A rapid method for the measurement of the oxoreductase activity of 11beta-hydroxysteroid dehydrogenase in granulosa-lutein cells from patients undergoing in-vitro fertilization.
The presence of 11beta-hydroxysteroid dehydrogenase (11beta-HSD; EC1.1.1.146), the enzyme responsible for the interconversion of cortisol and cortisone, in granulosa-lutein (GL) cells is associated with a poor outcome in in-vitro fertilization (IVF). We have developed a simple method of assessing the reductase component of 11beta-HSD in these cells which is sufficiently rapid to provide data on the enzyme's activity prior to embryo replacement. Cells were pooled from follicular aspirates and challenged with cortisone within 2 h of aspiration. Cortisol secretion was then measured by radioimmunoassay. Conversion of cortisone to cortisol was linear for up to 3 h and was completely inhibited by glycyrrhetinic acid, a specific 11beta-HSD inhibitor. Initial velocity rates were determined for eight cortisone concentrations (range 0.1-8 micromol/l), and the apparent Km calculated (1.6 +/- 0.4 micromol/l). There was no evidence of substrate/product inhibition and conversion of cortisone to cortisol was <2% in all experiments. In subsequent work, cells were challenged with cortisone (6 micromol/l) for 2 h. Cells challenged for 2 h immediately following purification from follicular aspirates produced varying amounts of cortisol (range 25-150 nmol/pooled follicles from each patient, n = 10 patients), while basal outputs were <6 nmol/l. Enzyme activity was also examined in cells on a per follicle basis from individual patients and found to vary considerably (e.g. 19, 53 and 36 nmol/l cortisol/1000 cells, three follicles). Having established the method for assessing 11beta-reductase activity within GL cells, we performed a small prospective study on a series of 20 patients examining the enzyme activity within 110 individual follicles. 11Beta-reductase activity varied greatly from patient to patient and from follicle to follicle ranging from <0.024-0.57 nmol cortisol/microg DNA but at present low patient numbers preclude a meaningful correlation between enzyme activity and pregnancy rate. In summary, we have developed a simple, rapid (<8 h) assay for detecting the reductase activity of 11beta-HSD in GL cells isolated from pooled or individual follicles. This procedure is sufficiently quick to aid in the choice of embryo for replacement.
Knaggs
P
P
Department of Medicine, Hope Hospital, Salford, UK.
Lambert
A
A
Proudfoot
F
F
Nickson
I
I
Hooper
M A
MA
Lenton
E
E
Robertson
W R
WR
D042842:11-beta-Hydroxysteroid Dehydrogenases; D003348:Cortisone / Q000378:metabolism; D005260:Female; D005307:Fertilization in Vitro; D006107:Granulosa Cells / Q000201:enzymology*; D006801:Humans; D006854:Hydrocortisone / Q000378:metabolism; D006913:Hydroxysteroid Dehydrogenases / Q000032:analysis* / Q000378:metabolism; D008184:Luteal Cells / Q000201:enzymology*; D011247:Pregnancy; D011446:Prospective Studies; D011863:Radioimmunoassay / Q000379:methods
D006913:Hydroxysteroid Dehydrogenases; D042842:11-beta-Hydroxysteroid Dehydrogenases; D003348:Cortisone; D006854:Hydrocortisone