Service Gynécologie-Obstétrique III, Clinique Universitaire Baudelocque, Hôpital Cochin, 123 bd Port-Royal, Paris 75014, France
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Abstract |
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Key words: estradiol/hyperstimulation/ovarian stimulation/percentiles/rFSH
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Introduction |
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In order to decide whether to proceed with an ongoing cycle and to define the so-called poor and high responders, Phelps et al. and Khalaf et al. used different cut-off E2 levels at day 46 and on the day of hCG administration (d-hCG) of the stimulation cycle. Poor responders, defined by E2 levels <75 pg/ml at day 4 (Phelps et al., 1998) or <50 pg/ml at day 6 (Khalaf et al., 2000
), were associated with low pregnancy and high cancellation rates. Follicular aspiration in patients with E2 levels <500 pg/ml at d-hCG yields less than five mature oocytes (Sharara and McClamrock, 1999
). On the other hand, high responders, defined as having E2 levels >3000 pg/ml at d-hCG, are more often subject to OHSS (Asch et al., 1991
; Morris et al., 1995
). However, the influence of high E2 levels on the outcome of IVF cycles is still controversial. Simon et al. suggested a detrimental effect of high E2 on endometrial receptivity (Simon et al., 1998
), but others found no adverse consequences (Sharara and McClamrock, 1999
; Levi et al., 2001
).
The use of E2 levels to differentiate poor, normal and high responders, and to predict IVF outcome, has been the subject of debate. In most studies, cut-off levels were chosen arbitrarily and depend on various factors. Percentile curves offer a more objective definition of these three categories of patients. To our knowledge, only one study has used percentile curves to adjust the doses of gonadotrophins to the ovarian response and to evaluate IVF outcome in stimulation cycles (Forman et al., 1988). In our centre, since 1989 we have routinely based COS on percentile E2 curves for long and short GnRH agonist protocols combining triptorelin and hMG (J.-R.Zorn, unpublished data). Gonadotrophin dose and ovulation induction by hCG are adjusted according to the pattern of E2 levels on the percentile curve of the corresponding protocol.
The objectives of the present study were: (i) to determine the ovarian response in COS with recombinant (r)FSH in a short GnRH agonist protocol by using E2 percentile curves; (ii) to define low, normal and high responders; and (iii) to predict the IVF outcome by using percentile E2 serum levels at day 5 and d-hCG.
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Materials and methods |
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Drugs and COS protocols
GnRH agonist
Triptorelin (Decapeptyl; Ipsen, Paris, France) was administered in a dose of 0.1 mg/day, starting 12 days after withdrawal bleeding.
rFSH
Follitropin-ß (Puregon; NV Organon, Paris, France) or follitropin- (Gonal-F; Serono, Geneva, Switzerland) were used during this study. A total of 787 cycles were stimulated with Gonal-F and 118 with Puregon.
hCG
hCG (Gonadotrophine Chorionique Endo; Organon) was used at a dose of 5000 IU/ampoule. Patients received norethisterone (NE) (Primolut Nor; Schering, Paris, France) 10 mg daily for 816 days starting from day 3 of a natural cycle. Triptorelin, 0.1 mg daily, was given for 615 days starting 4 days after the last day of NE. At day 3 of triptorelin treatment, rFSH was started at a daily dose of 36 ampoules (75 IU) per day. Generally, the initial dose for patients in group A was 3 ampoules, for group B, 4 ampoules and for group C, 6 ampoules. From day 5 of the stimulation cycle, doses of rFSH were adjusted according to the number of follicles found on ultrasound and to the E2 levels. hCG 10 000 IU was injected when two or more follicles >15 mm were found. Oocyte retrieval was performed transvaginally under ultrasound guidance.
Embryology assessments
After liquefaction at 37°C, motile sperm were selected through a two-step density gradient (Puresperm; JCD, Lyon, France). The pellet was washed and resuspended in human tubal fluid (HTF) culture medium (Irvine scientific, CA, USA) supplemented with 7.5% autologous female decomplemented serum. For the IVF procedure, oocytes were inseminated with 10 000 motile sperm in microdroplets of 30 µl of HTF with 7.5% serum under mineral oil and kept under 5% CO2 at 37°C. For ICSI, the cumulus cells around the oocytes were removed with 80 IU/ml hyaluronidase, and the oocytes were checked for their nuclear status. Only oocytes in metaphase II were microinjected and kept in HTF + 7.5% serum under 5% CO2 at 37°C. The oocytes were checked 1620 h after insemination or microinjection for evidence of fertilization. The oocytes were considered to be normally fertilized when two pronuclei were visible. Fertilized oocytes were kept in culture for an additional 24 h (HTF + 15% serum) and checked for cleavage. The embryos were evaluated according to the number of blastomas and the degree of fragmentation. Up to four embryos were transferred, and any remaining high grade embryos were cryopreserved.
Embryo transfer and luteal support
Two-day-old embryos were transferred using the Frydman catheter (CCD Paris, France). The number of embryos transferred was typically two, and occasionally up to four in patients >39 years old or when embryo quality was poor. Luteal support was administered in the form of vaginal progesterone (Utrogestan; Besins International, Paris, France) 800 mg per day for 15 days, and hCG 1500 IU was injected 48 h after the transfer in patients whose peak E2 levels were above the 90th percentile (>P90).
End points and outcome measures
The main outcome for plotting rFSH E2 patterns for the three different age groups was E2 levels from day 5 to d-hCG. IVF outcomes were expressed as the number of oocytes, number of embryos obtained, number of high grade embryos, E2 peak levels and pregnancy rates (PRs) at day 5 and d-hCG. High, normal and poor responders were defined as those women with E2 levels at day 5 and d-hCG >P90, between 10th and 90th percentile (P10P90) and below the 10th percentile (<P10) respectively. The P10 and P90 cut-offs were chosen based on previous reports for other fields of gynaecologyobstetrics (Lubchenco et al., 1963; Leroy and Lefort, 1971
).
Laboratory analysis
E2 determination was performed using an immunoenzyme assay by sequential competitive technique on an Immuno-1 multiparameter analyser (Bayer reference ES2). The capture antibody used was a rabbit polyclonal antibody against E2 conjugated to fluorescein. The competitive marker was constituted by E2 conjugated to alkaline phosphatase. Separation was performed using a dispersed solid phase that is achieved by magnetic particles linked to monoclonal antibody anti-fluorescein. The signal was read on the spectrophotometer at 405 and 450 nm. E2 standard USP in a saline buffer bovine albumin with sodium azoture was used for calibration. The molar conversion factor was 3.67 (pmol/l = pg/mlx3.67). The analytical range was from 0 to 3600 pg/ml (0 to 13 200 pmol/l). Samples with concentration values exceeding the maximal calibrator can be diluted in the zero calibrator or in an albumin buffer. Analytical sensitivity was 5 pg/ml, corresponding to twice the SD of the zero calibrator. The functional limit of detection at the 20% of inter-assay coefficient of variation (CV) was near 10 pg/ml (36.7 pmol/l). Inter-assays imprecision show CV <20% for a high concentration of E2 (>300 pg/ml), and near 5% for a low concentration of E2 (<30 pg/ml). Blood samples were collected in tubes without anticoagulant and separated by centrifugation. Assays were run on the day of sampling with 50 µl of serum. The method remained unchanged during the study period.
Statistical analysis
Results are expressed as means ± SD. Statistical comparisons were made by using the 2-test and analysis of variances when appropriate. Statistical significance was set at P < 0.05. Statview software (Abacus, F-4.5, 1999; Berkeley, CA, USA) was used for statistical analysis.
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Results |
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Discussion |
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For poor responders identified at day 5, P10 E2 were 50 pg/ml, which are similar to those previously described in IVF cycles stimulated with gonadotrophins, either with or without GnRH agonist. In the study by Phelps et al. E2 levels <75 pg/ml at day 4 yielded poor IVF outcomes (Phelps et al. 1998
), and for Khalaf et al. E2 levels <50 pg/ml at day 6 had higher cancellation rates and poorer IVF results (Khalaf et al., 2000
). Sharara and McClamrock defined poor responders as patients who had <500 pg/ml E2 at d-hCG (Sharara and McClamrock, 1999
). In the present study, it is of interest that poor responders at day 5 had better IVF outcomes than poor responders identified later at d-hCG for all groups of patients. This can be explained by the fact that doses of rFSH were increased in cases of poor response at day 5, and this increase obviously improved the ovarian response. This difference was not observed for normal responders, probably because of the unchanged initial dose of rFSH in these patients.
Phelps et al. considered high responders to be patients with E2 levels >200 pg/ml at day 4 (Phelps et al., 1998). Interestingly, none of our patients with E2 >P90 at day 5 was hospitalized for OHSS. This may be explained by the reduction of rFSH dose for those patients after day 5. For our high responders identified at d-hCG, P90 E2 levels were similar to those of high responders defined by others (Simon et al., 1998
; Sharara and McClamrock, 1999
). Generally, in our study, patients with E2 levels >P90 had significantly better IVF outcomes (expressed as numbers of oocytes, total embryos and high-grade embryos) than normal responders (P10P90) and, as expected, than poor responders. In the only study in which E2 percentile curves were used to predict IVF outcomes, Forman et al. showed lower PRs for patients with E2 >P90 (>2320 pg/ml) (Forman et al., 1988
). Of note is that this study was published before the GnRH agonist era. Our data are consistent with those previously published by Sharara and McClamrock (Sharara and McClamrock, 1999
).
The issue of deleterious effects of high E2 levels on uterine receptivity has been under debate for many years. High levels of steroid hormones induce morphological alterations of the endometrium (Garcia et al., 1984; Bladford et al., 1997
) and alter endometrial E2/progesterone ratios, which is associated with impairment of endometrial receptivity (Gidley-Baird et al., 1986
). In COS, a significant reduction of the nuclear receptors for progesterone and E2 has been demonstrated in both glands and stroma (Hadi et al., 1994
). Several studies have shown a negative effect of high E2 levels on pregnancy and implantation rates (Testart et al., 1986
; Pellicer et al., 1996
; Simon et al., 1998
). Moreover, Valbueña et al. found that a high E2 concentration affects embryonic adhesion (Valbueña et al., 2001
). However, most of these studies used small sample sizes and different definitions of high responders, or dealt with in-vitro models. Our results are from a large cohort of human subjects are consistent with previously reported data (Chenette et al., 1990
; Tarin et al., 1992
; Loumaye et al., 1997
; Sharara and McClamrock, 1999
; Levi et al., 2001
). According to our study, we did not find any deleterious effect of high E2 levels, and patients with E2 >P90 had better IVF outcomes in all group of patients. Our three cases of severe OHSS belonged to group A (<35 years) and were normal responders (d-hCG E2 = P10P90). No cases of severe OHSS were detected in patients with E2 >P90 at d-hCG, in contrast to previously published data which reported incidences of 0.94 and 2.9% of severe OHSS (Asch et al., 1991
; Jaffe et al., 1993
).
An important difference between our study and prior reports (Simon et al., 1998; Sharara and McClamrock, 1999
; Khalaf et al., 2000
) is the stimulation regimen. We cannot exclude the possibility that the type of gonadotrophin influences oocyte and embryo quality. Moreover, high responders with rFSH may behave differently. We used both types of rFSH (follitropin-
and -ß) and we do believe that this has affected our results, as previous reports have shown that there are no differences in the biological and clinical activity of the two rFSH hormone preparations (Brinsden et al., 2000
; Harlin et al., 2002
).
In conclusion, we believe that percentile curves can help practitioners to follow stimulation cycles and to adjust the dose of the stimulation regimen according to the ovarian response. Percentile curves have been used successfully in other fields of gynaecologyobstetrics, such as fetal ultrasonography, and can be useful in COS monitoring. Our high responders (E2 >P90) had better IVF outcomes than normal and poor responders in all age groups. More studies with larger sample sizes using the same protocols have to be performed for further confirmation of our results.
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Acknowledgements |
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Notes |
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References |
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Bladford, A.J., Najmabadi, S. and Paulson, R.J. (1997) Ultrastructural characteristics of the luteal phase endometrium in donors undergoing controlled ovarian hyperstimulation. Fertil. Steril., 67, 625630.[ISI][Medline]
Brinsden, P., Akagbosu, F., Gibbons, L.M., Lancaster, S., Gourdon, D., Pharm, D., Engrand, P. and Loumaye, E. (2000) A comparison of the efficacy and tolerability of two recombinant human follicle-stimulating hormone preparations in patients undergoing in vitro fertilizationembryo transfer. Fertil. Steril., 73, 114116.[ISI][Medline]
Chenette, P.E., Sauer, M.V. and Paulson, R.J. (1990) Very high serum E2 levels are not detrimental to clinical outcome if in vitro fertilization. Fertil. Steril., 54, 858863.[ISI][Medline]
Forman, R., Fries, N., Testart, J., Belaisch-Allart, J., Hazout, A. and Frydman, R. (1988) Evidence for an adverse effect of elevated serum E2 concentration on embryo implantation. Fertil. Steril., 49, 118122.[ISI][Medline]
Garcia, J.E., Acosta, A.A., Hsiu, J.G. and Jones, H.W. (1984) Advanced endometrial maturation after ovulation induction with human menopausal gonadotrophin/human chorionic gonadotrophin for in vitro fertilization. Fertil. Steril., 41, 3137.[ISI][Medline]
Gidley-Baird, A.A., ONeil, C., Sinosich, M.J., Porter, R.N., Pike, I.L. and Saunders, D.M. (1986) Failure of implantation in human in vitro fertilization and embryo transfer patients: the effects of altered progesterone/estrogen rations in humans and mice. Fertil. Steril., 45, 6975.[ISI][Medline]
Hadi, F.H., Chantler, E., Anderson, E., Nicholson, R., McClelland, R.A. and Seif, M.W. (1994) Ovulation induction and endometrial steroid receptors. Hum. Reprod., 9, 24052410.[Abstract]
Harlin, J., Aanesen, A., Csemiczky, G., Wramsby, H. and Fried, G. (2002) Delivery rates following IVF treatment, using two recombinant FSH preparations for ovarian stimulation. Hum. Reprod., 17, 304309.
Jaffe, S.B., Jaffe, L.H. and Jewelewicz, R. (1993) Incidence of severe ovarian hyperstimulation syndrome with extremely elevated serum estrogen levels. Gynecol. Obstet. Invest., 35, 222227.[ISI][Medline]
Khalaf, Y., Taylor, A. and Braude, P. (2000) Low serum E2 concentrations after five days of controlled ovarian hyperstimulation for in vitro fertilization are associated with poor outcome. Fertil. Steril., 74, 6366.[ISI][Medline]
Leroy, B. and Lefort, F. (1971) A propos du poids et de la taille des nouveaus-nés à la naissance. Rev. Fr. Gynecol., 66, 391395.
Levi, L.J., Drews, M.R., Bergh, P.A., Miller, B.T. and Scott, R.T. (2001) Controlled ovarian hyperstimulation does not adversely affect endometrial receptivity in in-vitro fertilization cycles. Fertil. Steril., 76, 670674.[ISI][Medline]
Loumaye, E., Engrand, P., Howles, C.M. and ODea, L. (1997) Assessment of the role of serum luteinizing hormone and E2 response to follicle-stimulating hormone on in vitro fertilization treatment outcome. Fertil. Steril., 67, 889899.[ISI][Medline]
Lubchenco, L.O., Hansman, C., Dressler, M. and Boyd, E. (1963) Intrauterine growth as estimated from liveborn birthweight data at 24 to 42 weeks of gestation. Pediatrics, 32, 793800.[Abstract]
McDonough, P. (1999) low and high respondersat what level of serum E2 do things start to get fuzzy? Fertil. Steril., 71, 584586.[ISI]
Morris, R.S., Paulson, R.J., Sauer, M.V. and Lobo, R.A. (1995) Predictive value of serum E2 concentrations and oocyte number in severe hyperstimulation syndrome. Hum. Reprod., 10, 811814.[Abstract]
Pellicer, A., Valbueña, D., Cano, F., Remohí, J. and Simón, C. (1996) Lower implantation rates in high responders: evidence for an altered endocrine milieu during the preimplantation period. Fertil. Steril., 65, 11901195.[ISI][Medline]
Phelps, J.Y., Levine, A.S., Hockman, T.N., Zacur, H.A., Wallach, E.E. and Hinton, E.L. (1998) Day 4 E2 levels predict pregnancy success in women undergoing controlled ovarian hyperstimulation for IVF. Fertil. Steril., 69, 10151019.[ISI][Medline]
Sharara, F.I. and McClamrock, H.D. (1999) High E2 levels and high oocyte yield are not detrimental to in vitro fertilization outcome. Fertil. Steril., 72, 401405.[ISI][Medline]
Simón, C., Garcia Velasco, J.J., Valbueña, D., Peinado, J.A., Moreno, C., Remohí, J. and Pellicer, A. (1998) Increasing uterine receptivity by decreasing E2 levels during the preimplantation period in high responders with the use of a follicle-stimulating hormone step-down regimen. Fertil. Steril., 70, 234239.[ISI][Medline]
Tarín, J.J., Sampaio, M.C., Calatayud, C., Castellvi, R.M., Bonilla-Musoles, F. and Pellicer, A. (1992) Relativity of the concept high responder to gonadotrophins. Hum. Reprod., 7, 1922.[Abstract]
Testart, J., Belaisch-Allart, J. and Frydman, R. (1986) Relationship between embryo transfer results and ovarian response and in vitro fertilization rates: analysis of 186 human pregnancies. Fertil. Steril., 45, 237243.[ISI][Medline]
Valbueña., D., Martin, J., de Pablo, J.L., Remohí, J., Pellicer, A. and Simón, C. (2001) Increasing levels of estradiol are deleterious to embryonic implantation because they directly affect the embryo. Fertil. Steril., 76, 962968.[ISI][Medline]
Submitted on December 5, 2001; resubmitted on March 20, 2002; accepted on July 17, 2002.