A randomized, double-blind clinical trial using fixed daily doses of 100 or 200 IU of recombinant FSH in ICSI cycles

Henk J. Out1,7, Ishay David2, Raphael Ron-El3, Shevach Friedler3, Eliezer Shalev4, Joel Geslevich4, Jehoshua Dor5, Adrian Shulman5, Zion Ben-Rafael6, Benjamin Fisch6 and Martha Dirnfeld2

1 Organon Laboratories, Cambridge Science Park, Cambridge, UK, 2 IVF Unit, Carmel Medical Centre, Haifa, 3 IVF Unit, Assaf Harofeh Medical Centre, Zerifin, 4 HaEmek Medical Centre, IVF Unit, Afula, 5 Department of Obstetrics and Gynaecology, Sheba Medical Centre, Tel-Hashomer, and 6 Rabin Medical Centre, Campus Beilinson, Petach Tiqva, Israel


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The effect of 100 and 200 IU per day recombinant FSH (rFSH) on numbers of oocytes retrieved and the total dose used in ovarian stimulation before intracytoplasmic sperm injection was investigated in a double-blind, randomized multicentre trial. A total of 91 women was treated with a low-dose protocol and 88 with a high-dose regimen at five centres. For each started cycle, significantly more oocytes were retrieved in the 200 IU group than in 100 IU group (12.0 versus 5.7, P < 0.001); total rFSH consumption was 1121 and 1875 IU in the low- and high-dose groups respectively. Significant variations were noted between centres with regard to numbers of oocytes collected per started cycle, ranging from 2.8 to 7.2 in the 100 IU group and from 9.0 to 19.1 in the high-dose group. Exploratory analyses of secondary outcomes suggested there were no differences in vital pregnancy rates per started cycle (19.2 versus 16.9%) and per embryo transfer (26.2 versus 19.3%) in the low- and high dose groups respectively. There were four hospitalizations due to ovarian hyperstimulation syndrome, all in the 200 IU group.

Key words: ICSI/IVF/Puregon/randomized clinical trial/recombinant FSH


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Recently, pleas have been made for milder ovarian stimulation protocols before IVF or intracytoplasmic sperm injection (ICSI). An editorial was published entitled: `Why delay the obvious need for milder ovarian stimulation?' (Edwards et al., 1997Go), while others (Felberbaum et al., 1998Go) introduced the term `soft protocol' to emphasize alternative, less aggressive, ways to obtain multiple oocytes in assisted reproduction technology. More recently, the advantages of milder ovarian stimulation were summarized (Fauser et al., 1999Go), and these included less patient discomfort, less complex and shorter stimulation regimens, fewer chances of short-term complications and of long-term health risks, and reduced cost. However, potential disadvantages might include fewer oocytes available for IVF, the technique becoming less programmable, and perhaps fewer embryos being available for cryopreservation (Fauser et al., 1999Go).

With the arrival of recombinant FSH (rFSH) the issue of milder regimens becomes even more relevant, given the higher potency of rFSH versus urinary FSH (Out et al., 1995Go; Bergh et al., 1997Go) and its perceived high acquisition costs (Meniru, 1999Go). The availability of lower presentation units of rFSH (50 IU, 100 IU) also facilitates clinical research as to the feasibility of low-dose protocols. Earlier, it was shown in observational studies that a 100 IU starting dose may be effective, thereby considerably reducing the total dose of administered gonadotrophins (Devroey et al., 1998Go; Bergh, 1999Go). However, very few double-blind, randomized clinical trials have been performed to show that these milder regimens do not jeopardize the chance for a successful birth and at the same time diminish the chances of side effects occurring. Recently, a double-blind comparison of fixed daily doses of 100 IU and 200 IU of rFSH was performed (Out et al., 1999Go). Significantly more oocytes were retrieved in the high-dose group, but the clinical pregnancy rates did not differ. In the current study, this clinical trial was repeated in a different subset of patients, namely those undergoing ICSI and in a different geographical location, Israel.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Study design
This was a prospective, randomized, double-blind, multicentre study comparing a fixed dose of 100 or 200 IU of rFSH (follitropin beta, Puregon®; NV Organon, Oss, The Netherlands). The objective of the study was to assess the efficacy and efficiency of these dosing regimens in down-regulated women undergoing ovarian stimulation prior to ICSI. The study was performed between May 1997 and June 1999 in five specialized infertility centres in Israel. The aim was to include 200 patients, with 100 patients in each treatment group (see Sample size considerations). The study was approved by the ethics committees of the individual study centres, and each subject provided their written informed consent before participating in the study. The study was conducted in compliance with the Declaration of Helsinki and according to the European Community note on Good Clinical Practice for trials on medicinal products in the European Community (CPMP Working Party on Efficacy of Medicinal Products, 1990Go).

Selection of patients
Inclusion criteria were as follows: at least 18 and at most 37 years of age at the time of screening; male infertility (total motile count <5x106 spermatozoa) solvable by ICSI using ejaculatory spermatozoa; normal regular cycles with a mean length of between 24 and 35 days; presence of two ovaries; good physical and mental health; body mass index between 18 and 29 kg/m2; and willing to provide written, informed consent.

Exclusion criteria were: female cause of infertility, except mild endometriosis or a mechanical factor; previous IVF or ICSI cycle(s) after which less than three oocytes were retrieved; previous IVF or ICSI cycle(s) with hospitalization due to ovarian hyperstimulation syndrome (OHSS); more than four previous IVF or ICSI cycles; total fertilization failure in a previous IVF or ICSI cycle; LH/FSH ratio at screening >=3; chronic cardiovascular, hepatic, renal or pulmonary disease; history of (within 12 months) or current abuse of alcohol or drugs; and administration of non-registered investigational drugs within 3 months prior to screening.

When all inclusion criteria and none of the exclusion criteria were met, the subject was considered to be eligible.

Study drugs and study procedures
Pre-treatment with intranasal buserelin (3x200 µg, Suprecur®; Hoechst, Frankfurt, Germany) for pituitary down-regulation was started in the midluteal phase. Recombinant FSH (Batch nos. CP 096025 and CP 096149) was supplied as lyophilized spheres in ampoules containing 50 or 100 IU FSH in-vivo bioactivity. For subcutaneous injection, two ampoules were reconstituted with 1 ml solvent. Human chorionic gonadotrophin (HCG) (Pregnyl®; NV Organon, Oss, The Netherlands) in doses of 5000 IU per ampoule was supplied to trigger ovulation. For i.m. injection of HCG, one ampoule was reconstituted with 1 ml solvent.

Demographic and other subject variables were obtained during the admission visit. In addition, the general medical and gynaecological histories were obtained, a general medical and gynaecological examination was performed, and an endocrinological, biochemical and haematological analysis of blood was performed. All general medical, biochemical and haematological measurements were performed according to routine procedures of the individual study centres. Eligible subjects were randomized by receiving a subject number from a randomization list corresponding with patient boxes in which the medication was kept. The 50 and 100 IU ampoules were indistinguishable one from another. The randomization was carried out in blocks of four and was computer-generated by using random numbers.

When down-regulation was achieved (defined as oestradiol serum concentrations <200 pmol/l), treatment with rFSH was started and continued until at least three follicles >=17 mm had developed. Dose adaptations were not allowed. HCG (5000 IU) was given to trigger ovulation. After oocyte retrieval and ICSI, a maximum of three embryos were replaced. Progesterone was given as luteal support according to the routine regimens of the centre.

Serum concentrations of oestradiol, progesterone, FSH and LH were measured at baseline (moment of down-regulation) and on the day of HCG injection, using local assays. Cycle monitoring included frequent vaginal ultrasound investigations and oestradiol measurements.

Study end-points
The primary efficacy end-point was the number of oocytes retrieved, and the primary efficiency end-point was the total dose of rFSH used.

Secondary variables included the number of follicles >=17, >=15, >=13 and >=11 mm at the day of HCG administration, serum concentrations of FSH, LH, progesterone and oestradiol at the day of HCG administration, treatment duration, embryo development rate, the number of transferable embryos, clinical pregnancy rate, implantation rate, miscarriage rate and vital pregnancy rate.

Retrieved oocytes were classified as oocytes with intact zona pellucida and clear cytoplasm, metaphase II oocytes (extrusion first polar body, no germinal vesicles), metaphase I oocytes (no polar body, no germinal vesicle) and germinal vesicles stage oocytes.

The number and quality of embryos were assessed according to the following criteria: type 1, all blastomeres of equal size without the presence of anucleate fragments; type 2, not all blastomeres of equal size, anucleate fragments present in <20% of volume; type 3, not all blastomeres of equal size, anucleate fragments present in >20% of volume; and type 4, the embryo is totally fragmented. Transferable embryos were defined as embryos of either type 1, 2 or 3.

The embryo development rate was defined as the number of transferable embryos divided by the total number of oocytes injected. The implantation rate was defined as the number of gestational sacs seen on ultrasound examination divided by the total number of embryos replaced. A vital pregnancy was defined as an intrauterine pregnancy with positive heart action. No strict definition of OHSS was given; in the analysis of the occurrence of this syndrome, its incidence was based on the fact that the investigator reported it as such.

Sample size considerations
With 100 subjects included in each treatment group, and assuming a SD of 450 IU of rFSH for the total dose used and a SD of 6.4 oocytes for the number of oocytes retrieved, a difference of 180 IU Puregon and 2.5 oocytes could be detected between the two treatment groups with a power of 80% using a two-sided t-test and a significance threshold of 5% (not adjusted for two primary outcomes).

Statistical analysis
The analysis of variance (ANOVA) was performed for non- dichotomous variables. If the ANOVA was not applicable, the Wilcoxon rank sum test was performed. To communicate the Wilcoxon results in clinically meaningful entities, Cochran's method (adjusted for centre) was used, and a pseudo-interaction test based upon midranks (Koch et al., 1990Go) was calculated to obtain an impression of the consistency of results across centres. Dichotomous variables were analysed by Mantel–Haenszel statistics, adjusted for centre.

The statistical analysis included all subjects who received at least one injection of rFSH.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Study population
A total of 180 women was randomized in five centres, 91 in the low-dose and 89 in the high-dose group. The aim to recruit 200 women was not reached because recruitment of patients was slower than anticipated. Ninety-one women started rFSH treatment in the low-dose group, and 88 in the high-dose group. The numbers of patients treated per centre were 8, 22, 35, 43 and 71.

Both groups had comparable demographic characteristics (Table IGo). The overall mean age was 27.5 years in both groups. The mean age in the low and high-dose groups at the five centres were 27.9 and 27.4, 28.2 and 28.0, 27.0 and 27.1, 25.8 and 27.3, and 26.7 and 26.9 years respectively. The cause of infertility was male in all cases. A tubal factor was also present in five subjects of the 100 IU group and two of the 200 IU group.


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Table I. Demographics and infertility characteristics of patients
 
All women were down-regulated at the start of rFSH treatment. Mean serum oestradiol concentrations before ovarian stimulation were 124.4 and 116.1 pmol/l in the 100 IU and 200 IU groups respectively. The corresponding LH concentrations were 2.0 and 2.4 IU/l.

Primary end-points
Results on the primary efficacy and efficiency end-points are listed in Tables II and IIIGoGo. For each started cycle, significantly more oocytes were retrieved in the 200 IU group (12.0 versus 5.7, P < 0.001). However, the total rFSH dose used was considerably lower in the 100 IU group (1121 versus 1875 IU, P < 0.001).


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Table II. Number of oocytes retrieved
 

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Table III. Total dose (IU) of recombinant FSH used
 
In subjects with oocyte retrieval, the number of oocytes recovered was 12.0 in the high-dose group compared with 6.9 in the low-dose group (P < 0.001). However, the overall statistical analysis of the number of oocytes retrieved was influenced seriously by the variability between treatment centres as well as between treatment groups within centres and, consequently, a test for centre interaction was statistically significant (P < 0.01).

Secondary end-points
Results on the secondary outcomes are listed in Table IVGo. The mean number of follicles >=11 mm on the day of HCG administration was 13.1 in the high-dose group and 10.6 in the low-dose group (P = 0.01). Correspondingly, significantly more follicles >=13 mm and >=15 mm had developed in the high-dose group. The numbers of follicles >=17 mm were similar in both groups (5.9 and 6.5 in the low-dose and high-dose treated women respectively). On average, the treatment duration was longer in the low-dose group (11.1 days) than in the high-dose group (9.4 days, P < 0.001).


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Table IV. Results (means adjusted for centre) on secondary parameters
 
Endocrinological parameters measured on the day of HCG administration showed significantly higher values in the 200 IU group for serum FSH (10.7 versus 6.8 IU, P < 0.001), serum oestradiol (5650 versus 4086 pmol/l, P < 0.01) and serum progesterone (4.5 versus 3.5 nmol/l, P < 0.001). LH concentrations did not differ (2.2 versus 2.5 IU/l).

Significantly more oocytes with intact zona pellucida and clear cytoplasm (11.2 versus 5.2, P < 0.001) and metaphase II oocytes (9.3 versus 4.6) were seen in the high-dose group. The number of metaphase I oocytes was lower in the 100 IU group (0.5 versus 1.1, P < 0.01), as was the number of germinal vesicle stage oocytes (0.3 versus 0.9, P < 0.001).

Spermatozoa used for ICSI had an average (± SD) count of 6.3 ± 13.6 x106/ml in the low-dose group, compared with 6.8 ± 13.8x106/ml (SD 13.8) in the high-dose group (P = NS). Progressive motility was 47.8 and 42.3% respectively (P = NS).

Significantly more transferable embryos were obtained in the 200 IU group (5.6 versus 3.7, P < 0.001). The overall embryo development rate was 64.6 and 60.3% in the low-dose and high-dose groups respectively. The centre-adjusted mean number of embryos transferred was 2.7 in both groups.

Two embryos more (2.9 versus 0.9) were frozen in the 200 IU group than in the 100 IU group.

The clinical pregnancy rates were not statistically significantly different in the two dose groups, both per started cycle (20.3 versus 24.7%) and per embryo transfer (27.6 versus 28.3%). The miscarriage rate in the 100 IU group appeared to be lower than that in the 200 IU group (1.5 versus 9.0% per embryo transfer), but the difference was not significant. Vital pregnancy rates per started cycle were not significantly different (19.2% in the 100 IU group versus 16.9% in the 200 IU group). Per embryo transfer, the vital pregnancy rates were 26.2 and 19.3% in the low- and high-dose groups respectively (P = NS).

Cycle cancellations
Among 91 women who started in the low-dose group, 74 had an oocyte retrieval (81%) and 67 an embryo transfer (74%). In the high-dose group, 86 women out of 88 who started rFSH treatment had an oocyte retrieval (98%), and 78 (89%) had an embryo transfer.

Reasons for drop-out before the stage of embryo transfer differed between the two treatment groups. In the low-dose group, insufficient ovarian response (n = 14), premature LH surge and (or) progesterone too high (n = 2), poor quality oocytes at retrieval (n = 1), adverse event (n = 1), no fertilization (n = 2) and other causes (n = 4) were reported. In the high-dose group, the reasons for drop-out were risk for hyperstimulation (n = 1), insufficient ovarian response (n = 1), poor quality oocytes at retrieval (n = 1), adverse events (n = 2), no fertilization (n = 4) and other causes (n = 1).

Safety
Ovarian hyperstimulation syndrome was reported in four cases, all in the 200 IU group, and all patients were hospitalized (4.5%). One other patient in the 200 IU group was hospitalized because of an ectopic pregnancy. No reports of OHSS were made in the 100 IU group.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The results of this study showed that a dose–response relationship exists between gonadotrophin dose and the number of oocytes retrieved. Thus, when the daily dose of rFSH was doubled from 100 to 200 IU, more than twice as many oocytes per started cycle could be retrieved. However, with the 100 IU regimen—in which a total of 1121 IU of rFSH was used—it appeared possible to obtain 3.7 transferable embryos without any hospitalization due to OHSS and with a vital pregnancy rate per started cycle of 19.2%. A similar outcome was found in a previous double-blind trial which compared treatment with 100 and 200 IU rFSH (Out et al., 1999Go).

The benefit of using 200 IU to treat these women was that two extra embryos could be frozen. However, four hospitalizations due to OHSS occurred in that group, compared with none in the 100 IU group. Although this study does not prove that OHSS occurs significantly more often in women treated with 200 IU rFSH, caution is nevertheless warranted in this situation. In interpreting these data, it is important to realize that the average age of the women in the current study was relatively low at 27.5 years.

It has been shown in the past, that with a successful cryoprogramme, the augmentation in pregnancy rates achieved by replacing frozen–thawed embryos can be clinically relevant (Jones et al., 1997Go). From that perspective, a high number of embryos available for freezing is attractive. Although in the current study overall fresh pregnancy rates were not different between the groups, it is likely that the pregnancy rate per single ovarian stimulation cycle including frozen–thawed embryo replacements will be higher in the high-dose group. However, due to limited drug budgets as imposed by health authorities, insurance companies or hospitals, a low-dose alternative might be more acceptable.

It is difficult to define an `ideal' dose of rFSH based on the findings of the current study. Ovarian sensitivity for gonadotrophins varies among women, and an individualized approach is most likely to result in a sufficient number of oocytes with minimal side effects. In addition, more data are needed on the effects of other daily doses (e.g. 150 IU).

It is important to note that large inter-centre variations were observed in the primary outcome parameters. Even using the same protocol, the number of collected oocytes per started cycle ranged from 2.8 to 7.2 in the 100 IU group and from 9.0 to 19.1 in the high-dose group. This was not due to differences in age of the women treated. However, despite this centre–treatment interaction, the difference between the two groups in numbers of oocytes retrieved was highly significant.

Also, there was a discrepancy between the number of large follicles seen by using transvaginal ultrasonography on the day of HCG administration and the number of oocytes actually retrieved. In the 100 IU group, a mean of 9.6 follicles >=13 mm were seen on ultrasound, and only 6.9 oocytes were retrieved, whereas in the high-dose group 12.0 oocytes could be collected with 12.2 follicles >=13 mm on ultrasound. These data suggest that in women in the low-dose group the oocyte recovery might have been performed more efficiently, assuming that it should be possible to retrieve an oocyte from a follicle of at least 13 mm diameter. It is also possible that, due to difficult counting, the actual number of large follicles was underestimated because many are being seen on ultrasound.

Interestingly, the previously observed increased miscarriage rate in the 100 IU group (13.3 versus 1.2%; Out et al., 1999) was not seen in the current study. In fact, more miscarriages occurred in the 200 IU group (9.0 versus 1.5%), although this difference was not statistically significant. This shows that interpretation of the secondary end-points such as miscarriage rates should be made with caution, as these studies were not set up to address differences in these outcomes. Given the fact that many analyses were carried out, some—such as the tests on miscarriage rates—might have been statistically significant purely by chance.

Earlier, a dose–response relationship between rFSH and number of oocytes recovered was not shown in women between 37 and 39 years of age: increasing the daily dose from 150 IU to 250 IU did not result in more retrievable oocytes (Out et al., 2000Go). It has been shown that the magnitude of the ovarian response is predictive of IVF outcome. High responders attain greater numbers of recovered oocytes and embryos for transfer, and also experience a higher fresh pregnancy rate than low responders (Hall et al., 1999Go). This might be due to better possibilities of selecting the morphologically best embryos for transfer. However, in view of the data from the current study it is also possible that the increased pregnancy rates in high responders are due to a general favourable reproductive performance of these women rather than the distinct result of an ovarian stimulation regimen aimed at maximizing the number of retrievable oocytes.

In conclusion, this study has confirmed earlier observations (Devroey et al., 1998Go; Bergh, 1999Go; Out et al., 1999Go) that a substantial proportion of women undergoing ovarian stimulation before ICSI benefit from rFSH doses as low as 100 IU per day. This seems to be associated with a lower incidence of OHSS, as well as lower costs of treatment. The challenge for the future is to investigate patient characteristics that predict ovarian response before treatment, thereby enabling rational decisions to be made on the rFSH starting dose.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The authors would like to thank Drs I.Ben Shlomo, H.Nieuwenhuis, I.Calderon, A.Raziel and M.Schachter for their contributions to this study.


    Notes
 
7 To whom correspondence should be addressed at: Organon Laboratories, Cambridge Science Park, Milton Road, Cambridge CB4 0FL, UK. E-mail: h.out{at}organon.cge.akzonobel.nl Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Bergh, C. (1999) What are the benefits of recombinant gonadotrophins? Recombinant follicle stimulating hormone. Hum. Reprod., 14, 1418–1419.[Free Full Text]

Bergh, C., Howles, C.M., Borg, K. et al. (1997) Recombinant human follicle stimulating hormone (r-hFSH, Gonal-F) versus highly purified urinary FSH (Metrodin HP). Results of a randomized comparative study in women undergoing assisted reproductive techniques. Hum. Reprod., 12, 2133–2139.[Abstract]

CPMP Working party on efficacy of medicinal products. (1990) Good Clinical Practice for trials on medicinal products in the European Community. Pharmacol. Toxicol., 67, 361–372.[Medline]

Devroey, P., Tournaye, H., Van Steirteghem, A. et al. (1998) The use of a 100 IU starting dose of recombinant FSH (Puregon®) in in-vitro fertilization. Hum. Reprod., 13, 565–566.[Free Full Text]

Edwards, R.G., Lobo, RA. and Bouchard, P. (1997) Why delay the obvious need for milder forms of ovarian stimulation? Hum. Reprod., 12, 399–401.[ISI][Medline]

Fauser, B.C.J.M., Devroey, P., Yen, S.S.C. et al. (1999) Minimal ovarian stimulation for IVF: appraisal of potential benefits and drawbacks. Hum. Reprod., 14, 2681–2686.[Free Full Text]

Felberbaum, R.E., Ludwig, M. and Diedrich, K. (1998) Are we on the verge of a new era in ART? Hum. Reprod., 13, 1778–1780.[Free Full Text]

Hall, J.E., Welt, C.K. and Cramer, D.W. (1999) Inhibin A and inhibin B reflect ovarian function in assisted reproduction but are less useful at predicting outcome. Hum. Reprod., 14, 409–415.[Abstract/Free Full Text]

Jones, H.W., Jr, Out, H.J., Hoomans, E.H.M. et al. (1997) Cryopreservation: the practicalities of evaluation. Hum. Reprod., 12, 1522–1524.[Abstract]

Koch, G., Carr, G., Amara, I. et al. (1990) Categorical data analysis. In Berry, D.A. (ed.), Statistical Methodology in the Pharmaceutical Sciences, Volume 13, pp. 389–470, Dekker, New York.

Meniru, G.L. (1999) What are the benefits of recombinant gonadotrophins? Is Puregon a `good' or `super' drug? Hum. Reprod., 14, 1409–1411.[Free Full Text]

Out, H.J., Mannaerts, B.M.J.L., Driessen, S.G.A.J. and Coelingh Bennink, H.J.T. (1995) A prospective, randomized, assessor-blind, multicentre study comparing recombinant and urinary follicle-stimulating hormone (Puregon vs Metrodin) in in-vitro fertilization. Hum. Reprod., 10, 2534–2540.[Abstract]

Out, H.J., Lindenberg, S., Mikkelsen, A.L. et al. (1999) A prospective, randomised, double-blind clinical trial to study the efficacy and efficiency of a fixed dose of recombinant follicle stimulating hormone (Puregon®) in women undergoing controlled ovarian hyperstimulation. Hum. Reprod., 14, 622–627.[Abstract/Free Full Text]

Out, H.J., Braat, D.D.M., Lintsen, B.M.E. et al. (2000) Increasing the daily dose of recombinant follicle-stimulating hormone (Puregon®) does not compensate for the age-related decline in retrievable oocytes after controlled ovarian hyperstimulation. Hum. Reprod., 15, 29–35.[Abstract/Free Full Text]

Submitted on July 5, 2000; accepted on March 6, 2001.