Polycystic ovary syndrome (PCOS) is the most common cause of anovulatory infertility. Although the cause of the anovulation is unknown there are a number of endocrine candidates; hyperinsulinaemia, raised steroid production and increased levels of the sexual differentiative factor anti-Müllerian hormone (AMH). The latter is increasingly thought to be an inhibitor of folliculogenesis. Hyperinsulinaemia in PCOS is now widely treated with metformin and we have shown that this insulin sensitizer also inhibits steroid production by ovarian cells. The aim of this study was to investigate the mechanism of the steroid inhibition and to determine any possible effect of metformin on AMH production.
Granulosa-luteal cells were collected from IVF follicular aspirates. For aromatase studies, cells were incubated with 10−7 M (16 ng/ml) metformin, 10 ng/ml insulin or metformin+insulin for 48 h (n=3). Reverse transcribed aromatase mRNA was quantified using SYBR® Green I and MX4000 real-time cycler (Stratagene) and normalized to ß-actin. Insulin increased aromatase mRNA expression by a ratio of 2.73±3.15 over control, whereas metformin inhibited it by a ratio of 0.055±0.11 (P<0.05 using REST©). Interestingly, metformin/insulin combined significantly reduced the stimulation seen by insulin: 0.068±0.12 (P<0.05, using REST©), an effect also seen when aromatase was measured by activity assay. For the AMH studies, cells were incubated with 10−7 M metformin for 48 h. RNA was extracted from cells and AMH protein levels measured in cell-conditioned media by ELISA. Real-time qPCR revealed that metformin reduced the expression of AMH mRNA by 27±9% (n=4, P=0.05). AMH protein levels were reduced by a mean of 65±8% (n=4, P=0.002) compared to untreated cells.
In summary, metformin inhibits both basal and insulin-stimulated aromatase expression in granulosa cells and the expression and production of AMH. In addition to its insulin-sensitising action, both of these further activities of metformin would have an effect in terms of induction of ovulation in women with anovulatory PCOS.
06 - 07 Nov 2006
Society for Endocrinology