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PubMed2004

Acrosomal integrity and capacitation are not influenced by sperm cryopreservation in the giant panda.

Sperm cryopreservation and artificial insemination are important management tools for giant panda breeding and the preservation of extant genetic diversity. This study examined the influence of freeze-thawing on sperm function, specifically capacitation. Sperm from nine giant pandas were assessed before and after rapid (- 40 and - 100 degrees C/min) cryopreservation by incubation in HEPES-buffered Ham's F10 medium with and without the capacitation accelerators, 3-isobutyl-1-methylxanthine (IBMX) and dibutyryl cyclic AMP (dbcAMP). At 0, 3 and 6 h of exposure, aliquots were assessed for sperm motility traits and capacitation, defined as the proportion of sperm with intact acrosomes following exposure to solubilised zonae pellucidae (ursid or felid) or calcium ionophore subtracted from the proportion of sperm with intact acrosomes before exposure. Although mean+/-S.E.M. sperm motility post-thaw (56.1 +/- 3.9% at 0 h) was less (P < 0.05) than pre-freeze (71.7 +/- 6.0%), there was no difference (P > 0.05) in the proportion of acrosome-intact sperm (fresh, 93.0 +/- 1.7% versus cryopreserved-thawed, 81.7 +/- 4.7% at 0 h). Incidence of capacitation was greater (P < 0.05) in fresh sperm incubated with capacitation accelerators IBMX and dbcAMP (9 h: 50.9 +/- 1.1) compared with fresh sperm incubated without accelerators (9 h: 41.2 +/- 1.1%). Frozen-thawed sperm preincubated without accelerators underwent capacitation (49.6 +/- 1.1%) to a greater extent (P < 0.05) compared with these fresh counterparts. Thawed samples with (9 h: 45.9 +/- 1.4%) and without accelerators (9 h: 41.2 +/- 1.1%) did not differ (P > 0.05) during the 9-h incubation. We conclude that giant panda spermatozoa (1) undergo capacitation in vitro with or without chemical accelerators and (2) withstand a rapid cryopreservation protocol, including retaining normal acrosomal integrity and functional capacitation ability.

1-Methyl-3-isobutylxanthine↗
PubMed1995

[Investigations on egg yolk-free diluting medium for the cryopreservation of bull spermatozoa].

On the basis of various laboratory researches and field experiments with 959 alternating first inseminations the traditional tris-extender and a yolk-free medium for semen freezing on soy lecithin base were compared in studies using a split-sample technique. Concerning important criteria such as non-return-rate, conservation quality, and standard of hygiene and practicability, the alternative product was found at least of equal quality. The renunciation of chicken yolk is to be seen as a general advantage. Here, the development of cryomedia has been enriched by a useful and sensitive new component. Apart from being competitive this product is also the first to conform to future demands in standardization through ISO-norm.

Animals↗
PubMed2006

Update on semen technologies for animal breeding.

Despite the scale of the livestock breeding industry, where many millions of insemination doses are prepared each year, sperm preparation techniques are used infrequently in animal assisted reproduction compared with its human counterpart. However, some of the techniques used for human sperm preparation, for example, density gradient centrifugation, improve the quality of sperm preparations which is, in turn, reflected by an increased conception rate. The preparation technique separates motile spermatozoa with normal morphology and intact DNA from the total sperm population, leaving behind immature or senescent spermatozoa, morphologically abnormal ones and those with damaged DNA. Furthermore, the motile spermatozoa are removed from the seminal plasma which carries cells, cellular debris and reactive oxygen species, as well as pathogens. Gradient-prepared spermatozoa survive longer, either in liquid storage or when cryopreserved, and are free of bacteria and viral infectivity if prepared carefully. Preparation techniques such as density gradient centrifugation, or the simplified single layer centrifugation technique, have considerable potential for aiding sperm preparation from poor quality semen samples, such as may be obtained from unselected semen donors in captive breeding programmes, or from performance horses. Moreover, the removal of pathogens has important implications, both for disease control and for avoiding the use of antibiotics in semen extenders, which can be detrimental to sperm survival.

Animals↗
PubMed1991

Effect of cryopreservation on the frequency of chromosomal abnormalities and sex ratio in human sperm.

The effects of cryopreservation on the frequency and type of chromosomal abnormalities in human sperm were investigated. Employing a technique that enables direct visualization of human sperm chromosomes following in vitro penetration of hamster oocytes, sperm samples from 10 normal men were examined before and after freezing in liquid nitrogen. A total of 1,960 sperm karyotypes were analyzed, 1,132 before freezing and 828 after freezing. There was no significant difference in the frequency of structural chromosomal anomalies (10.5% prefreeze vs. 8.5% postfreeze), but there was a significant decrease in the frequency of numerical abnormalities (5.2% prefreeze vs. 3.0% postfreeze). However, there was a large excess of hypohaploid complements compared with hyperhaploid complements, suggesting that the hypohaploid complements were caused by technical artefact. A conservative estimate of aneuploidy, derived by doubling the hyperhaploid frequencies, did not differ before (0.4%) and after (0.4%) freezing. There was no evidence for interdonor variability in response to sperm cryopreservation for total chromosomal abnormalities, structural abnormalities, and sex ratios. The sex ratios were also not affected by cryopreservation and did not differ significantly from the theoretical 50%. It is concluded that cryopreservation does not affect the frequencies of chromosomal abnormalities or alter the sex ratio in human sperm, provided that an adequate cryoprotective buffer and freezing system is employed.

Adult↗
PubMed2007

In vitro pyrogen test--A new test method for solid medical devices.

Medical devices manufactured for implantation into humans must be free of any contamination with viable bacteria. However, remnants of dead bacteria and bacterial components alone may induce an inflammatory immune response. Pyrogen tests for such inflammatory contaminations are generally performed either by determining the content of lipopolysaccharide in rinsing solutions of batch samples by limulus amoebocyte lysate assay, by injecting the rinsing solutions into rabbits or by implanting batch samples into rabbits and measuring change of body temperature. In this study, we show that the in vitro pyrogen test (IPT), which measures the release of the inflammatory cytokine IL-1beta in fresh or cryopreserved human whole blood, can be used to assess the pyrogenic contamination of implantable medical devices. This test was used to check neurosurgical implants, namely aneurysm clips, as a proof of principle. Owing to the direct contact of the test material with the blood cells, this test does not require rinsing procedures, which have variable efficacy. The use of human blood ensures the detection of all substances that are pyrogenic for humans and reflects their relative potency. The safety of the products as delivered could be confirmed. The effects of sterilization and depyrogenization procedures on intentional pyrogenic contaminations of samples could be followed. This new application of the already internationally validated method promises to replace further rabbit pyrogen tests. It generates extremely sensitive results with an extended range of detectable pyrogenic contaminants.

Equipment and Supplies↗
PubMed2005

Relationship between the duration of sexual abstinence and semen quality: analysis of 9,489 semen samples.

OBJECTIVE: To evaluate the relationship between duration of sexual abstinence and various characteristics of normal and subnormal semen. DESIGN: A retrospective study based on computerized data. SETTING: Fertility and IVF unit at a university medical center. PATIENT(S): Nine thousand, four hundred eighty-nine semen samples from 6,008 patients were analyzed according to the World Health Organization (WHO) manual and grouped according to sperm concentration (10(6)/mL) into severe (0.2-4 x 10(6)), moderate (>4-10 x 10(6)), and mild (>10-19.99 x 10(6)) oligozoospermia, and normozoospermia (> or =20-250 x 10(6)) groups. MAIN OUTCOME MEASURE(S): In each group mean values of semen volume, sperm concentration, percentage of motile sperm and of normal morphology (according to WHO or Kruger criteria), total sperm count, and total motile sperm count per ejaculate were related to duration of abstinence. RESULT(S): Among the 3,506 oligozoospermic samples, the peak mean sperm motility of 30.3% was observed after 1 day of abstinence. Similarly, the mean percentage of normal morphology among mild-moderate oligozoospermic samples (n = 2,260) reached peak values of 7.4%-8.6% between 0-2 days of abstinence. The 5,983 normozoospermic samples showed a significant decrease in the percentage of sperm motility and normal morphology to mean values of 33.1% and 7.0%, respectively, on days 11-14 of sexual abstinence. CONCLUSION(S): Our data challenge the role of abstinence in male infertility treatments and suggest that to present the best possible semen samples, patients with male factor infertility should collect the semen after just 1 day of sexual abstinence. Patients presenting normal sperm analysis or sperm donors for cryopreservation purposes should be advised not to exceed 10 days of sexual abstinence.

Adult↗
PubMed1992

Low prevalence of HIV in high-risk seronegative homosexual men evidenced by virus culture and polymerase chain reaction.

OBJECTIVE: To assess the presence of covert HIV-1 infection. SETTING: High-risk seronegative homosexual men from the Pittsburgh portion of the Multicenter AIDS Cohort Study were examined for the presence of HIV-1 infection. PATIENTS, PARTICIPANTS: Ten men (group 1) were examined prospectively for the presence of HIV-1 in their freshly-obtained peripheral blood mononuclear cells (PBMC). Furthermore, cryopreserved PBMC from 26 men (group 2) at their first visit (1984-1985) were examined retrospectively for the presence of HIV-1. MAIN OUTCOME MEASURES: PBMC samples from groups 1 and 2 were examined for HIV-1 by polymerase chain reaction (PCR) using gag, env and strong-stop (long terminal repeat) specific primers. In addition, fresh PBMC samples from group 1 were examined for HIV-1 by virus culture. RESULTS: None of the 10 PBMC samples from group 1 were positive for virus culture and PCR. Only one of the 26 men from group 2 was positive for gag and strong-stop DNA sequences. This PCR-positive, seronegative subject was found to be negative for HIV-1 by PCR at follow-up visits up to 48 months later. None of 15 seronegative, low-risk homosexual men and 12 seronegative heterosexual men were found to be PCR-positive for HIV-1. However, six HIV-1-seropositive men were positive by PCR for gag, env, and strong-stop HIV-1 DNA sequences. CONCLUSIONS: These results suggest a low prevalence of covert HIV-1 infection in high-risk seronegative homosexual men in our geographic area.

Adult↗
PubMed1988

Growth of erythroid burst-forming units (BFU-E) in cultures of canine bone marrow and peripheral blood cells: effect of serum from irradiated dogs.

Erythroid burst-forming units (BFU-E) from canine bone marrow and peripheral blood could be grown in methylcellulose in the presence of an appropriate batch of fetal calf serum (FCS), transferrin, and erythropoietin (Epo). However, improved colony formation (size and number of bursts) was obtained when serum from total body irradiated dogs was present in the culture. This serum, obtained from dogs at day 9 after total body irradiation with a dose of 3.9 Gy, reduced markedly the Epo requirement of BFU-E. Furthermore, it allowed the omission of FCS from the culture medium if cholesterol and bovine serum albumin (BSA) were used as FCS substitutes. BFU-E concentrations were found to be rather different in the peripheral blood and in bone marrow samples from different sites (i.e., iliac crest, sternum, and humerus) of normal beagles. The studies further show that canine bone marrow BFU-E can be cryopreserved in liquid nitrogen.

Animals↗
PubMed1995

PCNA immunopositivity index as a substitute to 3H-thymidine pulse-labeling index (TLI) in methanol-fixed human lymphocytes.

PHA-stimulated human lymphocytes or myelogenous leukemia cells (strain K-562) were pulse labeled with 3H-thymidine and submitted to various fixation-permeabilization procedures. They were then immunostained with the 19A2, 19F4 or PC10 monoclonal antibody against the proliferating cell nuclear antigen (PCNA). The preparations were finally scored for the proportion of unlabeled, double-labeled and single PCNA or 3H-thymidine-labeled nuclei. Unstimulated lymphocytes were immunonegative in all the conditions tested, as also were stimulated lymphocytes checked with an isotype of the primary antibody. A specificity (Sp) and a sensitivity (Se) score was calculated to evaluate the recognition by PCNA staining of the S-phase cells, as defined by the 3H-labeling. The data show that in most instances the three antibodies recognized the 3H-labeled cells with high sensitivity, ie with few false negative, but with low specificity, ie with PCNA positivity extending to variable proportions of non-S-phase cells. By contrast, methanol fixation followed by a brief treatment with the detergent Triton X-100 and immunostaining with either 19F4 or PC10 (but not with 19A2) combined a high sensitivity and specificity scores of the recognition of the 3H-thymidine-labeled cells: PC10 gave a more intense and, hence, more readable reaction. PHA-stimulated lymphocytes that had been preserved at -20 degrees C as cytocentrifuged smears failed to show any immunopositivity for PCNA if not submitted to further fixation prior to the immunocytochemical assay. When methanol-Triton was used for this step, only PC10 gave positive immunoreaction, yet with a lower specificity score (Sp = 76%) than in cells submitted to this fixation-permeabilization procedure without prior cryopreservation (Sp = 91.7%). The PCNA index was measured in cryopreserved, methanol-fixed smears of lymphocytes from patients with various hematological diseases and was compared to the Ki-67 index established independently on a serial sample. A good correlation was found between the two indices (r = 0.79; P < 0.0001) with the PCNA index generally lower than or close to the Ki-67 index. This warrants a note of caution about the use of total (ie stable and labile) PCNA immunostaining to measure the growth fraction (GF), classically defined as the proportion of proliferating cells in a population. However, in the absence of an absolute reference marker for G0 cells, there is no reason to assume that the PCNA index would necessarily be a worse estimate of GF than the Ki-67 index.(ABSTRACT TRUNCATED AT 400 WORDS)

Autoradiography↗
PubMedDate not supplied

Enhancement of cellular immunity in melanoma patients immunized with a peptide from MART-1/Melan A.

PURPOSE: In this study, we tested the effectiveness of a melanoma-associated antigen-derived peptide, MART-1(27-35), in eliciting cellular immune responses in vivo in the context of a phase I active immunization protocol. This peptide (AAGIGILTV) corresponds to residues 27-35 from the nonmutated melanoma-associated antigen MART-1/Melan A and is recognized by most melanoma-specific, HLA-A*0201-restricted, tumor-infiltrating lymphocytes. To test the in vivo induction of cytotoxic T lymphocyte (CTL) sensitization, we compared CTL reactivity in vitro from peripheral blood mononuclear cell (PBMC) pools obtained before and after vaccination. PATIENTS AND METHODS: MART-1(27-35) was administered to HLA-A*0201 melanoma patients subcutaneously in an emulsification with incomplete Freund's adjuvant. A vaccination course included four inoculations of peptide at 3-week intervals. PBMC collected by leukapheresis and separated by Ficoll-Hypaque gradient before and after vaccination were analyzed in 18 patients by in vitro sensitization with MART-1(27-35). To induce MART-1(27-35)-specific CTL, PBMC were incubated with 1 microM peptide (on day 0) and interleukin-2 (IL-2) (300 IU/mL, on days 1 and 4 after each stimulation). At weekly intervals, cells were harvested and an aliquot was cryopreserved for later analysis. The remaining cells were replated and restimulated using irradiated autologous PBMC pulsed with 1 microM of relevant peptide. After three restimulations, all samples from one patient were tested simultaneously for HLA-A*0201-restricted anti-MART-1(27-35) reactivity by microcytotoxicity and cytokine (IFN-gamma) release assays. RESULTS: Toxicities were minimal and consisted of local irritation at the site of vaccine administration. None of the patients sustained a clinical response. The first eight patients were monitored by inducing CTL reactivity from PBMC obtained preimmunization and after two and four vaccinations. Only two prevaccination cultures were reactive to MART-1, compared with five and seven cultures from PBMC obtained after two and four vaccinations, respectively. Thus, an enhancement in cytotoxic activity could be detected in postvaccination CTL cultures, and serial vaccine administrations appeared to boost the detectability of cytotoxicity in vitro. For completeness, the analysis compared prevaccination with postvaccination PBMC cultures. Specific anti-MART-1(27-35) cytotoxicity (> or = 10 lytic units) could be detected in two prevaccination and 12 postvaccination cultures after two in vitro stimulations. In 15 postvaccination CTL cultures, a more than threefold increase in specific release of IFN-gamma was noted, compared with prevaccination. DISCUSSION: In vivo administration of a melanoma-associated antigen peptide, emulsified in incomplete Freund's adjuvant, could safely augment CTL reactivity against epitopes commonly expressed by melanoma cells. Although the enhancement of CTL reactivity did not achieve tumor regression, it is possible that the use of recombinant immunogens with increased immunomodulatory capabilities in future clinical trials could reach the threshold of CTL activation necessary for tumor regression.

Adult↗
PubMed2002

Flow cytometric CD34+ determination in stem cell transplantation: before or after cryopreservation of grafts?

Various attempts have been made to standardize and improve the reproducibility of flow cytometric determination of CD34+ hematopoietic progenitor cells. It is still not clear, however, whether the quantification of CD34+ cells in a stem cell graft should be done before or after cryopreservation. To address this issue, we investigated 78 unselected and 32 immunomagnetically selected autologous and allogeneic leukapheresis products (LA) before and after cryopreservation using pilot vials. Cell numbers were quantified within a Neubauer chamber, and CD34+ content was determined by flow cytometry; propidium iodide staining was used to exclude dead cells from analysis. Before freezing, the mean viable CD34 cell content in the unselected samples was 1.22% and increased after thawing to a mean of 2.16% of viable cells. Taking into account cell loss and cell death, the overall recovery of viable cells was 64.5%; all CD34+ cells could be recovered. Mean purity in the CD34-selected cell fraction was 85% (48-97) before and 91.3% (67-99) after thawing. The number of viable cells was 86.8% before and 86.1% after freezing with a 93.9% recovery of total cells. This leads to a mean 93.7% (SD +/- 23.1) recovery of viable cells and 100% (SD +/- 22.3) recovery of viable CD34+ cells. There was no significant difference in tolerance to freeze/thaw stress between cells from heavily pretreated autologous patients and healthy allogeneic donors. Our data show that freezing significantly increases the percentage of CD34(+) cells in unmanipulated LA, probably due to the death of granulocytes and mononuclear cells (MNCs). Nevertheless, the overall number of viable CD34+ cells in unselected as well as selected samples remains unchanged. Thus, CD34 data from different laboratories, for example, within multicenter trials, should be comparable independent of the different time points of acquisition.

Antigens, CD34↗
PubMedDate not supplied

[Cryopreserved neurons of a mollusc are capable of morphological differentiation in culture in vitro].

It was shown in culture in vitro that neurons isolated from the cryopreserved brain of adult molluscs Lymnaea stagnalis L. retain viability. Isolated brains were frozen in liquid nitrogen vapors at a rate of 400-500 degrees C/min in the presence of 2 M dimethylsulfoxide. The samples were then plunged into liquid nitrogen and stored from 1 month to 2 years. Upon thawing and removing the cryoprotectant, the neurons were isolated from the brain and then introduced into a cellular culture in vitro. It was shown that the thawed neurons were capable of regenerating new nerve processes similar to those formed by unfrozen neurons in the control.

Animals↗
PubMed2001

The outcome of intracytoplasmic injection of fresh and cryopreserved ejaculated spermatozoa--a prospective randomized study.

BACKGROUND: The overall aim of this prospective, randomized study was to compare the reproductive potential of fresh and frozen-thawed ejaculated spermatozoa from oligoasthenoteratozoospermic patients in an intracytoplasmic sperm injection (ICSI) procedure. METHODS: All patients consenting to participate in this study had a sperm sample frozen prior to the start of a cycle. Patients were randomized using a random number table to undergo ICSI with either fresh (group A, n = 118) or frozen-thawed (group B, n = 122) spermatozoa. All prognostic variables were equally distributed among the two groups. RESULTS: The pregnancy rate per started cycle was 29.7% in group A and 38.5% in group B, P > 0.05. A significant difference was observed in the rate of ongoing pregnancies between group A (23.7%) and group B (35.2%), P < 0.05. CONCLUSION: From our data we can conclude that cryopreservation of spermatozoa from men with poor sperm quality does not negatively affect fertilization and pregnancy rates after ICSI. A larger study will be needed to investigate whether the use of cryopreserved spermatozoa can be helpful in selecting the most vital spermatozoa for ICSI.

Adult↗
PubMed2004

Evaluation of an automated instrument for viability and concentration measurements of cryopreserved hematopoietic cells.

Two important parameters for determination of deleterious effects of cellular processing on hematopoietic progenitor cells are cell viability and concentration. The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins Hospital evaluated the Beckman Coulter Vi-Cell automated instrument for the measurement of these two parameters. Using 33 thawed hematopoietic progenitor cell samples, automated Vi-Cell viability results were compared to those obtained using the standard trypan blue manual method. In addition, cell concentrations from these samples were compared with results from the Model Z2 Coulter Counter. Chinese Hamster Ovary cells were used for the evaluation of Vi-Cell linearity at the Beckman Coulter Cellular Analysis Development Center. Significant correlation was obtained when the two methods were compared for both cell concentration and percentage viability (P < .0001). The results of the linearity study indicated that the Vi-Cell is linear from approximately 5 x 10(4) to greater than 1 x 10(7) cells/mL. The Vi-Cell uses sample volumes as low as 0.5 mL; cell diameters may be 2 to 70 microns. The Vi-Cell automated instrument offers many significant advantages for cell analyses in today's busy laboratory environment.

Animals↗
PubMed1998

A programme of semen cryopreservation for patients with malignant disease in a tertiary infertility centre: lessons from 8 years' experience.

The improved survival in recent years of young males suffering from cancer, and an understanding of the gonadotoxic effects of chemotherapy treatment, have motivated patients and clinicians to preserve fertility potential before embarking on adjuvant therapy. Among 231 men (mean age 28.0; range 15-56 years) diagnosed with malignant disease and referred to our unit for semen cryopreservation, 112 patients (49.8%) had reduced sperm quality of <10 x 10(6) motile spermatozoa per ejaculate; however, most had sufficient suitable spermatozoa for freezing. In 40 patients (17.3 %) the semen samples were not frozen because of complete azoospermia (n = 32) or only immotile sperm in the ejaculate (n = 2), while six men were unable to produce a single sample. Some 79 men had testicular tumours (group I), 121 suffered from haematological malignancy (leukaemia or lymphoma; group II), and 27 had cancer of different causes (group III). Men in group I had significantly lower (P < 0.001) sperm quality compared with groups II and III. There was no difference between patients with seminoma and non-seminoma tumours. In the haematological malignancy group there was no difference in sperm parameters between leukaemia (n = 12) and lymphoma (n = 77) patients, but patients with Hodgkin's lymphoma had significantly lower sperm quality compared with non-Hodgkin's lymphoma. Following chemotherapy, six couples attended the clinic for assisted conception treatment using the frozen semen. Two had successful intrauterine insemination cycles which each resulted in delivery of a healthy girl; one couple had conceived in their first in-vitro fertilization (IVF) attempt, followed by delivery of healthy twins. Two women conceived after intracytoplasmic sperm injection treatment and the sixth woman achieved only biochemical pregnancy after numerous IVF and frozen embryo replacement cycles. We recommend that a properly designed programme for semen cryopreservation for cancer patients should be developed in leading tertiary assisted conception centres, which have adequate facilities and experience for cryopreservation and can offer the whole range of appropriate assisted reproductive treatment and counselling.

Adolescent↗
PubMed1996

Testicular aspiration of sperm for intracytoplasmic sperm injection: a novel treatment for ejaculatory failure on the day of oocyte retrieval.

OBJECTIVE: To present a successful outcome of an IVF cycle complicated by failure to produce a sperm sample on the morning of oocyte retrieval. DESIGN: Case report. SETTING: Hospital-based IVF clinic. PATIENT: A couple suffering secondary infertility undergoing their first IVF and intracytoplasmic sperm injection (ICSI) cycle. INTERVENTION: Fine-needle testicular biopsy for sperm retrieval. MAIN OUTCOME MEASURES: Sperm retrieval, fertilization, pregnancy. RESULTS: Sufficient spermatozoa were obtained for ICSI. Ten metaphase II oocytes were injected with two embryos being transferred fresh and four cryopreserved. A single fetal heart was confirmed at 6-week sonar. CONCLUSION: Testicular aspiration of sperm for ICSI is a simple, inexpensive, and effective means of rescuing an IVF cycle in which the male partner unexpectedly has been unable to produce a sperm sample on the morning of oocyte collection.

Adult↗
PubMed2002

[Cryopreservation of ovarian tissue].

Ovarian tissue cryopreservation (OTCP) is a new procedure of medically assisted procreation, still at the experimental stage, whose primary aim is to store female gametes as sperm cryopreservation permits to do for male gametes. Ovarian tissue is removed very simply by laparoscopy. It survives well to freezing if the medium contains a cryoprotective agent and the rate of freezing is slow. In contrast, thawing must be rapid. There are three processes for the utilization of ovarian tissue after thawing. In vitro maturation and xenografting remain impossible for technical and ethical reasons. Autologous transplantation (orthotopic or heterotopic) of the tissue is therefore the only foreseeable method over the short term. Indications for OTCP must remain rare as long as no pregnancy has been obtained in human. At the present time, only female patients who would inevitably suffer the loss of their fertility should be able to take advantage of OTCP. Basically, this would mean women subjected to castrating anticancer therapy. It would seem reasonable to set the age limit at 35-years for carrying out OTCP. Lastly, female patients should be clearly informed that the method is still at the research stage, and in France samples must be taken in accordance with the laws governing clinical research.

Adult↗
PubMed2006

Application of cryopreserved human hepatocytes in trichloroethylene risk assessment: relative disposition of chloral hydrate to trichloroacetate and trichloroethanol.

BACKGROUND: Trichloroethylene (TCE) is a suspected human carcinogen and a common groundwater contaminant. Chloral hydrate (CH) is the major metabolite of TCE formed in the liver by cytochrome P450 2E1. CH is metabolized to the hepatocarcinogen trichloroacetate (TCA) by aldehyde dehydrogenase (ALDH) and to the noncarcinogenic metabolite trichloroethanol (TCOH) by alcohol dehydrogenase (ADH). ALDH and ADH are polymorphic in humans, and these polymorphisms are known to affect the elimination of ethanol. It is therefore possible that polymorphisms in CH metabolism will yield subpopulations with greater than expected TCA formation with associated enhanced risk of liver tumors after TCE exposure. METHODS: The present studies were undertaken to determine the feasibility of using commercially available, cryogenically preserved human hepatocytes to determine simultaneously the kinetics of CH metabolism and ALDH/ADH genotype. Thirteen human hepatocyte samples were examined. Linear reciprocal plots were obtained for 11 ADH and 12 ALDH determinations. RESULTS: There was large interindividual variation in the Vmax values for both TCOH and TCA formation. Within this limited sample size, no correlation with ADH/ALDH genotype was apparent. Despite the large variation in Vmax values among individuals, disposition of CH into the two competing pathways was relatively constant. CONCLUSIONS: These data support the use of cryopreserved human hepatocytes as an experimental system to generate metabolic and genomic information for incorporation into TCE cancer risk assessment models. The data are discussed with regard to cellular factors, other than genotype, that may contribute to the observed variability in metabolism of CH in human liver.

Adult↗