Disparities in reproductive outcomes according to the endometrial preparation protocol in frozen embryo transfer : The risk of early pregnancy loss in frozen embryo transfer cycles.

PURPOSE: The purpose of this study was to determine the effect of stimulated and artificial endometrial preparation protocols on reproductive outcomes in frozen embryo transfer (FET) cycles. METHODS: We performed a retrospective study of 1926 FET cycles over a 3.5-year period in the Fertility Unit at a University Hospital. Stimulated and artificial protocols were used for endometrial preparation. The embryos for FET were obtained from either in vitro fertilization or intracytoplasmic sperm injection cycles. Live birth rate and early pregnancy loss rates were retrospectively compared. In artificial protocols, oral or vaginal administration of oestradiol 2 mg two or three times a day was followed by vaginal supplementation with progesterone 200 mg two or three times a day. In stimulated protocols, recombinant follicle-stimulating hormone was administered from day 4 onward. Vaginal ultrasound was used for endometrial and ovarian monitoring. A pregnancy test was performed 14 days after FET. If it was positive, oestradiol and progesterone were administered up until the 12th week of gestation in artificial cycles. We defined early pregnancy losses as biochemical pregnancies (preclinical losses) and miscarriages. RESULTS: Data on 865 artificial cycles (45% of the total) and 1061 stimulated cycles (55%) were collected. Early pregnancy loss rate was significantly lower for stimulated cycles (34.2%) than for artificial cycles (56.9%), and the live birth rate was significantly higher for stimulated cycles (59.7%) than for artificial cycles (29.1%). CONCLUSION: In frozen embryo transfer, artificial cycles were associated with more early pregnancy loss and lower live birth rate than stimulated cycles.

Frequency dependence of viscous and viscoelastic dissipation in coated micro-cantilevers from noise measurement.

We measure the mechanical thermal noise of soft silicon atomic force microscope cantilevers. Using an interferometric setup, we obtain a resolution down to 10(-14) m Hz(-1/2) on a wide spectral range (3-10(5) Hz). The low frequency behavior depends dramatically on the presence of a reflective coating: almost flat spectra for uncoated cantilevers versus a 1/f like trend for coated ones. The addition of a viscoelastic term in models of the mechanical system can account for this observation. Use of Kramers-Kronig relations validate this approach with a complete determination of the response of the cantilever: a power law with a small coefficient is found for the frequency dependence of viscoelasticity due to the coating, whereas the viscous damping due to the surrounding atmosphere is accurately described by the Sader model.

Ribozyme targeting of the growth factor pleiotrophin in established tumors: a gene therapy approach.

The growth and metastasis of solid tumors relies on the activities of polypeptide growth factors to recruit stromal tissue and expand the tumor mass. Pleiotrophin (PTN) is a secreted growth factor with angiogenic activity that has been found to contribute to the growth and metastasis of tumors including melanoma. Here, we present a gene therapy approach of targeting PTN in established tumors using ribozymes. Tetracycline-regulated ribozyme expression vectors were used to deplete conditionally PTN mRNA from melanoma xenograft tumors in vivo. We found that tetracycline-mediated initiation of ribozyme expression in established tumors reduced further tumor growth. Next, we generated synthetic anti-PTN ribozymes that inhibit PTN-dependent colony formation of cells in soft agar. Intraperitoneal administration of these synthetic ribozymes into nude mice inhibited growth of PTN-positive, subcutaneous melanoma. Furthermore, PTN released from the tumors into the circulation of mice was reduced after ribozyme treatment. These data show that ribozyme targeting of rate-limiting tumor growth factors could provide an efficient tool for cancer therapy and that the efficacy may be reflected in the reduction of the serum levels of the targeted protein, PTN.

Low-level arsenite activates the transcription of genes involved in adipose differentiation.

In this study we analyzed gene expression in 3T3-F442A pre-adipocyte cells that differentiate in the presence of micro-molar arsenate concentration. Two concentrations of arsenite (As2O3, 0.25 micromol/L and 0.5 micromol/L) were applied for three days with and without insulin (170 nmol/L) and gene expressions were evaluated by quantitative RT-PCR. The genes included genes of oxidative-stress responses: heme-oxygenase-1 (HO1) and the hypoxia inducible factor 1a (HIF1alpha), genes of cell-cycle: c-jun and Kruppel like factor 5 (KLF5), and genes that play important roles in adipose determination: a peroxisome proliferator-activated receptor (PPARgamma) and a CCAAT/ enhancer binding protein (C/EBPalpha). Arsenite induced the expression of HO1, HIF1alpha, KLF5, PPARgamma and C/EBPalpha. These results suggest that under condition of oxidative stress arsenite induces genes that are required for adipose differentiation.

Cytogenetic monitoring of industrial radiographers using the micronucleus assay.

Industrial radiography is the process of using either gamma-emitting radionuclide sources or X-ray machines to examine the safety of industrial materials. Industrial radiographers are among the radiation workers who receive the highest individual occupational radiation doses. To assess occupationally induced chromosomal damage, we performed the cytokinesis-block micronucleus (CBMN) assay in peripheral lymphocytes of 29 male industrial radiographers, exposed to ionizing radiation for 12.8 years+/-11.2, in comparison with 24 gender-, age-, and smoking habits-matched controls. The CBMN assay was combined with fluorescent in situ hybridization with a pan-centromeric DNA probe in 17 exposed subjects and 17 controls randomized from the initial populations. The mean cumulative equivalent dose, recorded by film dosimeters, was 67.2 mSv+/-49.8 over the past 5 years. The mean micronucleated binucleated cell rate (MCR) was significantly higher in the industrial radiographers than in the controls (10.7 per thousand +/-5.2 versus 6.6 per thousand +/-3.1, P=0.009); this difference was due to a significantly higher frequency of centromere-negative micronuclei (C-MN) in exposed subjects than in controls (8.5 per thousand +/-4.9 versus 2.2 per thousand +/-1.6, P<0.001). The two populations did not significantly differ in centromere-positive micronuclei (C+MN) frequency. These findings demonstrate a clastogenic effect in lymphocytes of industrial radiographers. MCR significantly positively correlated with age in the two groups. After correction for the age effect, MCR did not correlate with duration of occupational exposure. No correlation between radiation doses and MCR, C-MN, and C+MN frequencies was observed. In addition to physical dosimetry records, the enhanced chromosomal damage in lymphocytes of industrial radiographers emphasizes the importance of radiation safety programs.

Acentromeric micronuclei are increased in peripheral blood lymphocytes of untreated cancer patients.

Increased micronucleated cell rates, dicentric chromosomes, and other chromosomal damages have been reported in lymphocytes of cancer patients prior to the initiation of chemotherapy, and/or radiotherapy. The cause of these chromosomal damages in these lymphocytes remains unclear. In the present work, we investigated whether these micronuclei mainly reflect structural or numerical chromosomal aberrations by applying the cytokinesis-blocked micronucleus (CBMN) assay in combination with fluorescent in situ hybridization (FISH) of a DNA centromeric probe on blood samples of 10 untreated cancer patients (UCPs), and 10 healthy subjects (HSs). Micronucleated binucleated lymphocyte rate was significantly increased in patients (mean+/-S.D.: 19.0 per thousand +/-14.1 versus 9.2 per thousand +/-4.6 in controls). Trinucleated cytokinesis-blocked cells were not significantly higher in patients than in controls. Acentromeric, centromeric, and multicentromeric micronucleus levels were two-fold higher in patients than in controls, but the difference was significant only with acentromeric micronuclei. The percentage of micronuclei containing one or more centromeres averaged 69.2, and 71.5% in patients, and controls, respectively. The percentage of micronuclei containing several centromeres was 44.7% in patients, and 54.6% in controls. Among centromere-positive micronuclei, the percentage of micronuclei containing several centromeres averaged 59.7% in patients, and 75.4% in controls. These results indicate that genetic instability in peripheral blood lymphocytes of UCPs occurs because of enhanced chromosome breakage. However, a substantial proportion of this genetic instability occurs because of defects in chromosome segregation.

The expression of genes induced in melanocytes by exposure to 365-nm UVA: study by cDNA arrays and real-time quantitative RT-PCR.

Ultraviolet A radiation (UVA; 320-400 nm) constitutes more than 90% of the terrestrial UV solar energy. This type of radiation generates reactive oxygen species and consequently induces DNA damage. UVA irradiation is now considered to be an important carcinogen agent especially in the development of melanoma. UVA radiation is known to activate several pathways in mammalian cells. We have used cDNA arrays to analyze differential gene expression in primary cultures of human melanocytes in response to 365-nm UVA. Among 588 genes studied, 11 were overexpressed. These genes included genes involved in cell cycle regulation (GADD45, CIP1/WAF1), in stress response (HSP70, HSP40, HSP86), in apoptosis (GADD153, tristetraproline) and genes encoding transcription factors (EGR-1, ETR-101, c-JUN, ATF4). This coordinate gene regulation was confirmed by real-time quantitative RT-PCR.

Oligoribonucleotides with 2'-O-(tert-butyldimethylsilyl) groups.

The chemical synthesis of oligoribonucleotides on solid support is routinely performed via the phosphoramidite method. However, the additional 2-OH function of the ribofuranosyl sugar requires suitable protection during oligoribonucleotide synthesis. This unit describes methods for 2-OH protection using the TBDMS group.

Pharmacokinetics and tissue distribution of a ribozyme directed against hepatitis C virus RNA following subcutaneous or intravenous administration in mice.

A nuclease resistant ribozyme targeting the 5' untranslated region (5' UTR) of hepatitis C virus (HCV) at site 195 has been identified. To investigate the therapeutic utility of this ribozyme, we evaluated the pharmacokinetics and tissue distribution with two labeled forms of this ribozyme. [(32)P]-labeled ribozyme was administered as a single subcutaneous (SC) or intravenous (IV) bolus at a dose of 10 mg/kg or 30 mg/kg in C57Bl/6 mice. Regardless of route of administration, peak liver concentrations achieved were greater than the concentration necessary to inhibit HCV-IRES-luciferase expression in cell culture. The ribozyme was well absorbed after SC administration (89%) and had an elimination half-life of 23 minutes. To show intracellular localization of the ribozyme in target tissue, a tetramethyl rhodamine (TMR)-labeled ribozyme was administered as a single SC or IV bolus at a dose of 30 mg/kg in C57Bl/6 mice. Mice treated SC or IV with TMR-labeled ribozyme had positive fluorescence in the liver from 15 minutes to 48 hours after dosing. Definite positive fluorescence was still present at 72 hours in the mice dosed via the IV route. At early time points (15 and 30 minutes postinjection), nuclear and possibly cytoplasmic fluorescence was present in the hepatocytes, and sinusoidal fluorescence was intense. At the later time points, fluorescence became more punctate. Abundant staining was often present in Kupffer cells. This study confirms the retention of ribozyme in liver cells and supports the potential of an anti-HCV ribozyme as a therapeutic agent for treatment of chronic hepatitis C.

Acute toxicology and pharmacokinetic assessment of a ribozyme (ANGIOZYME) targeting vascular endothelial growth factor receptor mRNA in the cynomolgus monkey.

The potential acute toxicity of a ribozyme (ANGIOZYME) targeting the flt-1 vascular endothelial growth factor (VEGF) receptor mRNA was evaluated in cynomolgus monkeys following i.v. infusion or s.c. injection. ANGIOZYME was administered as a 4-hour i.v. infusion at doses of 10, 30, or 100 mg/kg or a s.c. bolus at 100 mg/kg. End points included blood pressure, electrocardiogram (ECG), clinical chemistry, hematology, complement factors, coagulation parameters, and ribozyme plasma concentrations. ANGIOZYME was well tolerated, with no drug-associated morbidity or mortality. There was no clear evidence of ANGIOZYME-related adverse effects in this study. Slight increases in spleen weight and lymphoid hyperplasia were observed in several animals. However, these changes were not dose dependent. Steady-state concentrations of ANGIOZYME were achieved during the 4-hour infusion of 10, 30, or 100 mg/kg. Dose-dependent elimination of ANGIOZYME was observed, with faster clearance at the two highest doses. ANGIOZYME was slowly absorbed after s.c. administration, resulting in steady-state concentrations for the 9-hour sampling period. Monkeys in this toxicology study received significant plasma ANGIOZYME exposure by both the s.c. and i.v. routes.

Quantitative determination of a chemically modified hammerhead ribozyme in blood plasma using 96-well solid-phase extraction coupled with high-performance liquid chromatography or capillary gel electrophoresis.

Versatile bioanalytical assays to detect chemically stabilized hammerhead ribozyme and putative ribozyme metabolites from plasma are described. The extraction protocols presented are based on serial solid-phase extractions performed on a 96-well plate format and are compatible with either IEX-HPLC or CGE back-end analysis. A validation of both assays confirmed that both the HPLC and the CGE methods possess the required linearity, accuracy, and precision to accurately measure concentrations of hammerhead ribozyme extracted from plasma. These methods should be of general use to detect and quantitate ribozymes from other biological fluids such as serum and urine.

Inhibition of hepatitis C virus (HCV)-RNA-dependent translation and replication of a chimeric HCV poliovirus using synthetic stabilized ribozymes.

Ribozymes are catalytic RNA molecules that can be designed to cleave specific RNA sequences. To investigate the potential use of synthetic stabilized ribozymes for the treatment of chronic hepatitis C virus (HCV) infection, we designed and synthesized hammerhead ribozymes targeting 15 conserved sites in the 5' untranslated region (UTR) of HCV RNA. This region forms an internal ribosome entry site that allows for efficient translation of the HCV polyprotein. The 15 synthetic ribozymes contained modified nucleotides and linkages that stabilize the molecules against nuclease degradation. All 15 ribozymes were tested for their ability to reduce expression in an HCV 5' UTR/luciferase reporter system and for their ability to inhibit replication of an HCV-poliovirus (HCV-PV) chimera. Treatment with several ribozymes resulted in significant down-regulation of HCV 5' UTR/luciferase reporter expression (range 40% to 80% inhibition, P <.05). Moreover, several ribozymes showed significant inhibition (>90%, P <.001) of chimeric HCV-PV replication. We further show that the inhibitory activity of ribozymes targeting site 195 of HCV RNA exhibits a sequence-specific dose response, requires an active catalytic ribozyme core, and is dependent on the presence of the HCV 5' UTR. Treatment with synthetic stabilized anti-HCV ribozymes has the potential to aid patients who are infected with HCV by reducing the viral burden through specific targeting and cleavage of the viral genome.

A multicenter study of 4-chloro-m-cresol for diagnosing malignant hyperthermia susceptibility.

UNLABELLED: Standardization of the in vitro contracture test (IVCT) for malignant hyperthermia (MH) susceptibility has resulted in very rare false negative tests. However, false positive results stigmatizing the patient seem to be more frequent than false negative results and make supplementary tests desirable. This multicenter approach studied the usefulness of an IVCT with 4-chloro-m-cresol (4-CmC), a ryanodine receptor-specific agonist for a better definition of MH susceptibility. Diagnosis made by the standard IVCT was compared with the results of this 4-CmC test on muscle specimens of 202 individuals from 6 European MH centers. In the 4-CmC test, the results of the MH susceptible group differed significantly from both the MH normal and the MH equivocal group. 4-CmC revealed a qualitatively dose response-curve similar to caffeine. A correlation index of r = 0.79 for the concentration thresholds underlined the strong concordance of the caffeine and the 4-CmC effects. The optimal threshold concentration was determined to be 75 microM in the pooled data of all centers and is much lower than that of caffeine (2 mM), suggesting a more than 25-fold higher affinity of 4-CmC. The predictive value of 4-CmC is as high as that of caffeine and consequently higher than that of halothane. 4-CmC seems to be a suitable drug to refine diagnosis of MH susceptibility and could be used as an additional test substance. IMPLICATIONS: Although in vitro contracture testing for malignant hyperthermia diagnosis is well standardized, with a relatively high sensitivity and specificity, false test results cannot be excluded and may be associated with serious disabilities for the concerned individuals. In this multicenter study, 4-chloro-m-cresol was evaluated as a new test substance for the in vitro contracture testing. Its use improves the accuracy of in vitro diagnosis of malignant hyperthermia susceptibility.

Pharmacokinetics and tolerability of an antiangiogenic ribozyme (ANGIOZYME) in healthy volunteers.

The pharmacokinetics and tolerability of a chemically stabilized synthetic ribozyme (ANGIOZYME) targeting the Flt-1 VEGF receptor mRNA were evaluated in healthy volunteers. In a placebo-controlled, single-dose escalation study, ribozyme was administered as a 4-hour i.v. infusion of 10 or 30 mg/m2 or as a s.c. bolus of 20 mg/m2. Peak ribozyme plasma concentrations of 1.5 and 3.8 micrograms/mL were observed after the 10 and 30 mg/m2 i.v. infusions, respectively. When normalized to dose, AUC values as well as peak concentrations increased proportionally as the dose was increased from 10 to 30 mg/m2. Peak concentrations of 0.9 microgram/mL were observed approximately 3.25 hours after a 20 mg/m2 s.c. bolus of ribozyme. The dose-normalized AUCs obtained after s.c. dosing were compared to the mean dose-normalized AUC after i.v. dosing to estimate an absolute s.c. bioavailability (f) of approximately 69%. An average elimination half-life of 28 to 40 minutes was observed after i.v. administration, which increased to 209 minutes after s.c. administration. Only 4 of 12 reported adverse events were possibly related to administration of ribozyme (headache and somnolence). Thus, ribozyme administration was well tolerated after a single 4-hour i.v. infusion of up to 30 mg/m2 or a single s.c. bolus of 20 mg/m2.

Identification and characterization of a novel high affinity metal-binding site in the hammerhead ribozyme.

A novel metal-binding site has been identified in the hammerhead ribozyme by 31P NMR. The metal-binding site is associated with the A13 phosphate in the catalytic core of the hammerhead ribozyme and is distinct from any previously identified metal-binding sites. 31P NMR spectroscopy was used to measure the metal-binding affinity for this site and leads to an apparent dissociation constant of 250-570 microM at 25 degrees C for binding of a single Mg2+ ion. The NMR data also show evidence of a structural change at this site upon metal binding and these results are compared with previous data on metal-induced structural changes in the core of the hammerhead ribozyme. These NMR data were combined with the X-ray structure of the hammerhead ribozyme (Pley HW, Flaherty KM, McKay DB. 1994. Nature 372:68-74) to model RNA ligands involved in binding the metal at this A13 site. In this model, the A13 metal-binding site is structurally similar to the previously identified A(g) metal-binding site and illustrates the symmetrical nature of the tandem G x A base pairs in domain 2 of the hammerhead ribozyme. These results demonstrate that 31P NMR represents an important method for both identification and characterization of metal-binding sites in nucleic acids.

Use of DNA, RNA, and chimeric templates by a viral RNA-dependent RNA polymerase: evolutionary implications for the transition from the RNA to the DNA world.

All polynucleotide polymerases have a similar structure and mechanism of catalysis, consistent with their evolution from one progenitor polymerase. Viral RNA-dependent RNA polymerases (RdRp) are expected to have properties comparable to those from this progenitor and therefore may offer insight into the commonalities of all classes of polymerases. We examined RNA synthesis by the brome mosaic virus RdRp on DNA, RNA, and hybrid templates and found that precise initiation of RNA synthesis can take place from all of these templates. Furthermore, initiation can take place from either internal or penultimate initiation sites. Using a template competition assay, we found that the BMV RdRp interacts with DNA only three- to fourfold less well than it interacts with RNA. Moreover, a DNA molecule with a ribonucleotide at position -11 relative to the initiation nucleotide was able to interact with RdRp at levels comparable to that observed with RNA. These results suggest that relatively few conditions were needed for an ancestral RdRp to replicate DNA genomes.

Moieties in an RNA promoter specifically recognized by a viral RNA-dependent RNA polymerase.

RNAs 33 nucleotides in length can direct accurate initiation of subgenomic RNA synthesis by the brome mosaic virus RNA-dependent RNA polymerase (RdRp), provided that the native sequences are maintained at five positions: -17, -14, -13, -11, and the +1 initiation site. The functional groups in the bases of these essential nucleotides required to interact with RdRp were examined by using chemically synthesized RNAs containing base analogs at each of the five positions. Analysis using a template competition assay revealed that the mode of recognition for the initiation nucleotide (+1) is distinct from that of the other essential nucleotides in the promoter. Competition experiments also determined that three template nucleotides are sufficient for stable interaction with RdRp. These results identify base moieties in the brome mosaic virus subgenomic promoter required for efficient RNA synthesis and support the hypothesis that the recognition of a RNA promoter by a viral RdRp is analogous to the recognition of DNA promoters by DNA-dependent RNA polymerases.

Post-synthetically ligated ribozymes: an alternative approach to iterative solid-phase synthesis.

To improve the overall yield of ribozyme synthesis, a convergent approach, based on the post-synthetic formation of an amino linker between two half-ribozymes was investigated. Borane.pyridine-mediated reductive amination of 3'-phosphoglycaldehyde-5'-half-ribozymes with 5'-aminohexyl-3'-half-ribozymes generated the corresponding amino-linked ribozymes in yields > 77% on different scales. The investigation of a variety of reducing agents is discussed together with a kinetic analysis of the selected coupling reaction. These post-synthetically ligated ribozymes exhibited slightly reduced in vitro catalytic activity and cell efficacy.