1 Dipartimento Clinico di Emergenza Ostetrica, Ginecologica e Medicina della Riproduzione, Area Funzionale di Medicina della Riproduzione ed Endoscopia Ginecologica, Università degli Studi di Napoli `Federico II', Naples, 2 Istituto di Clinica Ginecologia, II Università di Napoli, Largo Madonna delle Grazie, 80128 Naples and 3 Centre for Reproductive Biology Clinica Villa del Sole,Via Manzoni, 15, 80126 Naples, Italy
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Abstract |
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Key words: gonadoptrophin/human oocyte/luteinizing hormone/pituitary/poor responders
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Introduction |
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The evidence for the role of LH during the early phases of folliculogenesis is difficult to establish unequivocally (Lèvy et al., 2000). However, several lines of evidence point to the hypothesis. The use of high concentrations of gonadotrophin-releasing hormone (GnRH) antagonist in combination with recombinant FSH (rFSH) produces a dose-dependent decline in serum oestradiol during stimulation, oocyte retrieval and pregnancies, together with an increase in abortions, suggesting that FSH alone is not sufficient for folliculogenesis (The Ganirelix dose-finding study group, 1998
). Furthermore, the ovarian response to controlled ovarian stimulation (COH) with rFSH is often reduced in women who required prolonged GnRH agonist treatment to achieve down-regulation, with a subsequently profound suppression of endogenous gonadotrophins (Fleming et al., 1998
; Ravhon et al., 2000
), although normal folliculogenesis has also been reported in some patients (Couzinet et al., 1988
; Ben-Chetrit et al., 1998
). In addition, a less profound pituitary down-regulation was achieved with the GnRH-agonist short protocol (Tasdemir et al., 1996
), and low dose GnRH-agonist protocols seem to improve the outcome in patients stimulated by rFSH with a known poor response to the `long protocol' (Feldberg et al., 1994
; Olivennes et al., 1996
; Surrey and Schoolcraft, 2000
), again suggesting that FSH alone is not sufficient for efficient folliculogenesis and oogenesis.
Pituitary down-regulation with GnRH agonist prior to COH with exogenous gonadotrophins [GnRH agonist `long protocol' (Hughes et al., 1992)] is a well established protocol for the treatment of many patient groups undergoing cycles of IVF. In young, normogonadotrophic women this protocol is the most commonly adopted COH protocol for IVF stimulation cycles worldwide (Tan et al., 1992
; Filicori, 1996
). According to the two cell, two gonadotrophin theory, the low concentrations of serum LH are sufficient to support follicular and oocyte development when the `long protocol' is used with urine-derived highly purified FSH or rFSH (Loumaye et al., 1997
; Sills et al., 1999
). However, in a subset of patients, the ovarian response to this protocol is suboptimal. This may be due to low LH activity caused either by low serum concentrations of LH (Noci et al., 1998
; Ludwig et al., 1999
) or low LH bioactivity (Schroor et al., 1999). Patients characterized by a poor response to the classical `long protocol' may benefit from the use of LH-containing gonadotrophin preparations (The European Recombinant Human LH Study Group, 1998
; Laml et al., 1999
; Fleming et al., 2000
; Lèvy et al., 2000
; Westergaard et al., 2000
). However, excessive concentrations of LH during folliculogenesis can also be detrimental (Chappel and Howles, 1991
; Yamashita et al., 1996
; Liu et al., 2000
), suggesting that the use of LH should be carefully controlled. In this study, we tested whether the addition of HMG during mono-therapy with rFSH improved the IVF outcome of normo-ovulatory normogonadotrophic, pituitary down-regulated patients characterized by a poor ovarian response to rFSH. We established criteria for the use of LH-containing preparations during COH protocols for IVF and show that these protocols, when applied to patients at risk of cycle cancellation during the `long protocol', increase the outcome significantly more than a simple increase of the FSH dose. The data establish the need for LH supplementation in specific patient groups, despite the lack of a priori indications.
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Materials and methods |
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Ovarian stimulation protocols
Pituitary desensitisation was induced with triptorelin (Decapeptyl 3.75 depot; Ipsen SpA, Milan, Italy) on the first day of the menstrual cycle. After 15 days, pituitary suppression was assessed by measuring serum oestradiol and LH concentrations; endometrial and ovarian status was also assessed by trans-vaginal ultrasound. If the concentration of serum oestradiol was 0.15 pmol/ml, serum LH was
1.5 IU/l, the thickness of the endometrium was
5 mm, and follicular development appeared to be arrested, then gonadotrophin administration was started. Patients with delayed suppression were excluded from the study. A fixed dose of 150 IU of rFSH (Gonal-F; Serono Pharma, Rome, Italy) was administered s.c. twice daily (at 8:00 am, and between 6:00 and 8:00 pm), as per our clinic routine. Serum oestradiol concentrations were measured and follicular growth was monitored with ultrasound on the fifth and the eighth day of the stimulation and thereafter on alternate days until HCG was administered. On the fifth day of stimulation, serum oestradiol was measured at 8:00 am and the evening rFSH dose was reduced to 75 IU in patients whose concentrations were >0.6 pmol/ml. On the eighth day of stimulation, patients with serum oestradiol concentrations
0.6 pmol/ml and ultrasound evidence of no follicles with a mean diameter >10 mm were randomized into two groups using random number tables (Figure 1
). In Group A (n = 20; mean age ± SD = 31.6 years ± 3.8), the evening rFSH dose was substituted by 150 IU of HMG (Menogon; Ferring S.p.A., Milan, Italy, see Figure 1
). The stimulation regime of Group B (n = 23; mean age = 30.4 ± 3.8) involved the increase of the evening rFSH dose to 225 IU (total rFSH daily dose 375 IU, Figure 1
). Patients characterized by a tripling of serum oestradiol concentrations between days 5 and 8 and >4 follicles >10 mm in diameter on day 8 constituted Group C (control group; n = 40; mean age = 30.1 ± 3.5, see Figure 1
). Nine patients with 14 follicles of diameter >10mm on day 8 were outside the criteria for inclusion in Groups A, B, or C and were therefore excluded from the present study. COH cycles were cancelled when <5 follicles of 12 mm in diameter were observed on day 12 of stimulation. When 3 follicles measured at least 17 mm in diameter, HCG (Profasi; Serono Pharma) was administered. Oocytes were retrieved by trans-vaginal ultrasound-guided aspiration 35 h after the HCG injection. Patients began 50 mg/day i.m. progesterone supplementation (Prontogest; AMSA S.r.l., Rome, Italy) on the day of oocyte retrieval.
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Statistical analysis
The results are reported as the mean ± SD. Data were analysed with the Statistica `98 version 5.1 statistics package (Stat. Soft. Inc., Tulsa, USA). One way analysis of variance (ANOVA) was used to determine the effect of the stimulation protocol on continuous variables. The post hoc Fisher least-significant-difference (PLSD) method was used to assess differences between groups. 2 statistics were used to compare discontinuous data. P < 0.05 was considered significant. The MannWhitney U-test was applied to test the significance of non-parametric distributions.
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Results |
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Discussion |
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We have tested whether LH supplementation during COH, as opposed to increasing the daily rFSH dose, can improve the outcome in normo-ovulatory normogonadotrophic patients characterized by a poor initial response after rFSH treatment. We showed that the use of LH-containing preparations caused an increase in success rates that matched the control group, i.e. normoresponders. A simple augmentation of the FSH daily dose, although rescuing the COH cycle, did not result in success rates equivalent to normoresponders, strongly suggesting that LH supplementation was necessary in these cases. Two hypotheses may explain these data. Firstly, LH concentrations after pituitary suppression in these patients may be unable to sustain multiple follicular growth. Secondly, the bioactivity of LH in some groups of patients could be subnormal. Examples of reduced LH bioactivity caused by the presence of different isoforms or altered subunits of this hormone have been reported in the literature (Huhtaniemi et al., 1999; Jiang et al., 1999
; Ropelato et al., 1999
). Whether reduced LH bioactivity is an indication for infertility in patients with normal serum LH concentrations has not yet been tested.
We examined whether a cut-off value of serum LH able to sustain multiple follicular growth existed by measuring serum LH on day 1 of stimulation (after pituitary suppression). However, we did not find any statistically significant differences between the three groups (see Table I). Our results suggest a small trend to lower LH suppression concentrations in the two study groups. If the first hypothesis proposed above is correct, LH concentrations may decrease to the critical value during COH, showing significant differences only on the eighth day of stimulation. In the present work, this hypothesis does not appear to be supported by the data. In the patients where serum LH concentrations were tested on day 8 of stimulation, no significant differences were found (see Table I
). If the immunoreactive concentrations of LH are not correlated with the requirement for LH during folliculogenesis suggested by the present data, the bioactivity of LH may play a role. Although immunoreactive LH and bioactive LH concentrations are correlated, differences are often observed (Schroor et al., 1999). We suggest that the combination of low serum LH and low LH bioactivity in certain patient groups may be the cause of the inadequate response to COH where rFSH is used alone.
In conclusion, our data demonstrates that LH supplementation in a subset of patients with an initial poor response to rFSH therapy can rescue COH cycles, increase the number of oocytes retrieved and increase peak oestradiol concentrations significantly more than a simple increase of the daily rFSH dose. The availability of recombinant LH will enable a greater degree of control over the LH dosage during COH without modifying the dosage of rFSH. It is not currently possible to establish an a priori indication for this protocol, however the protocol described in the present report is highly efficient in the rescue of COH cycles where day 8 indications otherwise suggest a poor outcome.
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Notes |
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References |
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Submitted on December 29, 2000; accepted on June 14, 2001.