The objective of this study was to determine whether beta human chorionic gonadotropin (hCG) (CGB) subunits and alpha hCG (CGA) subunits are expressed and the hCG dimer is produced in normal human cyclic endometrium. Endometrial specimens were collected for histological dating from women undergoing treatment in our division of human reproduction. RNA from normal secretory endometrium was extracted, and CGB and CGA gene expression was assessed by semiquantitative PCR. Adequate secretory endometrial specimens were homogenized using protease inhibitors. Proteins present in the supernatant were separated electrophoretically, and molecular hCG isoforms were detected by Western blot. The supernatant hCG concentrations were measured by ELISA. We characterized hCG and leukocytes in endometrial specimens by immunohistochemistry. Uterine flushing was performed to confirm endometrial hCG secretion into the uterine fluid. A full-length CGB mRNA encompassing the exon 1 promoter region and the structure exons 2 and 3 (including the C-terminal peptide) was expressed in normal secretory endometrial specimens (similar to CGA) during the early secretory phase of the menstrual cycle, up to an optimum at the midsecretory to late secretory phases. In homogenate supernatants obtained from normal secretory endometrium, hormone concentrations of dimeric hCG were approximately 5 mU per 10 mg of tissue, compared with considerably smaller concentrations of corresponding single free CGB subunit. Single chains of CGB, CGA, and dimeric molecular hCG isoforms were found in endometrial specimens by Western blot. Glandular endometrial hCG production is demonstrated immunohistochemically, with an increase toward the late secretory phase vs. the early secretory phase of the normal secretory menstrual cycle. However, glandular hCG release is diminished or absent in the dyssynchronous or missing endometrial secretory transformation. Endogenous endometrial hCG may be important for implantation and maintenance of pregnancy.

The aim of the present study is to investigate the impact of glyphosate on the microbiota and on the botulinum neurotoxin (BoNT) expression during in vitro ruminal fermentation. This study was conducted using two DAISY(II)-incubators with four ventilated incubation vessels filled with rumen fluid of a 4-year-old non-lactating Holstein-Friesian cow. Two hundred milliliter rumen fluid and 800 ml buffer solution were used with six filter bags containing 500 mg concentrated feed or crude fiber-enriched diet. Final concentrations of 0, 1, 10, and 100 µg/ml of glyphosate in the diluted rumen fluids were added and incubated under CO2-aerated conditions for 48 h. The protozoal population was analyzed microscopically and the ruminal flora was characterized using the fluorescence in situ hybridization technique. Clostridium botulinum and BoNT were quantified using most probable number and ELISA, respectively. Results showed that glyphosate had an inhibitory effect on select groups of the ruminal microbiota, but increased the population of pathogenic species. The BoNT was produced during incubation when inoculum was treated with high doses of glyphosate. In conclusion, glyphosate causes dysbiosis which favors the production of BoNT in the rumen. The global regulations restrictions for the use of glyphosate should be re-evaluated.

A sensitive enzyme immunoassay for the measurement of insulin in human sera on microtiter plates was established. The assay is based on the sandwich technique with guinea pig anti-insulin IgG adsorbed at microtiter plate wells, human insulin as standard and the same anti-insulin IgG labeled with horseradish peroxidase. Standards used cover a range from 0 to 1200 pmol/l with a detection limit of 10 pmol/l. Coefficients of variation between 3-7% for intraassay precision and 5-11% for interassay precision were obtained over the concentration range of 80-1000 pmol/l. The correlation of EIA-data with those of a commercially available double antibody radioimmunoassay (r = 0.98) could be expressed by the equation: EIA = 0.97 RIA - 57 pmol/l. Normal fasting serum insulin concentrations in healthy subjects ranged from 11-165 pmol/l. In subjects with potentially diminished basal values concentrations of 10-79 pmol/l were determined. The insulin response in oral glucose tolerance tests of children was discussed, who had a constitutional tall stature or Turner's syndrome, respectively.

We have previously confirmed glandular cell CGB and CGA subunit mRNA gene expression as well as the expression of their dimeric and single-subunit human chorionic gonadotropin (hCG) proteins in normal secretory transformed endometrium. The objective of this study was to investigate the endometrial epithelial gene locus of the human hCG/LH gene cluster from CGB genes responsible for gene expression. For this study, endometrial specimens were selected from women characterized using our endometrium score and hCG staining index that had normal secretory transformed endometrium and optimal hCG staining. Using full-length CGB mRNA sequence analysis, we found that epithelial CGB is (co)expressed as the product of gene locus CGB7 and CGB6 (48%), as single CGB7 (42%), or to a lower percentage as single CGB6 (10%). In addition to known differences between these genes and CGB5, the nucleotide sequence of the mRNA differs between CGB7 and CGB6 in the untranslated promoter region and in translated exon 2. Immunohistochemical results show that endometrial joint CGB7 and CGB6, single CGB7, and single CGB6 mRNA expression lead to the release of endometrial hCG. Genespecific antibodies for CGB7 reveal secretory endometrial hCG production, which is not observed for gene-specific CGB5 antibodies, whereas the placenta is positive for CGB5 and negative for CGB7 antibody as revealed by immunohistochemistry and Western blot hCG isoform analysis. Only endometrial CGB7 expression seems to be supported specifically by secretory endometrial transcription factors. In conclusion, epithelial hCG is expressed and produced as CGB7 and/or CGB6 but not CGB5, and it is produced together with CGA as a secretory transformation marker in the normal secretory phase endometrium.endometrium, female reproduction tract, human chorionic gonadotropin (hCG/hCG receptor), menstrual cycle, uterus

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