Ovulation induction with low dose alternate day recombinant follicle stimulating hormone (Puregon)

H.M. Buckler1, W.R. Robertson2, A. Anderson1, M. Vickers1 and A. Lambert2,3

1 Department of Medicine, Hope Hospital, Salford and 2 Nuffield Department of Obstetrics and Gynaecology, John Radcliffe Hospital, Headington, Oxford OX3 9DU, UK


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We investigated whether a recombinant follicle stimulating hormone (FSH) (Puregon®) can be administered less frequently and at lower doses during ovulation induction than is current practice. Patients (20–35 years, body mass index <30 kg/m2) with infertility and chronic anovulation secondary to polycystic ovarian syndrome and resistant to previous clomiphene treatment received (Puregon®; 100 IU, n = 17 patients, or 50 IU, n = 10 patients) on alternate days. After 2 weeks and in the absence of follicular recruitment, doses were increased stepwise at weekly intervals (50 IU/alternate days). Twenty-two cycles out of 27 were ovulatory. There were six pregnancies, five from Puregon® (100 IU) and one from Puregon® (50 IU); four pregnancies proceeded to term. The duration of stimulation (mean, range) with Puregon® (100 IU) was 16.4, 7–29 and Puregon® (50 IU) 19.1, 8–38 days. The gonadotrophin doses administered (mean; range) were 689, 200–1800 IU (Puregon® 50 IU) and 939, 400–2300 IU (Puregon® 100 IU). We conclude that low dose alternate day Puregon® treatment is suitable for this difficult patient group.

Key words: ovulation induction/Puregon®/recombinant FSH


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The number of follicles recruited and matured in any given natural or induced cycle is dependent on the amount of follicle stimulating hormone (FSH) administered, the duration of stimulation and the sensitivity of the ovary as outlined in the FSH `threshold' and `window' concepts for in-vivo follicular growth (Brown, 1987Go; Fauser and van Heusden, 1997Go). The aim of ovulation induction is to stimulate unifollicular development and produce an oocyte of high quality with a high probability of developing into a good quality embryo, a singleton pregnancy and ultimately a healthy baby. It clearly differs from the aim of in-vitro fertilization (IVF) treatment which is normally designed to promote multifollicular development and as such will usually employ higher doses of FSH than those used for ovulation induction. Moreover, in the latter group the threshold dose required for unifollicular development can vary considerably from patient to patient and consequently step-up regimens where the dose of FSH is systematically increased by relatively small increments over the treatment period have become the strategies of choice (van der Meer et al., 1994Go; White et al., 1996Go). However, the timing and amount of the starting dose has largely been determined pragmatically and has been dependent to some extent on the type and quality of the exogenous gonadotrophin preparation employed. For example, White et al. (White et al., 1996Go) were able successfully to induce ovulation in patients with polycystic ovary syndrome (PCOS) using a low starting dose (52.5 IU/day) of urinary gonadotrophins [human menopausal gonadotrophin (HMG) and pure FSH].

Although urinary preparations have been the mainstay of assisted reproduction programmes over the last two decades and are still the most widely used throughout the world, they are intrinsically difficult to control with regard to both batch to batch purity and FSH/luteinizing hormone (LH) biological quality and quantity (Rodgers et al., 1992Go, 1995Go). With the development of recombinant human gonadotrophin FSH preparations (Puregon®; Organon, Oss, The Netherlands; Gonal-F®; Serono, Welwyn Garden City, UK) these quality issues have been overcome and an increasingly large literature on their efficacy is now available for both IVF and ovulation induction (Recombinant Human FSH Study Group, 1995Go, 1998Go) and lower starting doses (100 IU/day) of recombinant FSH in IVF have been successfully employed (Devroey et al., 1998Go) in view of the apparent increased bioactivity of Puregon® over the urinary FSH preparation, Metrodin® (Out et al., 1995Go). Further, Puregon® is more efficient than urinary FSH in inducing ovulation in World Health Organization (WHO, 1973) group II women with clomiphene citrate-resistant chronic anovulation, as demonstrated by a lower total FSH dose and a shorter treatment period (Coelingh Bennink et al., 1998Go). Thus, the existence of pure, highly standardized recombinant FSH preparations of unlimited quantity coupled to the fact that follicles appear only to need FSH for their recruitment, growth and maturity (Schoot et al., 1992Go; Shoham et al., 1993Go; Thompson et al., 1995Go) is a firm basis for reliable and rigorous investigations into the mode of delivery and dose of FSH in assisted reproduction programmes.

We have investigated whether recombinant FSH can be administered less frequently and at lower doses in an ovulation induction programme. Our protocol was designed in view of the apparent increased biopotency of Puregon® over urinary products (Out et al., 1995Go) and in the light of the detailed information on the pharmacokinetics available on this product for women (Mannaerts et al., 1996Go). With a terminal elimination half-life for Puregon® of around 30–40 h (Mannaerts et al., 1996Go) we felt that an alternate day regimen for FSH (100 or 50 IU) administration would be adequate for unifollicular development. We chose to examine this regimen in a difficult anovulatory patient group having clomiphene resistant PCOS. Some of these data have been presented in a preliminary form elsewhere (Buckler et al., 1998aGo, bGo).


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients
A total of 17 non-obese patients (body mass index <30 kg/m2, aged 20–35 years) with infertility and chronic anovulation secondary to polycystic ovarian syndrome was recruited to take part in this study. All patients gave their informed consent and the study was approved by the Salford and Trafford Ethics Committee. The patients were diagnosed as having PCOS by clinical and ultrasonographic criteria (Adams et al., 1986Go). All patients had polycystic ovaries on pelvic ultrasonography and all had biochemical evidence of hyperandrogenaemia (either raised total testosterone >2.4 nmol/l, or raised free androgen index with low sex hormone binding globulin concentrations <20 nmol/l or both). LH/FSH ratio was not taken into account since there is some controversy over its reliability as a diagnostic criterion for PCOS. The patients had a long history (>2 years) of oligomenorrhoea with cycle length >6 weeks and a detailed ovulation study performed over a time period of 6 weeks had failed to provide evidence of ovulation. In addition, all patients had failed to respond previously to 3–6 cycles of clomiphene citrate therapy. (Clomiphene citrate was administered from days 2–6; all patients started with a dose of 50 mg/day which was increased to 100 mg/day when they failed to ovulate.)

The patients received Puregon® on alternate days daily from the start of a withdrawal bleed induced by a course of a progestogen (dydrogesterone 10 mg twice daily for 10 days). All 17 patients recruited received the 100 IU protocol initially and of these, 10 received the 50 IU alternate day start dose regimen following a rest period of at least a month and a further induced withdrawal bleed. Blood was taken three times weekly during stimulation for the measurement of oestradiol, LH, FSH, inhibin A and inhibin B. Blood sampling occurred 12–24 h after the last treatment dose. Decisions about ongoing treatment were made on the basis of ultrasound scanning and oestradiol measurements. If Puregon® failed to stimulate follicular development (no dominant follicle >10 mm in diameter) after 2 weeks, doses were increased stepwise at weekly intervals (50 IU/alternate days). If a follicle emerged the dose of FSH was maintained until it was at least 17 mm in diameter. A single dose of HCG 5000 IU i.m. (Serono) was given to trigger ovulation and couples were advised to have intercourse on the day HCG was given and then daily for 5 days. Luteal support was given in the form of the progestagen, gestone (100 mg) administered on days 4 and 8 following HCG administration. All male partners had a normal semen analysis.

Oestradiol assay
Serum oestradiol concentrations were measured by Delfia kit (Wallac, Milton Keynes, UK) according to the manufacturer's protocol. In our hands this assay has a limit of detection of 50 pmol/l and intra- and interassay coefficients of variation (CV)(as determined from a precision profile constructed using >500 samples) of <10 and <13% over the range 100–1200 pmol/l.

LH and FSH assays
Serum samples were assayed for LH and FSH using the appropriate Delfia time-resolved fluorescence immunoassay kits following the manufacturer's instructions. In our laboratory, the limits of detection of the serum assays were 0.6 IU/l (LH) and 1 IU/l (FSH) and the intra- and interassay CV (as determined from a precision profile constructed using >500 samples) were <6 and <15% for all assays respectively over the range 1–250 IU/l.

Inhibin A assay
Inhibin A was measured by enzyme-linked immunosorbent assay (ELISA) using a commercial kit (Serotech, Kidlington, Oxford, UK). The assay has minimal cross-reaction with inhibin B, pro-{alpha}C or activins. In our hands the limit of detection was 4 ng/l and intra- and interassay CV (based on analysis of >500 samples) was <6 and <16% over the working range of 4–500 pg/ml.

Inhibin B assay
Inhibin B was measured by ELISA using a commercial kit (Serotech). The assay has minimal cross-reaction with inhibin pro-{alpha}C or activins, and approximately 1% cross-reaction with inhibin A. In our laboratory the limit of detection is 16 ng/l and intra- and interassay CV (based on analysis of >500 samples) was <6 and <13% over the working range of 16–1000 pg/ml.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Of the 27 cycles of treatment, 22 were ovulatory and there were six clinical pregnancies, five from Puregon® (100 IU) and one from Puregon® (50 IU). Of the five pregnancies resulting from treatment with Puregon® (100 IU), there have been three singleton live births but two women miscarried at 6 and 12 weeks gestation respectively. The pregnancy resulting from treatment with Puregon® (50 IU) resulted in the birth of healthy girl.

Of the 17 Puregon® (100 IU) treated cycles, one was abandoned due to over-stimulation (four follicles >17 mm at day 35) and two patients failed to stimulate. Two out of the 10 patients from the Puregon® (50 IU) group failed to stimulate. All completed cycles were uni-ovulatory with the exception of one cycle in the Puregon® (100 IU) group which gave rise to two follicles and one patient had three follicles following Puregon® 50 IU. Four out of eight patients ovulated after treatment with Puregon® 50 IU for <2 weeks and one patient became pregnant after treatment with Puregon® 50 IU for 2 weeks plus one 100 IU dose.

The duration of stimulation was similar in the two groups (Table IGo); the dose of gonadotrophin administered appeared to be lower in the Puregon (50 IU) group but this difference was not statistically significant due to low patient numbers. At the time of HCG administration endometrial thickness was similar in both groups (Table IGo).


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Table I. Outcome parameters of patients treated with Puregon® (100 IU) or Puregon® (50 IU). Data are presented as mean; range
 
Plasma concentrations of oestradiol, FSH, inhibin A and B on the day of HCG administration for those cycles which resulted in unifollicular development are shown in Table IIGo. FSH concentrations, measured 12–24 h after administration of the Puregon® injection (100 or 50 IU), did not increase above basal endogenous values. Oestradiol, inhibin A and B concentrations were variable at the time of HCG administration within each group but no differences were observed between the groups. The fold increases in the hormone concentrations from basal to peak values at the time of HCG administration (Table IIGo) were significant (P < 0.05) for oestradiol and inhibin A in both groups but the apparent increase of inhibin B in the Puregon (50 IU) group was not significant, probably due to low patient number.


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Table II. Serum hormone concentrations (median; range) and their fold changes (mean ± SD) measured before gonadotrophin administration (basal) and on the day of HCG administration of patients treated with Puregon® (100 or 50 IU) who had unifollicular development
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In this study we successfully achieved unifollicular development in a group of anovulatory, infertile women with PCOS who had failed to conceive during previous clomiphene citrate therapy. Ovulation was achieved using a recombinant FSH preparation, Puregon®, in an alternate day injection protocol and with much lower doses of FSH than those conventionally used. The present study extends an earlier study (White et al., 1996Go) in which a starting dose of 52.5 IU/day of urinary gonadotrophins (usually HMG, occasionally pure FSH treatment) was continued for up to 14 days before being increased in small stepwise increments if necessary. In that study 109/209 patients became pregnant with only seven multiple pregnancies (all twins) and no cases of severe hyperstimulation syndrome. The excellent pregnancy rates achieved, both in our study and in that of White et al. (1996) indicate that the oocytes matured by these treatment regimens were of high quality and it is likely that these low-dose treatment regimens result in the maturation of only the most FSH-receptor rich follicle. Very recently a daily starting dose of 50 IU of recombinant FSH (Puregon®) has been employed to induce ovulation in a group of 11 patients with clomiphene-resistant PCOS (Hayden et al., 1999Go). Six out of the 11 patients ovulated, of whom two conceived and four had their cycles cancelled due to over-stimulation. Our data with 100 IU Puregon® on alternate days clearly established that this dose is very effective in inducing ovulation but the 50 IU protocol gave rise to similar results to those seen by Hayden et al. (1999). Whether the alternate day regimen employed in our trial was of itself important was not tested by our protocol. However, it is possible that there are benefits to this approach as the ovulation and pregnancy rates achieved appeared to be somewhat better in our patients treated with 100 IU on alternate days compared with those reported by Hayden et al. (1999) in an essentially similar patient group. However, as the patient numbers were small in both studies, the relative benefits of daily vis à vis alternate day regimens and the optimal dose required with either approach need further investigation and we are presently performing this study with a larger number of patients. Of note, of 17 patients treated over the same period with the urinary preparation, Normegon® (FSH:LH ratio 3:1) in a step-up regimen (75 IU daily which was increased by 37.5 IU after 2 weeks if no dominant follicle >10 mm was present), all except one had uni-ovulatory cycles and three became pregnant. Some of these patients had previously received Puregon®. Interestingly, although the duration of stimulation was similar to the Puregon®-treated patients, the mean total dose of gonadotrophin administered to the Normegon®-treated patients was higher (P < 0.05) than in the Puregon® groups.

We found that FSH concentrations, measured 12–24 h after administration of Puregon® (100 or 50 IU), did not increase above basal endogenous values. It may be that the relative decrease in FSH concentrations which must have occurred during the 24 h prior to the next injection in our patient group led to atresia of follicles poor in FSH receptors thus enhancing the likelihood of unifollicular development and decreasing the risk of over-stimulation in a patient group prone to ovarian hyperstimulation syndrome.

Oestradiol, inhibin A and B concentrations were variable both basally and at the time of HCG administration within each group but no differences were observed between the groups due to the large patient-to-patient variation. Basal concentrations of inhibin B were slightly lower in our PCOS patients than in those reported by Lockwood et al. (1998) in a similar group of patients.

Both oestradiol and inhibin A concentrations increased markedly during gonadotrophin treatment and these data are compatible with the concept that inhibin A is a marker of the maturity of the dominant follicle (Lockwood et al., 1996Go). Concentrations of inhibin A and B during treatment were similar to values reported during the follicular phase of the normal menstrual cycle (Groome et al., 1996Go; Muttukrishna et al., 1996Go). We conclude that from an endocrine point of view, the treatment regimens employed in this study match a normal menstrual cycle in terms of both timing and hormone parameters.

The role, if any, of the gonadotrophins in PCOS is unclear. Gonadotrophin secretory abnormalities including an elevated baseline LH and LH:FSH ratio have been reported with variable prevalence (Conway et al., 1989Go; Franks, 1989Go). It has been suggested that a rapid frequency of gonadotrophin releasing hormone (GnRH) secretion may play a key role in the gonadotrophin defect in PCOS patients (Taylor et al., 1997Go). In the present study we have demonstrated that infertile patients with PCOS, whose endogenous FSH is unable to promote normal follicular development, respond rapidly (in <2 weeks in most cases) to produce unifollicular ovulation even to a small dose of Puregon® which was undetectable in blood over endogenous FSH. One possible interpretation of these observations is that the small follicles in the ovaries of patients with PCOS respond to the glycoform mixture of the recombinant FSH and that the endogenous FSH glycoforms recruit them but do not then promote growth and development of the dominant follicle. Unfortunately little is known about the glycoform quality of FSH in PCOS although the LH is markedly less acidic (Ding and Huhtaniemi, 1991Go). However, we have characterized the mixture of glycoforms in Puregon® and those which are present in the blood during the different stages of the normal menstrual cycle (Lambert et al., 1995Go; Harris et al., 1996Go; Anobile et al., 1998Go; Horsman et al., 1998Go). We are currently investigating the possibility that a different FSH glycoform mixture predominates in PCOS and is responsible for recruitment, as has been suggested by others (ChristinMaitre et al., 1996Go).

We conclude that Puregon® successfully induces uni-ovulation in patients with PCOS when injected on alternate days and that this treatment regimen induces a physiological and biochemical milieu similar to that prevailing during a normal menstrual cycle.


    Acknowledgments
 
The authors thank Organon UK and Salford Royals NHS Trust for financial support.


    Notes
 
3 To whom correspondence should be addressed Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on June 17, 1999; accepted on September 20, 1999.