Articles Use of recombinant FSH and recombinant LH in multiple follicular stimulation for IVF: a preliminary study

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1 RBMOnline - Vol 3. No Reproductive BioMedicine Online webpaper 2001/226 on web 15 October 2001 Articles Use of recombinant FSH and recombinant LH in multiple follicular stimulation for IVF: a preliminary study Dr Franco Lisi was born in 1955 in L Aquila, Italy. He became a general doctor in 1979 and specialist in Obstetrics and Gynaecology in 1983 at the University of Rome. Until 1987 he worked mainly in Obstetrics after which he became totally engaged in assisted reproduction and in-vitro fertilization. Since 1991 he has been Clinical Director of Biogenesi, the service of assisted reproduction of Clinica Villa Europa in Rome. Dr Franco Lisi F Lisi 1,4, L Rinaldi 1, S Fishel 1,2, R Lisi 1, G Pepe 1, M G Picconeri 1, A Campbell 1,2, P Rowe 3 1 BIOGENESI, Casa di Cura Villa Europa all EUR, Via Eufrate 27, Eur, Roma 00144, Italy 2 CARE (Centres for Assisted Reproduction), Park Hospital, Sherwood Lodge Drive, Arnold, Nottingham NG5 8RX, UK 3 School of Pharmacy and Chemistry, John Moore s University, Byrom Street, Liverpool, L3 3AF, UK 4 Correspondence: Tel ; Fax ; flisi@tin.it Abstract Twelve patients (17 cycles) who needed >3000 IU of recombinant FSH (rfsh) on previous follicular stimulation attempts for IVF using rfsh (group A) underwent further attempts (a total of 12 cycles) using rfsh supplemented with rlh from day 7 of stimulation (group B). There was no significant difference in the total quantitative administration of rfsh (mean: 4759 versus 4800 IU rfsh), days of stimulation (14.0 versus 12.6), number of M2 oocytes per patient (3.76 versus 4.17) or embryo morphology between group A and group B respectively. However, there was a significant increase in the incidence of fertilization (60.9 versus 86.0%; P = 0.006) and clinical (ongoing) pregnancies (1 versus 6; P = 0.022). The higher incidence of fertilization increased the mean number of embryos transferred per patient, although in this small group the data was not significant (1.75 versus 2.71). This preliminary clinical trial suggests that in poor responders superimposing rlh upon a down-regulated cycle stimulated with rfsh improves relevant outcome data; and this preliminary trial has encouraged the need for a larger and more detailed study. Keywords: follicle growth, human, IVF, ovarian stimulation, recombinant FSH and LH Introduction 190 Human FSH for clinical use was first extracted from pituitary glands and the first pregnancies were reported in 1960 (Gemzell et al., 1960; Lunenfeld et al., 1962). Subsequently, urinary extracts of gonadotrophins from postmenopausal women were shown to be safe and effective and became the standard for 30 years (WHO, 1973) at either FSH:LH ratio of 1:1 (prepared as human menopausal gonadotrophin; HMG) or in the preparation of FSH HP (high purity) with only traces of LH (<0.1 IU LH/1000 IU FSH). During the 1990s, recombinant DNA technology permitted the introduction of rfsh, a preparation totally devoid of LH activity, for clinical use (Germond et al., 1992; Mannaerts et al., 1993; Porchet et al., 1993: Weinbauer et al., 1994). Recombinant FSH is well tolerated without side-effects, its plasma half-life is similar to that of the native hormone and no antirecombinant FSH antibodies have been noted (Schoot et al., 1994). rlh has been available for clinical trials since 1993 and was first used in addition to rfsh to induce ovulation in female hypopituitary-hypogonatotrophic patients (WHO group I of anovulation; Agrawal et al., 1997) as an alternative to conventional HCG for ovulation induction (Chandrasekher et al., 1994). Hull et al. (1994) reported that rfsh did not facilitate sufficient steroidogenesis even in large follicles, and addition of low dose rlh was required to induce adequate oestradiol secretion for characteristic mucus secretion, endometrial growth and pregnancy. However, the amount of rlh required to support rfsh-induced follicular development

2 was unclear. The European Recombinant Human LH Study Group (1998) reported that 75 IU per day is sufficient for the majority of women. The first pilot study investigating the importance of endogenous LH in IVF embryo transfer (Devroey et al., 1994) concluded that even after deep pituitary suppression, endogenous LH concentrations are sufficient to induce adequate steroidogenesis. Scott Sills et al. (1999) utilized FSH- HP and FSH-HP + rlh in gonadotrophin-releasing hormone analogue (GnRHa) down-regulated patients and they concluded that appropriate endogenous basal LH exists, obviating the need for supplemental LH. This group reported better results in women stimulated with FSH-HP compared with a group of women treated with FSH-HP + rlh. However, Laml et al. (1999) studied a group of six normogonadotrophic female poor responders recommending the supplementation of rlh to rfsh for follicular stimulation in such patients. A pilot study was therefore conducted of 12 patients who had been poor responders, in 17 cycles of treatment, supplementing rfsh stimulation with rlh. The results demonstrated apparent benefits of this therapy and are reported below. Materials and methods Patients Twelve patients (17 cycles: 10 ICSI and 7 IVF) who, during follicular stimulation with rfsh, required >3000 IU to reach follicular maturity (group A) returned for subsequent cycles supplemented with rlh (group B). Ten of the patients had never achieved a pregnancy, while two had a baby conceived by artificial reproduction techniques several years previously. This group of patients had a mean basal FSH measured on day 3 of a natural cycle of 12.2 miu/ml (range 7 19) and a mean age of 36.1 years. Down-regulation was as in the first cycles and was induced with triptoreline 0.1 mg (Ipsen, Paris, France) subcutaneously from the mid-luteal phase of the previous cycle (day 21) for 3 weeks before starting rfsh stimulation. On day 7 of stimulation, 75 IU rlh (Luveris; Serono, Geneva, Switzerland) was administered to supplement the ongoing rfsh administration until the day of HCG administration (10,000 IU; Profasi, Serono). Oocytes were recovered 36 h after HCG administration. Of the ICSI cases, three had thawed testicular spermatozoa previously recovered by TESE. Oocyte retrieval and IVF embryo transfer were carried out in accordance with usual clinical practice. For luteal support, all patients received 50 mg i.m. progesterone (Gestone; Amsa srl, Rome, Italy) daily. Data on oestrogen concentrations during gonadotrophin stimulation, number and size of follicles, and on bioactive LH before and during the stimulation cycles are not shown but will form part of a future more extensive study report. Statistics Results were analysed by Student s t-test and χ 2 test. The data for follicle sizes was subjected to an analysis of covariance, with day number as a covariate, using the General Linear Modelling facility within Minitab (Minitab Release 13.1; Minitab Inc., Pennsylvania, USA). No statistical attempt was made to compensate for possible skewness of the data or non-normal distribution because of the preliminary nature of this study. Results Patients from group A needed a mean of 4759 IU of rfsh (range ) to reach follicular maturity, compared with a mean of 4800 IU of rfsh (range ) for group B; the number of ampoules of rfsh was adjusted on a daily basis. The mean days of stimulation were 14.0 and 12.6 in group A and group B respectively. The circulating oestradiol on the day of HCG administration was elevated in group B (3101 pmol/l; range ) when compared with group A (2084 pmol/l; range ). The rate of growth and number of follicles in relation to the inclusion of LH was assessed, recognizing the combination of limited numbers of patients and hence a low power analysis. Under these circumstances, any effect of LH was not apparent. In group A, 64 MII eggs were recovered in total compared to 50 in group B. Table 1 demonstrates the incidence of fertilization, cleavage and pregnancy. There was a significant increase in the percentage of feritilization in group B (86.0%) compared with group A (60.9%), and a trend towards an increased percentage of cleavage and grade 1 score for embryo morphology in group B patients. Due to the increased percentage of fertilization, Table 1. Outcome results for the same patients using rfsh alone or in combination with rlh for follicular stimulation after downregulation Group A Group B P-value (rfsh alone) (rfsh + rlh) Cycles MII 64 (85.7% 50 (85.3% of of oocytes) oocytes) MII/cycle (mean ± SD) 3.76 ± ± PN Fertilization (%) a Embryos Cleavage (%) Grade I 15 (50.0%) 26 (66.7%) Grade II 13 (43.3%) 7 (17.9%) Grade III 2 (6.7%) 6 (15.4 %) Grade IV 0 0 Transfers No. embryo transfers Mean no. embryos transferred/patient bhcg positive a Clinical pregnancies b 1 (5.9%) 6 (50.0%) c FH b /embryo transferred (%) MII = metaphase II a χ 2 test. b FH = fetal heart. c Fisher s Exact test. 191

3 192 however, more embryos were available for patients in group B when compared with patients in group A. In group A, one clinical pregnancy was obtained compared with six in group B. Discussion FSH and LH are both required for normal follicular development in mammals; the precise contribution of LH in this process and its interaction with FSH is not totally clear (Chappel et al., 1991; Shoham et al., 1993; Andersen et al., 1999). Sullivan et al. (1999) found that once FSH has initiated follicular growth, either FSH or LH is capable of sustaining follicular oestradiol production and the maturing follicle may continue to develop in the presence of diminishing FSH concentrations by acquiring the capacity to respond to LH. In human physiology, the synergistic activity of FSH and LH is responsible for the selection of a dominant follicle, and increased oestrogen secretion as a consequence of increased aromatase activity induced in the granulosa cells of the maturing follicle causes a feedback inhibition of FSH secretion (Steinkampf et al., 1987; Zeleznik et al., 1987); this determines maturation arrest of less mature follicles, while the dominant follicle continues development in the presence of diminishing FSH concentration. In the period called FSH high responsiveness, FSH-induced LH receptors develop on the granulosa cells, marking the point at which mature follicles become responsive both to FSH and LH: this protects the mature follicle or follicles from diminishing FSH as they acquire responsiveness to LH. This hypothesis has been proposed as a prophylaxis for hyperstimulation syndrome, i.e. reducing FSH and augmenting LH administration in the last days of follicular stimulation to initiate follicle atresia of the less mature follicles (Chandrasekher et al., 1994; Sullivan et al., 1999). Therefore, human rlh may promote periovulatory maturation of a select cohort of follicles compared with a longacting HCG bolus, which may be particularly relevant in a patient with a history of high risk of ovarian hyperstimulation syndrome. For 60 years the synergistic role of FSH and LH in oestrogen production has promulgated the two-cell, two-gonadotrophin theory, involving theca and granulosa cells (Fevold, 1941). In this hypothesis, at the pre-antral and antral stages LHdependent theca cells convert cholesterol to androgens, and the FSH-dependent granulosa cells convert the androgens to oestrogens. However, once ovarian follicles exceed approximately 10 mm, granulosa cells acquire LH receptors (Zeleznik and Hillier, 1984) that permit adequate LH-derived stimulation of granulosa cells in the presence of declining FSH. Folliculogenesis can proceed independently of FSH activity as evidenced by an existing paracrine/autocrine mechanism involving LH-induced granulosa cell aromatization and inhibin production; inhibin enhancing LH-stimulated theca cell androgen synthesis (Hillier et al., 1994, Smyth et al., 1993). Thus, once a follicle exceeds 10 mm, the two-cell, twogonadotrophin theory might be redundant. However, the clinical relevance of folliculogenesis, such as oocyte maturation/quality and sustainable pregnancies, in the absence of FSH or LH remains to be evaluated (Sullivan et al., 1999; Lévy et al., 2000). Nevertheless, the dependence upon both FSH and LH during mid to late folliculogenesis is essential for normal/optimal physiological function, and the role of LH, especially in profoundly down-regulated normo-gonadotrophic and gonadotrophin-deficient women, is becoming increasingly apparent (Schoot et al., 1994; Balash et al., 1995; European Recombinant Human LH Study Group, 1998; Ganirelix Dose- Finding Study Group, 1998; Filicori, 1999; Filicori et al., 1999). The adequate circulating concentration of LH is unknown, although a serum concentration of 2.5 IU/l of LH has been described as sufficient to maintain adequate androgen substrate and maintain follicular aromatase activity (Sullivan et al., 1999). Reduced production of oestradiol and reduced pregnancy rate has been observed in LH-deficient women (Westergaard et al., 1996; Agrawal et al., 1998); which corresponds to the observed oestradiol production on the day of HCG administration in this study; group A having a lower oestradiol than group B. According to Andersen et al. (1999), HCG/LH activity is needed in follicular stimulation of immature mice, and its activity is very much dose dependent, with 5% of the FSH activity allowed for maximal results remaining constant while increasing the HCG/LH activity to 50% of the FSH activity. LH is believed to be essential for nuclear maturation in in-vitro maturing oocytes; Cortvrindt et al. (1998) reported that the maximum number of Met II oocytes were obtained after 13 days in-vitro growth when LH was added to the maturation medium. Weston et al. (1996) compared the developmental potential of cryostored macaque embryos produced by IVF following stimulation with rfsh alone versus 1:1 combination of rfsh + rlh; they found no difference in the ability of embryos to cleave and produce hatched blastocysts, but the embryos derived from the rfsh alone displayed reduced postthaw viability and slower developmental rates compared to the rfsh + rlh group. In the patients in this study the number of recovered Met II oocytes was similar in the two groups, but the quality of the oocytes might be reflected in the increased incidence of fertilization, grade 1 embryo morphology, pregnancy and implantation in group B compared to group A. Serum LH concentration after down-regulation often is below the critical point of 2.5 IU/l. Laml et al. (1999) reported a mean LH concentration of 1.58 IU/l (range ) in six patients who were down-regulated, indicating insufficient circulating LH for adequate folllicle/oocyte maturation. Hull et al. (1998) identified the need for circulating rfsh above an administered threshold of 150 IU/day to prevent reduction of non-pulsatile LH concentration. Two distinct components of LH secretion have been suggested, a GnRH-dependent phasic secretion and a GnRH-independent tonic basal secretion; Hull et al. (1998) proposed that only the GnRH-independent tonic is affected by levels of circulating FSH. Whether this is variable between women or an absolute biochemical threshold is unclear. However, we believe this observation is of paramount importance for our clinical practice as there is a group of women deficient in GnRH-dependent phasic LH as a consequence of down-regulation. The group of patients in this study differed from the group reported by Laml et al. (1999); in this study, administration of rlh was commenced on day 7 of rfsh stimulation. This rationale was based on the report of Yamoto et al. (1992), who indicated that LH receptors are present on the granulosa cells by the mid-follicular phase (day 7) and increase throughout the

4 late follicular phase. The precise size at which the follicle becomes responsive to LH is unknown but the granulosa cells acquire LH receptors and become responsive to LH around mm (Zeleznik and Hillier, 1984). Administration of rlh at day 7, if too early, will not be detrimental, simply ineffective. A recent study by Scott Sills et al. (1999) compared two groups of down-regulated patients, one group receiving FSH HP (high purity) alone and the other FSH HP + rlh. In this study they found no differences in mean length of stimulation, total number of oocytes retrieved, oestradiol concentrations, rate of fertilization or embryo morphology. A distinction between our study and the Scott Sills paper is that we re-examined the same patients who had previously required >3000 IU FSH to elicit an acceptable follicular response, and the mean FSH on cycle day 3 of a spontaneous cycle was 12.2 miu/ml. In the Scott Sills paper the group of patients administered rlh were distinct from those recruited for rfsh only, and their mean FSH values were 6 ± 2 and 7 ± 2 miu/ml respectively; indeed, any history of endocrine pathology or more than three prior gonadotrophin treatments for IVF were part of the exclusion criteria in the study by Scott Sills. However, the fact that a high number of pregnancies are regularly obtained after an apparent deep GnRH agonist down-regulation using rfsh indicates that many women do not need exogenous LH support; but, studies are needed to identify those women that might require LH (rlh, or low dose HCG) support to rfsh stimulation. Numerous larger clinical trials are now needed to confirm such benefits and to establish (i) a quantitative evaluation of the efficacy rlh administration, (ii) the types of patients that might profit from additional therapy, and (iii) the stage of the follicular phase at which rlh should be administered for maximum benefit; this is part of a clinical research programme at our Unit. References Agrawal R, West C, Conway GS et al Pregnancy after treatment with three recombinant gonadotrophins. Lancet 349, Agrawal R, Conway GS, Engmann L 1988 Implication of using follicle-stimulating hormone preparations depleted of luteinizing hormone to achieve follicular growth in in vitro fertilization. Gynecological Endocrinology 12, Andersen CY, Ziebe S, Xia Guoliang et al Requirement for human gonadotropin and recombinant human luteinizing hormone for follicular development and maturation. 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