A double-blind, randomized study to compare recombinant human follicle stimulating hormone (FSH; Gonal-F®) with highly purified urinary FSH (Metrodin® HP) in women undergoing assisted reproductive techniques including intracytoplasmic sperm injection

R. Frydman, C.M. Howles, F. Truong and for The French Multicentre Trialists*


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This prospective, double-blind, randomized, multicentre study compared the efficacy and safety of recombinant human follicle stimulating hormone (r-hFSH; Gonal-F®) versus highly purified urinary FSH (u-hFSH HP; Metrodin® HP) in women undergoing ovarian stimulation for in-vitro fertilization, including intracytoplasmic sperm injection. A total of 278 patients began a long gonadotrophin-releasing hormone agonist protocol, then 139 received r-hFSH and 139 u-hFSH HP, 150 IU/day administered s.c., for the first 6 days of treatment. On day 7, the dose was adjusted, if necessary, according to ovarian response. Human chorionic gonadotrophin (HCG, 10 000 IU, s.c.) was administered once there was more than one follicle 18 mm in diameter and two others >=16 mm. Oocyte retrieval was performed 36–38 h after HCG injection: 128 patients (92%) receiving r-hFSH and 113 (81%) receiving u-hFSH HP had at least one oocyte retrieved. Among patients receiving r-hFSH, there was a significantly higher mean (± SD) number of oocytes retrieved (11.0 ± 5.9 versus 8.8 ± 4.8 with u-hFSH HP; P = 0.002) and mean number of embryos obtained (5.1 ± 3.7 versus 3.5 ± 2.9 with u-hFSH HP; P = 0.0001). With r-hFSH, significantly fewer FSH treatment days (11.7 ± 1.9 versus 14.5 ± 3.3) and 75 IU ampoules (27.6 ± 10.2 versus 40.7 ± 13.6) were required than with u-hFSH HP (P = 0.0001). Embryo replacement on day 2–3 after oocyte retrieval resulted in 36 liveborn children in the Gonal-F® group and 33 in the Metrodin HP® group (not significant). There were seven cases (5.0%) of ovarian hyperstimulation syndrome in the r-hFSH group and three (2.2%), in the u-hFSH HP group (not significant). It is concluded that r-hFSH is more effective than u-hFSH in inducing multiple follicular development.

Key words: ICSI/IVF/multiple follicular development/r-hFSH/u-hFSH HP


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In-vitro fertilization (IVF) of human oocytes followed by the replacement of embryos in the uterine cavity is a well-established treatment for female infertility attributable to damaged Fallopian tubes, endometriosis or unexplained causes. In addition, it has been widely applied in the treatment of andrological infertility. However, IVF may fail in couples with severe male factor infertility and, in some cases, standard IVF treatment cannot be carried out if the number of motile spermatozoa present in the ejaculate is too low. Recently, several procedures of assisted fertilization have been introduced. Palermo and colleagues reported the first successful use of intracytoplasmic sperm injection (ICSI) for treating male factor infertility (Palermo et al., 1992Go). The use of ICSI has been further investigated and several studies have now shown that the technique is more effective than either subzonal insemination or routine IVF for treating severe male factor infertility (Palermo et al., 1993Go; Van Steirteghem et al., 1993Go).

In order to obtain more than one embryo for replacement in IVF and thus improve the chances of a live birth (Waterstone et al., 1991Go; Nijs et al., 1993Go; Staesson et al., 1993), it is necessary to stimulate the growth and maturation of several follicles. Such multiple follicular development is induced by daily administration of follicle stimulating hormone (FSH) and has been proposed since the 1980s.

Advances in purification techniques have led to the clinical availability of highly purified urinary FSH (u-hFSH HP; Metrodin HP®; Serono Laboratories, Aubonne, Switzerland) with a specific activity of >9000 IU FSH/mg protein, which can be characterized and controlled from batch to batch by physico-chemical techniques. Because of its high purity, u-hFSH HP can be administered s.c. (Le Cotonnec et al., 1993Go; Howles et al., 1994Go; Wikland et al., 1994Go), yielding a similar pharmacodynamic profile to u-hFSH.

Recently, a recombinant hFSH preparation (r-hFSH, Gonal-F®; Serono) has become available (Chappel et al., 1992Go; Olijve et al., 1996Go). As only a mammalian cell can glycosylate the FSH protein correctly, r-hFSH is produced in genetically engineered Chinese hamster ovary (CHO) cells to ensure full biological activity (Chappel et al., 1992Go; Howles, 1996Go). hFSH manufacture by recombinant DNA technology, however, ensures that production is not only independent of urine collection, but is also biochemically pure (Loumaye et al., 1995Go) with a specific activity of ~10 000 IU FSH/mg protein thereby ensuring local tolerance to injections and low immunogenicity when administered s.c. (Redfearn et al., 1995Go; Albano et al., 1996Go).

However, controversy over the most adequate gonadotrophin preparation for induction of follicular maturation in IVF still exists. The starting dose of FSH in normally ovulating women undergoing ovarian stimulation for IVF is usually between 150 and 225 IU FSH per day, depending on factors such as age and previous ovarian response (Habu-Heija et al., 1995Go; Thornton, 1996Go). However, it has recently been suggested, on the basis of a small, non-comparative study, that a starting dose of 100 IU r-hFSH may be sufficient (Devroey et al., 1998Go). In the present study, the ovarian response to stimulation with r-hFSH or u-hFSH HP at a starting dose of 150 IU per day for a fixed period of 6 days was assessed.

This study was designed to compare the efficacy and safety of r-hFSH with u-hFSH HP, both administered s.c., in stimulating multiple follicular development in women down-regulated with a gonadotrophin releasing hormone (GnRH) agonist and undergoing assisted reproductive technology. A previous trial that was assessor-blinded reported a significant difference in efficacy between these two treatments, in favour of the recombinant product (Bergh et al., 1997Go). However, this is the first reported double-blind comparison of these two drugs.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patient population
Patients were recruited between December 1995 and December 1996, and were eligible for inclusion in the study if they satisfied the following criteria: (i) aged between 18 and 38 years, with regular spontaneous menstrual cycles of 25–35 days; (ii) FSH and luteinizing hormone concentrations in the normal local range during the early follicular phase, with prolactin <20 ng/ml, testosterone <3.5 nmol/l and fewer than 10 follicles <10 mm diameter on each ovary on pelvic ultrasound; (iii) infertility due to any of the following: tubal factors, mild endometriosis (classification stage I or II of the American Society of Reproductive Medicine), male factor, or unexplained factor; (iv) two functioning ovaries and normal uterine cavity; (v) not more than three previous assisted reproduction attempts and no treatment with clomiphene citrate or gonadotrophins within 1 month of the treatment cycle in the study; (vi) willingness to participate and comply with the protocol for the duration of the study; (vii) written informed consent.

Exclusion criteria were as follows: presence of clinically significant systemic disease or any contraindication to being pregnant and/or carrying a pregnancy to term; a body mass index (BMI) >30 kg/m2; a previous history of severe ovarian hyperstimulation syndrome (OHSS); a history of a poor response to gonadotrophin therapy (defined as fewer than three mature follicles in a previous attempt) or abnormal gynaecological bleeding of undetermined origin; a male partner with azoospermia and/or leukospermia (<2x106/ml) or clinical signs of infection detected in a semen analysis within the last 6 months.

The study was approved by the French national Ethics Committee.

Design
This was a double-blind, randomized, prospective, multicentre, phase IV study comparing the efficacy and safety of r-hFSH and u-hFSH HP in women undergoing ovulation induction for conventional IVF and ICSI. The study was conducted in seven centres in France.

Starting on day 20 of the previous cycle or on the first day of the current cycle, eligible patients received GnRH agonists depot or daily: triptorelin (Decapeptyl®; Ipsen Paris, France), 3 mg, or leuprorelin (Enantone®; Takeda, Puteaux, France), 3.75 mg, depot; triptorelin (Decapeptyl®; Ipsen), 0.1 mg, daily s.c.; intranasal nafarelin 200 µg to 400 µg (Synarel®; Monsanto, Boulogne, France) or buserelin 0.6 mg, daily s.c. (Suprefact®; Hoechst, Frankfurt, Germany). Pituitary down-regulation was confirmed by an ultrasound scan showing no evidence of ovarian activity, endometrial thickness <7 mm and/or serum oestradiol <=50 pg/ml. The patient was then randomized into one of the two treatment arms (r-hFSH or u-hFSH HP) and received treatment according to her identification number. A single manufactured batch of r-hFSH (no. 19302115) and three batches (nos. 17364095, 17361076, B 3624 ) of u-hFSH HP were used in the study. All patients in the u-FSH arm received the same batch of treatment. However, in some patients more than 40 ampoules were necessary, so they received in addition a few ampoules from the other batches. There was no difference in the results emanating from the different batches.

Both drugs were administered s.c. at a starting dose of 150 IU per day during the first 6 days. The ovarian response was assessed on days 7 and 9 (by ultrasound scan and measurement of serum oestradiol level) and FSH dose was titrated if necessary. Gonadotrophins and GnRH agonists were administered until the criteria for triggering final follicular maturation (one follicle >=18 mm in diameter and at least two follicles >=16 mm) were met. The last injection was given within 24 h of the HCG injection (Profasi®, Serono; 10 000 IU s.c.), which triggered ovulation.

Oocytes were retrieved 36–38 h after HCG administration, then were assessed and fertilized in vitro. For ICSI, the cumulus cells were removed and an assessment of oocyte nuclear maturity was made. Depending upon the rate of embryo cleavage, embryos were replaced on day 2–3 after ovum retrieval. Additional embryos were frozen.

The luteal phase was supported by natural progesterone (Utrogestan, 400 mg/day; Besins–Iscovesco) administered per os, beginning the day of embryo transfer and continuing either until a negative pregnancy test was obtained or for at least 12 days. The patient was followed up and the treatment outcome (menstruation or clinical pregnancy) was recorded. All patients underwent only one treatment cycle in this protocol.

Parameters evaluated
The primary endpoint was the total number of oocytes retrieved from patients who received HCG. The following parameters were also recorded in each group: number of ampoules of FSH 75 IU used during the stimulation; number of patients who required an increase in their FSH dose; number of days of FSH stimulation; number of follicles >12 mm in diameter on the day of HCG administration (i.e. the number of follicles responding to FSH treatment); oestradiol concentration during stimulation; number of oocytes retrieved per number of follicles >12 mm on day of HCG; fertilization rate; number of two-pronuclear (2PN) fertilized oocytes; number of cleaved embryos; number of embryos replaced or frozen; number of clinical pregnancies, miscarriage, multiple pregnancies and live births.

The incidence and severity of OHSS was recorded and graded as mild, moderate or severe (Schenker and Weinstein, 1978Go).

Assessments
Plasma oestradiol was measured according to the routine method established in each centre. Follicular size was measured using vaginal ultrasound (Wikland et al., 1994Go), by taking the widest diameter in two planes.

Sample size
The clinical equivalence of r-hFSH and u-hFSH HP would be declared if the difference in the mean number of oocytes retrieved (the primary endpoint) between the two groups was less than three. To demonstrate this equivalence with a probability of 95%, using a two-sided 90% confidence interval for difference in the mean number of oocytes retrieved, 212 evaluable patients were required. Since the secondary efficacy criteria (number of embryos and pregnancy rate) were strongly dependent on the primary endpoint, it was not considered necessary to calculate the numbers of subjects required for their evaluation.

Statistics
The analysis was performed on an intention-to-treat basis, taking into account all randomized patients. All computations were performed using SAS version 6.12 for Windows (SAS Institute, Cary, NC, USA).

The comparability of the two treatment groups on inclusion was checked for all the demographic data, and the history and clinical examination data, using the {chi}2-test, Fisher's exact test or the Student's t-test, depending on the sample size and characteristics of the variable. Descriptive statistics (mean, SD, median, range) have been reported for the efficacy criteria.

The main objective of this study was to show the equivalence of r-hFSH and u-hFSH HP in the primary efficacy endpoint — that is, the number of oocytes retrieved from patients who received HCG. Data were analysed using a two-way analysis of variance (ANOVA) in order to take into account any effects of the different treatment centres. Other efficacy parameters were analysed by ANOVA (quantitative parameters) or using the Cochran–Mantel–Haenzsel test (dichotomous parameters). The incidences of adverse events in the two groups were compared using Fisher's exact test or the {chi}2-test, depending on the smallest cell size.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients
A total of 278 patients (139 in each treatment group) was recruited to the study and underwent pituitary down-regulation. All the inclusion criteria were fulfilled except in three patients (one patient was 39 years and 9 months old, one had had more than three previous assisted reproduction attempts and one had a BMI that was marginally too high). As these protocol deviations were not major and the FSH and LH concentrations were normal, these patients were retained in the analysis. All patients who started FSH therapy (classed as the `intent-to-treat' group) were analysed irrespective of any protocol violations.

No significant differences were found between the demographic characteristics of the two groups (Table IGo) suggesting that they were adequately randomized. The main causes of infertility were tubal and male factors. There was a trend for more patients with tubal factor in the r-hFSH group and more patients with male factor infertility in the u-hFSH group. Additional analysis was undertaken to adjust for this trend (see below).


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Table I. Demographic characteristics of the patients enrolled and randomized to treatment with recombinant human follicle stimulating hormone (r-hFSH; Gonal-F®) and urinary human (u-h)FSH HP (Metrodin® HP) (values are means ± SD)
 
Patients achieving criteria for IVF
A total of 278 patients was given GnRH agonist administered in a long protocol. All patients received FSH stimulation once they had demonstrated pituitary down-regulation. Following FSH treatment, 130 of the patients given r-hFSH (93.5%) and 116 of those taking u-hFSH HP (83.5%) received HCG. Oocytes were retrieved in 128 (92.1%) patients in the r-hFSH group and 113 (81.3%) patients in the u-hFSH HP group (P = 0.008) and at least one embryo was transferred in 116 (83.5%) patients in the r-hFSH group and 98 (70.5%) patients in the u-hFSH HP group (P = 0.01).

Efficacy results
The primary efficacy criterion of the number of oocytes retrieved showed a significant difference in favour of r-hFSH (Table IIGo): the mean number of oocytes retrieved was 11.0 ± 5.9 for r-hFSH compared with 8.8 ± 4.8 for u-hFSH HP (P = 0.002). There were also significant differences between the two groups in several of the secondary efficacy parameters (Table IIGo). The number of follicles 12–18 mm diameter on the day of HCG administration was significantly greater following r-hFSH treatment (12.1 ± 5.2 compared with 10.4 ± 4.6 for u-hFSH HP; P = 0.005). In addition, fewer days of FSH stimulation were required with r-hFSH to reach the criteria for triggering follicle maturation than with u-hFSH HP (11.7 ± 1.9 versus 14.5 ± 3.3 respectively; P = 0.0001). Correspondingly, the total dose of FSH required to reach these criteria was lower for r-hFSH than u-hFSH HP (27.6 ± 10.2x75 IU ampoules, compared with 40.7 ± 13.6; P = 0.0001). Only 56.2% of patients in the r-hFSH group required an increase in dosage after the first 6 days of treatment, compared with 85.3% of those receiving u-hFSH HP (P = 0.001).


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Table II. Stimulation characteristics of patients receiving human chorionic gonadotrophin (HCG) (values are means ± SD)
 
Serum oestradiol measurements were carried out in most of the patients throughout treatment. Between FSH treatment day 1 and the day of HCG administration, the oestradiol concentration rose from 60 to 2349pg/ml and 28 to 1662 pg/ml in the r-hFSH and u-hFSH groups respectively. This difference reflects the number of growing follicles in each group (Figure 1Go).



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Figure 1. Serum oestradiol concentration (mean ± SEM) during the stimulation period. HCG = human chorionic gonadotrophin.

 
Embryological characteristics and pregnancies
Table IIIGo shows the embryological characteristics of patients in the two treatment groups. On day 2 after fertilization, there were significantly more cleaved embryos in the r-hFSH group than in the u-hFSH HP group (5.0 ± 3.7 per patient compared with 3.5 ± 2.9; P = 0.0001). On average 2.3 ± 1.1 and 2.0 ± 1.2 embryos respectively were transferred on day 2 or 3 after fertilization. A total of 74.6% of patients in the r-hFSH group and 56.9% of those in the u-hFSH HP group had two or three embryos transferred 2–3 days after fertilization. Much smaller proportions of 5.4% and 8.6% respectively had four or five embryos transferred.


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Table III. Embryological characteristics in patients receiving human chorionic gonadotrophin (values are means ± SD)
 
It should be noted that when statistical analysis was undertaken to adjust for the difference in infertility indication trend between the two groups (see above), the number of embryos was significantly higher when infertility was due to non-male factor. When this factor was taken into account, the difference in number of embryos between the two treatment groups was still significant (P = 0.0001). However, there was no difference between fertilization rate or number of embryos cryopreserved.

Any supernumerary embryos were frozen. Of 651 embryos obtained in the r-hFSH group, 164 (25%) were frozen, compared with 68 (17%) out of 401 in the u-hFSH group.

The initial pregnancy rate (excluding biochemical) per started cycle was 32/139 (23.0%) for r-hFSH and 38/139 (27.3%) for u-hFSH HP (not significant, Table IVGo). Among the 38 pregnancies in the u-hFSH group, one was extra-uterine and one was obtained after intra-uterine insemination. The number of ongoing pregnancies was 25 in both treatment groups; however, 40% of pregnancies among r-hFSH-treated patients were multiple, compared with 36% of those receiving u-hFSH HP (not significantly different). The number of miscarriages was eight in the r-hFSH group compared with 11 in the u-hFSH group (not significant). The number of liveborn children in both groups was similar (36 versus 35 respectively; one pregnant patient in the Metrodin HP® group was lost to follow-up).


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Table IV. Embryo transfer and pregnancy
 
Safety results
The proportion of patients (36 patients in the Gonal-F® group versus 29 patients in the Metrodin HP® group) presenting adverse events was comparable between study arms.

Similarly there was no significant difference for the incidence of OHSS: seven patients (5.0%) receiving r-hFSH compared with three patients (2.2%) receiving u-hFSH HP. In patients receiving r-hFSH, none of the cases of OHSS was severe, whereas there was one severe case in the u-hFSH HP group, in which the patient required hospitalization. The other most frequently reported adverse events concerned pregnancy and local reactions to injections. Concerning serious adverse events, all except one in each group occurred after diagnosis of pregnancy (eight in the Gonal-F® group versus 18 in the Metrodin HP® group) and concerned miscarriages or threat of premature delivery.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The results of this study demonstrated that r-hFSH was significantly more effective than u-hFSH HP in stimulating the development of mature oocytes in preparation for assisted reproduction. This was chosen as the primary efficacy endpoint instead of the number of follicles (Recombinant FSH Study Group, 1995) because it may sometimes be difficult to assess the number of growing follicles in a stimulated ovary due to `crowding'. Some clinical units only measure the number of mature follicles, thus leading to a large discrepancy at oocyte recovery. However, in the present study, there was a strong relationship between follicles observed and number of oocytes retrieved (91.4% in the Gonal-F® vs 86.4% in the Metrodin HP® group, not significant).

The primary efficacy result was supported by significant differences in the secondary efficacy parameters, also in favour of r-hFSH. Thus, the number of days of treatment and the dose of FSH required to reach criteria for triggering follicle maturation were both lower for r-hFSH than u-hFSH HP. All patients in the present study received FSH at a starting dose of 2x75 IU ampoules for the first 6 days, with subsequent increases if necessary. Fewer patients in the r-hFSH group required an increase in dosage after 6 days, compared with those receiving u-hFSH HP. A possible explanation for the protracted response in the u-hFSH HP group is that the starting dose of 150 IUI was too low, and that the dose correction made on day 7, when 85% of patients had their doses increased, was instigated too late, leading to the development of a smaller cohort of mature follicles by the time of administration of HCG.

It is also important to note that Gonal-F® administration results in a higher percentage of patients reaching the criteria for HCG administration, oocyte retrieval or embryo transfer. The number of cancelled cycles should be a parameter to be taken into account in economic discussions on one hand and for psychological reasons for the patient on the other hand.

Treatment with r-hFSH resulted in significantly more embryos than with u-hFSH HP, but the majority of transfers involved two or three embryos. The number of embryos in culture at the time of transfer has recently been shown to be a strong predictor of pregnancy outcome (Templeton et al., 1998Go). In this analysis of over 40000 treatment cycles, it was demonstrated that the probability of a live birth was significantly higher if a woman had more than four embryos available in the culture dish even though only a maximum of three was replaced. Thus a cohort of good quality embryos is a strong contributor to a successful outcome.

The results of this double-blind study confirm those obtained in earlier prospective open label and single-blind multicentre studies comparing r-hFSH with urinary hFSH (u-hFSH; Out et al., 1995) and u-hFSH HP (Bergh et al., 1997Go). In each of these studies, significantly more oocytes were retrieved following r-hFSH treatment than after u-hFSH treatment. Pregnancy rates were, again, the same for the two treatment groups. However, patients receiving r-hFSH had more embryos cryopreserved, compared with those in the u-FSH treatment groups and Out et al. (1995) showed that when frozen embryo cycles were included in the analysis, ongoing pregnancy rates were significantly in favour of r-hFSH. Indeed, a meta-analysis of three comparative trials found that, when ongoing pregnancies from cryopreserved embryos were taken into account, the treatment difference was 6.4% (P = 0.011 in favour of r-hFSH). In the study by Bergh et al. (1997) and the current trial population, a significantly larger number of cleaved embryos was produced following r-hFSH compared with u-hFSH HP treatment and, therefore, more embryos were available for cryopreservation. However, the number of transfers of cryopreserved embryos in both these studies so far is too small to allow an assessment of those data.

The number of ampoules used was significantly lower in the Gonal-F® group compared to the Metrodin HP® group; this difference was found by Bergh et al. (1997). Moreover, the French national IVF register (FIVNAT 1998Go) has also reported that in current practice, there was a significant difference in terms of ampoules consumption in favour of r-hFSH.

The most significant side-effect of FSH stimulation is OHSS. In the present study, there was no significant difference in the incidence of OHSS in patients receiving r-hFSH (two mild and five moderate cases) compared with u-hFSH HP (two mild and one severe cases). It has been demonstrated that the number of oocytes retrieved and the ongoing pregnancy rate are risk factors for development of the syndrome (Delvigne et al., 1993Go). Thus, careful monitoring of treatment and a low dose of FSH are desirable to minimize the risk of this syndrome.

Local reactions to injections accounted for 30% of adverse events; these events could be decreased by using less solvent and by administering the product slowly.

The greater effectiveness of r-hFSH over urinary FSH products in stimulating follicular development may be due to a number of mechanisms. It has been suggested that differences in the isoform or glycosylation profiles between the recombinant and urinary products, or the presence of FSH-inhibitory substances in urinary FSH may play a role in this (Out et al. 1995Go). Thus, the reliable consistency of r-hFSH from one batch to another contribute to its greater efficacy.

In conclusion, this study confirms that r-hFSH HP is more effective than u-hFSH HP in inducing ovulation in women undergoing assisted reproductive treatment. Patients given the recombinant product required fewer days of treatment and a lower total dose of FSH to reach the criteria for HCG administration than those receiving u-hFSH HP. The safety profile of r-hFSH was found to be similar to that of u-hFSH HP, and the incidence of OHSS in both groups was low.


    Acknowledgments
 
We would like to thank all investigators and embryologists who participated in this study, Dr Mai Nguyen for writing the protocol and Pascale Pinck, CRA in charge of this study.


    Notes
 
* The French Multicentre Trialists: Pr R.Frydman, Hopital A. Beclère, Clamart; C.M.Howles, Ares Serono, Geneva, Switzerland; F.Truong, Serono, France, Boulogne; Dr F.Olivennes, Dr C.Righini, Hopital A. Beclère, Clamart; Dr Avril, Clinique St Antoine, Bois-Guillaume; Dr Camier, Centre Hospitalier–Maternité C. Desmoulins, Amiens; Dr Cornet, Clinique de la Dhuys, Bagnolet; Dr Carles, Hôpital de la Conception, Marseille; Dr Moreau, CMCO, Schiltigheim; Dr Ragage, Maternité Ste Croix, Metz; Dr Sage, Clinique Belledone, Grenoble, Dr Thébault, Hôpital Américain, Neuilly. Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on February 1, 1999; accepted on November 12, 1999.