Induction of preovulatory luteinizing hormone surge and prevention of ovarian hyperstimulation syndrome by gonadotropin-releasing hormone agonist*

FERTILITY AND STERILITY

Vol. 56, No. 2, August 1991

Printed on acid-free paper in U.S.A.

Copyright c 1991 The American Fertility Society

Induction of preovulatory luteinizing hormone surge and prevention of ovarian hyperstimulation syndrome by gonadotropin-releasing hormone agonist* Joseph Itskovitz, M.D., D.Sc. t:l: Rafael Boldes, M.D. t Jacob Levron, M.D.t

Yohanan Erlik, M.D.t Luna Kahana, Ph.D.§ Joseph M. Brandes, M.D.t

Rambam Medical Center, Technion Israel Institute of Technology, Faculty of Medicine, and the Central Endocrine Laboratory, Linn Medical Center, Haifa, Israel

Objective: To use gonadotropin-releasing hormone agonist (GnRH -a) instead of human chorionic gonadotropin (hCG) to induce oocyte maturation for in vitro fertilization (IVF). Design: Pituitary and ovarian responses to GnRH -a and the outcome of IVF were studied prospectively. Data from patients injected with hCG were analyzed retrospectively. Setting: Program of IVF at the Ran!bam (Governmental) Hospital, Haifa, Israel. Patients and Interventions: One or two doses of buserelin acetate 250 to 500 p.g were administered to six patients with moderate response (Estradiol [E2 ], 1,494 ± 422 [±SD] pg/mL) and 8 patients with exaggerated response (E 2 , 7,673 ± 3,028 pg/mL) to gonadotropin stimulation. Progesterone (P) and E 2 were administered for luteal support. Main Outcome Measures: Gonadotropin-releasing hormone agonist effectively triggered luteinizing hormone (LH)/follicle-stimulating hormone (FSH) surge. Mature oocytes were recovered in all patients. Luteal E 2 and P were lower than in patients injected with hCG. No signs of ovarian hyperstimulation syndrome were observed. Results: Serum LH and FSH rose over 4 and 12 hours, respectively, and were significantly (P < 0.05) elevated for 24 hours. Of all mature oocytes, 67% fertilized and 82% cleaved. Four pregnancies were obtained. Conclusions: A bolus of GnRH -a is able to trigger an adequate midcycle LH/FSH surge, resulting in oocyte maturation and pregnancy. Our preliminary results also suggest that it allows a more accurate control of ovarian steroid levels during the luteal phase and may prevent the clinical manifestation of ovarian hyperstimulation syndrome. Fertil Steril 56:213, 1991

Gonadotropin medications have been used successfully for three decades to stimulate ovarian follicular development in anovulatory patients and, more re-

Received November 6, 1990; revised and accepted April 16, 1991. * Presented in part at the International Symposium on GnRH Analogues in Cancer and Human Reproduction, Geneva, Switzerland, February 18 to 21, 1988, and at the 6th World Congress of In Vitro Fertilization and Alternative Assisted Reproduction, Jerusalem, Israel, April2 to 7, 1989_ t Department of Obstetrics and Gynecology, Rambam Medical Center. :j: Reprint requests: Joseph Itskovitz (Eldor), M.D., D.Sc., Department of Obstetrics and Gynecology, Rambam Medical Center, Haifa 31096, Israel. §The Central Endocrine Laboratory, Linn Medical Center. Vol. 56, No.2, August 1991

cently, in normal ovulatory women for the purpose of in vitro fertilization (IVF). 1•2 In these patients, endogenous luteinizing hormone (LH) surge infrequently occurs and therefore the administration of human chorionic gonadotropin (hCG) is required to induce oocyte maturation and ovulation.3•4 Although similar in action to LH, hCG does not provide a physiological stimulation that is identical to the endogenous LH surge. Furthermore, hCG therapy, because of its longer half-life time compared with LH, is associated with a sustained luteotropic effect, multiple corpora lutea (CL) development, and supraphysiological serum levels of estradiol (E2 ) and progesterone (P) throughout the luteal phase. A high estrogen (E) level has been implicated in implantation failure and early embryonic loss in humans and animals.5•6 ltskovitz et al.

GnRH-a-induced LH/FSH surge

213

Table 1

Diagnosis, Stimulation Protocol, and the Number of Mature Fertilized Oocytes Diagnosis

Stimulation protocol

Dosage of buserelin acetate

No. of mature fertilized oocytes

I'll

Group A patients (normal responders) 1 2 3 4 5 6

Mechanical Mechanical Mechanical Male factor PCOD Mechanical, PCOD, male factor

FSH/hMG CC/hMG FSH/hMG FSH/hMG CC/hMG FSH/hMG

500 500 500 500 500 500

X2 X2 X2 X2 X2 X2

3/3 6/6 10/12 2/11 9/10 1/7

Mechanical Mechanical Mechanical Unexplained PCOD, male factor Mechanical Mechanical, male factor PCOD, male factor

FSH/hMG FSH/hMG FSH/hMG CC/hMG hMG FSH/hMG FSH/hMG hMG

250 250 250 500 500 250 250 250

X2 X2 X2 X1 X1 X1 X1 X1

18/20 22/25 13/19 9/13 16/24 26/28 11/20 0/13

Group B patients (high responders) 7

8 9 10 11 12 13 14

Patients who disclose multiple follicular development and high serum E 2 levels (> 1,500 pg/mL) before hCG is given are prone to develop ovarian hyperstimulation syndrome, the most seriously encountered complication related to gonadotropin therapy. 2•7 Although the pathophysiology of the syndrome is not known, it is clearly related to the existence of multiple CL and the sustained luteotropic effect of hCG. Withholding injection of hCG may prevent the development of ovarian hyperstimulation syndrome but results in failure to ovulate and conceive. The potent gonadotropin-releasing hormone analog (GnRH-a) induces a sustained release of LH and FSH from the pituitary that may last, depending on the dose administered and the phase of the menstrual cycle, for several hours.s-11 This initial stimulatory effect is followed by a decline of pituitary response to further GnRH-a stimulation.9 In view of the potential clinical benefits derived from its dual effect on the secretion of gonadotropins, we have used the GnRH-a as an alternative to hCG to stimulate endogenous LH/follicle-stimulating hormone (FSH) surge in patients undergoing ovarian stimulation with gonadotropins for the purpose of IVF. In these patients, GnRH -a effectively triggered LH/ FSH surge that resulted in the recovery of mature, fertilizable oocytes. However, despite the existence of multiple preovulatory follicles, circulatory levels of E 2 and P during the luteal phase were close to those of the nonstimulated menstrual cycle, and no signs or symptoms of ovarian hyperstimulation syn214

ltskovitz et al.

GnRH-a-induced LH/FSH surge

drome were observed in patients at high risk to develop this syndrome. MATERIALS AND METHODS Patients and Protocols

Fourteen women (age 30.0 ± 4.6 years, mean ± SD) undergoing ovarian stimulation for IVF were included in this study. Table 1 summarizes the clinical diagnosis and ovarian stimulation protocols in individual patients. In the FSH/human menopausal gonadotropins (hMG) protocol (n = 9), two ampules ofFSH (Metrodin; Teva, Petah-Tikva, Israel) containing 75 IU of human urinary FSH and <1 IU of LH in each ampule and two ampules of hMG (Pergonal; Teva) containing 75 IU of FSH and 75 IU of LH were injected on days 3 and 4 of the cycle. This was followed by injection of two ampules of hMG on subsequent days. In the hMG-only protocol (n = 2), patients received two ampules of hMG from day 3 of the cycle. In the clomiphene citrate (CC)/hMG protocol (n = 3), a tablet of CC (Ikaclomine; Teva), 50 mg two times daily, was given from days 5 to 10, and two ampules of hMG were injected from day 8 of the cycle and thereafter. Patients were monitored during the follicular phase with daily determination of serum E 2 , P, and gonadotropin levels, and with transvaginal scanning of ovarian follicles. Buserelin acetate (D-Ser[TBU] 6 -des-Gly-NHl0 GnRH ethylamide, Superfact; Hoechst AG, FrankFertility and Sterility

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Figure 1 Hormonal levels (mean ± SEM) before and after the injection of two doses ofbuserelin acetate, 500 J.l.g, in six normalresponder patients. Baseline is the 3rd day of the menstrual cycle. Serum LH and FSH were significantly (P < 0.05) higher than on time 0 for a period of 24 hours.

furt, Germany) was injected subcutaneously to induce an endogenous LH/FSH surge when at least one follicle reached a diameter of 16 mm. In group A, which included six normal-responder patients, a dose ofbuserelin acetate, 500 f.Lg, was injected in two successive doses 12 hours apart. Group B included eight high-responder patients; in this group a double dose of buserelin, 250 f.Lg, 12 hours apart was injected into three patients, and a single dose of buserelin acetate 250 to 500 f.Lg was injected into five patients. Transvaginal ultrasound-guided aspiration of ovarian follicles was carried out 36 hours after the first GnRH-a injection. Oocytes were cultured in Ham's F-10 (Gibco, Paisley, Scotland) medium supVol. 56, No.2, August 1991

plemented with 7.5% human fetal cord serum and were inseminated with 50,000 to 100,000 sperm/mL 3 to 6 hours after retrieval. The oocytes were then examined for fertilization 18 to 20 hours after insemination, zygotes were cultured in Ham's F-10 medium supplemented with 15% fetal cord serum, and cleaved embryos were transferred to the uterus 42 to 48 hours after insemination. Serum samples for the determination of E 2 ·and P were collected during the luteal phase, and patients were supplemented with E and P to maintain serum E 2 and P levels at about 200 to 400 pg/mL and 15 to 20 ng/mL, respectively. Luteal phase serum levels of E 2 and P were compared retrospectively with those measured in a group of 14 control normalresponder IVF patients injected with an ovulatory dose of hCG 10,000 IU and supplemented with P 25 to 50 mg/d throughout the luteal phase. The average age was 31.1 ± 4.9 years. The indications for IVF were mechanical infertility (n = 8), unexplained infertility (n = 2), male infertility (n = 2), polycystic ovarian disease (PCOD), and mechanical infertility (n = 1), and PCOD and male infertility (n = 1). For ovarian stimulation, 10 patients were injected with FSH/hMG, and 4 patients received hMG only. Exogenous E was provided as oral E 2 valerate (Progyluton; Schering AG, Berlin, Germany), and P was provided by intramuscular injection of P in oil (Gestone; Paines and Byrne, Greenford, United Kingdom). Patients were examined, and the pelvis was scanned transvaginally at the midluteal and late luteal phase to determine the size of the ovaries and the existence of the free peritoneal fluid. Hormonal Assays

Serum E 2 and P were assayed using a commercially available radioimmunoassay (RIA) kit (CoatA-Count; Diagnostic Product Corporation, Los Angeles, CA). Serum LH and FSH were measured by RIA (Amerlex M; Amersham International, Amersham, United Kingdom). The intra-assay and interassay coefficients of variation were 6% and 10% for E 2 , 8% and 11% for P, 9% and 16% for LH, and 6% and 8% for FSH, respectively. Statistical Analysis

The paired Student's t-test with Bonferroni's correction for multiple measurement was used to determine statistical significance (Figs. 1, 2, and 3). Probability < 0.05 was considered significant. Itskovitz et al.

GnRH-a-induced LH/FSH surge

215

200

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of GnRH-a had no effect on LH release, at least as reflected in the serum levels of LH 2 hours after its injection, suggesting that pituitary desensitization to GnRH -a had already occurred 12 hours after the first injection. Serum E 2 levels at the time of GnRH -a injection averaged 1,494 ± 422 (±SD) pg/mL (range, 732 to 2,131 pg/mL). The LH/FSH surge was temporally associated with the attainment of peak E 2 levels and the initiation of a rapid rise of P during the 1st 12 hours. Serum E 2 then gradually fell, and P plateaued during the 24 hours preceding follicle aspiration. After follicle aspiration, a second rise of P and a continuous fall of E 2 were observed, reflecting an acute shift in ovarian steroidogenesis in favor of P.

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RESULTS

i

i

Pituitary and Ovarian Response to GnRH-a 2

Normal Responders

Serum LH, FSH, E 2 , and P before and after the injection of two successive doses of buserelin acetate 500 f.Lg into six normal responders (group A) are shown in Figure 1. The injection of GnRH -a resulted in an acute release of LH and FSH. Serum LH and FSH rose over 4 hours and 12 hours, with an incremental ratio (peak/basal) of 12.4 and 2.6, respectively. The LH surge could be divided into two phases: a short ascending phase (4 hours) and a long descending phase (32 hours). The second injection 216

Itskovitz et al.

4

e

a

10

12

Days Alter Follicle Aspiration

GnRH-a-induced LH/FSH surge

Figure 3 Serum E 2 and P levels (mean ± SEM) throughout the luteal phase in normal responders injected with either hCG (0- 0, n = 14) or GnRH-a (group A) (e -- e, n = 6) and in high responders injected with GnRH-a (group B) (0 -- 0, n = 8). Control normal responders treated with hCG were supplemented with P in oil 25 to 50 mg/d from day 2 until day 12. Normal and high responders injected with GnRH-a were supplemented with E 2 valerate and Pin oil (see Tables 2 and 3 for details). Serum E 2 was lower (P < 0.05) from day 4 until day 8, and on day 6, in normal and high responders injected with GnRH-a, respectively, than in normal responders injected with hCG. Serum P was lower (P < 0.05) from day 2 until day 8, and from day 4 until day 8, in normal and high responders treated with GnRH-a, respectively, than in normal responders injected with hCG.

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Table 2

Luteal Phase Support and Hormonal Levels in Patients Injected With GnRH-a Luteal support Progesterone

E 2 valerate

Midluteal serum level" Daily dose

Group A patients {normal responders) 1 2 3 4 5b 6 Group B patients (high responders) 7 8 9 10

llb

12. 13 14

Day started

25 to 50 25 50 50 25 to 100

4 8 2 3 2

50 25 to 75 50 to 100 25 to 75 50 50 50

3 3 4 2

3 3 2

• Day 6 of the luteal phase. b Patients conceived after transfer of fresh embryos.

Daily dose

Day started

2to 4 2 2 to 4 2

4

2to4

8 2 3 5

2 3 2 2to 4 2 2 2

6 5 6 6 4 5 5

E2

p

pg/mL

ng/mL

102 317 195 137 283 628

13 17 22 20 40 33

149 413 337 173 128 200 138 1,500

26 13 17 17 16 11

14 40

• Patient conceived after transfer of cryopreserved embryos.

(n = 5) or double (n = 3) dose of GnRH-a are illustrated in Figure 2. Serum E 2 at the time of GnRH-a injection averaged 7,673 ± 3,028 pg/mL and ranged from 5,050 to 13,130 pg/mL. Serum LH rose 20-fold over 4 hours and then fell to baseline after 36 hours. Serum E 2 and P levels were higher than those measured in normal responders, but the pattern and dynamics of the hormonal changes were similar. There were no differences in the hormonal response among the different regimens of buserelin acetate therapy.

to the uterus of 13 patients resulted in three pregnancies (one late abortion at 16 weeks [patient no. 5], one premature delivery of twins at 30 weeks with survival of one normally developing infant, [patient no. 8], and one term delivery of healthy infant [patient no. 11]). Forty-four embryos were frozen. Until now, 16 embryos were thawed and transferred to the uterus of three patients during a natural menstrual cycle, resulting in one term delivery of normal infant (patient no. 12).

Oocytes and Embryos

The Luteal Phase

Mature oocytes were recovered in all patients (Table 1). A mean of 8.2 ± 3.4 (range, 3 to 12) and 20.2 ± 5.4 (range, 13 to 28) mature oocytes per patient were retrieved from the normal- and high-responder groups, respectively. Of all oocytes recovered, 11.9% were judged to be immature and 9.7% were atretic. Of the mature oocytes 67.3% (142/211) fertilized normally; only 2.7% (4/146) were polyspermic. In patient no. 14, none of the mature oocytes fertilized, most likely because of abnormal sperm profile. In patient no. 6, only one of seven mature oocytes fertilized despite an apparently normal sperm profile. In this patient, a low fertilization rate was also observed in two previous IVF attempts. Of the normally fertilized mature oocytes, 82.4% (117 /142) cleaved, and the transfer of 60 embryos

Table 2 summarizes the dosage of P and E 2 valerate administered and the midluteal phase serum P and E 2 levels in individual patients. Figure 3 illustrates serum E 2 and P changes throughout the luteal phase in normal and high-responder patients. In 14 control, normal responders, serum E 2 on the day of hCG injection averaged 1,182 ± 562 pg/mL (range, 515 to 2,200 pg/mL). It fell after follicle aspiration and then rose to a peak midluteal level of 1,145 ± 662 pg/mL (range, 350 to 2,541 pg/mL) (Fig. 3). Serum Prose progressively after follicle aspiration and reached a midluteal phase peak of 87.5 ± 49.7 ng/mL and then fell to 15.5 ± 4.7 ng/mL on day 10 (Fig. 3). Serum E 2 and P were relatively stable throughout the luteal phase and were lower (P < 0.05) in

Vol. 56, No.2, August 1991

ltskovitz et al.

GnRH-a-induced LH/FSH surge

217

both the normal and high responders treated with GnRH-a than those treated with hCG (Fig. 3). In 12 patients, the administration of E 2 valerate and P was required to maintain midluteal and late luteal phase serum E 2 and P levels between 200 and 400 pgfmL and 15 and 20 ng/mL, respectively, (Table 2 and Fig. 3). In patients 6 and 14 (both diagnosed as having PCOD), no steroid supplementation was required, and in patient 2 supplementation was initiated only on day 8 of the luteal phase (Table 2). In patients who conceived after embryo transfer, P in oil 50 to 100 mg/d was administered throughout the first trimester of pregnancy. None of the patients developed any signs or symptoms of ovarian hyperstimulation syndrome. Transvaginal sonography disclosed only slightly to moderately enlarged multicystic ovaries with no, or only a minimal amount of peritoneal fluid.

DISCUSSION

This study shows that a bolus midcycle administration of GnRH -a triggered an endogenous LH/ FSH surge that was effective in the induction of oocyte maturation in vivo and enabled the recovery of mature fertilizable oocytes in patients undergoing ovarian stimulation for the purpose of IVF. The dynamics of pituitary gonadotropin secretion and the pattern of circulating E 2 and P at the periovulatory interval were qualitatively similar to those observed in the natural menstrual cycleP Despite the development of multiple preovulatory follicles, luteal phase serum E 2and P were lower than those detected in ovarian-stimulated patients injected with hCG and were close to the levels observed in the natural menstrual cycle. The normal spontaneous LH surge of the natural menstrual cycle is characterized by a short ascending phase (14 hours), a peak plateau phase (14 hours), and a long descending phase (20 hours)P The GnRH-a-induced LH surge was similar in magnitude to the natural LH surge, but it consisted only of a short ascending limb (>4 hours) and a long descending limb (>20 hours) (Fig. 1). This GnRH-ainduced LH surge was, however, effective in inducing the final stage of follicular maturation and the initiation and completion of oocyte maturation in vivo. Although meiosis resumption was most likely initiated and completed by the GnRH-a-induced LH surge, it is also possible that the stimulus for oocyte maturation was provided or enhanced by a direct effect of the GnRH-a on the oocyte. A direct stimulating action of GnRH or its agonist on oocyte 218

Itskovitz et al.

GnRH-a-induced LH/FSH surge

maturation in the rat and monkey has been demonstrated.13-15 The GnRH-a-induced FSH surge was similar in its pattern and duration to that observed in the natural cycle (Fig. l)P The physiological role, if any, of the FSH surge in the human has not been defined. It is not known if the use of GnRH -a, because of its release of pituitary FSH, has any advantage over the use of hCG devoided of FSH-like activity. The presence of midcycle FSH surge is not an obligatory one because apparently normal oocyte maturation and ovulation occur after the injection of hCG, when it might be expected that endogenous peripheral FSH levels would not be elevated. However, studies in rats have shown a role for FSH in the maturation of the oocyte-cumulus complex through the suppression of the intercellular coupling of the cumulus cells and the promotion of the secretion of glycosaminoglycans from granulosa cells. 16·17 Similar to the natural menstrual cycle, 12 the initiation of the GnRH-a-induced LH surge was temporally associated with a rapid rise of serum P and the attainment of peak E 2 levels during the 1st 12 hours after the injection of GnRH-a (Figs. 1 and 2). This was followed by a transient suppression of P biosynthesis and a gradual fall in E 2 levels during the 24 hours preceding follicle aspiration. After follicle aspiration, a second acute rise of P and continuous fall of E 2 were observed, reflecting the transition from the follicular to the luteal phase. This apparently normal follicular-luteal shift in steroidogenesis was, however, associated with luteal phase serum E 2 and P levels that were significantly lower than those normally observed in gonadotropin-stimulated patients injected with an ovulatory dose of hCG (Fig. 3). In fact, the administration of E and P was required in most patients to maintain serum E 2 and P levels above those detected in the luteal phase of the normal natural cycle, although as many as 28 mature oocytes were aspirated in the high responders (Table 2). After ovulation, the CL is dependent on pituitary LH. 18·19 It is possible that the administration of a high dose of GnRH -a altered the pattern of pulsatile or basal pituitary LH secretion during the luteal phase, resulting in reduced luteotropic support of pituitary LH. Alternatively, GnRH-a could inhibit ovarian steroidogenesis by direct action on the ovary. Specific binding of GnRH to human luteal tissue and the presence of high affinity GnRH receptors in human granulosa cells have been recently reported. 20·21 Furthermore, inhibitory effects of GnRH-a on P production by human and rat granulosa or luteal cells have also been demonstrated. 22- 25 Fertility and Sterility

Common factors associated with the development of ovarian hyperstimulation syndrome are multiple follicles and high E 2 levels on the day of hCG injection. Remarkably, none of the high responders developed any signs or symptoms of ovarian hyperstimulation, despite the presence of many mature follicles and extremely high levels of E 2 (as high as 13,130 pg/mL) before GnRH-a injection. Our preliminary results suggest that the use of GnRH -a instead of hCG, for the first time provides a means by which the risk of developing ovarian hyperstimulation syndrome after ovulation induction can be reduced or even eliminated. Further studies are required to determine the efficacy of this therapeutic regimen in reducing the risk of ovarian hyperstimulation syndrome in women showing exaggerated response to exogenous gonadotropins. The possible adverse effects of these extremely high levels of E 2 at the periovulatory period even in the absence of frank ovarian hyperstimulation syndrome, on blood coagulation and electrolyte homeostasis need to be examined. Some controlled ovarian stimulation protocols for IVF include adjunctive GnRH-a therapy during the follicular phase to prevent premature LH surge and early luteinization. Because of the resultant gonadotroph desensitization to GnRH, induction of endogenous LH/FSH surge with GnRH-a may not be possible in cycles that include adjunctive GnRH-a therapy. The use of GnRH -a instead of hCG also enables the development of new therapeutic manipulations of the hormonal levels throughout the luteal phase in patients undergoing ovarian stimulation with gonadotropins. Luteal phase support and the desired circulation levels of E 2 and P could be managed more accurately, thus avoiding the excessive levels of circulation Es, which have been implicated in the high rate of early embryonic loss and implantation failure in patients undergoing ovarian stimulation for IVF or other assisted reproductive techniques. 5 •6 A potential disadvantage of this therapeutic schedule in patients who have conceived lies in the need for prolonged luteal support with P until the time of the luteal-placental shift in P biosynthesis. Further studies are needed to determine the optimal ovulatory dose of GnRH-a and to examine its effects on CL function. The use of GnRH-a has other theoretical advantages over hCG for ovulation induction. The short GnRH-a-induced LH surge, rather than the extended surge of hCG, could provide a more physiological ovulatory stimulus. This time-limited stimulus can be restricted to the few follicles that are more mature, and thus a lower frequency of multiple Vol. 56, No. 2, August 1991

pregnancies could be expected. Whether normal ovulation can occur in vivo in patients undergoing ovarian stimulation for the purpose of ovulation induction remains to be examined. REFERENCES 1. Gemzell C: Induction of ovulation with human gonadotropins. Recent Prog Horm Res 21:179, 1965 2. Rabau E, David A, Serr DM, Mashiach S, Lunenfeld B: Human menopausal gonadotropins for anovulation and sterility: results of 7 years of treatment. Am J Obstet Gynecol 98:92, 1967 3. Ferraretti AP, Garcia JE, Acosta AA, Jones GS: Serum luteinizing hormone during ovulation induction with human menopausal gonadotropin for in vitro fertilization in normally menstruating women. Fertil Steril40:742, 1983 4. Shenken RS, Hodgen GD: Follicle-stimulating hormone-induced ovarian hyperstimulation in monkeys: blockade of the luteinizing hormone surge. J Clin Endocrinol Metab 57:50, 1983 5. Gidley-Baird AA, O'Neill C, Sinosich MJ, Porter RN, Pike IL, Saunders DM: Failure of implantation in human in vitro fertilization and embryo transfer patients: the effects of alteredprogesterone/estrogen ratios in human and mice. Fertil Steril 45:69, 1986 6. Forman R, Fries N, Testart J, Belaisch-Allart J, Hazout A, Frydman R: Evidence for an adverse effect of elevated serum estradiol concentrations on embryo implantation. Fertil Steril 49:118, 1988 7. Golan A, Ron-El R, Herman A, Soffer Y, Weinraub Z, Caspi E: Ovarian hyperstimulation syndrome: an update review. Obstet Gynecol Surv 44:430, 1989 8. Dericks-Tan JSE, Hammer E, Taubert HD: The effect of D-Ser(TBU) 6 -LH-RH-EA10 upon gonadotropin release in normally cyclic women. J Clin Endocrinol Metab 45:597, 1977 9. Lemay A, Faure N, Labrie F, Fazekas ATA: Gonadotroph and corpus luteum responses to two successive intranasal doses of a luteinizing hormone-releasing hormone agonist at different days after the midcycle luteinizing hormone surge. Fertil Steril 39:661, 1983 10. Monroe SE, Henzl MR, Martin MC, Schriock E, Lewis V, Nerenberg C, Jaffe RB: Ablation offolliculogenesis in women by a single dose of gonadotropin-releasing hormone agonist: significance of time in cycle. Fertil Steril 43:361, 1985 11. ltskovitz J, Boldes R, Barlev A, Erlik Y, Kahana L, Brandes JM: The induction of LH surge and oocyte maturation by GnRH analogue (buserelin) in women undergoing ovarian stimulation for in vitro fertilization. Gynecol Endocrinol 2(Suppl 2):165, 1988 12. Hoff JD, Quigley ME, Yen SSC: Hormonal dynamics at midcycle: a reevaluation. J Clin Endocrinol Metab 57:792, 1983 13. Hillenzsjo T, LeMaire WJ: Gonadotropin-releasing hormone agonist stimulate meiotic maturation of follicle-enclosed rat oocytes in vitro. Nature 287:145, 1980 14. Dekel N, Shalgi R: Fertilization in vitro of rat oocytes undergoing maturation in response to a GnRH analogue. J Reprod Fertil 80:531, 1987 15. Lefevre B, Gougeon A, Peronny H, Testart J: A gonadotropinreleasing hormone agonist and an activator of protein kinase C improve in vitro oocyte maturation in Macaca fascicularis. Gamete Res 21:193, 1988 16. Moor RM, Osborn JC, Cran OG, Walters DE: Selective effect of gonadotropins on cell coupling, nuclear maturation and ltskovitz et al.

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21. Popkin R, Bramley TA, Currie A, Shaw RW, Baird DT, Fraser HM: Specific binding of luteinizing hormone releasing hormone to human luteal tissue. Bichem Biophys Res Commun 114:750, 1983 22. Parinaud J, Beaur A, Bourreau E, Vieitez G, Pontonnier G: Effect of a luteinizing hormone-releasing hormone agonist (Buserelin) on steroidogenesis of cultured human preovulatory granulosa cells. Fertil Steril50:597, 1988 23. Maruo T, Otani T, Mochizuki M: Antigonadotropic actions of GnRH agonist on ovarian cells in vivo and in vitro. J Steroid Biochem 23:765, 1985 24. Tureck RW, Mastroianni L, Blasco L, Strauss JF III: Inhibition of human granulosa cell progesterone secretion by a gonadotropin-releasing hormone agonist. J Clin Endocrinol Metab 54:1078, 1982 25. Reddy PV, Azhar S, Menon KNJ: Multiple inhibitory actions of luteinizing hormone-releasing hormone agonist on luteinizing hormone/human chorionic gonadotropin receptor mediated ovarian responses. Endocrinology 107:930, 1980

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