Exploiting LH in ovarian stimulation

RBMOnline - Vol 12. No 2. 2006 221-233 Reproductive BioMedicine Online; www.rbmonline.com/Article/1970 on web 15 December 2005

Review Exploiting LH in ovarian stimulation Dr Carlo Alviggi obtained the MD, specialty in Obstetrics and Gynaecology, and PhD degrees at the Faculty of Medicine, University of Naples ‘Federico II’, Italy. He has collaborated with various departments in Imperial College, London, the Italian National Research Council, Naples, the University of California, Los Angeles, and the University of Athens, Greece. This collaborative network resulted in various publications including new hypotheses on the pathogenesis of pelvic endometriosis. Recently he has specialized in reproductive medicine at the Fertility Unit of the University of Naples ‘Federico II’. Dr Alviggi has published extensively and given many invited lectures at international meetings in his field, as well as serving as ad-hoc reviewer for international journals. Dr Alviggi’s current research interests include the pathogenesis of pelvic endometriosis and the genetics of human reproduction. Dr Carlo Alviggi C Alviggi1, A Mollo, R Clarizia, G De Placido Dipartimento di Scienze Ostetriche Ginecologiche Urologiche e Medicina della Riproduzione–Università degli Studi di Napoli ‘Federico II’, via S. Pansini 5, 80131, Naples, Italy 1 Correspondence: via S. Pansini 5, 80131, Naples, Italy. Tel: +39-081 7462699; Fax: +39 081 7463747; e-mail: [email protected]

Abstract During intermediate–late phases of human folliculogenesis, LH plays a key role in promoting steroidogenesis and growth of the leading follicle. Ovarian stimulation for assisted reproduction techniques usually consists of administering exogenous FSH in a low LH environment. Although an impairment in LH-dependent paracrine activities would be expected, multiple follicular growth is efficiently achieved in almost all patients. Thus, there appears to be a discrepancy between classical folliculogenesis models and data from IVF. This study examines the ‘interface’ between basic endocrinological and clinical evidence, in an attempt to answer two questions: is there an LH therapeutic window, and if there is, how can this be exploited in the practice of assisted reproduction? It also reviews the evidence that specific subgroups of women may benefit from LH supplementation during ovarian stimulation. Keywords: assisted reproductive techniques, ceiling, LH, ovarian stimulation, recombinant gonadotrophin, threshold

Introduction Role of LH in folliculogenesis The ‘two cells–two gonadotrophins’ model highlighted the role of LH in promoting androgen production and release throughout folliculogenesis (Fevold, 1941; Hillier et al., 1994). According to this model, 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, the activities and proliferation of which 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 in the granulosa compartment at the intermediate follicular phase (Erickson et al., 1979; Shima et al.,, 1987; Hillier 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.

FSH and LH induce local production of the soluble molecule B inhibin, and growth factors. Among these, insulin 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). Locally produced peptides, rather than oestrogens, are known to be the key factors regulating primate follicle growth and development (Rabinovici et al., 1989; Pellicer et al., 1991; Zelinski-Wooten et al., 1993, 1994; Shetty et al., 1997). Thus, both gonadotrophins and the autocrine–paracrine system contribute to the complex mechanisms governing follicular growth and selection. The finding that gonadotrophins also regulate granulosa cell activity suggests that LH is also 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., 2002a). In this scenario, the oocyte undergoes several changes. In the 85 days preceding ovulation, it doubles in size thanks to small

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Review - Exploiting LH in ovarian stimulation - C Alviggi et al. molecules, amino acids and nucleotides, which derive from cumulus–oophorus cells and reach the oocyte via junction systems and follicular fluid. Granulosa-deriving signals are also involved in the regulation of oocyte transcriptional activities, which peak at the tertiary follicle stage. As a consequence, the gamete progressively accumulates heterogeneous material, including polypeptides and RNA macromolecules, which are crucial for successive steps of its development and during the initial stages of zygotic life. Because gonadotrophins directly regulate granulosa cell activities, it could be argued that LH indirectly regulates variables involved in the final step of oocyte maturation and early embryo survival (Elder and Dale, 2000; Fair, 2003). Hence, LH seems to exert two roles during folliculogenesis. One is described in the ‘two cells–two gonadotrophins’ model, and comprises induction of androgen production. This activity is exerted in the theca compartment throughout the follicular phase. The second begins in a still undetermined (Willis et al., 1998; Filicori, 2003a) stage of the intermediate follicular phase, 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. For instance, ovarian thecal/ interstitial androgen synthesis seems to be enhanced via a FSHstimulated paracrine mechanism (Smyth et al., 1993), which indicates that these paracrine activities increase LH activity in the theca compartment. 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.

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Interestingly, LH has been implicated in a third process during folliculogenesis. The selection of the leading follicle is related to the progressive decrease in FSH concentration: secondary follicles are deselected for their lack of FSH receptors, dominance being a higher receptivity to low FSH concentrations. It has been suggested that LH may contribute to the deselection of non-dominant follicles. This idea derives from the response of the leading follicle to the mid-cycle surge. A rapid increase in serum LH concentration blocks granulosa cell mitosis, and allows oocyte meiosis to resume (Shoham et al., 1995). Within a few hours, cumulus–oophorus cells also undergo morphological and functional modifications that lead to their luteinization. This suggests that LH can modulate the progression of secondary follicles. Preclinical evidence showed that developing follicles have specific requirements for exposure to LH, beyond which normal maturation ceases (Hillier 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 depend upon follicular stage, being higher in larger follicles and relatively lower in smaller ones. Should this be the case, an increasing LH concentration would promote leading follicle progression (being below its ceiling) and degeneration of secondary ones (by overcoming their ceiling). How LH favours the degeneration of non-leading follicles is still unclear, but a body of evidence indicates that active and androgen-related mechanisms are involved in this process (McNatty et al.,, 1979; Hillier et al., 1980; Jia et al., 1985; Hillier, 1987; Magoffin, 2003). 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).

LH and ovarian stimulation The gonadotrophin-releasing hormone agonist (GnRHa) long protocol is a well-established strategy for ovarian stimulation in young, normogonadotrophic women (Hughes et al., 1992). In this protocol, exogenous FSH is administered only when a GnRHa mediated suppression of the hypothalamus–pituitary–gonadal 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 GnRHa formulation, dose and mode of administration (Westergaard et al., 2001). Although the post-suppression decline of LH concentrations is variable, concentrations ranging between 0.5 and 2.5 IU/l are usually observed. These concentrations often fall to <0.5 IU/l during the intermediate late stages of stimulation. Thus, multiple follicular growth is induced without exogenous LH and in a low endogenous LH environment. Nevertheless, an adequate ovarian response is achieved in almost all patients. Interestingly, in patients with severe FSH and LH deficiency [World Health Organization (WHO) group I/hypogonadotrophic hypogonadal women], serum LH concentrations ≥1.2 IU/l are necessary to provide adequate LH support to FSH-induced follicular development (Hemsey et al., 2001; Loumaye and O’Dea, 2002). In these patients, monotherapy with FSH resulted in developmentally deficient follicles, which displayed low production of oestradiol and inability to luteinize and rupture in response to a human chorionic gonadotrophin (HCG) stimulus. Thus, there appears to be a discrepancy between IVF practice and classical folliculogenesis models and hypogonadotrophic hypogonadism. This 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 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). 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.

Review - Exploiting LH in ovarian stimulation - C Alviggi et al. In conclusion, the discrepancies between spontaneous folliculogenesis and ovarian stimulation seem to be related to LH concentrations and secretion patterns. This study will explore this paradox on the basis of clinical evidence. It will address the following questions: (i) Does a persistently low LH environment result in adequate multiple follicular growth? (ii) If yes, is it true for all women? (iii) Conversely, are there subsets of women who can benefit from LH supplementation during ovarian stimulation? The last section will evaluate whether there is an ‘LH ceiling’ by examining the results of clinical trials. Finally, the question of whether an LH ceiling can be exploited to modulate ovarian response to exogenous gonadotrophins will be discussed.

The GnRHa long protocol: is a low LH environment consistent with optimal ovarian/IVF outcome? Evidence from observational studies Many groups have examined the relationship between LH serum concentrations and ovarian/IVF outcome in normogonadotrophic women undergoing ovarian stimulation in a GnRHa long protocol. The review is limited to observational trials with r-hFSH (Table 1). In 1998, Fleming et al. identified an LH cut-off of 0.5 IU/l between stimulation days 7–9 (days S7–9) to classify 61 women (age <37 years) as ‘normal’ (≥5 IU/ l) and ‘low’ LH (<0.5 IU/l) patients. This value was chosen on the basis of assay reliability parameters. The starting daily dose of r-hFSH was 225 IU. Data were prospectively collected and

analysed a posteriori. The only significant difference in ovarian outcome was the amount of oestradiol secreted/follicle on the day of HCG (10.4 ± 4.3 versus 7.2 ± 3.6 pg/mm, in women with ‘normal’ and ‘low’ LH respectively; P <0.05). The fertilization rate was also significantly higher in the ‘normal’ LH group (P < 0.05, Table 2). Interestingly, these differences did not seem to affect the rate of blastocyst formation on day 6 of culture (77.0 versus 67.0% in the ‘normal’ and ‘low LH’ groups respectively). No information concerning IVF and reproductive outcome of these women was provided. Westergaard et al. (2000) were the first to identify an inverse relationship between LH concentrations during ovarian stimulation and IVF outcome. They retrospectively evaluated 200 patients (age <40 years) who had received GnRHa buserelin from the mid-luteal phase of the menstrual cycle. The starting dose of r-hFSH was 225 IU/day. LH concentrations were measured on day 8 of r-hFSH administration. Again, an LH cut-off of 0.5 IU/l was identified on the basis of assay reliability parameters and literature data. The oestradiol serum concentration on day 8 of r-hFSH administration was significantly higher (P <0.0001) in 102 women with a ‘normal’ LH concentration (i.e. ≥0.5 IU/l) than in those with a ‘low’ LH concentration (2908 ± 225 versus 1349 ± 101 respectively). There were no other significant between-group differences in ovarian and embryologic parameters. However, the ‘low LH’ patients had a significantly higher (P <0.005) percentage of early pregnancy losses compared with the ‘high LH’ group (Table 2). In a similar design, Balasch et al. (2001), retrospectively analysed 144 normogonadotrophic women (mean age 34.0 ± 0.4

Table 1. Features of observational studies correlating LH serum concentrations during ovarian stimulation and ovarian/IVF outcome in women undergoing the gonadotrophin-releasing hormone agonist long protocol. Reference

No. of

Analogue

patients

Starting

Dose

Day of LH determination

LH cutoff (IU/l)

Cut-off selection criteria

200 μg intranasally 4 times daily 0.5 mg . daily for 14 days

7–9

0.5

8 s.c

0.5

7

0.5, 0.7, 1.0

Assay reliability parameters Assay reliability parameters, evidence from literature ROC curves

Periovulatory mean 8

3.0

Arbitrary

0.5, 1.0, 1.5

Arbitrary

day

Fleming et al., 1998 (prospective)

61

Buserelin

Not specified

Westergaard et al., 2000 (retrospective)

200

Buserelin

Midluteal phase

Balasch et al., 2001 (retrospective)

144

Leuprolide

Midluteal phase

Esposito et al., 2001 (retrospective)

166

Leuprolide

Midluteal phase

Humaidan et al., 2002 (retrospective)

207

Buserelin

Midluteal phase

1 mg daily s.c. then 0.5 mg 0.5 mg daily s.c. then 0.25 mg 0.8 mg s.c. daily for 12–20 days

ROC = receiver-operating characteristic.

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Review - Exploiting LH in ovarian stimulation - C Alviggi et al. Table 2. Results of observational studies correlating LH serum concentrations during ovarian stimulation and ovarian/IVF outcome in women undergoing the gonadotrophin-releasing hormone agonist long protocol. Reference

Cut-off Oocytes Fertiliz- Transgroup retation ferred rieved (%) embryos

Days of stimulation

FSH ampoules

Biochemical pregnancies

Clinical pregnancies n (%)

Early pregnancy loss (%)

Positive Clinical HCG pregloss nancy (%) loss (%)

Fleming et al., 1998

<0.5 >0.5

10.28 13.07

76a 84b

2.05 2.20

– –

– –

– –

– –

– –

– –

– –

Westergaard et al., 2000

<0.5 >0.5

12.7 14.1

62 64

1.6 1.7

9.9 9.9

30.0 30.1

25 33

13 3

45a 9b

36a 3b

Balasch et al., 2001

<1 ≥1 ≤0.7 >0.7 <0.5 ≥0.5

9.1 9.5 8.3 9.6 7.7 9.5

78 74 79 75 75 76

2.7 2.7 2.8 2.6 2.7 2.7

11.1 10.8 11.0 10.9 11.1 10.9

34.9 33.8 34.9 33.0 36.1 34.0

– – – – – –

16 (80) 42 (81) 9 (82) 49 (80) 4 (66) 54 (82)

20 19 18 20 33 18

– – – – – –

– – – – – –

Esposito et al., 2001

<3 >3

13.2 11.9

52a 58b

2.8 2.5

11.8 11.6

56.5 54.7

51 (46) 20 (43)

44 (40) 20 (43)

22 20

– –

– –

Humaidan et al., 2002

<0.5 8.7 0.51–1.0 9.8 1.01–1.5 10.1 >1.51 10.4

46ac 54ab 63b 38c

1.8 1.9 1.9 1.8

13.7 13.9 13.3 12.9

30.33a 29.9a 26.1ab 23.78b

10 (42)ab 9 (38) 57 (53)ac 50 (46)a 22 (56)ac 17 (44) 12 (32)b 9 (24)b

– – – –

20 16 2 25

4 2

1 2 0 0

abc

Values with different letters P < 0.05. – = Not reported. HCG = human chorionic gonadotrophin.

years, range 23–42 years). Pituitary down-regulation had been achieved with mid-luteal administration of leuprolide acetate. All patients received 450 and 300 IU of r-hFSH on stimulation days 1 and 2 respectively. Because women had been included on the basis of positive pregnancy tests, only comparisons of ovarian and embryological parameters and probability of ongoing pregnancy were reliable. LH serum concentration was measured on day 7 of ovarian stimulation. Receiver-operating characteristic (ROC) curves were constructed a posteriori to identify the most predictive LH concentrations. The ‘low’ and ‘normal’ LH groups were stratified first on the basis of a LH cut-off of 0.5, then of 0.7, and finally of 1.0 to ≥1.0 IU/l. There were no significant differences in ovarian response, IVF/ intracytoplasmic sperm injection (ICSI) outcome, implantation rate or pregnancy outcome between the groups at any of the LH cut-off values examined.

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Esposito et al. (2001) conducted a retrospective analysis of 166 normogonadotrophic subjects who had received GnRHa leuprolide acetate from the mid-luteal phase. The starting r-hFSH daily dose ranged between 300 and 450 IU. They used a relatively high LH cut-off, i.e. 3.0 mIU/ml, that was derived from the means of four or five serum LH measurements obtained on stimulation days 5–12. The fertilization rate was significantly higher (P <0.05) in women with a ‘high’ LH concentration (≥3 mIU/ml) (Table 2). There were no other significant differences in ovarian, IVF and pregnancy outcomes between the ‘low’ and ‘high LH’ groups.

The largest prospective analysis of the relationship between endogenous LH concentrations during ovarian stimulation and ovarian/IVF outcomes available so far was conducted by Humaidan et al. (2002). GnRHa buserelin was administered from the mid-luteal phase. The starting dose of r-hFSH was tailored to between 100 and 375 IU/day, and 207 women (age <40 years) were monitored. LH concentrations were measured on stimulation day 8. Three cut-off values were arbitrarily identified: 0.5, 1.0, and 1.5 IU/l. Thus, patients were divided on the basis of their LH concentrations: 24 had LH ≤0.5 IU/l, 108 between 0.51 and 1.0 IU/l, 38 between 1.01 and 1.5 IU/l, and 37 ≥1.51 IU/l. The fertilization rate was significantly higher in the two middle groups compared with the flanking groups (Table 2). There were no significant differences in implantation and pregnancy rates in the groups with LH < 0.5 IU/l and the two intermediate groups. In contrast, the implantation rate was significantly higher in the LH ≤0.5 IU/l groups versus the LH ≥ 1.51 IU/l group. The clinical pregnancy rate was significantly higher (P < 0.05) in the two intermediate groups versus the ≥1.51 IU/l group. Finally, there were no intergroup differences in the abortion rate. An overall analysis of the data showed a trend to a better ovarian/IVF outcome in women with LH concentrations between 0.51 and 1.5 IU/l, thereby providing clinical evidence of an ‘optimal LH range’ during ovarian stimulation. In conclusion, based on clinical data, it still remains to be established whether a low LH environment during ovarian

Review - Exploiting LH in ovarian stimulation - C Alviggi et al. stimulation affects ovarian IVF and pregnancy outcome in women undergoing the GnRHa long protocol combined with r-hFSH. Thus, no LH cut-off value able to identify women requiring LH supplementation can be recommended. 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.

Interventional studies: effect of exogenous LH on ovarian/IVF outcomes There is a large body of data on r-hFSH monotherapy versus combined exogenous FSH and LH in women undergoing the GnRHa long protocol. It is noteworthy that LH is usually administered in the form of human menopausal gonadotrophin (HMG), or recombinant human LH (r-hLH). HMG is an agglomerate of gonadotrophins and aspecific proteins extracted from the urine of post-menopausal women, in which an FSH/ LH content of 1:1 is usually expected. In practice, each vial contains 75 IU of extractable FSH and 75 IU of ‘LH activity’, in the form of both extractive LH and HCG (Stokman et al., 1993; Giudice et al., 2001). Conversely, r-hLH vials contain 75 IU of pure molecule. Interestingly, some authors have proposed the administration of LH activity in the form of HCG during ovarian stimulation for intrauterine insemination and ICSI (Filicori et al., 1999, 2002a). Van Wely et al. (2003) conducted a meta-analysis of four randomized controlled trials (RTC) comparing r-hFSH and HMG protocols in young normogonadotrophic patients undergoing a GnRHa long protocol. They found no statistically significant difference in the main outcome measures (ongoing pregnancy rates and live birth rate) or in the secondary outcomes (gonadotrophin dose, cancellation rate, number of oocytes retrieved, implantation and clinical pregnancy rates). The authors concluded that larger randomized trials are needed to evaluate differences between r-hFSH and HMG. One of the four RCT examined in the meta-analysis (European and Israeli Study Group, 2002) compared the outcome of r-hFSH versus highly purified HMG (HP-HMG). The study showed no statistically significant difference in ongoing pregnancy rates between groups. The same authors (Platteau et al., 2004) also analysed their data according to IVF and ICSI cycles, and found a significantly higher ongoing pregnancy rate with HPHMG in IVF but not in ICSI cycles. These data demonstrate the importance of developing RCT in which outcome parameters are examined according to the fertilization method. Thanks to the availability of both recombinant gonadotrophins, exogenous LH can now be administered without modifying the exogenous FSH cumulative dose. The possibility of tailoring FSH/LH ratios led investigators to re-explore the hypothesis that appropriately calibrated LH supplementations could improve the ovarian/IVF outcomes. Three RCT of FSH versus FSH plus r-hLH regimens in normogonadotrophic women undergoing the GnRHa long protocol have been conducted. Sills et al. (1999) studied a total of 31 cycles in 30 women (age 30–41 years): leuprolide acetate was given daily from the midluteal phase. Seventeen patients were randomly allocated to the group receiving 150–450 IU of highly purified FSH (FSH-HP). The remaining 13 women (14 cycles) received the same dose of

FSH-HP plus one ampoule of r-hLH (75 IU) from stimulation day 1. There was no significant difference in any of the outcome parameters. The authors concluded that residual endogenous LH concentrations after GnRHa suppression can sustain normal follicular growth, obviating the need for exogenous LH supplementation. A large multicentre, randomized trial was published by Marrs et al. in 2004. A total of 431 ICSI patients (age 18−40 years) undergoing the mid-luteal GnRHa long protocol (leuprolide acetate ‘daily’) 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 stimulation day 6; n = 212). No significant difference in the number of metaphase II oocytes retrieved (primary end-point) or in the cumulative pregnancy rate was found. Conversely, 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 supplementation in 231 normogonadotrophic women, aged <40 years, undergoing the GnRHa long protocol with daily buserelin from the mid-luteal phase. Stimulation was initiated with r-hFSH (starting dose: 150–300) in all women: from day 8 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 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 pharmacokinetics studies (le Cotonnec et al., 1998a,b), indicate the need for RCT to evaluate the efficiency of higher doses. In conclusion, one meta-analysis of studies comparing r-hFSH with HMG and three RCT comparing r-hFSH with r-hFSH associated with r-hLH, in women undergoing the GnRHa long protocol are available. Cumulative data analysis seems to suggest that exogenous LH administration is not associated with any significant improvement of the IVF/ICSI outcome when compared with r-hFSH monotherapies. More specifically, data from r-hLH trials suggest that LH, given at doses of 75 IU/day from stimulation day 1 or 150 IU/day from stimulation days 6 or 8, represents an adjunctive cost that is not counterbalanced by any clinical advantage, at least in normogonadotrophic, ‘good prognosis’ patients.

Are there subgroups of women who may benefit from LH supplementation? Women of advanced reproductive age After the analysis of cumulative data (see previous section), 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 <0.05) in the r-hFSH plus r-hLH group in patients aged ≥35 years at their first assisted reproduction cycle (Table 3). This finding suggested that LH supplementations might be beneficial in women at an advanced reproductive age. Humaidan et al. (2004) also stratified their study population according to age,

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Review - Exploiting LH in ovarian stimulation - C Alviggi et al.

Table 3. Randomized controlled trials showing advantages of LH supplementation in subgroups of women undergoing gonadotrophin-releasing hormone agonist long protocol. Reference

Criterion for giving LH

De Placido et al., 2001

Statistically significant outcome differences

Patients who benefited from LH supplementation

Poor response to r-hFSH r-hFSH (n = 23) in the ongoing cycle1 r-hFSH, then HMG 150 IU from day 8 (n = 20) Normal responders2 (n = 40)

Peak oestradiol (pmol/ml)

Poor responders to r-hFSH in the ongoing cycle

Marrs et al., 2004

Unselected women

r-hFSH (n = 219) r-hFSH + r-hLH 75 IU from day S6 (n = 212)

Pregnancy (%) 22.5a in women 45.8b >35 years at their first cycle

Humaidan et al., 2004

Unselected women

r-hFSH (n = 115)

Implantation (%)

13.3a,d Pregnancy 23.5a,d Patients >35 31.2b,e per ET (%) 46.5b,e years

Ferraretti et al., 2004

Hypo-response to r-hFSH in the ongoing cycle3

r-hFSH (n = 50) 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)

Implantation (%)

14.1a 36.8b

r-hFSH (n = 58) r-hFSH + r-hLH 150 IU from day 8 (n = 59) Normal responders to r-hFSH6 (n = 112)

Oocytes retrieved (n)

De Placido et al., 2005

Groups

r-hFSH + r-hLH (2:1) from day S8 (n = 116)

‘Steady response’ to r-hFSH in the ongoing cycle5

4.3a 7.8b 8.9b

Oocytes retrieved (n)

5.9a 11.3b 11.8b

Patients >35 years

Pregnancy 24.4b per ET (%) 54.0a

7.4c

11.0b

35.4b

41.0ab

6.1a 9.0b 10.5c

Mature 4.7a oocytes (n) 7.8b 9.0b

Hypo-responders to r-hFSH in the ongoing cycle

Steady responders to r-hFSH in the ongoing cycle

Poor responders to r-hFSH = serum oestradiol level ≤0.06 pmol/ml and no follicle > 10 mm at USG on day S8. Normal responders to r-hFSH = tripling serum oestradiol levels 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 E2 ≥100 pg/ml but no increase in the E2 level and in the follicular size between days S7 and S10. 4 Normal responders to r-hFSH = No need to increase the FSH dose during the ovarian stimulation. 5 Steady responders to r-hFSH = More than 5 follicles, but no follicles > 10 mm at the USG scan on day S8, E2 levels < 180pg/ml between days S5 and S8. 6 Normal responders to r-hFSH = Tripling E2 levels between days S5 and S8, more than 4 follicles > 10 mm on day S8. abc Values with different superscript letters within a reference are significantly different P < 0.05. d,e Individuals >35 and <35 years old respectively. ET = embryo transfer; HMG = human menopausal gonadotrophin. 1 2

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Review - Exploiting LH in ovarian stimulation - C Alviggi et al. using a cut-off of 35 years. Among women who did not receive r-hLH supplementation, pregnancy and implantation rates were significantly lower in those ≥35 years (P < 0.05); this difference did not occur in the r-hLH supplemented group (Table 3). Although based on a-posteriori stratification processes, the above data identify at least one subset of women, those aged >35, who may benefit from LH supplementation. This is in line with models of human folliculogenesis in which LH activity is locally enhanced by paracrine activities. There is evidence that ovarian paracrine activity decreases with age (Hurwitz and Santoro, 2004).

Women with an abnormal response to r-hFSH In about 10–15% of young women, ovarian response to the GnRHa long protocol and r-hFSH remains sub-optimal, despite the presence of normal FSH or LH circulating concentrations. Nevertheless, an impairment of LH-dependent mechanisms has been invoked in these cases. On this basis, several groups have evaluated the efficacy of LH supplementation in women selected according to specific profiles of ovarian response to standard r-hFSH doses. In 2001, Lisi et al. conducted 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. No other enrolment criterion was reported. Women had a mean basal FSH of 12.2 IU/l and a mean age of 36.1 years. Re-stimulation entailed the addition of 75 IU of r-hLH from stimulation day 7 to the standard r-hFSH regimen. As in the first cycle, all women received triptorelin 0.1 mg daily, from the mid-luteal phase (GnRHa 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 < 0.05) in r-hLH supplemented cycles. Despite small sample size and the methodological limits of non-randomized trials, this study provided the first clinical evidence that a suboptimal response to r-hFSH may be significantly improved by LH supplementation. De Placido et al. (2001) found that in about 10–12% of normogonadotrophic patients, an initial response (i.e. at least five 2–9mm 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 LH-dependent 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 stimulation day 8 were randomized to receive LH 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 stimulation days 5–8 in association with >4 follicles >10 mm on stimulation day 8) served as a nonrandomized control population. All women received triptorelin 3.75 mg (depot depot preparation) on the first day of spontaneous menstruation. After pituitary desensitization, a starting dose of 300 IU of r-hFSH was administered. First dose adjustment was performed on stimulation day 5. The mean number of oocytes retrieved was significantly higher in women treated with HMG supplementation than in those who received r-hFSH ‘step up’ (Table 3). Moreover, the ovarian outcome of the HMG group was comparable with that observed in ‘normal responders’, suggesting that LH supplementation was able to ‘rescue’ this apparently abnormal response to r-hFSH. Interestingly, when serum LH concentrations were measured on stimulation day 8, before allocation to treatment groups, there was no statistically significant difference between woman whose ovarian outcome improved with HMG and normal responders to r-hFSH (0.27 ± 0.06 versus 0.39 ± 0.35 mIU/ml, respectively). These findings revealed the existence of a subgroup of hypo-responders who benefit more from LH supplementation than from an increase in the daily r-hFSH dose. Using 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. GnRHa 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 150–300 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 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 RCT (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 depot preparation) was administered on the first day of spontaneous menstruation. The starting dose of rhFSH 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 an 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 stimulation days 5 and 8, more than 4 follicles >10 mm on stimulation day 8) 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

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Review - Exploiting LH in ovarian stimulation - C Alviggi et al. the r-hFSH step-up protocol (Table 3). Moreover, the mean number of mature oocytes of r-hLH group was similar to that observed in ‘normal responders’. Although power analysis was not performed for these categorical variables, it is also noteworthy that implantation and ongoing pregnancy rates were similar in ‘steady responders’ treated with r-hLH and ‘normal responders’ (14.2 and 32.5% versus 18.1 and 40.2% respectively). Conversely, both parameters were significantly lower (P < 0.05) in ‘steady responders’ treated with step-up r-hFSH (10.0 and 22.0%) than in ‘normal responders’. Also in this study, endogenous LH serum concentrations on stimulation day 8 (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 GnRHa 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 stimulation days 7 and 10 (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 pregnancyper-embryo transfer and implantation rates were significantly higher in group B than in groups A and C, and did not differ from normal responders (Table 3). Consistent with other studies (De Placido et al., 2001, 2004, 2005), there were no significant differences in LH circulating concentrations on stimulation day 7 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 either ‘poor responders’, because at least 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. For 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.

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Interestingly, neither De Placido et al. (2001, 2005) nor Ferraretti et al. (2004) found a correlation between circulating concentrations of endogenous LH and initial response to r-hFSH. Thus, there is a discrepancy between endogenous LH concentration and LH requirement. This may be explained in various ways. Firstly, LH circulating concentrations may not be representative of local hormonal activity, which is also related to receptor status and paracrine networks. During critical phases of folliculogenesis, LH concentrations may fall below the threshold of single follicles: this phenomenon could be counteracted by FSH-dependent mechanisms in almost all patients. Conversely, in some women, local variables may interfere with adaptive mechanisms and render the lack of LH critical. Alternatively, incongruence between LH serum concentration and exogenous LH requirement may be related to genetic variables (De Placido et al., 2004; Ferraretti et al., 2004). Some polymorphic variants of the FSH receptor are associated with a poor ovarian response to exogenous FSH (Simoni et al., 2002; de Castro et al., 2004). In such cases, an initial sub-optimal response to r-hFSH would be rescued by LH, which is able to substitute FSH activity during the intermediate-late stages of folliculogenesis (Filicori et al., 2003a). Finally, it is possible that LH is less biologically active in women who benefit from exogenous LH. In other words, serum concentrations of the ‘immunoreactive’ molecule may be not representative of the bioactivity of the hormone (Mitchell et al., 1995; Huhtaniemi et al., 1999; Jiang et al., 1999; Ropelato et al., 1999; Themmen and Huhtaniemi, 2000). This hypothesis was tested in a pilot study of the incidence of v-βLH, a common polymorphic form of LH, in normogonadotrophic women (<37 years) with different profiles of ovarian response to the GnRHa long protocol and r-hFSH (Alviggi et al., 2005). This LH variant (Trp8Arg and Ile15Thr of the β subunit) has a shorter half-life than the native molecule, which in turn may affect its half life in vivo (Pettersson, 1994). The incidence of this variant in the heterozygotic form ranges between 5.0 and 41.9% in the general population (Nilsson et al., 1997). Although the reproductive potential seems to be conserved, carriers of this variant have a higher incidence of ovulatory and menstrual disorders (Ramanujam et al., 1999). Interestingly, three carriers were identified (two heterozygous and one homozygous) among 10 hypo-responders, who required >3500 IU of r-FSH to produce a minimum six oocytes, whereas no variant was found among 22 normal responders (cumulative dose < 2000 IU). Thus, it is conceivable that v-βLH may be less effective in supporting FSH-stimulated multiple-follicular growth thereby resulting in a sub-optimal ovarian response to standard ovarian stimulation regimens and in higher drug consumption. Should these epidemiological observations be confirmed, the effect of LH supplementation in these carriers should be evaluated.

Is there a trace of an ‘LH ceiling’ in the clinical trials? Results of observational studies The first clinical evidence of a negative correlation between LH serum values and oocyte competence was reported by Stanger and Yovich in 1985. In 62 patients undergoing IVF and treated with clomiphene citrate (CC), CC + HMG or HMG alone, they found a significant reduction of the fertilization rate of mature oocytes in patients whose basal LH values

Review - Exploiting LH in ovarian stimulation - C Alviggi et al. were greater than 1 SD above the mean. Ten pregnancies were achieved among 59 women who underwent embryo transfer. No pregnancy occurred in patients with elevated LH values. Howles et al. (1986) reported data on the urinary output of LH of 200 IVF women undergoing ovarian stimulation with CC (from stimulation day 2 to 6) and HMG (2 ampoules from stimulation day 5). Patients who did not become pregnant had a significantly higher (P <0.01) u-LH output (0.22 ± 0.01 IU/h) in the two days before HCG administration [pregnant (ongoing) women had an u-LH output of 0.17 ± 0.01 IU/h]. Interestingly, non-pregnant women with frozen spare embryos had the same u-LH concentrations as those who became pregnant. These concentrations were significantly lower than those observed in non-pregnant patients with no frozen embryos. Since only good quality embryos can be frozen, the authors concluded that the LH environment rather than endometrial receptivity influenced oocyte quality. In this scenario, observational and prospective studies were conducted to try to relate LH serum concentrations to reproductive outcome. In this context, polycystic ovarian syndrome (PCOS), a clinical condition associated with reduced oocyte competence, and an increased risk of abortion, could be considered a good in-vivo model. In PCOS patients, serum LH concentrations are significantly elevated, probably due to enhanced amplitude and frequency of GnRH pulses: concentrations above the 95th percentile of normal occur in about 60% of cases (van Santbrink, 1997; Laven et al., 2002). Homburg et al. (1988) measured basal LH concentration in 54 PCOS women treated with pulsatile GnRH. Forty-one patients ovulated and 27 conceived. Early pregnancy loss occurred in nine women. Basal LH concentration was lower in patients who conceived than in those who did not (median and range: 12.4, 1.3–29.0 versus 19.0, 3.5–50.0 IU/l respectively). Moreover, women whose pregnancy progressed had significantly lower LH concentrations than those who had early abortion (median and range: 9.6, 1.3–29.0 versus 17.9, 7.0–29.0 IU/l respectively). It was concluded that high LH concentrations during the follicular phase in women with PCOS exert a deleterious effect on conception rates and may contribute to early pregnancy loss. In contrast, there is evidence that metformin, an insulin sensitizer, is effective in correcting anovulation and menstrual disorders, and in lowering miscarriage rates (Jakubowicz et al., 2002; Nestler et al., 2002), which suggests that reproductive failure results from elevated insulin rather than high LH concentrations. In conclusion, the issue of heterogeneity of variables renders PCOS a questionable model with which to assess the relationship between LH activity and disorders of folliculogenesis. Other studies have focused on the link between LH concentrations and reproductive outcome in subsets of women during stimulated and natural cycles. Shoham et al. (1990) reported data from 28 patients who presumably ovulated after CC treatment. These authors identified two patterns of hormonal secretion during stimulation. There was a ‘normal’ response in 17 patients, and a significantly higher LH/FSH ratio from stimulation day 9 until HCG administration in the remaining 11 patients. Five pregnancies were achieved in the ‘normal response’ group. No woman with an abnormal gonadotrophin response to CC become pregnant. These data support the idea that exposure to high LH concentrations during the follicular phase may affect the fertilization rate. Regan

et al. (1990) prospectively investigated 193 women with a regular spontaneous cycle. Patients were divided in two groups according to basal LH concentrations: pregnancy was achieved in 130 out of 147 (88%) subjects with LH <10 IU/l (‘normal LH group’), and in 31 out of 46 (67%) women with LH >10 IU/l (‘high LH group’). The miscarriage rates were 12% and 65% in the two groups respectively. The results of this set of studies reinforced the concept that high LH concentrations may affect reproductive outcome: depending on the phase of follicular development, the ‘abnormal’ environment may induce atresia or early luteinization, leading to impaired oocyte competence (Chappel and Howles, 1991). However, most data derive from observational studies in which the subsets of women were heterogeneous and not always well defined. In addition, most of the studies were carried out in the pre-GnRHa era, which means in a ‘spontaneous’ LH environment (i.e. 2.0–7.0 IU/l). More recently, the relationship between LH concentrations and IVF/ovarian outcome in women undergoing a standard GnRHa long protocol has been investigated (Balasch et al., 2001; Humaidan et al., 2002). This pharmacologically induced environment is characterized by LH concentrations relatively lower than those observed during spontaneous cycles (i.e. 0.5–2.5 IU/l). In addition, this strategy is often associated with administration of r-hFSH, which is free from exogenous LH activity. Thus, deleterious effects consequent to high LH concentrations would not be expected with hFSH treatment. Humaidan et al. (2002) divided 207 IVF/ICSI patients (age < 40 years) undergoing the GnRHa long protocol plus r-hFSH according to their LH concentrations on stimulation day 8. Three LH cut-off values were arbitrarily defined: 0.5, 1.0, and 1.5 IU/l. Women with LH concentrations ≥ 1.51 IU/l had significantly lower fertilization and clinical pregnancy rates than women with concentrations between 0.5 and 1.5 IU/l (Table 2). Balasch et al. (2001) obtained different results in a retrospective evaluation of 144 normogonadotrophic women (mean age 34.0 ± 0.4 years, range 23–42 years) treated with the GnRHa long protocol plus r-hFSH. Serum concentrations of LH serum were measured on day S7. Three cut-off values were established from ROC curves: 0.5, 0.7 and 1.0 IU/l. There was no significant relationship between ovarian response, IVF/ICSI outcome, implantation rate and pregnancy outcome in any of the groups at any LH cut-off value examined (Table 2).

Interventional studies: can exogenous LH be used to modulate follicular growth? Data from spontaneous and stimulated cycles prompted the notion that exposure to high LH concentrations during folliculogenesis may alter follicle/oocyte development (Stanger and Yovich, 1985, Howles et al., 1986; Regan et al., 1990). However, slight increases in LH concentration during standard ovarian stimulation protocols are compatible with quantitatively normal multiple-follicular growth. More recently, the idea of using LH to ‘modulate’ the response to ovarian stimulation has emerged. Filicori et al. (2002b) demonstrated that during late ovarian stimulation stages, LH activity in the form of low-dose HCG, besides promoting leading follicle growth, favoured the degeneration of small follicles independently of FSH administration. In that RCT, 40 normogonadotrophic

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Review - Exploiting LH in ovarian stimulation - C Alviggi et al. normoovulatory women (BMI 20–25 kg/m2) undergoing the GnRHa long protocol for intrauterine insemination were evaluated. All women received 3.75 mg of depot triptorelin during the mid-luteal phase. Ovarian stimulation was started 14 days thereafter with 150 IU/day r-hFSH. On stimulation day 8, women were randomized into four groups (10 per group) as follows: group A received monotherapy with 150 IU/day r-hFSH; group B, 50 IU/day r-hFSH and50 IU/day HCG; group C, 25 IU/day r-hFSH and 100 IU/day HCG; and group D, 200 IU/day HCG alone. Treatment was successfully completed in 70% of women stimulated with different HCG doses (groups B–D). Interestingly, these patients had a significant decrease (P < 0.001) in the mean number of small follicles (<10 mm), but no significant reduction in the number of large (>14 mm) follicles when compared with group A. The authors concluded that the ‘regulatory’ effects of LH on the number of small follicles may be a means to prevent ovarian hyperstimulation syndrome.

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The idea that the LH regulatory effect could be calibrated to favour mono-ovulation during ovarian stimulation has been evaluated in two RCT (Loumaye et al., 2003a; Hugues et al. 2005). This working hypothesis was based on the assumption that there is an LH ceiling and that it depends on follicle developmental stage: mature follicles are more resistant than immature ones (‘higher ceiling’). Thus, a similar LH environment may favour the development of mature follicles and arrest the progression of small follicles (Hillier et al., 1980; Hillier, 1987, Smyth et al., 1993). In an attempt to identify the optimal timing and dosage of LH, Loumaye et al. (2003a) conducted a two-arm, double-blind trial. Arm A consisted of 20 women aged 18–39 years with a clinical history of hypogonadotrophic hypogonadism (WHO group I type anovulation). All patients were initially treated with r-hFSH (starting dose 112.5 IU/day) and r-hLH (225 IU/day); when at least one follicle reached a mean diameter of 10–13 mm, patients were randomized to continue treatment with: (i) both drugs (n = 8), (ii) r-hLH alone (n = 6) or (iii) r-hFSH alone (n = 6). r-hLH was administered at the same dose as in the initial r-hFSH phase (i.e. 225 IU/ day). After initial adjustments, the daily r-hFSH dose was not modified after randomization. The mean number of follicles >11 mm on the day of HCG administration was 6.0 ± 2.3 in the r-hFSH/r-hLH group, 1.5 ± 0.7 in the r-hLH/placebo group, and 4.2 ± 0.3 in the r-hFSH/placebo group. Differences were statistically significant between the r-hLH/placebo and r-hFSH/ placebo groups (P = 0.017) and between the r-hLH/placebo and r-hFSH/r-hLH groups (P = 0.003). Seventeen women, aged 18–39 (BMI < 35 kg/m2), infertile due to ovulatory dysfunction (WHO group II anovulation), were enrolled in arm B. Eligibility for this group was evaluated during ovarian stimulation: women with a hyper-response to FSH treatment (>4 follicles of >8 mm and <13 mm in diameter, no larger follicles and an endometrial thickness of >8 mm) were included: FSH treatment was stopped and patients were randomly allocated to three treatment groups: (i) a daily s.c. injection of 225 IU/day r-hLH (n = 4), (ii) 450 IU/day r-hLH (n = 8) or (iii) placebo (n = 5). The mean number of follicles >11 mm was 4.6 ± 1.8 for the placebo group, 2.5 ± 1.9 for the r-hLH 225 IU group, and 4.2 ± 1.4 in the r-hLH 450 IU group (there were non-significant differences for all pairwise comparisons). The authors concluded that r-hLH alone can trigger follicular growth arrest in a significant number of patients, which confirms the existence of an LH ceiling during late follicular maturation.

The same group recently conducted a RCT in WHO II anovulatory women to test the hypothesis that LH may promote the dominance of a leading follicle when administered in the late follicular phase (Hugues et al., 2005). As in arm B of the previous study, eligibility was assessed during FSH (FSH-HP or r-hFSH) administration: women showing hyper-responsiveness (at least three follicles 11–15 mm in diameter, but no follicle >15 mm) at any point during stimulation were randomized to receive placebo (n = 30) or rLH at different doses: i) 6.8 μg (150 IU, n = 32); ii) 13.6 μg (300 IU, n = 28); iii) 30 μg (660 IU, n = 28); iv) 60 μg (1325 IU, n = 29). A daily dose of 37.5 IU of r-hFSH in addition to placebo or r-hLH was administered in each case, from the day of the randomization until HCG administration, whatever the amount of FSH given during the period prior to randomization. The primary efficacy endpoint of the study was the proportion of patients who received HCG and who had exactly one follicle ≥16 mm in diameter on that day. The proportion of patients with exactly one follicle ≥16 mm was 13.3% in the placebo group and 32.1% in the 30 μg r-hLH group (P = 0.048). The authors concluded that an r-hLH dose of 30 μg seems to be appropriate to control development of the follicular cohort during FSH stimulation. The results of these studies support the concept that LH administered during the late follicular phase can ‘modulate’ multiple follicular growth. However, there are several discrepancies between these studies. Although data about the effects of different forms of LH activity are scarce, it should be noted that HCG potency seems to be approximately six times greater than that of LH (Stokman et al., 1993; Filicori et al., 2003a). Should this be the case, 200 IU of HCG should be about as active as 1200 IU r-hLH. Interestingly, in the study conducted by Filicori et al. (2002b), 200 IU of HCG given from stimulation day 8 had no detrimental effect on large follicles (i.e. ≥10 mm), but it hastened the demise of small follicles (i.e. <10 mm). Conversely, according to Hugues et al. (2005), an approximately half-potent LH dose (i.e. 660 IU/day) significantly increased the proportion of women developing a single-dominant follicle (i.e. ≥16 mm), whereas the highest r-hLH dose (i.e. 1325 IU/day) did not affect the number of small follicles. These discrepancies can be related to basic and methodological aspects: (i) although a potency proportion can be proposed, r-LH and HCG remain different gonadotrophin preparations; (ii) the studies concerned substantially different subsets of patients, i.e., normo-ovulatory women undergoing intrauterine insemination for unexplained infertility and/or male factor (Filicori et al., 2002b) and WHO group I (Loumaye et al., 2003a) and II anovulatory subjects (Loumaye et al., 2003a, Hugues et al., 2005); (iii) the endogenous LH environment was different during ovarian stimulation (GnRHa long protocol; Filicori et al., 2002b and no pituitary suppression; Loumaye et al., 2003a; Hugues et al., 2005). Finally, differences in the timing of LH administration can be also invoked (Filicori et al., 2003b; Loumaye et al., 2003b).

Concluding remarks Recent studies have led to a revision of the classical concepts of folliculogenesis and to a better understanding of the role of exogenous gonadotrophins in reproductive medicine. The main discrepancy among experimental models is that LH seems to be a critical regulator of follicular growth in some conditions,

Review - Exploiting LH in ovarian stimulation - C Alviggi et al. and almost unnecessary in others. This observation should be evaluated in the light of the evidence that LH exerts three functions during folliculogenesis: it co-operates with FSH in promoting steroidogenesis during follicular growth; it directly supports the development of leading follicles and regulates oocyte maturation from the intermediate–late stages of the process; and finally, it favours monoovulation by modulating multiple follicular progression. The advent of recombinant gonadotrophins (FSH and LH) provided the means to investigate these contradictory observations and to identify new pieces in the complex mosaic of folliculogenesis. One of these pieces is represented by the effects of different LH environments on several endocrine and reproductive parameters. Randomized clinical trails demonstrate that an adequate response to ovarian stimulation is achieved even when GnRHa long protocols are associated with monotherapy with r-hFSH. Despite a sub-physiological LH environment, the capacity to induce steroidogenesis and promote follicular growth is preserved and/or adequately counteracted by other activities (i.e. FSH). This, together with the idea that LH-related mechanisms of modulation of multiple follicular progression are not required during ovarian stimulation, clinical evidence demonstrating no benefit from the routine administration of LH-containing drugs in IVF women, and the argument that slight increases in LH concentrations may also affect the success rate of these procedures, has led to the use of r-hFSH monotherapy, at least in unselected women. However, there is now clear evidence that specific subgroups of women undergoing the GnRHa long protocol plus FSH benefit from LH supplementation. More specifically, adequately timed, calibrated LH administration significantly improves the ovarian/IVF outcome in patients >35 years and those showing an initial abnormal ovarian response to r-hFSH monotherapy. Finally, data concerning the use of LH activity to modulate ovarian response are scarce and come from heterogeneous studies. Consequently, there is a need for RCT designed to explore the modulating role of diverse LH activities with respect to specific aims, i.e., prevention of ovarian hyperstimulation syndrome or achievement of monoovulation, in large and homogeneous study populations.

Acknowledgements We are indebted to Jean Gilder for editing the text. We are grateful to Drs Ida Strina, Mariateresa Varricchio and Antonio Ranieri for discussions regarding this review. This study was partially supported by grants from MIUR (Programmi di Ricerca Scientifica di Rilevante Interesse Nazionale – annualità 2004 Prot. 2004061475–003).

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