ABSTRACT
<p><b>UNLABELLED</b>Objective To explore the effects of sperm DNA integrity rate, acrosome integrity rate and acrosome reaction rate on the outcomes of rescue intracytoplasmic sperm injection (ICSI).</p><p><b>METHODS</b>This retrospective analysis was conducted among 97 infertile couples receiving rescue ICSI due to failure of in vitro fertilization procedures in our Reproductive Medicine Center. Of these 97 women, 41 had clinical pregnancy and 56 did not, and the effects of sperm DNA integrity rate (estimated by DNA fragmentation index, DFI), acrosome integrity rate and acrosome reaction rate on rescue ICSI outcomes were analyzed.</p><p><b>RESULTS</b>No significant difference was found in paternal age, testosterone value, testicular volume, FSH, female patient' age or the number of eggs retrieved between the two groups (P>0.05), but the infertility years was significantly shorter in the pregnancy group than in the non-pregnancy group (P<0.05). The fertilization rate and cleavage rate were similar between the two groups (P>0.05), but the good embryo rate was significantly higher in the pregnancy group (P<0.05). The sperm DNA integrity or acrosome reaction rate did not differ significantly between the two groups (P>0.05), but the acrosome integrity rate was significantly higher in the pregnancy group (P<0.05). The sperm DNA integrity rate, acrosome integrity or acrosome reaction rate were not correlated with the fertilization rate, cleavage rate or good embryo rate (P>0.05). The pregnancy rate, twin and single fetus rates were 42.3%, 10.3% and 32.0% in this cohort after recue ICSI, respectively.</p><p><b>CONCLUSION</b>Rescue ICSI is an effective treatment after failed in vitro fertilization procedure, and sperm acrosome integrity rate is associated with the outcome of rescue ICSI.</p>
Subject(s)
Female , Humans , Male , Pregnancy , Acrosome , Pathology , Acrosome Reaction , DNA Fragmentation , Fertilization , Fertilization in Vitro , Infertility , Pregnancy Rate , Retrospective Studies , Sperm Injections, IntracytoplasmicABSTRACT
Objective To comparative analysis the intracytoplasmic sperm injection (ICSI)result and rate of sperm DNA in-tegrity (DNA fragmentation index,DFI)about testicular and epididymis sperm.Methods Totally 183 obstructed azoospermia pa-tients were choosed to use ICSI.80 cycles by PESA and 103 cycles by TESA,compared two groups of sperm DNA integrity rate and ICSI outcome.Results Sperm DNA integrity rate,fertilization rate,cleavage rate,good-qualityembryo rate and pregnancy rate com-pared with no difference by ICSI(P >0.05).Conclusion DNA integrity rate and ICSI outcomes of the testis and epididymis sperm have no significant differences,clinicians can be based on personal experiences or patients,wills to select sperm for ICSI.
ABSTRACT
Trehalose, a compatible solute, is widely used in food, cosmetics, pharmaceutical products and organ transplantation. Nowadays, trehalose is mostly produced by enzymatic synthesis with many secondary products and lowpurity. In this study, high amount of trehalose was produced by recombinant E. ccli fermentation. First, a bifunctional trehalose gene TPSP was amplified from genome of C. hutchinscoii. Second, an expression vector pTac-HisA containing TPSP was constructed and transformed into the host E. coli. Expression of this bifunctional enzyme-TPSP converted glucose to trehalose. The result suggested that TPSP from C. hutchinsonji has been successfully expressed in E. ccoi. High amount of extracellular trehalose generated from glucose by whole-cell catalysis and After optimization, the production of trehalose in shake flasks was improved to 1.2 g/L and the relative conversion rate reached 21%. The production in bioreactor reached 13.3 g/L and the relative conversion rate reached 48.6%. It is the first time to realize the functional expression of the bifunctional enzyme-TPSP of C. hutchinsonii in E. coli and achieved the conversion form glucose to trehalose. This study laid a foundation for industrial large-scale production of trehalose.
Subject(s)
Bioreactors , Catalysis , Escherichia coli , Genetics , Glucose , Glucosyltransferases , Industrial Microbiology , Organisms, Genetically Modified , TrehaloseABSTRACT
Total mRNA was extracted from lymphocytes separated from the peripheral blood of allergic patients, and then variable region of heavy chain (VH) and variable region of light chain (VL) cDNA library were constructed by RT-PCR. Human scFv templates for rabbit reticulocyte lysate ribosome display were assembled by primers and linker peptide (Gly4Ser)3. mRNA bound in antibody-ribosome-mRNA complexes was recovered using in-situ single primer RT-PCR, and three rounds of anti-IgE scFv DNA were enriched. The target DNA fragments were double enzyme digested and ligated into plasmid pET22b (+), followed by transformation in E. coli Rosseta (DE3). Positive clones were screened using clone PCR, Dot blotting and antigen ELISA. The correct lengths of VH (400 bp) and VL (710 bp) PCR products were obtained. The expected 1,000 bp ribosome display templates were also observed in agarose gel electrophoresis. After three rounds of ribosome display target sequences were effectively enriched, leading to a library of 10(13) members. Antibodies with the highest ELISA value for IgE were generated in the strain pET-IgE-6. A human anti-IgE scFv library was successfully constructed as described herein. Ribosome display using single primer in-situ RT-PCR as the recovery procedure effectively enriched target sequences. Anti-IgE scFv with high affinity and specificity were identified. The prepared human anti-IgE scFv fragment might be self-developed to a lead drug for treating asthma. Our study provides an alternative method for rapid discovery of human antibodies of therapeutic importance.