An attempt to potentiate the ovarian superstimulatory response in cattle by co-treatment with an aromatase inhibitor

Highlights

• Treatment with an aromatase inhibitor delayed the onset of estrus and decreased the variation in the onset of estrus.

• Treatment with an aromatase inhibitor did not affect the number of antral follicles at wave emergence.

• Co-treatment with an aromatase inhibitor and follicle stimulating hormone did not potentiate the superstimulatory.

Abstract

Letrozole is used for the treatment of subfertility in women undergoing ovarian superstimulation, but the mechanism of action has not been investigated critically. The objective was to test the hypothesis that treatment with letrozole will potentiate the superstimulatory response following gonadotropin treatment by increasing the number of follicles present at ovarian follicular wave emergence in cattle. In Experiment 1, ovarian follicular wave emergence was synchronized among beef heifers (n = 8) by transvaginal ultrasound-guided follicle ablation. On Day 0 (wave emergence), a letrozole-releasing device (LRD) was placed intravaginally for 5 days, followed again by transvaginal follicle ablation on Day 5. The number of follicles ≥3 mm was recorded by transrectal ultrasonography on Days 0 and 6.5 (i.e., pre- vs. post-LRD treatment). In Experiment 2, non-lactating dairy cows were assigned randomly to one of two groups (n = 15/gp) after follicle ablation-induced synchronization of wave emergence (Day 0), and given either an LRD or sham device for 5 days. Superstimulatory treatment was initiated on Day 0, consisting of 8 doses of 50 mg of porcine FSH im at 12 h intervals, and luteolytic doses of prostaglandin on Days 3 and 3.5. The LRD/sham devices were removed on Day 3.5, GnRH was given im on Day 5, estrus response was determined on Days 5 and 6, and the ovarian response was recorded by ultrasonography on Days 0, 3.5, 5, 6.5, and 12. In Experiment 1, no difference was detected in the number of antral follicles at wave emergence pre- vs. post-LRD treatment (23.2 ± 3.2 vs. 23.5 ± 3.8 follicles; P = 0.67; mean ± SEM). In Experiment 2, the interval from prostaglandin treatment to estrus was longer (50.3 ± 1.1 vs. 40.7 ± 2.0 h; P < 0.001) and less variable (residuals: 3.1 ± 0.5 vs. 6.7 ± 0.9 h; P < 0.01) in the LRD vs. sham group. The proportion of ovulations (number of CL on Day 12 over the number of follicles ≥3 mm on Day 0) did not differ (0.65 ± 0.02 vs. 0.70 ± 0.02; P = 0.15) nor did the number of CL on Day 12 (15.9 ± 2.5 vs. 19.0 ± 2.0; P = 0.32) between the LRD and sham groups. In summary, treatment with letrozole did not increase the number of antral follicles at wave emergence or the superstimulatory response, but increased precision in the interval to estrus and may be useful for artificial insemination at a fixed time in superstimulatory protocols.

Introduction

Letrozole is a third-generation non-steroidal aromatase inhibitor that specifically and reversibly inhibits the enzyme P450_aromatase in a dose-dependent manner [1]. The aromatase enzyme is essential for the conversion of androstenedione and testosterone into estrone and estradiol, respectively. Letrozole (FEMARA®) is available commercially, labeled for use as an adjuvant or first-line treatment for hormone-dependent breast cancer in post-menopausal women [2]; however, several reports have been published on the use of letrozole in women undergoing controlled ovarian hyperstimulation (i.e., superstimulation) for the treatment of subfertility [[3], [4], [5]].

Although not examined critically, the putative effect of letrozole in the treatment of subfertility in women is based on the concept that estradiol suppresses gonadotropin release through negative feedback effects on the hypothalamo-pituitary axis and that the reduction of circulating estradiol by means of aromatase inhibition is thought to relieve the suppressive effects on follicle-stimulating hormone (FSH) release, resulting in the development of more than one ovarian follicle to a pre-ovulatory size [6]. While this hypothesis remains to be tested in women, a second mechanism has been proposed for the apparent stimulatory effect of aromatase inhibitor treatment based on an increase in intrafollicular concentrations of testosterone and androstenedione as a result of a reduced conversion of androgens to estrogens [3]. In Rhesus monkeys, treatment with testosterone for 3–10 days increased FSH-receptor mRNA on granulosa cells [7,8], and in androgen receptor knock-out mice, androgen treatment attenuated follicular atresia, increased expression of granulosa cell FSH-receptors, and enhanced FSH-induced antral follicle development in vitro [9]. In clinical studies, daily androgen treatment for ≥12 weeks before FSH superstimulation improved in vitro fertilization (IVF) outcome in women classified as poor-responders [10], and daily co-administration of letrozole and FSH during ovarian stimulation increased both the number of oocytes recovered and the number of blastocysts produced following IVF compared to FSH treatment alone [11].

In a series of studies using the bovine model to examine the effects of aromatase inhibitors, results did not support the hypothesis that letrozole treatment increases circulating FSH concentrations, but treatment did elevate circulating LH concentrations and altered follicular wave dynamics in a fashion that may be useful for ovulation synchronization [12]. The use of the bovine model to test the effects of aromatase inhibitor treatment as an adjunct to gonadotropin treatment for ovarian superstimulation has not been reported.

Over the last 20 years, the average number of transferable embryos produced per donor has increased an estimated 13–22%, to about 6.5 per superstimulation [[13], [14], [15]]. The increase in transferable embryos may be attributed to protocol adaptation, such as initiating gonadotropin treatment at the onset of wave emergence [16], selection of donors with a high antral follicle count [17], and extending the duration of superstimulatory treatment from 4 to 7 days [15,18]. Factors that may increase the responsiveness of individual follicles to exogenous FSH have not been investigated in cattle.

Based on the effects of non-steroidal aromatase inhibitors reviewed above, we hypothesized that co-treatment with letrozole would potentiate the follicular responsiveness to gonadotropin treatment during ovarian superstimulation. The objectives of the present study were to determine if treatment with the aromatase inhibitor, letrozole, will increase the number of follicles present at the time of follicular wave emergence (Experiment 1) and whether letrozole will potentiate the superstimulatory response to FSH treatment (Experiment 2) in cattle.