1 Institut Clínic of Gynecology, Obstetrics and Neonatology and 2 Hormonal Laboratory, Faculty of MedicineUniversity of Barcelona, Hospital ClínicInstitut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain
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Abstract |
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Key words: FSH/inhibin B/IVF/low responders/ovarian response
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Introduction |
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However, healthy women with comparable weights and basal hormonal concentrations, and a normal menstrual cycle may react differently to the same dose of gonadotrophins (van Hooff et al., 1993; Edwards and Brody, 1995
). Normally, the first evaluation of the ovarian response during a treatment cycle comprises vaginal ultrasound scanning of ovarian follicles and serum oestradiol determination a few days after ovarian stimulation is initiated. When the ovarian response by this evaluation is insufficient, it seems logical to anticipate a low response and to increase the gonadotrophin dose. Increasing the dose in the course of an assisted reproductive treatment cycle, however, does not enhance ovarian response in many patients but leads to cycle cancellations which further increase the cost and duration of therapy (van Hoof et al., 1993).
Interestingly, a recent study investigated the use of the serum oestradiol concentrations obtained shortly after the initiation of gonadotrophin stimulation in order to provide an early marker of ovarian responsiveness and assist in deciding whether to proceed with an ongoing cycle (Phelps et al., 1998). It was concluded that oestradiol concentrations obtained on the fourth day of gonadotrophin therapy are highly predictive of successful ovulation induction and pregnancy outcome in cycles using luteal phase leuprolide acetate (Phelps et al., 1998
).
With increasing understanding of the control of synthesis and secretion of the inhibins and their potential endocrine role in the human menstrual cycle, attention has turned to the possibility that this family of peptides may provide a more direct index of ovarian reserve and improved predictors of assisted reproductive treatment outcome (Hughes et al., 1990; Balasch et al., 1996
; Lockwood et al., 1996
; Seifer et al., 1997
; Hall et al., 1999
). In the females, the pattern of secretion of the inhibins suggests that early follicular phase inhibin B may reflect the number of follicles present at baseline, whereas inhibin A may indicate follicle maturity (Groome et al., 1994
, 1996
; Hayes et al., 1998
). Also, a recent study investigating serial changes in plasma concentrations of inhibin A and inhibin B in women undergoing ovarian stimulation with FSH in down-regulated cycles of IVF treatment, showed that ovarian production of dimeric inhibin A and B are gonadotrophin dependent, and therefore these hormones were postulated as potential new markers for monitoring the effects of ovarian stimulation (Lockwood et al., 1996
).
On the above evidence, the present study was undertaken to investigate the usefulness of inhibin A, inhibin B and serum oestradiol concentrations obtained on the fifth day of gonadotrophin therapy in predicting ovarian response and assisted reproductive treatment outcome in women undergoing ovarian stimulation under pituitary desensitization. The fifth day of gonadotrophin therapy was used because ovarian stimulation is routinely started on Thursday in our assisted reproductive treatment programme.
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Materials and methods |
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All patients had both ovaries with no previous ovarian surgery and normal ovulatory function according to midluteal plasma progesterone concentrations and regular menses. For the specific purpose of this study all subjects had blood samples drawn on day 3 of their cycle within 3 months of the IVF/ICSI attempt for assay of basal inhibin B, FSH and oestradiol, as well as inhibin A and inhibin B determinations on the fifth day of gonadotrophin therapy which were measured at the completion of the study.
Stimulation regimen
Ovarian stimulation was carried out with FSH under pituitary suppression with gonadotrophin-releasing hormone (GnRH) agonist. In all women, pituitary desensitization was achieved by s.c. administration of leuprolide acetate (Procrin; Abbott Laboratories, Madrid, Spain) (1 mg daily, which was reduced to 0.5 mg after ovarian arrest was confirmed) started in the midluteal phase of the previous cycle. Gonadotrophin stimulation of the ovaries was started when serum oestradiol concentrations declined to <50pg/ml and a vaginal ultrasonographic scan showed an absence of follicles >10mm diameter. On days 1 and 2 of ovarian stimulation, six ampoules/day of highly purified FSH (Neo-Fertinorm; Serono SA, Madrid, Spain) were administered s.c. On days 3 and 4 of ovarian stimulation, two ampoules per day of FSH were administered to each patient. From day 5 onwards, FSH was administered on an individual basis according to the ovarian response. The criteria for human chorionic gonadotrophin (HCG) administration were the presence of two or more follicles >18 mm in diameter with 5 follicles measuring
14 mm in association with a consistent rise in serum oestradiol concentration. Oocyte aspiration was performed with vaginal ultrasonography 3536 h after HCG administration (5000 IU i.m.; Profasi; Serono SA). The maturational status of the oocytes was recorded according to the criteria of Veeck (1988). Up to four embryos per patient were replaced and the luteal phase was supported with additional doses of HCG. The cycle was cancelled when there were <3 follicles with diameter >14 mm after 89 days of gonadotrophin therapy or when requirements for HCG injection were not accomplished following 4 to 5 additional treatment days.
Hormone analyses
Hormones were measured using commercially available kits. FSH serum concentrations were measured by an immunoenzymatic assay with two monoclonal antibodies (Immuno 1; Technicon, Bayer, Tarrytown, NY, USA) and data are expressed in terms of International Reference Preparation (IRP) 78/549. The sensitivity of the assay was 0.1 IU/l and the interassay coefficient of variation was 2.7%. Oestradiol concentrations in serum were estimated by a competitive immunoenzymatic assay (Immuno 1; Technicon, Bayer). The sensitivity was 10 pg/ml and the inter-assay coefficient of variation was 5%. Dimeric inhibin B was measured by a solid-phase sandwich enzyme-linked immunosorbent assay which used two monoclonal antibodies (Serotec, Oxford, UK). The first monoclonal antibody was specific for the ßB subunit of inhibin; the second was directed to the -subunit and coupled to alkaline phosphatase. The assay utilized an immunopurified mixture of inhibin forms from human follicular fluid calibrated against recombinant 32 kDa inhibin B. The sensitivity of the assay was 15 pg/ml and the intra-assay and inter-assay coefficients of variation were <11% and 15%, respectively. 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 the Student's t-test, the MannWhitney U-test, and 2-test were used as appropriate. A multivariate logistic regression model was performed to assess the joint effect of variables and to estimate the sensitivity and specificity values for all possible combinations of the study variables. The discrimination attained between the study groups (cancelled versus punctured assisted reproductive treatment cycles, and pregnancy versus non pregnancy cycles) was evaluated with receiver-operating characteristic (ROC) analysis (Hanley and McNeil, 1982
; Zweig and Campbell, 1993
). Sensitivity, specificity, and the area under the ROC curve (AUCROC) which is a quantitative measure of accuracy, were obtained for each model. 95% confidence intervals (CI) were calculated for each of the estimates. The models' AUCROC values were compared using the method of Hanley and McNeil (1982). Areas of 1.0 and 0.5 denote no overlapping and no discrimination, respectively, between groups. Data were analysed by the Statistical Package for Social Sciences (SPSS; Chicago, IL, USA) and MedCalc Software (Mariakarke, Belgium).
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Results |
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Tables II and III show logistic regression analysis and individual probability estimates for basal FSH, oestradiol and inhibin B and serum concentrations of oestradiol, inhibin A and inhibin B on day 5 of gonadotrophin therapy. Logistic regression analysis showed that the association for day 5 inhibin B (with an accuracy, predictive value of ovarian response, of 91.03%; sensitivity, 94%; specificity, 81%) with cancellation rate was significant, independent of, and stronger than the effects of any other hormone variable (alone or in combination) investigated. The analysis of the impact of the remaining hormone variables (alone or in combination) within the groups defined by day 5 serum inhibin B values did not further delineate any group with significantly higher or lower chances of cancellation (data not shown). As presented in Table III
, however, day 5 inhibin B was not a better predictor of pregnancy than the other hormone variables studied on this day.
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Discussion |
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Normal baseline hormonal values, however, never guarantee that an endocrine system is functioning normally. Indeed, patients with baseline values in the normal range may have diminished ovarian reserve (Farhi et al., 1997; Kim et al., 1998
). Thus, a number of provocative tests have been devised to indirectly assess ovarian reserve and identify patients who might not be detected by basal hormone screening alone. These include the clomiphene citrate challenge test and the GnRH agonist stimulation test (Scott and Hofmann, 1995
). However, whether these indirect provocative tests are more informative of ovarian reserve than basal FSH remains unclear (Galtier-Dereure et al., 1996
). On the other hand, neither basal hormonal measurements nor such dynamic tests provide direct information concerning the responsiveness of the ovaries to the exogenous gonadotrophins used in ovarian stimulation for assisted reproductive treatment. In fact, recent studies propose the clinical response to gonadotrophin treatment as the more direct way to assess ovarian reserve and the better predictor of fertility potential (Roest et al., 1996
; Farhi et al., 1997
).
According to a recent report, oestradiol concentrations obtained shortly after the initiation of gonadotrophin therapy offer an easily assessable dynamic test of ovarian reserve in assisted reproductive treatment (Phelps et al., 1998). This was explained on the basis that oestradiol concentrations obtained early after the initiation of ovarian stimulation directly reflect follicular activity and ovarian responsiveness to the ongoing regimen of gonadotrophin stimulation (Phelps et al., 1998
). The present study confirms and expands that previous study (Phelps et al., 1998
) where neither basal hormone concentrations nor inhibins were investigated. According to the results of the current study, day 5 oestradiol is a better predictor of cancellation in assisted reproductive treatment cycles than basal FSH, inhibin B and oestradiol, as well as day 5 inhibin A serum concentrations. Day 5 inhibin B, however, seems to have a higher predictive value than day 5 oestradiol. Furthermore, day 5 inhibin B but not oestradiol was found to be directly correlated with variables of ovarian response and oocyte and embryological outcome, while day 5 inhibin A concentrations were correlated with the number of mature follicles observed in the late follicular phase. Therefore, it may be postulated that serum inhibin response to ovarian stimulation is a more sensitive and early index of declining ovarian function than oestradiol, and inhibin B measurements could enable more accurate monitoring of the ovarian response to gonadotrophin therapy in assisted reproductive treatment cycles. The following observations support this contention.
First, previous work indicating that ovarian inhibin and oestradiol production are controlled by different mechanisms (Hughes et al., 1990; Pellicer et al., 1994
). Second, recent investigations suggesting that the source of inhibin B secretion is different from that of inhibin A and oestradiol (Pellicer et al., 1994
; Welt et al., 1999b
). Third, recent studies using both GnRH-deficient patients and the administration of GnRH antagonist to normal cycling women showing that inhibin B secretion serves as the earliest index of FSH-dependent growth of immature follicles (Welt et al., 1997
, 1999b
). Fourth, FSH pharmacokineticpharmacodynamic studies demonstrating that serum inhibin is an early index of follicular development, being the first pharmacodynamic marker to increase (and also the first to decline when FSH concentrations decreased) as compared with oestradiol serum concentration and total follicular volume (Porchet et al., 1994
). Fifth, previous reports (Lockwood et al., 1996
; Anderson et al., 1998
) showing that inhibin B concentrations rose earlier and more markedly than inhibin A during gonadotrophin stimulation, thus indicating that inhibin B is a useful marker of follicular activity from smaller follicles at the time of recruitment and selection. In contrast, peak serum concentrations of inhibin A were reached in the peri-ovulatory phase when concentrations of inhibin B were already falling.
On the above evidence, it has been suggested that inhibin B may be more sensitive to the stimulus of FSH than inhibin A and oestradiol in the early phase of the menstrual cycle (Burger et al., 1998). Thus, the rise in serum inhibin B concentrations during ovarian stimulation with FSH is detectable before the increase in oestradiol and inhibin A and before follicular growth is evident on ultrasound (Lockwood et al., 1996
; Anderson et al., 1998
). Differences in the time course and peak values of those hormones would then explain the higher predictive properties of day 5 inhibin B measurement for ovarian performance in assisted reproductive treatment cycles. However, a second possibility exists, which is that day 5 inhibin B is a better predictor of follicular and oocyte outcome than day 5 oestradiol. It has been demonstrated that the decrease in inhibin B secretion is the earlier marker of declining reproductive potential which is linked to both quantitative and qualitative changes in oocytes (Seki et al., 1997
; Welt et al., 1999a
). The present study, in which aged-matched women were used and correlations between hormone measurements and variables of ovarian response and oocyte and embryological outcome were calculated, adds new evidence to the subject.
In the study by Phelps et al. (1998) oestradiol was found as an additional tool for predicting pregnancy. The results of the current study indicate that day 5 oestradiol, inhibin A and inhibin B have similar predictive properties on pregnancy outcome after assisted reproductive treatment. Although statistical significance was achieved with day 5 hormone measurements in the logistic regression analysis (Table III), the strength of the association between each predictor hormone and the presence or absence of the pregnancy, was very poor as indicated by odds ratios and 95% CI approaching unity as well as AUCROC indicating poor discrimination. Therefore, this is lacking clinical significance. In this respect it is to be noted that in the study by Phelps et al. (1998) patients becoming pregnant had higher oestradiol concentrations on the fourth day of gonadotrophin stimulation than women failing to become pregnant. However, the former group of patients was also significantly younger than the latter and age was not analysed as an independent risk factor for pregnancy in that study (Phelps et al., 1998
). Hormone measurements may actually be better predictors of ovarian response to stimulation than age in assisted reproductive treatment cycles, but age is a stronger predictor of pregnancy rate (Commenges-Ducos et al., 1998
; Sharif et al., 1998
; Hall et al., 1999
).
In conclusion, inhibin B concentrations obtained early in the follicular phase during ovarian stimulation under pituitary suppression for assisted reproductive treatment are highly predictive of ovarian response. As much as 86% of cycles having day 5 inhibin B 141 pg/ml underwent transvaginal oocyte retrieval as compared with only 7% of those cycles with day 5 inhibin B concentrations <141 pg/ml. This early assessment of ovarian stimulation response could help to decide early cancellation thus avoiding further cost and therapy. In addition, day 5 inhibin B values were correlated directly with oocyte and fertilization outcome although they were not useful in the prediction of pregnancy after assisted reproductive treatment. This may be due to the influence of factors such as endometrial response, which could not be controlled or assessed, a fact stressed in previous reports concluding that inhibin tests are helpful to evaluate ovarian function but are less useful in their prediction of the reproductive outcome (Hughes et al., 1990
; Corson et al., 1999
; Hall et al., 1999
).
Finally, as previously stressed by others (Galtier-Dereure et al., 1996; Corson et al., 1999
), considering that day 3 FSH determination has clinical relevance, is simple for the patient, inexpensive for the community, not time-consuming for the medical team and so easily repeatable, further work is warranted before widespread usage of the inhibin B assay which is technically challenging and not readily available. Thus, there is no international assay standard for inhibin B yet (both human recombinant and follicular fluid standards are being used) which may explain discordance between different studies and makes comparison of results between laboratories difficult.
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Notes |
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References |
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Submitted on December 29, 1999; accepted on April 7, 2000.