1 Institut Clínic of Gynecology, Obstetrics and Neonatology and 2 Hormonal Laboratory, Faculty of Medicine, University of Barcelona, Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain
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Abstract |
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Key words: assisted reproduction/early pregnancy loss /FSH/LH/ovarian stimulation
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Introduction |
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Human menopausal gonadotrophin (HMG) containing different FSH/LH ratios has been used effectively for ovulation induction in IVF patients, and for years this was the only preparation available for clinical use. Because of advances in the urinary purification techniques that have become available, HMG was purified further so that, in the early 1980s, purified FSH (with <1% LH contamination) was made possible, whereas in the 1990s highly purified FSH (with <0.1% LH contamination) became a therapeutic option for ovarian stimulation. More recently, biotechnology has made available for the first time a recombinant human FSH preparation (Howles, 1996; Olijve et al., 1996
) which is completely devoid of LH activity.
As previously stressed (Hull et al., 1994; Daya, 1998
; FIVNAT, 1999
; Fleming et al., 2000
), it is clear that changes in stimulation regimens with widespread use of urinary or recombinant FSH preparations, without LH supplementation, has not led to a drop in overall programme success rates, and it has been suggested that there is no need to explore the requirement for LH supplementation further (Hull et al., 1994
; Daya, 1998
). However, when considering the relative roles of FSH and LH preparations in folliculogenesis, it has been stressed that the potential benefit of adding LH to FSH-only regimens might depend on the extent to which the endogenous plasma LH is suppressed by concomitant GnRH analogue therapy (Hillier, 1994
).
Whereas the relative importance of FSH and LH in inducing follicular growth and maturation in the human is still being investigated, considerable debate exists as to whether the LH activity contained in HMG preparations could affect the outcome of assisted reproduction treatment in GnRH agonist down-regulated women (Hull et al., 1994; Westergaard et al., 1996
; Loumaye et al., 1997
; Daya, 1998
; Söderström-Anttila, 1998
; Filicori, 1999
; Lévy et al., 2000
). Thus, the concentrations of residual endogenous LH remaining during full-dose GnRH agonist pituitary suppression are certainly sufficient to achieve adequate follicular maturation during ovarian stimulation with purified human urinary FSH or recombinant FSH (Lévy et al., 2000
). However, it has been suggested that GnRH agonist down-regulation in some normogonadotrophic women may result in profound suppression of LH concentrations, impairing adequate oestradiol synthesis (Westergaard et al., 1996
; Fleming et al., 1998
, 2000
; Janssens et al., 2000
), fertilization rates (Westergaard et al., 1996
) or the final clinical treatment outcome by increasing the risk of early pregnancy loss (Westergaard et al., 2000
).
The amount of LH activity actually necessary for a normal follicle and oocyte development is not known, but is likely to be very low, since <1% of follicular LH receptors need to be occupied in order to allow normal steroidogenesis (Chappel and Howles, 1991; Lévy et al., 2000
). Threshold values of serum LH of <1,
0.07 and <0.5 IU/l in the mid-follicular phase (days 78) of gonadotrophin ovarian stimulation cycles, in down-regulated women have been proposed to diagnose the group of `profoundly' suppressed LH patients (Chappel and Howles, 1991
; Fleming et al., 2000
; Westergaard et al., 2000
).
Following the recently published papers on this subject (Fleming et al., 2000; Westergaard et al., 2000
), we have examined in our own clinic the impact of hormonal parameters on the outcome of assisted reproduction with a retrospective analysis of prospectively collected material. The specific aims of this study, where receiver-operating characteristic (ROC) analysis was used, were: (i) to assess further the usefulness of mid-follicular serum LH concentration as a predictor of ovarian response, IVF outcome, implantation, and the outcome of pregnancy in pituitary-suppressed patients undergoing assisted reproduction; and (ii) to define the best threshold value, if any, to discriminate between women with `low' and `normal' LH concentrations.
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Materials and methods |
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Stimulation regimen
Ovarian stimulation was carried out with FSH under pituitary suppression with GnRH agonist. In all women, pituitary desensitization was achieved by s.c. administration of leuprolide acetate (Procrin; Abbott Laboratories, Madrid, Spain). This treatment was started in the midluteal phase of the previous cycle and given 1 mg daily, then reduced to 0.5 mg after ovarian arrest was confirmed. Gonadotrophin stimulation of the ovaries was started when serum oestradiol concentrations declined to <50 pg/ml and a vaginal ultrasonic scan showed an absence of follicles >10 mm diameter. On days 1 and 2 of ovarian stimulation, 6 and 4 ampoules/day of recombinant human FSH (75 IU per ampoule) (Gonal-F, Serono SA, Madrid, Spain) respectively, were administered subcutaneously. On days 3 and 4 of ovarian stimulation, two ampoules per day of FSH were administered to each patient. From day 5 onward, FSH was administered on an individual basis according to the ovarian response as assessed by sequential transvaginal ultrasonography and serum oestradiol measurements. Human chorionic gonadotrophin (HCG; 5000 IU) (Profasi; Serono SA) was administered i.m. when a consistent rise in serum oestradiol concentration was associated with the presence of two or more follicles >18 mm in diameter with 4 follicles measuring
14 mm. Oocyte aspiration was performed with vaginal ultrasonography 3536 h after HCG administration. The maturational status of the oocytes and the embryo grading were recorded according to published criteria (Veeck, 1986
); embryos of Veeck grades 1 or 2 were considered high quality. Two days later up to three embryos per patient were replaced. Two additional doses of 2500 IU of HCG on the days of embryo transfer and 3 days later were given to supplement the luteal phase in all patients.
Pregnancy outcome
The following categorization of pregnancy outcome was made: (i) biochemical pregnancy: four women showed serum ß-HCG concentrations >50 IU/l 1314 days after embryo transfer and 7 days later but no intrauterine or extrauterine pregnancy could be demonstrated by ultrasonography and menses ensued; (ii) spontaneous abortion: ten women had increasing concentrations of ß-HCG on days 1314 and 2021 after embryo transfer, and pregnancy was confirmed by ultrasonic demonstration of an intrauterine gestational sac but the gestation subsequently spontaneously terminated; (iii) ongoing pregnancy: this category comprised 58 patients achieving >20 weeks gestation.
For the evaluation of the results, the following groups of IVF/ICSI outcome were considered: (i) no conception (n = 72); (ii) conception (n = 72) which included ongoing pregnancy (n = 58) and biochemical (n = 4) and spontaneous abortions (n = 10); (iii) ongoing pregnancy (n = 58); (iv) early pregnancy loss (n = 14) which included biochemical (n = 4) and first trimester spontaneous abortions (n = 10).
Hormone analyses and ultrasonography
For this study, serum concentration of LH was measured retrospectively on S7 in all treatment cycles studied. This was done using frozen blood samples which were examined in one run. Hormones were measured using commercially available kits as reported previously (Balasch et al., 1998; Fábregues et al., 2000
; Peñarrubia et al., 2000
). Oestradiol concentrations in serum were estimated by a competitive immunoenzymatic assay (Immuno 1, Technicon; Bayer, Tarrytown, NY, USA). The sensitivity was 10 pg/ml and the interassay coefficient of variation (CV) was 5%.
FSH and LH serum concentrations were measured by an immunoenzymatic assay with two monoclonal antibodies (Immuno 1, Technicon; Bayer) and data are expressed in terms of IRP 78/549 and 68/40 respectively. The sensitivity of the assays was 0.1 IU/l for FSH and 0.3 IU/l for LH, and interassay CV were 2.7 and 3.1% respectively. The intra-assay CV of the LH assay with varying concentrations of LH were found to be (mean concentration, intra-assay CV): (i) 1.13 IU/l, 1.76%; (ii) 0.66 IU/l, 2.12%; (iii) 0.53 IU/l, 3.10%; (iv) 0.32 IU/l, 3.85%. There was not cross-reactivity of LH with free alpha subunit. Since it has been stressed (Fleming, 1999) that a statistically normal distribution of concentrations of serum LH should be observed within the `measurable' range (>0.3 IU/l in our study) in order to validate an LH assay, we tested this point in our study population. Using the one-sample KolmogorovSmirnov test which is a test of the goodness of fit between a distribution of observed frequencies and a theoretical frequency distribution, we found that LH values were normally distributed in the whole study population, the non-conception group, and pregnant patients (data not shown). This supports the validity of the present investigation. Total ß-HCG was measured by a solid-phase, two-site chemiluminescent enzyme immunometric assay standardized against the Third International Standard 75/537 (Immulite, Diagnostic Products Co., Los Angeles, CA, USA), with a detection limit of 2 IU/l. The interassay CV was 5.8%.
Ultrasonic scans were performed using a 5 mHz vaginal transducer attached to an Aloka sector scanner (Model SSD-620; Aloka Co. Ltd, Tokyo, Japan).
Statistics and probability testing
For statistical analysis Student's t-test, the MannWhitney U-test, Fisher's exact test and 2-test were used as appropriate. Results are expressed as mean ± SE, and as medians and interquartile (25 and 75%) ranges. P < 0.05 was considered significant. The discrimination attained between two study groups (conception versus non-conception assisted reproduction treatment cycles, and ongoing pregnancy versus early pregnancy loss groups) was evaluated with ROC analysis (Hanley and McNeil, 1982
; Zweig and Campbell, 1993
). ROC curves are plots of all the sensitivity and specificity pairs which are possible for all levels of a particular parameter. They are constructed by plotting the false positive rate or (100 specificity) on the x-axis. The y-axis shows the true positive rate or sensitivity. The best threshold value discriminating between two conditions is the value located at the greatest distance from the diagonal.
Calculation of the area under the ROC curve (AUCROC) provides the quantitative measure of accuracy, i.e. the ability of a particular parameter (e.g. LH concentrations on S7) to discriminate between two conditions (e.g. ongoing pregnancy versus early pregnancy loss). An ROC curve representing a parameter with no discrimination at all is a 45° diagonal line from the left lower corner (0% true positive rate and 0% false positive rate) to the upper right corner (100% true positive rate and 100% false positive rate) with an area under the curve of 0.5. A parameter with no overlap between the two conditions will discriminate perfectly and has an ROC curve passing along the y-axis to the upper left corner (100% true positive rate and 0% false positive rate) to end again in the upper right corner with an area under the curve of 1.0.
Data were analysed by Statistics Package for Social Sciences (SPSS, Chicago, IL, USA) statistical software. The Medcalc software (Mariakerke, Belgium) was used to construct ROC curves.
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Results |
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Discussion |
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Two very recent studies exploring the effects of suppression of LH during ovarian stimulation (days 78) in normogonadotrophic women undergoing assisted reproduction (Fleming et al., 2000; Westergaard et al., 2000
) have added further to the controversy. Westergaard et al. (2000) reported that as many as 49% of women stimulated with recombinant FSH under pituitary suppression had low concentrations of LH (<0.5 IU/l) in the mid-follicular phase. In comparison with the normal LH group, these women had serum oestradiol concentrations significantly lower on S8. A total of 64 positive pregnancy tests were recorded and the proportion of them in the two groups (30 versus 34% per started cycle in the low and normal LH groups respectively) was similar. However a 5-fold higher risk of early pregnancy loss (biochemical pregnancies and clinical spontaneous abortions) was seen in the low LH group (45 versus 9%). It has been reported that 26% of women treated with highly purified or recombinant FSH and GnRH agonist had suppressed LH concentration (
0.7 IU/l) on S7 (Fleming et al. 2000
). Patients with suppressed LH had much lower oestradiol concentrations at this point and at HCG administration in cycles treated with either urinary or recombinant FSH. However, the gross ovarian response, as became evident by FSH dose demands, duration of stimulation, and also oocyte and embryo yields and embryo cryopreservation, were influenced only in cycles treated with urinary FSH.
The present study adds further to the debate. First, the proportion of `profoundly' suppressed women in our study population (31, 15 and 7% of patients had mid-follicular LH serum concentration <1, 0.7 and <0.5 respectively) was lower than previously reported (Fleming et al., 2000
; Westergaard et al., 2000
). Second, no significant effect of LH suppression on oestradiol biosynthesis by the follicle was observed. Oestradiol serum concentrations on S7 were not significantly different when `low' and `normal' LH groups delineated by the three different LH thresholds were compared. Although, as expected, oestradiol concentration at S7 was somewhat lower in the `low' LH groups, this did not imply a negative influence of low LH activity on the ovary. The following facts support this contention: (i) peak oestradiol concentrations reached after ovarian stimulation in IVF cycles depends both on the number of growing follicles and the presence of sufficient LH action capable of stimulating androgen substrate production from theca cells to be transformed into oestrogen by FSH-stimulated aromatase activity in granulosa cells (Hillier, 1994
); (ii) oestradiol serum concentrations at the end of ovarian stimulation (HCG injection day) were similar in `low' and `normal' LH groups; (iii) final follicular development and oocyte yield were also similar; (iv) duration of stimulation and total dose of recombinant FSH were similar irrespective of the S7 LH threshold considered.
The above discrepancies between previous studies and ours with respect to ovarian steroidogenesis could be explained on the basis of the GnRH agonist used. A number of studies in the literature have shown that different GnRH agonists have yielded satisfactory results in IVF patients. For hydrophobic GnRH agonists, however, there is a correlation between the hydrophobicity of substituents in position 6 and potency; in general, the potency increases with increasing hydrophobicity (Conn and Crowley, 1991). Thus, they may have different effects on different human cells. Both in-vitro (Miró et al., 1992
) and in-vivo (Balasch et al., 1992
) studies have shown that the inhibitory effect on ovarian steroidogenesis and follicular development is more evident with the more potent buserelin used by Fleming et al. (2000) and Westergaard et al. (2000) than with leuprolide acetate used in our study. Fleming et al. (2000) reported that with the more potent recombinant FSH treatment, suppression of LH failed to show the impact upon gross ovarian responses that were evident with highly purified urinary FSH. In fact, the same recombinant FSH preparation was used in Fleming's and our studies.
In concordance with previous reports (Fleming et al., 2000; Westergaard et al., 2000
) no differences were observed in our study with respect to ovarian response and IVF/ICSI outcome in `low' and `normal' LH groups irrespective of the LH threshold considered according to those values proposed in the literature. In agreement with the study by Fleming et al. (2000) but in contrast with data from Westergaard et al. (2000), we found that serum LH measurements in the mid-follicular phase did not predict implantation and the outcome of pregnancy. It is notable that in those previous studies (Fleming et al., 2000
; Westergaard et al., 2000
) the threshold values for LH (
0.7 and <0.5 respectively) were established according to the detection limit of LH assays used. We have, on the other hand, used a statistical method using ROC curve analysis to test the usefulness of mid-follicular LH concentration to discriminate between conception versus non-conception cycles and ongoing prenancies versus early pregnancy losses. In ROC curve analysis, many efficiencies of all decision levels can be calculated, resulting in an overall quantification of accuracy which is not affected by the prevalence of a condition (Hanley and McNeil, 1982
; Zweig and Campbell, 1993
). Thus, our analysis shows clearly that mid-follicular LH concentrations are not predictive of implantation or early pregnancy loss after IVF/ICSI. In fact, a marked overlapping was found for mid-follicular LH concentrations in the present study when non-conception, conception, ongoing pregnacy, and early pregnancy loss groups were compared.
In conclusion, the present study has shown that serum LH measurements, in the mid-follicular phase during ovarian stimulation with recombinant FSH under pituitary suppression with leuprolide in normogonadotropic women undergoing assisted reproduction, cannot predict ovarian response, IVF/ICSI outcome, implantation, and the outcome of pregnancy. Therefore, our results do not support the need for additional exogenous LH supplementation in down-regulated women receiving a recombinant FSH-only preparation. Finally, irrespective of the role of the agonist, we would stress that researchers who are going to carry out analyses on LH concentrations and the outcome of assisted reproduction should adopt rigorous statistical methods for the evaluation of their results.
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Notes |
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References |
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Submitted on February 19, 2001; accepted on April 26, 2001.