Effect of GnRH-agonist downregulation on serum AMH levels: a prospective cohort study with repeated measurements P. Drakopoulos1, A. van de Vijver1, J. Parra2, E. Anckaert1, J. Schiettecatte1, J. Smitz1, C. Blockeel1, M. Hund3, W. D. J. Verhagen- Kamerbeek3, Y. He4, H. Tournaye1, N. P. polyzos5,6,7 1Centre for Reproductive Medicine, Universitair Ziekenhuis Brussel, Vrije Universiteit Brussel, Brussels, Belgium; 2Department of Statistics, Universitair Ziekenhuis Brussel, Vrije Universiteit Brussel, Brussels, Belgium; 3Roche Diagnostics International Ltd, Rotkreuz, Switzerland; 4Roche Diagnostics GmbH, Penzberg, Germany; 5Department of Reproductive Medicine, Dexeus University Hospital, Barcelona Spain; 6Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Brussels, Belgium; 7Faculty of Health, Aarhus University, Aarhus, Denmark

Study design Prospective cohort study performed at a center for reproductive medicine in a tertiary university hospital (July 2015 – March 2016) Primary endpoint: effect of a GnRH-agonist on serum AMH concentration after 7 and 14 days of treatment. †Baseline measurement taken before treatment was initiated SC, subcutaneous; UZB, Universitair Ziekenhuis Brussel

Patient demographics and baseline characteristics All patients (n=52) Mean ± SD age, years 35.8 ± 3.6 Mean BMI ± SD, kg/m2 23.5 ± 3.6 Race, n (% of total study population) White Asian Black Other 48 (92.3) 1 (1.9) 2 (3.9) Smoking status, n (% of total study population Yes No Missing 4 (7.7) 47 (90.4) Mean ± SD AMH at baseline, ng/mL 1.65 ± 0.8 Mean AFC ± SD at baseline 11.3 ± 4.3 AFC class, n (% of total study population) 0–7 8–15 >15 6 (11.5) 35 (67.3) 5 (9.6) AFC, antral follicle count; AMH, anti-Müllerian hormone; BMI, body mass index; SD, standard deviation

Box plots of serum AMH concentrations at baseline, and Days 7 and 14 of GnRH-agonist treatment (n=52) Serum AMH concentrations followed a v-shape from baseline through Day 7 to 14 ***P<0.001 versus baseline; Circles represent individual patient data; crosses are the mean value; horizontal lines summarize the median (within the box) and the first and third quartiles; whiskers are 1.5 x the interquartile range. AMH, anti-Müllerian hormone

Median % change in concentration (95% CI) [P value*] Percentage changes in serum concentrations of AMH, FSH, LH, E2 and progesterone (n=52) Hormone Median % change in concentration (95% CI) [P value*] Day 7 vs baseline Day 14 vs baseline Day 14 vs Day 7 AMH –14.9 (–23.0, –10.1) [<0.05] 17.4 (10.4, 35.9) [<0.001] 48.3 (34.7, 57.4) FSH –19.8 (–27.5, 3.04) [NS] –7.31 (–25.0, 22.6) 5.47 (–6.97, 22.6) LH –9.96 (–17.6, 17.2) –43.2 (–55.4, –33.1) –47.6 (–52.2, –41.0) E2 –28.6 (–60.6, 25.3) –94.8 (–95.9, –93.4) –90.5 (–94.5, –81.0) Progesterone –69.2 (–85.5, 0.168) –95.8 (–97.7, –94.5) –90.9 (–94.7, –79.1) Significant decreases in serum LH, E2 and progesterone at Day 14 confirm the ability of GnRH-agonist treatment to downregulate pituitary function *Wilcoxon signed rank test, median of the percentage change Statistical significance for AMH was defined as P<0.05; for other hormones P<0.0125 (Bonferroni correction) AMH, anti-Müllerian hormone; E2, estradiol; FSH, follicle stimulating hormone; LH, luteinizing hormone; NS, not significant

Individual serum AMH concentrations for the four aliquots measured for each patient per visit: baseline, and Days 7 and 14 (n=52) 4 3 2 1 BL 7 14 Serum AMH (ng/mL) 5 6 9 10 11 12 13 15 16 17 18 19 20 21 22 23 24 26 27 28 29 30 31 32 33 34 35 36 37 40 41 43 44 45 46 47 48 49 50 51 52 53 54 57 SJ1 SJ2 Elecsys® AMH assay demonstrated excellent precision across the four aliquots measured for each serum sample Circles represent the four aliquots for each sample at each visit BL, baseline

Conclusions GnRH-agonist treatment had a statistically significant effect on serum AMH concentrations in women with infertility who were planning to undergo ovarian stimulation. In a clinical setting, these changes may lead to an inaccurate prediction of ovarian reserve. Serum AMH should therefore be measured before starting GnRH- agonist treatment to allow for reliable assessment of ovarian reserve and prediction of response to ovarian stimulation.