AMH, the fetal testicular sexual differentiative factor, is now implicated in adult ovarian function. Antral follicle immunohistochemistry demonstrated AMH protein and message, the staining peaking around 4 mm. Interestingly, AMH-knockout mice have increased FSH sensitivity. Our aim was to measure AMH in follicular fluid and cell-conditioned medium and AMHRII in normal ovaries from women undergoing TAH/BSO.
Follicles were dissected intact, follicular fluid aspirated and granulosa and theca cultured ±gonadotrophins. Granulosa luteal cells (GLC) were harvested from women undergoing IVF. AMH in fluid and media was measured by ELISA (DSLabs). Characterisation of the assay for media use included: parallel dilution of samples with the standard curve, good recovery and no loss with repeated freeze/thaw cycles. The detection limit was 0.025 ng/ml. AMHRII expression was assessed by PCR of RNA extracted from granulosa, GLC and theca cells.
Follicular fluid AMH concentrations were mean (range) 4 (0.316)ng/ml (n=318). Levels declined exponentially with increasing follicle size, being undetectable in follicles >9 mm. AMH in granulosa-conditioned medium ranged from undetectable to 1.7 ng/ml (n=17) with levels again falling with follicle size: mean at 5 mm=1.47, mean at 10 mm=0.17 ng/ml and undetectable above 10 and in GLC. Levels in stroma and theca were below or at the detection limit. Incubation with FSH or LH (5 ng/ml) had no consistent effect. AMHRII was present in granulosa, GLC and theca.
In summary, the DSLabs AMH ELISA is suitable for use with ovarian cell culture medium. AMH is produced primarily by granulosa cells in the human ovary and there is a rapid decline in production as follicles approach 10 mm in diameter. These data add further weight to AMH having an important role in follicle selection. As all compartments of antral follicles express the receptor, further elucidation of the action of AMH in the ovary is now essential.
01 - 05 Apr 2006
European Society of Endocrinology