Fertility and Sterility
Volume 95, Issue 1 , Pages 317-319, January 2011

Microdose gonadotropin-releasing hormone agonist in the absence of exogenous gonadotropins is not sufficient to induce multiple follicle development

Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology and Infertility, University of Southern California Keck School of Medicine, Los Angeles, California

Received 6 April 2010; received in revised form 19 July 2010; accepted 21 July 2010. published online 01 September 2010.

Article Outline

Because the effectiveness of the “microdose flare” stimulation protocol often is attributed to the dramatic endogenous gonadotropin release induced by the GnRH agonist, the aim of this study was to determine whether use of microdose GnRH agonist alone could induce multiple ovarian follicle development in normal responders. Based on these data, the duration of gonadotropin rise is approximately 24 to 48 hours and is too brief to sustain continued multiple follicle growth.

Key Words: Microdose flare, controlled ovarian hyperstimulation, in vitro fertilization, GnRH agonist, poor responders, ovarian stimulation

 

Gonadotropin-releasing hormone agonists (GnRH-a) are used widely as adjuncts to ovarian stimulation for IVF. Administration of GnRH-a causes an initial stimulatory effect resulting from a surge of endogenous gonadotropins, followed by down-regulation of the gonadotropin-gonadal axis (1). To take advantage of the agonistic effect, the “flare protocol” initiates the GnRH-a in the early follicular phase, allowing the surge of endogenous gonadotropins to drive follicular stimulation. As low as one fiftieth the standard luteal dose was shown to induce comparable gonadotropin release 2, 3, and, with the goal of achieving maximal gonadotropin release rather than rapid down-regulation, the “microdose flare” protocol uses low-dose GnRH-a 4, 5.

The microdose flare protocol has been reported to yield lower cancellation rates, higher serum E2 levels, and improved ongoing pregnancy rates among poor responders 6, 7, 8 and is now considered one of the most aggressive regimens for ovarian stimulation. High doses of exogenous gonadotropins typically are added on the third day of agonist, but the effective stimulation largely has been attributed to endogenous gonadotropin release (9).

We previously measured gonadotropin levels in poor responders stimulated with the microdose flare protocol and confirmed that they are significantly higher than those achieved with gonadotropins alone (10). However, the magnitude and duration of gonadotropin rise in response to low-dose GnRH-a without exogenous gonadotropins has not been reported. Animal studies have demonstrated that very low doses of GnRH-a in the absence of supplemental exogenous gonadotropins achieve FSH levels that surpass the threshold required to achieve multiple follicle growth (11). In this study, we hypothesized that low doses of GnRH-a administered alone in the early follicular phase would result in an increase in gonadotropin levels that could induce multiple follicle development among normal responders. We aimed to determine the magnitude and duration of increase in circulating gonadotropin levels and the effect on ovarian follicle development.

Institutional Review Board approval was obtained before initiating subject recruitment. We aimed to enroll five patients from a single university-based infertility practice between the ages of 18 and 42 years with tubal factor infertility. Patients with evidence of diminished ovarian reserve (day 3 FSH ≥20 mIU/mL) were not eligible for participation.

Subjects were enrolled on menstrual cycle day 3. Baseline FSH, E2 levels, and transvaginal ultrasound findings were recorded. All subjects were instructed to start an oral contraceptive (OC) pill (desogestrel 0.15 mg and ethinyl E2 30 μg), to be taken daily for 14 days. On discontinuation of the OC pill, subjects were asked to return for blood draw and transvaginal ultrasound examination. Subjects then were instructed to administer 40 μg of leuprolide acetate (LA) SC every 12 hours starting on the fourth day after the last OC pill. Subjects returned for daily blood sampling and transvaginal ultrasound examination approximately 30 minutes after the morning dose of LA. Serum FSH, E2, LH, and P levels were recorded, and all ovarian follicles were measured in two dimensions daily for 14 days from the initiation of LA.

Follicle-stimulating hormone, LH, and P levels were measured with use of the Immulite chemiluminescent system (Immulite 2000; Siemens, Deerfield, IL), and E2 levels were determined by the radioimmunoassay (Pantex, Santa Monica, CA) after extraction. All interassay and intra-assay coefficients of variation were <10%.

Curves created by serial determinations of hormone values were plotted for each subject to characterize the magnitude and duration of rise over a 14-day period compared with baseline values. Total number and maximum mean diameters of ovarian follicles also were plotted. Data were analyzed with use of repeated-measures analysis of variance with the a priori hypothesis that FSH levels would be elevated significantly the day after initiation of LA compared with baseline levels and that multiple follicle growth would be observed on ultrasound examination. Differences were considered statistically significant at P values <.5.

Five subjects who met inclusion criteria were enrolled and completed the study. The mean age of the subjects was 31.6 ± 5.3 years (range 27–39 years), mean day 3 FSH level was 5.9 ± 2.0 mIU/mL (range 4.0–9.1 mIU/mL), and mean BMI was 24.4 ± 4.4 (range 20.9–25.8). None of the subjects previously had undergone ovarian stimulation, and all were expected to demonstrate normal response.

Mean FSH levels were 5.9 mIU/mL and 3.9 mIU/mL, at baseline and on discontinuation of OC pills, respectively. At study visit 2, approximately 24 hours after initiation of LA, mean FSH levels rose significantly to 19.4 ± 7.6 mIU/mL (range 11–31.7 mIU/mL) (P=.007). At 48 hours after initiation of LA (study visit 3), there was a significant relative decline in serum FSH concentrations to 11.2 ± 4.4 mIU/mL (range 7.9–18.5 mIU/mL, P=.01). Levels of FSH were not significantly different from baseline after 3 days of LA (by study visit 4 and onward) (P=.07). Curves created by serial FSH values depict a plateau occurring around the sixth day of LA (Fig. 1A). On average, serum LH concentrations exhibited similar patterns of significant elevation for 24 to 48 hours followed by a rapid decline and plateau by day 6 of LA (Fig. 1B).

  • View full-size image.
  • Figure 1 

    (A–C) Curves created by serial determinations of hormone values were plotted for each subject to characterize the magnitude and duration of rise over 14 days compared with baseline values. (A) Follicle-stimulating hormone (FSH) levels over time. (B) Luteinizing hormone (LH) levels over time. (C) Estradiol levels over time. (D) Mean follicle diameter of largest follicle. Total number and maximum mean diameters of ovarian follicles were plotted over a 14-day period. All visible ovarian follicles were measured in two dimensions daily for a total of 14 days from the initiation of LA. All curves started at “study visit 0,” which corresponded to baseline values obtained on cycle day 3. “Study visit 1” reflected values obtained on discontinuation of the OC pills. Study visits 2 through 14 represented daily morning values after initiation of LA.

Serum E2 levels also rose significantly for 24 to 48 hours after LA initiation followed by a relatively rapid decline. However, late follicular phase E2 values deviated from the patterns described by the gonadotropins in that two subjects had rising E2 concentrations, achieving peak levels of 1,389 pg/mL (subject 3) and 898 pg/mL (subject 5) at day 14 (Fig. 1C). These findings were attributable to the development of single dominant follicles seen on ultrasound scan for both subjects. Mean follicle diameters measured 27 mm (subject 3) and 31 mm (subject 5) (Fig. 1D). Ultrasound findings in the remaining three subjects did not demonstrate dominant follicle development, consistent with the observed plateau in serum E2 levels. No subject had multiple ovarian follicles develop >12 mm in diameter. Serial ultrasound examinations did not detect evidence of ovulation among the five subjects. In addition, serum P levels remained low throughout the duration of the study, averaging 0.5 ± 0.5 ng/mL on day 14 (range 0.2–1.2 ng/mL).

This is the first study to our knowledge to isolate the endocrine and ovarian response to clinically relevant microdoses of GnRH-a in the absence of confounding by exogenous gonadotropins. We demonstrated that, after pretreatment with OCs, 40 μg twice daily causes a dramatic rise in endogenous gonadotropins in the absence of supplemental exogenous gonadotropins. We found results similar to those reported previously in pharmacokinetic studies that used higher doses of GnRH-a (100 μg daily), with serum FSH levels peaking at approximately 20 mIU/mL (1). Thus, our study supports the findings of previous studies demonstrating that low doses are capable of inducing a surge of endogenous gonadotropins similar in magnitude to that with higher doses.

It has been theorized that FSH concentrations must rise above a distinct threshold to stimulate the antral follicles to begin the process of development (12). In a natural menstrual cycle, the luteal-to-follicular phase transition is characterized by an increase in FSH levels (13), which surpasses the threshold necessary to initiate follicular recruitment. For patients undergoing IVF, supraphysiologic doses of exogenous gonadotropins are used, demonstrating that FSH elevations higher than the natural threshold 14, 15 and persisting for longer durations than in the natural cycle (16) result in the maturation of multiple follicles.

The importance of the duration, as opposed to the magnitude of FSH increase above a critical threshold, has been emphasized in studies of follicular development (16). In our study, the duration of gonadotropin rise was approximately 24 to 48 hours, and, despite the dramatic magnitude of gonadotropin rise, no subject achieved multiple follicular development, indicating that the endogenous flare was too brief to sustain continued follicle growth. These findings thereby support the theory that duration of gonadotropin rise may be more important than magnitude in stimulating multiple follicle development. In addition, the reported improvement in ovarian response with the microdose flare protocol is likely more attributable to the supplemental exogenous gonadotropin than previously believed.

Hormone profiles among our study subjects demonstrated that, overall, pituitary suppression occurred by day 6 of GnRH-a administration and that very low doses were largely effective in preventing the premature LH surge. In the early development of the microdose flare protocol, a very low dose was selected with the aim of achieving maximal endogenous gonadotropin release rather than optimal down-regulation (3). At that time, it was unclear whether such low doses were enough to induce pituitary suppression 17, 18. In our study, all subjects exhibited gonadotropin profiles consistent with pituitary suppression by day 6 of GnRH-a administration. However, in two subjects, rising E2 levels and single dominant follicles on ultrasound scan developed despite continued exposure to the GnRH-a. These results suggest that microdoses of GnRH-a, though effective in preventing the premature LH surge, do not achieve complete inhibition of pituitary-ovarian function.

In summary, the use of microdoses of GnRH-a alone causes a dramatic rise in endogenous gonadotropins, lasting approximately 24 to 48 hours, but does not stimulate development of multiple ovarian follicles in normal responders. Very low doses are effective in preventing the premature LH surge but do not appear to achieve complete pituitary suppression. Further studies are needed to better characterize the range of responses to low doses of GnRH-a in the absence of exogenous gonadotropins in both normal and poor-responder populations.

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References 

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 K.C. has nothing to disclose. R.F. has nothing to disclose. K.B. has nothing to disclose. K.C. has nothing to disclose. R.P. has nothing to disclose.

PII: S0015-0282(10)02207-7

doi:10.1016/j.fertnstert.2010.07.1081

Fertility and Sterility
Volume 95, Issue 1 , Pages 317-319, January 2011

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