Anti-Müllerian hormone, progesterone, and estrogen at breeding season onset and their association with reproductive performance in multiparous ewes.

Background: Ovarian reserve is established during fetal life and progressively declines with age, a process accompanied by increased follicular atresia, impaired steroidogenic capacity of granulosa cells, and consequent alterations in circulating anti-Müllerian hormone (AMH), estradiol, and progesterone levels. These changes may compromise ovulatory efficiency, uterine receptivity and luteal function, thereby influencing conception rates and the risk of early pregnancy loss. Because ewes combine a long reproductive lifespan with follicular dynamics that resemble those of women, they are of interest as a large-animal model of ovarian ageing; in women, declining follicular reserve and falling AMH are established markers of reduced fertility. This study evaluated the association between serum AMH, progesterone and estrogen concentrations measured at the onset of the breeding season and subsequent reproductive performance in multiparous ewes. Methods: Forty-four multiparous kangal ewes aged 6-8 years, with a history of multiple prior parturitions, were used as study material. Blood samples were collected at the onset of the breeding season to determine serum AMH, estrogen, and progesterone concentrations. Animals were monitored throughout the breeding season for time to conception and pregnancy outcome, and were subsequently allocated into six groups: Group 1 (n=8, conceived in the 1st cycle), Group 2 (n=7, conceived in the 2nd-3rd cycle), Group 3 (n=5, conceived in the 2nd-3rd cycle but experienced pregnancy loss), Group 4 (n=8, conceived in the 4th cycle), Group 5 (n=8, conceived in the 5th-8th cycle), and Group 6 (n=8, failed to conceive throughout the breeding season). Hormone concentrations were compared among groups, and Spearman correlation analysis was performed to assess pairwise hormonal relationships. Results: Serum AMH concentrations were significantly higher in Group 1 compared with Group 3 (P<0.01), Group 5 (P<0.01), and Group 6 (P<0.05), and in Group 2 compared with Group 3 (P<0.01) and Group 5 (P<0.01). No significant differences in AMH were observed between the remaining group pairs. Serum progesterone and estrogen concentrations did not differ significantly among groups (P>0.05). Across the entire cohort, progesterone and estrogen demonstrated a moderate positive correlation (Spearman's ρ = 0.387), indicating that animals with higher progesterone concentrations tended to have higher estrogen concentrations. This association remained significant after Benjamini-Hochberg correction within the whole-cohort correlation family (P = 0.010; q = 0.030), whereas no significant correlation was detected between AMH and progesterone (ρ = -0.248, P = 0.104) or between AMH and estrogen (ρ = -0.146, P = 0.346). None of the exploratory within-group correlations survived correction for multiple testing. Conclusions: Among the three hormones evaluated, AMH was the only one that differed significantly among reproductive outcome groups; progesterone and estrogen showed no significant differences, although the study was powered to detect only large effects and smaller but biologically meaningful differences cannot be excluded. These findings are consistent with the proposed value of the ewe as a model of human ovarian ageing, a parallel that currently rests on phenotypic rather than established mechanistic similarity, and indicate that AMH measured before mating is associated with subsequent reproductive performance in aged multiparous ewes. Prospective longitudinal studies using species-specific cohorts are warranted to better characterize the timeline and underlying mechanisms of ovarian aging.