Follicular development and hormone concentrations following recombinant FSH administration for anovulation associated with polycystic ovarian syndrome: prospective, randomized comparison between low-dose step-up and modified step-down regimens

Juan Balasch,1, Francisco Fábregues, Montserrat Creus, Bienvenido Puerto, Joana Peñarrubia and Juan A. Vanrell

Institut Clínic of Gynecology, Obstetrics and Neonatology, Faculty of Medicine-University of Barcelona, Hospital Clínic-Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The present study compared ovarian performance and hormone concentrations, after ovulation induction, in polycystic ovarian syndrome (PCOS) patients, using recombinant human FSH (rhFSH) in low-dose step-up and modified step-down regimens. Twenty-six women with clomiphene citrate-resistant chronic anovulatory infertility were treated with rhFSH in two consecutive cycles according to two different low-dose regimens: (i) the classic chronic low-dose step-up protocol, the starting dose being 75 IU; (ii) a modified step-down protocol where the starting dose was 300 IU followed by 3 days free of treatment, then rhFSH 75 IU daily was given and stepwise dose increments were performed exactly the same as in the step-up method. Each woman received both treatment approaches, in a randomized order, with an interval of >=1 month between treatments. The total number of follicles that were >10, >14 and >17 mm in diameter on the day of human chorionic gonadotrophin (HCG) administration, and thus cycles with HCG cancelled, were significantly increased with the step-up approach. The total number of rhFSH ampoules tended to be higher with the step-down schedule despite the fact that both the mean duration of treatment and the threshold dose were similar with the two low-dose approaches. A physiological step-down approach for ovulation induction in PCOS patients may be more appropriate in order to avoid multifollicular cycles than the step-up approach.

Key words: low-dose FSH/ovulation induction/polycystic ovary/recombinant FSH/step-down regimen


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Gonadotrophin therapy is widely used for ovulation induction in clomiphene citrate-resistant patients with polycystic ovarian syndrome (PCOS), which is the commonest cause of anovulatory infertility (Hull, 1987Go; Hill, 1988Go). Since the early 1960s, these patients have been treated with urinary gonadotrophins in a conventional high dose step-up protocol which has proven to be effective in terms of overall pregnancy rates (Fauser and van Heusden, 1997Go). However, because of the high sensitivity of polycystic ovaries to exogenous gonadotrophin treatment, the major problem associated with this protocol is the development of multiple follicles leading to multiple pregnancy and/or ovarian hyperstimulation syndrome (Fauser and van Heusden, 1997Go).

Since the medical and social implications of a rising rate of multiple gestations have emerged, the need to re-evaluate the use of gonadotrophins for ovulation induction in PCOS patients has become imperative, thus leading to the implementation of low-dose treatment programmes which have been used both in step-up and step-down regimens (Franks and Gilling-Smith, 1994Go; Fauser and van Heusden, 1997Go).

The present report was undertaken to compare ovarian performance and hormone concentrations after ovarian stimulation in PCOS using recombinant human (rh)FSH in two different low-dose gonadotrophin regimens, the classic chronic step-up protocol and a modified step-down protocol. While rhFSH has proved to be highly efficacious for ovulation induction in PCOS patients when used in the standard step-up regimen (Balasch et al., 1998Go; Coelingh-Bennink et al., 1998Go), it appears that no previous report has investigated its use in the step-down approach.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients
A total of 26 women with anovulatory infertility due to PCOS were included in the present study, which was approved by our internal ethics committee. The mean (± SE) age of the patients was 31.1 ± 0.6 years and their mean duration of infertility was 4.1 ± 0.5 years. They presented with oligomenorrhoea or amenorrhoea, the mean basal LH/FSH ratio was 2.3 ± 0.35 and their mean basal androstenedione and free testosterone concentrations were 298 ± 24 ng/dl (normal values 60–200 ng/dl) and 5.8 ± 0.7% (normal values 0.3–3.8%) respectively. Their mean body mass index was 26.8 ± 0.7 kg/m2, and all of them had the ultrasonographic appearance of polycystic ovaries (Adams et al., 1986Go). Normal male partner semen parameters, a normal hysterosalpingogram or laparoscopy, and no history of pelvic surgery and/or pelvic inflammatory disease were recorded prior to ovulation induction in these patients. All of them either had failed to ovulate with clomiphene citrate or had not conceived after at least three ovulatory cycles on this treatment at doses <=200 mg/day for 5 days.

Study protocol
Each woman received both step-up and step-down low-dose rhFSH treatment protocols in a randomized order, with an interval of at least 1 month between treatment cycles. Treatment with s.c. rhFSH (Gonal-F; Serono SA, Madrid, Spain) was commenced on day 3 of a spontaneous cycle or of induced uterine bleeding after a baseline ultrasound examination. The chronic low-dose step-up regimen consisted of administration of a starting dose of 75 IU rhFSH per day and increased, if necessary, by increments of 37.5 IU. The first increase in daily dose was performed after 14 days of therapy only if there was no evidence of an ovarian response on ultrasound (i.e. no follicle >10 mm in diameter). Further dose adjustments were performed if necessary after a period of 7 days. This stepwise increase was continued until ovarian activity was seen on ultrasound; then, the same dose (i.e. the threshold dose) was continued until follicular diameter was >17 mm. Human chorionic gonadotrophin, 10 000 IU i.m. (HCG; Profasi; Serono SA) was then given to induce ovulation. The injection of HCG was withheld if four or more follicles >14 mm in diameter were present.

In the modified step-down protocol, patients received 4 ampoules (300 IU) of rhFSH on cycle day 3 and no treatment was given on the next 3 days (cycle days 4-6). rhFSH therapy was reinitiated on cycle day 7 (treatment day 5) by administering 1 ampoule per day of rhFSH after pertinent ultrasound scanning of the ovaries had been performed. This dose was maintained until cycle day 9 (i.e. 1 week after treatment was started) and then the protocol was exactly the same as that in the low-dose step-up approach. Thus, each woman was her own control for ovarian performance and hormone concentration studies.

Ovarian response monitoring
Ovarian response to rhFSH administration was monitored by vaginal ultrasound scanning and serum oestradiol measurements. Monitoring of ovarian response in the step-up regimen was performed according to the well-established principles of this chronic low-dose therapy (Homburg and Howles, 1999Go), i.e. weekly until ovarian response was observed on ultrasound. For the specific purpose of this study, monitoring of ovarian response in the step-down regimen was performed on treatment days 5 (cycle day 7) and 8 (cycle day 10) and repeated thereafter as in the step-up protocol. Oestradiol was measured by a competitive immunoenzymatic assay (Immuno 1; Bayer, Tarrytown, NY, USA). The sensitivity of the assay was 10 pg/ml and the interassay coefficient of variation was 5%. Ultrasonic scans were performed with a 5 mHz vaginal transducer attached to an Aloka sector scanner (model SSD-620; Aloka, Tokyo, Japan).

Sample size and statistical analysis
The primary end-point of this study was to compare ovarian performance and hormone concentrations (but not pregnancy rate) using two different low-dose regimens in order to avoid multifollicular cycles. Thus, before initiation of the study, power calculations were performed to determine the required number of patients for the detection of differences in the number of dominant follicles on the day of HCG administration, comparing both low-dose regimens. The only previous study on the subject by van Santbrink and Fauser (1997) reported a 32% difference regarding monofollicle growth in favour of the step-down protocol versus the step-up regimen. On this basis, assuming a power of 80% to detect such a difference between regimens in the proportion of unifollicular cycles obtained with a type I risk of 0.05, at least 25 women acting as their own controls were needed.

Data were analysed by Statistics Package for Social Sciences statistical software using the Wilcoxon matched-pairs signed-ranks test and the {chi}2-test as appropriate. Results are expressed as means with SE.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A total of 29 eligible patients were entered into the study. Fifteen of them were randomly allocated to the step-up treatment group first and 14 to the step-down group. There were two and one pregnancies in the step-up and step-down treatment groups respectively, during the first cycle of therapy. Exclusion of these three cycles, however, did not influence results. Thus, in total, the responses of 26 patients to both low-dose gonadotrophin regimens were evaluated.

All treatment cycles were ovulatory according to ultrasonographic data, basal body temperature recording, and the length of the luteal phase. No woman developed ovarian hyperstimulation syndrome. Comparative results of the two rhFSH treatment modalities are summarized in Table IGo. The total quantity of FSH used to induce ovulation in PCOS patients was higher, albeit not statistically significant, with the step-down method despite the fact that both the mean duration of treatment and the threshold dose were similar with the two approaches. Monofollicular cycles (i.e. only one follicle developed that reached >17 mm in diameter) were obtained in 46 and 54% of treatment cycles in the step-up and the step-down methods respectively (not significant). The key difference between the step-up and the step-down approaches was the significant reduction in the total number of growing follicles (>10 mm), as well as in the number of follicles that were mature (>14 mm) and >17 mm on the day of HCG injection. Accordingly, the number of cycles in which HCG withheld because of ovarian hyper-response was significantly higher in the step-up group. In agreement with final follicular development, oestradiol serum concentrations on the HCG day of administration were 32% higher with the step-up protocol, though this was not statistically significant.


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Table I. Ovarian response to treatment with the two regimens of low-dose recombinant human (rh)FSH
 
Pertinent data on follicular dynamics and oestradiol concentrations during rhFSH treatment in both study groups are presented in Table IIGo. Neither ultrasonography nor oestradiol serum determination were indicative of follicular growth on treatment day 5 (cycle day 7) with the step-down regimen. In addition, both follicular development and oestradiol serum concentrations were similar in the two regimens of low-dose rhFSH on treatment day 8 (cycle day 10). This clearly suggests that ovarian response monitoring may be the same for both treatment regimens.


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Table II. Follicular dynamics and oestradiol concentrations during ovarian stimulation with the two regimens of low-dose recombinant human FSH
 
There were two and one clinical gestations in the step-up and step-down treatment groups respectively, during the second cycle of gonadotrophin therapy (i.e. after cross-over was done). Thus, overall fecundity rate was 14.3 and 7.4% for the step-up (four pregnancies among 28 treated cycles) and the step-down (two pregnancies in 27 treated cycles) regimens respectively.


    Discussion
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 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The most important principle in ovulation induction is to provide as close as possible a physiological restoration of cyclical ovarian function; in particular, the aim should be to achieve the ovulation of a single follicle. Multiple follicular development is a complication which is characteristic of ovulation induction with exogenous gonadotrophins, particularly in women having PCOS who are highly sensitive to gonadotrophin stimulation (Franks and Gilling-Smith, 1994Go). In fact, ~75% of iatrogenic multifetal pregnancies are due to ovulation induction while the remaining 25% are the product of assisted reproductive techniques (Evans et al., 1995Go; Corchia et al., 1996Go).

Chronic, low-dose gonadotrophins have been widely used over the last decade in a step-up regimen where the starting dose of FSH is 75 IU daily which is gradually (half-ampoule per day) increased at 1–2 week intervals in an attempt to slowly and prudently surpass the individual FSH threshold for follicular recruitment (Franks and Gilling-Smith, 1994Go; Fauser and van Heusden, 1997Go). Low-dose step-up gonadotrophin therapy, however, may still lead to overstimulated cycles with multiple follicular development in PCOS patients (Herman et al., 1993Go). Thus, the largest series published so far (Hamilton-Fairley et al., 1991Go; White et al., 1996Go) and based on 934 treatment cycles indicates that ovulation of a single dominant follicle is attained in only ~50% of started gonadotrophin treatment cycles; 20% of them were abandoned before completion because more than three large follicles developed. This is in agreement with data in the present study when using the step-up regimen.

On the other hand, a low-dose step-down schedule, in which a large dose of gonadotrophin (150–225 IU) is given for an initial 2 or 3 days in order to mimic physiological secretion of endogenous FSH release, has been reported (Mizunuma et al., 1991Go; van Santbrink and Fauser, 1997Go). This is followed either by an approach similar to the step-up protocol (Mizunuma et al., 1991Go) or by a progressive decrease (every 3 days) of daily FSH dose to a minimum of 75 IU/day in order to obtain a subthreshold dose designed to maintain only the growth of the lead follicle (van Santbrink and Fauser, 1997Go). The first option is associated with a low rate (35%) of single dominant follicular development (Mizunuma et al., 1991Go) while the latter results in more monofollicular cycles (defined as not more than one follicle >=16 mm on the day of HCG administration) than the step-up protocol (van Santbrink and Fauser, 1997Go), but it demands more intense monitoring and its reproducibility may be difficult to achieve mainly because of the long half-life of FSH preparations (Baird, 1996Go; Franks and Hamilton-Fairley, 1996Go).

The present study, in which a modified low-dose step-down method of rhFSH administration was used for the first time, shows a significant reduction of multifollicular development which can lead to cycle cancellation as compared with the classic step-up approach. Interestingly, results in the present study preclude the need for a more stringent monitoring of follicular growth with the modified step-down approach than in the step-up method. The rationale for using this approach is as follows. First, the FSH concentration rises at the beginning of the natural cycle and this is essential for follicular recruitment (Hillier, 1994Go). Second, elevation of FSH concentrations above the threshold in the early follicular phase does not affect dominant follicle development (Hillier, 1994Go). Third, because of the terminal half-life of rhFSH the maximal effect of a given dose cannot be observed before 3–4 days (Le Cotonnec et al., 1994Go; Shoham and Insler, 1996Go). Fourth, gonadotrophin protocols for ovulation induction using an intermittent dose have been applied successfully (McFaul et al., 1989Go; Hardiman and Ginsburg, 1996Go). Finally, the step-down method has proven to be clinically useful (van Santbrink and Fauser, 1997Go; Fauser and van Heusden, 1997Go).

In the previous comparative study (van Santbrink and Fauser, 1997Go) of the two low-dose regimens, however, after a conventional initial high dose of gondotrophin was given, a gradual reduction of the dose was employed in the step-down approach. In contrast, in the present study a period of coasting was used. Coasting is feasible as the half-life of rhFSH is ~36 h (Le Cotonnec et al., 1994Go). One argument used to support this approach is that coasting can rescue cycles prone to develop ovarian hyperstimulation syndrome thus indicating that a pronounced decrease in serum FSH concentrations prevents the further development of medium-sized follicles, whereas large follicles continue to mature (Fauser and van Heusden, 1997Go). However, during coasting a sudden drop of oestradiol concentration can occur that leads to cycle cancellation (Sher et al., 1995Go). This has been observed by us, both in ovulation induction in anovulatory women and during ovarian stimulation (unpublished data). Therefore, in order to avoid cancellation a high starting dose was applied to mimic the natural cycle thus promoting follicular recruitment, to be followed later by small stepwise increments if necessary which may be critical in determining follicular maturation (Franks and Hamilton-Fairley, 1996Go). The above notwithstanding, the quantity of FSH required to induce ovulation was somewhat lower in the current study with the low-dose step-up method. This implies that further refinement of the initial high starting dose could be employed, but using the same coasting period. Also, further studies are warranted in order to compare the two low-dose regimens used in terms of pregnancy rates.

A feature of the current study is that, in contrast with a previous report comparing the low-dose step-up and step-down dose approaches (van Santbrink and Fauser, 1997Go), each patient acted as her own control; women who became pregnant in their first cycle, and thus did not receive the second treatment, were not included in the analysis of ovarian response. PCOS is a heterogeneous condition with distinct endocrine features. The FSH threshold varies for individual patients, thus suggesting variable abnormalities (Baird, 1996Go; Fauser and van Heusden, 1997Go). Therefore, the use of the same gonadotrophin drug applied to different treatment protocols in the same patient as previously reported (Couzinet et al., 1988Go; Shoham et al., 1991Go; Balasch et al., 1995Go, 1998Go) seems the more appropriate study design when ovarian performance and hormone concentrations but not pregnancy rate are to be compared. In fact, with cross-over trials any variability in treatment response due to individual subject characteristics (i.e. between-subject variability) is eliminated because statistical analysis is based on within-subject comparison (Daya, 1999Go).

In conclusion, the present study and the other few reports in the literature indicate that a physiological step-down approach for ovulation induction in PCOS patients women may be more appropriate in order to avoid multifollicular cycles and thus HCG cancellation than the step-up approach. However, further studies are necessary to improve this stimulation method in order to obtain the optimum clinical efficacy in terms of pregnancy rates with the minimal gonadotrophin requirements. In this respect it is to be stressed that daily injections of FSH are not really needed due to the long half-life of present FSH preparations (Buckler et al., 1999Go).


    Notes
 
1 To whom correspondence should be addressed at: Institut of Gynecology, Obstetrics and Neonatology, Hospital Clínic,C/Casanova 143, 08036-Barcelona, Spain. E-mail: jbalasch{at}medicina.ub.es Back


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 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on July 31, 2000; accepted on January 18, 2001.