Graham Burford, Ph.D. Albert Bernard, B.Sc. Bernard Bentick, M.R.C.O.G. Robert W. Shaw, M.D.t Claire A. Iflland, M.B.

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1 FERTILITY AND STERILITY Copyright <> 1988 The American Fertility Society Printed in U.S.A. A randomized comparative study of purified follicle stimulating hormone and human menopausal gonadotropin after pituitary desensitization with Buserelin* for superovulation and in vitro fertilization Bernard Bentick, M.R.C.O.G. Robert W. Shaw, M.D.t Claire A. Iflland, M.B. Graham Burford, Ph.D. Albert Bernard, B.Sc. Academic Department of Obstetrics and Gynaecology, Royal Free Hospita~ London, United Kingdom Twenty patients entered a randomized, crossover study of purified follicle-stimulating hormone (pure-fsh) or human menopausal gonadotropin (hmg) superovulation, 2 ampules per day after pituitary desensitization with the luteinizing hormone-releasing hormone (LH-RH) analogue Buserelin (D-Ser tbu6 LH-RH 1-9 ethylamide) nasal spray. There were no cycles cancelled. Six patients conceived (five on pure-fsh, one on hmg). There were 24.2 ± 2.5 (mean± standard error of the mean [SEM)) ampules ofpure-fsh and 24.3 ± 3.6 ampules of hmg stimulation required. There were similar numbers of preoperation follicles: 6.9 ± 1.0 on hmg and 6.6 ± 1.1 on pure-fsh, of oocytes collected; 8.5 ± 1.4 on hmg and 5.8 ± 1.4 on pure-fsh, and of pre-embryos achieved; 5.1 ± 0.9 on hmg and 3.4 ± 1.0 on pure-fsh; on either treatment. The fertilization rate on hmg was 60% and on pure-fsh was 55%. Pre-embryo transfer rates were 3.2 ± 0.3 in the hmg group and 2.7 ± 0.4 in the pure-fsh group. There were no differences in serum FSH, LH, estradiol, or progesterone levels between the hmg and pure-fsh groups. Mean± SEM luteal phase length was 10.6 ± 0.4 days in the nonpregnant cycles. Fertil Steril50:79, 1988 Since Trounsen et al. 1 reported higher pregnancy rates with increasing numbers of pre-embryos transferred, superovulation strategies have aimed to achieve at least three or four pre-embryos suitable for transfer. However, superovulating women with functional pituitary glands may provoke an unpredictable and adverse response, resulting in cyst formation, premature luteinization, follicular asynchrony, and early (unexpected) ovulation. 2 3 In Received October 16, 1987; revised and accepted March 14, * Buserelin, Hoechst UK Ltd., Pharmaceutical Division, Hounslow, Middlesex, United Kingdom. t Reprint requests: Professor Robert W. Shaw, Department of Obstetrics and Gynaecology, The Royal Free Hospital, Pond Street, London NW3 2QG, United Kingdom. contrast, women with hypothalamic or pituitary amenorrhea respond well to ovulation induction with high pregnancy rates. 4 Thus, if a temporary state of hypogonadotropic hypogonadism is induced, then subsequent stimulation, either for uniovulation or superovulation, should achieve goodquality stimulation and avoid these adverse responses. Luteinizing hormone-releasing hormone (LH-RH) analogues have been demonstrated to induce reversible hypogonadotropic hypogonadism, 6-7 and an easily administered preparation such as a nasal spray combines a convenient route of administration with rapid excretion. This study was commenced to determine whether pituitary suppression resulted in a favorable hormonal milieu during subsequent stimulation and so produced improved oocyte quality Bentick et al. Buserelin with FSH and hmg stimulation 79

2 compared with previously adverse stimulation cycles. In addition, the hypothesis that both endogenous pituitary luteinizing hormone (LH) release and exogenous LH administration were unnecessary for follicular growth was to be tested. MATERIALS AND METHODS Twenty patients, mean age 34.8 (SEM ± 1.4) years, were recruited from the in vitro fertilization (IVF) program at the Royal Free Hospital, London. Seventeen had previously developed problems: cyst formation (three), follicular asynchrony (three), poor-quality oocytes (six), or premature ovulation (five) when on conventional superovulation therapy with clomiphene citrate (CC) 150 mg daily days 2 to 6 and hmg 2 ampules daily days 5 to 9 but excluding any patient who had previously developed fewer than three follicles and couples with male factors in their infertility. The number of previous treatment cycles (number of patients) were 5(1), 3(4), 2(6), and 1(6). The infertility diagnoses were tubal (17), unexplained (2), and endometriosis (1), and all couples had been infertile for more than 4 years. A baseline ultrasound scan was performed in the midluteal phase (day 21 or 22) with a Diasonics DRF 100 mechanical sector scanner with a 3.5 MHz abdominal transducer (Sonotron Ltd., Bedford, England), then Buserelin (D-Ser tbu6 LH-RH 1-9 ethylamide, Hoechst UK Ltd., Pharmaceutical Division, Hounslow, Middlesex, England) intranasal spray was commenced at a dose of g X 5 daily (every 4 hours, omitting the 3 A.M. dose) This start date was ascribed day 1 of treatment. On day 15, if menses had commenced and a further ultrasound scan showed no cyst formation, stimulation was commenced with hmg (Pergonal, Serono Laboratories [UK] Ltd., Welwyn Garden City, Herts, England) or pure-fsh (Metrodin, Serono Laboratories (UK) Ltd., Welwyn Garden City, Herts, England) at a fixed dose of 2 ampules daily. This was designated day + 1 of stimulation. The LH-RH analogue therapy was continued until the time of human chorionic gonadotropin (hcg) administration. Prospective monitoring with ultrasound scanning was performed daily or on alternate days from day +8 of stimulation onward. The dose of gonadotropin was doubled for 2 days if there were no follicles ~8 mm on day +8. Stimulation was continued until the mean follicular diameter (MFD) of the leading follicle was ~15 mm. HCG (Profasi, Serono Laborato- 80 Bentick et al. Buserelin with FSH and hmg stimulation ries (UK) Ltd., Welwyn Garden City, England) 5000 IU was given intramuscularly when the mean diameter of the leading follicle was ~ 17 mm and there were ~3 follicles of mean diameter ~15 mm. Oocyte collection was performed 34 hours after hcg by laparoscopy in all but three cycles in which transurethral ultrasound guided collections were performed. Laboratory procedures were as previously described. 8 A maximum of four pre-embryos was transferred. There was no additional luteal phase support given. Blood was taken on days 1, 2, 4, 6, 8, 11, 13, and 15 (until the end of pituitary desensitization) and stimulation days (immediately before the gonadotropin injection) + 1, +2, +4, +6, +8, and + 10, then daily until the day of hcg injection. Luteal phase adequacy was assessed by taking blood on days 1, 2, 3, 4, 7, 10, and 14 after oocyte collection. All serum samples were stored at -20 C until assayed for FSH, LH, 17,8-estradiol (E 2) and progesterone (P). ASSAYS All assays were performed in duplicate. FSH tmd LH were measured with a solid-phase radioimmunoassay (RIA) as previously described, 9 with interassay and intra-assay coefficients of variation of 8.1% and 3.0% for FSH and 6.4% and 1.4% for LH. E2 was analyzed with a nonextraction RIA kit (Code ER-155, Steranti Research Ltd., St. Albans, Herts, UK) with interassay and intra-assay coefficients of variation of 6.6% and 5.4%, and P was measured with the Coata-count progesterone 125 I kit (Diagnostic Products Ltd., Wallingford, Oxon, UK; imported from Diagnostic Products Corporation, Los Angeles, CA) with interassay coefficient of variation 7.6%. RESULTS The commencement of Buserelin in the midluteal phase produced an initial stimulated release of FSH and LH, resulting in increased granulosa lutein cell production of E2 and P and persistent corpus luteum function until suppression of both gonadotropins was achieved. Baseline levels of FSH were obtained after 8 days and baseline LH levels after 11 days of Buserelin administration (Fig. 1). Menses were thereby delayed for up to 6 days beyond the woman's expected date of menses (11 to 13 days after the start of Buserelin) in this study. Menstruation occurred after luteolysis, and at this time early follicular phase serum P ( <2 Fertility and Sterility

3 , a-.. ~ Figure 1 Serum FSH, LH, E 2, and P during the desensitization phase (up to day 15) and during the stimulation phase (from day + 1) up the point of hcg injection in the 13 patients who completed both arms of the study. nmol/l[0.6 pg/ml]) levels and postmenopausal E2 levels ( <100 pmol/l[27 pg/ml]) had been achieved. Initially during the study, those patients who menstruated but still had functional ovarian cysts commenced stimulation immediately (two cases), but later on in the study, those patients with persistent cysts were treated by transurethral ultrasound-guided aspiration of these cysts before starting stimulation (three cases) because in the first two cases, despite achievement of menses and low E 2 and P levels, the commencement of exogenous gonadotropins caused further increase in cyst size. Six patients achieved a clinical pregnancy, and their results were analyzed separately from the nonconception cycles. One patient had a spontaneous abortion and completed both arms of the study. Two patients did not complete the crossover arm of treatment. Thirteen patients thus completed the crossover study. In these patients, during gonadotropin stimulation, serum LH levels were similar whether on hmg or pure-fsh treatment and showed no significant increase above those baseline levels achieved after suppression. However, serum FSH levels rose sharply to similar values on both hmg and pure FSH therapy. Serum P levels remained within the early follicular phase range ( <2 nmol/l) all through the stimulation phase on both arms of the study right up until the time of hcg injection (Table 1). During stimulation, E 2 levels rose sharply after day +4 to similar levels on hmg and pure-fsh, peak E 2 values reflecting numbers' of mature follicles (Fig. 1,,Table 2). There was no significant difference in peak E 2 levels per follicle Table 1 Comparison of Clinical Results HMG group Pure FSH group Pregnancy group Total (Mean± SEM) Total (Mean± SEM) Total (Mean± SEM) Number starting Buserelin 13 Number starting Stimulation 13 Duration of stimulation day (11.1 ± 0.9) Operation day (14.8 ± 0.8) Total ampules (24.3 ± 3.6) Follicles pre-hcg <15mm 58 (4.5 ± 0.9) :2:15mm 74 (5.7 ± 1.0) Total 132 (10.2 ± 1.6) Follicles preop <15mm 46 (3.5 ± 1.4) :2:15mm 90 (6.9 ± 1.0) Total 136 (10.5 ± 1.8) Follicles aspirated 128 (9.8 ± 1.6) Oocytes collected 111 (8.5 ± 1.4) Patients with nil fertilization 0 Embryos <4 cells 35 (2.7 ± 0.7) :2:4 cells 31 (2.4 ± 0.5) Total 66 (5.1 ± 0.9) Transfers 13 Embryos transferred 42 Embryos/transfer (11.4 ± 0.8) (12.3 ± 1.1) (14.8 ± 0.7) (15.7 ± 1.3) (24.2 ± 2.5) (24.2 ± 2.1) 77 (5.9 ± 1.1) 40 (6.7 ± 1.4) 54 (4.2 ± 0.8) 34 (5.7 ± 1.1) 131 (10.1 ± 0.9) 74 (12.4 ± 1.3) 42 (3.2 ± 0.6) 13 (2.2 ± 0.9) 86 (6.6 ± 1.1) 52 (8.7 ± 1.0) 128 (9.8 ± 1.0) 65 (10.8 ± 0.9) 99 (7.6 ± 0.7) 47 (7.8 ± 1.2) 75 (5.8 ± 1.4) 39 (6.5 ± 1.3) (1.4 ± 0.9) 10 (1.7 ± 0.8) 24 (1.9 ± 0.4) 17 (2.8 ± 0.5) 42 (3.2 ± 1.1) 27 (4.5 ± 0.7) Bentick et al. Buserelin with FSH and hmg stimulation 81

4 Table 2 Serum FSH, LH, E2 and P Levels Before Commencing Buserelin, Before Stimulation, and Pre-hCG in the 13 Patients Who Completed Both Arms of the Study Plus Those Who Achieved Pregnancy Hormone LH Treatment day Treatment group Mean± SEM lull 1 day Buserelin HMG 4.7 ± 1.1 started Pure-FSH 5.6 ± 0.9 Pregnancy 8.9 ± day stimulation HMG 2.2 ± 0.3 commenced Pure-FSH 2.6 ± 0.4 Pregnancy 3.3 ± 0.5 Pre-hCG HMG 2.5 ± 0.3 Pure-FSH 2.3 ± 0.2 Pregnancy 2.7 ± 0.4 FSH E2 p Mean± SEM Mean ± SEM Mean ± SEM pmol/l pmol/l nmol/l nmol/l lull [pglml] [pglml] [nglml] [nglml] 2.1 ± ± ± 4.8 [189] [33.5] [6.4] [1.5] 1.9 ± ± ± 6.8 [173] [17. 7] [8.1] [2.1] 2.2 ± ± ± 3.7 [139] [7.1] [6.0] [1.2] 2.2 ± ± ± 0.2 [18.8] [1.9] [0.2] [0.1] 1.7± ± ± 0.2 [15.8] [1.6] [0.3] [0.1] 1.9 ± ± ± 0.5 [16.6] [3.3] [0.5] [0.2] 6.6 ± ± ± 0.2 [1586] [278] [0.5] [0.1] 5.9 ± ± ± 0.4 [1283] [431] [0.3] [0.1] 5.8 ± ± ± 0.5 [1360] [437] [0.4] [0.2] ~15 mm MFD, mean ± SEM 1410 ± 125 pmol/l (384 ± 34 pg/ml) on hmg, and 1140 ± 216 pmol/l (310 ± 59 pg/ml) on pure-fsh. The amount and duration of stimulation were identical on either regimen, as were the number and size of follicles recruited (Table 1). There were no significant differences in numbers of oocytes collected, fertilization, cleavage, or pre-embryo transfer rates, though unexpectedly, no oocytes fertilized in three cycles on pure-fsh treatment (Table 1). Five pregnancies occurred on the pure-fsh arm and one on the hmg arm. In the nonconception cycles, the mean luteal phase was 10.8 ± 0.5 days on the pure-fsh arm and 10.6 ± 0.5 days on the hmg arm. The levels of serum Pin the luteal phase (excluding pregnancy cycles) are shown in Figure 2, with high levels in the early luteal phase and then luteal insufficiency from day OP + 10 but no differences between the hmg- and pure-fsh-treated groups. DISCUSSION Suppression of pituitary gonadotropin release in women using LH-RH analogues has now been well described for the subcutaneous and intranasal routes and used to maintain a situation of hypoes- 82 Ben tick et al. Buserelin with FSH and hmg stimulation trogenism for the treatment of endometriosis and to induce reduction of fibroids Furthermore, pituitary desensitization and down regulation have been found useful in ovulation induction for patients with polycystic ovarian syndrome 12 and for superovulation for IVF The major disadvantages are that the treatment cycle is prolonged to at least 5 weeks and the duration and amount of gonadotropin stimulation are likely to be at least double that used in regimens of CC and gonadotropins without pituitary desensitization. Commencement oflh-rh analogue in the midluteal phase initially causes an increased release of LH and FSH with ~ ; * Q) 8) 60 ~ PAEHCG PAEOP OP 2 OP+4 OP+-5 OP.a OP 10 Cycle Day Figure 2 Luteal phase P levels in the 13 patients who completed both arms of the study, excluding 1 pregnancy cycle. Fertility and Sterility

5 consequent stimulation of corpus luteum function. Pulsatile gonadotropin secretion is abolished by 8 days of administration of Buserelin. 8 When suppression levels of both gonadotropins are achieved, luteolysis then occurs. Menstruation signals the achievement of clinical hypothalamic-pituitaryovarian axis suppression as demonstrated in this study and thus avoids the need for repeated hormonal assays at this stage. In this group of patients, the clinical sign of menstruation occurred by the 13th day of Buserelin treatment, but other patients have required up to 22 days of Buserelin before menstruation occurred (unpublished data).15 Continuation of the LH-RH analogue until hcg injection maintains pituitary suppression during the stimulation phase and prevents premature luteinization. Stimulation with either pure-fsh or hmg is well controlled and continued until adequate follicle sizes are achieved without concern about an endogenous gonadotropin surge. In this group of patients, mature follicles developed in every cycle, there being no significant difference in the numbers obtained with the use of either pure-fsh or HMG. E2 synthesis was equal and adequate, on either arm of the study, and the maximum E 2 level per mature follicle was within the range reported as appropriate by Hillier et al.14 This indicates that pulsatile LH secretion is not required for follicular growth and that the low levels of LH present during stimulation are adequate for thecal production of androgen substrates (notably androstenedione) required by granulosa cells for estrogen synthesis.1a-18. Failure to see a rise in serum LH on stimulation in the hmg-treated group merely reflects the sample timing and half-life of LH, whereas significant increases above baseline in FSH are expected because of the relatively long half-life offsh; similar levels of serum FSH achieved reflected the same dose of FSH used in each group. There was no difference in the number of mature (MFD ~ 15 mm) or small follicles achieved on either regimen. There were no statistically significant differences in the numbers of follicles aspirated, oocytes collected and fertilized, cleavage rates, or pre-embryos transferred between the two groups: the trend to better results on hmg stimulation was considered to be due to recurrent problems of restricted theater operating time and IVF laboratory quality control that particularly affected the pure-fsh group. It might be said that the absence of exogenous LH in the pure-fsh group had some adverse effect on the follicle or oocyte, manifested as poor cleavage rates, but five out of the six pregnancies occurred on pure-fsh and these patients did not cross over to hmg stimulation because of the pregnancy. The rate of recovery of pituitary gonadotropin release when Buserelin was stopped at the time of hcg injections could not be evaluated in this study. Several in vitro and in vivo reports that confirm Buserelin binding to the corpus luteum suggest that luteal insufficiency could be a problem However, in this study, luteal P levels were usually sufficient to maintain integrity of the endometrium until such time as implantation and humoral preembryonic signals would be expected to have been established, leading to pre-embryonic control of corpus luteum function. That further exogenous support of the corpus luteum is not essential is evidenced by our successful pregnancies established in these studies. In conclusion, both hmg and pure-fsh used after pituitary down regulation with LH-RH analogue achieve adequate ovarian stimulation, preducing equal numbers of mature follicles and similar peripheral blood levels of FSH, LH, E2, and P. Premature luteinization is avoided; thus, oocyte pick-up can be planned for a convenient day and time. In an IVF unit without the benefit of prospective hormone assays and with constraints upon the use of operating facilities, this regimen is potentially advantageous both as a routine stimulation protocol and for those with previous cycle problems such as premature luteinization, follicular asynchrony, and cyst formation. Acknowledgments. We should like to thank Dr. Patrick Magill, Ph.D. (Hoechst UK Ltd., Hounslow, Middlesex, England), for supplying the Buserelin and Dr. Ellis Snitcher, M.B. (Serono Laboratories [UK] Ltd., Welwyn Garden City, Herts, England), for supplying the Metrodin used in this study. This study was approved by the Royal Free Hospital Ethical Committee and by the Medical Research Council/Royal College of Obstetricians Voluntary Licensing Authority. REFERENCES 1. Trounson AO, Leeton JF, Wood C, Webb J, Wood J: Successful human pregnancies by in-vitro fertilisation and embryo transfer in the controlled ovulatory cycle. Science 212:681, Stanger JD, Yovich JL: Reduced in-vitro fertilisation of human oocytes from patients with raised basal luteinising hormone levels during the follicular phase. Br J Obstet Gynaecol 92:385, 1985 Bentick et al. Buserelin with FSH and hmg stimulation 83

6 3. MacGregor AH, Johnson JE, Bunde CA: Further clinical experience with clomiphene citrate. Fertil Steril 19:616, Lunenfeld B: Therapy with gonadotrophins. In Advances in Gynaecological Endocrinology. Edited by HS Jacobs. Proceedings of the Sixth Study Group of the Royal College of Obstetricians and Gynaecologists London, Royal College of Obstetricians and Gynaecologists, 1979, p Lemay A, Maheux R, Faure N, Jean C, Clement J, Fazekas AT A: Reversible hypogonadism induced by a luteinizing hormone-releasing hormone (LH-RH) agonist (Buserelin) as a new therapeutic approach for endometriosis. Fertil Steril 41:863, Shaw RW, Frazer HM, Boyle H: Intranasal LHRH in the treatment of women with endometriosis. Br Med J 287:1167, Shaw RW, Kerr-Wilson RHJ, Fraser HM, McNeilly AS, Howie PW, Sandow J: Effect of an intranasal LHRH agonist on gonadotrophins and hot flushes in post-menopausal women. Maturitas 7:161, Shaw RW, Ndukwe G, Imoedemhe DAG, Bernard A, Burford G, Bentick B: Endocrine changes following pituitary desinsitisation with LHRH agonist and administration of purified FSH to induce follicular maturation. Br J Obstet Gynaecol 94:682, Ferguson KM, Hayes M, Jeffcoate SL: A standardised multicentre procedure for plasma gonadotrophin radio-immunoassay. Ann Clin Biochem 19:358, Matta W, Shaw RW, Nye M: Studies of the effect of a luteinising hormone releasing hormone (LHRH) agonist (Buserelin) in patients with uterine leiomyomata. Br J Obstet Gynaecol 93:1194, Franssen AMHW, Rolland R, Willemsen WNP: LHRH ag- onist treatment of uterine leiomyomas: preliminary observations. Br J Clin Pract 41(Suppl. 48):59, Fleming R, Haxton MJ, Hamilton MPR, McCune GS, Black WP, McNaughton MC, Coutts JRT: Successful treatment of infertile women with oligomenorrhoea using a combination of an LHRH agonist and exogenous gonadotrophins. Br J Obstet Gynaecol 92:369, Porter RN, Smith W, Craft IL, Abdulwahid NA, Jacobs HS: Induction of ovulation for in-vitro fertilisation using Buserelin and gonadotrophins (Letter). Lancet 2:1284, Hillier SG, Parsons JH, Margara RA, Winston RML, Crofton ME: Serum oestradiol and preovulatory follicular development before in-vitro fertilisation. J Endocrinol 101:113, Bentick B, Ulland CA, Shaw RW: Unpublished data. 16. McNatty KP, Makris A, DeGrazia C, Osathanondh R, Ryan KJ: The production of progesterone, androgens, and oestrogens by granulosa cells, thecal tissue and stromal tissue from human ovaries in vitro. J Clin Endocrinol Metab 49:687, McNatty KP, Osathanondh R, Ryan KJ: Effects of luteinizing hormone on steroidogenesis by thecal tissue from human ovarian follicles in vitro. Steroids 36:53, Baird DT: Factors regulating the growth of the preovulatory follicle in the sheep and human. J Reprod Fertil69:343, Popkin R, Bramley TA, Currie A, Shaw RW, Baird DT, Fraser HM: Specific binding of luteinizing hormone-releasing hormone to human luteal tissue. Biochem Biophys Res Comm 114:750, Sheehan KL, Casper RF, Yen SSC: Induction of luteolysis by luteinizing hormone-releasing factor (LRF) agonist: sensitivity, reproducibility, and reversibility. Fertil Steril 37:209, Bentick et al. Buserelin with FSH and hmg stimulation Fertility and Sterility

Prospective study of short and ultrashort regimens of gonadotropinreleasing hormone agonist in an in vitro fertilization program

FERTILITY AND STERILITY Copyright 1992 The American Fertility Society Printed on acid-free paper in U.S.A. Prospective study of short and ultrashort regimens of gonadotropinreleasing hormone agonist in