Negative impact of high doses of follicle-stimulating hormone during superovulation on the ovulatory follicle function in small ovarian reserve dairy heifers†

Abstract

When women with small ovarian reserves are subjected to assisted reproductive technologies, high doses of gonadotropins are linked to high oocyte and embryo wastage and low live birth rates. We hypothesized that excessive follicle-stimulating hormone (FSH) doses during superovulation are detrimental to ovulatory follicle function in individuals with a small ovarian reserve. To test this hypothesis, heifers with small ovarian reserves were injected twice daily for 4 days, beginning on Day 1 of the estrous cycle with 35, 70, 140, or 210 IU doses of Folltropin-V (FSH). Each heifer (n = 8) was superovulated using a Williams Latin Square Design. During each superovulation regimen, three prostaglandin F2α injections were given at 12-h interval, starting at the seventh FSH injection to regress the newly formed corpus luteum (CL). Human chorionic gonadotropin was injected 12 h after the last (8th) FSH injection to induce ovulation. Daily ultrasonography and blood sampling were used to determine the number and size of follicles and corpora lutea, uterine thickness, and circulating concentrations of estradiol, progesterone, and anti-Müllerian hormone (AMH). The highest doses of FSH did not increase AMH, progesterone, number of ovulatory-size follicles, uterine thickness, or number of CL. However, estradiol production and ovulation rate were lower for heifers given high FSH doses compared to lower doses, indicating detrimental effects on ovulatory follicle function.

Keywords: anti-Müllerian hormone; bovine; estradiol; number of ovulatory follicles; ovulation rate; ovulatory follicle function; superovulation.

Figure 1

Effect of different doses of FSH on circulating concentrations of AMH and AFC for heifers with a small ovarian reserve. Beginning on Day 1 of the estrous cycle, near the beginning of the first follicular wave, heifers were superovulated with twice-daily injections of each of four different doses (35 IU ●, 70 IU □, 140 IU ○, 210 IU ♦) of FSH (Folltropin-V) for 4 days (depicted by arrows), using a Williams Latin Square Design as explained in ‘Materials and Methods’. Thus, each heifer was superovulated for a total of four times with 21–24 days between the superovulation regimens. Symbols represent means (±SEM) for the same eight heifers. As depicted by arrows, three prostaglandin F_2α (PG) injections were given, 12 h apart, starting on Day 4 of the FSH treatment, to regress the corpus luteum. A single hCG injection (arrow) was given 12 h after the last FSH injection on Day 5 of the treatment to induce ovulation. AMH concentrations were determined every 24 h throughout Folltropin-V treatment but not post-hCG. The number of antral follicles ≥3 to <10 mm in diameter (AFC) was determined daily by ovarian ultrasonography until 3 days after the hCG injection, when AFC was determined every other day until the end of the study. As depicted within each figure, the results of the Type III ANOVA indicate whether significant (P ≤ 0.05) differences existed in overall AMH concentrations and AFC in heifers during the different days of treatment (Day) and among the FSH doses (Dose), and among heifers treated with the different FSH doses for any day during the FSH treatment period (Day*Dose). Asterisks indicate that means differ (^*P < 0.05, ^**P < 0.01, ^***P < 0.001) when compared with the 210 IU FSH dose.

Figure 2

Effect of different doses of FSH on ovulatory-size follicle number and estradiol concentrations for heifers with a small ovarian reserve. The heifers were superovulated, beginning on Day 1 of the estrous cycle with four different doses (35 IU ●, 70 IU □, 140 IU ○, 210 IU ♦) of FSH (Folltropin-V, arrows depict injections) and treated with PG and hCG (depicted by arrows) as explained in the legend for Figure 1. Symbols depict means (±SEM) for the same eight heifers. A number of ovulatory-size follicles (≥10 mm) were determined by daily ultrasonography measurements and then every other day, starting 3 days after the hCG injection. Estradiol concentrations were determined at 24-h intervals during FSH treatments and at 48-h intervals post hCG. As depicted within each figure, the results of the Type III ANOVA indicate whether significant (P ≤ 0.05) differences existed in the overall number of ovulatory-size follicles and circulating estradiol concentrations in heifers during the different days of treatment (Day) and among the FSH doses (Dose), and among heifers treated with the different FSH doses for any day during the treatment period (Day*Dose). Asterisks indicate that means differ (^*P < 0.05, ^**P < 0.01, ^***P < 0.001) when compared with the 210 IU FSH dose.

Figure 3

Effect of different doses of FSH on corpora lutea number, progesterone concentrations and uterine thickness for heifers with a small ovarian reserve. Heifers were superovulated, beginning on Day 1 of the estrous cycle with four different doses (35 IU ●, 70 IU □, 140 IU ○, 210 IU ♦) of FSH (Folltropin-V, arrows depict injections) and treated with PG and hCG (depicted by arrows) as explained in the legend for Figure 1. Symbols depict means (±SEM) for the same eight heifers. A number of corpora lutea and uterine thickness were determined by daily ultrasonography measurements and then every other day, starting 3 days after the hCG injection. Progesterone concentrations were determined every 24 h during FSH treatment and every 48 h after the hCG injection. As shown within each figure, the results of the Type III ANOVA indicate whether significant (P ≤ 0.05) differences existed in the overall number of corpora lutea, progesterone concentrations and uterine thickness for heifers during the different days of treatment (Day) and among the FSH doses (Dose), and among heifers treated with the different FSH doses for any day during the treatment period (Day*Dose).

Figure 4

Effect of different doses of FSH on ovulation rate for heifers with a small ovarian reserve. Heifers were superovulated, beginning on Day 1 of the estrous cycle with four different doses (35, 70, 140, 210 IU) of FSH (Folltropin-V, arrows depict injections) and treated with PG and hCG (depicted by arrows) as explained in the legend for Figure 1. Bars depict means (±SEM) for the same eight heifers. Ovulation rate was calculated by dividing the number of corpora lutea on Day 7, post-hCG (Figure 3B) by the number of ovulatory-size follicles present at the time of hCG (Figure 2A). As depicted within the figure, results of the Type III ANOVA indicated that a significant (Dose = P = 0.03) difference existed in ovulation rate for heifers treated with the different doses of FSH. Asterisks indicate that the pooled mean (±SEM) for ovulation rates for the 140 and 210 IU doses was lower (^***P < 0.001) compared with the pooled mean (±SEM) for the 35 and 70 IU doses.