Stimulation of endogenous surge of luteinizing hormone with gonadotropin-releasing hormone analog after ovarian stimulation for in vitro fertilization

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1 FERTILITY AND STERILITY Copyright" 1991 The American Fertility Society Vol. 55, No.2, February 1991 Printed on acid-free paper in U.S.A. Stimulation of endogenous surge of luteinizing hormone with gonadotropin-releasing hormone analog after ovarian stimulation for in vitro fertilization Daniel A. G. Imoedemhe, M.R.C.O.G.* Alejandro B. Sigue, B.S.M.T. Edgardo Luis A. Pacpaco, B.S.M.T. Arturo B. Olazo, B.S.M.T. Human Reproductive Biology Unit, Soliman Fakeeh Hospital, Jeddah, Saudi Arabia The effect of induction of preovulatory endogenous surge of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) with intranasal administration of GnRH-analog (GnRH-a) in an in vitro fertilization (IVF) program is reported. The use of GnRH-a resulted in a significantly better percentage of replaceable embryos (91 % versus 85%). The pregnancy rate was 51 % in comparison with 32% in control cycles in which follicular maturation was achieved by human chorionic gonadotropin administration. There was no significant difference in the postoocyte recovery serum progesterone patterns between the two groups. Our results indicate that the induction of endogenous LH and FSH surge with GnRH -a may be successfully employed for final follicular maturation after ovarian suprastimulation without affecting the outcome of IVF adversely. Apart from being a more physiological approach to oocyte maturation, it also has potential economic and clinical advantages. Fertil Steril55:328, 1991 The successful use of suppressive doses of gonadotropin-releasing hormone analog (GnRH-a) in combination with pure follicle-stimulating hormone (FSH) and or human menopausal gonadotropin (hmg) injections for superovulation in an in vitro fertilization (IVF) is well documented. 1-3 In all the available reports to date, human chorionic gonadotropin (hcg) is used for final oocyte maturation and subsequent timing of oocyte recovery irrespective of the stimulation protocol. Endogenous luteinizing hormone (LH) surge, the natural mechanism for final oocyte maturation, is circumvented by this practice, thus enabling the surgeon to schedule oocyte recovery at convenient times ofthe working day. Although no adverse effect on the outcome of treatment has been demonstrated as a Received August 14, 1989; revi$ed and accepted October 1, * Reprint requests: Daniel A. G. Imoedemhe, M.R.e.O.G., Human Reproductive Biology Unit, Soliman Fakeeh Hospital, P.O. Box 2537, Jeddah 21461, Saudi Arabia. result ofthe use ofhcg, it is apparent that the follicle is denied the full complement of endogenous gonadotropin endocrine milieu required for final maturation. These include a combined surge of LH and FSH in the midcycle. Although unlike LH, the function of the surge in FSH is not clear, it is known that the conversion of plasminogen to plasmin and deposition of hyaluronic acid matrix-processes necessary for cumulus expansion-are enhanced by FSH. 4,5 Furthermore, a rise in FSH in the midcycle ensures adequate complement of granulosa cell LH receptors, which is necessary for optimum luteinizing and hence luteal function. The purpose of this study was to evaluate the immediate endocrine accompaniment and clinical outcome of treatment after induction of endogenous LH surge using GnRH-a nasal spray in women undergoing IVF. MATERIALS AND METHODS The study included 70 women who underwent 77 IVF treatment cycles. They all had regular men- 328 Imoedemhe et al. GnRH-a induction of endogenous lh in ivf Fertility and Sterility

2 strual cycles of between 27 and 31 days. Their age ranged between 24 and 35 years (mean 28.9 years). Only women with tubal disease or unexplained infertility as causes of infertility were included. The duration of infertility ranged from 8 to 20 years (mean 11.3 years). Ovarian stimulation was carried out with injections of pure FSH or hmg in doses of 150 to 225 units daily from day 2 of the menstrual cycle for 4 to 5 days followed by 150 units daily until the morning of administration of either hcg or Gn RH -a. Follicular monitoring was carried out as previously described. 6 Briefly, daily ultrasound (US) scans were done starting from day 9 of the menstrual cycle. Daily serum estradiol (E2) and LH were done from day 9 of the menstrual cycle until the day of admission when 6 hourly serum LH estimations were commenced. Patients were admitted for oocyte recovery if two or more follicles of at least 18 mm mean diameter were present and the endocrine profile had been satisfactory. Patients were then allocated to received either hcg or Gn RH -a on admission. Blood samples were obtained for LH, FSH, E 2, and progesterone (P) assays one hour (-1 hr) before as well as just before administration of either hcg or GnRH-a. Subsequently, blood samples were obtained at + 1, +2, +12, +36, +60, +84, and hours, respectively, also for serum LH, FSH, E2, and P determination. At 0 hour intramuscular injection of 5,000 units of hcg was administered in 46 treatment cycles, whereas in 31 cycles 100 ILg of GnRH-a nasal spray (Suprefact; Hoechst Pharmaceutical, Middlesex, United Kingdom) was administered. A second 100-ILg dose of GnRH-a was administered 8 hours later. Oocyte recovery was carried out at 34 to 36 hours by US-guided follicular aspiration 6 after a preoperative US scan. The IVF laboratory techniques were as previously described. 6,7 Embryo replacement was carried out 48 to 50 hours after oocyte insemination. Embryos were classified as replaceable if at least two regular blastomeres were present. A maximum of six embryos were replaced per patient because cryopreservation facility was then not available on our program. Luteal phase support was commenced on the day after embryo replacement with 400 mg of P vaginal pessaries (Cyclogest 400; Hoechst) at night in all 77 cycles. Pregnancy was confirmed by a rising serum,8-hcg 12 days after embryo replacement and clinically by US evidence of an intrauterine gestation sac with fetal heart activity at 6 weeks from the 1st day of the last menstrual period. Table 1 Comparison ofivf-et Cycles: HCG Versus Endogenous GnRH-a Induced LH Surge for Oocyte Maturation No. offollicle ~ 1.5 cm/ cycle" No. of oocytes recovered No. of oocytes fertilized Fertilization rate (%) No. ofreplaceable embryos No. of embryos replaced Embryos replaced/cycle" No. of pregnancies Pregnancy rates (%) Pregnancy loss HCG (n = 36) 377 (819 ± 3.24) (84.9)b ± (33.3)b "Values are means ± SD. b Values in parentheses are percents. Hormone Assays GnRH-a (n = 31) 245 (790 ± 4.18) (90.8)b ± (37.5)b Serum concentration of LH and FSH were determined by a solid-phase dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA; LKB Wallac, Oy, Finland) as previously described.s-lo Standards used were calibrated for LH against the first International Reference Preparation (IRP) for human pituitary LH (code 68/40) and FSH against the record IRP for human pituitary (code 78/549). The interassay and intra-assay coefficient of variations (CVs) were 3.0% and 3.5% for FSH and 4.0% and 5.5% for LH, respectively. Serum E2 and P concentrations were determined by a competitive solid-phase fluoroimmunoassay based on competition between europium-labeled E2 or P and sample E2 or P for polyclonal anti-e2 or anti-p antibodies derived from rabbits as previously described8-l0 using DELFIA kits. The interassay and intra-assay CVs were 9.6% and 6.5% for E2 and 9% and 10.5% for P, respectively. Statistical Analysis The Student's t-test for comparison of means and x2 test with Yates correction for continuity where applicable were used for statistical analysis. A P value of <0.05 was regarded as significant. RESULTS The number of follicles of ~ 1.5 mm mean diameter were comparable (8.19 ± 3.24 versus 7.90 ± 4.18) per cycle, but the oocyte recovery rate was significantly higher in patients having induction of endogenous LH surge (90.6% versus 82.5%; P < ) (Table 1). In the GnRH-a group, 99 Vol. 55, No.2, February 1991 Imoedemhe et al. GnRH-a induction of endogenous lh in ivf 329

3 E2 P (A) pg/ml ng/rnl ~. " I::: i. 0 i FSH LH ~ ! " (B) E ~;::::::±=:===i FSH LH ~-fl~0r-+,i--+t2~+'1~2-+136~+6~0~+~8~4-+~'08 TIME - tdjrs Figure 1 (A), P (Pregnant, A - A; Nonpregnant, /:; - /:;) and E2 (Pregnant, e---e; Nonpregnant, 0---0). (B), FSH (Pregnant, A - A; Nonpregnant, /:; - /:;) and LH (Pregnant, e---e; Nonpregnant, 0---0). (46.6%) of the oocytes recovered were graded as mature, 117 (52.7%) were of borderline maturity, and 6 (2.7%) were regarded as immature. The corresponding values for the heg group were 114 (36.6%),183 (58.8%), and 14 (4.5%). The percentage of oocytes graded as mature in those receiving GnRH-a group was significantly higher than in those who received heg (P < 0.05). The fertilization rates were comparable (89.4% versus 88.7%), but the number of replaceable embryos were significantly higher in cycles when endogenous surge of LH had been induced (90.8% versus 84.9%; P < 0.05). The mean number of embryos replaced per cycle were similar in both groups (4.43 ± 1.40 versus 4.71 ± 1.28). The pregnancy rates of 51.6% versus 32.6% were not significantly different. Figures 1 and 2 illustrate the endocrine patterns in the conception and nonconception cycles of the respective study groups. There was no spontaneous surge of LH, defined as a rise of ~ 180% of the mean of the preceding four values of LH recorded in the study. The mean pre-gnrh-a and heg serum levels were not significantly different. A significant rise in serum LH was obtained in the 1st hour after GnRH -a administration and remained elevated up to +12 hours later. The mean LH peak was attained at +2 hours in the conception cycles as compared with + 12 hours in the nonconception cycles. The mean values of LH at + 1, +2, and 36 hours were significantly higher in the conception as compared with nonconception cycles (P < 0.025, P < 0.05, and P < 0.025, respectively). The pattern of immunoreactive LH detected in serum after heg administration is shown in Figure 2. Significantly higher mean baseline serum FSH were recorded in the conception cycles of both Gn RH-a and heg study groups at -1 and 0 hours compared with nonconception cycles within the respective groups: in the GnRH -a group, P < at -1 and 0 hour, respectively, and in the heg group, P < 0.05 and P < There was no significant difference between the mean serum FSH levels of the conception cycles of the two groups at these times. After the administration of GnRH -a, a rise in the mean serum level was recorded in both conception and nonconception cycles and the patterns followed closely that described for LH but to a lesser magnitude. The mean FSH values were significantly higher in the conception cycles at +1, +2, and + 12 hours compared with the values attained in the nonconception cycles at those times: P < , P < , and P < 0.05, respectively. EO P (A) pg/ml ogj ,. m p Endocrine Profile... FSH LB (B) TIME - HOURS Figure 2 (A), P (Pregnant, A - A; Nonpregnant, /:; - /:;) and E2 (Pregnant, e---e; Nonpregnant, 0---0). (B), FSH (Pregnant, A - A; Nonpregnant, /:; - /:;) and LH (Pregnant, e---e; Nonpregnant, 0---0). 330 Imoedemhe et al. GnRH-a induction of endogenous lh in ivf Fertility and Sterility

4 No rise in FSH level was recorded in the group receiving hcg. The levels in the conception and nonconception cycle remained comparable after hcg administration. There was no significant difference in the mean serum E2 between the conception and nonconception cycles in both study groups: 2,624 ± 1,616 versus 3,190 ± 1,216pg/mL (mean± SD) GnRH-aand 2,216 ± 1,612 versus 2,095 ± 1,599 pg/ml (mean ± SD) hcg group. No significant difference in mean E2 was observed between the conception cycles of the two groups or when the conception cycle of the GnRH -a was compared with the nonconception cycle of the hcg group. There was, however, a significantly higher mean serum E2 in the nonconception GnRH -a cycles as compared with either the conception and the nonconception cycles in the hcg cycles; P < 0.05 and P < 0.01, respectively. The serum P patterns were similar in both study groups, and no discernible significant difference was observed in the study. Pregnancy Outcome Spontaneous abortion occurred in 5 (33.3%) of the pregnancies in the hcg group. Four of these occurred before 12 weeks' gestation, whereas the other occurred at 21 weeks' gestation. There were 6 (37.5%) spontaneous abortions in the GnRH-a group, and they all occurred before the 12th week of gestation. There was no significant difference in the pregnancy loss rate. Fifteen live normal babies (6 singletons, 3 sets of twins, and a set of triplets) have been delivered by the remaining 10 mothers receiving hcg. In the GnRH-a group, the 11 remaining women have delivered a total of 15 babies (7 singletons and 4 sets of twins). No physical or neurological abnormality was observed in any of the 30 babies at birth. DISCUSSION The data presented indicated for the first time that preovulatory endogenous surge of gonadotropins can be successfully induced with GnRH-a nasal spray in the suprastimulated cycles without adverse effect to the outcome of IVF -embryo transfer (ET) treatment cycles. Although earlier reports ll - 13 have suggested that GnRH -a may have luteolytic effects, we failed to observe, at least in the early postoocyte recovery period, any difference in the patterns of P production between those receiving hcg and those receiving GnRH -a for induction of endogenous LH surge for final oocyte maturation. Whether an adverse effect of GnRH-a exists beyond +108 hours is a subject for further studies. However, Fleming et al. 14 have reported successful pregnancies and no shortening of the luteal phase after administration of GnRH -a up to 6 days after hcg injection in this study as compared with Hompes et al./ 5 who using twice as much GnRH -a, found shortening of the phase. This would suggest a dose-dependent effect of GnRH-a on the luteal phase. We therefore believe that the 200 J,Lg total dose utilized in our study, although adequate for inducing gonadotropin surge, is insufficient to induce luteolysis. The possible effectiveness of even much lower doses of Gn RH -a to induce adequate gonadotropin surge for oocyte maturation is open to further studies. It is our opinion that the induction of an endogenous LH and FSH surge after suprastimulation of the ovary simulates the natural sequence of endocrine events leading to the final maturation of the oocyte. The rise in FSH along with LH would allow for the optimization of granulosa cell LH receptors and hence may result in an improved luteal function. Weare unable to explain the rather delayed and truncated LH and FSH response to GnRH-a in the nonpregnant group. Further studies are needed to clarify the significance of this pattern of response. This method of preovulatory oocyte maturation apart from being more physiological has some other attraction of clinical and economic relevance. These include the convenience of administration because the nasal spray can be self-administered without supervision. Its administration is also atraumatic and hence more acceptable to the patient. It is also economically more cost-effective. Another advantage ofthis method is its application in patients with potential hyperstimulation after ovarian suprastimulation. 16 Because ovarian hyperstimulation syndrome is virtually unknown in the absence of hcg/ 7 we suggest that in patients in which there is the possibility of this occurring, follicular maturation can be completed by inducing an endogenous surge of gonadotropin with GnRHa. This would allow oocytes to be collected, fertilized, and cryopreserved for utilization in future cycles without the risk of hyperstimulation syndrome. Significant savings in cost of ovarian stimulation medication could also be made by this practice rather than cancellation of such cycles. In conclusion, we report for the first time the induction of endogenous LH and FSH surge with Gn RH -a nasal spray for final oocyte maturation after ovarian suprastimulation for IVF -ET treatment. Vol. 55, No.2, February 1991 Imoedemhe et al. GnRH -a induction of endogenous lh in ivf 331

5 These initial results do not indicate an adverse effect of GnRH -a on corpus luteum function in the immediate postovulatory period. Apart from the more physiological maturation of the oocyte, the technique has significant clinical, economic, and research value. Further studies to determine the lowest optimum dose required for adequate gonadotropin response are required. REFERENCES 1. Fleming R, Coutts JRT: Induction of multiple follicular growth in normally menstruating women with endogenous gonadotropin suppression. Fertil Steril 45:226, Shaw RW, Ndukwe G, Imoedemhe DAG, Bernard A, Burford G, Bentick B: Endocrine changes following pituitary desensitisation with LHRH agonist and administration of purified FSH to induce follicular maturation. Br J Obstet Gynaecol 94:682, Howles CM, Macnamee MC, Edward RG: Short term use of an LHRH agonist to treat poor responders entering an in vitro fertilization program. Hum Reprod 2:655, Stickland S, Beers WH: Studies on the role of plasminogen activator in ovulation. In vitro response of granulosa cells to gonadotropins, cyclic nucleotides and prostagladins. J Bioi Chern 251:5694, Eppig JJ: FSH stimulates hyaluronic acid synthesis by 00- cytes-cumulus complexes from mouse preovulatory follicle. Nature 281:483, Imoedemhe DAG, Wafik AH, Chan RCW: In vitro fertilization in women with "frozen pelvis" clinical outcome of treatment. Fertil Steril 49:268, Imoedemhe DAG, Mahgoub OA, Wafik AH, Chan RCW, Sigue AB, Reyes VV: The in vitro fertilization program at the Soliman Fakeeh Hospital, Jeddah, Saudi Arabia. J In Vitro Fert Embryo Transfer 5:52, Soini E, Kojola H: Time resolved fluorometer for lanthanide chelates-a new generation of non isotopic immunoassay. Clin Chern 29:65, Hemmila I, Dakubu S, Mukkala VW, Siitari H, Lovgren T: Europeum as label in time-resolved immunofluorometric assays. Anal Biochem 137:335, Lovgren T, Hemmila I, Petterson K, Eskola JV, Bertoff E: Determination of hormone by time-resolved fluoroimmunoassay. Talanta 31:909, Popkin R, Bramley TA, Currie A, Shaw RW, Baird DT, Frazer HM: Specific binding of luteinizing hormone releasing hormone to human luteal tissue. Biochem Biophys Res Commun 144:750, Asch RH, Siler-Khodr TM, Smith AG, Schally AV: Luteolytic effect of D-TRp6-luteinizing hormone releasing hormone in rhesus monkey (Macaca mulatta). J Clin Endocrinol Metab 52:565, Sheenan KL, Casper RF, Yen SSC: Induction of luteolysis by luteinizing hormone-releasing factor (LRF) agonist: sensitivity, reproducibility and reversibility. Fertil Steril 37:209, Fleming R, Haxton MJ, Hamilton MPR, McCune GS, Black WP, MacNaughton MC, Coutts JRT: Successful treatment of infertile women with oligomenorrhoea using a combination of an LHRH -agonist and exogenous gonadotropin. Br J Gynaecol 92:369, Hompes PGA, Van Weissenbrach MM, Burger CW, Schoemaker J: The additional use of Buserelin in HMG-HCG ovulation induction in PCO: a double blind controlled study. In Gonadotropin Down-Regulation in Gynaecological Practice, Edited by R Allan. New York, Liss Inc publishers, 1986, p Imoedemhe DAG, Chan RCW, Sigue AB, Pacpaco E, Olazo A: Gonadotropin releasing hormone analog in the management of potential ovarian hyperstimulation syndrome in an in vitro fertilization program. Unpublished data 17. Glass RH: Infertility. In Reproductive Endocrinology: Physiology, Pathology and Clinical Management, Edited by SSC Yen, RB Jaffe. Philadelphia, W.B. Saunders Co., 1978, p Imoedemhe et al. GnRH-a induction of endogenous lh in iuf Fertility and Sterility