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
3 To whom correspondence should be addressed at: Institut Clínic of Gynecology and Obstetrics, Hospital Clínic, C/Casanova 143, 08036 Barcelona, Spain. Email: jbalasch{at}ub.edu
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Abstract |
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Key words: AMH/IVF/low responders/ovarian reserve/ovarian response
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Introduction |
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Many tests have been developed to screen for diminished ovarian reserve. Traditional methods used to predict prospectively response to ovarian stimulation have included mainly the measurement of baseline cycle day 3 serum concentrations of hormones such as FSH, estradiol and inhibins, or ultrasonographic tests such as pretreatment ovarian volume and the number of early antral follicles (Bukman and Heineman, 2001). On their own, however, normal baseline values are not a guarantee that an endocrine organ is functioning normally, and non-response to ovarian stimulation in normogonadotropic, normogonadal women has been reported (Wallach, 1995
; Farhi et al., 1997
). As recently stressed (Tarlatzis et al., 2003
), despite the plethora of predictive tests for low ovarian response, the poor responder is revealed definitely only during ovarian stimulation. Therefore, the ability to predict ovarian response early in the course of controlled ovarian hyperstimulation would provide the opportunity of obtaining essential information to assist in deciding whether to proceed with an ongoing cycle. Recent studies by us (Peñarrubia et al., 2000
) and others (Phelps et al., 1998
; Eldar-Geva et al., 2000; Fawzy et al., 2002
) have addressed this issue and concluded that estradiol and inhibin B levels obtained after 34 days of gonadotropin therapy rather than baseline values were highly predictive of ovarian response in assisted reproduction treatment cycles.
Recently, anti-Müllerian hormone (AMH), also referred to as Müllerian-inhibiting substance, has been proposed as a novel marker for predicting ovarian response to gonadotropin stimulation (Seifer et al., 2002; van Rooij et al., 2002
; Gruijters et al., 2003
). AMH is a member of the transforming growth factor
superfamily of growth and differentiation factors. It was identified as a factor which, being synthesized by testicular Sertoli cells, induces regression of the Müllerian ducts during male fetal development. In females, AMH is only expressed by the ovary, and mRNA studies in rat and mouse species revealed specific expression of AMH in granulosa cells of early growing, preantral and small antral follicles but not in non-atretic large antral follicles and all atretic follicles (Baarends et al., 1995
; Gruijters et al., 2003
). It has been shown that AMH affects two important regulatory steps during folliculogenesis in female mice (Durlinger et al., 2002
; Gruijters et al., 2003
). At initial recruitment, AMH inhibits recruitment of primordial follicles into the growing pool, whereas at cyclic recruitment AMH lowers the FSH sensitivity of follicles. In these ways, AMH plays an essential role in regulating ovarian follicular growth in rodents. Notably, very recent data indicate that AMH expression in the human follows a similar pattern as compared to the mouse and rat, thus suggesting important roles for AMH in human folliculogenesis (Weenen et al., 2004
).
It has been shown that human female serum contains measurable levels of AMH during the reproductive life (Lee et al., 1996). AMH serum levels decline with increasing female age in normo-ovulatory women (de Vet et al., 2002
) and are more strongly correlated with the number of early antral follicles than the usual hormone markers such as FSH, LH, estradiol and inhibin B on cycle day 3 (Fanchin et al., 2003a
). Thus, AMH has been proposed as a marker for ovarian ageing (de Vet et al., 2002
; van Roij et al., 2002
; Fanchin et al., 2003a
). In fact, poor ovarian response to controlled ovarian hyperstimulation in assisted reproduction cycles, which is considered as indicative of ovarian ageing (Beckers et al., 2002
), has been demonstrated to be associated with reduced early follicular phase AMH serum levels (Seifer et al., 2002
; van Rooij et al., 2002
; Fanchin et al., 2003b
).
On the above evidence, the present study was undertaken to investigate the usefulness of baseline cycle day 3 AMH levels and AMH serum concentrations obtained on the fifth day of gonadotropin therapy in predicting ovarian response and pregnancy in women undergoing ovarian stimulation under pituitary desensitization for assisted reproduction. The fifth day of gonadotropin therapy was used because ovarian stimulation is routinely started on Thursday in our assisted reproduction programme and the first evaluation of the ovarian response is performed on Monday (i.e. after 4 days of gonadotropin treatment).
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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. In our assisted reproduction programme, basal FSH, LH and estradiol serum levels are routinely measured in the early follicular phase within the 3 months preceding IVF/ICSI treatment, and estradiol serum concentrations on the fifth day of gonadotropin therapy are routinely used to evaluate ovarian response. For the specific purpose of this study all subjects had serum AMH determinations on day 3 of their cycle within 3 months of the IVF/ICSI attempt and on the fifth day of gonadotropin therapy during the IVF/ICSI index cycle, which was measured on completion of the study in frozen blood samples.
Stimulation regimen
All patients received standard ovarian stimulation with FSH under pituitary suppression with GnRH agonist, according to a protocol previously reported (Peñarrubia et al., 2003). In all women, pituitary desensitization was achieved by s.c. administration of triptorelin acetate (Decapeptyl 0.1 mg; Ipsen Pharma, Barcelona, Spain; 0.1 mg daily, which was reduced to 0.05 mg after ovarian arrest was confirmed) started in the mid-luteal phase of the previous cycle. Gonadotropin stimulation of the ovaries was started when serum estradiol concentrations declined to <50 pg/ml and a vaginal ultrasonographic scan showed an absence of follicles >10 mm diameter. On days 1 and 2 of ovarian stimulation, 450 IU and 300 IU/day of recombinant human FSH (Gonal-F; Serono, Madrid, Spain), respectively, were administered subcutaneously. On days 3 and 4 of ovarian stimulation, 150 IU 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 estradiol measurements. The criteria for human chorionic gonadotropin administration (recombinant human HCG; 250 µg) (Ovitrelle; Serono) were the presence of two or more follicles >18 mm in diameter with
4 follicles measuring
14 mm in association with a consistent rise in serum estradiol concentration. 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, 1999
); embryos of Veeck grades 1 or 2 were considered high quality. Up to three embryos per patient were replaced and the luteal phase was supported with vaginal micronized progesterone. The cycle was cancelled when there were <3 follicles with diameter
14 mm after 89 days of gonadotropin therapy (early cancellation) or after 45 additional treatment days without attaining, or the imminent prospect of attaining, the criteria for HCG administration (late cancellation).
Pregnancy was diagnosed by increasing serum concentrations of -HCG after embryo transfer, and the subsequent demonstration of an intrauterine gestational sac by ultrasonography.
Hormone analyses and ultrasonography
Blood samples were drawn between 08:0010:00 h and processed within 2 h after withdrawal. For this study two serum aliquots were obtained. FSH, LH and estradiol were measured in one of the serum aliquots for clinical monitoring, and the second aliquot was stored at 20 °C for later measurement of AMH. Frozen serum samples from each patient for AMH measurement were examined in one run within 6 months of collection.
FSH, LH, estradiol and AMH in serum were measured using commercially available kits as reported previously (Balasch et al., 2001; Peñarrubia et al., 2003
; Pigny et al., 2003
). Estradiol concentrations in serum were estimated by a competitive immunoenzymatic assay (Immuno 1, Technicon; Bayer, Tarrytown, NY). 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 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. Total
-HCG was measured by a solid-phase, two-site chemiluminiscent enzyme immunometric assay standardized against the Third International Standard 75/537 (Immulite, Diagnostic Products Co., Los Angeles, CA) with a detection limit of 2 IU/l. The inter-assay CV was 5.8%. Serum AMH levels were determined in duplicate using an ultrasensitive enzyme-linked immunosorbent assay (ELISA) (Immunotech-Coulter, Marseilles, France) according to the supplier's instructions. Results are expressed in pmol/l using human recombinant AMH as a standard. The detection limit of this assay using the ultrasensitive protocol is 0.7 pmol/l. Intra- and inter-assay coefficients of variation were <5.5% and <9%, respectively.
Ultrasonic scans were performed using a Toshiba Eccocee SAA-340A/EF unit (Toshiba Co., Tokyo, Japan) equipped with a 57 MHz endovaginal probe (PVF-641VT).
Statistics and probability testing
For statistical analysis the MannWhitney U test, the Wilcoxon matched-pairs signed ranks test and 2-test were used as appropriate. Results are expressed as mean ± standard error of mean (SEM). P < 0.05 was considered significant. The discrimination attained between two study groups (cancelled vs non-cancelled cycles, and conception vs non-conception cycles) was evaluated with receiver-operating characteristic (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 cut-off 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. AMH serum concentrations) to discriminate between two conditions (e.g. cancelled vs punctured cycles). Sensitivity, specificity and the AUCROC 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). 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. Thus, an AUCROC whose CI includes 0.5 means no discrimination. A parameter with no overlap between the two conditions will discriminate perfectly and has a 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 analyzed by Statistics Package for Social Sciences (SPSS version 10.0, Chicago, IL).
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Results |
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To analyze the diagnostic accuracy of both basal and day 5 AMH and determinations to discriminate between cancelled versus control cycles and pregnancy versus non-pregnancy cycles, the AUCROC values determined with ROC analysis for each hormone measurement are shown in Table II. The AUCROC for day 5 AMH in predicting the likelihood of cancellation in an assisted reproduction treatment programme was significantly higher than that for basal AMH measurement. The ROC curve analysis was used to determine the best threshold values for day 5 AMH serum concentrations in predicting ovarian response (Figure 1). The best criterion value discriminating between control and cancelled cycles was
4.9 pmol/l (sensitivity 53%, specificity 96%). Diagnostic accuracy of estradiol serum concentration measured on stimulation day 5 to discriminate between cancellation and no cancellation was similar to that afforded by day 5 AMH measurement (Table II). When the likelihood of pregnancy was analyzed, the AUCROC for basal AMH was similar to that for day 5 AMH, and none of them was useful in their prediction of the reproductive outcome (Table II; Figure 1). Similarly, basal and day 5 estradiol serum levels were not discriminatory for pregnancy and non-pregnancy cycles.
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Discussion |
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Traditional methodology used to assess ovarian reserve has consisted of baseline serum levels of hormones such as FSH, estradiol and inhibins, and chronological age (Scott and Hofmann, 1995; Sharara and Scott, 1997
; Karande and Gleicher, 1999
; Bukman and Heineman, 2001
). Also, 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 (Scott and Hofmann, 1995
; Sharara and Scott, 1997
; Bukman and Heineman, 2001
). However, neither basal hormone measurements nor such dynamic tests provide direct information concerning the responsiveness of the ovaries to exogenous gonadotropins used in ovarian stimulation for assisted reproductive treatment. Thus, despite the validity of all these tests, there still remain patients who respond poorly to stimulation despite having normal tests of ovarian reserve. This supports the idea that ovarian reserve is not a simple static anatomic number of follicles but rather a dynamic process, the mechanism of which is not yet fully understood (Lass, 2001
). In fact, it has been recently stressed that the ideal ovarian reserve test is the response of the ovaries to a normal or standard ovarian stimulation protocol (Karande and Gleicher, 1999
; Tarlatzis et al., 2003
). Therefore, an early marker of ovarian responsiveness after the initiation of gonadotropin therapy would assist in deciding whether to proceed with an ongoing cycle. Ultimately, this will decrease the cost of continued monitoring and medication for patients in whom therapy will most likely fail.
The current study shows for the first time that AMH serum concentration obtained in the early follicular phase during ovarian stimulation with gonadotropins under pituitary suppression for assisted reproductive treatment is a better predictor of cancelled cycle than basal AMH. Thus, this report adds new data to recent work proposing cycle day 3 AMH measurement as a new marker for ovarian ageing and poor ovarian response to gonadotropin therapy in assisted reproduction cycles (de Vet et al., 2002; Seifer et al., 2002
; van Roij et al., 2002
; Fanchin et al., 2003a
,b) and supports the idea that dynamic tests seem better predictors of ovarian response than basal testing (Bukman and Heineman, 2001
). A feature of the present investigation is that patients were matched for age, BMI, basal FSH and indication for assisted reproductive treatment. It has been reported that serum levels of AMH in normo-ovulatory women decrease over time and decrease with advancing age before changes occur in currently known aging-related variables (de Vet et al., 2002
). Both BMI and cause of infertility may influence ovarian response to gonadotropins (Crosignani et al., 1994
; Roseboom et al., 1995
; Tinkanen et al., 1999
; Loh et al., 2002
) and it has been reported that AMH serum levels tend to be lower in obese than in nonobese women (Pigny et al., 2003
). Basal serum FSH levels are an indication of biological age (Sharara and Scott, 1997
) and FSH may be involved in the ontogenesis of AMH. However, data available in the literature in the latter respect are contradictory. Thus, it has been reported that FSH may down-regulate the AMH and AMH type II receptor expression in adult rat ovaries (Baarends et al., 1995
). Conversely, follicles from AMH knockout mice have been shown to be more sensitive to FSH than those from the wild type (Durlinger et al., 2001
). On the other hand, there are studies showing that AMH may foster FSH-induced follicular growth (McGee et al., 2001
) whereas it is well established that FSH is a positive regulator of testicular AMH gene expression in adults (Lukas-Croisier et al., 2003). Therefore, our study design included matching for these variables, allowing the analysis of AMH as an independent marker of ovarian response. Although cohort studies can be more efficient than casecontrol studies, the latter can be accomplished in a shorter period of time, mainly when patients matching by pre-established criteria are used (Cramer, 1994
; Schulz and Grimes, 2002
). Performing IVF studies within a short time frame clearly contributes to precluding any bias due to possible changes in IVF/ICSI laboratory techniques. In this regard, a cohort study controlling for those well pre-established potential confounding variables would require too long a period of follow-up.
AMH serum levels obtained after 4 days of gonadotropin treatment were significantly lower than baseline AMH concentrations in both groups of patients studied. This is in agreement with recent work showing that serum AMH levels decline gradually during multiple follicular maturation, probably reflecting the dramatic reduction in the number of small antral follicles due to controlled ovarian hyperstimulation (Fanchin et al., 2003b). These data suggest that AMH is preferentially secreted by small antral follicles and provide support to the hypothesis that differentiation of granulosa cells during follicular growth is likely to alter their ability of expressing AMH (Baarends et al., 1995
; Fanchin et al., 2003b
).
The question as to why AMH serum levels on stimulation day 5 would be a better predictor of ovarian response than basal AMH remains to be established for several reasons. First, the reduction in AMH serum levels observed during ovarian stimulation may be due to several factors such as a negative role of exogenous FSH administration or the supraphysiological increase in estradiol levels (La Marca et al., 2004). Moreover, as stated above, the decrease in AMH may also be the result of a gonadotropin follicular growth stimulation. Second, it remains unclear what the relative contributions of the primordial pool and the preantral and early antral follicles may have in determining the serum concentrations of AMH, and the question whether AMH expression is lost in the follicles that are selected for dominance remains unanswered (Seifer et al., 2002
; Weenen et al., 2004
). Finally, uncertainties persist with respect to the control of granulosa cell AMH production and the possible effects of controlled ovarian hyperstimulation with exogenous gonadotropins on peripheral AMH levels (Fanchin et al., 2003a
). However, even though it is still questioned whether AMH is a marker of primordial follicles or later stages of follicle development or both, its serum level appears as a reliable marker of the ovarian follicle pool (Durlinger et al., 2002
; Pigny et al., 2003
). In addition, it is also possible that AMH function involves a role in selection of follicles that do not undergo atresia, and in preventing premature follicle maturation (Baarends et al., 1995
). Thus, it may be postulated that an ovarian dynamic test in the form of AMH detemination during gonadotropin treatment in the index cycle and reflecting the glandular response to stimulation may be a better marker of the overall follicular pool and activity of the ovary and AMH-related functions than basal AMH.
There are several potential limitations to the use of AMH as a marker of assisted reproduction treatment outcome. First, a wide range of serum AMH concentrations was found in the whole population studied both at baseline and on day 5 of gonadotropin therapy. This is in keeping with previous studies where the serum AMH concentration in normal prepubertal girls and normal adult women ranged from 0.7 to 73.9 pmol/l and 0.7 to 74.7 pmol/l, respectively (Long et al., 2000). The wide range in values obtained for AMH would explain the low sensitivity (53%) of the best criterion value discriminating between control and cancelled cycles obtained in the present study. It could be argued that cancelled patients developing 12 follicles in response to prolonged gonadotropin treatment (late cancellation group) could still have a chance for pregnancy and thus results could be different if other cancellation criteria were used. However, from a practical point of view, it is considered that four oocytes are needed to reach an average of two embryos available for transfer (Bancsi et al., 2002
; Klinkert et al., 2004
). Thus, the collection of <4 oocytes at retrieval or cancellation of the cycle due to insufficient follicular growth (<3 developing follicles in response to exogenous gonadotropins) is the most widely used definition of poor response and it was used in many other studies because of the poor prognosis in such cases (Hanoch et al., 1998
; Hugues and Cedrin-Durnerin, 1998; Surrey et al., 1998
; De Placido et al., 2000
; García-Velasco et al., 2000
; Bancsi et al., 2002
; El Toukhy et al., 2002
; Klinkert et al., 2004
). Furthermore, in the current study, basal and day 5 AMH serum levels were similar in the early cancellation and late cancellation groups.
Second, while cycle day 3 FSH measurement (which is the most routinely used ovarian reserve test) has clinical relevance, is inexpensive for the community and is not time-consuming for the medical team and so is easily repeatable, AMH assay is technically challenging and not readily available. Thus, there is as yet no international assay standard for AMH, which may explain discordance between different studies and makes comparison of results between laboratories difficult. In addition, the results of the current study indicate that stimulation day 5 estradiol serum level has similar predictive properties for ovarian performance in assisted reproductive treatment cycles as day 5 AMH determination. Third, as previously stressed, the pregnancy rate is the only important end-point of an ovarian reserve test (Bukman and Heineman, 2001). In this regard it is noteworthy that neither basal nor day 5 AMH or estradiol serum measurements were able to predict pregnancy in our study. This is in agreement with previous work by us and others stressing that hormone measurements may be helpful to evaluate ovarian response to stimulation but are less useful in their prediction of reproductive outcome (Commenges-Ducos et al., 1998
; Sharif et al., 1998
; Hall et al., 1999
; Creus et al., 2000
; Fábregues et al., 2000
; Peñarrubia et al., 2000
; van Rooij et al., 2003
).
Finally, antral follicle counts were not considered in the present investigation but it has been reported that the number of antral follicles as counted early in the follicular phase provides better prognostic information on the occurrence of poor response during hormone stimulation for IVF than does the patient's chronological age and the currently used endocrine markers (Scheffer et al., 1999; Bancsi et al., 2002
, Bancsi et al., 2004
). In addition, recent reports found a tight relationship between the AMH serum level and the antral follicular count assessed by ultrasonography in regularly menstruating infertile women studied at baseline day 3, before undergoing assisted reproduction (Van Rooij et al., 2002
; Fanchin et al., 2003a
). Therefore, the antral follicular count before ovarian stimulation may yield the same information and will better prevent unnecessary costs and effort.
In conclusion, AHM serum concentrations in the fifth day of gonadotropin therapy in women undergoing ovarian stimulation under pituitary desensitization for assisted reproduction have a higher predictive value of ovarian response than basal AMH. This early assessment of ovarian stimulation reponse could help to decide early cancellation, thus avoiding further cost and therapy. However, the predictive capacity of day 5 AMH was not better than that provided by day 5 estradiol. Furthermore, neither basal nor day 5 AMH or estradiol measurements were useful in the prediction of pregnancy after assisted reproductive treatment. Therefore, definite clinical applicability of AMH determination as a marker of IVF outcome remains to be established.
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Acknowledgements |
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Submitted on October 5, 2004; accepted on December 7, 2004.