1 Department of Medicine, Hope Hospital, Salford and 2 Nuffield Department of Obstetrics and Gynaecology, John Radcliffe Hospital, Headington, Oxford OX3 9DU, UK
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Abstract |
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Key words: ovulation induction/Puregon®/recombinant FSH
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Introduction |
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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., 1992, 1995
). 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, 1995
, 1998
) and lower starting doses (100 IU/day) of recombinant FSH in IVF have been successfully employed (Devroey et al., 1998
) in view of the apparent increased bioactivity of Puregon® over the urinary FSH preparation, Metrodin® (Out et al., 1995
). 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., 1998
). 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., 1992
; Shoham et al., 1993
; Thompson et al., 1995
) 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., 1995) and in the light of the detailed information on the pharmacokinetics available on this product for women (Mannaerts et al., 1996
). With a terminal elimination half-life for Puregon® of around 3040 h (Mannaerts et al., 1996
) 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., 1998a
, b
).
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Materials and methods |
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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 1224 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 1001200 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 1250 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-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 4500 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-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 161000 pg/ml.
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Results |
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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 I); 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 I
).
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Discussion |
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We found that FSH concentrations, measured 1224 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., 1996). 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., 1996
; Muttukrishna et al., 1996
). 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., 1989; Franks, 1989
). 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., 1997
). 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, 1991
). 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., 1995
; Harris et al., 1996
; Anobile et al., 1998
; Horsman et al., 1998
). 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., 1996
).
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.
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Acknowledgments |
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Notes |
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References |
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Submitted on June 17, 1999; accepted on September 20, 1999.