Follicular fluid levels of vascular endothelial growth factor and its receptors and pregnancy outcome of women participating in intracytoplasmic sperm injection cycles.

PURPOSE: The intracytoplasmic sperm injection (ICSI) outcome is depended mainly on oocyte quality. Cytokines and their receptors play a critical role in oocyte maturation, fertilization, and embryo implantation. The purpose of the study was to study the levels of vascular endothelial growth factors (VEGFA, VEGFR1, VEGFA) in follicular fluids (FF) women participating in ICSI-in vitro fertilization (IVF) cycles in relation to cycle's outcome. MATERIAL AND METHODS: One hundred and fifty three samples of 70 women participating in ICSI cycles were classified in three infertility groups: male factor, female factor, and low responders. For controlled ovarian stimulation in male and female factor group, the long agonist protocol with leuprolide and recombinant follicle stimulating hormone (FSH) was employed, while the antagonist cetrorelix was used in low responders. Cytokines levels were evaluated with enzyme-linked immunosorbent assay (ELISA). RESULTS: In a total of 153 samples, the overall pregnancy rate was 51.6%, the higher one observed in female factor group (59% vs. 37.5% and 28.6% in male a factor and low responders group, p = 0.013. VEGFR2 differed statistically significantly between the two groups, being higher in the pregnancy group [median (IQR): 5,630 (4,870 - 6,651) vs. 4938 (4,068 - 6,020) in the non-pregnancy group, p = 0.003]. There were significant correlations between VEGF receptors, differentiated depending on infertility groups. CONCLUSIONS: The VEGFA/VEGFR2 system is important in human reproduction and the association pattern between VEGFA receptors may serve as a marker for ICSI outcome. Examination for spermatozoa functional defects may increase pregnancy rate in male factor group.

Open versus closed vitrification of blastocysts from an oocyte-donation programme: a prospective randomized study.

The use of open carriers for embryo vitrification has raised safety concerns and therefore vitrification in closed systems has been proposed. However, the drop in the cooling rate emerges as a major drawback. The objective of the present study was to compare the efficiency of vitrification in open versus closed conditions. Blastocysts were randomly allocated either to open ultra-rapid vitrification (group I) or closed aseptic vitrification (group II). In group I, blastocysts were exposed to two solutions of ethylene glycol/dimethylsulphoxide (10%/10% and 20%/20%), while in group II, blastocysts were pretreated with a solution of lower concentration (5%/5%). A total of 208 and 224 vitrification-warming cycles were performed for groups I and II, respectively. Both groups were equal in terms of maternal age, sperm parameters and number and quality of blastocysts vitrified, warmed and transferred per cycle. Importantly, there was no significant difference between the groups in the analysed outcomes; embryo survival rate (84.1% versus 82.1%), clinical pregnancy rate (45.9% versus 42.4%), implantation rate (25.6% versus 24.5%), cycle cancellation rate (6.7% versus 8.5%) and live birth rate (41.2% versus 41.0%). These data suggest that ultra-rapid vitrification may be replaced by aseptic vitrification without affecting clinical efficiency.

The ε4 allele of the APOE gene is associated with more severe peripheral neuropathy in type 2 diabetic patients.

We examined the association between the ε4 allele of the apolipoprotein E gene and severity of peripheral neuropathy in 234 patients with type 2 diabetes mellitus (T2DM). Based on the Neuropathy Disability Score (NDS), patients were divided into group A (NDS ≤ 6: mild or no neuropathy) and group B (NDS > 6: severe neuropathy). In each group, patients were further divided into ε4 carriers and non-ε4 carriers. In multivariate analysis, a more than 5-fold increased risk of severe neuropathy was associated with ε4 carrier status (adjusted odds ratio [aOR]: 5.26, 95% confidence interval [CI]: 2.24-12.31, P = .0001). The other significant risk factors for severe neuropathy included male gender (aOR: 2.08, 95% CI: 1.05-4.14, P = .036), diabetes duration (aOR: 1.05, 95% CI: 1.00-1.09, P = .039), and hemoglobin A1c (aOR: 1.32, 95% CI: 1.05-1.66, P = .020). In conclusion, the ε4 carrier status appears to be associated with severe peripheral neuropathy in T2DM.

High levels of HCV core+1 antibodies in HCV patients with hepatocellular carcinoma.

The core region of the hepatitis C virus (HCV) genome possesses an overlapping ORF that has been shown to encode a protein, known as the alternate reading frame protein (ARFP), F or core+1. The biological role of this protein remains elusive, as it appears to be non-essential for virus replication. However, a number of independent studies have shown that the ARFP/F/core+1 protein elicits humoral and cellular immune responses in HCV-infected individuals and interacts with important cellular proteins. To assess the significance of the core+1 humoral response in HCV-infected patients, we examined the prevalence of anti-core+1 antibodies in sera from patients with hepatocellular carcinoma (HCC) in comparison with chronically HCV-infected individuals without HCC. We produced two HCV core+1 histidine-tagged recombinant proteins for genotypes 1a (aa 11-160) and 1b (aa 11-144), as well as a non-tagged highly purified recombinant core+1/S protein (aa 85-144) of HCV-1b. Using an in-house ELISA, we tested the prevalence of core+1 antibodies in 45 patients with HCC in comparison with 47 chronically HCV-infected patients without HCC and 77 negative-control sera. More than 50 % of the serum samples from HCC patients reacted with all core+1 antigens, whereas <26 % of the sera from the non-HCC HCV-infected individuals tested positive. No core+1-specific reactivity was detected in any of the control samples. In conclusion, the high occurrence of anti-core+1 antibodies in the serum of HCC patients suggests a role for the ARFP/F/core+1 protein in the pathogenesis of HCC.

Apolipoprotein E and first-ever ischaemic stroke in Greek hospitalized patients.

The association between apolipoprotein E (ApoE) polymorphism and stroke is still controversial. This study investigated the potential association between ApoE genotypes and stroke subtypes, and risk factors for ischaemic stroke in Greek patients hospitalized with their first-ever ischaemic stroke. One hundred patients (70 men and 30 women; mean age +/- SD 60.7 +/-9.8 years) were included in the study. The control group comprised 96 age- and sex-matched healthy volunteers. Cerebral infarction was classified as atherothrombotic, cardioembolic or lacunar small-vessel stroke. The three common ApoE alleles (E2, E3 and E4) were determined using the semi-nested polymerase chain reaction. No significant difference in the ApoE alleles was found between patients and controls. Similarly, there was no significant association between ApoE alleles and stroke subtypes, common risk factors for ischaemic stroke and neck vessel stenosis. Although the sample size was small, these results do not support a role for ApoE polymorphism in the pathogenesis of ischaemic stroke.

Racial differences in allelic distribution at the human pulmonary surfactant protein B gene locus (SP-B).

Variable numbers of composite repetitive motifs are found in different individuals within intron 4 of the surfactant protein B (SP-B) gene (Biochem J. 1995;305:583). This study tests the hypothesis that the distribution of SP-B alleles differs among racial/ethnic groups. A total of 412 SP-B alleles were analyzed: 206 from Caucasian, 68 from African-American, and 138 from Nigerian individuals. Twelve groups of alleles (A-L) carrying 3 to 18 motifs were found. The distribution of the 12 alleles in the Caucasian group differs from that found in the Nigerian (p < .001) and African-American (p < .001) populations. The overall distribution of alleles between the African-American and the Nigerian populations were not statistically different. Specific alleles were also present in different proportions among the groups studied. For example, the most common allele (allele E) in all three populations is present at a significantly higher frequency in Caucasians than in the other two populations, but its frequency does not differ from the Nigerian and African-American groups. A less frequent allele, H, also differs significantly when Caucasians are compared with each of the other two populations, but the frequency of this allele is comparable between the African-American and Nigerian populations. To assess the importance of having comparable racial composition between the control and the case groups, a group of African-Americans with respiratory distress syndrome (RDS) (n = 40) was compared with the African American and the Caucasian groups studied above. No significant difference was observed between the racially matched groups but a significant difference (p = .006) was observed between the racially mixed groups. The results indicate that the distribution of SP-B alleles differs between the racial groups but not between the ethnic groups studied. Thus, racial composition of the groups under study is important when considering whether particular alleles at this locus predispose to inherited disorders.

Dinucleotide repeats in the human surfactant protein-B gene and respiratory-distress syndrome.

Pulmonary surfactant, a lipoprotein complex, is essential for normal lung function, and deficiency of surfactant can result in respiratory-distress syndrome (RDS) in the prematurely born infant. Some studies have pointed towards a genetic contribution to the aetiology of RDS. Because the surfactant protein B (SP-B) is important for optimal surfactant function and because it is involved in the pathogenesis of pulmonary disease, we investigated the genetic variability of the SP-B gene in individuals with and without RDS. We identified a 2.5 kb BamHI polymorphism and studied its location, nature and frequency. We localized this polymorphism in the first half of intron 4 and found that it is derived by gain or loss in the number of copies of a motif that consists of two elements, a 20 bp conserved sequence and a variable number of CA dinucleotides. Variability in the number of motifs resulting from either deletion (in 55.3% of the cases with the variation) or insertion (44.7%) of motifs was observed in genomic DNAs from unrelated individuals. Analysis of 219 genomic DNAs from infants with (n = 82) and without (n = 137) RDS showed that this insertion/deletion appears with significantly higher frequency in the RDS population (29.3 as against 16.8%, P < 0.05).

Surfactant protein C: hormonal control of SP-C mRNA levels in vitro.

We have studied hormonal regulation of the surfactant protein C (SP-C) in fetal 18-dah rat lung explants. SP-C mRNA was detected in Northern blots with a specific rat SP-C cDNA probe and quantified by densitometry. Treatment of the explants with dexamethasone resulted in a dose-dependent increase of the SP-C mRNA level. Transcriptional assays have shown that the regulation of SP-C mRNA by dexamethasone involves a transcriptional step. Administration of the cAMP analogues, 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) or dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP), produced a dose-dependent increase of SP-C mRNA levels, with maximum stimulation observed at 200 microM. The thyroid hormone T3 had no effect on SP-C mRNA levels, whether administered alone or in combination with dexamethasone. Variation in the effects of the above hormones on three surfactant protein mRNAs, SP-A, SP-B and SP-C, indicates that the hormonal regulation of the surfactant proteins is a complex process and that each gene is, in part, differentially regulated.

Hormonal effects on the surfactant protein B (SP-B) mRNA in cultured fetal rat lung.

Glucocorticoids, triiodothyronine (T3), and cyclic adenosine monophosphate (cAMP) have been shown previously to modulate phosphatidylcholine and surfactant protein A (SP-A) synthesis in fetal rat lung explant cultures. In this report, we have examined the hormonal regulation of the rat surfactant protein B (SP-B) mRNA to determine whether SP-B expression is coordinately regulated with the surfactant phospholipids or with SP-A. Dexamethasone (1 to 200 nM) and cAMP (200 microM) had a stimulatory effect on SP-B mRNA levels, whereas T3 tended to inhibit the accumulation of SP-B mRNA. In combination experiments, treatment with dibutyryl-cAMP (200 microM) and dexamethasone (100 nM) resulted in about a 22-fold increase, whereas dexamethasone or dibutyryl-cAMP alone produced 18- and 2-fold increases, respectively. When the cAMP analogue 8-bromo-cAMP (200 microM) was used in combination with dexamethasone, there was no significant difference between the combined effect and that of dexamethasone alone. T3 treatment, however, resulted in a significant reduction of the dexamethasone-induced stimulation from about a 22-fold to a 14-fold increase. Tissue in situ hybridization showed that dexamethasone stimulated the levels of SP-B mRNA in cells from both the alveolar and bronchiolar epithelium. These data indicate that there are differences in the hormonal regulation of the components of surfactant, suggesting that they are independently regulated.

Regulation of surfactant protein A mRNA by hormones and butyrate in cultured fetal rat lung.

We have previously shown that dexamethasone, triiodothyronine (T3) and dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP) stimulate phosphatidylcholine (PC) synthesis in fetal rat lung explants in culture. There are also additive interactions between these agents with regard to PC synthesis. In this study we examined the regulation of surfactant protein A (SP-A) mRNA in fetal rat lung in culture. Dexamethasone increased SP-A mRNA in the explants in a dose-dependent fashion (1-200 nM), but T3 did not. Whereas 8-bromo-cAMP increased SP-A mRNA, a decrease was observed with dibutyryl cAMP. These findings support the view that at least some of the genes involved in the synthesis of the various components of surfactant are independently regulated. Since we observed differences in the effects of a cAMP analogue which contained butyrate and one that did not, explants were then cultured with Na butyrate, a known regulator of gene expression. A significant decrease in SP-A mRNA was observed at mM concentrations. Exposure of the explants to alpha-aminobutyric acid, a butyric acid analogue which is elevated in the blood of infants of diabetic mothers, resulted in a significant decrease in SP-A mRNA at a concentration 1/25 of that required for Na butyrate. This observation raises the question of whether the decreased SP-A levels reported in fetuses of diabetic mothers may, at least in part, be related to this metabolite.

Transgenic regulation of moth chorion gene promoters in Drosophila: tissue, temporal, and quantitative control of four bidirectional promoters.

Bidirectional chorion gene promoter regions from three silkmoth species, Bombyx mori, Antheraea pernyi, or Antheraea polyphemus (members of two different moth families), were tested for their ability to transcriptionally activate a bacterial marker gene (chloramphenicol acetyltransferase) in transformant Drosophila. Relatively short 5' flanking DNA fragments (272-367 bp) of chorion gene pairs are sufficient to confer a high degree of tissue and choriogenic stage specificity of expression to the marker gene. Thus, significant conservation of molecular interactions controlling transcription during choriogenesis is observed between the distantly related orders, Lepidoptera and Diptera. However, quantitative and fine temporal regulation in the Drosophila host does not fully parallel the in situ regulation in moths, indicating that some regulatory protein-DNA interactions have diversified in the approximately 250 million years since the last common ancestor of these insect groups. Limited in vitro mutagenesis of a B. mori promoter DNA has shown that a central 189-bp region includes elements sufficient for the qualitative specificity of chorion-specific expression. The same experiments have shown that a previously identified essential element, centered on the TCACGT hexamer, is not sufficient for chorion-specific expression: an additional essential element or elements are found farther upstream, within a 112-bp DNA region. Comparisons of silkmoth and Drosophila chorion gene promoter sequences have identified some candidates for cis-acting elements involved in the developmental regulation of chorion gene expression.