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PubMed1999

Fertility potential of individual sperm donors.

OBJECTIVE: To study the fertility potential of individual semen donors with ejaculates of optimal as compared to suboptimal quality. METHODS: 363 semen donations were obtained from 11 donors between January 1993 and September 1997. 270 samples were cryopreserved and 1,399 straws obtained from 120 ejaculates were used in 495 insemination cycles. RESULTS: 52 pregnancies were achieved in 128 recipients (40.6%). No significant differences were found between donors of high fecundability and those of low fecundability regarding sperm parameters. The mean donor fecundability index was 10.5%. The optimal and suboptimal samples yielded similar results. Donors with suboptimal semen quality had an increased number of samples rejected after thawing. CONCLUSION: Traditional semen analysis parameters do not differentiate high from low fecundability donors. Suboptimal results of the semen analysis should not exclude potential donors from an artificial insemination donor (AID) program.

Cryopreservation↗
PubMed2003

[Azoospermia : In vitro fertilization results, using testicular biopsy and cryopreserved spermatozoa].

Testicular sperm extraction (TESE) by open testicular biopsy, has become, since the introduction of the ICSI in 1992, the corner stone in treatment for azoospermia. This study reports the outcome of patients with open testicular biopsy for ICSI procedure indicated by azoospermia. 16 patients with azoospermia (12 obstructive and 4 non obstructive). The open testicular biopsy was performed prior the controlled ovarian hyperstimulation (COH) protocols of their couples, in two of the 16 patients no sperm were found in the biopsy, and the other 14 patients were admitted to 18 ICSI cycles, and the testicular samples were cryopreserved until oocyte capture. The controlled ovarian hyperstimulation (COH) was individualized in each case in accordance to the characteristics of each patient: 12 patients were used the long luteal phase GnRH agonist protocol, ultrashort protocol (Flare up) in one patient, GnRH antagonist in four, and the last one with endometrial preparation for cryopreserved embryo transfer. The mean of oocytes recovered was 11.71 +/- 7.24 oocytes per patient with a recovery rate of 72.64%. The fertilization rate was 49.35 +/- 29.24 and were transfered an average of 3 embryos per patient with a range between 1 and 5 embryos. In 18 cycles, 7 pregnancies occurred, four singles, one twin, one triple and one biochemical pregnancy. In conclusion the testicular sperm extraction (TESE) by open testicular biopsy is a good option for patients with azoospermia usually it achieves an adequate sample of testicular tissue with high possibilities of sperms recovery, in our study we obtained appropriate sperms in fourteen of sixteen patients for the ICSI procedure with good fertilization and pregnancy rates.

Adult↗
PubMed1999

Higher-quality human sperm in a sample selectively attach to oviduct (fallopian tube) epithelial cells in vitro.

OBJECTIVE: To determine if sperm attachment to oviduct epithelial cells (OEC) in vitro is selective for higher quality sperm and if the system requires homologous species OEC. DESIGN: Controlled prospective study with outcomes assayed by a technician blind to sperm treatment groups. SETTING: An academic research laboratory. PATIENT(S): Experiment 1: normospermic donors with children (4 donors, 7 ejaculates). Experiment 2: cryopreserved donor samples (4 donors). INTERVENTION(S): Semen collection by masturbation after 48 hours of abstinence. MAIN OUTCOME MEASURE(S): Experiment 1: sperm assays of motility, morphology, membrane integrity, and capacitation status. Experiment 2: sperm chromatin (DNA) integrity and condensation. RESULT(S): Experiment 1: sperm not attaching to OEC had lower motility, more membrane disruptions, and more acrosome reactions than did control sperm. This selectivity was equivalent for sperm in coculture with all OEC types. Experiment 2: sperm attached to OEC had fewer abnormalities in chromatin structure compared with sperm that were not attached. CONCLUSION(S): Selective attachment of functionally superior sperm to OEC is likely important during sperm reservoir formation in vivo and may be exploitable in vitro as a method to isolate high-quality sperm for clinical procedures. Such a system does not require human origin OEC.

Cryopreservation↗
PubMed1995

Effect of cryopreservation on semen quality in patients with testicular cancer.

OBJECTIVES: Current techniques in cryopreservation of human semen substantially decrease sperm quality. In addition, the pregnancy rate using cryopreserved sperm obtained from testicular cancer patients is lower than when sperm from normal fertile men is used. However, it is still unclear whether cryopreserved sperm from these patients is inherently defective or if the sperm loses its motility after thawing. This study was undertaken to assess the effect of cryopreservation on the quality and motion characteristics of semen from patients with testicular cancer before definitive therapy compared with semen from normal volunteers. METHODS: We compared the sperm quality before and after cryopreservation in samples from 34 patients with testicular cancer and 30 normal volunteers who were referred for sperm banking over a 7-year period. The effects of cancer stage and histologic type on various semen parameters were also examined. A computer-assisted semen analysis was performed before and after cryopreservation on each specimen. The nitrogen vapor technique using Test yolk buffer with glycerol as a cryoprotective agent was used for cryopreservation. The motile sperm count and motion characteristics (motility, velocity, linearity, amplitude of the lateral head movement, motility index) were analyzed before and after cryopreservation and compared between the groups. RESULTS: Semen quality did not significantly differ among patients with Stage I, II, or III cancer. However, semen quality tended to be poorer at higher cancer stages. In general, semen quality was better among patients with pure seminomas than with pure embryonal tumors; quality was worst among patients with mixed germ cell tumors. However, 71.4% of patients with mixed tumors presented with Stage III disease, whereas all patients with seminomas presented with Stage I disease. Significant differences were also seen in prefreeze motility (median, 42% [interquartile range, 24 to 51] versus 60.5% [range, 49 to 73]; P = 0.0004) and motile sperm count (6.7 x 10(6)/mL [range, 3.4 to 14.4] versus 50.0 [range, 24.6 to 72.0]; P = 0.0001) in patients compared with controls, respectively. The motile sperm count and percent motility significantly decreased in both patients and controls after cryopreservation (P = 0.0001). However, the percentage decline in motile sperm count and motion characteristics after cryopreservation did not differ significantly between patients and controls (P > 0.01). CONCLUSIONS: We conclude that the effect of cryopreservation on sperm quality in patients with testicular cancer is identical to its effect on sperm from normal fertile men. Differences in values after preservation are explained by poor semen characteristics before freezing; semen quality declines with more extensive disease. Stage I patients also had poorer quality than control subjects. Thus, we recommend that routine sperm banking be encouraged among all patients with testicular cancer before the initiation of specific medical treatment. We also recommend that future efforts be focused on improving the technique of sperm banking.

Adolescent↗
PubMed2001

Decrease of L-selectin expression on human CD34+ cells on freeze-thawing and rapid recovery with short-term incubation.

In the homing process of hematopoietic progenitor cells (HPCs) to bone marrow, several adhesion molecules play important roles. However, HPCs are subjected to dramatic alteration of freeze-thawing that could affect these molecules. In this study, we investigated the effect of cryopreservation on the expression of adhesion molecules on HPCs. The expression of different adhesion molecules on CD34+ cells was examined by flow cytometry using an immunofluorescence technique. Changes in expression before and after cryopreservation, and that of L-selectin on addition of dimethylsulfoxide (DMSO) or with serum incubation, were investigated. The relationship between expression and function was also determined using a transmigration assay system. L-selectin expression on CD34+ cells derived from mobilized peripheral blood (MPB), bone marrow (BM), and umbilical cord blood (CB) was significantly decreased from 37% to 23%, 48% to 11%, and 67% to 19%, respectively, by the freeze-thawing process, while the expression of other adhesion molecules was not appreciably changed. Within 30 minutes of incubation with DMSO, L-selectin expression on CD34(+) cells significantly decreased from 65% to 22%. Furthermore, this was completely prevented by the matrix metalloproteinase (MMP) inhibitor, KB-R8301, indicating that the loss of L-selectin was due to shedding mediated by an MMP. To determine if L-selectin expression can be upregulated after cryopreservation, thawed samples were cultured overnight with serum. Values were observed to return to or rise higher than those of the fresh samples, this being particularly rapid and pronounced when the CD34+ cells were cocultured with the human BM stromal cell line, HS-27A. Therefore, this adhesion molecule could possibly be restored in vivo after transplantation in a way similar to the in vitro case. Despite considerable damage to HPCs during cryopreservation, changes in these cells are reversible, in line with successful restoration of hematopoiesis after transplantation.

Antigens, CD34↗
PubMed2004

Decrease in glutathione content in boar sperm after cryopreservation. Effect of the addition of reduced glutathione to the freezing and thawing extenders.

Although glutathione content in boar spermatozoa has been previously reported, the effect of reduced glutathione (GSH) on semen parameters and the fertilizing ability of boar spermatozoa after cryopreservation has never been evaluated. In this study, GSH content was determined in ejaculated boar spermatozoa before and after cryopreservation. Semen samples were centrifuged and GSH content in the resulting pellet monitored spectrophotometrically. The fertilizing ability of frozen-thawed boar sperm was also tested in vitro by incubating sperm with in vitro matured oocytes obtained from gilts. GSH content in fresh semen was 3.84 +/- 0.21 nM GSH/10(8) sperm. Following semen cryopreservation, there was a 32% decrease in GSH content (P < 0.0001). There were significant differences in sperm GSH content between different boars and after various preservation protocols (P = 0.0102 ). The effect of addition of GSH to the freezing and thawing extenders was also evaluated. Addition of 5 mM GSH to the freezing extender did not have a significant effect on standard semen parameters or sperm fertilizing ability after thawing. In contrast, when GSH was added to the thawing extender, a dose-dependent tendency to increase in sperm fertilizing ability was observed, although no differences were observed in standard semen parameters. In summary, (i) there was a loss in GSH content after cryopreservation of boar semen; (ii) addition of GSH to the freezing extender did not result in any improvement in either standard semen parameters or sperm fertilizing ability; and (iii) addition of GSH to the thawing extender resulted in a significant increase in sperm fertilizing ability. Nevertheless, future studies must conclude if this is the case for all boars. Furthermore, since addition of GSH to the thawing extender did not result in an improvement in standard semen parameters, this suggests that during the thawing process, GSH prevents damage of a sperm property that is critical in the fertilization process but that is not measured in the routine semen analysis.

Animals↗
PubMed1995

Cytogenetic analysis of thyroid tumors after cryopreservation.

Current cytogenetic evaluation of solid tumors is performed on fresh tissue specimens requiring on-call tissue culture facilities. The application of cryopreservation to tumor samples prior to cytogenetic analysis allows collection of tumors to a desired sample size. We evaluated methods of cryopreservation for their effects on growth potential from 11 benign thyroids and one papillary thyroid cancer. Mitotic indices and thyroglobulin expression applying imunocytology were analyzed. Compared to fresh tumors, the revived tumor samples showed unaltered thyroglobulin expression. A statistically significant (p < 0.004) prolongation to develop mitotic activity occurred in samples received after the freezing of dispase digested tissues, but not in samples frozen as thinly cut pieces. In addition, the data show that cytogenetic analysis at the 400-band level can be achieved in cryopreserved thyroid tissues.

Carcinoma, Papillary↗
PubMed1999

Are cytokines and growth factors responsible for the detrimental effects of hydrosalpingeal fluid on pregnancy rates after in vitro fertilization-embryo transfer?

OBJECTIVE: To characterize the secretion of cytokines and growth factors in hydrosalpingeal fluid. DESIGN: Retrospective analysis. SETTING: Hospital-based infertility practice. PATIENT(S): Ten infertile women who underwent laparoscopic aspiration of their hydrosalpingeal fluid before salpingectomy or neosalpingostomy. INTERVENTION(S): Samples were cryopreserved, then thawed and centrifuged to remove cellular debris. MAIN OUTCOME MEASURE(S): The supernatants were analyzed for the presence of human interferon-gamma, epidermal growth factor, transforming growth factor-beta2, and tumor necrosis factor-alpha by quantitative enzyme immunoassay kits. RESULT(S): Interferon-gamma and transforming growth factor-beta2 were not detected in any of the hydrosalpingeal fluid samples. Epidermal growth factor was present in 5 of 10 hydrosalpingeal fluid samples, with a mean (+/- SE) concentration of 26.7+/-11.4 pg/mL. Tumor necrosis factor-alpha was detected in 7 of 10 samples, with a mean (+/- SE) concentration of 6.2+/-3.6 pg/mL. Three of the 10 samples contained both tumor necrosis factor-alpha and epidermal growth factor. CONCLUSION(S): For the first time, we described the absence of interferon-gamma and transforming growth factor-beta2, and the presence of epidermal growth factor and tumor necrosis factor-alpha in human hydrosalpingeal fluid. Because the fundamental role of the human fallopian tube is secretory in nature, the alteration in substances secreted from the tubal epithelium that reflux into the uterine cavity may explain the deleterious effects that hydrosalpingeal fluid has on pregnancy rates after IVF-ET.

Body Fluids↗
PubMed2003

Lack of detectable human immunodeficiency virus type 1 superinfection during 1072 person-years of observation.

We examined consecutive protease (PR) and reverse transcriptase (RT) sequences from human immunodeficiency virus (HIV) type 1-infected individuals, to distinguish changes resulting from sequence evolution due to possible superinfection. Between July 1997 and December 2001, >/=2 PR and RT samples from 718 persons were sequenced at Stanford University Hospital. Thirty-seven persons had highly divergent sequence pairs characterized by a nucleotide distance of >4.5% in PR or >3.0% in RT. In 16 of 37 sequence pairs, divergence resulted from the loss of mutations during a treatment interruption or from the gain of mutations with reinstitution of treatment. tat and/or gag sequencing of HIV-1 from cryopreserved plasma samples could be performed on 15 of the 21 divergent isolate pairs from persons without a treatment interruption. The sequences of these genes, unaffected by selective drug pressure, were monophyletic. Although HIV-1 PR and RT genes from treated persons may become highly divergent, these changes usually are the result of sequence evolution, rather than superinfection.

California↗
PubMed1995

Density separation of umbilical cord blood and recovery of hemopoietic progenitor cells: implications for cord blood banking.

Umbilical cord blood (CB) has been evaluated as a potential source of hematopoietic stem cells suitable for clinical use in the transplantation setting. Previous reports have documented a significant loss of progenitor cells by any manipulation other than cryopreservation. We have evaluated the feasibility of fractionating and cryopreserving CB samples with minimal loss of progenitor cells. We have compared various separation procedures based on different density gradients in the attempt to obtain the highest depletion of red blood cells (RBC) while maintaining the highest recovery of progenitor cells. We compared three different densities of Percoll (1.069 g/ml, 1.077 g/ml, 1.084 g/ml), sedimentation over poligeline (Emagel ) and sedimentation over poligeline followed by separation over Ficoll/Hypaque (F/H). Separated samples (n = 25) were analyzed for recovery of CD34+ cells and progenitor cells (CFU-GEMM, BFU-E, CFU-GM). Separation by sedimentation over poligeline followed by F/H allowed the highest depletion of RBC (hematocrit of the final cellular suspension 0.4 +/- 0.1%) while maintaining high recovery of CD34+ cells (85.3 +/- 5.6%) and total recovery for CFU-GEMM, BFU-E and CFU-GM. After cryopreservation, recovery of clonogenic progenitors was 82% for CFU-GEMM, 94% for BFU-E, 82% for CFU-GM and 90% for colony-forming units (CFUs) after five weeks of long-term culture (LTC). We further evaluated the effect of stem cell factor (SCF) on the in vitro growth of hemopoietic progenitors and on replating efficiency.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigens, CD↗
PubMed1996

Density separation and cryopreservation of umbilical cord blood cells: evaluation of recovery in short-and long-term cultures.

The clonogenic capacity of human umbilical cord blood (UCB) has been evaluated in several studies, which have shown high numbers of primitive hematopoietic progenitor cells. Recently, UCB progenitor cells were demonstrated to possess significant advantages over bone marrow (BM) in terms of proliferative capacity and immunologic reactivity. Therefore, UCB has been considered an attractive source of hematopoietic stem cells for both research and clinical applications. Previous reports have documented a significant loss of progenitor cells by any manipulation other than cryopreservation. We have evaluated the feasibility of fractionating and cryopreserving UCB samples with minimal loss of progenitor cells. We compared separation over three different densities of Percoll (1.069, 1.077 and 1.084 g/ml), sedimentation over poligeline (Emagel), and sedimentation over poligeline followed by separation over Ficoll/Hypaque (F/H). Separated samples (n = 25) were analyzed for recovery of CD34+ cells and progenitor cells (CFU-GEMM, BFU-E, CFU-GM). Separation by sedimentation over poligeline followed by F/H allowed the highest depletion of RBCs (hematocrit of the final cellular suspension 0.4 +/- 0.1%), while maintaining a high recovery of CD34+ cells (85.3%) and total recovery of CFU-GEMM, BFU-E and CFU-GM. After cryopreservation, recovery of clonogenic progenitors was 82% for CFU-GEMM, 94% for BFU-E, 82% for CFU-GM and 90% for colony-forming units after 5 weeks of longterm culture. Moreover, the presence of Stem cell factor significantly increased CFU-GEMM (14 +/- 4 vs. 49 +/- 5, p < or = 0.0005) and CFU-GM (112 +/- 18 vs. 178 +/- 19, p < or = 0.025), but not B FU-E (42 +/- 7 vs. 53 +/- 7, p < or = 0.375) growth. In conclusion, RBC depletion of UCB can be accomplished with minimal loss of committed and primitive hematopoietic progenitors. This procedure may have important implications in the large-scale banking of UCB and in ex vivo expansion/gene therapy protocols.

Antigens, CD34↗
PubMedDate not supplied

Glass wool filter preparation of cryopreserved spermatozoa.

Oligozoospermic and asthenozoospermic semen ejaculates, as well as cryopreserved sperm samples prepared by the wash and swim-up procedure often result in unsatisfactory sperm recovery rates. In this study the glass wool filter and the wash and swim-up preparation procedures were compared on the basis of their 'effective' (number of live sperm per millilitre) recovery rates. The glass wool filter procedure consistently produced significantly (P = 0.0002) higher viable sperm concentrations, making it the preferred method for the preparation of cryopreserved sperm to be used in assisted reproduction techniques. The use of this preparation procedure has also been shown to have no adverse affect on the fertilizing potential of human spermatozoa in our unit.

Cryopreservation↗
PubMed2002

Declared hepatitis C screening strategies in blood recipients in French hospitals.

AIMS OF THE STUDY: In spite of official recommendations and measures in France, screening strategies of hepatitis C performed in the field of transfusion are not clearly known. The aim of this study is to describe the screening strategies before and after the current year of the transfusion in blood recipients in several French medical departments and hospitals. MATERIALS AND METHODS: A qualitative study using the key informant technique was carried out. A sample of 179 departments and 64 hospitals in charge of patients transfused with low or high-volumes of homologous blood products was constituted. The key informants were asked about the number of homologous blood products, the number of recipients transfused in the hospital, the volume of transfusion performed, the existence of a single defined screening strategy, the time of prescription of the biological tests (before or after transfusion), the tests performed on cryopreserved blood samples, and the indications of the transfusion. RESULTS: The main screening strategy was HCV serology (second or third generation of enzyme immunoassays) with transaminase assessments before and after transfusion in 14% of the declared screening strategies. Screening tests were more frequently prescribed after transfusion, in at least 64% of the declared screening strategies according to the volume of transfusion. HCV serology was the common test prescribed in 61 and 50% of the screening strategies for low and high-volume transfusion respectively. The screening strategies showed a large heterogeneity combining HCV serology, transaminase assessment, before or after transfusion. CONCLUSION: A great heterogeneity of screening strategies was found. The most frequent was HCV serology with transaminase assessment before and after transfusion. Recommendations on screening strategies are needed in order to limit practice heterogeneity. This study will help building a cost-efficacy model in order to guide public health decision making.

Alanine Transaminase↗
PubMed1995

TEST-yolk media and sperm quality.

Media prepared with egg yolk and two buffers, TES and Tris, called TEST-yolk have been shown to have beneficial effects on the survivability, fertilizing capacity and storage potential of human spermatozoa. Egg yolk lipoproteins are the critical compounds for the beneficial effects, with a synergistic effect due to the TES-Tris buffer component. TEST-yolk media have been used in the sperm penetration assay, the hemizona assay, sperm preparation for clinical in-vitro fertilization, artificial insemination with homologous spermatozoa, cryopreservation, sperm samples with a positive antisperm antibody test, and preparation for techniques requiring capacitated spermatozoa. Few harmful consequences of TEST-yolk have been reported, although controlled trials are required to evaluate therapeutic effects in the treatment of male infertility.

Cryopreservation↗
PubMed1990

Comparison of semen quality in young and mature Holstein bulls measured by light microscopy and flow cytometry.

Random samples of cryopreserved, milk-extended semen, collected from 20 Holstein bulls at about 14 mo of age (young) and again at about 4 yr of age (mature), were evaluated at thawing and during 3-h incubation to compare semen quality of young versus mature bulls. Evaluation by differential interference contrast microscopy showed greater proportions of cytoplasmic droplets in semen from young versus mature bulls. Mature bulls exhibited greater proportions of intact acrosomes in freshly thawed semen than did young bulls. Evaluation of sperm chromatin structure by flow cytometry after staining with acridine orange showed lower values for mature versus young bulls, indicating resistance of DNA in nuclear chromatin to acid denaturation increased with age. Correlations between ages for most sperm morphology, acrosome integrity, and flow cytometry variables were high and positive. Nonreturn rate for young bulls was positively related to morphologically normal sperm and acrosomal integrity and negatively related to flow cytometry traits. Results suggest semen quality of young bulls was related to subsequent quality as mature bulls. With flow cytometry, differences were detected between semen samples that were not evident with light microscopy.

Acrosome↗
PubMed1998

Coculture of human sperm with bovine oviduct epithelial cells decreases sperm chromatin structural changes seen during culture in media alone.

OBJECTIVE: To compare sperm chromatin structural changes seen in media only culture or in coculture with bovine oviduct epithelial cells. DESIGN: Three freshly ejaculated and three cryopreserved sperm samples in media culture or in oviduct epithelial cell coculture. Sperm in each treatment were evaluated by the sperm chromatin structure assay during a 72-hour time course. SETTING: An academic research laboratory. PATIENT(S): Normospermic donors with children. INTERVENTION(S): Semen collection through masturbation after 48 hours of abstinence. MAIN OUTCOME MEASURE(S): The sperm chromatin structure assay using flow cytometry to detect the susceptibility of sperm in either treatment to denaturation of DNA in situ. RESULT(S): The sperm chromatin structure assay data differed for sperm type (fresh or cryopreserved), over time, and between treatments within 6 hours of culture. In oviduct epithelial cell coculture, fresh sperm chromatin structure assay values for fresh sperm were stable, whereas in control medium higher chromatin degeneration levels were seen by 10 hours. For cryopreserved sperm, chromatin degeneration had increased by 1 hour postthaw in both treatments, although levels were higher in the control treatment thereafter. CONCLUSION(S): Sperm chromatin structural changes occur over time in culture. Such changes were observed within 2 hours for cryopreserved sperm. Coculture of sperm with oviduct epithelial cells results in a stabilizing effect for sperm against chromatin changes.

Animals↗
PubMed2004

Viability and histologic structure of porcine valves after cryopreservation.

BACKGROUND: Increased awareness of the limitations of current cardiac valve substitutes has generated a renewed interest in the use of allograft valves. The effects of currently used preservation techniques on the viability of the valve leaflets and the longevity of the implantation however remain controversial. The objective of this study is to analyze the influence of ischemic time, sterilization methods with or without fungicides, and storage procedures on the viability of the valve leaflets and on the histologic structure of the arterial wall, valve leaflet, and myocardium. METHODS: The tissue sources were hearts from 40 pigs with 1 hour of warm ischemic time. The aortic and pulmonary valves were dissected after 2 or 24 hours of cold ischemic time. They were stored in antibiotic solution for 20 hours at 4 degrees C with or without an antifungal agent. The samples were cryopreserved using a programmed temperature decrease method. After 1 week of storage in a liquid nitrogen tank, either in a gas or a liquid phase, the cardiac valves were slowly thawed and examined. RESULTS: Pulmonary valves showed greater viability than aortic valves. Decreased cellular viability was observed independent of cold ischemic time, treatment with amphotericin B, or the storage method used. Treatment with or without amphotericin B had no influence on cellular viability. Conversely it was observed that there was greater cellular viability among those valves stored in a liquid phase. As far as the histologic structure of the valve is concerned we did not observe any influence either in the treatment with amphotericin B or the storage method used although it was observed that reduction of the cold ischemic time minimized histologic injury. CONCLUSIONS: Optimization of preservation methods may decrease the negative effects of cryopreservation on cell viability and histologic structure of the valve.

Animals↗