Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Open Access

Peer-reviewed

Research Article

Prediction of Reproductive Outcomes According to Different Serum Anti-Müllerian Hormone Levels in Females Undergoing Intracystoplasmic Sperm Injection

  • Santiago Brugo Olmedo,

    Affiliation Centro Médico, Seremas, Buenos Aires, Argentina

    ⨯
  • Sabrina De Vincentiis,

    Affiliation Centro Médico, Seremas, Buenos Aires, Argentina

    ⨯
  • Evelyn De Martino,

    Affiliation Centro Médico, Seremas, Buenos Aires, Argentina

    ⨯
  • Patricia Bedecarrás,

    Affiliation Centro de Investigaciones Endocrinológicas “Dr. César Bergadá” (CEDIE)CONICET – FEI – División de Endocrinología, Hospital de Niños R. Gutiérrez, Buenos Aires, Argentina

    ⨯
  • Ana María Blanco,

    Affiliation Centro de Estudios Bioquímicos, Andrológicos y Ginecológicos, Buenos Aires, Argentina

    ⨯
  • Analía Freire,

    Affiliation Centro de Investigaciones Endocrinológicas “Dr. César Bergadá” (CEDIE)CONICET – FEI – División de Endocrinología, Hospital de Niños R. Gutiérrez, Buenos Aires, Argentina

    ⨯
  • Mariano G. Buffone,

    Affiliations Centro Médico, Seremas, Buenos Aires, Argentina, Instituto de Biología y Medicina Experimental (IBYME-CONICET), Buenos Aires, Argentina

    ⨯
  • Rodolfo A. Rey

    rodolforey@cedie.org.ar

    Affiliation Centro de Investigaciones Endocrinológicas “Dr. César Bergadá” (CEDIE)CONICET – FEI – División de Endocrinología, Hospital de Niños R. Gutiérrez, Buenos Aires, Argentina

    ⨯

Abstract

Background and aim of the study

Serum anti-Müllerian hormone (AMH) is a reliable marker of ovarian reserve, and it has been shown to be correlated with reproductive outcomes in grouped analyses. However, practical data is scarce for the physician and the patients to predict these outcomes in an individual couple according to serum AMH measured prior to assisted reproduction technology (ART) procedures.

Study Design

To address this question, we performed an analytic observational study including 145 females undergoing intracytoplasmic sperm injection (ICSI) in a single center. Results were analyzed according to serum AMH; subgroup analyses were performed by grouping patients according to patient’s age and FSH levels.

Results

The risk of cycle cancelation decreased from 64% in patients with serum AMH ≤3 pmol/L (0.42 ng/mL) to 21% with AMH ≥15 pmol/L (2.10 ng/mL). Cycle cancelation occurred in approximately two-thirds of the patients with AMH ≤ 3 pmol/L irrespective of the FSH level. However, with higher AMH values the risk of cycle cancelation decreased more significantly in patients with normal FSH. The rate of good response increased from almost null in patients with AMH ≤3 pmol/L to 61% in those with AMH ≥15 pmol/L. The positive correlation between good response and AMH was also significant, but with lower absolute rates, when patients were grouped according to their age or FSH levels. Pregnancy rate increased moderately, but significantly, from 31% with AMH ≤3 pmol/L to 35% with AMH ≥15 pmol/L.

Conclusions

We provide estimates of reproductive outcomes according to individualized values of serum AMH, in general and in subgroups according to patient’s age or serum FSH, which are helpful for the clinician and the couple in their decision making about starting an assisted reproductive treatment.

Citation: Brugo Olmedo S, De Vincentiis S, De Martino E, Bedecarrás P, Blanco AM, Freire A, et al. (2013) Prediction of Reproductive Outcomes According to Different Serum Anti-Müllerian Hormone Levels in Females Undergoing Intracystoplasmic Sperm Injection. PLoS ONE 8(9): e75685. https://doi.org/10.1371/journal.pone.0075685

Editor: Renato Pasquali, Azienda Policlinico S. Orsola-Malpighi, Italy

Received: February 11, 2013; Accepted: August 19, 2013; Published: September 17, 2013

Copyright: © 2013 Brugo Olmedo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was partially funded by CONICET (Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina) to cover the cost of AMH assay reagents. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding received for this study.

Competing interests: The authors have read the journal's policy and have the following conflicts: P. Bedecarrás and R.A. Rey have received honoraria from CONICET (Argentina) for technology services using the AMH ELISA.

Introduction

The human ovary contains a limited population of primordial follicles, set approximately 20 weeks post-conception, when the ovary follicle reserve achieves its maximum size. Thereafter, the ovarian reserve decreases, having an impact on natural fertility which clearly declines after the age of 30 years [1,2]. The age of women giving birth is increasing worldwide due to diverse social reasons. Consequently, a growing number of couples is facing age-related infertility problems and seek medical assistance. Assisted reproductive technology (ART) outcomes have improved over the years but are limited by the ovarian response to hyperstimulation used in treatment protocols. On the other hand, ART treatments are expensive for healthcare systems or private practices and represent a stressful situation for the couple. Therefore, the necessity exists for optimization of the evaluation of the ovarian reserve in order to minimize the uncertainty of the outcome of ART procedures. Age and circulating FSH levels, which have classically been used to predict the fertility potential, lack precision under specific clinical circumstances, especially because there is a considerable variability regarding the ovarian reserve amongst women of the same age [3].

The ovarian reserve is determined by the number of primordial follicles present in the ovary and their quality. Reliable markers of oocyte quality are yet to be developed, and direct assessment of the pool of primordial follicles present in the ovaries is not feasible. However, the number of antral follicles represents a good estimator of the primordial follicle pool and, therefore, of the quantitative aspect of the ovarian reserve [4,5]. In the recent years, anti-Müllerian hormone (AMH) has been shown to represent a reliable marker of the ovarian reserve [6] and of the response to ovarian stimulation [7–10]. A member of the TGFβ superfamily initially believed to be a fetal testis hormone [11], AMH is also produced in the ovary essentially by the granulosa cells of primary and small antral follicles [12,13]. Serum AMH levels are clearly correlated with the granulosa cells mass, ranging from undetectable in normal post-menopause [14] and in Turner syndrome patients with absence of gonadal tissue [15] to very high levels in patients with polycystic ovary syndrome [16,17] or granulosa cell tumors [18]. A particular advantage of AMH as a marker of ovarian reserve is its insignificant variation during the menstrual cycle [19–21], which does not restrict AMH measurement to a particular stage of the cycle. The first in-house AMH immunoassays developed in 1990 [22–24] were replaced by commercial assays after 1998 [18,25,26]. The two different commercially available AMH assays used in the following 10 years showed clear differences in the reported levels, mainly in the low female range [27], which complicates the interpretation of the results for the clinician. Furthermore, the large number of studies assessing the performance of serum AMH as a predictor of the ovarian reserve and a prognostic factor for the outcome of ART treatments have mostly used only one cutoff value, as summarized in a recent meta-analysis [5], thus dividing the population into two groups: one below the cutoff value usually of homogeneously poor prognosis, and another one above the cutoff value which is extremely heterogeneous. Furthermore, although the effect of age and of other reproductive variables on serum AMH has been acknowledged for years, most of the studies have analyzed serum AMH in the studied samples as a whole, or after introducing complex correction factors in the statistical analysis which hamper a simple interpretation of the AMH value observed in an individual patient presenting to the clinician.

The adequate identification of the responsiveness potential specific for the serum AMH level in each patient before entering an ART treatment may be most helpful for the couple in order to decide whether to start treatment and for the clinician in the proper management regarding the stimulation protocol. The objective of this work was to provide the clinician with a reliable tool to predict the most commonly used reproductive outcomes in women undergoing intracytoplasmatic sperm injection (ICSI) before starting the procedure. To fulfill this objective we assessed the clinical value of different serum levels AMH in predicting the rates of: cycle cancelation, good response to ovarian stimulation, syngamy, cleavage, implantation and clinical pregnancy. The assessment was performed separately according to patients’ age (25-37 and 38-43 yr) and to serum FSH (normal or elevated).

Methods

Study subjects and design

Subjects.

We performed an observational study, retrospectively collecting data from 145 consecutive women, aged 25 to 43 years-old, undergoing ICSI at Seremas Institute (Buenos Aires, Argentina) between August 2009 and October 2010. The study was approved by the Seremas Institutional Review Board. The need for written informed consent was waived by the Institutional Review Board because, owing to the retrospective observational design used, study results could not modify any clinical decision made at the moment of ICSI procedures.

Ovarian stimulation.

All patients were submitted to a standard GnRH agonist protocol, and underwent controlled ovarian hyperstimulation with recombinant FSH (Gonal-F; Serono Laboratories, Switzerland). Ovulation was induced with highly purified hCG (Ovidrel, Serono). Daily FSH doses and timing of hCG administration were adjusted according to the usual criteria of follicle maturation [28]. Follicle count was performed by ultrasonography using a transvaginal probe.

Serum hormone levels.

Serum was obtained at first visit for AMH measurement, and during ovarian stimulation on day 3 (d3) for FSH and E2 measurement and on the day of hCG administration (d-hCG) for E2 measurement. AMH was measured at the Centro de Investigaciones Endocrinológicas, Hospital Ricardo Gutiérrez (Buenos Aires, Argentina), using an ultrasensitive enzyme-linked immunoassay specific for human AMH (EIA AMH/MIS®, Immunotech, Beckman-Coulter Co., Marseilles, France, ref. A11893), recently validated by our group [29]. FSH and estradiol (E2) were measured by chemiluminescence using Access® technology (Beckman Coulter Inc.).

Intracytoplasmic sperm injection (ICSI).

Conventional ICSI, as previously described [30], was conducted ~5 hours post oocyte aspiration. Motile sperm were isolated using the swim up technique. Around 2 µl of sperm were placed in 7% polyvinylpyrrolidone and a sperm was injected into each oocyte using standardized techniques. The embryos were cultured in a single droplet containing 20 µl of medium and incubated at 37°C under controlled conditions (5% CO2, 5% O2 and 90% N2). All embryo transfers were performed 72 hours after oocyte aspiration. Since reproductive outcomes may be associated with cycle rank (i.e. the number of previous ART cycles a patient had already been submitted to) patients were included only if they were in their first three attempts.

Outcome measures.

The main outcome measure was the absolute risk (risk rate) of cycle cancelation (number of patients with cancelled cycles divided by the total number of patients in whom a cycle was initiated), good response to ovarian stimulation (defined as ≥ 5 oocytes retrieved at the time of aspiration), syngamy, cleavage at 48 h, multinucleated embryos, implantation (defined as the number of gestational sacs observed on ultrasound) and clinical pregnancy (defined as the presence of fetal heart activity detected by ultrasound at 6 weeks) for each of the following serum AMH levels: 3, 6, 9, 12 and 15 pmol/L. Secondary outcome measures were the predictive values of: patient’s age, serum FSH and estradiol on d3, and serum estradiol and follicle count by ultrasound assessment on d-hCG.

Statistical analyses

Data distribution was assessed for normality using the Shapiro-Wilk test. Data of the risk rates of cycle cancelation, good response to ovarian stimulation, syngamy, cleavage at 48 h, multinucleated embryos, implantation and clinical pregnancy are presented with their 95% confidence intervals. Patient’s age, basal serum AMH, FSH and estradiol, and serum estradiol and follicle count by ultrasound assessment on the day of ovulation induction by hCG administration are presented as the median and interquartile range. Comparisons between 2 groups were made using an unpaired t test, except when a non-Gaussian distribution was found, where a Mann-Whitney test was used. Areas under the ROC curves were calculated for age and hormone levels to estimate the predictive values for the rates of: cycle cancelation, good response to ovarian stimulation, syngamy, cleavage at 48 h, multinucleated embryos, implantation and clinical pregnancy. Analyses wer performed for the whole group, and independently according to age, where the study cohort was divided into 2 groups (25-37 yr and 38-43 yr), or according to FSH levels, where the study cohort was divided into 2 groups (FSH within the normal reference range and FSH above the reference range). The absolute risk values were compared using a one-sided Fisher’s exact test. The level of significance was set at P <0.05. All statistical analyses were performed using GraphPad Prism version 5.01 for Windows (GraphPad Software, San Diego, CA, USA).

Results

Cycle cancelation

In the whole group analyzed (Table 1), age and FSH was significantly higher, and AMH was lower in patients with cancelled cycles, as expected. No difference was observed in E2. When patients were grouped by age (Table 2), the significantly lower AMH and higher FSH values were still observed in patients aged 38-43 yr with cancelled cycles. When patients were classified according to their FSH levels (Table 3), lower AMH levels were observed in patients with cancelled cycles irrespective of their FSH levels. Serum AMH showed excellent areas under the ROC curves to predict cycle cancelation in the whole group and in the age and FSH subgroups (Table 4). The absolute risk of cycle cancelation was inversely correlated to serum AMH in the whole group (Figure 1, A) and in the subgroups (Figure 1, B-E). The risk of cycle cancelation in the whole group decreased from 64% in patients with serum AMH ≤3 pmol/L (0.42 ng/mL) to 43% in patients with AMH 6 pmol/L (0.84 ng/mL), 29% with AMH 9 pmol/L (1.26 ng/mL), 26% with AMH 12 pmol/L (1.68 ng/mL) and 21% with AMH ≥15 pmol/L (2.10 ng/mL). When related to patients with AMH <3 pmol/L (0.42 ng/mL), the relative risk of cycle cancelation was 0.42 in patients with AMH between 6-9 pmol/L (0.84-1.26 ng/mL) and 0.37 in patients with AMH >15 pmol/L (>2.1 ng/mL) (Table 5). Interestingly, cycle cancelation occurred in approximately two-thirds of the patients with AMH ≤3 pmol/L irrespective of the FSH level (Figure 1, D and E). However, with higher AMH values the risk of cycle cancelation decreased more significantly in patients with normal FSH.

Non cancelledCancelledP
n11431
Age (yr)34 (32-38)38 (36-40)0.003
AMH (pmol/L)14.4 (8.6-24.9)3.7 (<2.5-11.1)< 0.001
FSH (IU/L)7.3 (5.9-9.2)11.4 (7.5-16.8)0.016
E2 (pg/ml)47 (29-71)43 (31-75)0.871

Table 1. Characteristics of the whole study population with cancelled and non-cancelled cycles.

Results are reported as median and interquartile range.
Comparisons between groups were made using a Mann-Whitney test.
CSV
Download CSV
25-37 yr38-43 yr
Non cancelledCancelledPNon cancelledCancelledP
n83163115
Age (yr)33 (31-35)35 (31-37)0.25039 (38-41)39 (38-40)0.985
AMH (pmol/L)14.7 (8.9-27.2)8.7 (3.5-19.9)0.07112.6 (7.5-22.5)2.5 (<2.5-4.2)< 0.001
FSH (IU/L)7.0 (6.1-9.2)8.7 (7.4-13.8)0.0607.8 (5.3-9.4)13.4 (7.1-22.7) 0.020
E2 (pg/ml)45 (28-72)44 (27-76)0.99349 (35-72)43 (33-79)1.000

Table 2. Characteristics of the whole study population with cancelled and non-cancelled cycles, grouped by age.

Results are reported as median and interquartile range.
Comparisons between groups were made using a Mann-Whitney test.
CSV
Download CSV
Normal FSHHigh FSH
Non cancelledCancelledPNon cancelledCancelledP
N8692822
Age (yr)34 (31-37)37 (33-40)0.24636 (33-38)38 (37-39)0.039
AMH (pmol/L)16.3 (9.5-26.3)6.4 (3.3-17.0)0.01711.9 (5.5-15.0)<2.5 (<2.5-5.1)0.005
FSH (IU/L)6.7 (5.5-7.9)7.6 (5.6-9.2)0.29210.8 (9.6-13.2)16.8 (13.7-23.8) 0.004
E2 (pg/ml)48 (34-75)71 (33-110)0.34844 (23-68)40 (30-63)0.922

Table 3. Characteristics of the whole study population with cancelled and non-cancelled cycles, grouped by FSH levels.

Results are reported as median and interquartile range.
Comparisons between groups were made using a Mann-Whitney test.
CSV
Download CSV
All25-37 yr38-43 yrNormal FSHHigh FSH
Age0.683 ± 0.0710.623 ± 0.1210.504 ± 0.1000.618 ± 0.1030.753 ± 0.103
AMH0.813 ± 0.0650.695 ± 0.1080.928 ± 0.0550.744 ± 0.1030.848 ± 0.080
FSH0.726 ± 0.0750.704 ± 0.0920.765 ± 0.1060.608 ± 0.1020.857 ± 0.059
E20.514 ± 0.0800.502 ± 0.1080.502 ± 0.1270.626 ± 0.1370.515 ± 0.109

Table 4. Areas under the ROC curve (± standard error) for age and hormone levels to assess the value of serum AMH as a predictor of cycle cancelation in the study population.

CSV
Download CSV
thumbnail
Download:
Figure 1. Absolute risk (risk rate) of cancelled cycles as a function of AMH levels.

Patients were grouped according to age or FSH levels. Rates are shown for serum AMH at 3, 6, 9, 12 and 15 pmol/L (equivalences for AMH in ng/mL are given below the X axis of each graph). Dotted lines represent the 95% confidence interval.

https://doi.org/10.1371/journal.pone.0075685.g001

Cycle cancelationGood response
RR95%CIPRR95%CIP
3.0-5.9 pmol/L0.610.24-1.540.3955.000.68-36.680.155
6.0-8.9 pmol/L0.420.19-0.950.0128.841.33-58-890.006
9.0-11.9 pmol/L0.430.19-0.990.0368.001.17-54.520.009
12.0-14.9 pmol/L0.400.18-0.900.00411.331.73-74.30<0.001
≥ 15 pmol/L0.370.17-0.81<0.00111.311.73-73.99<0.001

Table 5. Relative risk (RR) of cycle cancelation and good response (RR was considered as 1 in patients with AMH < 3 pmol/L).

CSV
Download CSV

Oocyte retrieval

The number of oocytes retrieved in non-cancelled cycles increased progressively in correlation with serum AMH up to an AMH level of 40 pmol/L (5.6 ng/mL) and plateaued thereafter (Figure 2). The increase was observed irrespective of age or serum FSH, yet with a lower absolute number of oocytes in patients aged 38-43 years or with elevated FSH.

thumbnail
Download:
Figure 2. Number of oocytes retrieved at the time of aspiration as a function of AMH levels (non-linear regression).

Patients were grouped according to age or FSH levels.

https://doi.org/10.1371/journal.pone.0075685.g002

Serum AMH showed the most significant differences between patients with good and poor response to stimulation, i.e. retrieval of 5 or more oocytes (Tables 6, 7 and 8). Among basal serum hormone determinations, AMH also showed areas under the ROC curves of high performance to predict a good response (Table 9).

Good responsePoor responseP
n8331
Age (yr)33 (31-37)37 (34-40)0.002
AMH (pmol/L)15.9 (11.3-28.0)4.6 (2.5-9.8)< 0.001
FSH (IU/L)7.3 (5.9-8.7)10.5 (6.4-16.8)0.003
E2 (pg/ml)46 (29-75)45 (28-70)0.667
E2-dhCG1574 (1196-2273)773 (521-1059)0.002
Follicle count7 (5-10)3 (2-4)< 0.001

Table 6. Age, hormone levels and follicle count in the whole study population with good and poor response to controlled ovarian hyperstimulation.

Results are reported as median and interquartile range.
Comparisons between groups were made using a Mann-Whitney test.
CSV
Download CSV
25-37 yr38-43 yr
Good responsePoor responsePGood responsePoor responseP
n62162115
Age (yr)33 (31-35)35 (33-37)0.01639 (38-40)40 (38-41)0.241
AMH (pmol/L)18.3 (12.1-33.5)5.8 (2.8-11.1)< 0.00116.9 (9.5-24.3)3.7 (<2.5-7.5)< 0.001
FSH (IU/L)7.0 (6.1-8.7)7.69 (6.0-12.6)0.1027.8 (5.3-8.1)14.5 (6.8-19.0)0.008
E2 (pg/ml)45 (29-76)45 (27-73)0.82553 (36-80)46 (27-66)0.410
E2-dhCG1751 (1199-2504)646 (400-1025)0.0031664 (1303-2254)894 (555-1168)0.023
Follicle count8 (5-10)3 (2-4)< 0.0017 (5-9)3 (2-4)0.019

Table 7. Age, hormone levels and follicle count in patients with good and poor response to controlled ovarian hyperstimulation, grouped by age.

Results are reported as median and interquartile range.
Comparisons between groups were made using a Mann-Whitney test.
CSV
Download CSV
Normal FSHHigh FSH
Good responsePoor responsePGood responsePoor responseP
n68171514
Age (yr)31 (33-37)37 (34-41)0.02934 (31-37)38 (37-39)0.007
AMH (pmol/L)16.9 (11.1-28.3)8.7 (3.6-11.2)< 0.00113.2 (11.7-15.7)2.7 (<2.5-5.8)< 0.001
FSH (IU/L)6.7 (5.6-7.9)6.9 (5.3-7.7)0.7610.1 (9.1-10.9)16.8 (13.2-21.2)< 0.001
E2 (pg/ml)46 (32-73)57 (38-89)0.33748 (24-76)40 (25-49) 0.359
E2-dhCG1600 (1207-2478)894 (761-1059)0.0041288 (1009-1916)433 (111-1090) 0.045
Follicle count8 (6-10)3 (2-4)< 0.0015 (5-10)3 (2-4)0.054

Table 8. Age, hormone levels and follicle count in patients with good and poor response to controlled ovarian hyperstimulation, grouped by FSH levels.

Results are reported as median and interquartile range.
Comparisons between groups were made using a Mann-Whitney test.
CSV
Download CSV
All25-37 yr38-43 yrNormal FSHHigh FSH
Age0.683 ± 0.0710.623 ± 0.1210.504 ± 0.1000.669 ± 0.0730.793 ± 0.085
AMH0.813 ± 0.0650.695 ± 0.1080.928 ± 0.0550.783 ± 0.0650.938 ± 0.041
FSH0.726 ± 0.0750.704 ± 0.0920.765 ± 0.1060.525 ± 0.0800.976 ± 0.023
E20.514 ± 0.0800.502 ± 0.1080.502 ± 0.1270.587 ± 0.0880.602 ± 0.108

Table 9. Areas under the ROC curve (± standard error) for age and hormone levels to assess the value of serum AMH as a predictor of good response to controlled ovarian hyperstimulation in the study population.

CSV
Download CSV

The rate of good response increased with serum AMH: it was almost null in patients with AMH ≤3 pmol/L (0.42 ng/mL) in both age groups (Figure 3) and rose up to 61% in those patients with AMH 15 pmol/L (2.10 ng/mL). When related to patients with AMH <3 pmol/L (0.42 ng/mL), the chances of good response were approximately eight-fold higher in patients with AMH between 6-12 pmol/L (0.84-1.68 ng/mL) and eleven-fold higher in patients with AMH >15 pmol/L (>2.1 ng/mL) (Table 5). The positive correlation between good response and AMH was also significant, but with lower absolute rates, when patients were grouped according to their age (Figure 3, B-C) or FSH levels (Figure 3, D-E).

thumbnail
Download:
Figure 3. Absolute risk (risk rate) of good response to ovarian stimulation, defined as ≥ 5 oocytes retrieved at the time of aspiration, as a function of AMH levels.

Patients were grouped according to age or FSH levels. Rates are shown for serum AMH at 3, 6, 9, 12 and 15 pmol/L (equivalences for AMH in ng/mL are given at the bottom of the figure). Dotted lines represent the 95% confidence interval.

https://doi.org/10.1371/journal.pone.0075685.g003

Syngamy, cleavage, implantation and pregnancy rates

No significant correlation was found between AMH levels and the rates of syngamy (Spearman r -0.080, P=0.313), cleavage at 48 h (Spearman r -0.004, P=0.623), multinucleated embryos (Spearman r -0.062, P=0.457) or implantation (Spearman r -0.053, P=0.557).

The rate of pregnancy showed an increase in correlation with serum AMH in the whole group. Serum AMH was lower and age was higher in patients with a higher pregnancy rate when the analysis was performed in the whole group (Table 10). Interestingly, this was also observed in patients with high FSH (Tables 11 and 12). A significantly positive correlation between pregnancy rate and AMH was observed in the whole group (Spearman r0.894, P=0.020, Figure 4 A), and in patients >38 yr (Figure 4 C) or with high FSH (Figure 4 E).

PregnancyNonpregnancyP
n3589
Age (yr)33 (31-37)36 (33-39)0.028
AMH (pmol/L)15.4 (12.0-32.6)11.6 (5.9-21.5)0.015
FSH (IU/L)7.1 (5.2-9.1)7.4 (6.2-9.7)0.289
E2 (pg/ml)47 (29-70)49 (32-71)0.964
E2-dhCG1463 (1021-1944)1391 (803-2264)0.936
Follicle count8 (5-10)7 (4-9)0.094

Table 10. Age, hormone levels and follicle count in the whole study population according to the achievement of clinical pregnancy.

Results are reported as median and interquartile range.
Comparisons between groups were made using a Mann-Whitney test.
CSV
Download CSV
25-37 yr38-43 yr
PregnancyNonpregnancyPPregnancyNonpregnancyP
n2759830
Age (yr)32 (31-34)34 (32-36)0.06739 (38-41)40 (39-41)0.263
AMH (pmol/L)14.9 (12.1-33.3)13.9 (7.5-22.8)0.09216.4 (7.7-21.9)7.6 (3.7-15.4)0.113
FSH (IU/L)7.6 (5.7-9.3)6.9 (5.6-8.7)0.5155.3 (4.5-7.9)8.1 (7.1-14.5)0.015
E2 (pg/ml)37 (29-67)50 (33-69)0.49564 (49-77)43 (31-79)0.135
E2-dhCG1443 (876-1927)1411 (923-2467)0.4561669 (1285-2229)1243 (712-1862)0.171
Follicle count8 (6-10)7 (4-10)0.2508 (4-10)5 (3-9)0.238

Table 11. Age, hormone levels and follicle count in patients, grouped by age, according to the achievement of clinical pregnancy.

Results are reported as median and interquartile range.
Comparisons between groups were made using a Mann-Whitney test.
CSV
Download CSV
Normal FSHHigh FSH
PregnancyNonpregnancyPPregnancyNonpregnancyP
n2768821
Age (yr)34 (31-38)35 (32-38)0.51333 (31-34)38 (37-39)< 0.001
AMH (pmol/L)15.9 (12.0-32.6)14.7 (7.8-25.1)0.18015.0 (7.1-36.6)5.7 (<2.5-11.9)0.008
FSH (IU/L)6.3 (4.8-7.9)6.7 (5.6-7.8)0.3569.9 (9.1-10.9)13.1 (10.4-18.3)0.014
E2 (pg/ml)53 (30-80)50 (32-75)0.81131 (29-64)45 (27-65)0.647
E2-dhCG1463 (944-1950)1467 (923-2467)0.5231586 (1150-2029)1242 (506-1765)0.097
Follicle count8 (6-10)7 (4-9)0.1127 (4-10)4 (3-10)0.535

Table 12. Age, hormone levels and follicle count in patients, grouped by FSH levels, according to the achievement of clinical pregnancy.

Results are reported as median and interquartile range.
Comparisons between groups were made using a Mann-Whitney test.
CSV
Download CSV
thumbnail
Download:
Figure 4. Absolute risk (risk rate) of clinical pregnancy rates as a function of AMH levels.

Patients were grouped according to age or FSH levels. Rates are shown for serum AMH at 3, 6, 9, 12 and 15 pmol/L (equivalences for AMH in ng/mL are given at the bottom of the figure). Dotted lines represent the 95% confidence interval.

https://doi.org/10.1371/journal.pone.0075685.g004

Discussion

One of the most critical aspects before starting an ART procedure is the initial evaluation of the female’s capacity to produce healthy and developmental competent oocytes. Serum AMH has become a standard determination to evaluate the ovarian reserve. In the present study, we provide practical data for the clinician and the couple to easily predict the odds for cycle cancelation, good response to stimulation and pregnancy, according to the level of circulating AMH in a random sample obtained prior to initiating the ART procedure. On the basis of the data provided in Figure 1, the clinician can give practical counseling: risk of cycle cancelation attains approximately two-thirds of the couples if serum AMH is 3 pmol/L, 1/3 if AMH is 9 pmol/L and 1/5 if AMH is 15 pmol/L. Further discrimination can be made according to patient’s age or serum FSH. Similarly, as shown in Figure 3, less than one-twentieth of the attempts will yield a good response to stimulation when serum AMH is 3 pmol/L, but the rate increases to approximately 1/5 when AMH is 6 pmol/L, approximately ½ when AMH is 9-12 pmol/L and almost 2/3 when AMH is 15 pmol/L. Many authors have previously analyzed the association between serum AMH and reproductive outcomes in women undergoing ART treatments, as summarized in a recent systematic review [5]. Most of these studies provide correlation coefficients or sensitivity and specificity levels for a given AMH cutoff, using adequate statistical procedures to avoid confounders. Although the strength of the association has been unequivocally proven, correlation coefficients or more complicate calculations do not represent practical tools for straightforward application in clinical practice. In the present work we have used a practical approach and provide useful and easily applicable results of serum AMH levels to predict the risk of cycle cancelation and the chances of good response to controlled ovarian hyperstimulation and pregnancy in females undergoing an ART procedure, according to their age or FSH level. Some studies have reported predictive values for poor response and pregnancy rates, but only using 1 or 2 cutoff levels of serum AMH [20,27,31–36]. Nelson and colleagues [37] reported more discriminated prediction rates of oocyte retrieval and live births, for five different serum AMH ranges. Here, we provide detailed results for several reproductive outcomes helping to predict the success rate of many steps of the ART procedure. One particularly relevant, from a practical standpoint, is the prediction of the risk of cycle cancelation, which has not been analyzed by previous publications.

Our results are in line with those previously reported by other authors showing that AMH is a useful marker for predicting cycle cancelation [38] and poor response to ovarian stimulation [5], representing a better predictor than the other classically associated parameters such as FSH, estradiol and age [39]. Interestingly, our data show the special importance of serum AMH in women aged >38 yr or with high serum FSH, and have immediate application for the clinician and the patient in their decision prior to start hormonal stimulation. For example, in women 38-43 yr seeking for assisted reproduction treatment, an AMH value ≥ 15 pmol/L (2.10 ng/mL) indicates a risk of 38% to result in a cycle cancelation, and the risk increases to 83% if AMH is ≤ 3 pmol/L (0.42 ng/mL). Also, different AMH values are helpful for decision making in both patients with normal or elevated FSH levels.

It is well established that AMH is correlated with the number of oocytes retrieved at the time of aspiration [40]. Our results are consistent with this observation, independently of the patient’s age. Furthermore, from a practical standpoint, we show that AMH is particularly useful to predict ovarian response to stimulation, as shown by the best areas under the ROC curves. Only E2-dhCG showed a better area under the ROC curve; yet, it cannot be used as a predictor before starting the stimulation protocol.

In our hands, AMH was not as powerful as a predictor of oocyte quality in terms of fertilization rate, embryo development and implantation. Although we found a correlation between serum AMH and pregnancy rate in the whole group, the effect magnitude was modest, in line with the controversial results reported by other groups showing no [41–43] or moderate usefulness of AMH in predicting embryo development [8,44]. However, it was interesting to note that AMH was more discriminant in the high-risk groups, i.e. in the patients >38 yr or with high FSH.

In conclusion, in an individual patient seeking for assisted reproductive technology treatment, the serum AMH level measured in a previous cycle can be used to predict the risk of cycle cancelation and the chances of good ovarian response, when analyzed together with patient’s age or serum FSH. Serum AMH has a significantly better predictive value than FSH and follicle count particularly in women > 38 yr. Furthermore, serum AMH is useful to predict reproductive outcomes in patients with a mild to moderate increase in FSH levels. Although our study design does not allow us to conclude that an infertility treatment should be interrupted on the basis of low AMH, our results add to the existing evidence, and provide practical information, on the usefulness of serum AMH level to help clinicians and patients estimate the chances of good treatment outcomes before initiating the stimulation protocol. AMH evaluation is not routinely performed prior to stimulation and should be incorporated into the initial screening of the patient.

Author Contributions

Conceived and designed the experiments: SBO SDV MGB RAR. Performed the experiments: SBO SDV EDM PB AMB RAR. Analyzed the data: SBO SDV AF MGB RAR. Contributed reagents/materials/analysis tools: SBO SDV EDM PB AMB RAR. Wrote the manuscript: SBO SDV MGB AF RAR.

References

  1. 1. te Velde ER, Pearson PL (2002) The variability of female reproductive ageing. Hum Reprod Update 8: 141-154. doi:https://doi.org/10.1093/humupd/8.2.141. PubMed: 12099629.
  2. 2. Nelson SM, Messow MC, McConnachie A, Wallace H, Kelsey T et al. (2011) External validation of nomogram for the decline in serum anti-Müllerian hormone in women: a population study of 15,834 infertility patients. Reprod Biomed Online 23: 204-206: S1472-S6483. 11)00272-0 . PII .
  3. 3. Broekmans FJ, Knauff EA, te Velde ER, Macklon NS, Fauser BC (2007) Female reproductive ageing: current knowledge and future trends. Trends Endocrinol Metab 18: 58-65. S1043-. doi:https://doi.org/10.1016/j.tem.2007.01.004. PubMed: 17275321. 2760(07)00005-7 . PII .
  4. 4. Hendriks DJ, Kwee J, Mol BW, te Velde ER, Broekmans FJ (2007) Ultrasonography as a tool for the prediction of outcome in IVF patients: a comparative meta-analysis of ovarian volume and antral follicle count. Fertil Steril 87: 764-775. S0015-. doi:https://doi.org/10.1016/j.fertnstert.2006.11.006. PubMed: 17239869. 0282(06)04377-9 . PII .
  5. 5. Broer SL, Mol BW, Hendriks D, Broekmans FJM (2009) The role of antiMüllerian hormone in prediction of outcome after IVF: comparison with the antral follicle count. Fertil Steril 91: 705-714. doi:https://doi.org/10.1016/j.fertnstert.2007.12.013. PubMed: 18321493.
  6. 6. Broekmans FJ, Kwee J, Hendriks DJ, Mol BW, Lambalk CB (2006) A systematic review of tests predicting ovarian reserve and IVF outcome. Hum Reprod Update 12: 685-718. doi:https://doi.org/10.1093/humupd/dml034. PubMed: 16891297.
  7. 7. Feyereisen E, Méndez Lozano DH, Taieb J, Hesters L, Frydman R et al. (2006) Anti-Müllerian Hormone: clinical insights into a promising biomarker of ovarian follicular status. Reprod Biomed Online 12: 695-703. doi:https://doi.org/10.1016/S1472-6483(10)61081-4. PubMed: 16792844.
  8. 8. La Marca A, Sighinolfi G, Radi D, Argento C, Baraldi E et al. (2010) Anti-Müllerian Hormone (AMH) as a predictive marker in assisted reproductive technology (ART). Hum Reprod Update 16: 113-130. doi:https://doi.org/10.1093/humupd/dmp036. PubMed: 19793843. dmp036 . PII .
  9. 9. Broer SL, Dólleman M, Opmeer BC, Fauser BC, Mol BW et al. (2011) AMH and AFC as predictors of excessive response in controlled ovarian hyperstimulation: a meta-analysis. Hum Reprod Update 17: 46-54. doi:https://doi.org/10.1093/humupd/dmq034. PubMed: 20667894. dmq034 . PII .
  10. 10. Anderson RA, Nelson SM, Wallace WH (2012) Measuring anti-Müllerian hormone for the assessment of ovarian reserve: when and for whom is it indicated? Maturitas 71: 28-33. doi:https://doi.org/10.1016/S0378-5122(12)70112-3. PubMed: 22119275.
  11. 11. Josso N, Picard JY, Rey R, di Clemente N (2006) Testicular Anti-Müllerian Hormone: History, Genetics, Regulation and Clinical Applications. Pediatr Endocrinol Rev 3: 347-358. PubMed: 16816803.
  12. 12. Vigier B, Picard JY, Tran D, Legeai L, Josso N (1984) Production of anti-Müllerian hormone: another homology between Sertoli and granulosa cells. Endocrinology 114: 1315-1320. doi:https://doi.org/10.1210/endo-114-4-1315. PubMed: 6546716.
  13. 13. Rey R, Sabourin JC, Venara M, Long WQ, Jaubert F et al. (2000) Anti-Müllerian hormone is a specific marker of Sertoli- and granulosa-cell origin in gonadal tumors. Hum Pathol 31: 1202-1208. doi:https://doi.org/10.1053/hupa.2000.18498. PubMed: 11070112.
  14. 14. Rey RA, Lhommé C, Marcillac I, Lahlou N, Duvillard P et al. (1996) Antimüllerian hormone as a serum marker of granulosa cell tumors of the ovary: comparative study with serum alpha-inhibin and estradiol. Am J Obstet Gynecol 174: 958-965. doi:https://doi.org/10.1016/S0002-9378(96)70333-2. PubMed: 8633676.
  15. 15. Hagen CP, Aksglaede L, Sørensen K, Main KM, Boas M et al. (2010) Serum levels of anti-Müllerian hormone as a marker of ovarian function in 926 healthy females from birth to adulthood and in 172 Turner syndrome patients. J Clin Endocrinol Metab 95: 5003-5010. doi:https://doi.org/10.1210/jc.2010-0930. PubMed: 20719830. jc.2010-0930 . PII .
  16. 16. Fallat ME, Siow Y, Marra M, Cook C, Carrillo A (1997) Müllerian-inhibiting substance in follicular fluid and serum: a comparison of patients with tubal factor infertility, polycystic ovary syndrome, and endometriosis. Fertil Steril 67: 962-965. doi:https://doi.org/10.1016/S0015-0282(97)81417-3. PubMed: 9130910.
  17. 17. Pigny P, Jonard S, Robert Y, Dewailly D (2006) Serum Anti-Müllerian Hormone as a Surrogate for Antral Follicle Count for Definition of the Polycystic Ovary Syndrome. J Clin Endocrinol Metab 91: 941-945. PubMed: 16368745.
  18. 18. Long WQ, Ranchin V, Pautier P, Belville C, Denizot P et al. (2000) Detection of minimal levels of serum anti-Müllerian hormone during follow-up of patients with ovarian granulosa cell tumor by means of a highly sensitive enzyme-linked immunosorbent assay. J Clin Endocrinol Metab 85: 540-544. doi:https://doi.org/10.1210/jc.85.2.540. PubMed: 10690852.
  19. 19. Cook CL, Siow Y, Taylor S, Fallat ME (2000) Serum Müllerian inhibiting substance levels during normal mestrual cycles. Fertil Steril 73: 859-861. doi:https://doi.org/10.1016/S0015-0282(99)00639-1. PubMed: 10731554.
  20. 20. La Marca A, Stabile G, Artenisio AC, Volpe A (2006) Serum anti-Müllerian hormone throughout the human menstrual cycle. Hum Reprod 21: 3103-3107. doi:https://doi.org/10.1093/humrep/del291. PubMed: 16923748.
  21. 21. Hehenkamp WJK, Looman CWN, Themmen APN, de Jong FH, te Velde ER et al. (2006) Anti-Müllerian Hormone Levels in the Spontaneous Menstrual Cycle Do Not Show Substantial Fluctuation. J Clin Endocrinol Metab 91: 4057-4063. doi:https://doi.org/10.1210/jc.2006-0331. PubMed: 16804046.
  22. 22. Josso N, Legeai L, Forest MG, Chaussain JL, Brauner R (1990) An enzyme linked immunoassay for anti-Müllerian hormone: a new tool for the evaluation of testicular function in infants and children. J Clin Endocrinol Metab 70: 23-27. doi:https://doi.org/10.1210/jcem-70-1-23. PubMed: 1688440.
  23. 23. Hudson PL, Dougas I, Donahoe PK, Cate RL, Epstein J et al. (1990) An immunoassay to detect human mullerian inhibiting substance in males and females during normal development. J Clin Endocrinol Metab 70: 16-22. doi:https://doi.org/10.1210/jcem-70-1-16. PubMed: 2294129.
  24. 24. Baker ML, Metcalfe SA, Hutson JM (1990) Serum levels of mullerian inhibiting substance in boys from birth to 18 years, as determined by enzyme immunoassay. J Clin Endocrinol Metab 70: 11-15. doi:https://doi.org/10.1210/jcem-70-1-11. PubMed: 2403569.
  25. 25. Rey RA, Belville C, Nihoul-Fékété C, Michel-Calemard L, Forest MG et al. (1999) Evaluation of gonadal function in 107 intersex patients by means of serum antimüllerian hormone measurement. J Clin Endocrinol Metab 84: 627-631. doi:https://doi.org/10.1210/jc.84.2.627. PubMed: 10022428.
  26. 26. Cook CL, Siow Y, Brenner AG, Fallat ME (2002) Relationship between serum müllerian-inhibiting substance and other reproductive hormones in untreated women with polycystic ovary syndrome and normal women. Fertil Steril 77: 141-146. doi:https://doi.org/10.1016/S0015-0282(01)02944-2. PubMed: 11779604.
  27. 27. Fréour T, Mirallié S, Bach-Ngohou K, Denis M, Barrière P et al. (2007) Measurement of serum Anti-Müllerian Hormone by Beckman Coulter ELISA and DSL ELISA: Comparison and relevance in Assisted Reproduction Technology (ART). Clin Chim Acta 375: 162-164. doi:https://doi.org/10.1016/j.cca.2006.06.013. PubMed: 16860302.
  28. 28. Fanchin R, Schonäuer LM, Righini C, Frydman N, Frydman R et al. (2003) Serum anti-Müllerian hormone dynamics during controlled ovarian hyperstimulation. Hum Reprod 18: 328-332. doi:https://doi.org/10.1093/humrep/deg043. PubMed: 12571169.
  29. 29. Grinspon RP, Ropelato MG, Bedecarrás P, Loreti N, Ballerini MG et al. (2012) Gonadotrophin secretion pattern in anorchid boys from birth to pubertal age: pathophysiological aspects and diagnostic usefulness. Clin Endocrinol (Oxf) 76: 698-705. doi:https://doi.org/10.1111/j.1365-2265.2011.04297.x. PubMed: 22098623.
  30. 30. Olmedo SB, Rawe VY, Nodar FN, Galaverna GD, Acosta AA, Chemes HE (2000) Pregnancies established through intracytoplasmic sperm injection (ICSI) using spermatozoa with dysplasia of fibrous sheath. Asian J Androl 2: 125-130. PubMed: 11232789.
  31. 31. Muttukrishna S, Suharjono H, McGarrigle H, Sathanandan M (2004) Inhibin B and anti-Müllerian hormone: markers of ovarian response in IVF/ICSI patients? BJOG: An International Journal of Obstetrics and Gynaecology 111: 1248-1253. doi:https://doi.org/10.1111/j.1471-0528.2004.00452.x. PubMed: 15521870.
  32. 32. Muttukrishna S, McGarrigle H, Wakim R, Khadum I, Ranieri DM et al. (2005) Antral follicle count, anti-Müllerian hormone and inhibin B: predictors of ovarian response in assisted reproductive technology? BJOG 112: 1384-1390. doi:https://doi.org/10.1111/j.1471-0528.2005.00670.x. PubMed: 16167941.
  33. 33. Peñarrubia J, Fábregues F, Manau D, Creus M, Casals G et al. (2005) Basal and stimulation day 5 anti-Müllerian hormone serum concentrations as predictors of ovarian response and pregnancy in assisted reproductive technology cycles stimulated with gonadotropin-releasing hormone agonist-gonadotropin treatment. Hum Reprod 20: 915-922. PubMed: 15665015.
  34. 34. Ebner T, Sommergruber M, Moser M, Shebl O, Schreier-Lechner E et al. (2006) Basal level of anti-Müllerian hormone is associated with oocyte quality in stimulated cycles. Hum Reprod 21: 2022-2026. doi:https://doi.org/10.1093/humrep/del127. PubMed: 16679324.
  35. 35. McIlveen M, Skull JD, Ledger WL (2007) Evaluation of the utility of multiple endocrine and ultrasound measures of ovarian reserve in the prediction of cycle cancellation in a high-risk IVF population. Hum Reprod 22: 778-785. PubMed: 17114197.
  36. 36. Smeenk JMJ, Sweep FCGJ, Zielhuis GA, Kremer JAM, Thomas CMG et al. (2007) AntiMüllerian hormone predicts ovarian responsiveness, but not embryo quality or pregnancy, after in vitro fertilization or intracyoplasmic sperm injection. Fertil Steril 87: 223-226. doi:https://doi.org/10.1016/j.fertnstert.2006.06.019. PubMed: 17081531.
  37. 37. Nelson SM, Yates RW, Fleming R (2007) Serum anti-Müllerian hormone and FSH: prediction of live birth and extremes of response in stimulated cycles--implications for individualization of therapy. Hum Reprod 22: 2414-2421. doi:https://doi.org/10.1093/humrep/dem204. PubMed: 17636277. dem204 . PII .
  38. 38. Fridén B, Sjöblom P, Menezes J (2011) Using anti-Müllerian hormone to identify a good prognosis group in women of advanced reproductive age. Aust N Z J Obstet Gynaecol 51: 411-415. doi:https://doi.org/10.1111/j.1479-828X.2011.01374.x. PubMed: 21988118.
  39. 39. Jayaprakasan K, Campbell B, Hopkisson J, Johnson I, Raine-Fenning N (2010) A prospective, comparative analysis of anti-Müllerian hormone, inhibin-B, and three-dimensional ultrasound determinants of ovarian reserve in the prediction of poor response to controlled ovarian stimulation. Fertil Steril 93: 855-864. doi:https://doi.org/10.1016/j.fertnstert.2008.10.042. PubMed: 19046583.
  40. 40. Seifer DB, MacLaughlin DT, Christian BP, Feng B, Shelden RM (2002) Early follicular serum müllerian-inhibiting substance levels are associated with ovarian response during assisted reproductive technology cycles. Fertil Steril 77: 468-471. doi:https://doi.org/10.1016/S0015-0282(01)03201-0. PubMed: 11872196.
  41. 41. Hazout A, Bouchard P, Seifer DB, Aussage P, Junca AM et al. (2004) Serum antimullerian hormone/mullerian-inhibiting substance appears to be a more discriminatory marker of assisted reproductive technology outcome than follicle-stimulating hormone, inhibin B, or estradiol. Fertil Steril 82: 1323-1329. doi:https://doi.org/10.1016/j.fertnstert.2004.03.061. PubMed: 15533354.
  42. 42. Silberstein T, MacLaughlin DT, Shai I, Trimarchi JR, Lambert-Messerlian G et al. (2006) Mullerian inhibiting substance levels at the time of HCG administration in IVF cycles predict both ovarian reserve and embryo morphology. Hum Reprod 21: 159-163. doi:https://doi.org/10.1093/oxfordjournals.humrep.a002489. PubMed: 16123085.
  43. 43. Lekamge DN, Barry M, Kolo M, Lane M, Gilchrist RB, Tremellen KP (2007) Anti-Müllerian Hormone as a predictor of IVF outcome. Reprod Biomed Online 14: 602-610. doi:https://doi.org/10.1016/S1472-6483(10)61053-X. PubMed: 17509203.
  44. 44. Lie Fong S, Baart EB, Martini E, Schipper I, Visser JA et al. (2008) Anti-Müllerian Hormone: a marker for oocyte quantity, oocyte quality and embryo quality? Reprod Biomed Online 16: 664-670. doi:https://doi.org/10.1016/S1472-6483(10)60480-4. PubMed: 18492370.