Late-follicular androgen and estrogen rise associated with clinical pregnancy inIVF/ICSIwith granulosa cell studies

This study aimed to characterize phase-specific dynamics of androstenedione and estradiol across IVF/ICSI treatment, identify predictors of reproductive outcomes, and examine the effects of follicle-stimulating hormone (FSH), IGF1, and estradiol on steroidogenesis in human granulosa-like tumor (KGN) cells. In women undergoing IVF/ICSI (10 successful and 19 unsuccessful), serum hormones were measured before recombinant FSH stimulation (Phase 1) and during the mid-follicular (Phase 2) and ovulatory (Phase 3) phases, with follicular fluid (FF) collected at Phase 3. Mechanistic experiments were conducted in KGN cells (n = 3) treated for 24 h. Successful pregnancy was associated with greater increases in androstenedione and estradiol from Phase 2 to Phase 3 and higher Phase 3 steroid-to-basal gonadotropin ratios. Elevated Phase 1 androstenedione and estradiol were negatively correlated with the number of retrieved oocytes, mature oocytes, and/or embryos. Phase 3 serum androstenedione (cutoff ≥ 2.382 ng/mL; AUC = 0.708, P = 0.038) and FF aromatase (cutoff ≤ 0.378 ng/mL; AUC = 0.727, P = 0.007) predicted clinical pregnancy. In KGN cells, IGF1 stimulated early steroidogenesis (HSD3B2 mRNA expression and progesterone levels), FSH enhanced both early and late pathways (HSD3B2 and CYP19A1 mRNA expression together with progesterone, androstenedione, and aromatase levels), and supraphysiological estradiol suppressed downstream steroidogenesis (CYP19A1 mRNA expression and aromatase and progesterone levels). In conclusion, a favorable pattern with low early-follicular and high late-follicular androstenedione and estradiol was associated with IVF/ICSI success, whereas high early androstenedione and estradiol appeared detrimental. In KGN cells, supraphysiological estradiol without androgen substrate suppressed downstream steroidogenesis, consistent with the adverse effects of excessive early estradiol exposure, supporting granulosa cell contribution to local androgen availability. ## Lay summary Fertility treatment does not always lead to pregnancy. Hormones produced by the ovaries play a key role in pregnancy success. However, it is not clear how hormonal changes over time affect pregnancy. To address this gap, we followed up women undergoing fertility treatment and measured key reproductive hormones, including androstenedione (A4) and estradiol (E2), at different stages of the treatment cycle. We also used human ovarian cells to understand how E2 influences hormone production. We found that women who became pregnant showed a clear rise in A4 and E2 just before ovulation, while women who did not become pregnant had higher hormone levels at the beginning of the cycle. Our study in human ovarian cells supported these results, showing that excessive early A4 and E2 exposure could reduce later hormone production. Our findings may improve the prediction of treatment success and support the development of better fertility care strategies.