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PubMed1993

Detection of Chlamydia trachomatis in semen of artificial insemination donors by the polymerase chain reaction.

OBJECTIVE: To assess the feasibility of detecting Chlamydia trachomatis in cryopreserved donor semen by a specific, direct polymerase chain reaction (PCR). DESIGN: Cryopreserved donor semen was tested for the presence of C. trachomatis by a specific PCR, directly applied to semen without prior DNA purification. SETTING: Tertiary care fertility center in a teaching hospital and university-based laboratory for molecular pathology. PARTICIPANTS: Cryopreserved semen from 30 donors was investigated. These semen samples had previously given negative results in cell culture for C. trachomatis. Two different ejaculates of each donor, cryopreserved with an interval of 2 years, were retrospectively analyzed. INTERVENTIONS: None. MAIN OUTCOME MEASURE: The presence of C. trachomatis as demonstrated by PCR. RESULTS: In 3 of 30 donors C. trachomatis was detected in both ejaculates, whereas in 2 additional donors only one of the two samples tested positive. Additional samples from 2 positive donors, together with samples from 3 negative donors, were studied more extensively, to test the reproducibility and reliability of PCR results. All ejaculates of the donors, previously positive for C. trachomatis by PCR, indeed appeared to be positive, whereas the samples of the negative donors remained negative. CONCLUSIONS: The direct PCR is a reliable, sensitive, and valuable method for detection of C. trachomatis in semen. The incidence of contamination of donor semen with C. trachomatis in the donor population in this study stresses the need for rigorous screening of donor semen before artificial insemination, preferably using a sensitive method such as the PCR.

Adult↗
PubMed1997

Artificial stimulation of cryopreserved human spermatozoa by sodium nitroprusside, 2-chloroadenosine, and 2-deoxyadenosine.

OBJECTIVE: We analyzed the effects of sodium nitroprusside (SNP), 2-chloroadenosine (2-CLA), and 2-deoxyadenosine (2-DA) on sperm motility and motion characteristics of cryopreserved human spermatozoa. METHODS: Thawed semen samples from healthy donors (n = 10) were incubated with the stimulants for 0, 30, 60, 120, and 180 min. The final concentrations used were SNP 25, 50, and 100 nM; 2-CLA 12.5, 25, and 50 microM, and 2-DA 0.5, 1, and 2.5 mM. Sperm motility and changes in motion characteristics were measured on a computer-assisted semen analyzer. RESULTS: Compared with control (0 min, no stimulant), a significant improvement in percentage motility was generally seen with all three stimulants even at 180 min. The sperm motility improved at concentrations of 50 and 100 nM for SNP (except at 120 min), 25 and 50 microM for 2-CLA, and at all concentrations of 2-DA. Other sperm motion characteristics increased to varying extent with the stimulants. Of the three stimulants, all concentrations of 2-DA resulted in significant increases in curvilinear velocity, average path velocity, and amplitude of lateral head displacement at 60 min of incubation. CONCLUSIONS: All three stimulants significantly increased percentage motility for extended intervals of time. 2-DA also improved sperm motion characteristics, though these changes were less uniform with 2-CLA. Motility stimulation by these chemicals may be beneficial in the treatment of cryopreserved and oligozoospermic sperm specimens for use in assisted reproduction.

2-Chloroadenosine↗
PubMed1998

The first births and ongoing pregnancies associated with sperm cryopreservation within evacuated egg zonae.

This new procedure principally aims to avoid a second or possibly multiple surgical procedures for sperm extraction from the male partner in cases of limited amounts of sperm cells, where normal freeze-thaw protocols would fail. Patients (n = 34) diagnosed as azoospermic, extreme oligozoospermic, or oligoasthenozoospermic underwent the process of sperm cryopreservation within evacuated egg zonae. Other samples were allocated to conventional sperm freezing. Sperm samples were acquired using testicular sperm extraction (TESE), microepididymal sperm aspiration (MESA), or fresh ejaculate. Subsequently, five of these 34 couples have undergone in-vitro fertilization (IVF) and achieved normal fertilization using post-thawed spermatozoa frozen under zonae pellucidae in conjunction with intracytoplasmic sperm injection (ICSI). The average fertilization rate for the post-thaw injected spermatozoa was 65%. This is comparable with the regular fertilization rate of 65% for combined MESA and TESE using fresh spermatozoa. All patients underwent embryo transfer. The average implantation rate per embryo was 31%; nearly the same for regular MESA/TESE ICSI cycles (32%). The first pregnancy associated with this procedure concluded with the full term delivery of healthy twin girls on July 18, 1997. The remaining four thaw procedures resulted in another twin delivery, an ongoing singleton gestation, a negative pregnancy test and a biochemical pregnancy respectively.

Adult↗
PubMed2005

Evaluation of DNA damage in rainbow trout (Oncorhynchus mykiss) and gilthead sea bream (Sparus aurata) cryopreserved sperm.

Cryopreservation causes several types of damage to spermatozoa, such as loss of plasma membrane integrity and functionality, loss of motility, and ATP content, resulting in decrease of fertility rates. This spermatozoal damage has been widely investigated for several marine and freshwater fish species. However, not much attention has been paid to the nuclear DNA. The objective of this study was to determine the degree to which cryopreservation induces spermatozoal DNA damage in two commercially cultured species, rainbow trout (Oncorhynchus mykiss) and gilthead sea bream (Sparus aurata), both of which could benefit from the development of cryopreservation strategies on a large scale. We have used the single-cell gel electrophoresis, commonly known as Comet assay to detect strand breaks in DNA. This technique was performed on fresh and cryopreserved sperm from both species. In rainbow trout there was a significant increase in the averages of fragmented DNA and Olive tail moment after cryopreservation (11.19-30.29% tail DNA and 13.4-53.48% Olive tail moment in fresh and cryopreserved sperm, respectively), as well as in the proportion of cells with a high percentage of DNA fragmentation. For gilthead sea bream there were no significant differences in the percentage of tail DNA between the control samples and sperm diluted 1:6 and cryopreserved (28.23 and 31.3% DNA(t), respectively). However, an increase in the sperm dilution rate produced an increase in the percentage of DNA fragmentation (41.4%). Our study demonstrates that cryopreservation can induce DNA damage in these species, and that this fact should be taken into account in the evaluation of freezing/thawing protocols, especially when sperm cryopreservation will be used for gene bank purposes.

Animals↗
PubMed1987

Monoclonal rat anti-human lymphocyte antibody Campath-1 binds to T and B lymphocytes but effectively lyses only T cells.

Binding and human complement-mediated T and B lymphocyte lysis were investigated in bone marrow samples obtained from 15 normal donors and 12 patients with a variety of malignant disorders undergoing marrow cryopreservation prior to autologous bone marrow transplantation. All marrow samples were obtained during remission except for one patient with neuroblastoma. The mononuclear cell fractions were collected and the distribution of B cell restricted markers (surface Ig and GP-70) and T cell surface markers (Leu-1 and rosettes with sheep red blood cells) were studied before and after marrow purging with Campath-1, a monoclonal rat anti-human lymphocyte antibody, and autologous serum as complement. Effective lysis of T lymphocytes (greater than 99.5%) was documented in all cases. However, although effective binding of Campath-1 to B lymphocytes was uniform, no effective lysis occurred. The data suggest that effective lysis of mature T lymphocytes can be accomplished while leaving normal B lymphocytes intact.

Antibodies, Monoclonal↗
PubMed2002

Whole blood cryopreservation in epidemiological studies.

Standardized and cost-effective biological sample collection, processing, and storage procedures are needed in large-scale epidemiological studies to provide material for testing a broad range of etiological hypotheses. One component of sample collection in the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial involves shipment of blood in acid-citrate-dextrose anticoagulant to a central processing laboratory, where 10% DMSO is added, and whole blood aliquots are cryopreserved. A single technician is able to routinely process 50-60 samples/day. Tests conducted to evaluate potential uses of cryopreserved whole blood showed successful EBV transformation (>90%, up to 20 months of storage). In addition, lymphocytes maintained good viability and stable T-cell:B-cell ratios, and T cells maintained the capacity to proliferate in response to solid phase anti-CD3/CD28 plus interleukin 2. Whole blood cryopreservation is a cost-effective approach to large-scale storage of viable cells in epidemiological studies.

Aged↗
PubMed1999

The effect of cryopreservation on the survivability, viability and motility of epididymal African buffalo (Syncerus caffer) spermatozoa.

The effect of cryopreservation on the viability and motility of epididymal African buffalo spermatozoa was studied in samples obtained from 17 and 13 animals in 1995 and 1996, respectively. Cryopreservation significantly reduced the viability and motility of the epididymal spermatozoa. The average percentage of live (+/- SE) spermatozoa declined significantly from 90.4 +/- 2.0% (1995) and 84.4 +/- 1.1% (1996) in fresh epididymal samples, to 57.0 +/- 2.0% and 56.3 +/- 1.1%, respectively, in frozen-thawed samples. The acrosomal integrity (+/- SE) of spermatozoa declined from 89.3 +/- 2.3% (1995) and 93.3 +/- 2.2% (1996) to 50.2 +/- 2.3% and 37.5 +/- 2.2%, respectively. In 1995, this effect was largely associated with the thermal equilibration prior to cryopreservation.

Acrosome↗
PubMedDate not supplied

Individual cryopreservation with dimethyl sulfoxide and polyvinylpyrrolidone of ejaculates and pooled semen of three avian species.

Artificial insemination (AI) has been used for avian reproduction due to the discovery of cryoprotectants extending its usefulness both in production of domestic fowl and conservation of wild species. The goal of this study was to assess the effect on domestic and wild fowl pooled semen and individual ejaculate cryopreservation with dimethyl sulfoxide (DMSO) and polyvinylpyrrolidone (PVP). Twenty ejaculates and twenty samples of pooled semen of roosters, pheasants and hawks were frozen in media containing DMSO or PVP. DMSO and PVP cryopreservation are equally effective both for ejaculates and pooled semen. Even PVP is a good alternative since no significant difference was found when compared to DMSO. The fertilizing capacity of fresh and cryopreserved pooled semen was analyzed through AI of hens and female pheasants. Similar fertility rates using DMSO, PVP or frozen-thawed samples demonstrated that reproduction is possible through the use of cryopreserved semen. In the case of female pheasants, the same values were obtained with both cryopreserved and fresh semen.

Animals↗
PubMedDate not supplied

A simplified testing system to evaluate performance after transplantation of human skin preserved in glycerol or in liquid nitrogen.

We have designed and tested a mouse recipient model for evaluation and direct comparison of skin-preservation procedures. Glycerolized skin was compared with cryopreserved and fresh cadaveric skin. Human skin samples were grafted on Balb/c mice, and primary take was evaluated after 4 and 7 days. The results demonstrate that although all grafted specimens were initially accepted as indicated by gross observations, histologic differences were evident and significant. Cryopreserved skin grafts performed better than did glycerolized skin even after a transplantation period as short as 4 days; this difference became even more pronounced after 7 days. Both methods of preservation provided a less successful product than did fresh viable cadaveric skin. However, for very short periods of grafting the performance of glycerolized skin might be considered adequate (79% compared with cryopreserved). Because the present study used an immunocompetent xenogeneic model, it is possible that the period of adequate allogeneic grafting will be prolonged in patients who are immunosuppressed. The present model is simple to use, requires a minimum of maintenance and expertise, and has inherent clinical relevance, because it is concerned primarily with the clinical performance of the skin. Thus it may be used as a quality control test for banked skin.

Animals↗
PubMed2001

The activity in ex vivo expansion of cord blood myeloid progenitor cells before and after cryopreservation.

A total of 50 human umbilical cord blood (UCB) samples were studied. The hematopoietic stem/progenitor (CD34+) populations were isolated from UCB mononuclear cells (MNC) by means of immunomagnetic separation. Double immunofluorescent staining of UCB CD34+ cells revealed that there was a high proportion (82.33 +/- 4.47%) of CD34+ cells co-expressing CD13, while the percentage of CD34+ CD33+ cells was much lower (22.17 +/- 3.35%). In contrast, for co-expressing lymphoid differentiation antigens, the proportion of CD34+CD38+ cells (38.34 +/- 6.09%) was relatively higher than that of CD34+CD10+ cells (11.52 +/- 1.24%) or CD34+CD2+ cells (9.84 +/- 2.30%). For stimulating the ex vivo expansion of UCB progenitor cells, no single hematopoietic growth factor (HGF) was efficacious when used alone, while combination of 4 HGFs, such as GM-CSF, G-CSF, IL-3, and SCF could induce a 55-fold increase in the myeloid progenitor cells, day-14 CFU-GM, in a short term of 7 days' liquid culture. Cryopreservation of UCB as MNC preparations at -196 degrees C could satisfactorily retain the number and activity of CD34+ cells. After thawing, a high recovery rate of about 80% CD34+ cells was obtained. When suspended in liquid cultures containing a combination of 4 HGFs, as shown above, the frozen cord blood progenitor cells could be well expanded, reaching a >50-fold increase in day-14 CFU-GM, which was very similar to that of the fresh UCB samples. In addition, a similar result was also seen in CFU-GEMM, indicating that after cryopreservation the recovered UCB progenitor cells retain an intact clonogeneic ability capable of efficiently responding to hematopoietic growth factors for ex vivo expansion.

ADP-ribosyl Cyclase↗
PubMedDate not supplied

Feasibility of refreezing human spermatozoa through the technique of liquid nitrogen vapor.

OBJECTIVE: To assess the feasibility of refreezing human semen using the technique of liquid nitrogen vapor with static phases. MATERIALS AND METHODS: Twenty samples from 16 subjects who required disposal of their cryopreserved semen were thawed, corresponding to 6 cancer patients and 10 participants in the assisted reproduction (AR) program. Samples were refrozen using the technique of liquid nitrogen vapor with static phases, identical to the one used for the initial freezing, and thawed again after 72 hours. We assessed the concentration of motile spermatozoa, total and progressive percent motility and spermatic vitality, according to criteria of the World Health Organization (WHO), as well as spermatic morphology according to the strict Kruger criterion, after the first and after the second thawing. RESULTS: We observed a significant decrease in all the parameters evaluated between the first and the second thawing. Median values for the concentration of motile spermatozoa decreased from 2.0x10(6)/mL to 0.1x10(6)/mL (p < 0.01); total percent motility from 42% to 22.5% (p < 0.01); progressive percent motility from 34% to 9.5% (p < 0.01); vitality from 45% to 20% (p < 0.01); and morphology from 5% to 5% (p = 0.03). There was no significant difference in the spermatic parameters between the cancer and assisted reproduction groups, both after the first and after the second thawing. We observed that in 100% of cases there was retrieval of motile spermatozoa after the second thawing. CONCLUSION: Refreezing of human semen by the technique of liquid nitrogen vapor allows the retrieval of viable spermatozoa after thawing.

Adult↗
PubMed2006

MHC class I and class II molecules are expressed in both human and mouse prostate tumor microenvironment.

BACKGROUND: There has been a determined search for therapies specifically aimed at eradicating tumor cells while leaving normal host cells unaffected. This goal can potentially be accomplished by engaging tumor antigen-specific T-cell repertoire to attack the tumor. A pre-requisite for a successful T-cell-mediated attack against tumors or pathogens is that the target tissues express major histocompatibility complex (MHC) molecules. Using newer anti-MHC class I and MHC class II antibody reagents, we re-examined the expression of MHC in both human and mouse prostate tumors and their microenvironments. METHODS: Using immunocytochemistry, we examined the expression of MHC class I, class II, and CD3 molecules on cryopreserved human and mouse prostate tumor samples. RESULTS: MHC class I molecules are expressed by the entire spectrum of different stages of both human and mouse prostate tumor cells. Additionally, cells of the hematopoietic lineage, dispersed in the tumor microenvironment, showed significant expression of MHC class II molecules. Human prostate tumors also show a significant infiltrate of CD3+ T cells. CONCLUSIONS: Expression of MHC class I and class II molecules within the prostate tumor microenvironment are consequential for T-cell-mediated immunotherapeutic approaches against prostate cancer.

Aged↗
PubMed2003

Tumor load in patients with follicular lymphoma post stem cell transplantation may correlate with clinical course.

The purpose of this study was to evaluate if the tumor load, as determined by a real-time quantitative PCR (RQ-PCR) assay, correlated with the clinical course of follicular lymphoma patients after stem cell transplantation (SCT). Cryopreserved bone marrow and/or peripheral blood samples obtained at different time intervals after SCT from 11 patients (seven allogeneic, T-cell depleted/four autologous) were tested for tumor load, as defined by t(14;18) positive cells/total cells, using RQ-PCR. None of the six patients who remained in remission had samples with a tumor load >0.01% after SCT, although fluctuating tumor loads of </=0.01% were observed in three of these patients. In contrast, four of the five patients (three allogeneic/two autologous) with relapsed/progressive disease had increasing tumor loads of >0.01% after SCT (0/6 vs 4/5, P<0.02, Fisher's exact). Our results suggest that RQ-PCR measurable tumor load >0.01% after SCT may correlate with relapsed/progressive disease. Prospective studies with greater numbers of cases are indicated to better determine the critical tumor load that predicts poor outcome after SCT with RQ-PCR.

Adult↗
PubMed1989

Transmission of sexually transmitted diseases by donor semen.

Therapeutic insemination by donor (TID) is being used with increasing frequency. Because many diseases, some of which are lethal, can be transmitted through semen, the American Fertility Society established guidelines for use of donor sperm. They limit TID to cases of male infertility or hereditary/genetic disorders. Donor selection requires good health and absence of genetic abnormalities; criteria for semen including normal sperm motility, concentration, and normal morphology, and blood screening for infectious agents. Human immunodeficiency virus (HIV) testing should be performed initially in donors for fresh semen inseminations. If positive, the assay is verified with a Western blot test; if negative, the donor should be screened at 6-month intervals. Frozen samples should not be used until the 180 day reevaluation of the donor. Many studies show higher pregnancy rates using fresh rather than frozen semen samples for insemination. New methods of cryopreservation minimize the deleterious effects of freezing. If these effects, namely decreased sperm motility and impaired penetration ability, are eliminated, pregnancy rates can be expected to rise. Frozen semen is preferable because it allows time for sexually transmitted diseases to manifest themselves and for specimens from those donors to be rejected prior to use.

Cryopreservation↗
PubMed1996

Novel CD44 messenger RNA isoforms in human thyroid and breast tissues feature unusual sequence rearrangements.

CD44 is a family of cell surface proteins implicated in adhesion interactions and tumor metastasis. Multiple CD44 mRNA isoforms arise from alternative splicing of variant exons (termed v1-v10). We recently discovered a novel CD44 mRNA isoform in human papillary thyroid cancers featuring a junction between subsegments of exons 4 and 13 (v8). The sequence ACAG was repeated at both the donor and acceptor sites in the genomic DNA (G. Ermak et al., Cancer Res., 56: 1037-1042, 1996). We used reverse transcription-PCR to characterize expression of this isoform in a panel of thyroid lesions. In addition, we assayed three cryopreserved human breast cancers and two samples of normal breast tissue (from female subjects who had undergone cosmetic mammoplasty) to determine whether a similar isoform is present in breast carcinomas. Levels of the novel isoform were up-regulated in 88% of the goiters, adenomas, and papillary cancers, but were undetectable in cases of thyroiditis and absent or low-level in four samples of normal thyroid tissue. The three breast cancers each yielded a 546-bp PCR product that was not detected in normal breast tissue. The PCR product from one of the breast cancers was cloned, and sequence analysis revealed a novel mRNA isoform featuring a junction between exon 3 and an internal site within exon 13 (v8). The sequence GCTTCAG was repeated at both the donor and acceptor sites in the genomic DNA. These results show that human thyroid and breast tissues contain novel CD44 mRNA isoforms featuring unusual rearrangements at repeated sequences. Further studies are warranted to determine whether the expression of this class of isoforms correlates with growth status.

Breast↗
PubMed1985

Reduced penetration of zona-free hamster ova by cryopreserved human spermatozoa.

The effect of cryopreservation on human sperm fertilizing potential was assessed by using the human sperm penetration into zona-free hamster ovum test. Semen samples from 12 fertile men were compared before and after cryopreservation for motility, sperm penetration rate, and number of sperm cells incorporated per oocyte. In fresh samples sperm concentration was 102 +/- 51 X 10(6) cells/ml, motility 66 +/- 14%, penetration rate 77.8 +/- 19%, and sperm incorporation 4.3 +/- 3.9 sperm per ovum. Frozen-thawed sperm cells showed a marked reduction of 61 +/- 21% (p less than 0.001) in motility. Penetration rate was reduced by 53 +/- 34% (p less than 0.01), and sperm incorporation dropped by 50 +/- 28% (p less than 0.05). Despite this substantial reduction in all three parameters, 75% of the samples maintained a penetration rate exceeding 14%, which is the lower limit for fertile semen. For the individual subject the decrease in sperm motility did not reflect actual fertility potential as expressed by its ability to penetrate zona-free hamster ova. These findings are related to morphological and biochemical changes in frozen-thawed semen and apparently are correlated with the decrease in pregnancy rates after cryopreservation. This test may be a valuable supplement to routine microscopic semen analysis for semen cryopreservation candidates.

Animals↗
PubMed1980

Lymphocyte surface markers and cytotoxicity following cryopreservation.

The effects of cryopreservation (CP) on lymphocyte subpopulation distribution and functional activity in blastogenic and cytotoxicity assays were tested. Peripheral blood lymphocytes (PBL) from 12 healthy human donors were obtained by Ficoll-Hypaque separation. Half of each sample was tested fresh, while the other half was cryopreserved and then thawed and tested the same day. Each sample of CP-PBL was compared to fresh PBL from the same donor in simultaneous assays. Following CP there was a significant reduction in the percentage of E, EA gamma, and EA mu rosette-forming cells with a reciprocal increase in EAC rosette-forming cells. The blastogenic response to alloantigens was stable following CP while blastogenesis in unstimulated control cultures was significantly reduced. Mixed lymphocyte culture (MLC)-induced cell-mediated lympholysis (CML) was consistently and significantly diminished by CP. Cytotoxicity in 4 h chromium release NK (K562), ADCC, and LDCC assays was also significantly diminished by CP. In contrast, cytotoxicity was unaffected in an 18 h cytotoxicity assay against adherent cultured melanoma target cells.

Cell Survival↗
PubMed1989

Re-establishing a life cycle of Schistosoma mansoni from cryopreserved larvae.

To study the feasibility of re-establishing a life cycle of Schistosoma mansoni (NMRI strain) from cryopreserved larvae, schistosomules were suspended in the cryoprotectant 1,2-ethanediol and cryopreserved in liquid nitrogen. Mice were injected intramuscularly with samples thawed after 3 days, 3 wk, or 6 mo in liquid nitrogen storage. Two to 5% of the cryopreserved larvae and approximately 18% of corresponding unfrozen control larvae developed into adult worms. Infectivity did not decrease as a function of storage time. The adult worms showed no structural damage or changes in overall size and morphology when examined by light and transmission electron microscopy. Female worms derived from cryopreserved larvae had the same or slightly elevated egg production as controls, but tissue egg distributions were comparable. Subsequent passages through Biomphalaria glabrata snails and mice revealed no difference in snail prepatent death rate, percentage of snails infected, cercarial production per snail, or cercarial infectivity.

Animals↗