Articles Use of recombinant LH in a group of unselected IVF patients

RBMOnline - Vol 5. No 2. 104 108 Reproductive BioMedicine Online; www.rbmonline.com/article/642 on web 6 June 2002 Articles Use of recombinant LH in a group of unselected IVF patients 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 1,3, L Rinaldi 1, S Fishel 1,2, R Lisi 1, GP Pepe 1, MG Picconeri 1, A Campbell 1,2 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 Correspondence: e-mail: flisi@tin.it Abstract The aim of this study was to evaluate the use of recombinant luteinizing hormone (rlh) supplementation in an unselected group of IVF patients undergoing follicular stimulation with recombinant follicle stimulating hormone (rfsh) after pituitary down-regulation. Group A comprised 122 cycles administered rfsh and rlh while group B included 331 cycles using rfsh only during the same period of treatment. There was no significant difference in any of the endocrine, embryological and outcome parameters measured. The implantation rate of 14.2% for group A compared with 9.8% for group B showed a positive trend (P = 0.055), but for patients in whom LH concentration was <1.0 IU/l at down-regulation or required excessive FSH stimulation, there was an increased incidence of implantation if rlh was supplemented. It was concluded that the addition of exogenous rlh to an unselected group of down-regulated patients stimulated with rfsh appears to offer little benefit, but in the event of profound LH down-regulation or requirement for excessive exogenous FSH (>2500 IU), the rate of implantation might be improved. Keywords: follicular stimulation, IVF, recombinant gonadotrophins, recombinant LH 104 Introduction Most women undergoing IVF are normo-ovulatory but are exposed to pituitary desensitization (down-regulation) and exogenous gonadotrophins for follicular stimulation to enhance the chances of a successful outcome following their IVF treatment. Women who had regular cycles might become poor responders to exogenous follicular stimulation; and some women with unexplained secondary infertility might produce poor quality eggs and embryos. Until the introduction of recombinant technology, first using rfsh and, more recently rlh, urinary products containing a variety of other substances were used. The regulation of folliculogenesis and ovulation in the natural cycle is controlled by an exquisite orchestration of hormonal events, involving various feedback mechanisms within endocrine and paracrine systems, and diurnal variation regulated via the hypothalamic hypophyseal ovarian access. The use of exogenous, even partially purified, urinary products provides little opportunity to study the effects of a particular gonadotrophin in isolation. The combined use of down-regulation by gonadotrophin-releasing hormone (GnRH) analogues and exogenous recombinant gonadotrophins has provided an opportunity to investigate the requirement for FSH and LH, alone or combined, during follicular stimulation. The biochemistry, pharmacokinetics and pharmacodynamics of recombinant gonadotrophins have been thoroughly discussed elsewhere (Ulloa-Aguirre and Timossi, 2000; Hugues et al., 2001; Rísquez, 2001; Trinchard-Lugan et al., 2002). An increasing number of studies demonstrate that both FSH and LH from mid-phase folliculogenesis are essential for optimal physiological function; furthermore, especially in patients who are profoundly down-regulated, LH appears to be essential (Schoot et al., 1994; Balasch et al., 1995; De Placido et al., 2001; European Recombinant Human LH Study Group, 1998; Ganirelix Dose-Finding Study Group, 1998; Fleming et al., 1998; Filicori, 1999; Filicori et al., 1999; Westergaard et al., 2000), although this has been disputed (Balasch et al., 2001). The use of rfsh and rlh was examined in an unselected group of patients undergoing IVF, and the findings are presented below.

Materials and methods Patients All patients began treatment during a set period ( treatment run ) in which patients were allocated rfsh only or rfsh and rlh on the basis of randomization by allocation of treatment to every third patient. This was deemed an acceptable approach, as there is no cyclical pattern amongst the subjects, and this provided the required spread of patients over an extensive period to minimize other potential effects, such as in the laboratory. Exclusion criteria were a body mass index <18 or >35, an abnormal karyotype or any known endocrinopathy/illness. Patients were included if there was evidence of tubal damage and endometriosis, but their frequency was not significantly different between all groups studied. Patients had no evidence of abnormal menstrual cycles and evaluation of basal hormone concentrations occurred on day 3 of the spontaneous cycle. The down-regulation and follicular stimulation regimen has been published previously (Lisi et al., 2001). Briefly, downregulation was induced with triptoreline 0.1 mg (Ipsen, France) subcutaneously from the mid-luteal phase of the previous cycle (day 21) for 3 weeks before starting gonadotrophin stimulation. All patients were commenced on a daily dose of 150 IU rfsh; those in group A were supplemented daily with 75 IU rlh (Luveris; Serono, Geneva, Switzerland) from day 7 of stimulation. As Luveris was not available for purchase for the duration of this study, Serono kindly provided it to the patients. Table 1. Basal and peripheral levels of gonadotrophins and ovarian steroids for group A and group B. Group A (n = 122) (rfsh and rlh) Group B (n = 331)(rFSH) Basal FSH (miu/ml) 9.1 ± 0.65 7.7 ± 0.47 Basal LH (miu) 4.5 ± 0.43 4.5 ± 0.39 Basal oestradiol (pmol/l) 233.1 ± 13.6 237.9 ± 12.8 Basal prolactin (nmol/l) 322.2 ± 17.9 343.4 ± 21.3 Down-regulated LH (miu) 2.2 ± 0.27 2.6 ± 0.20 Down-regulated FSH (miu/ml) 5.5 ± 0.41 4.6 ± 0.38 Down-regulated oestradiol (pmol/l) 109.8 ± 9.3 122.2 ± 12.4 Maternal age (years) 34.8 ± 0.48 34.7 ± 0.28 Table 2. Outcome data following treatment of group A and group B patients. Group A (n = 122) Group B (n = 331) (rlh and rfsh) (rfsh) No. of days of stimulation 12.57 ± 0.15 12.77 ± 0.27 Total FSH (IU) 2998 ± 112 3201 ± 198 Terminal oestradiol (pmol/l) 1221 ± 58 1072 ± 36 Mean no. oocytes per case 7.0 ± 0.32 6.97 ± 0.20 Proportion of M2 oocytes 0.840 ± 0.016 0.810 ± 0.012 Oestradiol:M2 ratio 855.0 ± 56.5 827.4 ± 43.7 Proportion of M2 oocytes fertilized 0.607 ± 0.023 0.591 ± 0.014 Proportion of 2PN zygotes 0.597 ± 0.022 0.571 ± 0.013 Proportion of grade 1 embryos 0.505 ± 0.31 0.480 ± 0.021 Proportion of grade 1 + 2 embryos 0.903 ± 0.019 0.882 ± 0.013 Proportion cleaved (2PN) 0.836 ± 0.016 0.853 ± 0.024 Mean no. embryos transferred 2.71 ± 0.087 2.5 ± 0.057 Implantation rate (%) 41/288 (14.2) 74/753 (9.8) a Biochemical pregnancy (%) 36/122 (29.5) 83/331 (25.1) Deliveries (%) 30/122 (24.6) 62/331 (18.7) M2 = metaphase II; PN = pronuclear. a P = 0.055. 105

The rational for commencing rlh on day 7 is based on the twocell, two-gonadotrophin model involving a transition from FSH-mediated acquisition of granulosa cell LH receptors at a follicular size around 10 mm (Zeleznik and Hillier, 1984) to LH stimulation of folliculogenesis independent of FSH activity after this stage (Hillier et al., 1994; European Recombinant Human LH Study Group, 1998; Filicori, 1999). This is a particularly significant feature in the spontaneous cycle in which rising concentrations of serum LH and falling concentrations of FSH occur at the time of dominant follicle selection, as demonstrated in sheep (Campbell, 1999). Patients in group B were maintained on rfsh only. If additional stimulation, or a reduction in stimulation, was required, this was achieved by modulating the amount of rfsh only; for group A patients, rlh was maintained at a constant dose. Ovulation induction was achieved by administration of human chorionic gonadotrophin (HCG) injection (Profasi 10,000 IU; Serono, Geneva, Switzerland). Follicle growth and endometrial thickness were tracked by ultrasound scanning from day 7 of stimulation to HCG administration. Oocytes were recovered 36 h after HCG administration. Patients underwent either IVF or intracytoplasmic sperm injection (ICSI), but there was no difference in the data between these insemination procedures (data not presented). For luteal support, all patients received 50 mg i.m. progesterone (Gestone; Amsa srl., Rome, Italy) daily. Embryology Oocyte retrieval and IVF embryo transfer were carried out in accordance with usual clinical practice, as published previously (Fishel et al., 1995). Table 3. Growth of follicles for groups A and B and the pregnant versus non-pregnant patients. Regression coefficient of follicle growth between days 7 15 (± SEM) Group A (rlh and FSH) 1.119 ± 0.045 Group B (rfsh) 1.172 ± 0.043 Non-pregnant 1.145 ± 0.034 Biochemical pregnancies 1.130 ± 0.047 Clinical pregnancies 1.138 ± 0.055 Statistics A variety of statistical techniques were used to analyse the data. Continuous random variables such as hormone concentrations were analysed by means of conventional analysis of variance, which also permitted adjustments for confounding variables. Data consisting of proportions were analysed by means of logistic regression methods, where the dependent variable was the logistic transform of the proportion of interest, which was then regressed against potential explanatory factors. Typically, the proportion might be that of mature eggs, and the type of stimulation was a potential explanatory variable. The findings were confirmed by Fisher s exact test. The growth of follicles was summarized by average values of the linear regression coefficients, which had been calculated for each patient. Results There was no significant variation in basal or down-regulated concentrations of FSH, LH or oestradiol, basal prolactin, or patient age (Table 1). Analysis of the cause of infertility, previous IVF history, endometrial thickness during the monitoring phase and number of days of ovarian suppression were not significantly different between the groups (data not shown). Other parameters such as the total amount of rfsh administered, the number of days of stimulation, the terminal oestradiol concentration, the mean number of oocytes per case, the proportion of metaphase II (M2) oocytes per patient or the oestradiol:m2 ratio, the rate of fertilization of M2 oocytes, the morphology and cleavage of embryos, and the overall biochemical pregnancy miscarriage and delivery rates, were not significantly different (Table 2). There was no difference within the groups when comparing IVF or ICSI as the insemination procedure and the effect of rlh. In group A, 14.2% of embryos implanted compared with 9.8% for group B, perhaps indicating a trend (P = 0.055; Table 2). Analysis of follicular development showed that the addition of rlh did not affect the number, size and rate of follicular growth during the 9 days of monitoring (Table 3). The regression coefficients displayed in Table 3, which quantify the growth per day of the follicles between days 7 and 15, were remarkably similar. Thus there was no evidence of a difference Table 4. Implantation rates and oestradiol:m2 ratio for patients in groups A and B when down-regulation LH <1.0 miu/ml, or basal FSH >10 miu/ml or rfsh administration >2500 IU. Down-reg LH <1.0 miu/ml Basal FSH >10 miu/ml FSH >2500 IU Group A Group B Group A Group B Group A Group B Oestradiol:M2 ratio 1121.9 ± 78.6 918.5 ± 85.2 990.8 ± 30.6 626.6 ± 41.4 792.9 ± 17.6 897.2 ± 19.3 Implantation rate (%) 4/26 (15.4) 0/38 a (0) 2/21 (9.5) 0/19 (0) 5/7 (71.4) 1/14 b (7.1) 106 M2 = metaphase II oocytes a P < 0.05; b P < 0.001.

in the growth rate between the treatment groups A and B, or between groups determined by pregnancy status. Oestradiol:M2 oocyte ratios and implantation rates were also examined for the subset of patients with LH concentrations <1.0 miu/ml (n = 22) at down-regulation, those patients whose basal FSH was >10 miu/ml (n = 25), and a few patients requiring >2500 IU rfsh (n = 8). In this sub-set of patients there was no difference in age or FSH concentrations as measured on day 3 of a spontaneous cycle (for the <1.0 IU and >2500 IU rfsh groups). There was a significant increase in the implantation rate of group A patients when downregulation LH was <1.0 IU/l. The four patients successfully implanted all delivered uneventfully. There was also a significant rate of implantation in three group A patients (n = 7 embryos) who had >2500 IU exogenous FSH (P < 0.001). These resulted in a set of twins and a singleton (Table 4). Discussion Previously, the use of rlh had been examined in conjunction with rfsh for follicular stimulation after down-regulation in patients requiring excessive rfsh (>2500 IU) (Lisi et al., 2001, 2002). In these patients, the addition of rlh appeared to improve embryo quality, implantation and delivery rates. In this study, an unselected group of patients was examined. A possible increase in the implantation rate was observed in all patients given rlh, but there was a significant effect of the use of rlh on implantation in patients with peripheral LH concentrations <1.0 IU/l at down-regulation. The data presented here support previous suggestions that the general use of purified or recombinant FSH preparations without LH supplementation does not lead to a drop in overall success rates (Hull et al., 1994; Daya, 1998; FIVNAT, 1999; Fleming et al., 2000). However, it has been postulated that GnRH agonist down-regulation might result in profound suppression of peripheral LH, impairing adequate oestradiol synthesis (Westergaard et al., 1996; Fleming et al., 1998, 2000; Janssens et al., 2000), resulting in decreased fertilization rates and increasing the risk of early pregnancy loss (Westergaard et al., 1996, 2000). This has been disputed in a recent study by Balasch et al. (2001). These authors used receiver-operating characteristics (ROC) to examine serum LH on day 7 of stimulation as a prognosticator of IVF outcome after downregulation and stimulation with rfsh. Balasch et al. (2001) found no difference in the median and range of LH values for conception and non-conception cycles, pregnancy and miscarriage; they concluded that there was no requirement for exogenous LH supplementation. Part of the difference between the conclusions of these authors and those of previous studies can be attributed to the type of GnRH agonist and its degree of hydrophobicity, which correlates to the degree of pituitary desensitization (Conn and Crowley, 1991; Miró et al., 1992). Balasch et al. (2001) used leuprolide acetate compared with buserelin, as used by Fleming et al. (2000) and Westergaard et al. (2000). Balasch et al. (2001) also suggest that their ROC curve analysis approach reduces inaccuracies in overall quantification of outcome data that might be affected by the prevalence of a particular condition. It is debatable whether this approach is of more value, but it should be considered in future prospective analyses. In agreement with the current investigation, none of the above studies found any difference in ovarian response and endocrine parameters, indicating no overt signal for an improvement in outcome when rlh is utilized. However, it is important to note that immunoreactive LH and bioactive LH may vary (Schroor et al. 1999) and might also be a cause of variable results. Chappel and Howles (1991) postulated that <1% of follicular LH receptors need to be occupied to permit normal steroidogenesis; hence, if biologically active threshold values of <1 IU/l serum LH in the mid-follicular phase (day 7 8) are indicative of profound suppression of LH, the effect measured in this and other studies might be significant. The recent study by Balasch et al. (2001) did not assess biologically active LH in their samples, as indeed they were apparently not examined in the studies above. This is perhaps an area of consideration for future studies, although the quantitative distinction between immunoreactive and biologically active LH, and patient variation, remains to be elucidated. Clinical situations arise in which LH is naturally inactive or absent, such as Kallmann s syndrome. In these patients, treatment with purified rfsh results in multiple follicular development but deficient ovarian steroidogenesis; however, there is disagreement regarding whether the presence or absence of LH induces fewer (Shoham et al., 1993) preovulatory follicles, or makes no difference (Balasch et al., 1995). It is clear that there is a range of heterogeneity in the case of hypogonadotrophic hypogonadism syndromes, hence the various published discrepancies when measuring serum and follicle oestradiol concentrations, inhibin concentrations, endometrial thickness, reoccurrence of ovulation, rate of fertilization and embryo cryo-survival rates (Shoham et al., 1993; Schoot et al., 1994; Balasch et al., 1995; Kousta et al., 1996). Careful inclusion criteria are essential for all prospective clinical studies. However, in previous and more recent studies in a specific group of patients who previously required excessive rfsh to achieve follicular stimulation (Lisi et al., 2001, 2002), the improved outcome results indicated a subset of patients that might benefit from LH inclusion. This was confirmed in this study in three patients requiring >2500 IU FSH to complete stimulation, in whom five out of seven embryos implanted if rlh was administered, compared with those in group B, who required >2500 IU FSH. Therefore, it is important to continue investigations to establish whether there are groups of patients who, for hitherto unknown reasons, would benefit from supplementation of LH during follicular stimulation. In conclusion, this study of 454 cycles indicates that the addition of rlh to an unselected group undergoing downregulation and follicular stimulation with rfsh offers little benefit in terms of all the parameters measured, except for a possible improvement in the rate of embryo implantation. However, if the patient is profoundly down-regulated in terms of LH concentrations <1.0 IU/l, or if excessive amounts of FSH (>2500 IU) have been required to complete stimulation, supplementation with LH appears to be beneficial. Future studies will need to elucidate if there is a requirement for sequential use of rlh and rfsh that might provide a more physiological and safer option for follicular stimulation in a specific subset of patients. 107

108 Acknowledgements We gratefully acknowledge the statistical support of Dr Eurof Walters, Cambridge, UK. References Balasch J, Miro F, Burzaco I et al. 1995 The role of luteinising hormone in human follicle development and oocyte fertility: evidence from in-vitro fertilisation in a woman with longstanding hypogonadotrophic hypogonadism and using recombinant human follicle stimulating hormone. Human Reproduction 10, 1678 1683. Balasch J, Vidal E, Peñarrubia J et al. 2001 Suppression of LH during ovarian stimulation: analysing threshold values and effects on ovarian response and the outcome of assisted reproduction in down-regulated women stimulated with recombinant FSH. Human Reproduction 16, 1636 1643. Campbell BK 1999 The modulation of gonadotrophic hormone action on the ovary by paracrine and orthocrine factors. Anatomia Histologia Embryologia 28, 247 251. Chappel S, Howles C 1991 Re-evaluation of the roles of luteinizing hormone and follicle-stimulating hormone in the ovulatory process. Human Reproduction 6, 1206 1212. Conn PM, Crowley,WF 1991 Gonadotropin-releasing hormone and its analogues. New England Journal of Medicine 324, 93 103. Daya S 1998 hmg versus FSH: is there any difference? In: M Filicori, C Flaminigi (eds) Ovulation Induction: Update. 1998. Parthenon, New York, pp. 183 192. De Placido G, Mollo A, Alviggi C et al. 2001 Rescue of IVF cycles by hmg in pituitary down-regulated normogonadotrophic young women characterised by a poor initial response to recombinant FSH. Human Reproduction 16, 1875 1879. European Recombinant Human LH Study Group 1998 Recombinant human luteinizing hormone (LH) to support recombinant human follicle-stimulating hormone (FSH) induced follicular development in LH- and FSH- deficient anovulatory women: a dose finding study. Journal of Clinical Endocrinology and Metabolism 83 N.5, 1507 1514. Filicori M 1999 The role of luteinizing hormone in folliculogenesis and ovulation induction. Fertility and Sterility 71, 405 414. Filicori M, Cognigini GE, Taraborrelli S et al. 1999 Luteinizing hormone activity supplementation enhances follicle-stimulating hormone efficacy and improves ovulation induction outcome. Journal of Clinical Endocrinology and Metabolism 84, 2659 2663. Fishel SB, Lisi F, Rinaldi L et al. 1995 Systematic examination of immobilizing spermatozoa before intracytoplasmic sperm injection in the human. Human Reproduction 10, 497 500. FIVNAT 1999 Dossier FIVNAT 99 (Bilan de l année 98). Fleming R, Lloyd F, Herbert M et al. 1998 Effects of profound suppression of luteinizing hormone during ovarian stimulation on follicular activity, oocyte and embryo function in cycles stimulated with purified follicle stimulating hormone. Human Reproduction 13, 1788 1792. Fleming R, Rehka P, Deshpand EN et al. 2000 Suppression of LH during ovarian stimulation: effects differing cycle stimulated purified urinary FSH and recombinant FSH. Human Reproduction 15, 1440 1445. Ganirelix Dose-Finding Study Group 1998 A double-blind, randomised, dose-finding study to assess the efficacy of the gonadotrophin-releasing hormone antagonist Ganirelix (Org 37462) to prevent premature luteinizing hormone surges in women undergoing ovarian stimulation with recombinant follicle stimulating hormone (Puregon). Human Reproduction 13, 3023 3031. Hiller SG, Whitelaw PF, Smyth CD 1994 Follicular oestrogen synthesis; the two-cell, two-gonadotrophin model revisited. Molecular and Cellular Endocrinology 100, 51 54. Hugues J-N, Bstandig B, Bry-Gauillard H et al. 2001 Comparison of the effectiveness of recombinant and urinary FSH preparations in the achievement of follicular selection in chronic anovulation. Reproductive Biomedicine Online 3, 195 198. Hull ML, Corrigan E, Piazzi A et al. 1994 Recombinant human luteinizing hormone: an effective new gonadotropin preparation. Lancet 344, 334 335. Janssens RMJ, Lambalk CB, Wermiden JPW et al. 2000 Dosefinding study of triptorelin acetate for prevention of a premature LH surge in IVF: a prospective, randomised, double-blind placebo-controlled study. Human Reproduction 15, 2333 2340. Kousta AE, White DM, Biazzi A et al. 1996 Successful induction of ovulation and completed pregnancy using recombinant luteinizing hormone and follicle stimulating hormone in a woman with Kallman s syndrome. Human Reproduction 11, 70 71. Lisi F, Rinaldi L, Fishel S et al. 2001 Use of recombinant FSH and recombinant LH in multiple follicular stimulation for IVF: a preliminary study. Reproductive BioMedicine Online 3, 190 194. Lisi F, Rinaldi L, Fishel S et al. 2002 Use of recombinant FSH (Gonal F) and recombinant LH (Luveris) for multiple follicular stimulation in patients with suboptimal response for in vitro fertilisation. Fertility and Sterility (in press). Miró F, Sampai O, Tarìn J 1992 Steroidogenesis in vitro of human granulosa-luteal cells pretreated in vivo with two gonadotropin releasing hormone analogues employing different protocols. Gynecological Endocrinology 6, 77 84. Rísquez F 2001 Induction of follicular growth and ovulation with urinary and recombinant gonadotrophins. Reproductive BioMedicine Online 3, 54 72. Schoot DC, Harlin J, Shoham Z et al. 1994 Recombinant folliclestimulating hormone and ovarian response in gonadotropindeficient women. Human Reproduction 9, 1237 1242. Schroor E, Van Weissenbruch M, Engelbert M et al. 1999 Bioactivity of luteinising hormone during normal puberty in girls and boys. Hormone Research 51, 230 237. Shoham Z, Jacobs HS, Insler V 1993 Luteinizing hormone: its role, mechanism of action, and detrimental effect when hypersecreted during the follicular phase. Fertility and Sterility 5, 1153 1161. Trinchard-Lugan I, Khan A, Porchet HC, Munafo A 2002 Pharmacokinetics and pharmakodynamics of recombinant human chorionic gonadotrophin in healthy male and female volunteers. Reproductive BioMedicine Online 4, 106 115. Ulloa-Aguirre A, Timossi C 2000 Biochemical and functional aspects of gonadotrophin-releasing hormone and gonadotrophins. Reproductive BioMedicine Online 1, 48 62. Westergaard LG, Erb K, Laurse S et al. 1996 The effect of human menopausal gonadotropin and highly purified, urine-derived follicle stimulating hormone on the outcome of in-vitro fertilisation in down regulated normogonadotropic women. Human Reproduction 11, 1209 1213. Westergaard LG, Laurse SB, Anderson CY 2000 Increased risk of early pregnancy loss by profound suppression on luteinizing hormone during ovarian stimulation in normogonadotrophic women undergoing assisted reproduction. Human Reproduction 15, 1003 1008. Zeleznik AJ, Hillier SG 1984 The role of gonadotropins in the selection of the preovulatory follicle. Clinical Obstetrics and Gynaecology 27, 927 940.