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Who needs LH in ovarian stimulation?
RBMOnline - Vol 12 No 5. 2006 599–607 Reproductive BioMedicine Online; www.rbmonline.com/Article/2146 / on web 20 March 2006
/UTLOOK 7HO NEEDS ,( IN OVARIAN STIMULATION $R #ARLO !LVIGGI OBTAINED THE -$ SPECIALTY IN /BSTETRICS AND 'YNAECOLOGY AND 0H$ DEGREES AT THE &ACULTY OF -EDICINE 5NIVERSITY OF .APLES @&EDERICO )) )TALY (E HAS COLLABORATED WITH VARIOUS DEPARTMENTS IN )MPERIAL #OLLEGE ,ONDON THE )TALIAN .ATIONAL 2ESEARCH #OUNCIL .APLES THE 5NIVERSITY OF #ALIFORNIA ,OS !NGELES AND THE 5NIVERSITY OF !THENS 'REECE 4HIS COLLABORATIVE NETWORK RESULTED IN VARIOUS PUBLICATIONS INCLUDING NEW HYPOTHESES ON THE PATHOGENESIS OF PELVIC ENDOMETRIOSIS (E HAS SPECIALIZED IN REPRODUCTIVE MEDICINE AT THE &ERTILITY 5NIT OF THE 5NIVERSITY OF .APLES @&EDERICO )) $R !LVIGGI HAS PUBLISHED EXTENSIVELY AND GIVEN MANY INVITED LECTURES AT INTERNATIONAL MEETINGS IN HIS l ELD AS WELL AS SERVING AS AD HOC REVIEWER FOR INTERNATIONAL JOURNALS $R !LVIGGIS CURRENT RESEARCH INTERESTS INCLUDE THE PATHOGENESIS OF PELVIC ENDOMETRIOSIS AND THE GENETICS OF HUMAN REPRODUCTION $R #ARLO !LVIGGI # !LVIGGI 2 #LARIZIA ! -OLLO ! 2ANIERI ' $E 0LACIDO $IPARTIMENTO DI 3CIENZE /STETRICHE 'INECOLOGICHE 5ROLOGICHE E -EDICINA DELLA 2IPRODUZIONEn5NIVERSITÌ DEGLI 3TUDI DI .APOLI @&EDERICO )) .APLES )TALY #ORRESPONDENCE VIA 3 0ANSINI .APLES )TALY 4EL &AX E MAIL ALVIGGI UNINAIT
!BSTRACT LH plays a key role in the intermediate−late phases of folliculogenesis. Although ovarian stimulation is efficiently achieved in most cases by the administration of exogenous FSH alone, specific subgroups of women may benefit from LH activity supplementation during ovarian stimulation. Some authors have found improved outcome with LH activity supplementation in advanced reproductive age women. Experience suggests that in about 10−12% of young normogonadotrophic patients treated with a gonadotrophin-releasing hormone agonist (GnRH-a) long protocol plus recombinant FSH human (r-hFSH), a ‘steady response’ is observed. In this subgroup of women, a higher number of oocytes is retrieved when daily LH activity supplementation is given from stimulation day 8, if compared with the standard FSH dose increase. Another subgroup of patients who may benefit from LH activity supplementation are those at risk for poor ovarian response treated with GnRH antagonist. Recent data demonstrate that in these women, when GnRH is administered in a flexible protocol, the concomitant addition of recombinant human LH improves the number of mature oocytes retrieved, when compared with the standard GnRH-a flare-up protocol. Thus, well calibrated LH administration improves the ovarian outcome in patients >35 years, in those showing an initial abnormal ovarian response to r-hFSH monotherapy, and in ‘low prognosis’ women treated with GnRH antagonists. Keywords: assisted reproductive techniques, GnRH antagonists, LH, ovarian stimulation, recombinant gonadotrophins
,( AND PHYSIOLOGICAL FOLLICLE GROWTH According to the ‘two cells−two gonadotrophins’ model (Fevold, 1941; Hillierr et al., 1994), LH exerts its activity in theca cells, which form the involucres of the growing follicles and express enzymatic pathways of androgen synthesis. Theca involucres surround the granulosa cells, whose activities and proliferation are directly regulated by FSH. This hormone induces the expression of the aromatase enzyme, which in turn converts theca-deriving androgens into oestradiol. This theory reinforced the notion that granulosa and theca cells are distinct compartments regulated by FSH and LH respectively. However, it was subsequently found that LH receptors are detectable on the granulosa compartment at the intermediate follicular phase (Erickson et al., 1979; Shima et al., 1987; Hillierr et al., 1994; Filicori et al., 2003a), at a time when blood concentrations of LH increase. Therefore, it appears that LH regulates both granulosa and theca cells.
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FSH and LH induce the local production of the soluble molecule inhibin B and growth factors. Among these, insulinlike growth factors (IGF)-I and II, which are expressed by both granulosa and theca cells throughout folliculogenesis, are important in promoting follicular maturation (Zhou and Bondy, 1993; Huang et al. 1994). Thus, gonadotrophins and the autocrine−paracrine system contribute to the complex mechanisms governing follicular growth and selection. The finding that both gonadotrophins also regulate granulosa cell activity suggests that LH is involved in inducing and maintaining this paracrine system of biochemical factors by acting on the theca and granulosa compartments. These findings may explain the observation that FSH activity can be totally substituted by LH once granulosa cells express adequate amounts of LH receptors (Zeleznik and Hillier, 1984; Filicori et al., 2002). Hence, LH seems to play two roles during folliculogenesis. One is exerted in the theca compartment and consists of induction of
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Outlook - LH in ovarian stimulation - C Alviggi et al.
androgen production. The second begins during the intermediate follicular phase (Willis et al., 1998; Filicori et al., 2003a), involves granulosa cells, and consists of inducing the local production of various molecules. These factors promote the growth of granulosa cells, which in turn regulate oocyte maturation. These two mechanisms are closely related and probably support each other. According to the so-called ‘spare receptor hypothesis’ (Chappel and Howles, 1991), at a time when inhibin B and IGF-1 are adequately secreted, androgen synthesis and release are optimal even with <1% of LH receptors occupied.
paracrine activities, including the production of inhibins and IGF-1, may favour adaptation mechanisms by enhancing theca sensitivity to LH. In this context, a low LH environment would be advantageous, whereas during stimulation, LH-induced suppression of small follicles is not required. Furthermore, during the early−intermediate stages of follicular growth, low LH concentrations have been associated with a more physiological endometrial proliferation, which in turn seems to synchronize this compartment for successive embryo implantation (Kolibianakis et al., 2004).
LH has been implicated in a third process during folliculogenesis. In particular, it has been suggested that this hormone may contribute to negatively select non-dominant follicles. This idea was initially based on the observation that, following the mid-cycle LH surge, granulosa cell mitosis is blocked, and oocyte meiosis is resumed (Shoham m et al., 1995). Preclinical evidence showed that developing follicles have specific requirements for exposure to LH beyond which normal maturation ceases (Hillierr et al., 1994). This finding gave rise to the concept of an ‘LH ceiling’, meaning that each follicle would have an upper limit of stimulation. The LH ceiling may be higher in larger follicles and lower in smaller ones. As a consequence, an increasing LH concentration would promote leading follicle progression (being below its ceiling) and degeneration of secondary ones (by overcoming their ceiling). According to others, a dynamic interplay between LH secretion and receptor expression by different ovarian compartments governs the selection of dominant follicles; because small follicles in granulosa cells do not express LH receptors, LH may indirectly promote their degeneration (Filicori et al., 2003a,b).
Surprisingly, during ovarian stimulation, a low LH environment seems to be appropriate also during more advanced stages of folliculogenesis, when LH-dependent paracrine activities seem to be crucial for follicular growth and oocyte maturation. Also in this case, FSH may be important. In fact, it may make granulosa cells more responsive to low LH concentrations by enhancing their expression of LH receptors. Furthermore, this supraphysiological FSH environment may also balance the lack of LH by inducing compensatory paracrine activities in granulosa cells.
,( AND OVARIAN STIMULATION The gold standard for ovarian stimulation in young, normogonadotrophic women is the gonadotrophin-releasing hormone agonist (GnRH-a) long protocol (Hughes et al., 1992). Exogenous FSH is administered only when a GnRH-a mediated suppression of the hypothalamus−pituitary−gonad axis is achieved. Moreover, monotherapy with recombinant human FSH (r-hFSH), which is free of LH activity, is used in most cases. The degree of pituitary suppression also depends on the GnRH-a formulation, dose and mode of administration (Westergaard d et al., 2001). Following post-suppression decline, LH concentrations usually range between 0.5 and 2.5 IU/l. These concentrations often fall by <0.5 IU/l during the intermediate late stages of stimulation. Thus, multiple follicular growth is often induced in a low endogenous LH environment. Nevertheless, an adequate ovarian response is achieved in almost all patients.
A hiatus appears to exist between IVF practice and information deriving from folliculogenesis models and hypogonadotrophic hypogonadism. In particular, in the former situation, follicular growth normally occurs even when LH concentrations are below 0.5 IU/l. In contrast, there is evidence that in women with hypogonadotrophic hypogonadism, LH concentrations of 1.2 IU/l are required in order to achieve adequate ovarian response to r-hFSH. These discrepancies can be attributed to biological events that involve steroidogenic dynamics and follicular growth. Regarding stimulated cycles, the above-mentioned ‘spare receptor hypothesis’ (Chappel and Howles, 1991) can be invoked: even when pituitary desensitization is achieved, resting circulating concentrations of LH are able to occupy an adequate percentage of receptors and to elicit sufficient androgen release. FSH-dependent
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2ELATIONSHIPS BETWEEN ,( SERUM CONCENTRATIONS AND OVARIAN)6& OUTCOME Some observational trials (Fleming et al., 1998; Westergaard d et al., 2000; Balasch h et al., 2001; Esposito et al., 2001; Humaidan et al., 2002) with r-hFSH have examined the relationship between LH serum concentrations and ovarian/IVF outcome in normogonadotrophic women undergoing ovarian stimulation in a GnRH-a long protocol. Univocal LH cut-off value able to identify women requiring LH activity supplementation could not be found by those studies. Differences in patient selection criteria, clinical end-points, serum LH assays, and LH cut-off value may account for discrepancies among results. Hence, there is a need for adequately sized, prospective observational trials.
%XOGENOUS ,( AND OVARIAN)6& OUTCOMES IN UNSELECTED PATIENTS There is a large body of data on r-hFSH monotherapy versus combined exogenous FSH and LH [in the form of human menopausal gonadotrophin (HMG) or recombinant molecule] in women undergoing the GnRH-a long protocol. Van Wely et al. (2003) conducted a meta-analysis of four randomized controlled trials (RTC) comparing r-hFSH and HMG treatment in normogonadotrophic patients undergoing a GnRH-a long protocol. The authors failed to show any statistically significant difference in the main outcome measures (ongoing pregnancy rates and live birth rate). Three RTC of FSH versus FSH plus r-hLH regimens in normogonadotrophic women undergoing the GnRH-a long protocol have been published (Table 1). Sills et al. (1999) studied 31 cycles in 30 women (age 30–41 years). Seventeen patients received 150–450 IU of highly purified FSH (FSHHP). The remaining 13 women (14 cycles) were stimulated with
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Outlook - LH in ovarian stimulation - C Alviggi et al.
the same dose of FSH-HP plus one ampoule of r-hLH (75 IU) from the first day of stimulation (S1). There was no significant difference in any of the outcome parameters. A large multicentre, randomized trial was published by Marrs et al. in 2004. A total of 431 intracyoplasmic sperm injection (ICSI) patients (age 18−40 years) undergoing the mid-luteal GnRH-a long protocol were randomized to receive either 225 IU/day of r-hFSH (n = 219) or the same dose of r-hFSH plus r-hLH (150 IU/day, from day S6; n = 212). No significant difference in the number of metaphase II oocytes retrieved (primary end-point) or in the cumulative pregnancy rate was found. In contrast, the mean number of embryos transferred was significantly higher in the r-hLH supplemented group (2.9 ± 0.6 versus 2.8 ± 0.7, P < 0.05). Humaidan et al. (2004) studied the effects of LH activity supplementation in 231 normogonadotrophic women, aged <40 years. Stimulation was initiated with r-hFSH (150–300 IU): from day S8, patients were randomized to continue r-hFSH monotherapy (n = 115) or to receive r-hFSH plus r-hLH in a ratio of 2:1 (n = 116). Also in this instance, the first cumulative analyses failed to show any significant difference in terms of endocrinology, ovarian response and pregnancy outcome between groups. It should be stressed that in those trials, the daily r-hLH dose ranged between 75 and 150 IU in almost all patients. These clinical observations, together with pharmacokinetic studies (le Cotonnec et al., 1998a,b), indicate the need for RCT to evaluate the efficiency of higher doses.
In conclusion, cumulative data analysis is insufficient to prove that LH administration is associated with any significant improvement in the IVF/ICSI outcome parameters, including implantation and pregnancy rates, when compared with r-hFSH monotherapies, at least in unselected groups.
Drakakis et al. (2005), in an RCT (n = 46), has recently tested the effects of LH-activity supplementation (in the form of HMG in young normogonadotrophic women undergoing standard GnRH-a long protocol. Patients in group A (n = 22) received a starting dose of 200 IU of r-hFSH/day. In group B (n = 24), ovarian stimulation began with one ampoule/day of HMG in association with a daily dose of 150 IU of r-hFSH. Patients in group B showed a P <0.05) in the mean number of statistically significant increase (P mature oocytes (7.3 ± 2.9 versus 10.7 ± 3.4), fertilized oocytes (6.5 ± 1.8 versus 7.9 ± 3.0), and transferable embryos (6.0 ± 2.8 versus 7.7 ± 3.1). Nevertheless, previous meta-analysis of RCT (Van Wely et al., 2003) comparing HMG stimulation with r-hFSH monotherapy in women undergoing GnRH-a long protocol failed to show any significant difference in ovarian and IVF outcomes between protocols.
Although based on a-posteriori stratification processes, the above data identify at least one subset of women, those aged >35 years, who may benefit from LH activity supplementation. In fact, there is evidence that ovarian paracrine activity decreases with age (Hurwitz and Santoro, 2004).
%XOGENOUS ,( AND OVARIAN)6& OUTCOMES IN ADEQUATELY SELECTED PATIENTS 7OMEN OF ADVANCED REPRODUCTIVE AGE Marrs et al. (2004) stratified their patients by age and number of previous treatment cycles. With an intention-to-treat adjustment, the clinical pregnancy rate was significantly higher (P P < 0.05) in the r-hFSH plus r-hLH group in patients aged ≥35 years at their first assisted reproduction cycle. In addition, Humaidan et al. (2004) stratified their study population according to age. Among women who did not receive rLH supplementation, pregnancy and implantation rates were significantly lower in those ≥35 years (P P < 0.05); this difference did not occur in the r-hLH supplemented group.
7OMEN WITH AN ABNORMAL RESPONSE TO R H&3( Clinical evaluation of the ongoing ovarian response seems to be an accurate parameter for indicating the use of LH. Several groups have studied the efficacy of LH activity supplementation in women selected according to specific profiles of ovarian response to standard r-hFSH doses (Table 2). In 2001, Lisi et al. conducted
Table 1. Randomized controlled trials testing recombinant-human (r-hLH) supplementation in normogonadotrophic unselected women. Trial
Study design
Pituitary downregulation
Groups
Results
Sills et al., 1999
Prospective, randomized
GnRH-a daily from mid-luteal phase
No significant differences
Marrs et al., 2004
Multicentre, prospective
GnRH-a daily from mid-luteal phase
150–450 IU/day of FSH-HP (n = 17) versus FSH-HP plus 75 IU/day r-hLH throughout ovarian stimulation (n = 14) 225 IU/day of r-h-FSH (n = 219) versus r-hFSH plus 150 IU/day r-hLH from day S6 (n = 212)
Humaidan et al., 2004
Prospective, randomized
GnRH-a daily from mid-luteal phase
150–300 IU day of r-h-FSH (n = 115) versus r-hFSH plus r-hLH (2:1 ratio) from day S8 (n = 116)
Mean number of embryos transferred significantly higher in the r-hLH supplemented group No significant differences
GnRH-a = gonadotrophin-releasing hormone agonist; FSH-HP = highly purified FSH.
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a ‘self-control study’ of the effect of r-hLH supplementation in 12 patients who, during previous stimulation with r-hFSH, required >3000 IU to reach follicular maturity. Re-stimulation entailed the addition of 75 IU of r-hLH from day S7 to the standard r-hFSH regimen. As in the first cycle, all women received triptorelin 0.1 mg daily, from the mid-luteal phase (GnRH-a long protocol). There was no difference in the total consumption of r-hFSH, days of stimulation or number of MII oocytes per patient between the two cycles of the study. However, the incidence of fertilization (86.0 versus 60.9%) and clinical pregnancies (50.0 versus 5.9%) was significantly higher (P P <0.05) in r-hLH supplemented cycles. De Placido et al. (2001) found that in about 10−12% of normogonadotrophic patients, an initial response (i.e. at least five 2−9 mm follicles in each ovary) during the first days of stimulation is followed by a plateau in which there is no significant increase in follicular size or oestradiol production in the next 3−4 days of stimulation. This profile of initial ovarian response to r-hFSH is referred to as ‘steady response’ (De Placido et al., 2005), and usually leads physicians to increase the r-hFSH dose. De Placido and colleagues (2001) conducted a prospective randomized trial to determine whether this clinical condition could be related to excessive pituitary suppression and to impairment of LHdependent mechanisms. Women (age <37 years, basal FSH ≤10 IU/l) who had no follicle with a mean diameter >10 mm and oestradiol serum concentrations ≤180 pg/ml on day S8 were randomized to receive LH activity supplementation (n = 20) in the form of HMG (150 IU/day) or an increase in the r-hFSH daily dose (maximum daily dose of 375 IU; n = 23). In order not to modify the daily FSH administration, the r-hFSH dose was reduced to 150 IU in women of the HMG group. Forty women matched for age and body mass index (BMI) and with an initial adequate response to r-hFSH (i.e. a tripling of serum oestradiol concentration between days S5−8 in association with more than four follicles >10 mm on day S8) served as a non-randomized control population. All women received triptorelin 3.75 mg (depot preparation) on day 1 of spontaneous menstruation. After pituitary desensitization, a starting dose of 300 IU of r-hFSH was administered. First dose adjustment was performed on day S5. The mean number of oocytes retrieved was significantly higher in women treated with HMG supplementation than in those who received r-hFSH ‘step-up’. Moreover, the ovarian outcome of the HMG group was comparable with that observed in ‘normal responders’, suggesting that LH activity supplementation was able to ‘rescue’ this apparently abnormal response to r-hFSH. Interestingly, when serum LH concentrations were measured on day S8, there was no statistically significant difference between women whose ovarian outcome improved with HMG and normal responders to r-hFSH. These findings revealed the existence of a subgroup of hypo-responders who benefit more from LH activity supplementation than from an increase in the daily r-hFSH dose.
In a preliminary dose-finding design, De Placido et al. (2004) evaluated the efficacy of r-hLH supplementation in women displaying an initial ‘steady response’ to r-hFSH. GnRH-a administration and the criteria for ‘steady-response’ were identical to those used in the earlier study (De Placido et al., 2001). Patients (age <37 years, basal FSH ≤10 IU/l) received a starting r-FSH dose ranging between 150 and 300 IU/day. Women displaying a steady response were randomized on day 8 to receive a daily r-hLH dose of 75 (n = 23) or 150 IU (n = 23). The control population consisted of ‘normal’ responders to r-hFSH (n = 46). The mean number of oocytes retrieved (primary end-point) and
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the percentage of mature oocytes in women treated with 150 IU of r-hLH (9.65 ± 2.16, 79.0%) were similar to those observed in ‘normal responders’ (10.65 ± 2.8, 82.5%), and were significantly higher than those of subjects receiving 75 IU [6.39 ± 1.53, 65.7% (P <0.001 and P <0.05 respectively)]. The effectiveness of r-hLH in ‘steady responders’ was then evaluated in a larger multicentre RTC (De Placido et al., 2005) with the r-hFSH ‘step-up protocol’ as reference standard. A total of 229 IVF/ICSI cycles performed in seven Italian units were analysed. In all patients (age <37 years, basal FSH ≤10 IU/l), triptorelin 3.75 mg (depot preparation) was administered on day 1 of spontaneous menstruation. The starting dose of r-hFSH was 225 IU/day. ‘Steady responders’ were identified on day 8 (oestradiol serum concentrations <180 pg/ml and at least six follicles ranging between 6 and 10 mm, but no follicle with a mean diameter >10 mm) and randomized to receive either r-hLH supplementation of 150 IU/day (n = 59), or an increase of 150 IU in the daily r-hFSH dose (n = 58; r-hFSH ‘step-up’ protocol). Also in this case, an age/BMI-matched population of ‘normal responders’ (tripling oestradiol concentrations between days S5 and S8, more than four follicles >10 mm on day S8) was selected as a control group (n = 112). The number of cumulus−oocyte complexes (primary end-point) and mature oocytes retrieved was significantly higher in women receiving r-hLH than in those treated with the r-hFSH step-up protocol. Moreover, the mean number of mature oocytes of r-hLH group was similar to that observed in ‘normal responders’. Also in this study, endogenous LH serum concentrations on day S8 (before randomization) did not differ between ‘steady responders’ undergoing r-hLH supplementation and normal responders (median: 0.7, range: 0.1–3.6 IU/l; median: 0.7 range: 0.1–4.0 IU/l respectively). Ferraretti et al. (2004) conducted an RCT on 184 patients (age <38 years) undergoing the GnRH-a long protocol. Patients with normal initial follicular recruitment (>10 antral follicles ≥8 mm in diameter and oestradiol ≥100 pg/ml) with the fixed starting dose of recombinant FSH (150–300 IU), but showing a plateau in follicular growth between days S7 and S10 (no increase in follicle size or in oestradiol concentration) were randomized as follows: group A (n = 54) received an increase in the daily dose of r-hFSH; group B (n = 54) received 75–150 IU of r-hLH in addition to the increased dose of FSH; group C (n = 26) received HMG; group D consisted of 54 age-matched patients with an optimal response (no need to increase the FSH dose). The mean number of oocytes retrieved was significantly lower in group A (8.2) versus the other groups (11.1, 10.9, 9.8 in groups B, C, and D respectively). Furthermore, the pregnancy-per-embryo transfer and implantation rates were significantly higher in group B than in groups A and C, and did not differ from normal responders. Consistent with other studies (De Placido et al., 2001, 2004, 2005), there were no significant differences in LH circulating concentrations on day S7 among groups (0.99 ± 0.7, 1.02 ± 0.9, 1.3 ± 1.0, 0.93 ± 0.6 mIU/ml, in the four groups respectively). It should be noted that in this study, in those by De Placido et al. (2001, 2004, 2005) and in that reported by Lisi et al. (2001), patients with an initial sub-optimal response to ovarian stimulation who underwent an r-hFSH ‘step-up’ protocol required a mean cumulative dose >4000 IU. In contrast, women treated with r-hLH had a significantly lower consumption of r-hFSH. Thus, there is a subset of normogonadotrophic women who cannot be classified as either ‘poor responders’, because at least
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Outlook - LH in ovarian stimulation - C Alviggi et al.
Table 2. Studies testing recombinant-human LH (r-hLH) supplementation in normogonadotrophic adequately selected women*. Criterion for giving LH
Lisi et al., 2001
De Placido et al., 2001
De Placido et al., 2004
Ferraretti et al., 2004
De Placido et al., 2005
Groups
>3000 IU r-hFSH Self control r-hFSH requirement in (n = 12) r-hFSH + previous cycles r-hLH (75 IU) from day S7 Poor response to r-hFSH (n = 23) r-hFSH in the r-hFSH, then HMG 150 ongoing cycle1 IU from day 8 (n = 20) Normal responders2 (n = 40) Poor response to r-hFSH + r-hLH 150 IU r-hFSH in the from day 8 (n = 23) ongoing cycle1 r-hFSH + r-hLH 75 IU from day 8 (n = 23) Normal responders to r-hFSH2 (n = 46) Hypo-response to r-hFSH (n = 50) r-hFSH in the ongoing cycle3 r-hFSH + r-hLH (75– 150 IU) from day S10 (n = 54) r-hFSH + HMG from day S10 (n = 26) Normal responders to r-hFSH4 (n = 50) ‘Steady response’ r-hFSH (n = 58) to r-hFSH in the r-hFSH + r-hLH 150 IU ongoing cycle5 from day 8 (n = 59) Normal responders to r-hFSH6 (n = 112)
Statistically significant outcome differences
Patients who benefited from LH activity supplementation
Fertilization (%)
60.9a 86b
Clinical pregnancies (%)
50a 5.9b
Hypo-responders to r-hFSH in previous cycles
Peak oestradiol (pmol/ml)
4.3a 7.8b 8.9b
Oocytes retrieved (n)
5.9a 11.3b
Poor responders to r-hFSH in the ongoing cycle
11.8b Oocytes retrieved (n)
9.65a
79a
6.39b
65.7b
10.65a
82.5a
Implantation 14.1a (%) 36.8b
Oocytes retrieved (n)
Mature oocytes (%)
Pregnancy per 24.4b embryo transfer (%) 54.0a
7.4c
11.0b
35.4b
41.0ab
6.1a 9.0b 10.5c
Mature oocytes (n)
4.7a 7.8b
‘Steady responders’ to r-hFSH in the ongoing cycle
Hypo-responders to in the r-hFSH ongoing cycle
‘Steady responders’ in the r-hFSH ongoing cycle
9.0b
*
All studies are randomized controlled trials except that by Lisi et al. which is a ‘self-control’ study. Poor responders to r-hFSH = serum oestradiol concentrations ≤0.06 pmol/ml and no follicle >10 mm at ultrasound (USG) on day S8. 2 Normal responders to r-hFSH = tripling serum oestradiol concentrations between days S5 and S8, >4 follicles >10 mm at USG on day S8. 3 Hypo-responders to r-hFSH = >10 antral follicles ≥8 mm in diameter and oestradiol ≥100 pg/ml but no increase in the oestradiol concentration and in the follicular size between days S7 and S10. 4 Normal responders to r-hFSH = no need to increase the FSH dose during ovarian stimulation. 5 Steady responders to r-hFSH = more than five follicles, but no follicles >10 mm at the USG scan on day S8, oestradiol concentrations <180 pg/ml between days S5 and S8. 6 Normal responders to r-hFSH = tripling oestradiol concentrations between days S5 and S8, more than four follicles >10 mm on day S8. abc Values with different letters P < 0.05. r-h FSH = recombinant human FSH; HMG = human menopausal gonadotrophin. 1
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five oocytes are usually retrieved, or ‘normal responders’, because of the high cumulative r-hFSH requirement and reduced oocyte number and competence. These patients have a sub-optimal IVF outcome and seem to benefit from r-hLH administration. Thus, a clinical history of high r-hFSH consumption during ovarian stimulation should suggest the use of r-hLH-containing drugs for re-stimulation. In case of a first ovarian stimulation cycle, early identification of women who require a high r-hFSH dose may result in timely integration with r-hLH, which, in turn, may rescue the ovarian response and improve the ovarian IVF outcome, thereby avoiding a number of re-stimulations. Should these data be confirmed, the ovarian response to r-hFSH (in ongoing or previous cycles) may be a practical, reliable marker of women requiring r-hLH treatment.
0ATIENTS TREATED WITH 'N2( ANTAGONISTS GnRH antagonist (GnRH-ant) administration induces a fast and profound pituitary suppression, with a clear advantage in terms of premature LH surge avoidance. Nevertheless, LH activity is quickly and dramatically reduced in the phase in which this hormone activity is crucial: follicle which have been recruited in a physiological FSH and LH environment are dramatically deprived of their LH sustenance. Some authors (Cédrin-Durnerin n et al., 2004) have investigated the efficacy of an LH activity supplementation in GnRH-ant treated patients. They performed an RCT in 218 potential normal responders (age <38 years, no history of poor ovarian response). Ovarian stimulation was initiated with r-hFSH (150–300 IU/ day). Cetrorelix (3 mg, single dose) was given when at least one follicle >14–16 mm was found at ultrasound. Women of the group A received a daily injection of 75 IU r-hLH from the day of cetrorelix to HCG administration. Patients randomized to group B continued monotherapy with r-hFSH. Authors found a statistically significant increase in the oestradiol serum concentrations on the day of HCG in the r-hLH group (1476 ± 787 versus 1012 ± 659 pg/ml, P < 0.001). No statistically significant difference in any of the IVF outcome parameters was found between groups. Griesingerr et al. in 2005 performed an RCT in 127 normogonadotrophic infertile patients aged <39 years. Women were randomized in two groups on cycle day 2: group A received a daily dose of 150 IU of r-hFSH. Group B was treated with 150 IU of r-hFSH plus 75 IU of r-hLH per day. Cetrorelix (0.25 mg/ day) was administered from day S6 until HCG administration in all women. Primary endpoint was the number of days of gonadotrophin treatment, which was not shortened by LH administration. Serum oestradiol and LH concentrations on the day of HCG were significantly higher in the r-hLH-supplemented group (1924.7 ± 1256.4 versus 1488.3 ± 824.0 pg/ml, P <0.03, and 2.1 ± 1.4 versus 1.4 ± 1.5 IU/l, P <0.01 respectively). No other statistically significant difference in the outcome parameters was found. Taken together, these data seem to suggest that LH supplementation, in the form of r-hLH or HMG, may optimize follicle steroidogenesis in women treated with GnRH-ant. Nevertheless, current evidence is insufficient to evaluate the effect of such a treatment on implantation and pregnancy rates, at least in unselected patients.
Other groups have recently investigated the efficacy of LH activity supplementation in women at risk for poor ovarian response treated with GnRH-ant. Chung et al. (2005) conducted a retrospective
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cohort study in IVF patients who displayed less than four follicles in prior cycles and/or had basal FSH concentrations ranging between 10.1 and 18.0 IU/l. One hundred and forty one women receiving ganirelix (0.25 mg/day) when at least one follicle reached 12–14 mm until the day of HCG were evaluated. Patients were a posteriori divided in two groups according to gonadotrophins adopted. The first group consisted of 75 women stimulated with no LH activity supplementation. The other group included 66 subjects receiving LH activity supplementation in the form of HMG. Patients who did not receive LH activity supplementation displayed a significantly higher number of oocytes retrieved (8.8 ± 0.6 versus 6.6 ± 0.4, P = 0.004). Interestingly, when women were stratified on the basis of age, in those aged ≥40 years a significantly lower number of oocytes retrieved was observed in the HMG group (8.6 ± 0.6 versus 6.4 ± 0.4, P = 0.003). Similarly, the number of 2 pronucleate embryos was significantly lower in the LH activity supplemented women (5.2 ± 0.5 versus 3.8 ± 0.3, P = 0.03). In contrast, no significant difference in the outcome parameters was found in the younger patients. Although admitting the observational character of the study, the authors concluded that exogenous LH is not essential or beneficial in poor responders who are being treated with GnRH-ant, and could even be detrimental in older patients with a history of poor response. De Placido et al. (2006) recently conducted an RCT to test the efficacy of an LH activity supplementation to a flexible GnRHant protocol in patients at risk of poor ovarian response, having as control reference standard the GnRH-a flare up protocol. A total of 133 patients at risk for poor ovarian responsiveness (basal FSH >9 IU/l and/or age >37 years) were randomized in two groups. All the patients received a daily dose of r-hFSH of 300 IU starting on the second day of the cycle. In the antagonist group (n = 67), 0.125 mg/day of the GnRH-ant cetrorelix were administered for 2 days, starting when at least one follicle ≥14 mm was present; thereafter the GnRH-ant full dose of 0.25 mg/day was administered until the day of the HCG administration. Starting on the same day of GnRH-ant administration, a daily dose of 150 IU of r-hLH was also added until the day of the HCG. Controls (agonist group, n = 66) received daily a dose of triptorelin of 0.1 mg subcutaneously starting on the same day of r-hFSH administration. A dose of 150 IU/day of r-hLH was added when at least one follicle reached 14 mm. No premature LH surge was observed. No significant difference was observed in the total number of oocytes retrieved between groups (6.7 ± 2.5 versus 7.1 ± 2.9). A statistically significant difference was found in the number of mature oocytes (5.1 ± 2.2 in the group A and 4.2 ± 1.7 in group B, P <0.01). Thus, a stimulation regimen providing gradual increase in the GnRH-ant dose, in association with exogenous LH activity in form of r-hLH, seems to be able to significantly improve the ovarian response in women at risk for poor ovarian response, when compared with standard GnRH-a short protocol. This finding may be due to the fact that the study protocol offers a quite physiological and stable hormonal environment.
#ONCLUSIONS Physio-endocrinological observations indicate a key role for LH in follicle growth and maturation, particularly in the selection of the dominant follicle. Nevertheless, clinical data from ovarian stimulation practice seem to suggest that most
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Outlook - LH in ovarian stimulation - C Alviggi et al.
normogonadotrophic women achieve adequate multi-follicular growth by means of administration of FSH alone. In such women, after pituitary down-regulation, the residual amount of LH seems to be capable of sustaining the local follicular activities needed for growth and dominance. In other patients, lack of LH activity after GnRH-a down-regulation seems to compromise multiple follicular growth, usually resulting in an ‘idiopathic’ poor response to recombinant FSH. Those patients are in most cases treated with an increase in the FSH daily dose. Several RCT have demonstrated that these adequately selected hypo-responders to standard FSH monotherapy may benefit from LH activity supplementation, which seems to be more effective than over-consumption of FSH vials. Clinical response to r-hFSH more than basal LH concentration seems to be predictive of exogenous LH requirement during ovarian stimulation (De Placido et al., 2001, 2004, 2005), supporting the idea that a discrepancy between immuno-reactive and bioactive LH may exist (Huhtaniemi et al., 1999; Jiang et al., 1999; Ropelato et al., 1999). This observation is also consistent with data from Humaidan n et al. (2002), who stratified women undergoing ovarian stimulation according to LH concentration after pituitary downregulation with a GnRH-a. The authors found that patients with LH concentrations ≥1.51 IU/l had significantly lower fertilization and pregnancy rates than women with concentrations between 0.5 and 1.5 IU/l. In a subsequent study, the same group (Humaidan et al., 2004) showed that women with higher LH concentrations (i.e. >1.99 IU/l) benefited from LH activity supplementation. The hypothesis that carriers of a less bio-active LH may require higher gonadotrophin doses and/or benefit from LH activity supplementation during ovarian stimulation is supported by other lines of evidence. In this context, an association between ovarian resistance to r-FSH monotherapy and presence of an LH polymorphism (V-beta LH) in normogonadotrophic women undergoing GnRH-a long protocol has been recently suggested (Alviggi et al., 2005). Meta-analysis of RCT comparing r-hFSH monotherapy with FSH-HP in women undergoing standard GnRH-a long protocol has been published by Daya in 2002. The author concluded that patients treated with r-hFSH had a significantly higher pregnancy rate. A similar observation was also reported by De Placido et al. (2000) in poor responders. Taken together, these findings seem to suggest that exiguous LH contamination of the FSHHP preparations exerts detrimental effects on follicular/oocyte maturation. This hypothesis is not consistent with data indicating that LH activity supplementation may be useful, at least in some normogonadotrophic women (De Placido et al. 2001, 2004, 2005; Ferraretti et al., 2004). Possible explanations for these discrepancies may be related to the fact that RCT examined by Daya were performed in unselected normogonadotrophic patients. Conversely, in those by De Placido et al. and by Ferraretti et al., LH activity and r-hLH were administered in women selected on the basis of their initial response to r-hFSH. Moreover, the study published by De Placido et al. was not comparable with these RCT due to both methodological aspect and study population. In fact, it was a self control study performed in poor responder to FSH-HP who were re-stimulated with r-FSH. In addition, only bad prognosis subjects had been included (i.e. high basal FSH and pathological response to exogenous FSH stimulation test). Finally, although low LH amounts may be present in FSH-HP, it should not be comparable with whole LH activity contained in both r-hLH and HMG.
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A well-known condition associated with scarce follicular recruitment is advanced reproductive age. Recent data indicate that r-hLH supplementation is able significantly to improve ovarian response to r-hFSH in women aged ≥35 years (Humaidann et al., 2004; Marrs et al., 2004). This clinical evidence may be linked to an age dependent decrease in the number of functional LH receptors and/or in the biological activity of endogenous LH, which in turn may increase resistance to LH-mediated processes (Mitchell et al., 1995; Vihko et al., 1996). Another possible explanation may be related with models of human folliculogenesis suggesting that LH activity is locally enhanced by paracrine activities, including growth factors and cytokines. These variables may account for the maintenance of an adequate follicular growth and steroidogenesis even when LH concentrations are very low. There is evidence that ovarian paracrine activity also decreases with age (Hurwitz and Santoro, 2004). Another subset of women who can benefit from LH activity supplementation are those treated with GnRH-ant. In these patients, a peculiar pattern of pituitary suppression is observed (Ganirelix Dose-Finding Study Group, 1998). More specifically, a rapid decline of serum concentrations of both LH and oestradiol usually follows the administration of the drug. Some authors found that in normal responders undergoing GnRH-ant, oestradiol production is significantly higher when exogenous LH, in form of both r-hLH (Cédrin-Durnerin n et al., 2004) and HMG (Griesinger et al., 2005) is added to r-hFSH. Nevertheless, both studies failed to demonstrate any other significant improvement in IVF outcome between women receiving r-hFSH monotherapy and those treated with r-hFSH and LH supplementation. In women at risk of poor ovarian response, Chung et al. (2005) even found a worse IVF outcome when LH activity was added. In contrast, recent data (De Placido et al., 2006) showed an improved IVF outcome when a flexible protocol with GnRH-ant and r-hLH supplementation was employed in women at risk of poor ovarian response, when compared with the standard flare-up protocol. Discrepancies between trials may be due to methodological issues, including study design and different selection criteria. In conclusion, available data seem to suggest that LH activity is able to improve steroidogenesis in women undergoing GnRHant pituitary suppression. In addition, the establishment of a more regular and physiological LH environment may lead to improved oocyte maturation in women at risk for poor ovarian response. Nevertheless, the effect of such treatment on implantation and pregnancy rates should be evaluated in larger RTC. The advent of recombinant gonadotrophins gave the chance to test the differential actions of exogenous FSH and LH, when given together or independently. There is now concrete evidence that r-hLH supplementation improves ovarian response to ovarian stimulation in at least three adequately selected subgroups of patients; (i) hypo-responders to r-hFSH; (ii) advanced reproductive age women; (iii) patients at risk for poor responsiveness treated with GnRH antagonists (Table 3).
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Outlook - LH in ovarian stimulation - C Alviggi et al.
Table 3. Subgroups of women needing LH. Subgroup
Studiesa
Group characteristics
Hypo-responders to r-hFSH
De Placido et al., 2001, 2004, 2005; Ferraretti et al., 2004
Women with an initial response (i.e. at least five 2–9 mm follicles in each ovary) during the first days of stimulation followed by a plateau in which there is no significant increase in follicular size or oestradiol production in the next 3–4 days of stimulation Women who required >3000 IU to reach follicular maturity in previous cycles Patients aged ≥35 years
Lisi et al., 2001 Advanced reproductive age GnRH antagonists treated
Marrs et al., 2004; Humaidan et al., 2004 De Placido et al., 2006b
Patients showing basal FSH >9 IU/l and/or age >37 years, treated with a GnRH-antagonist flexible protocol
r-h FSH = recombinant human FSH; GnRH = gonadotrophin-releasing hormone. a For details of all study designs and results except De Placido et al., 2006, see Tables 1 and 2. b RCT on 133 patients at risk for poor ovarian responsiveness (basal FSH >9 IU/l and/or age >37 years). Antagonist group (n = 67): 0.125 mg/day of GnRH-antagonist for 2 days, starting when at least one follicle ≥14 mm was present; thereafter the GnRH- antagonist full dose of 0.25 mg/day until the day of the HCG. Starting on the same day of GnRH- antagonist, a daily dose of 150 IU of r-hLH was also added until the day of the HCG. Agonist group (n = 66): flare-up protocol with daily triptorelin 0.1 mg; 150 IU/day of r-hLH were added when at least one follicle reached 14 mm. Statistically significant higher number of mature oocytes (primary endpoint) in the antagonist group (5.1 ± 2.2 versus 4.2 ± 1.7, P <0.01) was observed.
2EFERENCES
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an initial inadequate response to recombinant FSH (rFSH) after pituitary downregulation. Clinical Endocrinology (Oxford) 60, 637–643. De Placido G, Mollo A, Alviggi C et al. 2001 Rescue of IVF cycles by HMG in pituitary down-regulated normogonadotrophic young women characterized by a poor initial response to recombinant FSH. Human Reproduction 16, 1875–1879. De Placido G, Alviggi C, Mollo A et al. 2000 Recombinant follicle stimulating hormone is effective in poor responders to highly purified follicle stimulating hormone. Human Reproduction 15, 17–20. Drakakis P, Loutradis D, Kallianidis K et al. 2005 Small doses of LH activity are needed early in ovarian stimulation for better quality oocytes in IVF−ET. European Journal of Obstetrics and Gynecology and Reproductive Biology 121, 77–80. Erickson GF, Wang C, Hsueh AJW 1979 FSH induction of functional LH receptors in granulosa cells cultured in a chemically defined medium. Nature 279, 336–338. Esposito MA, Barnhart KT, Coutifaris C et al. 2001 Role of periovulatory luteinizing hormone concentrations during assisted reproductive technology cycles stimulated exclusively with recombinant follicle-stimulating hormone. Fertility and Sterility 75, 519–524. Ferraretti AP, Gianaroli L, Magli MC et al. 2004 Exogenous luteinizing hormone in controlled ovarian hyperstimulation for assisted reproduction techniques. Fertility and Sterility 82, 1521–1526. Fevold H 1941 Synergysm of follicle stimulating and luteinizing hormones in producing estrogen secretion. Endocrinology 28, 33–36. Filicori M, Cognigni GE, Pocognoli P et al. 2003a Current concepts and novel applications of LH activity in ovarian stimulation. Trends in Endocrinology and Metabolism 14, 267–273. Filicori M, Cognigni GE, Ciampaglia W 2003b What clinical evidence for an LH ceiling? Human Reproduction 18, 1556–1557. Filicori M, Cognigni GE, Taraborrelli S et al. 2002 Intracytoplasmic sperm injection pregnancy after low-dose human chorionic gonadotrophin alone to support ovarian folliculogenesis. Fertility and Sterility 78, 414–416. Fleming R, Lloyd F, Herbert M et al. 1998 Effects of profound suppression of luteinizing hormone during ovarian stimulation on
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preponderance of basic luteinizing hormone (LH) isoforms accompanies inappropriate hypersecretion of both basal and pulsatile LH in adolescents with polycystic ovarian syndrome. Journal of Clinical Endocrinology and Metabolism 84, 4629– 4636. Shima K, Kitayama S, Nakano R 1987 Gonadotropin binding sites in human ovarian follicles and corpora lutea during the menstrual cycle. Obstetrics and Gynecology 69, 800–806. Shoham Z, Schacter M, Loumaye E et al. 1995 The luteinizing hormone surge – the final stage in ovulation induction: modern aspects of ovulation triggering. Fertility and Sterility 64, 237–251. Sills ES, Levy DP, Moomjy M et al. 1999 A prospective, randomized comparison of ovulation induction using highly purified folliclestimulating hormone alone and with recombinant human luteinizing hormone in in-vitro fertilization. Human Reproduction 14, 2230–2235. Van Wely M, Westergaard LG, Bossuyt PM et al. 2003 Human menopausal gonadotropin versus recombinant follicle stimulation hormone for ovarian stimulation in assisted reproductive cycles. Cochrane Database of Systematic Reviews 1, CD003973. Vihko KK, Kujansuu E, Morsky P et al. 1996 Gonadotropins and gonadotropin receptors during the perimenopause. European Journal of Endocrinology 134, 357–361. Westergaard LG, Erb K, Laursen SB et al. 2001 Human menopausal gonadotropin versus recombinant follicle-stimulating hormone in normogonadotropic women down-regulated with a gonadotropinreleasing hormone agonist who were undergoing in vitro fertilization and intracytoplasmic sperm injection: a prospective randomized study. Fertility and Sterility 76, 543–549. Westergaard LG, Laursen SB, Andersen CY 2000 Increased risk of early pregnancy loss by profound suppression of luteinizing hormone during ovarian stimulation in normogonadotrophic women undergoing assisted reproduction. Human Reproduction 15, 1003–1008. Willis DS, Watson H, Mason HD et al. 1998 Premature response to luteinizing hormone of granulosa cells from anovulatory women with polycystic ovary syndrome: relevance to mechanism of anovulation. Journal of Clinical Endocrinology and Metabolism 83, 3984–3991. Zeleznik AJ, Hillier SG 1984 The role of gonadotropins in the selection of the preovulatory follicle. Clinical Obstetrics and Gynecology 27, 927–940. Zhou J, Bondy C 1993 Anatomy of the human ovarian insulin-like growth factor system. Biology of Reproduction 48, 467–482.
Paper based on contribution presented at the International Serono Symposium ‘How to improve ART outcome by gamete selection’ in Gubbio, Italy, October 7–8, 2005. Received 15 November 2005; refereed 6 December 2005; accepted 6 February 2006.
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/UTLOOK 7HO NEEDS ,( IN OVARIAN STIMULATION $R #ARLO !LVIGGI OBTAINED THE -$ SPECIALTY IN /BSTETRICS AND 'YNAECOLOGY AND 0H$ DEGREES AT THE &ACULTY OF -EDICINE 5NIVERSITY OF .APLES @&EDERICO )) )TALY (E HAS COLLABORATED WITH VARIOUS DEPARTMENTS IN )MPERIAL #OLLEGE ,ONDON THE )TALIAN .ATIONAL 2ESEARCH #OUNCIL .APLES THE 5NIVERSITY OF #ALIFORNIA ,OS !NGELES AND THE 5NIVERSITY OF !THENS 'REECE 4HIS COLLABORATIVE NETWORK RESULTED IN VARIOUS PUBLICATIONS INCLUDING NEW HYPOTHESES ON THE PATHOGENESIS OF PELVIC ENDOMETRIOSIS (E HAS SPECIALIZED IN REPRODUCTIVE MEDICINE AT THE &ERTILITY 5NIT OF THE 5NIVERSITY OF .APLES @&EDERICO )) $R !LVIGGI HAS PUBLISHED EXTENSIVELY AND GIVEN MANY INVITED LECTURES AT INTERNATIONAL MEETINGS IN HIS l ELD AS WELL AS SERVING AS AD HOC REVIEWER FOR INTERNATIONAL JOURNALS $R !LVIGGIS CURRENT RESEARCH INTERESTS INCLUDE THE PATHOGENESIS OF PELVIC ENDOMETRIOSIS AND THE GENETICS OF HUMAN REPRODUCTION $R #ARLO !LVIGGI # !LVIGGI 2 #LARIZIA ! -OLLO ! 2ANIERI ' $E 0LACIDO $IPARTIMENTO DI 3CIENZE /STETRICHE 'INECOLOGICHE 5ROLOGICHE E -EDICINA DELLA 2IPRODUZIONEn5NIVERSITÌ DEGLI 3TUDI DI .APOLI @&EDERICO )) .APLES )TALY #ORRESPONDENCE VIA 3 0ANSINI .APLES )TALY 4EL &AX E MAIL ALVIGGI UNINAIT
!BSTRACT LH plays a key role in the intermediate−late phases of folliculogenesis. Although ovarian stimulation is efficiently achieved in most cases by the administration of exogenous FSH alone, specific subgroups of women may benefit from LH activity supplementation during ovarian stimulation. Some authors have found improved outcome with LH activity supplementation in advanced reproductive age women. Experience suggests that in about 10−12% of young normogonadotrophic patients treated with a gonadotrophin-releasing hormone agonist (GnRH-a) long protocol plus recombinant FSH human (r-hFSH), a ‘steady response’ is observed. In this subgroup of women, a higher number of oocytes is retrieved when daily LH activity supplementation is given from stimulation day 8, if compared with the standard FSH dose increase. Another subgroup of patients who may benefit from LH activity supplementation are those at risk for poor ovarian response treated with GnRH antagonist. Recent data demonstrate that in these women, when GnRH is administered in a flexible protocol, the concomitant addition of recombinant human LH improves the number of mature oocytes retrieved, when compared with the standard GnRH-a flare-up protocol. Thus, well calibrated LH administration improves the ovarian outcome in patients >35 years, in those showing an initial abnormal ovarian response to r-hFSH monotherapy, and in ‘low prognosis’ women treated with GnRH antagonists. Keywords: assisted reproductive techniques, GnRH antagonists, LH, ovarian stimulation, recombinant gonadotrophins
,( AND PHYSIOLOGICAL FOLLICLE GROWTH According to the ‘two cells−two gonadotrophins’ model (Fevold, 1941; Hillierr et al., 1994), LH exerts its activity in theca cells, which form the involucres of the growing follicles and express enzymatic pathways of androgen synthesis. Theca involucres surround the granulosa cells, whose activities and proliferation are directly regulated by FSH. This hormone induces the expression of the aromatase enzyme, which in turn converts theca-deriving androgens into oestradiol. This theory reinforced the notion that granulosa and theca cells are distinct compartments regulated by FSH and LH respectively. However, it was subsequently found that LH receptors are detectable on the granulosa compartment at the intermediate follicular phase (Erickson et al., 1979; Shima et al., 1987; Hillierr et al., 1994; Filicori et al., 2003a), at a time when blood concentrations of LH increase. Therefore, it appears that LH regulates both granulosa and theca cells.
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FSH and LH induce the local production of the soluble molecule inhibin B and growth factors. Among these, insulinlike growth factors (IGF)-I and II, which are expressed by both granulosa and theca cells throughout folliculogenesis, are important in promoting follicular maturation (Zhou and Bondy, 1993; Huang et al. 1994). Thus, gonadotrophins and the autocrine−paracrine system contribute to the complex mechanisms governing follicular growth and selection. The finding that both gonadotrophins also regulate granulosa cell activity suggests that LH is involved in inducing and maintaining this paracrine system of biochemical factors by acting on the theca and granulosa compartments. These findings may explain the observation that FSH activity can be totally substituted by LH once granulosa cells express adequate amounts of LH receptors (Zeleznik and Hillier, 1984; Filicori et al., 2002). Hence, LH seems to play two roles during folliculogenesis. One is exerted in the theca compartment and consists of induction of
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Outlook - LH in ovarian stimulation - C Alviggi et al.
androgen production. The second begins during the intermediate follicular phase (Willis et al., 1998; Filicori et al., 2003a), involves granulosa cells, and consists of inducing the local production of various molecules. These factors promote the growth of granulosa cells, which in turn regulate oocyte maturation. These two mechanisms are closely related and probably support each other. According to the so-called ‘spare receptor hypothesis’ (Chappel and Howles, 1991), at a time when inhibin B and IGF-1 are adequately secreted, androgen synthesis and release are optimal even with <1% of LH receptors occupied.
paracrine activities, including the production of inhibins and IGF-1, may favour adaptation mechanisms by enhancing theca sensitivity to LH. In this context, a low LH environment would be advantageous, whereas during stimulation, LH-induced suppression of small follicles is not required. Furthermore, during the early−intermediate stages of follicular growth, low LH concentrations have been associated with a more physiological endometrial proliferation, which in turn seems to synchronize this compartment for successive embryo implantation (Kolibianakis et al., 2004).
LH has been implicated in a third process during folliculogenesis. In particular, it has been suggested that this hormone may contribute to negatively select non-dominant follicles. This idea was initially based on the observation that, following the mid-cycle LH surge, granulosa cell mitosis is blocked, and oocyte meiosis is resumed (Shoham m et al., 1995). Preclinical evidence showed that developing follicles have specific requirements for exposure to LH beyond which normal maturation ceases (Hillierr et al., 1994). This finding gave rise to the concept of an ‘LH ceiling’, meaning that each follicle would have an upper limit of stimulation. The LH ceiling may be higher in larger follicles and lower in smaller ones. As a consequence, an increasing LH concentration would promote leading follicle progression (being below its ceiling) and degeneration of secondary ones (by overcoming their ceiling). According to others, a dynamic interplay between LH secretion and receptor expression by different ovarian compartments governs the selection of dominant follicles; because small follicles in granulosa cells do not express LH receptors, LH may indirectly promote their degeneration (Filicori et al., 2003a,b).
Surprisingly, during ovarian stimulation, a low LH environment seems to be appropriate also during more advanced stages of folliculogenesis, when LH-dependent paracrine activities seem to be crucial for follicular growth and oocyte maturation. Also in this case, FSH may be important. In fact, it may make granulosa cells more responsive to low LH concentrations by enhancing their expression of LH receptors. Furthermore, this supraphysiological FSH environment may also balance the lack of LH by inducing compensatory paracrine activities in granulosa cells.
,( AND OVARIAN STIMULATION The gold standard for ovarian stimulation in young, normogonadotrophic women is the gonadotrophin-releasing hormone agonist (GnRH-a) long protocol (Hughes et al., 1992). Exogenous FSH is administered only when a GnRH-a mediated suppression of the hypothalamus−pituitary−gonad axis is achieved. Moreover, monotherapy with recombinant human FSH (r-hFSH), which is free of LH activity, is used in most cases. The degree of pituitary suppression also depends on the GnRH-a formulation, dose and mode of administration (Westergaard d et al., 2001). Following post-suppression decline, LH concentrations usually range between 0.5 and 2.5 IU/l. These concentrations often fall by <0.5 IU/l during the intermediate late stages of stimulation. Thus, multiple follicular growth is often induced in a low endogenous LH environment. Nevertheless, an adequate ovarian response is achieved in almost all patients.
A hiatus appears to exist between IVF practice and information deriving from folliculogenesis models and hypogonadotrophic hypogonadism. In particular, in the former situation, follicular growth normally occurs even when LH concentrations are below 0.5 IU/l. In contrast, there is evidence that in women with hypogonadotrophic hypogonadism, LH concentrations of 1.2 IU/l are required in order to achieve adequate ovarian response to r-hFSH. These discrepancies can be attributed to biological events that involve steroidogenic dynamics and follicular growth. Regarding stimulated cycles, the above-mentioned ‘spare receptor hypothesis’ (Chappel and Howles, 1991) can be invoked: even when pituitary desensitization is achieved, resting circulating concentrations of LH are able to occupy an adequate percentage of receptors and to elicit sufficient androgen release. FSH-dependent
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2ELATIONSHIPS BETWEEN ,( SERUM CONCENTRATIONS AND OVARIAN)6& OUTCOME Some observational trials (Fleming et al., 1998; Westergaard d et al., 2000; Balasch h et al., 2001; Esposito et al., 2001; Humaidan et al., 2002) with r-hFSH have examined the relationship between LH serum concentrations and ovarian/IVF outcome in normogonadotrophic women undergoing ovarian stimulation in a GnRH-a long protocol. Univocal LH cut-off value able to identify women requiring LH activity supplementation could not be found by those studies. Differences in patient selection criteria, clinical end-points, serum LH assays, and LH cut-off value may account for discrepancies among results. Hence, there is a need for adequately sized, prospective observational trials.
%XOGENOUS ,( AND OVARIAN)6& OUTCOMES IN UNSELECTED PATIENTS There is a large body of data on r-hFSH monotherapy versus combined exogenous FSH and LH [in the form of human menopausal gonadotrophin (HMG) or recombinant molecule] in women undergoing the GnRH-a long protocol. Van Wely et al. (2003) conducted a meta-analysis of four randomized controlled trials (RTC) comparing r-hFSH and HMG treatment in normogonadotrophic patients undergoing a GnRH-a long protocol. The authors failed to show any statistically significant difference in the main outcome measures (ongoing pregnancy rates and live birth rate). Three RTC of FSH versus FSH plus r-hLH regimens in normogonadotrophic women undergoing the GnRH-a long protocol have been published (Table 1). Sills et al. (1999) studied 31 cycles in 30 women (age 30–41 years). Seventeen patients received 150–450 IU of highly purified FSH (FSHHP). The remaining 13 women (14 cycles) were stimulated with
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the same dose of FSH-HP plus one ampoule of r-hLH (75 IU) from the first day of stimulation (S1). There was no significant difference in any of the outcome parameters. A large multicentre, randomized trial was published by Marrs et al. in 2004. A total of 431 intracyoplasmic sperm injection (ICSI) patients (age 18−40 years) undergoing the mid-luteal GnRH-a long protocol were randomized to receive either 225 IU/day of r-hFSH (n = 219) or the same dose of r-hFSH plus r-hLH (150 IU/day, from day S6; n = 212). No significant difference in the number of metaphase II oocytes retrieved (primary end-point) or in the cumulative pregnancy rate was found. In contrast, the mean number of embryos transferred was significantly higher in the r-hLH supplemented group (2.9 ± 0.6 versus 2.8 ± 0.7, P < 0.05). Humaidan et al. (2004) studied the effects of LH activity supplementation in 231 normogonadotrophic women, aged <40 years. Stimulation was initiated with r-hFSH (150–300 IU): from day S8, patients were randomized to continue r-hFSH monotherapy (n = 115) or to receive r-hFSH plus r-hLH in a ratio of 2:1 (n = 116). Also in this instance, the first cumulative analyses failed to show any significant difference in terms of endocrinology, ovarian response and pregnancy outcome between groups. It should be stressed that in those trials, the daily r-hLH dose ranged between 75 and 150 IU in almost all patients. These clinical observations, together with pharmacokinetic studies (le Cotonnec et al., 1998a,b), indicate the need for RCT to evaluate the efficiency of higher doses.
In conclusion, cumulative data analysis is insufficient to prove that LH administration is associated with any significant improvement in the IVF/ICSI outcome parameters, including implantation and pregnancy rates, when compared with r-hFSH monotherapies, at least in unselected groups.
Drakakis et al. (2005), in an RCT (n = 46), has recently tested the effects of LH-activity supplementation (in the form of HMG in young normogonadotrophic women undergoing standard GnRH-a long protocol. Patients in group A (n = 22) received a starting dose of 200 IU of r-hFSH/day. In group B (n = 24), ovarian stimulation began with one ampoule/day of HMG in association with a daily dose of 150 IU of r-hFSH. Patients in group B showed a P <0.05) in the mean number of statistically significant increase (P mature oocytes (7.3 ± 2.9 versus 10.7 ± 3.4), fertilized oocytes (6.5 ± 1.8 versus 7.9 ± 3.0), and transferable embryos (6.0 ± 2.8 versus 7.7 ± 3.1). Nevertheless, previous meta-analysis of RCT (Van Wely et al., 2003) comparing HMG stimulation with r-hFSH monotherapy in women undergoing GnRH-a long protocol failed to show any significant difference in ovarian and IVF outcomes between protocols.
Although based on a-posteriori stratification processes, the above data identify at least one subset of women, those aged >35 years, who may benefit from LH activity supplementation. In fact, there is evidence that ovarian paracrine activity decreases with age (Hurwitz and Santoro, 2004).
%XOGENOUS ,( AND OVARIAN)6& OUTCOMES IN ADEQUATELY SELECTED PATIENTS 7OMEN OF ADVANCED REPRODUCTIVE AGE Marrs et al. (2004) stratified their patients by age and number of previous treatment cycles. With an intention-to-treat adjustment, the clinical pregnancy rate was significantly higher (P P < 0.05) in the r-hFSH plus r-hLH group in patients aged ≥35 years at their first assisted reproduction cycle. In addition, Humaidan et al. (2004) stratified their study population according to age. Among women who did not receive rLH supplementation, pregnancy and implantation rates were significantly lower in those ≥35 years (P P < 0.05); this difference did not occur in the r-hLH supplemented group.
7OMEN WITH AN ABNORMAL RESPONSE TO R H&3( Clinical evaluation of the ongoing ovarian response seems to be an accurate parameter for indicating the use of LH. Several groups have studied the efficacy of LH activity supplementation in women selected according to specific profiles of ovarian response to standard r-hFSH doses (Table 2). In 2001, Lisi et al. conducted
Table 1. Randomized controlled trials testing recombinant-human (r-hLH) supplementation in normogonadotrophic unselected women. Trial
Study design
Pituitary downregulation
Groups
Results
Sills et al., 1999
Prospective, randomized
GnRH-a daily from mid-luteal phase
No significant differences
Marrs et al., 2004
Multicentre, prospective
GnRH-a daily from mid-luteal phase
150–450 IU/day of FSH-HP (n = 17) versus FSH-HP plus 75 IU/day r-hLH throughout ovarian stimulation (n = 14) 225 IU/day of r-h-FSH (n = 219) versus r-hFSH plus 150 IU/day r-hLH from day S6 (n = 212)
Humaidan et al., 2004
Prospective, randomized
GnRH-a daily from mid-luteal phase
150–300 IU day of r-h-FSH (n = 115) versus r-hFSH plus r-hLH (2:1 ratio) from day S8 (n = 116)
Mean number of embryos transferred significantly higher in the r-hLH supplemented group No significant differences
GnRH-a = gonadotrophin-releasing hormone agonist; FSH-HP = highly purified FSH.
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a ‘self-control study’ of the effect of r-hLH supplementation in 12 patients who, during previous stimulation with r-hFSH, required >3000 IU to reach follicular maturity. Re-stimulation entailed the addition of 75 IU of r-hLH from day S7 to the standard r-hFSH regimen. As in the first cycle, all women received triptorelin 0.1 mg daily, from the mid-luteal phase (GnRH-a long protocol). There was no difference in the total consumption of r-hFSH, days of stimulation or number of MII oocytes per patient between the two cycles of the study. However, the incidence of fertilization (86.0 versus 60.9%) and clinical pregnancies (50.0 versus 5.9%) was significantly higher (P P <0.05) in r-hLH supplemented cycles. De Placido et al. (2001) found that in about 10−12% of normogonadotrophic patients, an initial response (i.e. at least five 2−9 mm follicles in each ovary) during the first days of stimulation is followed by a plateau in which there is no significant increase in follicular size or oestradiol production in the next 3−4 days of stimulation. This profile of initial ovarian response to r-hFSH is referred to as ‘steady response’ (De Placido et al., 2005), and usually leads physicians to increase the r-hFSH dose. De Placido and colleagues (2001) conducted a prospective randomized trial to determine whether this clinical condition could be related to excessive pituitary suppression and to impairment of LHdependent mechanisms. Women (age <37 years, basal FSH ≤10 IU/l) who had no follicle with a mean diameter >10 mm and oestradiol serum concentrations ≤180 pg/ml on day S8 were randomized to receive LH activity supplementation (n = 20) in the form of HMG (150 IU/day) or an increase in the r-hFSH daily dose (maximum daily dose of 375 IU; n = 23). In order not to modify the daily FSH administration, the r-hFSH dose was reduced to 150 IU in women of the HMG group. Forty women matched for age and body mass index (BMI) and with an initial adequate response to r-hFSH (i.e. a tripling of serum oestradiol concentration between days S5−8 in association with more than four follicles >10 mm on day S8) served as a non-randomized control population. All women received triptorelin 3.75 mg (depot preparation) on day 1 of spontaneous menstruation. After pituitary desensitization, a starting dose of 300 IU of r-hFSH was administered. First dose adjustment was performed on day S5. The mean number of oocytes retrieved was significantly higher in women treated with HMG supplementation than in those who received r-hFSH ‘step-up’. Moreover, the ovarian outcome of the HMG group was comparable with that observed in ‘normal responders’, suggesting that LH activity supplementation was able to ‘rescue’ this apparently abnormal response to r-hFSH. Interestingly, when serum LH concentrations were measured on day S8, there was no statistically significant difference between women whose ovarian outcome improved with HMG and normal responders to r-hFSH. These findings revealed the existence of a subgroup of hypo-responders who benefit more from LH activity supplementation than from an increase in the daily r-hFSH dose.
In a preliminary dose-finding design, De Placido et al. (2004) evaluated the efficacy of r-hLH supplementation in women displaying an initial ‘steady response’ to r-hFSH. GnRH-a administration and the criteria for ‘steady-response’ were identical to those used in the earlier study (De Placido et al., 2001). Patients (age <37 years, basal FSH ≤10 IU/l) received a starting r-FSH dose ranging between 150 and 300 IU/day. Women displaying a steady response were randomized on day 8 to receive a daily r-hLH dose of 75 (n = 23) or 150 IU (n = 23). The control population consisted of ‘normal’ responders to r-hFSH (n = 46). The mean number of oocytes retrieved (primary end-point) and
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the percentage of mature oocytes in women treated with 150 IU of r-hLH (9.65 ± 2.16, 79.0%) were similar to those observed in ‘normal responders’ (10.65 ± 2.8, 82.5%), and were significantly higher than those of subjects receiving 75 IU [6.39 ± 1.53, 65.7% (P <0.001 and P <0.05 respectively)]. The effectiveness of r-hLH in ‘steady responders’ was then evaluated in a larger multicentre RTC (De Placido et al., 2005) with the r-hFSH ‘step-up protocol’ as reference standard. A total of 229 IVF/ICSI cycles performed in seven Italian units were analysed. In all patients (age <37 years, basal FSH ≤10 IU/l), triptorelin 3.75 mg (depot preparation) was administered on day 1 of spontaneous menstruation. The starting dose of r-hFSH was 225 IU/day. ‘Steady responders’ were identified on day 8 (oestradiol serum concentrations <180 pg/ml and at least six follicles ranging between 6 and 10 mm, but no follicle with a mean diameter >10 mm) and randomized to receive either r-hLH supplementation of 150 IU/day (n = 59), or an increase of 150 IU in the daily r-hFSH dose (n = 58; r-hFSH ‘step-up’ protocol). Also in this case, an age/BMI-matched population of ‘normal responders’ (tripling oestradiol concentrations between days S5 and S8, more than four follicles >10 mm on day S8) was selected as a control group (n = 112). The number of cumulus−oocyte complexes (primary end-point) and mature oocytes retrieved was significantly higher in women receiving r-hLH than in those treated with the r-hFSH step-up protocol. Moreover, the mean number of mature oocytes of r-hLH group was similar to that observed in ‘normal responders’. Also in this study, endogenous LH serum concentrations on day S8 (before randomization) did not differ between ‘steady responders’ undergoing r-hLH supplementation and normal responders (median: 0.7, range: 0.1–3.6 IU/l; median: 0.7 range: 0.1–4.0 IU/l respectively). Ferraretti et al. (2004) conducted an RCT on 184 patients (age <38 years) undergoing the GnRH-a long protocol. Patients with normal initial follicular recruitment (>10 antral follicles ≥8 mm in diameter and oestradiol ≥100 pg/ml) with the fixed starting dose of recombinant FSH (150–300 IU), but showing a plateau in follicular growth between days S7 and S10 (no increase in follicle size or in oestradiol concentration) were randomized as follows: group A (n = 54) received an increase in the daily dose of r-hFSH; group B (n = 54) received 75–150 IU of r-hLH in addition to the increased dose of FSH; group C (n = 26) received HMG; group D consisted of 54 age-matched patients with an optimal response (no need to increase the FSH dose). The mean number of oocytes retrieved was significantly lower in group A (8.2) versus the other groups (11.1, 10.9, 9.8 in groups B, C, and D respectively). Furthermore, the pregnancy-per-embryo transfer and implantation rates were significantly higher in group B than in groups A and C, and did not differ from normal responders. Consistent with other studies (De Placido et al., 2001, 2004, 2005), there were no significant differences in LH circulating concentrations on day S7 among groups (0.99 ± 0.7, 1.02 ± 0.9, 1.3 ± 1.0, 0.93 ± 0.6 mIU/ml, in the four groups respectively). It should be noted that in this study, in those by De Placido et al. (2001, 2004, 2005) and in that reported by Lisi et al. (2001), patients with an initial sub-optimal response to ovarian stimulation who underwent an r-hFSH ‘step-up’ protocol required a mean cumulative dose >4000 IU. In contrast, women treated with r-hLH had a significantly lower consumption of r-hFSH. Thus, there is a subset of normogonadotrophic women who cannot be classified as either ‘poor responders’, because at least
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Table 2. Studies testing recombinant-human LH (r-hLH) supplementation in normogonadotrophic adequately selected women*. Criterion for giving LH
Lisi et al., 2001
De Placido et al., 2001
De Placido et al., 2004
Ferraretti et al., 2004
De Placido et al., 2005
Groups
>3000 IU r-hFSH Self control r-hFSH requirement in (n = 12) r-hFSH + previous cycles r-hLH (75 IU) from day S7 Poor response to r-hFSH (n = 23) r-hFSH in the r-hFSH, then HMG 150 ongoing cycle1 IU from day 8 (n = 20) Normal responders2 (n = 40) Poor response to r-hFSH + r-hLH 150 IU r-hFSH in the from day 8 (n = 23) ongoing cycle1 r-hFSH + r-hLH 75 IU from day 8 (n = 23) Normal responders to r-hFSH2 (n = 46) Hypo-response to r-hFSH (n = 50) r-hFSH in the ongoing cycle3 r-hFSH + r-hLH (75– 150 IU) from day S10 (n = 54) r-hFSH + HMG from day S10 (n = 26) Normal responders to r-hFSH4 (n = 50) ‘Steady response’ r-hFSH (n = 58) to r-hFSH in the r-hFSH + r-hLH 150 IU ongoing cycle5 from day 8 (n = 59) Normal responders to r-hFSH6 (n = 112)
Statistically significant outcome differences
Patients who benefited from LH activity supplementation
Fertilization (%)
60.9a 86b
Clinical pregnancies (%)
50a 5.9b
Hypo-responders to r-hFSH in previous cycles
Peak oestradiol (pmol/ml)
4.3a 7.8b 8.9b
Oocytes retrieved (n)
5.9a 11.3b
Poor responders to r-hFSH in the ongoing cycle
11.8b Oocytes retrieved (n)
9.65a
79a
6.39b
65.7b
10.65a
82.5a
Implantation 14.1a (%) 36.8b
Oocytes retrieved (n)
Mature oocytes (%)
Pregnancy per 24.4b embryo transfer (%) 54.0a
7.4c
11.0b
35.4b
41.0ab
6.1a 9.0b 10.5c
Mature oocytes (n)
4.7a 7.8b
‘Steady responders’ to r-hFSH in the ongoing cycle
Hypo-responders to in the r-hFSH ongoing cycle
‘Steady responders’ in the r-hFSH ongoing cycle
9.0b
*
All studies are randomized controlled trials except that by Lisi et al. which is a ‘self-control’ study. Poor responders to r-hFSH = serum oestradiol concentrations ≤0.06 pmol/ml and no follicle >10 mm at ultrasound (USG) on day S8. 2 Normal responders to r-hFSH = tripling serum oestradiol concentrations between days S5 and S8, >4 follicles >10 mm at USG on day S8. 3 Hypo-responders to r-hFSH = >10 antral follicles ≥8 mm in diameter and oestradiol ≥100 pg/ml but no increase in the oestradiol concentration and in the follicular size between days S7 and S10. 4 Normal responders to r-hFSH = no need to increase the FSH dose during ovarian stimulation. 5 Steady responders to r-hFSH = more than five follicles, but no follicles >10 mm at the USG scan on day S8, oestradiol concentrations <180 pg/ml between days S5 and S8. 6 Normal responders to r-hFSH = tripling oestradiol concentrations between days S5 and S8, more than four follicles >10 mm on day S8. abc Values with different letters P < 0.05. r-h FSH = recombinant human FSH; HMG = human menopausal gonadotrophin. 1
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five oocytes are usually retrieved, or ‘normal responders’, because of the high cumulative r-hFSH requirement and reduced oocyte number and competence. These patients have a sub-optimal IVF outcome and seem to benefit from r-hLH administration. Thus, a clinical history of high r-hFSH consumption during ovarian stimulation should suggest the use of r-hLH-containing drugs for re-stimulation. In case of a first ovarian stimulation cycle, early identification of women who require a high r-hFSH dose may result in timely integration with r-hLH, which, in turn, may rescue the ovarian response and improve the ovarian IVF outcome, thereby avoiding a number of re-stimulations. Should these data be confirmed, the ovarian response to r-hFSH (in ongoing or previous cycles) may be a practical, reliable marker of women requiring r-hLH treatment.
0ATIENTS TREATED WITH 'N2( ANTAGONISTS GnRH antagonist (GnRH-ant) administration induces a fast and profound pituitary suppression, with a clear advantage in terms of premature LH surge avoidance. Nevertheless, LH activity is quickly and dramatically reduced in the phase in which this hormone activity is crucial: follicle which have been recruited in a physiological FSH and LH environment are dramatically deprived of their LH sustenance. Some authors (Cédrin-Durnerin n et al., 2004) have investigated the efficacy of an LH activity supplementation in GnRH-ant treated patients. They performed an RCT in 218 potential normal responders (age <38 years, no history of poor ovarian response). Ovarian stimulation was initiated with r-hFSH (150–300 IU/ day). Cetrorelix (3 mg, single dose) was given when at least one follicle >14–16 mm was found at ultrasound. Women of the group A received a daily injection of 75 IU r-hLH from the day of cetrorelix to HCG administration. Patients randomized to group B continued monotherapy with r-hFSH. Authors found a statistically significant increase in the oestradiol serum concentrations on the day of HCG in the r-hLH group (1476 ± 787 versus 1012 ± 659 pg/ml, P < 0.001). No statistically significant difference in any of the IVF outcome parameters was found between groups. Griesingerr et al. in 2005 performed an RCT in 127 normogonadotrophic infertile patients aged <39 years. Women were randomized in two groups on cycle day 2: group A received a daily dose of 150 IU of r-hFSH. Group B was treated with 150 IU of r-hFSH plus 75 IU of r-hLH per day. Cetrorelix (0.25 mg/ day) was administered from day S6 until HCG administration in all women. Primary endpoint was the number of days of gonadotrophin treatment, which was not shortened by LH administration. Serum oestradiol and LH concentrations on the day of HCG were significantly higher in the r-hLH-supplemented group (1924.7 ± 1256.4 versus 1488.3 ± 824.0 pg/ml, P <0.03, and 2.1 ± 1.4 versus 1.4 ± 1.5 IU/l, P <0.01 respectively). No other statistically significant difference in the outcome parameters was found. Taken together, these data seem to suggest that LH supplementation, in the form of r-hLH or HMG, may optimize follicle steroidogenesis in women treated with GnRH-ant. Nevertheless, current evidence is insufficient to evaluate the effect of such a treatment on implantation and pregnancy rates, at least in unselected patients.
Other groups have recently investigated the efficacy of LH activity supplementation in women at risk for poor ovarian response treated with GnRH-ant. Chung et al. (2005) conducted a retrospective
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cohort study in IVF patients who displayed less than four follicles in prior cycles and/or had basal FSH concentrations ranging between 10.1 and 18.0 IU/l. One hundred and forty one women receiving ganirelix (0.25 mg/day) when at least one follicle reached 12–14 mm until the day of HCG were evaluated. Patients were a posteriori divided in two groups according to gonadotrophins adopted. The first group consisted of 75 women stimulated with no LH activity supplementation. The other group included 66 subjects receiving LH activity supplementation in the form of HMG. Patients who did not receive LH activity supplementation displayed a significantly higher number of oocytes retrieved (8.8 ± 0.6 versus 6.6 ± 0.4, P = 0.004). Interestingly, when women were stratified on the basis of age, in those aged ≥40 years a significantly lower number of oocytes retrieved was observed in the HMG group (8.6 ± 0.6 versus 6.4 ± 0.4, P = 0.003). Similarly, the number of 2 pronucleate embryos was significantly lower in the LH activity supplemented women (5.2 ± 0.5 versus 3.8 ± 0.3, P = 0.03). In contrast, no significant difference in the outcome parameters was found in the younger patients. Although admitting the observational character of the study, the authors concluded that exogenous LH is not essential or beneficial in poor responders who are being treated with GnRH-ant, and could even be detrimental in older patients with a history of poor response. De Placido et al. (2006) recently conducted an RCT to test the efficacy of an LH activity supplementation to a flexible GnRHant protocol in patients at risk of poor ovarian response, having as control reference standard the GnRH-a flare up protocol. A total of 133 patients at risk for poor ovarian responsiveness (basal FSH >9 IU/l and/or age >37 years) were randomized in two groups. All the patients received a daily dose of r-hFSH of 300 IU starting on the second day of the cycle. In the antagonist group (n = 67), 0.125 mg/day of the GnRH-ant cetrorelix were administered for 2 days, starting when at least one follicle ≥14 mm was present; thereafter the GnRH-ant full dose of 0.25 mg/day was administered until the day of the HCG administration. Starting on the same day of GnRH-ant administration, a daily dose of 150 IU of r-hLH was also added until the day of the HCG. Controls (agonist group, n = 66) received daily a dose of triptorelin of 0.1 mg subcutaneously starting on the same day of r-hFSH administration. A dose of 150 IU/day of r-hLH was added when at least one follicle reached 14 mm. No premature LH surge was observed. No significant difference was observed in the total number of oocytes retrieved between groups (6.7 ± 2.5 versus 7.1 ± 2.9). A statistically significant difference was found in the number of mature oocytes (5.1 ± 2.2 in the group A and 4.2 ± 1.7 in group B, P <0.01). Thus, a stimulation regimen providing gradual increase in the GnRH-ant dose, in association with exogenous LH activity in form of r-hLH, seems to be able to significantly improve the ovarian response in women at risk for poor ovarian response, when compared with standard GnRH-a short protocol. This finding may be due to the fact that the study protocol offers a quite physiological and stable hormonal environment.
#ONCLUSIONS Physio-endocrinological observations indicate a key role for LH in follicle growth and maturation, particularly in the selection of the dominant follicle. Nevertheless, clinical data from ovarian stimulation practice seem to suggest that most
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normogonadotrophic women achieve adequate multi-follicular growth by means of administration of FSH alone. In such women, after pituitary down-regulation, the residual amount of LH seems to be capable of sustaining the local follicular activities needed for growth and dominance. In other patients, lack of LH activity after GnRH-a down-regulation seems to compromise multiple follicular growth, usually resulting in an ‘idiopathic’ poor response to recombinant FSH. Those patients are in most cases treated with an increase in the FSH daily dose. Several RCT have demonstrated that these adequately selected hypo-responders to standard FSH monotherapy may benefit from LH activity supplementation, which seems to be more effective than over-consumption of FSH vials. Clinical response to r-hFSH more than basal LH concentration seems to be predictive of exogenous LH requirement during ovarian stimulation (De Placido et al., 2001, 2004, 2005), supporting the idea that a discrepancy between immuno-reactive and bioactive LH may exist (Huhtaniemi et al., 1999; Jiang et al., 1999; Ropelato et al., 1999). This observation is also consistent with data from Humaidan n et al. (2002), who stratified women undergoing ovarian stimulation according to LH concentration after pituitary downregulation with a GnRH-a. The authors found that patients with LH concentrations ≥1.51 IU/l had significantly lower fertilization and pregnancy rates than women with concentrations between 0.5 and 1.5 IU/l. In a subsequent study, the same group (Humaidan et al., 2004) showed that women with higher LH concentrations (i.e. >1.99 IU/l) benefited from LH activity supplementation. The hypothesis that carriers of a less bio-active LH may require higher gonadotrophin doses and/or benefit from LH activity supplementation during ovarian stimulation is supported by other lines of evidence. In this context, an association between ovarian resistance to r-FSH monotherapy and presence of an LH polymorphism (V-beta LH) in normogonadotrophic women undergoing GnRH-a long protocol has been recently suggested (Alviggi et al., 2005). Meta-analysis of RCT comparing r-hFSH monotherapy with FSH-HP in women undergoing standard GnRH-a long protocol has been published by Daya in 2002. The author concluded that patients treated with r-hFSH had a significantly higher pregnancy rate. A similar observation was also reported by De Placido et al. (2000) in poor responders. Taken together, these findings seem to suggest that exiguous LH contamination of the FSHHP preparations exerts detrimental effects on follicular/oocyte maturation. This hypothesis is not consistent with data indicating that LH activity supplementation may be useful, at least in some normogonadotrophic women (De Placido et al. 2001, 2004, 2005; Ferraretti et al., 2004). Possible explanations for these discrepancies may be related to the fact that RCT examined by Daya were performed in unselected normogonadotrophic patients. Conversely, in those by De Placido et al. and by Ferraretti et al., LH activity and r-hLH were administered in women selected on the basis of their initial response to r-hFSH. Moreover, the study published by De Placido et al. was not comparable with these RCT due to both methodological aspect and study population. In fact, it was a self control study performed in poor responder to FSH-HP who were re-stimulated with r-FSH. In addition, only bad prognosis subjects had been included (i.e. high basal FSH and pathological response to exogenous FSH stimulation test). Finally, although low LH amounts may be present in FSH-HP, it should not be comparable with whole LH activity contained in both r-hLH and HMG.
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A well-known condition associated with scarce follicular recruitment is advanced reproductive age. Recent data indicate that r-hLH supplementation is able significantly to improve ovarian response to r-hFSH in women aged ≥35 years (Humaidann et al., 2004; Marrs et al., 2004). This clinical evidence may be linked to an age dependent decrease in the number of functional LH receptors and/or in the biological activity of endogenous LH, which in turn may increase resistance to LH-mediated processes (Mitchell et al., 1995; Vihko et al., 1996). Another possible explanation may be related with models of human folliculogenesis suggesting that LH activity is locally enhanced by paracrine activities, including growth factors and cytokines. These variables may account for the maintenance of an adequate follicular growth and steroidogenesis even when LH concentrations are very low. There is evidence that ovarian paracrine activity also decreases with age (Hurwitz and Santoro, 2004). Another subset of women who can benefit from LH activity supplementation are those treated with GnRH-ant. In these patients, a peculiar pattern of pituitary suppression is observed (Ganirelix Dose-Finding Study Group, 1998). More specifically, a rapid decline of serum concentrations of both LH and oestradiol usually follows the administration of the drug. Some authors found that in normal responders undergoing GnRH-ant, oestradiol production is significantly higher when exogenous LH, in form of both r-hLH (Cédrin-Durnerin n et al., 2004) and HMG (Griesinger et al., 2005) is added to r-hFSH. Nevertheless, both studies failed to demonstrate any other significant improvement in IVF outcome between women receiving r-hFSH monotherapy and those treated with r-hFSH and LH supplementation. In women at risk of poor ovarian response, Chung et al. (2005) even found a worse IVF outcome when LH activity was added. In contrast, recent data (De Placido et al., 2006) showed an improved IVF outcome when a flexible protocol with GnRH-ant and r-hLH supplementation was employed in women at risk of poor ovarian response, when compared with the standard flare-up protocol. Discrepancies between trials may be due to methodological issues, including study design and different selection criteria. In conclusion, available data seem to suggest that LH activity is able to improve steroidogenesis in women undergoing GnRHant pituitary suppression. In addition, the establishment of a more regular and physiological LH environment may lead to improved oocyte maturation in women at risk for poor ovarian response. Nevertheless, the effect of such treatment on implantation and pregnancy rates should be evaluated in larger RTC. The advent of recombinant gonadotrophins gave the chance to test the differential actions of exogenous FSH and LH, when given together or independently. There is now concrete evidence that r-hLH supplementation improves ovarian response to ovarian stimulation in at least three adequately selected subgroups of patients; (i) hypo-responders to r-hFSH; (ii) advanced reproductive age women; (iii) patients at risk for poor responsiveness treated with GnRH antagonists (Table 3).
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Table 3. Subgroups of women needing LH. Subgroup
Studiesa
Group characteristics
Hypo-responders to r-hFSH
De Placido et al., 2001, 2004, 2005; Ferraretti et al., 2004
Women with an initial response (i.e. at least five 2–9 mm follicles in each ovary) during the first days of stimulation followed by a plateau in which there is no significant increase in follicular size or oestradiol production in the next 3–4 days of stimulation Women who required >3000 IU to reach follicular maturity in previous cycles Patients aged ≥35 years
Lisi et al., 2001 Advanced reproductive age GnRH antagonists treated
Marrs et al., 2004; Humaidan et al., 2004 De Placido et al., 2006b
Patients showing basal FSH >9 IU/l and/or age >37 years, treated with a GnRH-antagonist flexible protocol
r-h FSH = recombinant human FSH; GnRH = gonadotrophin-releasing hormone. a For details of all study designs and results except De Placido et al., 2006, see Tables 1 and 2. b RCT on 133 patients at risk for poor ovarian responsiveness (basal FSH >9 IU/l and/or age >37 years). Antagonist group (n = 67): 0.125 mg/day of GnRH-antagonist for 2 days, starting when at least one follicle ≥14 mm was present; thereafter the GnRH- antagonist full dose of 0.25 mg/day until the day of the HCG. Starting on the same day of GnRH- antagonist, a daily dose of 150 IU of r-hLH was also added until the day of the HCG. Agonist group (n = 66): flare-up protocol with daily triptorelin 0.1 mg; 150 IU/day of r-hLH were added when at least one follicle reached 14 mm. Statistically significant higher number of mature oocytes (primary endpoint) in the antagonist group (5.1 ± 2.2 versus 4.2 ± 1.7, P <0.01) was observed.
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Paper based on contribution presented at the International Serono Symposium ‘How to improve ART outcome by gamete selection’ in Gubbio, Italy, October 7–8, 2005. Received 15 November 2005; refereed 6 December 2005; accepted 6 February 2006.
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