Recombinant follicle stimulating hormone is effective in poor responders to highly purified follicle stimulating hormone

G. De Placido, C. Alviggi1, A. Mollo, I. Strina, M.T. Varricchio and M. Molis

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, Italy


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Ovarian stimulation in cases of poor ovarian responsiveness is an important challenge in in-vitro fertilization (IVF) programmes. Despite improvements in oocyte number and quality, an ideal ovarian stimulation strategy has yet to be defined. Here, the results of ovarian stimulation with recombinant follicle stimulating hormone (rFSH) in 28 poor responders to highly purified FSH (FSH-HP) with high basal concentrations of FSH are reported. The protocols used on the FSH-HP and rFSH cycles were identical with the sole exception of the FSH preparation: triptorelin 0.1 mg/day (gonadotrophin-releasing hormone, GnRH-agonist short protocol) and the starting FSH dose of 300 IU/day were administered from day 2 of the menstrual cycle. Ovarian outcome was classified as `normal', `intermediate' and `poor', depending on the number of mature oocytes retrieved and the peak serum oestradiol concentration. Nine of the 28 subjects had an intermediate ovarian response to re-stimulation with rFSH. In the 26 patients who received human chorionic gonadotrophin on both cycles, re-stimulation resulted in a significant increase (P < 0.05) in the mean number of mature oocytes (2.4 ± 1.4 versus 1.7 ± 0.8), mean peak oestradiol concentration (606 ± 252 versus 443 ± 32 pg/ml) and fertilization rate (73.0 versus 53.3%). Four pregnancies were achieved. It is concluded that rFSH in a GnRH-agonist short protocol improves the ovarian outcome in poor responders to FSH-HP with high basal concentrations of FSH.

Key words: exogenous FSH ovarian reserve test/GnRH-agonist/in-vitro fertilization/poor responders/recombinant FSH


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
An adequate ovarian response to ovarian stimulation is a prerequisite for successful in-vitro fertilization (IVF). A poor ovarian response and the consequent low number and quality of oocytes retrieved are frequently associated with a sub-optimal IVF outcome (Ben Rafael and Feldberg, 1993Go). Among the variables correlated with a poor ovarian response to exogenous gonadotrophins are maternal age and serum concentrations of follicle stimulating hormone (FSH) in the follicular phase (Padilla et al., 1996Go). In particular, within the population of poor responders, women with elevated baseline FSH concentrations on menstrual cycle day 3 tend to respond poorly in subsequent cycles (Hershlag et al., 1990Go). Notwithstanding the various stimulation protocols that have been devised in the attempt to improve IVF outcome in poor responders to ovarian stimulation (Dor et al., 1995Go; Awonuga and Nabi, 1997Go; Schoolcraft et al., 1997Go; Lashen et al., 1998Go), the latter technique remains one of the most controversial and disappointing issues in reproductive medicine.

A series of studies has shown that recombinant FSH (rFSH), which is 99% pure and free from luteinizing hormone (LH) activity (Howles, 1996Go), improves ovarian outcome in women with normal ovarian responsiveness to urinary FSH (uFSH) (Bergh et al., 1997Go; Out et al., 1997Go), and in poor responders with normal concentrations of basal FSH (Raga et al., 1999Go). These clinical observations indicate that in such assisted reproduction techniques as IVF, rFSH results in the retrieval of a significantly greater number of oocytes, more embryos and higher pregnancy rates with respect to uFSH. These findings prompted the current investigation into whether rFSH is effective in subjects with high basal FSH levels who had responded poorly to a previous IVF cycle with highly purified FSH (FSH-HP).


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patient selection
Between January and September 1997, all candidates for IVF underwent the exogenous FSH ovarian reserve test (EFORT) (Fanchin et al., 1994Go). Subjects received 300 IU of FSH-HP (Metrodin HP; Serono Pharma, Rome, Italy) on the third day of the menstrual cycle at 8:00 a.m. Two blood samples were withdrawn from each patient immediately before and at 24 h after the injection.

The results of the EFORT were classified according to baseline serum FSH concentration and the post-exogenous FSH serum oestradiol increment. Based on previous experience (G.De Placido et al., unpublished data), the threshold value for basal FSH was set at 9 IU/l (95% confidence limit of our FSH assays; normal results: <=9 IU/l) and that for oestradiol increment was 25 pg/ml (conversion factor to SI units, 3.671; normal results: >=25 pg/ml), which was the lowest oestradiol increment value observed in women who had a `normal' ovarian response. The results of the EFORT were classified `normal' when both parameters were normal, `altered' when both were outside normal values, and `intermediate' when only one parameter was outside normal values. Seventy-six Caucasian patients [mean (± SD) age 35.7 ± 8.4 years] affected by tubal infertility and with an altered EFORT were selected for this study. Exclusion criteria were: a normal or intermediate EFORT; stage III–IV endometriosis; body mass index [BMI = weight (kg)/height (m2)x100] value >26; presence of only one ovary; and moderate or severe male factor.

Ovarian stimulation protocols
All 76 patients underwent ovarian stimulation with FSH-HP in a `flare-up' gonadotrophin-releasing hormone (GnRH)-agonist protocol three menstrual cycles after undergoing the EFORT. From the second day of the menstrual cycle, decapeptyl (Ipsen SpA, Milan, Italy) 0.1 mg/day s.c. was administered in association with 150 IU of FSH-HP (Metrodin HP; Serono Pharma, Rome, Italy) twice daily (given at 8:00 a.m. and between 6:00 and 8:00 p.m.).

The protocol was adjusted for each subject according to serum oestradiol concentration and number of follicles. Serum oestradiol concentrations were measured and follicular growth was monitored with ultrasonography on the fifth day of stimulation and on alternate days thereafter until human chorionic gonadotrophin (HCG) was administered. On the fifth day of stimulation, serum oestradiol was measured at 8:00 a.m.: the daily FSH-HP dose was not changed in patients whose oestradiol serum concentrations were in the range 50–160 pg/ml. The evening FSH-HP dose was increased to 225 IU or reduced to 75 IU when serum oestradiol concentrations were <50pg/ml or >160pg/ml respectively. Further changes in the FSH dose were made only when patients were at risk for ovarian hyperstimulation (Blankstein et al., 1987Go). The cycle was cancelled when, on day 9 of monitoring, serum oestradiol concentrations were not triplicated with respect to day 5 values and/or there were less than two follicles of 12 mm in diameter. Ovarian outcome was arbitrarily classified `normal' when serum concentrations of oestradiol on the day of HCG administration were >=1500 pg/ml and when at least six mature oocytes were retrieved. Ovarian response was diagnosed `poor' when the peak serum oestradiol concentration was <500 pg/ml and a maximum of three mature oocytes were retrieved. Ovarian outcome was classified `intermediate' when one of the two parameters was altered, i.e., when the peak serum oestradiol concentration was <1500 pg/ml, or when fewer than six mature oocytes were retrieved.

On the first cycle, 28 women had a poor ovarian outcome, 31 cycles were cancelled, and 16 had an intermediate response. Only one patient had a normal ovarian outcome. From 4–6 months after the first cycle of stimulation, the 28 poor responders underwent a second course of ovarian stimulation with rFSH (Gonal F; Serono Pharma) at a dose of 150 IU administered twice daily at 8:00 a.m. and between 6:00 and 8:00 p.m., from the second day of the menstrual cycle. The GnRH-agonist dose and the protocol adjustments used on both cycles were identical, with the sole exception of the FSH preparation. On both cycles, HCG (Profasi; Serono Pharma) 10 000 IU i.m., was administered when two follicles measured at least 16 mm. Oocytes were retrieved by transvaginal ultrasound-guided aspiration 35 h after the HCG injection. Patients began progesterone 50 mg in oil (Prontogest; AMSA srl, Rome, Italy) supplementation (50 mg/day i.m.) on the day of the oocyte retrieval.

Hormone measurement
Serum concentrations of oestradiol were measured using an enzyme-linked fluorescent assay technique (Vidas Estradiol II, BioMérieux sa, Lyon, France). The detection limit, defined as the lowest concentration of oestradiol significantly different from zero with a probability of 95%, was 9 pg/ml. The intra- and inter-assay coefficients of variation (CV) for low and high concentrations were <10%. Serum FSH was determined with an immunometric technique using a FSH-Amerlite kit (Kodak Clinical Diagnostics, Amersham, UK). The detection limit was 0.04 IU/l and the intra- and inter-assay CV were <8%.

Statistical analysis
The results are reported as the mean ± SD. The statistical analyses used were the paired Student's t-test (two tails; d.f. = 1) for continuous data, and {chi}2 cross-tabulations for categorical data. A P-value of <0.05 was considered significant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
When the 28 poor responders to FSH-HP were re-stimulated with rFSH, nine had an intermediate response; the cycle was cancelled in two women because of an insufficient oestradiol response. The remaining 17 patients had a poor ovarian response. As shown in Table IGo, in the 26 patients who were given HCG in both cycles, the rFSH ovarian stimulation cycle resulted in a significant increase (P <0.05) in the average number of mature oocytes retrieved (2.4 ± 1.4 versus 1.7 ± 0.8), and in mean serum concentrations of oestradiol on the day of HCG administration (606 ± 252 versus 443 ± 32 pg/ml). The fertilization rate also increased significantly (73.0 versus 53.3%). There were no significant differences in the average number of FSH ampoules administered (54.2 ± 9.3 versus 57.7 ± 9.4) or in days of stimulation (12.7 ± 1.1 versus 13.2 ± 1.2). Four pregnancies were achieved. Serum oestradiol concentrations on days 5 (68 ± 36 versus 49 ± 10) and 9 (303 ± 102 versus 257 ± 72) were also significantly higher after re-stimulation (Table IGo). The individual patient values of the main stimulation parameters obtained on both cycles are shown in Figures 1 and 2GoGo.


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Table I. Main parameters of the 26 patients receiving human chorionic gonadotrophin in both cycles
 


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Figure 1. Individual patient values of days of stimulation and ampoules of gonadotrophins (A), number of mature oocytes (B) and transferred embryos (C) of the 26 patients receiving human chorionic gonadotrophin in both cycles [{circ}, highly purified follicle stimulating hormone (FSH-HP) cycle; {triangleup}, recombinant FSH (rFSH) cycle].

 


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Figure 2. Individual values of day 5 (A), day 9 (B) and peak (C) oestradiol serum concentrations of the 26 patients receiving human chorionic gonadotrophin in both cycles ({circ}, FSH-HP cycle; {triangleup}, rFSH cycle).

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Increases in the number of oocytes obtained from patients with poor ovarian responsiveness treated with ovarian stimulation (Padilla et al., 1996Go; Faber et al., 1998Go) have often been at the expense of costly and/or sophisticated protocols and large amounts of medication. We use the GnRH-agonist short protocol as first-line treatment in patients at risk for sub-optimal ovarian outcome (in the present study, subjects with an altered EFORT). This is a less costly procedure because fewer ampoules of gonadotrophins and fewer GnRH-agonist injections are required. In addition, this protocol seems to improve ovarian outcome in patients at risk for poor ovarian responsiveness to ovarian stimulation (Padilla et al., 1996Go), and in women with a low response to at least one IVF treatment cycle with the long protocol (Tasdemir et al., 1996Go).

Various groups have examined the efficacy of rFSH versus uFSH in women undergoing assisted reproductive techniques (Bergh et al., 1997Go; Out et al., 1997Go; Raga et al., 1999Go). It appears that this is the first study of the effect of rFSH in association with a GnRH-agonist short protocol in women with poor ovarian responsiveness to uFSH and high basal FSH concentrations.

All patients (n = 28) re-stimulated with rFSH were at-risk for poor ovarian response (i.e. they had an altered EFORT) and had responded poorly to previous FSH-HP treatment. The significant increase in the number of mature oocytes in women taking rFSH is consistent with other studies comparing rFSH and urinary gonadotrophins in ovarian stimulation, in which patients had not been selected on the basis of ovarian responsiveness (Bergh et al., 1997Go; Out et al., 1997Go).

The significant increase in fertilization rate in rFSH versus FSH-HP cycles was not associated with a significant difference in the percentage of mature oocytes. Although oocyte quality cannot be assessed quantitatively, the fertilization rate may reflect aspects of quality (Land et al., 1996Go). Consequently, the significantly lower fertilization rates observed in poor versus normal responders (Jenkins et al., 1991Go; Land et al., 1996Go) might indicate that in poor responders, impaired follicular development in response to exogenous gonadotrophins can involve both oocyte quality and number.

A significant improvement in oocyte quality may be more evident in women with impaired follicular and/or oocyte maturation in response to ovarian stimulation than in normal responders. Therefore, the discrepancy in fertilization rate between this study and others could be because earlier studies did not involve poor responders with high basal FSH levels. This discrepancy may be explained in one of two ways. Firstly, rFSH is totally free of LH activity. There is evidence that high LH levels in the follicular phase can affect oocyte and embryo quality (Regan et al., 1990Go; Daya, 1995Go). It is feasible that the low amounts of LH found in uFSH have a greater effect on follicular development in women with poor ovarian responsiveness than in normal responders.

Secondly, rFSH preparations may have a different iso-hormone profile compared with uFSH preparations. In-vitro studies have provided evidence of higher receptor binding and signal transduction of more basic iso-hormones (Ulloa-Aguirre et al., 1988Go). With ageing, the FSH iso-hormone profile becomes more acid (Wide and Hobson, 1983Go); thus, the bioactivity of uFSH obtained from post-menopausal women may differ from that circulating in pre-menopausal women. Poor responders to ovarian stimulation may be more sensitive to differences in iso-form profiles.

The results reported herein suggest that rFSH can significantly improve the IVF outcome in poor responders to FSH-HP. However, prospective and randomized studies are needed to assess the real impact of rFSH on oocyte and embryo quality in these patients. In fact, in the current study it is not possible to exclude categorically that the previous stimulation could have conditioned the outcome of the subsequent cycle. Attempts were made to reduce this source of potential error by separating the cycles by a minimum of 4 months.

In conclusion, the significant increase in the average number of mature oocytes collected and in mean serum oestradiol concentrations, as well as the improved fertilization rate observed in this study, indicate that rFSH in a GnRH-agonist short protocol may improve the IVF outcome in poor responders to FSH-HP with high basal FSH levels.


    Acknowledgments
 
The authors thank Mrs Jean Gilder for helpful discussion of the paper and Dr Gianluca Ficca for his suggestions on statistical analysis of the data.


    Notes
 
1 To whom correspondence should be addressed at: via L. Caldieri 132, 80128 Napoli, Italy Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on April 8, 1999; accepted on October 11, 1999.