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At least 1,603 records · Page 89Linked to original sources
PubMed1998

Investigation of fertilizing capacity of cryopreserved spermatozoa from patients with cancer.

PURPOSE: There are few published reports concerning fertilization and pregnancy outcomes achieved with cryopreserved spermatozoa from cancer patients. Controversy exists regarding the value of sperm banking for these patients before therapy, whether the spermatozoa are viable after long-term storage and whether they can fertilize the ovum. We assess fertilization and pregnancy outcomes achieved with cryopreserved spermatozoa from cancer patients using assisted reproductive techniques. MATERIALS AND METHODS: We studied 10 cancer patients who transferred cryopreserved semen specimens from our sperm bank to outside in vitro fertilization programs for assisted reproductive technique. Of these patients 5 had Hodgkin's disease, 2 testicular cancer, 1 leukemia and 2 prostate cancer. The length of specimen storage ranged from 14 to 135 months (median 49, interquartile range 24 and 82). RESULTS: The median pre-freeze motility was 44% (interquartile range 36 and 55%) and the median total sperm count was 31.1 x 10(6) (interquartile range 6.3 and 53.9 x 10(6)). At 24 hours after banking the median post-thaw motility was 11% (interquartile range 6 and 35%) and the median total sperm count was 6.6 x 10(6) (1.2 and 17.1 x 10(6)). A total of 18 cycles of assisted reproductive technique were performed among 10 couples with an overall pregnancy rate of 50% per couple, with 2 deliveries, 1 ongoing pregnancy and 2 miscarriages. The pregnancy rate per cycle of in vitro fertilization and intracytoplasmic sperm injection was 36.4% with an implantation rate of 13%. CONCLUSIONS: These results indicate that poor quality cryopreserved spermatozoa from cancer patients, irrespective of the length of storage, may provide successful results with the latest micromanipulative techniques such as intracytoplasmic sperm injection.

Adolescent↗
PubMed1998

Gene expression by single Reed-Sternberg cells: pathways of apoptosis and activation.

Although Hodgkin's disease is highly responsive to treatments that cause apoptosis, it remains resistant to the physiological mechanisms intended to cause cell death. Presumably, the Reed-Sternberg cell defies endogenous apoptosis, persists, accumulates, and manifests the malignant disorder seen clinically. The Reed-Sternberg cell expresses several members of the tumor necrosis factor receptor superfamily. This family of receptors is involved in both activation and proliferation of cells, as well as either protection from or initiation of apoptosis in cells expressing these surface proteins. Signals from these receptors affect transcription. We reasoned that the activation state and resistance to apoptosis of Reed-Sternberg cells might be attributable to dysregulation of genes controling these processes. To determine gene expression by Reed-Sternberg cells, we developed a method of micromanipulation, global reverse transcription, and the reverse transcription-polymerase chain reaction and applied it to 51 single Reed-Sternberg cells and their variants from six cases of Hodgkin's disease. This report analyzes the gene expression of bcl-xs, bcl-xl, bax-alpha, bax-beta, fadd, fas, fas ligand (fas L), ice, TNF-alpha, TNF-beta, TNFR1, TNFR2, TRAF1, TRAF2, TRAF3, cIAP2, and tradd at the level of mRNA in the single Reed-Sternberg cells and their variants. The findings here suggest a molecular mechanism for the activated state and in vivo survival occurring in untreated Reed-Sternberg cells of Hodgkin's disease.

Apoptosis↗
PubMed1998

Single-cell analysis of the t(14;18)(q32;q21) chromosomal translocation in Hodgkin's disease demonstrates the absence of this translocation in neoplastic Hodgkin and Reed-Sternberg cells.

Using the polymerase chain reaction (PCR) technique and total DNA extracts of Hodgkin's disease (HD)-involved lymph nodes, the t(14;18)(q32;q21) translocation was detected in 37 of 115 (32.2%) cases studied. No correlation was found between the presence of this translocation and bcl-2 protein expression in Hodgkin and Reed-Sternberg (HRS) cells detected by immunohistochemistry in 58 of 96 (60.4%) cases. To identify the cells carrying the t(14;18) translocation, single-cell DNA from HRS cells isolated by micromanipulation from frozen tissue sections of lymph nodes was investigated by PCR amplification. Eleven cases showing a positive band of the same size in at least two of five PCR experiments performed on the same total DNA extract were selected for single-cell PCR. We postulated that this repeated successful amplification could be indicative of the presence of the t(14;18) translocation in the neoplastic HRS cells. Single cells from frozen tumor sections of the t(14;18)-positive OCI LY8 cell line grafted into nude mice served as a positive control. The bcl-2/JH rearrangement, involved in this translocation, could be amplified from single-cell DNA of the latter tumor, whereas, in all of the HD cases, HRS cells were found to be negative. We conclude that the t(14;18) translocation is not localized in HRS cells, but in nonmalignant B bystander lymphocytes, admixed with these neoplastic cells.

Animals↗
PubMed1998

[A new approach to the analysis of complex chromosomal rearrangements in cell hybrids].

The chromosomal complements of somatic cell pig-mink hybrids was determined by a new approach. This approach includes microdissection of metaphase chromosomes, generation of chromosome and region-specific DNA libraries, and fluorescence in situ hybridization of these libraries with pig lymphocyte chromosomes. The studied hybrid cells were shown to contain two small acrocentric chromosomes and a microchromosome of porcine origin. Identification of these chromosomes by differential GTG-staining was impossible. Chromosome isolation by a micromanipulation technique followed by DNA amplification in TOPO-DOP polymerase chain reaction provided chromosome-specific DNA libraries of the rearranged chromosomes. Based on these libraries, the labeled DNA probes were prepared and hybridized to pig chromosomes. This allowed us to determine the origin of the material contributing to the hybrid cell chromosomes. One of these chromosomes contained five pig chromosomal regions: 15cen-q2; 6q21-q23; 13q21; 13q22; 7q25-qter, while the other contained the following pig chromosomal regions: 4p12-p13; 16q12-q14; 12pter-p15. The microchromosome contained the Xp11-Xq11 region. The minimal size of the revealed chromosomal regions was about 3 to 4 x 10(6) bp. Segregation analysis of the thymidine kinase gene 1 (TK1), which was earlier localized to the pig 12p region, and the hybrid cell pig chromosomes in the hybrid subclones suggested that TK1 gene can be assigned to 12p15-pter. The results obtained demonstrate the efficiency of the applied approach in its detailed and reliable description of complex chromosomal rearrangements in hybrid clones, when differential chromosome staining failed to identify these chromosomes.

Animals↗
PubMed1998

The clonal relationship between nodular sclerosis Hodgkin's disease with a clonal Reed-Sternberg cell population and a subsequent B-cell small noncleaved cell lymphoma.

We evaluated the clonal relationship between a case of nodular sclerosis Hodgkin's disease (NSHD) and a small noncleaved cell (SNC) lymphoma that subsequently developed. Single Hodgkin and Reed-Sternberg (H-RS) cells were isolated from immunostained sections of the NSHD by micromanipulation, and the immunoglobulin heavy chain gene (IgH) complementarity determining region (CDR) III of the cells was amplified by the polymerase chain reaction (PCR). A clonal population of H-RS cells was found in the NSHD tumor. The nucleotide sequence of the clonal H-RS cells was compared with the clonal IgH CDRIII sequence from the SNC lymphoma. The two sequences were found to be unrelated. In addition, clone-specific primers and probes were designed from the two clonal IgH CDRIII sequences and used to investigate the presence of the respective clonal population in the NSHD tumor. The latter studies confirmed the presence of a major clonal H-RS cell population, as detected by the single cell assay, but cells corresponding to the SNC clone were not demonstrable by this highly sensitive technique. These findings suggest that the SNC arising in this case represents the development of the second neoplasm clonally unrelated to the preceding NSHD. They also support the recent findings that the H-RS cells in classical HD consist of a clonal population of B cells.

Adult↗
PubMed1998

[Needle testicular biopsy in the diagnosis of male infertility].

OBJECTIVES: Evaluation of testicular tissue is very important diagnostic procedures in cryptozoospermia and azoospermia. It is verified the patient for treatment (stimulation spermatogenesis), for micromanipulation ICSI or for insemination donor semen procedure. Surgical biopsy is very popular between andrologist and urologist, but needle biopsy seems to be easier and safer. DESIGN: The authors present their experience with needle testicular biopsy during the diagnosis patients with azoospermia or cryptozoospermia and the men with paraplegia after trauma and without ejaculation. MATERIAL AND METHODS: The study group included 63 biopsies in 58 men with azoospermia and severe oligoasthenozoospermia (cryptozoospermia) and 2 with paraplegia. The specimens were taken from 125 testes under general anesthesia (i.v-Diprivan, Propofol, Zeneca and Fentanyl) using biopsy needle from Hepafix B. Braun Melsungen, Germany. All procedures were performed as a day case. RESULTS: In 95% specimens were adequate for histopathological investigations and for planning the treatment. Only one complication (0, 8%) -small haematoma testis was observed. CONCLUSIONS: The needle biopsy of testicular tissue is sufficiency in histopathological examination, safe for patients and easy for urologists. The total cost is much more lower than cost of surgical biopsy.

Adult↗
PubMedDate not supplied

[The biopsy of preimplantation embryos obtained from donor pigs with follicle-stimulating hormone-induced superovulation].

We have examined possible use of the follicle-stimulating hormone (FSH) for the induction of superovulation in pigs and studied the effect of biopsy of preimplantation pig embryos on their survival in vitro. Superovulation was induced by injecting FSH twice daily over a period of four days for a total dose of 25 units. per animal. Pigs receiving pregnant mare serum gonadotropin (PMSG) according to the standard scheme served as the control. The experiments demonstrated that FSH produces significantly better estrus figures as compared with PMSG (100% and 60%, respectively; p < 0.05). The mean number of ovulations per donor was also better in the FSH group, i.e., 36.5 as compared with 17.3 in the control group (p < 0.05). The mean number of embryos obtained from one donor was higher in FSH treated animals as compared with the animals that received PMSG (29.2 vs. 19.3), however, this difference was not statistically significant. Biopsy of preimplantation pig embryos was conducted by a "manual" technique without a micromanipulator; middle blastocysts collected at days 5-6 after insemination were the most convenient for this operation. Embryos at these developmental stages contained a well developed inner cell mass, which allows separation of trophoectodermal cells only, thus minimizing damage to the embryo. The number of cells in dissected fragments did not depend on the stage of development, and even smallest fragments (4 blastomeres) were sufficient for genome analysis with the aid of PCR. The survival of embryos in vitro after biopsy practically did not differ from that of control intact embryos. Thus, we demonstrated the effective use of FSH for the induction of superovulation in pigs; we also determined the developmental stages which are most convenient for conducting biopsies and developed a technique for preimplantation biopsy, which allows genome analysis of embryos without any decrease of their survival in vitro.

Animals↗
PubMed1998

[Fetal cells in the maternal blood: a step towards non-invasive prenatal diagnosis? Review of the literature].

Prenatal diagnosis of genetic abnormalities requires nucleated fetal cells which are currently obtained by invasive techniques such as amniocentesis, chorionic villus sampling and percutaneous umbilical blood sampling. Each of these entails a risk to the foetus and sometimes to the mother. Nucleated fetal cells have been reported to be present in maternal blood. Recovery of fetal cells from maternal blood would allow a noninvasive prenatal diagnosis. Their rarity (1 fetal cell for 10(6) to 10(8) maternal cells) presents a technical challenge. Due to the small number of fetal cells, sensitive analysis techniques such as PCR and FISH are necessary. Some degree of fetal cells enrichment in the maternal blood sample often precedes the analysis. Different techniques are used for the enrichment: discontinuous density gradient, magnetic activated cell sorting, fluorescence activated cell sorting, micromanipulator.... Several prenatal diagnosis have already been performed from maternal venous blood samples: diagnosis of gender, RhD blood genotype, Duchenne muscular dystrophy and hemoglobinopathy by PCR, diagnosis of gender and chromosome aneuploidy by FISH. Many teams are working on this subject. It is difficult to compare the studies because the techniques of enrichment and analysis vary. We review the different strategies chosen for prenatal diagnosis from maternal blood and discuss the results.

Cell Nucleus↗
PubMed1998

Analyses of single B cells by polymerase chain reaction reveal rearranged VH with germline sequences in spleens of immunized adult rabbits: implications for B cell repertoire maintenance and renewal.

We used PCR to amplify rearranged VHDJH genes in single cells collected by micromanipulation from splenic germinal centers of immunized adult rabbits. In the course of the study, the objective of which was to analyze diversification of rearranged VHDJH sequences, we were surprised to find cells 7 and 10 days after immunization with rearranged VH1a2 as well as a-negative (y33 and x32) sequences that were identical or close to germline (10 or fewer changes). About 58% (82/140) of the sequences had unique CDR3 regions and were unrelated. In seven different germinal centers, we found one to four different clones with two to seven members. Clonally related cells underwent diversification by hypermutation and gene conversion. We found that contrary to published reports, adult rabbits indeed have newly diversifying B cell receptors in splenic germinal centers. The attractive idea that the rabbit, like the chicken, develops its B cell repertoire early in life and depends upon self-renewing cells in the periphery to maintain its B lymphocyte pool throughout life, is challenged by the current finding. Although a major population of B lymphocytes may be generated early in life, diversified extensively, and maintained by self-renewal in the periphery, some sources of cells with sequences close to germline do exist in adult rabbits and appear in the developing germinal centers. Although considerable repertoire diversity is generated in young rabbits, mechanisms for continued generation of B cell receptor diversity are retained in adult life, where they may confer survival advantage.

Amino Acid Sequence↗