) pg/ml. The average level of active TGF-beta2 in aqueous humor of 38 cataract (group II) eyes was 40+/-24 (median 41) pg/ml determined within 3 h after puncture. The average level of total TGF-beta2 in group III was 1,654+/-631 (median 1,542) pg/ml compared to 33+/-39 (median 28) pg/ml of active TGF-beta2 determined directly after sampling, yielding a ratio of 2% of active to total TGF-beta2. Levels of active TGF-beta2 in aqueous humor determined directly after sampling were 4.4 fold lower than those measured in frozen samples. Thus, samples meant for determining active TGF-beta2 levels should not be kept frozen.

Aged↗
PubMed2005

Immunological and echocardiographic evaluation of decellularized versus cryopreserved allografts during the Ross operation.

OBJECTIVE: Compare the immunological and echocardiographic data of decellularized versus cryopreserved allografts used for RVOT reconstruction during Ross operation. METHODS: From 16/01/03 thru 07/10/03, 20 Ross operations were performed using decellularized (n=11) or cryopreserved (n=9) allografts. Echocardiography was done at discharge, 1, 3, 6 and 12 months and annually thereafter. Samples for determination of antibodies against HLA class I and II were obtained preoperatively and at days 5, 10, 30, 90 and 180 postoperatively. These samples were tested by the ELISA method in LAT-M dishes (unspecific) for identification of circulating antibodies and the results expressed as mean sample values (Is=DO/cutoff). If positive, LAT-E (specific) was performed and PRA levels determined. RESULTS: There was no mortality. Cryopreserved allografts showed marked Is values elevations for class I and II antibodies which started at the first month and remained elevated up to 6 months. In contrast, of the patients receiving decellularized allografts, seven remained negative, two patients had only marginal elevation of class I antibodies and two patients showed abnormal elevations of PRA levels. This response happened earlier than in the cryopreserved group, starting on the 5th postoperative day and has returned to baseline levels in one case. Echocardiography showed mild, but significant, elevation of gradients in cryopreserved valves but none in the decellularized. CONCLUSIONS: Decellularized allografts had normal function up to 18 months and showed important reduction of the immunogenic response when compared to cryopreserved valves.

Adolescent↗
PubMed2001

Cryopreservation in aquarium fishes.

Few studies have addressed sperm cryopreservation in aquarium fishes (body sizes of 10 cm or less). There are several challenges inherent in developing cryopreservation procedures for these fishes. First, their small body size and sperm volume limit experimental replication and the numbers of treatments possible without pooling of samples. This hinders research, especially if many experimental variables are evaluated. The small sample volume necessitates identification of optimal sperm-to-egg ratios to maximize fertilization potential and places greater emphasis on increasing and maintaining sperm viability after thawing. Other technical problems include the use of 0.25-ml French straws, which increase difficulties in sample handling (automated straw fillers are more common for the 0.5-ml straw) and labeling. Sperm cryopreservation of live-bearing fishes (with internal fertilization) is essentially unexplored. The sperm of these fishes is sufficiently different in structure (e.g., head shape) and physiology (e.g., energy metabolism) from the sperm of other fishes that the need to develop specialized techniques is almost assured. The requirement for artificial insemination also introduces a new variable complicating the collection of data (e.g., assessing fertilization is not straightforward with internally held eggs). Cryopreservation in aquarium fishes will assist the development (e.g., through selective breeding), protection (e.g., through germplasm repositories), and distribution (e.g., through shipment of frozen sperm) of research lines and offers benefits for restoration of endangered species.

Journal Article↗
PubMed1990

[Use of sperm cryopreservation by tumor patients].

Impairment of spermatogenesis can be expected after cytostatic treatment or radiation for cancer. To improve fertility prognosis, semen samples are usually deep-frozen and stored in liquid nitrogen prior to such a therapy. However, after therapy this deep-frozen sperm is seldom used. Of 47 patients for whom we started cryopreservation of semen during the years 1983-1988, 20 men were no longer interested in further storage within 2 years, so that the deposits were destroyed. Up to now, none of the 27 samples still existing has been used for artificial insemination or in vitro fertilization (IVF). Possible reasons for this are discussed.

Adolescent↗