Hormonal measurements and ultrasound scans

On the third day of cycle preceding COH, each woman underwent blood sampling by venipuncture for serum AMH, and FSH measurement and a transvaginal ovarian ultrasound scan was performed for follicle measurement.

Serum levels of AMH and FSH were deter- mined using an automated multianalysis system with chemiluminescence detection (ACS-180, Bayer Diagnostics, Puteaux, France). Serum AMH levels were determined using a second generation enzyme-linked immunosorbent assay. Intra- and inter-assay coefficients of variation were <6% and <10%, respectively, with the lower detection limit at 0.13 ng/mL and linearity up to 21 ng/mL for AMH. For FSH, functional sensitivity was 0.1 mIU/mL, and intra-assay and inter-assay CV were 3 and 5%, respectively. Ultrasound scans were performed using a 3.7-9.3 MHz multifrequency transvaginal probe (RIC5- 9H, General Electric Medical Systems, France) by a single operator who was blinded to the results of hormone assays.

The objective of ultrasound examination was to evaluate the number and size of small antral follicles. Follicles measuring of 3-12 mm in mean diameter (mean of two orthogonal diameters) in both ovaries was considered.

To optimize the reliability of ovarian follicular assessment, the ultrasound scanner was equipped with a tissue harmonic imaging system, which allowed improved image resolution and adequate recognition of follicular borders. Intra-analysis CV for follicular and ovarian measurements was <5%, and their lower limit of detection was 0.1 mm. In an effort to evaluate the bulk of granulosa cells in both ovaries, we calculated the mean follicle diameter (cumulative follicle diameter divided by the number of follicles measured 3-12 mm in diameter in both ovaries) and the largest follicle diameter.