Outlook Tailoring FSH and LH administration to individual patients

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1 RBMOnline - Vol 11. No Reproductive BioMedicine Online; on web 20 July 2005 Outlook Tailoring FSH and LH administration to individual patients Since 1992, Dr Borini has been Clinical Director of the Assisted Reproduction Centre Tecnobios Procreazione, Bologna, Italy, and of a number of associated IVF clinics. After graduating in Medicine and Surgery at the University of Bologna, Bologna, Italy, in 1986, he completed his residency in Obstetrics and Gynaecology at the University of Bologna in He then attended the University of California Irvine, Irvine, California, as Research Fellow from October 1989 to May His research areas are embryo implantation, oocyte freezing and multiple induction of ovulation. Dr Borini has published more than 160 research papers and acts as Chairman of CECOS ITALY. Dr Andrea Borini Andrea Borini 1, Luca Dal Prato Tecnobios Procreazione, Centre for Reproductive Health, Bologna, Italy 1 Correspondence: Tecnobios Procreazione, Via Dante 15, I Bologna, Italy. borini@tecnobiosprocreazione.it Abstract Many clinical trials have been carried out to find the optimal gonadotrophin starting dose for IVF. The consensus for patients undergoing first treatment and <40 years old is a range stretching from 150 to 250 IU/day. The varying ovarian response to gonadotrophins may be due to factors such as age, basal FSH, number of antral follicles and body mass index, all of which should be taken into account before choosing the type of protocol and the amount of gonadotrophins to use. Increasing the dose of recombinant FSH does not compensate for the age-related decline in retrievable oocytes. Higher doses of gonadotrophins are required in overweight patients, but enhanced protocols are thought to only marginally improve live birth rates in obese women. The actual role of LH in controlled ovarian stimulation is still a matter of debate. A therapeutic window of LH concentrations, below which oestradiol production is inadequate and above which LH may be detrimental to follicular development has been described. Keywords: body mass index, FSH, individual dose, LH, ovarian response, ovarian stimulation Introduction Optimal ovarian response to stimulation is crucial for the efficiency of assisted reproduction procedures. When starting ovarian stimulation, however, it is sometimes very difficult to predict the ovarian response to gonadotrophin stimulation. Both ovarian hyperstimulation syndrome (OHSS) and poor response are undesired conditions. The aim of ovarian stimulation is to strike a balance between giving a dose high enough to allow the growth of 8 12 follicles while at the same time minimizing the risk of ovarian hyperstimulation syndrome. The different ovarian responses to gonadotrophins may be due not only to different follicle sensitivities to FSH and FSH pharmacodynamics, but also to other factors that can be predicted and evaluated before choosing the right kind of protocol for ovulation induction and gonadotrophin dosage. It has been recognized that clinical factors such as age, body weight and FSH basal concentrations may influence ovarian response (Cahill et al., 1994; Popovic-Todorovic et al., 2003b). Furthermore, women with polycystic ovarian syndrome (PCOS) have proven to be more sensitive to gonadotrophin stimulation with a tendency to respond excessively to stimulation and increased OHSS risk (MacDougall et al., 1993); such women therefore benefit from a reduced starting dose. Patients considered standard because they are aged below 40 years, with a regular menstrual cycle and normal basal FSH concentrations, may also present marked variability in ovarian response to treatment when a standardized dose is administered. There is considerable interest today with the advent of new recombinant gonadotrophins in trying to identify a standard fixed dose to use for multiple induction of ovulation. The fact that the new recombinant formulations are more bioactive than the traditional urinary gonadotrophins (Out et al., 1995; Bergh et al., 1997) and have greater batch to batch consistency, with less variable FSH bioactivity (Hugues et al., 2003; Bassett and Driebergen, 2005), means it is easier to try and tailor the dose 283

2 for individualized treatments. A limitation of these formulations remains their high cost. The cost effectiveness of urinary and recombinant gonadotrophins is still a matter of debate (Gerli et al., 2004). The present paper is an up-to-date review of the published studies that tried to individualize doses and kind of gonadotrophins in ovarian stimulation for IVF or ICSI. Standard patients Many clinical trials have been performed with a view to discovering the optimal gonadotrophin starting dose for assisted reproduction procedure, particularly following the introduction of recombinant FSH preparations (rfsh). The starting dose in patients undergoing their first treatment and younger than 40 years typically ranges from 100 to 250 IU/day, but there is no universal agreement on the most advantageous initial FSH dose. Furthermore, the commercial availability of two products based on 50 IU (follitropin β) and 75 IU (follitropin α) vials makes the choice of dose even more complicated. The interpretations of the results of the studies that compare different starting doses of gonadotrophins is not easy, because these medications may be used in the fixed, step-down or stepup fashion during COH. This choice may induce a variation in the starting dose of FSH and influence the total dose and the results of the treatment. Fortunately there are several randomized studies in the literature that reported on the use of a fixed protocol. Studies with agonist A series of studies compared the effect of either a 100 or 200 IU daily fixed-dose regimen of rfsh in IVF (Out et al., 1999) or ICSI (Out et al., 2001) (Table 1). Compared with 200 IU, the use of a 100 IU fixed dose proved less efficacious in terms of the number of oocytes retrieved and cycle cancellations due to low response, but more efficient in terms of the lower total dose of gonadotrophins used. The clinical pregnancy rate per started cycle was similar for both doses. The advantage of using 200 IU to treat these women was that more embryos could be frozen. This might result in superior pregnancy rates if the results of the frozen thawed embryo replacements are included, as previously described (Out et al., 1995; Jones et al., 1997). However, the higher incidence of side-effects, and especially the greater number of OHSS cases requiring hospitalization, means caution needs to be exercised in the choice of the higher dose. Increasing daily doses of rfsh from 150 to 250 IU in women between 30 and 39 years of age (Latin-American Puregon IVF Study Group, 2001) has proved to be of limited usefulness in increasing ovarian response. Only in the 37- to 39-year-old age group could an average of two additional oocytes be retrieved in the patients treated with 250 IU, while pregnancy rates were not influenced by gonadotrophin doses, as suggested also by similar studies comparing different rfsh doses (Out et al., 1999; Harrison et al., 2001; Out et al., 2001). A recent prospective randomized clinical trial (Yong et al., 2003) comparing 150 and 225 IU of rfsh (Gonal-F; Serono, Geneva, Switzerland) in a fixed-dose regimen for ovarian stimulation in IVF found a significant dose-response relationship only in women aged below 33 years (Tables 1 and 2). In younger women, 225 IU is more effective than 150 IU in terms of oocyte yield and reduced cycle cancellation, but requires a higher total dose of rfsh and increases OHSS risk. Studies with antagonist A recent randomized, double-blind, multicentre clinical trial compared starting doses of 150 and 200 IU of rfsh (follitropin β) in women undergoing treatment with GnRH antagonist ganirelix (Out et al., 2004). By increasing the starting dose from 150 to 200 IU of rfsh, slightly more oocytes were retrieved, but gonadotrophin consumption rose (nearly 500 IU) (Table 1). Table 1. Effect of different gonadotrophin starting doses used in assisted reproduction treatment in standard patients. Results are mean (or % when indicated). PR = pregnancy rate. Study No. of Analogue FSH (IU) FSH total Cancellation Retrieved PR/transfer patients regimen dose (IU) rate (%) eggs (%) 284 Out et al., Agonist 100 versus 1114 versus 24 versus versus 36.1 versus versus fixed 1931, P < , P < , P = NS Out et al., Agonist 100 versus 1121 versus 19 versus versus 12.0, 27.6 versus versus fixed 1875, P < P < , P = NS Latin-American 201 Agonist 150 versus 1589 versus 9 versus versus 10.2, 27.8 versus Puregon IVF Study versus fixed 2492, P < P = , P = NS Group, 2001 Yong et al., Agonist 150 versus 1897 versus 3.3 versus 6.3 versus 8.3, 15 versus 19 a, versus fixed 2595, 15.0 P < 0.05 P < 0.05 P = NS P < Out et al., Antagonist 150 versus 1541 versus 3.1 versus versus 11.9, 34 versus 27 a, versus fixed 2014, P < 0.05 P = P = NS a Clinical PR/started cycle; NS = not statistically significant.

3 Moreover, the fact that the number of good-quality embryos was the same in the low- and high-dose groups, with a similar pregnancy rate, indicates that the use of a higher daily dose is clinically of limited importance. Dose individualization based on predictive factors for ovarian response Studies have been performed to help identify predictive factors for ovarian response to gonadotrophin stimulation that might help clinicians tailor drug doses, including day 3 serum FSH (Toner et al., 1991; Cahill et al., 1994), day 3 serum inhibin B (Seifer et al., 1997), ovarian volume (Lass et al., 1997; Syrop et al., 1999), antral follicle count in early follicular phase (Chang et al., 1998; Ng et al., 2000), ovarian stromal blood flow velocity (Engmann et al., 1999), smoking habits (El-Nemr et al., 1998), day 3 serum anti-müllerian hormone (AMH) (Hazout et al., 2004). Some authors, using multiple regression analysis, described different ovarian responses according to the number of antral follicles on day 2 5 of the menstrual cycle, patient age, ovarian volume, total Doppler score on day 2 5 and smoking habits (Popovic-Todorovic et al., 2003b). Recently, a normogram based on the above parameters was proposed (Popovic-Todorovic et al., 2003a) as a clinical tool for setting the optimal rfsh dose to generate the right number of oocytes, said to be between 5 and 14. The study compared individual rfsh doses of IU/day (calculated using the rfsh dose normogram) with a standard dose of 150 IU/day. An appropriate response was found in 77.1% of patients in the study group, compared with 65.6% in the control group (P < 0.05). The percentage of patients in whom fewer than five oocytes were retrieved was 1.5% in the study group versus 10.7% in the control group (P < 0.05), while no significant difference was found between the groups in patients where more than 14 oocytes were retrieved (20.6 and 19.8%). A total of 86% of the individual dose patients did not require any dose adjustment on day 8, compared with 45% of the standard dose patients (P < 0.01). A higher ongoing pregnancy rate per initiated cycle was observed in the individual dose group (36.6 versus 24.4%, P < 0.01). One patient (0.8%) in the study group and four patients (3.1%) in the control group were hospitalised for OHSS. An individualized dose regimen in a standard population may therefore increase the number of patients with appropriate ovarian response and reduce the need for dose adjustment during ovarian stimulation. Age and basal FSH concentrations There is general agreement in clinical practice that the poorer ovarian response after stimulation due to ageing and/or high basal FSH concentrations might at least in part be overcome by increasing the dosage of gonadotrophin (Ubaldi et al., 2005). Table 2. Effect of different gonadotrophin starting doses used in assisted reproduction treatment in correlation with age or basal FSH concentrations. Results are mean (or % when indicated). PR = pregnancy rate. Population No. of FSH (IU) FSH total Cancellation Retrieved Pregnancy patients regimen dose (IU) rate (%) eggs rate Out et al., 2000 Age <33 16 versus 150 versus 1584 versus 10.6 versus 13.5 versus fixed d Age versus 1661 versus 9.4 versus 20.9 versus d Age versus 1888 versus 8.1 versus 12.4 versus d Latin-American Puregon Age <33 66 versus 150 versus 1521 versus 11.6 versus 26.8 versus IVF Study Group, fixed 2398 a e Age versus 1558 versus 9.2 versus 12.0 versus a e Age versus 1695 versus 6.9 versus 13.5 versus a e Yong et al., 2003 Age > versus 7.0 versus 225 fixed 11.8 b Age versus 6.4 Age versus 6.6 Harrison et al., 2001 FSH <8.5 IU 146 versus 150 versus 1500 versus 14 versus versus versus step up 1800 c 27 d FSH >8.5 IU 24 versus 300 versus 3600 versus 16 versus 29 9 versus 9 12 versus step up d a, b, c Comparisons with these superscripts are significantly different: a P < 0.01; b P < 0.05; c P < d Clinical PR/started cycle. e Clinical PR/transfer. 285

4 286 The practice of administering higher rfsh doses according to age is very common, though there are no data in this regard in the literature. The cut-off value for age is usually 35 years with patients <35 years receiving 150 IU/day while those >35 years are started on higher rfsh doses (usually IU/day). A study in 344 women undergoing their first IVF cycle (Sharif et al., 1998) found that increasing basal FSH concentrations and age were closely related not only to increased total urinary gonadotrophin dose, but also to fewer collected oocytes and lower pregnancy rate. A prospective, randomized, double-blind, multicentre study (Out et al., 2000) compared the effect of a 150 (67 patients) and 250 (71 patients) IU daily fixed-dose regimen of rfsh (Puregon; Organon, Oss, The Netherlands) on the number of oocytes retrieved and on the total dose used in down-regulated women between 30 and 39 years of age undergoing ovarian stimulation (Table 2). Considering the total population, the number of oocytes retrieved was similar in both dose groups. Patients in the year age bracket receiving the 250 IU dose produced on average 4.2 additional oocytes (14.8 versus 10.6), while in the year age bracket almost one additional oocyte was retrieved in patients receiving the 150 IU dose (8.1 versus 7.4). The total dose used was significantly lower in women treated with 150 IU daily (1727 versus 2701 IU, P < 0.001). No significant relationship was found between serum FSH concentrations in early follicular phase and the number of oocytes collected, or the total dose. Similar results were reported in the study by Yong comparing 150 IU and 225 IU follitropin α (Yong et al., 2003) in 124 women aged years. In older women (over 33 years), the number of oocytes retrieved in the two groups was similar. Total rfsh dose was higher, while cancellation rate due to insufficient ovarian response was lower in the 225-IU group. No significant differences were found between the two groups as regards fertilization rate, number of embryos formed and cryopreserved, and pregnancy rates (Table 2). Another study (Harrison et al., 2001) evaluated the efficacy of different starting doses of rfsh in 345 couples undergoing their first IVF or ICSI cycle and divided them into two groups according to basal FSH concentrations (Table 2). Patients with day 3 FSH concentrations below 8.5 IU/l began treatment with 150 IU (n = 146) or 200 IU (n = 151) rfsh. Total dose of the drugs used was significantly lower in 150 IU group, as was the number of ICSI metaphase II oocytes (62 versus 82%, P < 0.004). No other significant differences were found. The dosage was increased in 9% of the women on day 5. Patients with day 3 FSH concentrations >8.5 IU/l were treated with 300 IU (n = 24) or 400 IU (n = 24) rfsh. No significant outcome differences were found between the two subgroups. Pregnancy rates for patients with high basal FSH concentrations were half those of patients with low basal FSH concentrations. From these studies, it can be concluded that increasing the daily dose of recombinant FSH does not compensate for the agerelated decline in retrievable oocytes after ovarian stimulation. In older patients, as well as in women with high basal FSH concentratons, this practice leads to neither higher oocyte yield nor increased pregnancy rates. Antral follicle count Several studies have shown that the antral follicle count (AFC) is a better predictor of poor response than age and basal FSH (Hendriks et al., 2005). A recent randomized controlled trial (Klinkert et al., 2005) evaluated the effect of doubling the starting dose of gonadotrophins [150, (group I) or 300 IU (group II) of rfsh] on the ovarian response in 52 IVF patients with a low antral follicle count, but concluded that expected poor response patients, defined as patients with an AFC <5, are not likely to benefit from a higher starting dose of gonadotrophins in IVF. In fact, the groups were comparable regarding patient characteristics and outcome of the treatment. The median number of oocytes collected was three for both groups. The difference in the mean number of oocytes was 0.3 oocytes in favour of group I. Sixtyfive per cent of patients in group I experienced a poor response, and 62% in group II. The ongoing pregnancy rate was 8% in group I and 4% in group II. Body mass index (BMI) and ovarian response to gonadotrophins Underweight and overweight women may experience a reduction in fecundity, probably due to multiple metabolic and endocrine alterations. Several studies (Table 3) have investigated the effects of being underweight or overweight on infertility treatments (induction of ovulation or assisted reproduction), but the results are conflicting. Some of the studies showed no significant influence of obesity on the outcome of assisted reproduction procedures in relation to response to ovarian stimulation, pregnancy and delivery rates. Others reported a lower response to ovarian stimulation, requiring higher doses of gonadotrophins and fewer collected oocytes, lower pregnancy rates and increased risk of miscarriage in overweight versus normal women. A retrospective study on 368 normally cycling women treated with clomiphene citrate and human menopausal gonadotrophin (HMG) for IVF or gamete intra-fallopian transfer (GIFT) showed that neither peak serum oestradiol concentrations nor duration of stimulation were related to body mass index (BMI) or total body weight of these women. The mean number of oocytes aspirated from women with BMI below 19.1 was higher (6.4 ± 3.2 versus 4.8 ± 2.6) than in obese women (BMI >27.6), but fertilization rates were not different. It was concluded that factors other than BMI or total body weight have more important effects on response to ovarian stimulation in normal women (Lewis et al., 1990). A more recent retrospective study compared 76 obese patients (BMI >27.9) with 152 controls, and 35 underweight patients (BMI <19) with 70 controls (Lashen et al., 1999). The obese patients had lower peak oestradiol concentrations than their normal controls, despite receiving similar gonadotrophin doses (P = 0.009). There were no other differences between the obese patients and the underweight patients and their normal controls

5 (Table 3). The study concluded that the extremes of body mass index do not adversely affect the outcome of IVF embryo transfer treatment. Another retrospective study on 398 couples showed that being underweight and overweight negatively affects IVF parameters and may diminish the chances of pregnancy (Wittemer et al., 2000). The mean FSH/LH ratio increased significantly according to BMI (1.5 ± 1.1 for BMI <20, 1.7 ± 1.1 for BMI 20 25, 1.8 ± 0.9 for BMI and 2.3 ± 1.4 for BMI >28, P < 0.05). In long stimulating protocol cycles an increased consumption of gonadotrophins was observed together with a decrease in the number of collected oocytes when BMI was 25 kg/m 2 (Table 3). The same observations were made in short stimulation protocol cycles, with BMI <20 and 25 kg/m 2. The study, however, was unable to find any significant difference in clinical pregnancy and miscarriage rates between underweight, normal weight and overweight patients. Zullo et al. (1996) found a significant correlation between BMI, urinary human FSH (u-hfsh) dose and duration of stimulation in PCOS and non-pcos obese patients. For these patients, he suggested ovarian stimulation be started with a double dose of u-fsh. More recently (Fedorcsak et al., 2004), the effect of BMI on treatment outcome was examined in 5019 IVF or ICSI treatments, after accounting for differences in age and infertility diagnosis (Table 3). Higher BMI was related to the need for increased gonadotrophin, length of stimulation and cycle cancellation rate. Moreover, a negative correlation was observed between BMI and the number of collected oocytes, transferred embryos and embryo transfers performed. Obesity, however, did not seem to affect implantation rates though it was associated with an increased risk of early pregnancy loss occurring before 6 weeks gestation. Being underweight (BMI <18.5 kg/m 2 ) was not related to impaired outcome of IVF or ICSI. These findings confirm previous reports on the positive relationship between BMI and the risk of spontaneous abortion in women who become pregnant after assisted reproductive technology treatment (Wang et al., 2002). The cause of early miscarriage in obese women has not been clearly identified, but some studies suggest that the endocrine alterations associated with obesity may impair endometrial receptivity, corpus luteum function or early embryo development (Fedorcsak et al., 2004). The recent introduction of recombinant FSH in clinical practice has raised concern about the effect of obesity on the pharmacokinetics of gonadotrophins administered subcutaneously (s.c.) and the need for different doses depending on the method of administration. A study in seven obese women, who received a single injection of human menopausal gonadotrophins, showed that FSH absorption was higher in intramuscular (i.m.) versus s.c. administration (Dobbs et al., 1994). However, a recent study (Steinkampf et al., 2003) aimed at determining whether recombinant FSH should be administered i.m. or s.c. to obese women confirmed that the pharmacokinetic effect of FSH is reduced in obese women, whether administration is i.m. or s.c. It therefore appears that s.c. administration of rfsh is the best approach in women regardless of body mass. Table 3. Body mass index and ovarian response to gonadotrophins in assisted reproduction treatment. Results are mean (or % when indicated). PR = pregnancy rate; amp = ampoule. Wittemer et al., 2000 Lashen et al., 1999 Fedorcsak et al., 2004 Under-weight Normal Obese Obese Normal Under-weight Normal Under-weight Normal Obese No. of patients FSH total f dose (IU) amp a, amp a, amp a, amp b amp b amp b,c Cancelled g cycles (%) Peak 3141 e 4074 e oestradiol (nmol/l) Retrieved 11.8 a, 4.9 b 11.3 a, 10.3 a,d, f eggs 6.6 b 6.1 b PR/cycle (%) Miscarriage rate (%) Live birth h rate (%) a, b Long and short protocols respectively. c, d Significant difference between the three groups ( c P < 0.01; d P < 0.025). e P = f, g, h Linear trends respectively: P < 0.001; P < ; P <

6 288 In conclusion, though it is generally accepted that higher doses of gonadotrophins are required in overweight patients, enhanced protocols may in the end improve live birth rates only marginally in obese women after IVF or ICSI. LH supplementation in ovarian stimulation The role of LH in ovarian stimulation is still a matter of debate. According to the 2-cell, 2-gonadotrophin theory (Fevold, 1941) some LH activity is essential for optimal maturation and development of the follicle oocyte unit during ovulation induction treatments. The actual amount of LH activity needed for normal follicle and oocyte development is not known, but is probably very low since <1% of follicular LH receptors need to be occupied to elicit normal steroidogenesis (Chappel and Howles, 1991). On the other hand, overly high LH concentrations during the follicular phase may induce excess follicular androgen secretion, thus increasing follicular atresia and producing low quality embryos (Loumaye et al., 1989). A dose-finding study (European Recombinant Human LH Study Group, 1998) compared four doses (0, 25, 75, or 225 IU/day) of recombinant human LH (rhlh) in addition to a fixed dose of rhfsh (150 IU/day) in hypogonadotrophic hypogonadal women (WHO group I anovulation). Patients receiving the two higher doses (75 and 225 IU) had a significantly higher number of large follicles and higher oestradiol concentrations per follicle. Interestingly, the majority of women treated with 75 IU presented appropriate ovarian response, notwithstanding undetectable serum LH concentrations. It is a common practice in IVF treatment to induce pituitary desensitization prior to ovarian stimulation, mimicking a state of hypogonadotrophic hypogonadism, in order to avoid premature LH surge and luteinization. The concentrations of residual endogenous LH remaining during full-dose gonadotrophinreleasing hormone (GnRH) agonist pituitary suppression are certainly sufficient to ensure adequate follicular maturation during ovarian stimulation with purified human urinary FSH or recombinant FSH in most patients (Chappel and Howles, 1991; Loumaye et al., 1997; Levy et al., 2000). It has however been suggested that GnRH agonist downregulation in some normogonadotrophic women may result in overly profound suppression of LH secretion, impairing oestradiol synthesis (Fleming et al., 1998; Janssens et al., 2000) and IVF/pregnancy outcome by increasing the risk of early pregnancy loss (Westergaard et al., 2000). It has also been suggested recently that a direct action of LH on uterine LH receptors is required to enhance endometrial growth and uterine receptivity (Tesarik, 2003), and that the expression of endometrial oestrogens and progesterone receptors is altered in ovarian stimulation when GnRH agonist is used in the presence of low LH concentrations (Bourgain et al., 2002). In cases when pure or recombinant FSH is used for ovarian stimulation after GnRH agonist down-regulation, very low serum LH concentrations may therefore adversely affect IVF outcome. It is possible that some normogonadotrophic women who undergo profound down-regulation with GnRH agonist may benefit from preparations containing LH. Some authors have proposed different threshold values of serum LH in down-regulated women, in order to identify the group of profoundly suppressed LH patients whose ovarian response would significantly benefit from the administration of exogenous LH during stimulation with FSH: <0.1 IU/l (Chappel and Howles, 1991; Fleming et al., 1996), <0.7 IU/l (Fleming et al., 2000), <0.5 IU/l (Fleming et al., 1998) (Westergaard et al., 2000). In two prospective studies, Fleming observed the effect of profound suppression of LH during follicular phase on oestradiol serum concentrations and oocyte quality. He showed that in patients treated with purified urinary FSH there is a decrease in follicular fluid oestradiol concentrations, oocyte yield and fertilization while the follicular phase is longer when LH concentrations are <1 IU/l, indicating a reduction in normal follicular steroid metabolism (Fleming et al., 1996). These findings were confirmed in a subsequent study (Fleming et al., 1998) in women with more profoundly suppressed midfollicular phase LH (<0.5 IU/l), who had significantly fewer embryos available for cryopreservation. The rate of blastocyst formation, however, was normal, indicating that embryo development potential is not impaired by the degree of LH suppression. Filicori et al. (2001) compared highly purified FSH and HMG in normo-ovulatory GnRH agonist suppressed women (candidates for intrauterine insemination) and found a shorter treatment duration with lower gonadotrophin consumption in the HMGtreated group, underlining the facilitatory role of LH activity in ovulation induction. Other trials have recently confirmed that rlh supplementation may be more effective than increasing rfsh doses in patients with poorer initial ovarian response to rfsh (Table 4). A multicentre trial (De Placido et al., 2005) showed that initial ovarian response to rfsh may be suboptimal in approximately 12 14% of young normogonadotrophic women treated with depot GnRH agonist long protocol and a starting dose of rfsh of 225 IU. From day 8 of stimulation, these women were randomly allocated to two groups: women in group A (n = 65) received 150 IU of rlh in addition to rfsh, while those in group B (n = 65) received an increase of 150 IU in their daily dose of rfsh. A total of 130 normally responding women continued monotherapy with rfsh and served as a further control population. The mean number of cumulus-oocyte complexes retrieved in group A (9.0 ± 4.3) was significantly higher (P < 0.01) than in group B (rfsh 6.1 ± 2.6), but significantly lower than in group C (10.49 ± 3.7, P < 0.05). Implantation and pregnancy rates were significantly lower (P < 0.05) in the rfsh step-up group (10.5 and 29.3% respectively) compared with normal responders (18.1 and 47.3% respectively). Another study showed that serum LH concentrations could not predict the need for LH (Ferraretti et al., 2004). Hyporesponsiveness to FSH might be related to iatrogenic LH deficiency that, in turn, could affect oocyte competence. Addition of a small amount of recombinant LH is able to recoup oocyte competence and produce viable embryos. Whenever FSH needs to be increased during ovarian stimulation, exogenous LH should be added to enhance oocyte competence, though it will not necessarily increase their number. The use of recombinant LH seems to be more effective than urinary

7 Table 4. LH supplementation in ovarian stimulation in long luteal gonadotrophin releasing hormone agonist cycles for assisted reproduction treatment. PR = pregnancy rate; amp = ampoule; HP = highly purified. Population Study groups No. of Mean age FSH total Retrieved PR (%) patients (years) dose eggs Ben-Amor, 2000 Normal responders HPFSH amp HPFSH + rlh 75 IU amp Lisi et al., 2002 Normal responders rfsh IU rfsh + rlh 75 IU IU Marrs et al., 2004 Normal responders rfsh amp rfsh + rlh 150 IU amp Lisi et al., 2001 Previous poor rfsh IU 5.9 a responders rfsh + rlh 75 IU IU 50.0 a De Placido et al., Initial poor response rfsh increased amp b,d 6.1 e,g 29.3 h 2005 rfsh + rlh 150 IU amp b,c, 9.0 e,f 37.2 Normal responders rfsh fixed amp c,d 10.5 f,g 47.3 h Ferraretti et al., Initial poor response rfsh increased IU i,j 8.2 k,m 24.4 n 2004 rfsh+rlh IU IU i 11.1 k 54 n Normal responders rfsh fixed IU j 9.8 m 41 a n Comparisons with the same superscript letter are significantly different: a, f, h n P < 0.05; e,g P < 0.01; b d P < LH contained in HMG. In the trial by Ferraretti et al. (2004), women presenting hyporesponsiveness to FSH under GnRH agonist down-regulation were randomized into three groups: group A (n = 54) received an increased dosage of FSH; group B (n = 54) was administered recombinant LH in addition to the increased dose of FSH; group C (n = 22) was given additional FSH and LH using HMG as a combined drug. A total of 54 age-matched women not requiring increased FSH doses were included as control group (D). In group B, the pregnancy and implantation rates were statistically higher than groups A and C and did not differ from the control group for normal response. The live birth rate was similar in groups B and D but was half in groups A and C. There is no general agreement about the clinical usefulness of either defining profoundly suppressed women or adding LH to FSH. A study of 144 infertile women undergoing rfsh stimulation for IVF/ICSI (Balasch et al., 2001) showed that serum LH concentration on day 7 was unable to discriminate between conception and non-conception cycles. To assess the potential impact of low LH concentrations during ovarian stimulation on the outcome of IVF/ICSI treatment, the three threshold values of mid-follicular serum LH proposed in the literature (<1, 0.7, <0.5 IU/l) (Chappel and Howles, 1991) (Fleming et al., 1996, 1998, 2000; Westergaard et al., 2000) to distinguish women with low from normal LH were applied to study population. No significant differences were found as regards ovarian response, IVF/ICSI outcome, implantation, and pregnancy outcome between low and normal day 7 LH women as defined by the above threshold values. The results of this study do not point to the need for additional exogenous LH supplementation in down-regulated women receiving recombinant FSH-only preparation, a conclusion recently confirmed by a study analysing three threshold values of serum LH (1, 1.5, and 2 miu/ml) in day 1 and day 7 of stimulation (Cabrera et al., 2005) and which found no significant differences regarding ovarian response, fertilization rate, and pregnancy outcome. A recent trial compared the effect of a combination of r-hfsh (Gonal-F) and r-hlh (n = 212), starting on day 6 of FSH stimulation at a daily fixed dose of 150 IU r-hlh, with that of r-hfsh alone (n = 219) in women undergoing ICSI for male factor infertility (Marrs et al., 2004). The study showed that younger patients do not seem to benefit from an LHsupplemented ovarian stimulation protocol. These findings confirm a previous preliminary report where infertile women down-regulated with buserelin received either FSH (150 IU/ day) or FSH (150 IU/day) plus a supplementation of rlh (75 IU/ day) started when the leading follicle had reached 14 mm (mean diameter) and continued until the day of HCG administration (Ben-Amor, 2000) (Table 4). On the other hand the study of Marrs suggested that women aged over 34 years may benefit from using r-hlh in addition to r-hfsh. Women aged 35 years who were undergoing their first assisted reproduction cycle had significantly more embryos transferred in the rfsh + rlh group (3.1 ± 0.6 versus 2.8 ± 0.7, P = 0.04). This subgroup had also a significantly higher clinical pregnancy rate (P < 0.03) with an odds ratio (95%CI) of 3.13 ( ). Similarly, another study (Humaidan et al., 2004) showed that women aged >35 years responded to exogenous LH supplementation (rfsh/ rlh = 2/1) with significantly increased implantation rates (36.4 versus 13.3%, P < 0.05) and significantly reduced total FSH consumption as compared with the non-supplemented group (2225 IU versus 2797 IU, P < 0.05). Pulling all these data together, there is evidence for the existence of an LH threshold below which oestrogen concentrations will be insufficient for endometrial proliferation and corpus luteum formation. The idea of an LH ceiling has also been recently introduced, since high concentrations of LH have been associated with atresia of developing follicles in women with hypogonadotrophic hypogonadism or polycystic ovary disease (Shoham, 2002; Loumaye et al., 2003). There appears therefore to be a therapeutic window of LH concentrations, below which oestradiol production is inadequate and above which LH may be detrimental to follicular development. 289

8 290 The size of this window and the clinical implications were examined in an elegant study by Humaidan et al. that evaluated the effect of LH concentrations at stimulation day 8 on ovarian response and pregnancy outcome (Humaidan et al., 2002). A total of 207 normal women received pituitary down-regulation with GnRH agonists and ovarian stimulation with recombinant FSH. Based on LH concentrations at stimulation day 8 patients were divided into four groups: <0.5, , , >1.51 IU/l. Oestradiol concentrations on day 8 and oestradiol per oocyte retrieved presented a highly significant correlation with LH concentrations on day 8. The total consumption of exogenous FSH and duration of gonadotrophin stimulation were inversely related to LH concentrations at day 8 (P < 0.002). Fertilization and clinical pregnancy rates were higher in the two middle groups, with a reduced chance of clinical pregnancy when LH concentrations were <0.5 or >1.5 IU/l. It should be noted that only 12% of the patients presented LH concentrations <0.5 IU/l versus 48% in previous studies (Westergaard et al., 2000), though this difference may be explained by the different mode and doses of GnRH agonist used in the different trials. The concept of a window for LH requirement in ovarian follicular development has been confirmed by other trials. Tesarik and Mendoza (2002) investigated the effect of exogenous LH in oocyte donors stimulated with rfsh. In donors with profound pituitary suppression (LH concentrations <0.1 IU/l) before ovarian stimulation, when compared with stimulation with FSH alone, the addition of exogenous LH increased the number of mature oocytes and good quality embryos as well as implantation rates. In contrast, when LH concentrations at the beginning of stimulation were >1 IU/l, exogenous LH administration impaired oocyte and embryo quality and reduced the implantation rate in recipients, though it did increase the number of metaphase II oocytes. The literature therefore seems to suggest that there may be a role for LH co-administration in cycles treated with depot GnRH agonist and FSH, at least in selected groups of patients (Caglar et al., 2005), though the real efficacy of this approach in terms of take-home babies remains to be proven by future prospective randomized trials. It has been suggested that lowering the dose of depot GnRH agonist may reduce LH requirement in patients undergoing assisted reproduction. LH activity, from either endogenous stores or administered exogenously, has a significant impact on the ovarian response in GnRH agonist pretreated women during ovarian stimulation. GnRH agonist regime and dose have an important effect on residual LH activity in circulation. Ovarian response and pregnancy rates were related not just to the amount of GnRH agonist used, but also to the mode of administration (Westergaard et al., 2001). Intranasal versus s.c. administration of buserelin resulted in significantly less depressed midfollicular LH concentrations and higher pregnancy rates. The use of reduced doses of short-acting GnRH agonist has been shown to attenuate pituitary suppression (Janssens et al., 2000; Dal Prato et al., 2001), enough to prevent premature LH surges, though it does require less gonadotrophin and sometimes elicits higher oestradiol concentrations and oocyte yield (Feldberg et al., 1994). A very recent report showed that half dose (1.87 mg) depot triptorelin can also be successfully used in ovarian stimulation for IVF or ICSI (Dal Prato et al., 2004). In this study the lower level of pituitary desensitization elicited by half dose triptorelin induced higher LH concentrations, though there was no premature LH surge. Compared with full dose, there was lower consumption of gonadotrophins and a higher yield of oocytes and good quality embryos with a very good chance of implantation. In light of these data, the use of reduced dose of GnRH agonist may be simpler and cheaper than screening for women that might benefit from the addition of exogenous LH. In recent years, GnRH antagonists have been introduced in clinical practice as an alternative to GnRH agonists in ovarian stimulation for IVF. Dose-finding studies with this protocol have shown that low pregnancy rates are to be observed in women treated with very high antagonist doses, resulting in very low LH concentrations (Ganirelix Dose-Finding Study Group, 1998). Reports exist which have shown that there is no evident benefit from supplementing GnRH antagonist and rfsh-treated cycles with rlh (Cédrin-Durnerin et al., 2004; Dal Prato and Borini, 2005; Kolibianakis et al., 2005). Moreover, a recent trial reported that profound suppression of LH (<5 IU/l) at day 8 of stimulation is associated with a significantly higher chance of ongoing pregnancy (Kolibianakis et al., 2004). On the other hand, a very recent study re-evaluated the benefits of supplemented LH in GnRH antagonist cycles in high responders (Kol and Muchtar, 2005). The situation in GnRH antagonist cycles is probably different from GnRH agonist cycles. The use of antagonists reduces the amount of gonadotrophins required compared with agonists (Dal Prato and Borini, 2005), and this reduction is more significant in the flexible antagonist protocol (Al-Inany et al., 2005). Therefore, any data regarding the association of LH concentrations following antagonist administration and the chance of achieving an ongoing pregnancy cannot be compared with those obtained from GnRH agonist cycles. Conclusions The characteristics of each patient are very important for choosing the most appropriate ovarian stimulation protocol and the amount of gonadotrophins to be used. Several factors, such as age, basal FSH, number of antral follicles and body mass index, should be taken into account. Increasing the dose of recombinant FSH may be necessary in older women, but it does not compensate for the age-related decline in retrievable oocytes. Higher doses of gonadotrophins are required in overweight patients, but an enhanced protocol may bring only a little improvement of live birth rate in obese women. The actual role of LH in controlled ovarian stimulation is still a matter of debate. A therapeutic window of LH concentrations, below which oestradiol production is not adequate and above which LH may be detrimental to follicular development, has been described. Although the criteria for the identification or the most appropriate ovarian stimulation protocol have not yet been fully elucidated, clinicians have the imperative to optimize patient

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