Induction of multiple ovulation by pulsatile administration of gonadotropin-releasing hormone*

Transcription

1 FERTILITY AND STERILITY Copyright 1983 The American Fertility Society VoL 40, No.1, July 1983 Printed in UBA. Induction of multiple ovulation by pulsatile administration of gonadotropin-releasing hormone* James H. Liu, M.D.t Raphael Durfee, M.D. Ken Muse, M.D. t Samuel S. C. Yen, M.D., D.Sc.:j: Department of Reproductive Medicine, School of Medicine (T -002), and the General Clinical Research Center, University of California, San Diego, La Jolla, California Pharmacologic doses of gonadotropin-releasing hormone (GnRH) pulses (10 flg) were used for the induction of multiple follicular development in six normal cycling women. GnRH administration via an autoinfusion pump (intravenously at 60- to 120-minute intervals) was begun on day 5 after the onset of menses. During these stimulated cycles, two to five follicles of preovulatory size were detected by sonography in all subjects. In two subjects, follicle aspiration was performed with successful oocyte recovery. Assessment of the follicular fluid steroid concentrations from these follicles suggests that two of three (subject 5) and four of five (subject 6) follicles were mature. Gonadotropin levels of treated cycles were significantly higher than in normal cycles. Both follicular and luteal phases were of normal duration but were associated with significantly elevated estradiol and progesterone levels, respectively. Timely midcycle gonadotropin surges occurred by 12 to 14 in all cases. Thus, ovarian steroid feedback control of gonadotropin secretion can be overridden by larger pulses of GnRH, resulting in the development of multiple follicles. This technique may prove useful for in vitro fertilization. Fertil Steril40:18, 1983 Pulsatile administration of gonadotropin-releasing hormone (GnRH) for ovulation induction and pregnancy in hypogonadotropic amenorrhea Received February 10, 1983; accepted March 1, *Supported by NIH grant HD-12303, and in part by the UCSD General Clinical Research Center, NIH, Division of Research Resources, grant RR This research was conducted in part by the Clayton Foundation for Research,. California Division. tfellow in Reproductive Endocrinology. :j:senior Clayton Foundation Investigator. Reprint requests: Samuel S. C. Yen, M.D., D.Sc., Professor, Department of Reproductive Medicine (T-002l, University of California, San Diego, University Hospital, University of California Medical Center, 225 Dickinson Street, San Diego, California patients has been shown to be effective. 1-9 However, the possibility of using this technique for induction of multiple follicular development in normal cycling women has not been explored. It has been assumed that the negative feedback effects of ovarian estradiol (E 2 ) would modulate the GnRH-mediated gonadotropin release 10,11 and thus circumvent hyperstimulation and multiple follicular development. In our laboratory, 1- to 5-f.Lg doses of pulsatile GnRH administered to hypogonadotropic women consistently induced ovulation (n = 23) with only one dominant follicle. 9 Because the induction of multiple follicular maturation is desirable for in vitro fertilization, we began a prospective study designed to achieve multiple ovulation utilizing pulses of GnRH in normal cycling women. 18 Liu et al. Multiple ovulation induction with GnRH Fertility and Sterility

2 o I OOO~ E 200 CI a. 0 LH.-. FSH 0-0 GnRH 10 Itg/h, FOLLICULAR SIZE RI ii I TT I T E2... p CYCLE DAY E... CI c: Figure 1 Serum levels of LH, FSH, E 2, P, and mean follicular diameter as determined by ultrasound during a stimulated cycle in a representative subject. The arrows denote intramuscular administration of 1500 U human chorionic gonadotropin. 25 MATERIALS AND METHODS Six subjects, 29 to 43 years of age, with regular menstrual cycles (range, 26 to 33 ) volunteered for this study. This project was approved by the Human Subjects Committee, University of California, San Diego, and informed consent was obtained. All subjects were begun on pulsatile GnRH stimulation of 10,...g intravenously every 60 minutes (subject 2, every 120 minutes) via a portable autoinfusion pump (Autosyringe, Hooksett, NH) on the fifth day following the onset of menses. Follicular growth was monitored by ultrasound using three-dimensional measurements. Presumptive ovulation was defined as an acute decrease in follicular size with the appearance of fluid in the cul-de-sac.i2 After documentation of ovulation by ultrasound, either pulsatile GnRH delivery was continued or human chorionic gonadotropin was administered. In the latter case, 1500 U of human chorionic gonadotropin were Vol. 40, No.1, July 1983 given intramuscularly every third day for four doses, starting 2 after presumed ovulation. Preparation of the GnRH (generously supplied by J. Rivier, Ph.D., Salk Institute, La Jolla, CA) solution and infusion procedures have been described previously.s, 9 Subjects 5 and 6 underwent diagnostic laparotomy and laparoscopy, respectively, and provided an opportunity for oocyte retrieval. Follicle aspirations and irrigations were performed with a 12-gauge beveled needle and in many instances yielded bloodstained fluid with sheets of granulosa cells. Because of excessive loss of granulosa cells during vigorous aspiration in both subjects, their hormonal data following aspiration were excluded from analysis. Concentrations of luteinizing hormone (LH), follicle-stimulating hormone (FSH), E2, progesterone (P), and androstenedione (d4a) were measured in daily serum samples obtained throughout the cycle by specific radioimmunoassay.i3-i6 Follicular fluid samples were assayed for E2, P, and d 4A following appropriate dilutions. Statistical analyses were performed using the Student's t-test. RESULTS The pattern of gonadotropin and circulating E2 and P steroid concentrations during GnRH stimulation were similar for all subjects, and representative data are depicted in Figure 1. In response to pulsatile administration of GnRH, there was an acute increment of levels of LH and FSH on the second day. Thereafter, LH levels remained elevated until a spontaneous surge occurred at midcycle ( 12 to 14; Table 1). LH levels then declined, with subsequent elevations reflecting, in part, human chorionic gonadotropin administration (Fig. 1). The transverse mean of LH in the midfollicular phase was twofold greater than normal controls for this laboratory (28.4 ± 3.5 miu/ml versus 12.7 ± 4.5 mivlml, P < 0.05). FSH levels remained elevated until E2 concentrations increased and then returned to the normal follicular range (Fig. 1). Mean (± standard error) peak E2 levels of 585 ± 55 pg/ml (range, 389 to 772 pg/ml) were significantly greater (P < 0.01) than the values reported by this laboratory (370 ± 30 pg/ml)p E2 levels peaked on the day of the LH surge and dropped precipitously after the onset of the surge. Concomitant with the E2 decline was a gradual increase in P levels, eventually Liu et al. Multiple ovulation induction with GnRH 19

3 Table 1. Clinical and Laboratory Data of Menstrual Cycle Duration, LH Surges, Steroid Levels, and Ultrasound Findings in Six Subjects Undergoing Pulsatile GnRH Stimulation Normal cy- Subject Age cle length ± ± ± ± ± 1 afollicle aspiration. bvigorous aspiration. Treated cycle length LH surge Follicle status Steroid concentration Luteal phase No. Diameter Preovulatory Luteal peak E. P em pglml nglml a b a b reaching a mean peak level of 46.1 ± 8.2 ng/ml (range, 28.1 to 69.6 ng/ml; Table 1). In subjects 5 and 6, peak P levels were much lower, presumably secondary to excessive loss of granulosa cells. Cycle lengths were 25 to 30, with luteal phase durations of 13 to 17. With three-dimensional techniques, ultrasound revealed two to five follicles of preovulatory size (~ cm) in all subjects (Table 1). In both subjects 5 and 6, three follicles > 1.8 cm were observed. Presumptive ovulation occurred in all subjects not undergoing follicular aspiration. Changes consistent with corpus luteum formation characterized by a loss of clear demarcation of the follicular cyst wall and the appearance ofintrafol -licular echoes 18 were seen 1 to 2 after the LH surge. Follicular maturation was assessed by analysis of follicular fluid steroid concentrations (Table 2). Retrospectively, both aspirations were performed during the LH surge. Two of three follicles in subject 5 (L l and R) and four of five follicles in subject 6 (L l to L 4 ) contained relatively low levels of a 4 A and high levels of E2 (range, to 2.66 J.Lg/ml), the a 4 A:E2 ratio being markedly reduced. P concentrations ranged from 1.14 to Table 2. Follicle Diameter, Follicular Fluid Volume, and Steroid Concentration in Two Subjects Undergoing Follicular Aspiration Follicles Follicular fluid steroids Side Oocyte Mean diameter Fluid volume recovery E. 6,'A P 6,'A:E. a Subject 5 Ll L2 R Subject 6 Ll L2 La L4 L5 amolar ratio. bprocedural loss. cbloodstained fluid. em ml 6 3 c 4 b,c 1.2 c 1.2 c I Jlml Liu et a!. Multiple ovulation induction with GnRH Fertility and Sterility

4 fj.g/ml. From a total of eight follicles aspirated, three oocytes were recovered. No side effects were noted during the stimulated cycles. among follicles with a potential increase in pregnancies from in vitro fertilization techniques remains to be determined. DISCUSSION In the present study, we have demonstrated for the first time the ability of pharmacologic doses of GnRH to reproducibly induce multiple follicular development in women with normal menstrual cycles. These stimulated cycles were characterized by a normal follicular and luteal phase length with a spontaneous LH surge occurring by 12 to 14. Both E2 and P levels were elevated above the normal range and were consistent with multiple follicular development with subsequent ovulation and corpus luteum formation. To further demonstrate that these follicles were indeed preovulatory, we performed follicle aspirations in two subjects. In both subjects, luteal phase P levels were lower following vigorous aspiration when compared with those of non as pi rated cycles. 19 However, the presence of high concentrations of E2 and P and low concentrations of a4a in two of three and four of five follicles in subjects 5 and 6, respectively, is characteristic of healthy, preovulatory follicles The relatively high levels of a4a in the follicular fluid of follicles L2 (subject 5) and L5 (subject 6) suggest that these may represent atretic follicles. In contrast to previous studies in our laboratory, this study differed in two critical aspects in an effort to induce multiple follicular development. First, the dose of GnRH administered was increased to a pharmacologic level in an effort to overcome E2 feedback. Second, the initiation of GnRH stimulation was timed to reflect the findings of dizerega and Hodgen 23 and of dizerega et al.,24 who have shown in monkeys that follicular recruitment occurs before day 7, follicular selection occurring by 7 to 9. The frequency of GnRH administration appeared to be less critical, because multiple follicular development also occurred in one subject given GnRH every 120 minutes. While this study was in progress, Bogschelman et a1. 25 reported one case of a multiple pregnancy following GnRH ovulation induction. In summary, we have established that GnRH is capable of induction of multiple follicular development and ovulation by overriding E2 modulation on the pituitary. Whether GnRH will result in greater synchronization of oocyte maturation Vol. 40, No.1, July 1983 Acknowledgments. We would like to thank Del Crowe for aid in manuscript preparation, and Jill Aurand, Mary Moscinski, and J eft" Wong for technical assistance. REFERENCES 1. 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