1 Center for Obstetrics and Gynecology, 2 Department of Obstetrics and Gynecology, University of Valencia School of Medicine and 3 Hospital Clínico Universitario, Valencia, Spain
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: poor responder/recombinant FSH/reproductive performance
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Recently, FSH has been manufactured by means of recombinant DNA technology using a Chinese hamster ovary cell line transfected with the gene encoding human FSH (Van Wezenbeek et al., 1990). Recombinant FSH (rFSH) is purified up to 99%, does not contain any luteinizing hormone (LH) activity, and shows similar pharmacokinetic and pharmacodynamic properties to the natural human FSH (Out et al., 1995
).
The clinical assessment of rFSH in assisted reproduction technologies such as IVF has suggested that significantly more oocytes are retrieved, more embryos obtained, and higher pregnancy rates in infertile patients treated with rFSH compared to those treated with uFSH (Out et al., 1996; Frydman et al., 1998
).
It has been estimated that up to 15% of all patients treated for IVF are poor responders to stimulation with exogenous gonadotrophins (Pellicer et al., 1987) and therefore this has become a frequently encountered problem in all reproductive centres. These patients are characterized by low oestradiol concentrations combined with markedly reduced numbers of oocytes in spite of massive stimulation with gonadotrophins (Muasher, 1993
).
The precise mechanisms that determine ovarian response to gonadotrophins are only partly understood. However, an age-related decline in fecundity is observed as women progress through reproductive life and ovarian reserves decline. This is reflected by increasing basal FSH concentration in the early follicular phase of the menstrual cycle, in an effort by the pituitary gland to maintain the normal follicle response (Anasti, 1998). Consequently, basal FSH and oestradiol concentrations have been shown to be useful predictors of reproductive performance (Toner et al., 1991
; Evers et al., 1998
).
Patients within the normal age range for reproduction and diminished response to ovarian stimulation may have so-called `occult ovarian failure' (Cameron et al., 1988), indicated by basal or clomiphene-induced increase in serum FHS and oestradiol concentrations (Toner et al., 1991
; Evers et al., 1998
). However, a group of young low responders, having a normal basal FSH and oestradiol and no apparent reason for repeated low response to aggressive stimulation, does exist (Pellicer et al., 1994
). The management of these young infertile patients remains one of the most difficult and disappointing issues in reproductive medicine. Hence, the present prospective, randomized study was undertaken to evaluate the reproductive performance of young patients, who normally have a low response to ovarian stimulaton, despite normal basal FSH and oestradiol when treated with high doses of either rFSH or uFSH.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
This study was performed according to the Declaration of Helsinki and the European Community note on Good Clinical Practice for trials on medical products in the European Community (CPMP Working Party on Efficacy of Medicinal Products, 1990). The local ethics committee approved the study protocol, and written informed consent was obtained from all patients.
Study design
This was a prospective and randomized study comparing recombinant human FSH (Puregon ; Organon, Barcelona, Spain) and highly purified urinary FSH (Neofertinorm®; Serono laboratories, Madrid, Spain). The study was not blinded because it is very important in such studies to avoid `therapeutic suspicion' bias when evaluating treatment results. Such bias may be introduced in the both the application of the treatment and interpretation of results and is likely to occur when the investigators have a prior expectation of results. Therefore our clinicians (F.R. and F.B.-M.) were not blinded, and applied a rigid protocol in all patients. The other authors (E.M.C. and F.B.) were blinded to the patients' characteristics and treatment group, and performed the randomization, data analysis, and interpretation. The study was undertaken between January and June 1998.
The objective of the study was to assess the efficacy of rFSH in relation to uFSH HP (highly purified) for ovarian stimulation in poor responder patients with normal basal oestradiol and FSH concentrations undergoing IVFembryo transfer. Eligible subjects received a number from a randomized list.
Concentrations of FSH and oestradiol on cycle day 3 were determined in a spontaneous cycle preceding the cycle of ovarian stimulation in all patients. Serum FSH and oestradiol were analysed using a commercially available microparticle enzyme immunoassay (MEIA) kit (Abbot Laboratories, Abbot Park, IL, USA). Intra- and interassay coefficients of variation were respectively 4.1 and 8.5% for FSH, and 4.6 and 6.1% for oestradiol.
The ovarian stimulation protocol began with the administration of 300 IU/day of either rFSH (two vials of 150 IU rFSH, Puregon; Organon) or uFSH HP (four ampoules of 75 IU uFSH HP, Neo-fertinorm; Serono) depending on the randomization, along with two ampoules of HMG/day (HMG-lepori®; Farma-Lepori laboratories, Madrid, Spain) for the first 4 days (total starting dose was 450 IU/day of FSH plus 150 IU/day of LH). After day 4, the dose of HMG and FSH was adjusted on an individual basis according to follicular development as assessed by transvaginal ultrasound scanning and serum oestradiol concentrations (Pellicer et al., 1994). The criteria for human chorionic gonadotrophin (HCG) administration (10 000 IU, Profasi ; Serono) were the presence of at least two follicles
18 mm at greatest diameter, and serum oestradiol concentrations >2.94 nmol/l. FSH/HMG injections were discontinued on the day of HCG administration. Oocyte retrieval 3638 h after HCG administration, fertilization procedures, and embryo transfer were done according to the local standards (Pellicer et al., 1994
), both procedures being performed by the same author (F. B.-M.). Micronized vaginal progesterone (Progeffik®; Effik Laboratories, Madrid, Spain) pills (400 mg/day) were prescribed for luteal support.
The cancellation criterion was the presence of <4 follicles with a diameter of 16 mm after 12 days of ovarian stimulation (Hanoch et al., 1998
).
We recorded in both groups the patient's age and body mass index (BMI), number of days of ovarian stimulation, total FSH dose (IU) administered, number of oocytes retrieved, number of mature oocytes, cancelled cycles, number of good quality embryos achieved (when <20% of the embryo was fragmented) (Ubaldi et al., 1996), pregnancy and implantation rates.
Statistical analysis
Data are expressed as mean ± SEM. For statistical comparison between groups, analysis of variance (ANOVA) was applied. When homogeneity and normality (KolgomorovSmirnov) of the samples were not achieved, non-parametric statistical methods (KruskalWallis and Wilcoxon test) were employed for comparison. P < 0.05 was considered statistically significant. The analysis was carried out using the statistical package for social sciences (SPSS Inc., Chicago, IL, USA).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Both treatment groups were comparable in demographic and infertility characteristics (Table I). The main cause of infertility associated with the low response was tubal disease (47 and 40% for rFSH- versus uFSH-treated groups respectively). The mean duration of infertility for rFSH- and uFSH-treated groups was 4.6 and 4.8 years respectively.
|
|
The number of embryos transferred in both groups was limited to four. However, it was expected that the higher number of good quality embryos achieved in the rFSH group (Table II) would lead to higher pregnancy and implantation rates. These were in fact significantly (P < 0.01) higher in these patients.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The recent advent of pure rFSH and its better clinical performance than uFSH in assisted reproduction (Out et al., 1995, 1996
; Frydman et al., 1998
) could help to improve the reproductive performance of young low responder patients.
Results from the present study show that the treatment of this group of low responders with rFSH is effective in stimulating ovarian follicular development. Moreover, they have revealed that rFSH has a significantly better reproductive performance than uFSH in these patients.
The group of patients treated with rFSH had significantly shorter treatment as well as requiring a significantly lower total dose of FSH to induce successful ovulation induction. These data are in agreement with previous studies in normal ovulatory infertile patients, and, compared to uFSH, the total dose of FSH was significantly lower with rFSH over a significantly shorter treatment period (Out et al., 1995; Khalaf et al., 1998
).
Significantly more oocytes were retrieved in the group of patients treated with rFSH. Moreover, a significantly higher number of mature oocytes was achieved in this group of patients. This observation is similar to that reported in previous prospective, multicentre studies comparing rFSH and uFSH in normal ovulatory infertile patients (Out et al., 1995, 1996
; Frydman et al., 1998
; Placido et al., 1998
).
As could be expected by the significantly higher number of mature oocytes achieved in the group of patients treated with rFSH, significantly more good quality embryos were obtained in this group of patients. Therefore, the significantly higher pregnancy and implantation rate achieved in the patients treated with rFSH is not surprising. This also agrees with previous studies comparing rFSH and uFSH in an infertile population undergoing IVF (Hedon et al., 1995; Out et al., 1996
, 1997
; Frydman et al., 1998
).
Additionally, the cancellation rate was significantly reduced in the group of patients treated with rFSH. This important result is consistent with that previously reported in one of the largest prospective, randomized, clinical trials ever performed in IVF, where the cancellation rate was significantly higher in the group of patients treated with uFSH compared to the group treated with rFSH (Out et al., 1995).
Several mechanisms have been implicated as responsible for the decreased ovarian response: a decreased number of FSH receptors available within the granulosa cells (Zeleznik et al., 1981), the presence of an FSH receptor binding inhibitor in the follicular fluid (Lee et al., 1993
), an inappropriate vascular network responsible for the distribution of circulating gonadotrophins (Pellicer et al., 1994
), a defective signal transduction after FSH binding to its receptor (Hernandez et al., 1992
; Fauser and van Heusden, 1997
), and a diminished ovarian reserve (Pellicer et al., 1998
). However, the exact mechanisms that determine a low ovarian response to exogenous stimulation with gonadotrophins in this group of young patients remain only partly understood.
On the other hand, possible factors which might explain the higher efficacy of rFSH compared to uFSH in terms of reproductive performance in these patients include differences at the oligosaccharide moieties of the molecules, differences in the bioactive to immunoreactive ratios and isohormone distributions of recombinant and urinary FSH, or proteinaceous contaminants in the urinary product inhibiting FSH action (Matikainen et al., 1994; Out et al., 1995
).
rFSH contains more relatively basic isoforms and fewer relatively acidic isoforms than uFSH (De Leeuw et al., 1996). Basic isoforms are known to have a higher receptor affinity and intrinsic bioactivity than acidic isoforms, indicating the important role of these short-lived basic isoforms in the induction of follicular growth (De Leeuw et al., 1996
). Moreover, during a normal menstrual cycle, more basic isoforms are present during the mid follicular phase (Padmanabhan et al., 1988
), indicating that rFSH resembles more closely the composition of natural FSH isoforms during the mid follicular phase.
In comparative pharmacokinetic studies of recombinant and urinary FSH, it has been shown that the bioactivity of the rFSH tends to be higher than that of the uFSH, whereas the immunoreactivities display an opposite trend (Mannaerts et al., 1996). Due to the lower bioactive to immunoreactive ratio of uFSH, the difference in that ratio after rFSH and uFSH treatment tends to increase, indicating that there might be a shift in the isoform composition during the clearance of uFSH (Matikanen et al., 1994). This has been previously suggesting as one of the factors responsible for the higher efficacy of rFSH in terms of inducing multiple follicular growth (Out et al., 1996
).
In order for the granulosa cells, and therefore a follicle, to progress beyond the preantral stage, they must be stimulated by FSH. Certainly one of the most important effects of FSH is the stimulation of mitosis, which leads to increasing numbers of granulosa cells in the developing Graafian follicle. In this context, oestradiol is also a potent mitogen and most certainly interacts synergistically with FSH to stimulate granulosa cell proliferation (Erickson, 1986). A large multicentre trial has previously shown a significant increase in oestrogen concentrations in the group of women receiving rFSH as compared to those treated with uFSH (Out et al., 1995
). Additionally, recent studies in vitro have shown a dose- and time-dependent increase in granulosalutein cell oestrogen production after culture with rFSH, even in the absence of exogenous androgens (Földesi et al., 1998
), reinforcing the important role of rFSH in follicular development.
In summary, it can be concluded that in this prospective, randomized study, there were significant differences between rFSH and uFSH in the outcome of ovarian stimulation in infertile young women with poor response to conventional exogenous urinary gonadotrophins in the presence of normal basal FSH and oestradiol concentrations. Moreover, our study suggests that rFSH may represent the first choice of treatment to induce ovarian stimulation in these patients, and therefore this new technology provides the opportunity for good reproductive outcome. However, much research remains to done and future multicentre trials, including a larger series of patients, should confirm the results of the present study.
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cameron, I.T., O'Shea, F.C., Rolland, J.M. et al. (1988) Occult ovarian failure: A syndrome of infertility, regular menses, and elevated follicle-stimulating hormone concentrations. J. Clin. Endocrinol. Metab., 67, 11901194.[Abstract]
CPMP Working Party on Efficacy of Medical Products (1990) Good clinical practice for trial on medicinal products in the European Community. Pharmacol. Toxicol., 67, 361372.[Medline]
De Leeuw, R., Mulders, J., Voortman, G. et al. (1996) Structurefunction relationship of recombinant follicle stimulating hormone (Puregon). Mol. Hum. Reprod., 2, 361369.[Abstract]
Erickson, G.F. (1986) An analysis of follicle development and ovum maturation. Semin. Reprod. Endocrinol., 4, 233254.[ISI]
Evers, J.L.H., Slaats, P., Land, J.A. et al. (1998) Elevated levels of basal estradiol-17ß predict poor response in patients with normal basal levels of follicle-stimulating hormone undergoing in vitro fertilization. Fertil. Steril., 69, 10101014.[ISI][Medline]
Fauser, B.C.J.M. and van Heusden, A.M. (1997) Manipulation of human ovarian function: Physiological concepts and clinical consequences. Endocrine Rev., 18, 71106.
Farhi, J., Homburg, R., Ferber, A. et al. (1997) Non-response to ovarian stimulation in normogonadotrophic, normogonadal women: a clinical sign of impending onset of ovarian failure pre-empting the rise in basal follicle stimulating hormone levels. Hum. Reprod., 12, 241243.[Abstract]
Földesi, I., Beckwoldt, M. and Neulen, J. (1998) Oestradiol production by luteinized human granulosa cells: evidence of the stimulatory action of recombinant human stimulating hormone. Hum. Reprod., 13, 14551460.[Abstract]
Frydman, R., Avril, C., Camier, B. et al. (1998) A double-blind, randomized study comparing the efficacy of recombinant human follicle stimulating hormone (rhFSH) and highly purified urinary FSH (uhFSH) in inducing superovulation in women undergoing assisted reproductive techniques. Hum. Reprod., 13, Abstract book,94.
Hanoch, J., Lavy, Y., Holzer, H. et al. (1998) Young low responders protected from untoward effects of reduced ovarian response. Fertil. Steril., 69, 10011004.[ISI][Medline]
Healy, D.L., Trounson, A.O. and Andersen, A.N. (1994) Female infertility: causes and treatment. Fertil. Steril., 343, 15391544.
Hedon, B., Out, H.J., Hugues, J.N. et al. (1995) Efficacy and safety of recombinant FSH (Puregon) in infertile women pituitary-suppresed with triptorelin undergoing in-vitro fertilization: a prospective, randomized, assessor-blind, multicenter trial. Hum. Reprod., 10, 31023106.[Abstract]
Hernandez, E.R., Hurwitz, A., Vera, A. et al. (1992) Expression of the genes encoding the insulin-like growth factors and their receptors in the human ovary. J. Clin. Endocrinol. Metab., 74, 419425.[Abstract]
Khalaf, Y., Taylor, A., Pettigrew, R. et al. (1998) The relative clinical efficacy of recombinant human follicle stimulating hormone and the highly purified urinary FSH preparation. Hum. Reprod., 13, (Abstract Bk.) 191.
Land, J.A., Yarmolinskaya, M.I., Dumoulin, J.C.M. and Evers, J.L.H. (1996) High-dose human menopausal gonadotropin stimulation in poor responders does not improve in vitro fertilization outcome. Fertil. Steril., 65, 961965.[ISI][Medline]
Lee, D.W., Grasso, P., Dattatreyamurty, B. et al. (1993) Purification of a high molecular weight follicle-stimulating hormone receptor-binding inhibitor form human follicular fluid. J. Clin. Endocrinol. Metab., 77, 160168.
Mannaerts, M.J.J., Rombout, F., Out, H.J. and Bennink, H.C. (1996) Clinical profiling of recombinant follicle stimulating hormone (rFSH; Puregon): relationship between serum FSH and efficacy. Hum. Reprod. Update, 2, 153161.
Manzi, D.L., Thornton, K.L., Scott, L.B. and Nulsen, J.C. (1994) The value of increasing the dose of human menopausal gonadotropins in women who initially demonstrated a poor response. Fertil. Steril., 62, 251256.[ISI][Medline]
Matikainen, T., De Leeuw, R., Mannaerts, B. and Huhtaniemi, I. (1994) Circulating bioactive and immunoreactive recombinant human follicle-stimulating hormone (Org 32489) after administration to gonadotropin-deficient subjects. Fertil. Steril., 61, 6269.[ISI][Medline]
Muasher, S.J. (1993) Controversies in assisted reproduction-treatment of low responders. J. Assist. Reprod. Genet., 10, 112114.[ISI][Medline]
Out, H.J., Mannaerts, B.M.J.L., Driessen, S.G.A.J. et al. for The European Puregon Collaborative IVF Study Group (1995) A prospective, randomized, assessor-blind, multicenter study comparing recombinant and urinary follicle-stimulating hormone (Puregon vs Metrodin) in in-vitro fertilization. Hum. Reprod., 10, 25342540.[Abstract]
Out, H.J., Mannaerts, B.M.J.L., Driessen, S.G.A.J. et al. (1996) Recombinant follicle stimulating hormone (rFSH; Puregon) in assisted reproduction: More oocytes, more pregnancies. Results from five comparative studies. Hum. Reprod. Update, 2, 162171.
Out, H.J., Driessen, S.G.A., Mannaerts, B.M.J.L. et al. (1997) Recombinant follicle-stimulating hormone (follitropin beta, Puregon) yields higher pregnancy rates in in vitro fertilization than urinary gonadotropins. Fertil. Steril., 68, 138142.[ISI][Medline]
Padmanabhan, V., Land, L.L., Sonstain, J. et al. (1988) Modulation of serum follicle stimulating hormone bioactivity and isoform distribution by estrogenic steroids in normal women and in gonadal dysgenesis. J. Clin. Endocrinol. Metab., 67, 465473.[Abstract]
Pellicer, A., Lightman, A., Diamond, M.P. et al. (1987) Outcome of in vitro fertilization in woman with low response to ovarian stimulation. Fertil. Steril., 47, 812815.[ISI][Medline]
Pellicer, A., Ballester, M.J., Serrano, M.D. et al. (1994) Aetiological factors involved in the low response to gonadotrophins in infertile women with normal basal serum follicle stimulating hormone levels. Hum. Reprod., 9, 806811.[Abstract]
Pellicer, A., Ardiles, G., Neuspiller, F. et al. (1998) Evaluation of the ovarian reserve in young low responders with normal basal FSH levels using three-dimensional ultrasound. Fertil. Steril., 47, 812815.
Placido, G., Alviggi, C., Strina, I. et al. (1998) Recombinant FSH versus FSH-HP for COH in patients with poor ovarian responsiveness. Hum. Reprod., 13, Abstract book, 337.
Toner, J.P., Philput, C.B., Jones, G.S. et al. (1991) Basal follicle-stimulating hormone level is a better predictor of in vitro fertilization performance than age. Fertil. Steril., 55, 784791.[ISI][Medline]
Ubaldi, F., Camus, M., Smitz, J. et al. (1996) Premature luteinization in in vitro fertilization cycles using gonadotropin-releasing hormone agonist (GnRH-a) and recombinant follicle-stimulating hormone (FSH) and GnRH-a and urinary FSH. Fertil. Steril., 66, 275280.[ISI][Medline]
Van Hoof, M.H.A., Alberda, A.T., Huisman, G.J. et al. (1993) Doubling the human menopausal gonadotrophin dose in the course of an in-vitro fertilization treatment cycle in low responders: a randomized study. Hum. Reprod., 8, 369373.[Abstract]
Van Wezenbeek, P., Draaijer, J., Van Meel, F. et al. (1990) Recombinant follicle stimulating hormone. I. Construction, selection and characterization of a cell line. In Crommelin, D.J.A. and Schellekens, H. (eds), From Clone to Clinic, Developments in Biotherapy. Kluwe, Amsterdam, pp. 245251.
Zeleznik, A.J., Schuler, H.M. and Reichert, L.E. Jr (1981) Gonadotropin-binding sites in the rhesus monkey ovary: role of the vasculature in the selective distribution of human chorionic gonadotopin to the preovulatory follicle. Endocrinology, 109, 356362.[Abstract]
Submitted on August 11, 1998; accepted on January 18, 1999.