In situ hybridization of GnRH mRNA in the rat and the mink hypothalamus using biotinylated synthetic oligonucleotide probes.

The cellular localization of GnRH messenger RNA (mRNA) in the rat and the mink hypothalamus has been examined using a newly developed highly sensitive non-radioactive in situ hybridization procedure. Synthetic oligonucleotides labeled by addition of a biotin-21-dUTP tail at their 3' end can be used to detect GnRH mRNA in both species. Streptavidin-alkaline phosphatase revealed with nitroblue tetra-zolium-bromo-chloro-indolyl-phosphate as substrate makes possible detection of the biotinylated oligonucleotides. In the rat, our findings confirm results previously obtained using synthetic radioactive probes, and demonstrate the potency of and interest in using biotinylated oligonucleotides to identify related sequences of bases in tissues. The principle advantages include rapid signal detection, excellent spatial resolution, and low background. In the mink, the in situ hybridization method clearly confirms the characterization of GnRH-producing cells and also allows detection of GnRH cell bodies in conditions in which they are not detected by immunohistochemistry. Adaptation of the in situ hybridization to the detection of GnRH mRNA in species like the mink which shows seasonal reproductive activity is a crucial step. This method offers a new approach to problems as fundamental as changes in gene expression depending on photoperiod or under a variety of experimental conditions.

Ovarian reserve test with the gonadotrophin-releasing hormone agonist buserelin: correlation with in-vitro fertilization outcome.

A gonadotrophin-releasing hormone agonist stimulation test determination of follicle stimulating hormone (FSH) concentrations before and 2 h after buserelin injection was carried out in 78 in-vitro fertilization cycles, and compared with basal FSH concentrations to predict ovarian response. Ovarian response was quantified by the ratio of peak oestradiol concentration divided by the total dose of human menopausal gonadotrophin (HMG) administered, the most reproducible parameter in 11 patients who underwent two treatment cycles. Stimulation outcome was highly related to the buserelin test, the best prognostic indicator being the sum of FSH concentrations. However, basal FSH concentration achieved similar correlations, even in those patients aged > 35 years. Sensitivity, specificity, positive and negative predictive values of basal FSH concentration and sum of FSH concentrations were similar. Low basal concentration and sum of FSH concentrations were both associated with a better ovarian response. Construction of receiver operator characteristic curves demonstrated that basal FSH concentration was more informative than the sum of FSH concentrations. Finally, the sum of FSH concentrations did not increase the prediction of ovarian response variability. We conclude that the buserelin test is strongly predictive of stimulation outcome, but is no more informative than the usual screening. We suggest that the performance of other stimulation tests should be clearly compared with that of basal FSH concentration.