Studies on the human ovarian renin-angiotensin system: optimization of assay methodology and effects of follicular stimulants

ABSTRACT The renal and genital tracts share a common embryological origin; it is thus not surprising that tissues from both can synthesize renin. Preliminary studies showed extremely high concentrations of renin in follicular fluid (FRC) following ovarian stimulation for in-vitro fertilization. This necessitated complete revalidation of the renin assays and showed that data obtained using commercial kits were invalid. An assay protocol was developed using a 1:2 dilution of follicular fluid taken into EDTA (0·3 mol/l) and o-phenanthroline (0·05 mol/l). The assay was performed at pH 7·5 in the presence of excess exogenous (sheep) renin substrate, with incubation periods of 5, 10 and 15 min at 37 °C. This protocol resulted in the linear generation of angiotensin I (AI). Activation of inactive renin was performed using eightfold more trypsin than was required for plasma samples. Follicular renin substrate concentrations (FRS) were measured using the same assay methodology as used for measurement of plasma renin substrate concentrations (PRS). Storage of samples at −18 °C for up to 2 months was found not to affect the FRC, although repeated freeze-thaw cycles did. FRC and plasma renin concentrations (PRC) were very similar in 25 unstimulated control women, studied in the follicular phase of the menstrual cycle. Trypsin activation increased follicular total renin concentration (FTRC) more than plasma total renin concentration (PTRC) (P< 0·0001). FRS was slightly higher than PRS (P<0·02). Ovarian stimulation with clomiphene citrate (CC; six women) was without effect on these parameters. However, hyperstimulation with CC, human menopausal gonadotrophins (hMG) and human chorionic gonadotrophin (hCG) resulted in substantial increments in FRC and FTRC (P< 0·0001 for both) and somewhat smaller rises in PRC and PTRC (P<0·05; P < 0·0005). There was also a small rise in PRS (P< 0·0002), but no change in FRS. Treatment with buserelin, hMG and hCG was associated with similarly large increases in renin concentrations, and also increases in both FRS and PRS (P< 0·003; P<0·007) in comparison with samples from women stimulated with CC, hMG and hCG. Increased plasma renin activity has previously been reported in stimulated ovarian follicular fluid. Our data show clearly that this is primarily due to a rise in FRC and FTRC and not to a rise in FRS. The use of the anti-oestrogen CC alone for ovarian stimulation was without effect on the follicular renin-angiotensin system. Thus we suggest that it is the gonadotrophins themselves which stimulate renin production, presumably by the theca cells. The much smaller rise in PRC and PTRC may reflect the effects of an overspill into the systemic circulation or, less likely, effects of the gonadotrophins on renal renin production. There is no evidence for this latter suggestion. FRS was increased only slightly by ovarian stimulation and must be presumed to be rate-limiting in the generation of AI. Journal of Endocrinology (1990) 127, 513–521