1 IVF Unit, Department of Reproductive Medicine and Science 2 Department of Paediatrics, Obstetrics and Gynaecology and 3 Department of Clinical Chemistry, Imperial College School of Medicine, Hammersmith Hospital, London, UK
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Abstract |
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Key words: GnRHa stimulation test/inhibin B/IVF/oestradiol/ovarian response
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Introduction |
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Prognostic assessment of ovarian reserve has relied upon indirect markers of ovarian function, including age (Schwartz and Mayaux, 1982), clomiphene citrate challenge test (Navot et al., 1987
), basal follicle stimulating hormone (FSH) concentrations on day 2 or 3 of the menstrual cycle (Scott et al., 1989
; Toner et al., 1991
), FSH:luteinizing hormone (LH) ratio (Mukherjee et al., 1996
; Kim et al., 1997
), basal oestradiol concentration (Licciardi et al., 1995
) and ovarian volume (Lass et al., 1997
; Syrop et al., 1999
). There are limited data on direct measurement of ovarian reserve.
Inhibin A and inhibin B are dimeric polypeptides produced by granulosa cells. Inhibin A is secreted predominantly in the luteal phase and inhibin B secreted predominantly in the follicular phase (Groome et al., 1996). Inhibin B has been suggested as a direct biochemical marker of ovarian reserve (Balasch et al., 1996
; Klein et al., 1996
; Seifer et al., 1999
), and may prove to be a useful adjunct in discriminating among patients who are likely to respond to exogenous ovarian stimulation (Hall et al., 1999
). Seifer et al. (1999) found that declining ovarian reserve might be demonstrated by a decrease in basal inhibin B concentrations before a rise in basal FSH concentrations.
It has been proposed that a dynamic evaluation of an endocrine response may provide a more useful assessment than a basal hormone measurement (Ranieri et al., 1998). The administration of gonadotrophin-releasing hormone analogue (GnRHa) elicits an initial surge in LH, FSH and oestradiol. The early rise in oestradiol concentrations has been evaluated as a prognostic indicator of ovarian reserve (Padilla et al., 1990
; Winslow et al., 1991
; Avrech et al., 1996
; Galtier-Dereure et al., 1996
; Ranieri et al., 1998
). Ranieri et al. (1998) found that the increase in oestradiol concentration after GnRHa administration (oestradiol concentration on day 3 minus oestradiol concentration on day 2) (increase in oestradiol) was a better predictor than age, basal FSH concentration and FSH:LH ratio.
Plasma concentrations of inhibin B rise rapidly in the early follicular phase (Groome et al., 1996) and respond to exogenous FSH administration in the follicular phase (Burger et al., 1998
). The early response of inhibin B to ovarian stimulation may reflect the ovarian reserve and may help in predicting the ovarian response to stimulation during treatment. This study was designed to identify prospectively the value of basal and dynamic tests of inhibin B and oestradiol in predicting ovarian response to stimulation.
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Materials and methods |
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For patients undergoing their first IVF cycle the starting dose of rFSH was 150 IU daily in patients <36 years of age. Patients between 36 and 40 years old were started on 225 IU. Patients over 40 years old were started on 300 IU. Patients with ultrasound findings consistent with polycystic ovaries (PCO) (Adams et al., 1985) were started on half of the above doses. For patients who had a previous IVF cycle the starting dose of rFSH depended on the dose required in the previous cycle. Plasma oestradiol concentrations were measured 5 days after starting rFSH and then daily from the eighth day. The first scan was done on day 9 and then every 2 or 3 days as required. The dose of rFSH was adjusted from day 5 of stimulation according to ovarian response.
Human chorionic gonadotrophin (HCG) (10 000 IU) (Profasi®; Serono) was given only when at least three follicles had a mean follicular diameter 17 mm and plasma oestradiol concentration was >3000 pmol/l. Egg collection was performed ~36 h after the HCG injection and embryo transfer was done 23 days later. Two embryos were transferred unless there were adverse features. Adverse features included treatment of older age women, poor embryo morphology or multiple failed cycles. In those circumstances three embryos were transferred. Progesterone was given for 12 days after embryo transfer and then serum HCG was measured. A clinical pregnancy was confirmed by transvaginal scan 2 weeks later. Cycles were cancelled if the ovarian response was poor or excessive. Criteria for poor response were <3 follicles with a mean diameter
17 mm and/or plasma oestradiol <3000 pmol/l, or at an earlier stage of stimulation, poor ovarian response despite a high dose of gonadotrophins. Criteria for excessive response were oestradiol >15 000 pmol/l, or at an earlier stage of stimulation, development of a high number of follicles (over 30).
Oestradiol, FSH and LH were measured using the microparticle enzyme immuno-assay by the AXSYM system (Abbott Laboratories, Maidenhead, Berks, UK). LH and FSH were measured using the World Health Organization second international standard.
For the LH, FSH and oestradiol assays, the limits of assay detection were 0.5 IU/l, 0.37 IU/l and 73 pmol/l respectively, and the interassay coefficients of variation (CV) for the range of concentrations measured were 9.512.3%, 5.18.3% and 6.19.2% respectively. No intra-assay CV was calculated since these assays were automated and performed in the same appliance.
Inhibin B was analysed in duplicate using a double-antibody enzyme-linked immunosorbent assay (ELISA, Serotec, Kidlington, Oxford, Oxon, UK) and performed according to the manufacturer's protocol. The limit of assay detection was 15 pg/ml and the interassay and intra-assay CV for the range of concentrations measured were 6.110.1% and 5.86.5% respectively. The specificity and validity of the inhibin B assay has been previously reported (Groome et al., 1996).
Statistical analysis included Pearson product moment correlation coefficient (r) to examine the associations between variables. The coefficient of determination (r2) was calculated using linear regression analysis. Multiple linear regression analysis was used to determine the effect of multiple continuous variables. It should be noted that related variables were not used in the same model. Transformation of non-Gaussian parameters was performed when indicated. Values of P < 0.01 were considered significant. Analysis was performed using STATA 6 statistical package (Statacorp 1999, College Station, TX, USA).
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Results |
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Table I represents parameters related to the GnRHa stimulation test and ovarian response. Table II
demonstrates the accuracy of different parameters in predicting ovarian response. The ovarian response was defined in two ways: `number of oocytes/total rFSH dose'; and `square root (number of follicles/total rFSH dose)'. The best predictor in both cases was the dynamic assay detecting the increase in oestradiol concentrations between day 3 and day 2 (increase in oestradiol). Other variables that were significantly predictive in both groups were: oestradiol concentration on day 3, inhibin B concentrations on day 3 and the sum of inhibin B concentrations on day 2 and day 3 (sum of inhibin B). The commonly employed basal FSH concentration was significantly correlated with these parameters of ovarian response; however, the correlation was weaker. Age performed poorly as a predictive test. The correlation between increase in oestradiol and sum of inhibin B was very high with correlation coefficient of 0.69 (P < 0.0001).
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Discussion |
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It would also be extremely useful to have a test that predicts ovarian response and which could be used to determine the optimal gonadotrophin dose for an individual patient. Age and/or basal FSH concentrations have been well studied and found to be simple, inexpensive and easily applied (Schwartz and Mayaux, 1982; Scott et al., 1989
; Toner et al., 1991
; Scott and Hofmann, 1995
). However, even where age and basal FSH concentrations are evaluated routinely, a number of poor or high responder patients remain undetected, justifying a more accurate reliable prognosticator of ovarian reserve.
Ovarian response to gonadotrophin stimulation needs to be defined and quantified. Ovarian response may be manifest by the number of follicles, number of oocytes collected and the peak oestradiol concentration. The response is also related to the dose of gonadotrophin used. As patients use different daily doses of gonadotrophins for varying lengths of time, the total dose of gonadotrophins is considered when analysing the ovarian response. The parameters of the number of follicles and the number of oocytes (both divided by the total rFSH dose) were more comparable with different predictors of ovarian response than the peak oestradiol concentration. Therefore these two parameters of ovarian response were used.
Although the clinical pregnancy rate of 26.5% per embryo transfer is acceptable, the small number of pregnancies obtained is too small to allow for any meaningful statistical analysis. In addition only one patient was cancelled due to poor response and there were no cases of OHSS. It is therefore not possible to analyse the predictive value of these tests for cancellation rate in this series.
The current results agree with those of Ranieri et al. (1998) who found that increase in oestradiol following a GnRHa stimulation is the best single predictor of ovarian response and is superior to the parameters of age, basal FSH concentration, basal oestradiol concentration and FSH:LH ratio.
Basal concentrations of inhibin B have previously been found to be predictive of ovarian response in IVF treatment (Seifer et al., 1997; Hall et al., 1999
). However, the opinion is split regarding its value in predicting treatment outcome. Seifer et al. (1997) studied 156 women who underwent 178 cycles of IVF treatment. The adjusted odds ratio for clinical pregnancy for women with day 3 inhibin B >45 pg/ml versus those with inhibin B <45 pg/ml was 6.8. In a comparison between women who achieved pregnancy and women who failed to achieve pregnancy within three IVF cycles, Hall et al. (1999) found a higher mean inhibin B concentration in the pregnant women group. However, the difference was not significant and did not provide additional information over age and number of oocytes in predicting successful outcome. Corson et al. (1999) investigated whether FSH and inhibin B concentrations before and after clomiphene citrate administration predict outcome after various fertility treatments (surgery, ovulation induction and intrauterine insemination). They found that patients with inhibin B concentration below or above 45 pg/ml had the same pregnancy rate. They did not investigate whether inhibin B predicts the ovarian response to gonadotrophin administration and did not look at patients undergoing IVF treatment. It should be taken into consideration that there is no international assay standard for inhibin B yet, so comparisons of absolute inhibin B values between different studies remain a problem.
This study represents a novel approach using a dynamic assay for inhibin B. The predictive value of the dynamic test is superior to the basal inhibin B concentration in this study. It is also more accurate than basal concentration of FSH, basal oestradiol concentration, FSH:LH ratio and age.
Both oestradiol and inhibin B are produced by granulosa cells. When measuring the basal concentrations of these hormones in the early follicular phase different concentrations are considered as predictors of ovarian reserve. Higher inhibin B concentration predicts better ovarian reserve (Seifer et al., 1997); in contrast higher basal oestradiol concentration predicts lower ovarian reserve (Licciardi et al., 1995
). When gonadotrophin administration is commenced the secretion of both oestradiol and inhibin B increases and their serum concentrations rise (Hughes et al., 1990
).
The current data suggest that the dynamic assays of oestradiol and inhibin B have similar predictive properties for ovarian response to gonadotrophin stimulation (with oestradiol being slightly more accurate). Combining oestradiol and inhibin B slightly improves the power of prediction compared with using oestradiol alone. However, measuring oestradiol is much simpler and cheaper than measuring inhibin B and it seems that for clinical practice measuring only oestradiol in a dynamic test is reliable enough.
The GnRHa stimulation test can be used as an adjuvant to an IVF treatment cycle when GnRHa is started in the early follicular phase either in long or short protocol (Winslow et al., 1991; Avrech et al., 1996
; Ranieri et al., 1998
). When treatment is started in the mid-luteal phase the test can be performed before treatment.
Performing a GnRHa stimulation test allows for the accurate prediction of ovarian response to stimulation. Patients with diminished ovarian reserve may benefit from stimulation with a higher dose of gonadotrophin or possibly a modification in GnRHa administration. Conversely, patients with excessive response to GnRHa administration may benefit from a reduction in the gonadotrophin dose and closer follow-up during stimulation.
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Notes |
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References |
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Submitted on April 5, 2000; accepted on July 10, 2000.