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RESEARCH QUESTION: How stable is the pH of human preimplantation embryo culture media during IVF culture and is there variation in pH between batches of culture media? DESIGN: To evaluate pH stability, three batches of three culture media were incubated in triplicate without embryos (sham culture) at CO2 levels recommended by the manufacturers (5% or 6%) for 4 days. To evaluate differences in pH between batches, the pH of three batches of five culture media was measured in triplicate during 1 day of sham culture. Linear mixed models were used for the analysis. RESULTS: An increase in pH during 4 days of culture was found in all three culture media, but the observed increased values were within the generally accepted range for clinical practice (pH 7.2-7.4). One medium was pH 7.1 in the first 2 days, but this was within the range provided by the manufacturer for that medium. Three out of five analysed media showed batch variation in pH that exceeded the generally accepted range for clinical practice. CONCLUSIONS: A relevant difference in pH was found between batches of human preimplantation embryo culture media. This suggests that the CO2 level of incubators may need to be adjusted for new batches of culture medium based on measured pH, to anticipate batch variability and safely accommodate limited pH increase over time. This study was unable to identify the cause of the differences in pH between batches, and further investigation on a larger number of batches and other media seems warranted.
Assuntos
Meios de Cultura/normas , Técnicas de Cultura Embrionária , Humanos , Concentração de Íons de HidrogênioRESUMO
BACKGROUND: Smoking has a negative effect on fertility and sperm quality. This study was designed to evaluate the effects of smoking on sperm quality and the related parameters such as sperm concentration, morphology and motility. MATERIALS AND METHODS: This cross-sectional study was conducted on 180 infertile men with at least one year history of idiopathic infertility, who admitted to the Avicenna Infertility Center, Tehran, Iran. A complete history including smoking habits and other diseases was obtained and semen analysis was performed for all participants. Statistical analysis was performed using SPSS software version 16 and t test and Mann-whitney tests with a significance level of α= 0.05. RESULTS: Comparison of sperm parameters in the two groups of smoker and nonsmoker subjects showed that active smoking (p=0.04) and cigarette consumption even in small amounts (p=0.03) decreased sperm concentration, However, no significant correlation was detected between smoking status and morphology or motility of sperms. CONCLUSION: This study failed to find a significant correlation between sperm analysis and smoking status except for sperm concentration, which was significantly decreased in the active smokers ,even in those consuming small amounts of tobacco. This finding propounds that tobacco consumption may negatively affect fertility.
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OBJECTIVE: Synthetic fluorescent dyes that are conjugated to antibodies are useful tools to probe molecules. Based on dye chemical structures, their photobleaching and photostability indices are quite diverse. It is generally believed that among different fluorescent dyes, Alexa Fluor family has greater photostability than traditional dyes like fluorescein isothiocyanate (FITC) and Cy5. Alexa Fluor 568 is a member of Alexa Fluor family presumed to have superior photostability and photobleahing profiles than FITC. MATERIALS AND METHODS: In this experimental study, we conjugated Alexa Fluor 568 and FITC dyes to a mouse anti-human nestin monoclonal antibody (ANM) to acquire their photobleaching profiles and photostability indices. Then, the fluorophore/antibody ratios were calculated using a spectrophotometer. The photobleaching profiles and photostability indices of conjugated antibodies were subsequently studied by immunocytochemistry (ICC). Samples were continuously illuminated and digital images acquired under a fluorescent microscope. Data were processed by ImageJ software. RESULTS: Alexa Fluor 568 has a brighter fluorescence and higher photostability than FITC. CONCLUSION: Alexa Fluor 568 is a capable dye to use in photostaining techniques and it has a longer photostability when compared to FITC.
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We have employed a peptide-based antibody generation protocol for producing antibody against human nestin. Using a 12-mer synthetic peptide from repetitive region of human nestin protein devoid of any N- or O-glyco-sylation sequences, we generated a mouse monoclonal antibody capable of recognizing human, mouse, bovine, and rat nestin. A wide variety of nestin proteins ranging from 140-250 kDa was detected by this antibody. This antibody is highly specific and functional in applications such as ELISA, flow cytometry, immunocytochemistry, and Western blot assays.
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Gene expression profiling of ovarian carcinoma tissues has shown an increase of four-fold expression of SORT1 gene. Sortilin 1 (NTR-3) is a 95-100 kDa protein normally expressed in heart, brain, placenta, skeletal muscle, spinal cord, thyroid, and testis. However, its expression has never been reported in normal ovary. Here, we report expression of sortilin 1 in ovarian carcinoma tissues both at gene and protein levels. Sortilin 1 was expressed in all ovarian carcinoma patients (n=15) as well as ovarian carcinoma cell lines (n=5) regardless of their phenotypic characteristics. Non-malignant ovaries (n=6) did not express sortilin 1. The molecular basis for this ectopic expression is not yet clear. Our results showed a major cell surface expression of sortilin 1 rather than ER-Golgi compartment where it is mainly expressed. This finding may introduce sortilin 1 as a novel tumor marker for diagnosis of ovarian carcinoma and may signify its therapeutic value in targeted therapy.
