1 Dipartimento Universitario di Scienze Ostetriche Ginecologiche e Medicina della Riproduzione, Area Funzionale di Medicina della Riproduzione ed Endoscopia Ginecologica, Università degli Studi di Napoli Federico II, Napoli, 2 Istituto Materno Infantile, Università di Palermo, Palermo, 3 Biogenesi, Casa di Cura Villa Europa all'EUR, Roma, 4 Unità Operativa Ginecologia e Ostetricia, A.O.San Giovanni di Dio e Ruggi D'Aragona, Salerno, 5 Dipartimento di Scienze Chirurgiche Generali e Specialistiche, Sezione Ginecologia e Ostetricia A, Università di Bari, Bari, 6 Dipartimento Universitario di Scienze Ginecologiche, Ostetriche e della Riproduzione, II Università di Napoli, Napoli
7 To whom correspondence should be addressed at: via Luigi Caldieri 132, 80128, Napoli, Italy. Email: alviggi{at}unina.it
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Abstract |
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Key words: FSH/IVF/LH/recombinant human LH/poor responders
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Introduction |
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With the recent availability of recombinant human LH (rLH), clinicians have the opportunity of administering the two gonadotrophins independently. Thus, exogenous LH administration may be calibrated independently of rFSH. Our preliminary prospective randomized trial demonstrated that a daily rLH dose of 150 IU resulted in a significant increase in the mean number of oocytes retrieved when compared with 75 IU in women displaying steady response to rFSH (De Placido et al., 2004). The present multicentre, prospective, randomized trial was designed in order to compare the efficacy of supplementation with 150 IU/die of rLH versus an rFSH step-up protocol in young normogonadotrophic women with an initial ovarian steady response to monotherapy with rFSH undergoing a GnRH agonist long protocol.
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Materials and methods |
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Patients
Among candidates for IVFembryo transfer or ICSI cycles, only patients aged 1837 years, with menstrual cycles ranging from 24 to 35 days (intra-individual variability±3 days), basal FSH (day 3 of a spontaneous menstrual cycle) concentrations 9 IU/l, and hysteroscopic evidence of a normal uterine cavity within the last 6 months were included. In addition, only women undergoing a GnRH agonist long protocol followed by rFSH administration were enrolled.
The following exclusion criteria were adopted: body mass index [BMI = weight (kg)/height (m2)] <18.0 and >28.0, biochemical and/or ultrasonographic evidence of polycystic ovarian syndrome (PCOS), stage IIIIV endometriosis according to the rAFS (1985), chromosomal abnormalities, endocrinological and/or autoimmune disorders, more than two previously unsuccessful IVF or ICSI cycles, and presence of only one ovary. On this basis, a total of 1389 patients were initially considered as potentially eligible. On the eighth day of stimulation, 130 patients showing a steady response (see Ovarian stimulation protocols) were randomized to two groups to receive rLH supplementation (group A) or an increase in the rFSH dose (group B). One hundred and thirty patients with an initial normal response to the ovarian stimulation protocol constituted group C.
Thus, a total of 260 women were enrolled. Six patients in group A and seven in group B had their cycle cancelled for an insufficient ovarian response. Eighteen women in group C dropped out from the study for cycle cancellation due to high risk of ovarian hyperstimulation syndrome (n=11), and seven were protocol violators. Finally, 59 patients in group A, 58 in group B and 112 in group C were considered for efficacy analysis (Figure 1).
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Patients who displayed one to four follicles of diameter >10 mm or no follicle >10 mm but serum E2 levels 180 pg/ml on day 8 of stimulation were outside the criteria for inclusion in either groups A, B or C and were therefore excluded from the present study. The ovarian response was defined inadequate when less than four follicles of 12 mm in diameter were observed on day 12 of stimulation. On the basis of this criterion, six and seven stimulation cycles were cancelled in groups A and B respectively. In group C, seven women were not considered for the efficacy analysis because of protocol violation and 11 cycles were cancelled because of an increased risk for ovarian hyperstimulation syndrome. The ovulatory dose (10 000 IU i.m.) of hCG (Gonasi®; AMSA SRL, Italy) was administered when three follicles showed a mean diameter of
17 mm. Oocytes were retrieved by transvaginal ultrasound-guided aspiration 35 h after the hCG injection. Serum concentrations of LH were measured on the day of pituitary suppression assessment and on the eighth day of stimulation (just before randomization into groups A and B).
Luteal phase supplementation
Patients began progesterone, 50 mg in oil (Prontogest®; AMSA SRL), luteal phase supplementation with daily i.m. administration (50 mg/day) on the day of oocyte retrieval.
Hormone measurement
Serum concentrations of E2 and LH were measured using an enzyme-linked fluorescent assay technique (Vidas oestradiol II and Vidas LH respectively; BioMérieux SA, France). The detection limits, defined as the lowest concentration that is significantly different from zero with a probability of 95%, were 9.0 pg/ml for E2 and 0.1 IU/l for LH. The intra- and inter-assay coefficients of variation (CV) were <8% for both E2 and LH assays. Serum FSH was determined by an immunometric assay based on enhanced luminescence (Amerlite FSH assay; Amersham International PLC, Amersham Pharmacia Biotech, UK). The detection limit was 0.5 IU/l. The intra- and inter-assay CV were <7%.
Statistical analysis
The results are reported as the mean±SD. Data were analysed with the SPSS version 12.0 (SPSS Inc., USA). Normal distribution has been inferred to continuous variables whose skewness ranged between +1.0 and 1.0 in each single group. In such a case, one-way ANOVA was used to determine the effect of the stimulation protocol. The post hoc Fisher least-significant-difference method was used to assess differences between groups. The MannWhitney U-test was applied to test differences between groups for continuous variables with non-parametric distributions. 2-Statistics were used to compare discontinuous data. P<0.05 was considered statistically significant.
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Results |
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Mean serum E2 levels on the day of hCG administration were significantly higher in the group of patients receiving rLH (1778.9±778.6 pg/ml) when compared with group B (1248.0±472.4 pg/ml, P<0.001). Mean levels observed in the control group, group C (2376.5±722.8 pg/ml), remained significantly higher (P<0.001) than those measured in both groups A and B (Table II, Figures 2 and 3).
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Implantation rates (number of gestational sacs/number of transferred embryos) were 14.2, 10.5 and 18.1% in groups A, B and C respectively; the only statistically significant difference was observed between groups B and C (P<0.05). The cumulative pregnancy rate (positive serum hCG per started cycle) was also significantly lower in group B (29.3%) with respect to group C (47.3%, P<0.05), whereas in patients treated with rLH, the pregnancy rate was 37.2%. Abortion rates were 17.0, 22.0 and 13.2% in groups A, B and C respectively (not significant). The ongoing pregnancy rates (number of pregnancies reaching week 12/cycle) were 32.5, 22.0 and 40.2% in the three groups respectively. Also for this parameter the only statistically significant difference was revealed between groups B and C (P<0.05). Serum LH levels on the day of assessment of pituitary suppression and on day 8 of stimulation were comparable in the three groups (Table II).
When the IVF outcome was evaluated according to the actual method of fertilization, standard IVF or ICSI, women who underwent standard IVF in group C showed a significantly higher (P<0.05) implantation rate (21.2%) when compared with those of group B (7.7%).
Furthermore, the IVF patients' pregnancy rate in group C (58.1%) was significantly higher than that observed in both group A (28.6%) and group B (23.1%).
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Discussion |
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The hypothesis of the present study was that this particular profile of initial ovarian response is due to insufficient LH activity following desensitization with a depot GnRH agonist. If this hypothesis is correct, the addition of rLH would reverse this condition and consequently result in a similar ovarian response to that in normally responding patients. This is the reason why a further control population, represented by women treated in the same centres during the same period and showing a good profile of initial response to rFSH, was included.
A significant increase in the number of COC (primary endpoint) was observed in patients with ovarian steady response who received rLH in the course of COS, when compared with those undergoing an increase in rFSH dose. Not surprisingly, this parameter was significantly higher in patients with a good initial ovarian response (group C). Nevertheless, it is interesting to observe that the mean number of mature oocytes, implantation and pregnancy rates of these normal responders were similar to those observed in women treated with rLH. Furthermore, in both groups these parameters were significantly better than those observed in group B. This evidence supports the hypothesis that women with a steady response are potential normal responders, whose ovarian and IVF outcome is significantly improved by LH supplementation.
Results concerning the effects of this treatment on ovarian outcome are consistent with our previous data (De Placido et al., 2001): in that study, the administration of exogenous LH in the form of hMG rescued the ongoing COS cycle in the same subset of patients, resulting in a significant increase in the number of oocytes retrieved when compared with the classical rFSH step-up protocol. The relevance of confirming this observation by using rLH is related to the advantage that in this case no change in the rFSH regimen is required. Firstly, it is to be considered that according to our previous data (De Placido et al., 2004
), an rLH daily dose of 150 IU is more effective than 75 IU in improving the ovarian outcome in this subgroup of patients. Notwithstanding, in our present and previous studies, the administration of such a daily rLH dose resulted in a significant decrease in the cumulative rFSH amount and in a trend toward a reduction of the stimulation length. The hypothesis that a higher rLH daily dose (i.e. 225 IU) would have improved the ovarian outcome cannot be excluded. Nevertheless, considering our previous study (De Placido et al., 2004
), the daily dose of 150 IU seemed to be the most suitable in order to improve the ovarian response to that commonly reported in normal responders. Recent studies have underlined the relevance of LH activity during follicular growth (Filicori and Cognini, 2001
; Filicori, 2003
). Nevertheless, clinical trials have shown that rLH supplementation in unselected young normogonadotrophic patients undergoing COS with rFSH does not improve either the ovarian or the IVF outcome when compared with rFSH monotherapy (Sills et al., 1999
; Ben-Amor et al., 2000
; Marrs et al., 2004
). These data indicate that residual LH levels after GnRH agonist-mediated pituitary desensitization are usually adequate to support multiple follicular development when rFSH is administered, and prompted investigators to identify specific subgroups of women who may benefit from exogenous LH administration (Humaidan et al., 2004
). Lisi et al. (2001)
have recently reported that women who had required a high rFSH dosage in a previous cycle showed a significant increase in implantation and pregnancy rates in a subsequent stimulation performed with rFSH and rLH. In this context, it could be considered that in the present study patients with initial steady response who underwent treatment with increased doses of rFSH required a mean cumulative dose >4000 IU. Taken together, our results and data from Lisi et al. (2001)
seem to identify a specific subset of women who cannot be classified either as poor responders, due to the achievement of a normal ovarian response (i.e. more than five oocytes retrieved), or as normal responders, due to the high cumulative rFSH requirement. This subgroup shows suboptimal ovarian and IVF outcomes and seems to benefit from rLH administration. These considerations lead to the hypothesis that an early identification of women at risk for requiring a high rFSH dose may allow an adequately timed integration with rLH, which, in turn, may rescue the ovarian response and improve the IVF outcome. Thus, a certain number of re-stimulations may be potentially avoided. The question is which is the most reliable criterion for identifying subjects who require exogenous LH. The evidence that these patients benefit from an rLH supplementation leads to the hypothesis that endogenous levels of the hormone may fall below a hypothetical threshold in the course of COS due to the combined action of the GnRH agonist and rising levels of E2 (and/or other factors secreted from developing follicles). Interestingly, our studies failed to demonstrate any significant correlation between circulating levels of endogenous LH and initial response to rFSH: patients with initial steady response (groups A and B) and patients in group C had comparable serum LH levels on the day of the assessment of suppression after GnRH agonist administration and on the eighth day of stimulation (before the first rLH dose in group A). This finding is consistent with previous studies demonstrating that levels of LH circulating during COS do not correlate with any ovarian outcome parameter (Westergaard et al., 2000
; Balasch et al., 2001
). In addition, available data from the literature are controversial and insufficient to recommend LH supplementation in women undergoing IVF on the basis of the analysis of LH values during COS (Fleming et al., 1998
; Westergaard et al., 2000
; Balasch et al., 2001
; Humaidan et al., 2002
). The contradictory results reported by different investigators may be related to differences in the assays used to measure LH. A two-site immunoradiometric assay (IRMA) may be most accurate, but whatever assay is used there is no guarantee that the LH measured is actually bioactive. Additionally, the type of GnRH agonist and the mode of administration seem to play a role in ovarian response and each may affect the endogenous LH activity (Westergaard et al., 2001
).
A clinical history of requirement of high rFSH doses in a previous cycle may prompt the addition of rLH in a subsequent one. Analogously, our data also suggest that an ongoing stimulation cycle with specific characteristics of initially inappropriate ovarian response to rFSH may be rescued by rLH supplementation. Thus, following confirmation of these data, the ovarian response to rFSH (in ongoing or previous cycles) may reveal a practical and reliable criterion for identifying women to undergo rLH treatment.
The observation that subsets of women showing an improvement in their ovarian response by rLH administration display apparently normal circulating levels of the endogenous hormone remains to be explained. It could be hypothesized that in these subjects, a less biologically active LH is present. In other words, serum concentrations of the immunoreactive molecule may be not representative of the effective bioactivity of the hormone (Huhtaniemi et al., 1999; Jiang et al., 1999
; Ropelato et al., 1999
). Similar clinical findings have been previously reported in the literature. In particular, discrepancies between immunoreactive and bioactive LH are manifest as an age-related phenomenon in men (Mitchell et al., 1995
). The presence of a less bioactive LH may also explain the observation of the statistically significant increase (
50%) in the number of oocytes retrieved in women with a steady response treated with rLH when compared with the rFSH group. Another possible explanation may involve a change at the level of the receptor (Huhtaniemi et al., 1999
). Finally, it is to be underlined that the use of different GnRH agonist preparations and/or administration modalities may result in different degrees of endogenous LH suppression (Westergaard et al., 2001
). In particular, the use of depot preparation may lead to a higher degree of suppression, which in turn may favour the manifestation of a steady response. Conversely, it could be hypothesized that the use of daily s.c. or intranasal preparation could reduce the probability of such a phenomenon.
In conclusion, our results suggest that: (i) specific subgroups of young normogonadotrophic women showing an initial abnormal ovarian response to monotherapy with rFSH require an LH supplementation during COS; (ii) in this subset of women, the initial clinical response to rFSHmore so than to LH serum levelsduring COS seemed to be most predictive of the exogenous LH requirement; (iii) in agreement with previous findings, the eighth day of stimulation seemed to be appropriate for starting rLH supplementation. Finally, larger prospective randomized trials are required to evaluate the effects of rLH supplementation in these subsets of patients on IVF outcome, according to the method of fertilization (IVF or ICSI).
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Acknowledgements |
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Notes |
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References |
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Submitted on August 6, 2004; accepted on October 29, 2004.