1 Department of Reproductive Medicine, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, 2 Department of Internal Medicine and 3 Center of Reproductive Medicine, Erasmus MC, Dr. Molewaterplein 40, 3015 GD Rotterdam, The Netherlands
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Abstract |
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Key words: anti-Müllerian hormone/antral follicle count/IVF/ovarian reserve/poor response
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Introduction |
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Human female serum contains measurable amounts of AMH during the reproductive life span (Lee et al., 1996). Since AMH is solely produced in the growing ovarian follicles, serum levels may be used as a marker for ovarian reserve, representing the quantity and quality of the ovarian follicle pool (te Velde and Pearson, 2002
). Recent preliminary reports indeed indicate that AMH levels decline with increasing female age (de Vet et al., 2002
) and that initial AMH is associated with ovarian response in IVF patients with normal FSH levels (Seifer et al., 2002
).
In the present study we prospectively assessed the significance of AMH as a marker for ovarian response in a large unselected IVF population. In addition, the predictive performance of serum AMH levels towards poor response in relation to other ovarian reserve tests was investigated. Finally, as little is known concerning the regulation of AMH production in the human, we also investigated whether serum AMH levels are affected by a rise in endogenous FSH and LH induced by a single, high dose GnRH agonist administration.
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Subjects and methods |
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On day 3 of a spontaneous cycle within the 3 months preceding IVF treatment, patients underwent a transvaginal ultrasound examination to assess the number of antral follicles, measuring 25 mm, as described previously (Bancsi et al., 2002). On the same day a venous blood sample was obtained for the measurement of AMH, FSH, estradiol (E2) and inhibin B. Serum and plasma samples were centrifuged at 1700 g within 2 h and stored at 20°C until assayed.
In a subset of 23 patients a GnRH agonist stimulation test (GAST) was performed. These patients received 0.1 mg triptorelin (Decapeptyl®, Ferring, Hoofddorp, The Netherlands) s.c. directly after blood sampling on cycle day 3 and returned exactly 24 h later for a second blood sampling for measurement of AMH, FSH, E2 and inhibin B.
FSH and E2 were assessed in plasma with the AxSYM immunoanalyser (Abbott Laboratories, Abbott Park, IL, USA). The World Health Organization Second International Reference Preparation for human FSH (78/549) was used as a standard in the FSH assay. For FSH, interassay coefficients of variation were found to be 6.0, 6.6 and 8.0% at the levels of 5, 25 and 75 IU/l respectively (n = 46). The E2 assay is standardized to gas chromatography/mass spectrometry. Interassay variation of the E2 assay at 300, 1105 and 2626 pmol/l was 12.5, 7.5 and 4.9% (n = 29) respectively. Serum inhibin B levels were measured using an immuno-enzymometric assay (Serotec, Oxford, UK) as described by Groome et al. (Groome et al., 1996). Intra- and interassay coefficients of variation were <14.6 and <14.0% respectively. An ultra-sensitive immuno-enzymometric assay kit (Immunotech-Coulter, Marseille, France) was used for the estimation of AMH as described elsewhere (Long et al., 2000
). The limit of detection (defined as blank + 3 SD of blank) was 0.05 µg/l. Intra- and interassay coefficients of variation were <5 and 8% respectively. If a GAST was performed both samples were analysed in the same run.
The IVF treatment has been described in detail in a previous publication (van Kooij et al., 1996). In brief, patients started with leuprolide acetate (Lucrin®; Abbott, Hoofddorp, The Netherlands) in the midluteal phase to achieve pituitary desensitisation. After menstruation, the ovarian hyperstimulation started with a fixed dose protocol of 150 IU follitropin alpha (rFSH, Gonal-F®; Serono Benelux BV, The Hague, The Netherlands). After 7 days follicular growth was assessed by ultrasound and E2 measurement. If necessary the dose of rFSH was adjusted. When at least three leading follicles developed, 10 000 IU hCG, (Profasi®; Serono Benelux) was administered and 36 h later oocyte retrieval was performed. A maximum of two embryos was transferred in women <38 years of age. Above this age a maximum of three embryos was transferred. To support the luteal phase, either hCG (Profasi®) or micronized progesterone (Progestan®; Nourypharma BV, Oss, The Netherlands) was used.
The main outcome measures of the study were the number of oocytes retrieved and poor ovarian response. As described previously (Bancsi et al., 2002), poor response was defined as fewer than 4 oocytes at follicle puncture or as cancellation due to impaired (fewer than 3 follicles) or absent follicular growth in response to ovarian hyperstimulation. With a mean fertilization rate of 5060% a minimum of four oocytes is necessary to transfer at least two embryos. In the analysis of poor ovarian response, the group of normal responders also included patients with cancelled cycles due to an exaggerated response. Patients were considered high responders in case of collection of more than 20 oocytes at ovum retrieval or when the cycle was cancelled due to exaggerated response (more than 30 follicles in both ovaries and/or peak E2 >15 000 pmol/l). High response was considered a secondary outcome measure, and in the analysis of high response both the poor and normal responders are considered as one group.
Another secondary outcome measure was ongoing pregnancy, defined as a viable pregnancy assessed by ultrasound of at least 11 weeks gestation. Data from patients whose cycles were cancelled due to either risk of ovarian hyperstimulation syndrome (OHSS) or poor response (fewer than 3 follicles) to hormone stimulation were not included in the pregnancy analysis, because it cannot be excluded that such patients would have become pregnant if IVF were performed. However, patients with complete absence of follicle growth and E2 <200 pmol/l were considered to have a zero chance of pregnancy, and therefore data were on their cycles was included in the analysis of pregnancy.
Data were analysed with the Statistical Program for Social Sciences (SPSS Inc., Chicago, IL, USA). Values are presented as median and range. To compare normal with poor responders the MannWhitney test or 2-test was performed whenever appropriate. The correlation between different parameters is expressed as Spearmans correlation coefficient. Univariate and multivariate logistic regression with the main outcome measure poor response and secondary outcome measures of high response and ongoing pregnancy were performed. For each single variable used in the univariate analysis and for the models, the ability to discriminate between patients with a poor response and patients with a normal response was assessed by calculating the area under the receiver operating characteristics curves (ROCAUC) (Harrell et al., 1996
). The ROCAUC may vary between 0.5 (no discriminative power) to 1.0 (perfect discrimination). For the comparison of E2, inhibin B and AMH before and after the GAST a Wilcoxon signed rank test was used. Statistical significance was considered to be reached at P-value <0.05.
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Results |
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Patient and ovarian reserve test characteristics of the complete group and of normal and poor responders separately are presented in Table I. The 35 poor responders were somewhat older, and their AMH, FSH and inhibin B levels and antral follicle counts were statistically different from those in the 84 normal responders. As expected, patients with a poor response were more often treated for unexplained infertility. The number of oocytes retrieved in the normal and poor responders groups are also depicted in Table I
. While seven patients in the normal response group had their ovum retrieval cancelled because of the risk of OHSS, no ovum retrieval took place in 16 poor responders because of insufficient follicle growth (02 follicles). Normal responders showed higher median peak E2 levels during ovarian stimulation compared with poor responders [6695 (202718 300) pmol/l versus 1354 (1006570) pmol/l ; P < 0.001].
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To obtain further support for the notion that AMH is a measure of the ovarian follicle population and not of the gonadotrophic hormonal status of the patient, we performed a GAST in a subset of 23 patients. In these patients a significant rise of E2 and inhibin B values was observed as a response to endogenous FSH and LH elevation after s.c. injection of GnRH agonist, whereas no changes in AMH levels were seen (Table III).
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Discussion |
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Ovarian reserve comprises two elements: the size of the stock of primordial follicles and the quality of the oocytes (te Velde and Pearson, 2002). From the primordial follicle pool, primary follicles will start a maturation process and develop through secondary (preantral) follicles into the pool of antral follicles from which the monthly follicle to be ovulated is selected (Fauser and Van Heusden, 1997
). In rodents, AMH is produced immediately after the transition of primordial to primary follicles, and AMH expression disappears when follicles either are selected for ovulation or become atretic (Baarends et al., 1995
). In the human ovary, AMH protein expression is also seen in granulosa cells of follicles from the primary stage up to the larger antral stage when follicles have gained FSH dependence (Rajpert-De Meyts et al., 1999
). Since the size of the primordial follicle stock is difficult to measure directly, a marker that reflects all numbers of follicles that have made the transition from the primordial follicle pool to the growing pool may be a good indirect measurement. AMH might be such a marker, as it is involved in the regulation of primordial follicle recruitment (Durlinger et al., 1999
), an important mechanism for the depletion of the primordial follicle pool (Gougeon, 1996
) and it is produced by all follicle stages until FSH-dependency.
The relative contribution of the different follicle classes to the final serum level is unclear. Although antral follicles of 25 mm may produce more AMH as a reflection of their high granulosa cell number, the smaller size follicles may also contribute significantly to serum AMH on the basis of their larger number. The higher serum AMH levels in PCOS patients also cannot provide an answer to this issue, as their ovaries contain about twice as many preantral (primary and secondary) follicles and antral follicles (Hughesdon, 1982; Cook et al., 2002
).
There are indications that the production or secretion of AMH by granulosa cells is not under a stringent extraovarian hormonal control. In transgenic mice all follicle parameters in heterozygous AMH (/+) animals lie in-between those of the wild type AMH (+/+) and null AMH (/) animals, indicating that the production of AMH rather depends on the activity of the gene itself, and is not regulated by gonadotrophic hormones (Durlinger et al., 1999). In the present study we have obtained further support for this notion, since AMH levels did not change in response to an acute endogenous rise in FSH and LH (GAST). As expected, production of E2 and inhibin B are under direct regulation of FSH as indicated by the changes after GnRH agonist administration (Winslow et al., 1991
; Elting et al., 2001
). Possibly, a longer duration of FSH stimulation leads to an increase in the number of granulosa cells, with a concomitant rise in AMH production, although longer stimulation may also result in selection of follicles for dominance with concomitant decline in AMH production. In all, the GAST results support the candidacy of AMH as a marker of the growing follicle population.
The available tests for assessment of ovarian reserve all reflect, directly or indirectly, the size of the antral (25 mm) follicle pool. AFC is a direct ultrasound measure of this pool, whereas the initial inhibin B and E2 levels are considered to be greatly dependent on the number of antral follicles in the early menstrual days. FSH levels are regulated by a negative feedback action of these two granulosa cell products and hence are a more indirect reflection of antral follicle number.
We have recently shown that AFC gives the best prognostic information with regard to the occurrence of poor response in IVF (Bancsi et al., 2002). Moreover, the combination of the three ovarian reserve tests, AFC, inhibin B and FSH, in a multivariate logistic model appeared to improve the response prediction. In the present study AMH is found to have a predictive performance comparable with that of AFC. The multivariate analysis on the prediction of poor ovarian response to hyperstimulation in the study revealed that AMH will contribute independently to this prediction but only if AFC is removed from the analysis. Obviously, the high interrelation between the follicle numbers in earlier stages and the antral follicle number prohibits any additional information to be obtained from this test. Both models need external validation, which is currently being performed. In the prediction of high response, comparable discriminative performance was found for the variables AFC and AMH, supporting the close relationship between these variables and ovarian response.
The present study confirms and extends results of a recent study where lower serum AMH levels were found in patients having six or fewer retrieved oocytes compared with patients having 11 or more oocytes (Seifer et al., 2002). We studied a group of unselected patients having their first IVF treatment with a fixed dose of gonadotrophins. Therefore we could compare AMH as a predictor of ovarian reserve with the other endocrinological markers and AFC.
There are advantages of the use of AMH over AFC in the multivariate model for the prediction of ovarian response, since all predictive information is obtained with blood sampling and no extra ultrasound is needed. Furthermore, since there is no change in AMH levels in response to gonadotrophins, AMH can be measured throughout the cycle in contrast to the other parameters, which can only be determined during the early follicle phase, an advantage for both patients and clinicians. Obviously, AMH intra-cycle and cycle-to-cycle variation should be further analysed, but the small fluctuation in serum AMH levels at three different time points during the menstrual cycle (Cook et al., 2000), supports the feasibility of AMH assessment throughout the cycle.
Application of AMH (this study), AFC (Bancsi et al., 2002), inhibin B (Seifer et al., 1997
; Hall et al. 1999
) and FSH (Sharif et al., 1998
; Bancsi et al., 2000
) as a predictor of ongoing pregnancy appears to be limited in view of the fact that they only represent the quantitative aspect of ovarian reserve, whereas pregnancy is also dependent on the oocyte quality. Furthermore, it is also possible for patients with normal ovarian reserve not to become pregnant, for instance as a result of fertilization failure. Nevertheless, the ability to predict poor response may be a valuable tool for patient counselling, since poor responders have a lower probability of pregnancy. A further potential application for the prediction of poor response is the augmentation of the starting dose of gonadotrophins in predicted poor responders. It is not certain that this may lead to higher pregnancy rates (Land et al., 1996
), but randomized prospective data on this issue are still lacking. Also, patients normally denied IVF treatment because of advanced age might profit from response prediction. As patients >40 years of age have a better outcome in the case of a normal response (Roest et al., 1996
), those patients with a normal response prediction can proceed to treatment.
In conclusion, we have found that AMH serum levels are associated with ovarian response in IVF patients and may serve as a novel marker for ovarian reserve. The predictive value of AMH for poor ovarian response is comparable with that of AFC and therefore AFC could be replaced by AMH in the prediction of ovarian response to controlled ovarian hyperstimulation in IVF.
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Notes |
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References |
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Submitted on June 7, 2002; accepted on August 1, 2002.