help button home button Endocrine Society JCEM
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2007-2205
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kevenaar, M. E.
Right arrow Articles by Visser, J. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kevenaar, M. E.
Right arrow Articles by Visser, J. A.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*OMIM
*Protein*SNP
*UniGene
*Substance via MeSH
Related Collections
Right arrow Female Endocrinology
The Journal of Clinical Endocrinology & Metabolism Vol. 93, No. 4 1310-1316
Copyright © 2008 by The Endocrine Society

A Functional Anti-Müllerian Hormone Gene Polymorphism Is Associated with Follicle Number and Androgen Levels in Polycystic Ovary Syndrome Patients

Marlies E. Kevenaar, Joop S. E. Laven, Sharon Lie Fong, André G. Uitterlinden, Frank H. de Jong, Axel P. N. Themmen and Jenny A. Visser

Department of Internal Medicine (M.E.K., A.G.U., F.H.d.J., A.P.N.T., J.A.V.), Department of Obstetrics and Gynaecology, Division of Reproductive Medicine (J.S.E.L., S.L.F.), and Departments of Epidemiology and Biostatistics (A.G.U.) and Clinical Chemistry (A.G.U.), Erasmus MC, 3000 CA Rotterdam, The Netherlands

Address all correspondence and requests for reprints to: Jenny A. Visser, Ph.D., Department of Internal Medicine, Room Ee532, Erasmus MC, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands. E-mail: j.visser{at}erasmusmc.nl.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Context: The common characteristic of polycystic ovary syndrome (PCOS) is a disturbance in the selection of the dominant follicle, resulting in anovulation. In PCOS women, serum anti-Müllerian hormone (AMH) levels are elevated. Because AMH decreases FSH sensitivity in mice, the elevated AMH levels may contribute to the disturbed follicle selection in PCOS women.

Objective: The objective of the study was to investigate the role of the AMH signaling pathway in the pathophysiology of PCOS using a genetic approach.

Design: The association of the AMH Ile49Ser (rs10407022) and the AMH type II receptor –482 A>G (rs2002555) polymorphism with PCOS susceptibility and phenotype was studied in a large cohort of PCOS women.

Setting/Subjects: A total of 331 women with PCOS, 32 normoovulatory controls, and 3635 population-based controls were included.

Main Outcome Measures: Ovarian parameters, serum AMH, FSH, androgen, and estradiol levels were measured.

Results: Genotype and allele frequencies for the AMH Ile49Ser and AMH type II receptor –482 A>G polymorphism were similar in PCOS women and controls. However, within the group of PCOS women, carriers of the AMH 49Ser allele less often had polycystic ovaries (92.7 vs. 99.5%, P = 0.0004), lower follicle numbers (P = 0.03), and lower androgen levels, compared with noncarriers (P = 0.04). In addition, in vitro studies demonstrated that the bioactivity of the AMH 49Ser protein is diminished, compared with the AMH 49Ile protein (P < 0.0001).

Conclusions: Genetic variants in the AMH and AMH type II receptor gene do not influence PCOS susceptibility. However, our results suggest that the AMH Ile49Ser polymorphism contributes to the severity of the PCOS phenotype.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
Polycystic ovary syndrome (PCOS) is the most frequent endocrine disorder and most common cause of anovulation in women of reproductive age (1). According to the Rotterdam 2003 consensus (2), PCOS is characterized by two of the following three criteria: oligo- or anovulation, clinical or biochemical hyperandrogenism, and polycystic ovaries on ultrasound. The etiology of this very heterogeneous syndrome is still poorly understood. However, a major genetic component has been demonstrated, i.e. the heritability estimate in monozygotic twin sisters is 0.72 (3), and a candidate gene approach has been widely used to identify the molecular genetic mechanisms and the metabolic and/or biochemical pathways that are implicated in the etiology of PCOS (4). In particular, candidate genes involved in pathways that regulate gonadotropin secretion, affect androgen production and action, and influence insulin signaling have been considered (reviewed in Refs. 4 and 5). However, little is known about factors involved in early follicle development (6). An important regulator of folliculogenesis that may play a role in the pathophysiology of PCOS is anti-Müllerian hormone (AMH), also known as Müllerian inhibiting substance (7). AMH is produced by the granulosa cells of growing follicles in the ovary, and serum AMH levels correlate with the number of antral follicles as observed by transvaginal ultrasound (8). PCOS women display a 2- to 3-fold increase in serum AMH levels, compared with normoovulatory women, reflecting the increased number of small antral follicles (9, 10).

Although little is known about the role of AMH in the human ovary, studies in mice showed that AMH inhibits initial recruitment (11) and reduces FSH sensitivity of growing follicles (12). Given its comparable expression pattern in women and mice (13, 14), AMH may have similar roles in human ovarian folliculogenesis. Hence, the high AMH levels in women with PCOS may contribute to their aberrant follicle selection. Because AMH inhibits FSH-induced aromatase activity in in vitro cultured mouse (15) and human granulosa cells (16), AMH may also be responsible for the reduced aromatase activity in PCOS granulosa cells (7, 17) and contribute to the elevated androgen levels in PCOS women. Indeed, AMH serum levels are positively correlated with androgen levels in PCOS patients (9, 10), supporting the latter hypothesis.

Recently we showed that two genetic variants of AMH (Ile49Ser; rs10407022) and its specific type II receptor (AMHR2 –482 A>G; rs2002555) genes are associated with estradiol levels in normoovulatory women, suggesting that these polymorphisms modulate intraovarian FSH sensitivity and thereby aromatase activity (18). In this study, we investigated for the first time whether these genetic variants of the AMH signaling pathway are associated with the susceptibility or phenotype of PCOS in a large Dutch Caucasian cohort of PCOS women (n = 331). In addition, in vitro studies were performed to analyze the functional aspects of the AMH Ile49Ser polymorphism.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subjects

The local Medical Ethics Review Committee approved of this study, and informed consent was obtained from all participants. Dutch Caucasian patients attending our fertility clinic between 1993 and 2004, who fulfilled the definition of PCOS according to the Rotterdam criteria (2) were enrolled in this study (n = 407). Hyperandrogenism was defined as elevated (>4.5) free androgen index (testosterone x 100/SHBG). Polycystic ovaries were defined as 12 or more follicles (measuring 2–9 mm) per ovary, and the ovarian volume was considered to be increased above 10 ml (19). Standardized initial screening (clinical investigation, transvaginal ultrasound, and fasting blood withdrawal) was performed on a random cycle day between 0900 and 1100 h, irrespective of the interval between blood sampling and the preceding menses.

DNA was available of 394 PCOS women, and genotyping for the AMH and AMHR2 polymorphisms was successful in 385 women. In a subset of 331 women, AMH serum levels and ultrasound data were available.

For sonographic imaging, we used a 6.5-MHz vaginal transducer (model EUB-415; Hitachi Medical Corp., Tokyo, Japan). Ovarian volume and follicle number were assessed as described earlier (20). Serum hormone levels were assessed at the time patients were originally seen using the following assays: serum FSH, LH, and SHBG were measured by luminescence-based immunometric assays (Immulite 2000; Diagnostic Products Corp., Los Angeles, CA). Serum estradiol and testosterone were measured using RIAs (Diagnostic Products Corp.). Serum androstenedione was measured using the Immulite 2000. Inhibin B was measured using an enzyme-immunometric assay (Oxford BioInnovation, Oxford, UK). AMH levels were measured collectively in samples stored frozen using an in-house AMH ELISA (21), commercially available through Diagnostic Systems Laboratories (Webster, TX). We have previously shown that AMH immunoreactivity in serum is stable during storage and after repeated freeze thaw cycles (21). Intra- and interassay coefficients of variation were less than 3 and 5.8% for FSH, less than 3.5 and 7.1% for LH, less than 10.9 and 10.7% for androstenedione, less than 6.0 and 4.8% for SHBG, less than 10.2 and 8.8% for estradiol, less than 5.7 and 8.4% for testosterone, less than 7.0 and 15% for inhibin B, and less than 3.5 and 4% for AMH. Free testosterone levels were calculated using the equation according to Sodergard et al. (22, 23).

The control group consisted of 32 Dutch Caucasian normoovulatory women in whom the AMH and AMHR2 polymorphism were genotyped, as described previously (18). Inclusion criteria were a regular menstrual cycle (26–30 d), age between 20 and 36 yr, and normal body mass index (BMI) (18–25 kg/m2). Serum testosterone, serum AMH, and follicle number were assessed on d 3 of the menstrual cycle using the methods described above.

In addition, a large population-based cohort of the elderly, the Rotterdam study, was used to determine the allele and genotype frequency of the AMH and AMHR2 polymorphisms in the general Dutch Caucasian population. The design and rationale of this study have been described earlier (24). For the present study, only women were included (n = 3635).

Furthermore, allele frequencies of the AMH and AMHR2 polymorphisms in the PCOS group were compared with frequencies reported for Caucasians in the HapMap database (www.hapmap.org) (25).

Genotyping

Genomic DNA was extracted from peripheral blood using standard DNA extraction methods. Previously we have shown that the AMH Ile49Ser and AMHR2 –482 A>G polymorphism are both in complete linkage disequilibrium (D' = 1 and r2 = 1) with additional polymorphisms with an allele frequency greater than 10% located in the coding and noncoding regions of each gene, including 1 kb of the promoter region (18). The AMH Ile49Ser and AMHR2 –482 A>G genotypes were determined using Taqman allelic discrimination assays. For the AMH Ile49Ser polymorphism, an Assay-by-Design (Applied Biosystems, Nieuwerkerk aan den IJssel, The Netherlands) with the following probes was used: 5'-CTCCAGGCAtCCCACAA-3' and 5'-CCAGGCAgCCCACAA-3'. For the AMHR2 –482 A>G promoter single-nucleotide polymorphism, we used an Assay-on-Demand, Assay (ID C_1673084_10). Reactions were performed as described previously (18).

Recombinant human AMH (hAMH) production

The full-length hAMH cDNA was isolated from human testis and subcloned into the pcDNA3.1 expression vector (Invitrogen, Breda, The Netherlands) as described previously (14). Quick-change site-directed mutagenesis was performed according to the manufacturer (Stratagene, Amsterdam, The Netherlands) to introduce the Ile49Ser polymorphism. HEK293 cells were transfected with the hAMH-49Ile and hAMH-49Ser expression vectors. Cells transfected with the empty pcDNA3.1 vector served as control. Supernatants were collected under serum-free culture conditions and were concentrated approximately 40-fold using a Centriprep system (Millipore Corp., Amsterdam, The Netherlands), and the amount of AMH was measured by the in-house AMH ELISA as described previously (21).

Western blot

Western blot analysis was performed using the mouse monoclonal antibodies 5/6A and 9/6A (14, 26). Proteins from conditioned medium were separated using 12% PAGE under reducing conditions, transferred to nitrocellulose membranes, and incubated with the 5/6A or 9/6A antibody at a 1:1000 dilution, followed by a secondary Alexa Fluor-680 goat antimouse antibody (Molecular Probes, Invitrogen, Breda, The Netherlands) at a 1:15,000 dilution. Proteins were visualized using the Licor-Odyssey imaging system and blots were analyzed with the Odyssey software version 2.1 (LI-COR Biosciences, Westburg, Leusden, The Netherlands).

Cell culture and transfections

The mouse granulosa cell line KK-1 (27) (kind gift from Dr. I. Huhtaniemi, Institute of Reproduction and Developmental Biology, Imperial College of London, London, UK), and the human granulosa cell line COV434, derived from a human granulosa cell tumor but possessing many characteristics of normal granulosa cells (28, 29), were cultured in DMEM/F12 (Life Technologies, Inc., Invitrogen, Breda, The Netherlands) containing 10% fetal calf serum and penicillin (400 IU/ml) and streptomycin (0.4 mg/ml), and stably transfected with an AMHRII expression vector (30). For AMH-induced luciferase assays, KK-1 and COV434 cells were seeded at 20% confluency in 24-well plates and transfected with the BRE-Luc reporter plasmid (150 ng/well) (31) (kind gift from Dr. P. ten Dijke, Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands) using Fugene 6 transfection reagent (Roche Diagnostics, Almere, The Netherlands) The pRL-TK plasmid (Promega, Leiden, The Netherlands) served as an internal control to normalize for transfection efficiency. Twenty-four hours after transfection, cells were cultured for 2 h in medium containing 0.2% fetal calf serum followed by 16 h treatment with increasing concentrations of the hAMH variants. Luciferase activity was determined using the Dual-Glo luciferase assay (Promega) in the TOPCOUNT luminometer (Applied Biosystems).

Statistical analysis

AMH levels were compared between normoovulatory controls and PCOS women using one-way analysis of (co)variance, with adjustment for age and BMI. Within the PCOS cohort, Spearman’s correlation coefficient was used to correlate AMH serum levels with additional hormone levels and total follicle number.

In each group of women, genotype frequencies of the AMH and AMHR2 polymorphisms were tested for Hardy-Weinberg equilibrium proportions using the ARLEQUIN package (32). Differences in allele and genotype frequencies between cases and controls were tested using a {chi}2 test. For reasons of statistical power, carriers of the AMH 49Ser allele and carriers of the AMHR2 –482G allele were compared with noncarriers. If appropriate, hormone levels were log transformed to normalize their distribution. Within the PCOS group, one-way analysis of (co)variance was used to determine differences in continuous variables between genotype groups. Androgen-related traits and ovarian parameters were adjusted for age and BMI. Categorical parameters were analyzed using Fisher’s exact test. Subsequently, to correct for multiple testing, we obtained an empirical P value by permutation analysis using Haploview version 3.32 (33). The phenotypic status of each individual was permuted 10,000 times and association analysis was performed to obtain the test statistic under the null hypothesis of no association. The empirical P value was obtained as the proportion of the 10,000 replicates that had a P value less than or equal to the one obtained from the actual (unshuffled) data.

Prism software was used to fit the sigmoidal dose-response curves of the in vitro studies and to calculate the EC50 and maximal response values. To test differences in EC50 and maximal response between the AMH variants, the F test comparison method was used (GraphPad Prism 4.0 Software, GraphPad Inc., San Diego, CA).

Unless stated otherwise, analyses were performed using Statistical Package for Social Sciences, SPSS, version 11.0.1 (SPSS Inc., Chicago, IL). P ≤ 0.05 was considered to be significant.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
AMH serum levels in PCOS women

Clinical characteristics of the normoovulatory women and the PCOS women are shown in Table 1Go. As shown previously, PCOS patients had elevated AMH levels, compared with normoovulatory controls (Table 1Go). To provide insight into the relationship of AMH serum levels with androgen levels and follicle number in the PCOS cohort, correlation coefficients between AMH levels and these parameters were determined. In the PCOS cohort, serum AMH levels were positively correlated with total testosterone levels (r = 0.44, P < 0.001), free testosterone levels (r = 0.36, P < 0.001), androstenedione levels (r = 0.44, P < 0.001), LH levels (r = 0.31, P < 0.001), and total follicle number (r = 0.54, P < 0.001) but not with estradiol levels (r = 0.01, P = 0.80).


View this table:
[in this window]
[in a new window]

  		TABLE 1. Clinical characteristics of normoovulatory and PCOS women

 
PCOS risk by AMH and AMHR2 genotypes

Genotype and allele frequencies of the AMH Ile49Ser and the AMHR2 –482 A>G polymorphism in the 331 PCOS women did not differ from frequencies in the normoovulatory controls and the (postmenopausal) women of the Rotterdam study (Table 2Go). In addition, the allele frequencies for both polymorphisms in PCOS women were similar to allele frequencies of Caucasians in the HapMap database (www.hapmap.org) (25). The genotype distributions of the AMH Ile49Ser and the AMHR2 –482 A>G polymorphism were in Hardy Weinberg equilibrium proportions in both cases and controls (results not shown).


View this table:
[in this window]
[in a new window]

  		TABLE 2. Genotype distributions of AMH and AMHR2 polymorphisms in PCOS cases and controls

 
PCOS phenotype by AMH Ile49Ser genotype

Within the PCOS cohort, genotypes of the AMH Ile49Ser polymorphism were not associated with general characteristics, such as age, BMI and waist hip ratio (Table 3Go). However, carriers of the AMH 49Ser allele had polycystic ovaries less often, compared with noncarriers (92.7 vs. 99.5%, P = 0.0008), which remained significant after permutation analysis (P = 0.0004) (Table 3Go). Hence, 49Ser allele carriers also had a lower total follicle number, on average 5.4 follicles less (12%), compared with noncarriers (P = 0.03) (Table 3Go). The mean ovarian volume and the percentages of women with amenorrhea were not different between both genotype groups (Table 3Go).


View this table:
[in this window]
[in a new window]

  		TABLE 3. General characteristics and ovarian parameters of PCOS patients by AMH Ile49Ser and AMHR2 –482 A>G genotypes

 
AMH levels were similar between carriers and noncarriers of the AMH 49Ser allele, but AMH 49Ser allele carriers had almost 10% lower total testosterone (P = 0.05) and androstenedione levels (P = 0.04), compared with noncarriers (Table 4Go). However, SHBG levels were also lower in AMH 49Ser allele carriers, compared with noncarriers (P = 0.04), and hence, free testosterone levels were not significantly different between the AMH genotype groups (P = 0.17). FSH, LH, estradiol, and inhibin B levels were similar in both groups (Table 4Go).


View this table:
[in this window]
[in a new window]

  		TABLE 4. Hormone levels in PCOS patients by AMH Ile49Ser and AMHR2 –482 A>G genotypes

 
Functional analysis of the AMH Ile49Ser polymorphism

The results described above suggest that the AMH Ile49Ser polymorphism modulates AMH function. To determine whether this polymorphism had an effect on secretion and/or processing of AMH, Western blot analysis of supernatants from hAMH-49Ile and hAMH-49Ser expressing cells was performed using proregion-specific (Mab 9/6A) and mature region-specific (Mab 5/6A) antibodies. For both AMH variants, the N-terminal proregion, the C-terminal mature region and an additional cleavage band as a result of a potential second cleavage site were detected with comparable intensities (Fig. 1Go), suggesting similar processing. The observed incomplete processing of recombinant hAMH-49Ile and hAMH-49Ser is consistent with previous reports (34, 35). Introduction of an optimized cleavage site (RARR) resulted in fully cleaved AMH. Again, no differences in processing between hAMH-RARR-49Ile and hAMH-RARR-49Ser were observed (results not shown).


Figure 1
View larger version (11K):
[in this window]
[in a new window]

  		FIG. 1. Western blot analysis of human recombinant AMH variants, AMH-49Ile (Ile) and AMH-49Ser (Ser), using mouse monoclonal AMH antibodies. The antibody 5/6A recognized the C terminal approximately 12 kDa mature region of AMH, including the stable dimer. The antibody 9/6A recognizes the full-length N terminal approximately 57 kDa pro-region and a second subunit due to a possible second cleavage site (~40 kDa, indicated by arrowhead). In addition, these antibodies recognize the AMH precursor protein. No AMH protein is detected in the control medium (c). Relative molecular mass (kilodaltons) of the standards is indicated on the left. No difference was observed in the cleavage of the AMH variants.

 
To determine the effect of the Ile49Ser polymorphism on AMH bioactivity, a mouse granulosa cell line (KK-1) and a human granulosa cell line (COV434) were transiently transfected with the AMH-responsive luciferase reporter BRE-Luc and stimulated with hAMH-49Ile and hAMH-49Ser. Dose-response experiments in both cell lines revealed that stimulation with equal amounts of hAMH-49Ser resulted in a similar EC50 but a significantly lower maximal induction of luciferase activity, compared with stimulation with hAMH-49Ile (P < 0.0001) (Fig. 2Go, and results not shown). These observations suggest that the hAMH-49Ser constitutes an AMH protein with lower bioactivity.


Figure 2
View larger version (8K):
[in this window]
[in a new window]

  		FIG. 2. Dose-response analysis of the recombinant human AMH variants in mouse KK-1/AMHRII cells. KK-1/AMHRII cells were transiently transfected with a luciferase reporter plasmid and incubated with equal concentration ranges of rhAMH-49Ile (black line) or rhAMH-49Ser (gray line). Stimulation with rhAMH-49Ser protein resulted in a similar EC50 but a lower maximum response, compared with stimulation with rhAMH-49Ile (*, P < 0.0001). Data are expressed as relative luciferase units (RLU) and are the mean ± SEM of triplicates from a representative experiment that was performed at least three times with two independent batches of the recombinant AMH variants from independent cultures. Some error bars are too small to be visible in the graph.

 
PCOS phenotype by AMHR2 –482 A>G genotype

The AMHR2 –482 A>G polymorphism was not associated with age, BMI, and waist hip ratio (Table 3Go). Furthermore, no association of the AMHR2 genotypes with polycystic ovaries, follicle number, or ovarian volume was observed (Table 3Go). AMH serum levels, androgen levels, and other hormones also did not differ between the genotype groups (Table 4Go).


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
The present study was designed to investigate the functional role of AMH in PCOS using a genetic approach. The association of polymorphisms in genes of the AMH signaling pathway with PCOS susceptibility and phenotype was studied in a large cohort of PCOS women. We observed that polymorphisms in the AMH and AMHR2 gene did not contribute to the risk for PCOS. However, within the PCOS group, the AMH Ile49Ser polymorphism was associated with follicle number, androgen levels, and percentage of women exhibiting polycystic ovaries.

The main feature of follicular dysfunction in PCOS is the disturbed selection of the dominant follicle resulting in anovulation. Studies in mice have shown that AMH decreases FSH sensitivity in vivo and in vitro (12). Hence, the elevated AMH levels in PCOS women may contribute to the refractoriness to FSH-induced follicle differentiation, leading to the aberrant selection process. The observed association of the AMH Ile49Ser variant with follicular parameters supports this hypothesis. The 49Ser variant is associated with a lower follicle number and a lower PCO frequency, compared with the 49Ile variant, suggesting that the AMH 49Ser variant is less effective in reducing the individual FSH sensitivity of antral follicles. Indeed, our in vitro studies demonstrated that the bioactivity of the AMH 49Ser protein is diminished, compared with the AMH 49Ile protein.

The Ile49Ser polymorphism is located in the proregion of the AMH protein. It has been suggested that this domain is involved in protein stability and folding, and mutations within this proregion could affect biosynthesis or bioactivity of AMH. Indeed, mutations in the proregion of AMH can render the protein inactive as has been demonstrated in patients with persistent Müllerian duct syndrome (36). The AMH Ile49Ser polymorphism did not affect the processing of AMH but did affect its bioactivity. The presence of a serine at amino acid position 49 possibly alters the folding of the protein, rendering it slightly less bioactive, compared with the protein with an isoleucine at this position. However, EC50 values were not changed, suggesting that the binding/interaction of both variants with the AMHRII/type I receptor complex is not different but that the 49Ser variant induces weaker or altered conformational changes in the receptor complex that lead to a lower maximal transduction efficacy, compared with the 49Ile variant.

Although the etiology of PCOS is not clearly established, accumulating evidence suggests that PCOS results primarily from exposure of the fetal ovary to high androgen levels (37). Subsequently secondary genetic and environmental factors may interact with this underlying process and lead to the heterogeneity in the phenotype of PCOS (38). The absence of an association of the functional AMH Ile49Ser polymorphism with PCOS risk indicates that the AMH signaling pathway is not directly involved in the pathophysiology of PCOS. Nevertheless, the association of this polymorphism with follicle number suggests that AMH may be one of the factors modifying the final PCOS phenotype.

In addition, AMH may be responsible for the diminished induction of aromatase activity in PCOS granulosa cells (7). In a previous study, we observed that normoovulatory women carrying the AMH 49Ser allele had higher follicular phase estradiol levels, compared with women carrying the AMH 49Ile allele, also suggesting that the AMH 49Ser variant results in less inhibition of FSH-induced aromatase activity in normal granulosa cells (18).

In the present study, PCOS women carrying the 49Ser allele had lower androstenedione and testosterone levels, compared with noncarriers, also suggesting less inhibition of FSH-induced aromatase activity. Nevertheless, the AMH Ile49Ser polymorphism was not associated with estradiol levels, but this may be explained by the peripheral conversion of androgens, also contributing to final serum estradiol levels and thereby masking the subtle differences in follicular fluid estradiol levels. Indeed, serum AMH levels were also not correlated with serum estradiol levels in the PCOS cohort.

Interestingly, AMH is located in the same chromosomal region (19p13) as a promising locus for genetic susceptibility for PCOS [STS marker D19S884, chr 19p13.2 (39, 40)]. However, the AMH Ile49Ser polymorphism is not associated with PCOS risk, and given the large distance and the lack of linkage disequilibrium between the AMH Ile49Ser polymorphism and this marker (about 5850 kb) or any single-nucleotide polymorphisms in its region [based on HapMap database (25)], it is very unlikely that our findings with the AMH Ile49Ser polymorphism are related to the proposed candidate gene region on chromosome 19p13.2.

The AMHR2 –482 A>G polymorphism is associated with neither PCOS susceptibility nor the final phenotype. Although we observed in our previous study that the AMHR2 –482G allele was associated with higher follicular phase estradiol levels in premenopausal women, suggesting less inhibition of FSH-sensitivity by this AMHR2 variant (18), in PCOS women this effect may be masked by the elevated AMH levels.

Candidate gene studies in PCOS suffer from a lack of reproducibility between cohorts, which may be attributed to the different criteria used to define PCOS but also to several additional factors (4). First, most PCOS studies reported so far have been based on very small sample sizes, therefore lacking sufficient statistical power. In contrast, our cohort, which consists of 331 PCOS women, is among the largest studied in PCOS genetics. Second, in many studies only one or a few variants per gene have been tested, whereas it is critical to characterize the genetic variation of the entire candidate gene to unravel the etiology of genetically complex diseases such as PCOS (4). In our study, the analyzed polymorphisms in the AMH and AMHR2 gene both capture the common genetic variation in the gene, including 1 kb of the promoter region (18). Last but not least, the issue of multiple testing requires attention in association studies. Nevertheless, the association of the AMH Ile49Ser polymorphism with the PCO phenotype withstands correction for multiple testing using permutation analysis. In addition, the strong a priori rationale in combination with the functional evidence makes it very unlikely that our results could be explained by chance alone (41).

In conclusion, our results provide new insight into the role of AMH in the pathophysiology of PCOS. The observed association between the AMH Ile49Ser polymorphism and follicle number and androgen levels, together with the in vitro evidence of the functional effect of this polymorphism, strongly suggests that AMH contributes to the severity of the PCOS phenotype. Although the findings of the association study need to be replicated in additional cohorts, our results imply that the AMH Ile49Ser polymorphism is one of the genetic factors contributing to the complex etiology of PCOS.


   Acknowledgments
 
The authors thank O. Valkenburg for data collection of the PCOS cohort and Professor Dr. H. A. P. Pols for critical reading of the manuscript. Furthermore, the authors thank the participants of the three study cohorts and acknowledge all participating general practitioners and field workers in the research center of the Rotterdam Study in Ommoord, Rotterdam, The Netherlands.


   Footnotes
 
Disclosure Statement: M.E.K., J.S.E.L., S.L.F., A.G.U., F.H.d.J., and J.A.V. have nothing to disclose. A.P.N.T. consults for Diagnostic Systems Laboratories.

First Published Online January 29, 2008

Abbreviations: AMH, Anti-Müllerian hormone; AMHR2, AMH type II receptor; BMI, body mass index; hAMH, human AMH; PCOS, polycystic ovary syndrome.

Received October 2, 2007.

Accepted January 23, 2008.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

  1. Franks S 1995 Polycystic ovary syndrome. N Engl J Med 333:853–861[Free Full Text]
  2. The Rotterdam ESHRE/ASRM-Sponsored PCOS Consensus Workshop Group 2004 Revised 2003 consensus on diagnostic criteria and long-term health risks related to polycystic ovary syndrome (PCOS). Hum Reprod 19:41–47[Abstract/Free Full Text]
  3. Vink JM, Sadrzadeh S, Lambalk CB, Boomsma DI 2006 Heritability of polycystic ovary syndrome in a Dutch twin-family study. J Clin Endocrinol Metab 91:2100–2104[Abstract/Free Full Text]
  4. Urbanek M 2007 The genetics of the polycystic ovary syndrome. Nat Clin Pract Endocrinol Metab 3:103–111[CrossRef][Medline]
  5. Escobar-Morreale HF, Luque-Ramirez M, San Millan JL 2005 The molecular-genetic basis of functional hyperandrogenism and the polycystic ovary syndrome. Endocr Rev 26:251–282[Abstract/Free Full Text]
  6. Franks S 2006 Candidate genes in women with polycystic ovary syndrome. Fertil Steril 86(Suppl 1):S15
  7. Visser JA, de Jong FH, Laven JSE, Themmen APN 2006 Anti-Mullerian hormone: a new marker for ovarian function. Reproduction 131:1–9[Abstract/Free Full Text]
  8. de Vet A, Laven JSE, de Jong FH, Themmen APN, Fauser BCJM 2002 Antimullerian hormone serum levels: a putative marker for ovarian aging. Fertil Steril 77:357–362[CrossRef][Medline]
  9. Pigny P, Merlen E, Robert Y, Cortet-Rudelli C, Decanter C, Jonard S, Dewailly D 2003 Elevated serum level of anti-mullerian hormone in patients with polycystic ovary syndrome: relationship to the ovarian follicle excess and to the follicular arrest. J Clin Endocrinol Metab 88:5957–5962[Abstract/Free Full Text]
  10. Laven JSE, Mulders AGMGJ, Visser JA, Themmen APN, De Jong FH, Fauser BCJM 2004 Anti-Mullerian hormone serum concentrations in normoovulatory and anovulatory women of reproductive age. J Clin Endocrinol Metab 89:318–323[Abstract/Free Full Text]
  11. Durlinger ALL, Kramer P, Karels B, de Jong FH, Uilenbroek JTJ, Grootegoed JA, Themmen APN 1999 Control of primordial follicle recruitment by anti-Mullerian hormone in the mouse ovary. Endocrinology 140:5789–5796[Abstract/Free Full Text]
  12. Durlinger ALL, Gruijters MJG, Kramer P, Karels B, Kumar TR, Matzuk MM, Rose UM, de Jong FH, Uilenbroek JTJ, Grootegoed JA, Themmen APN 2001 Anti-Mullerian hormone attenuates the effects of FSH on follicle development in the mouse ovary. Endocrinology 142:4891–4899[Abstract/Free Full Text]
  13. Durlinger ALL, Visser JA, Themmen APN 2002 Regulation of ovarian function: the role of anti-Mullerian hormone. Reproduction 124:601–609[Abstract]
  14. Weenen C, Laven JSE, Von Bergh AR, Cranfield M, Groome NP, Visser JA, Kramer P, Fauser BCJM, Themmen APN 2004 Anti-Mullerian hormone expression pattern in the human ovary: potential implications for initial and cyclic follicle recruitment. Mol Hum Reprod 10:77–83[Abstract/Free Full Text]
  15. di Clemente N, Goxe B, Remy JJ, Cate R, Josso N, Vigier B, Salesse R 1994 Inhibitory effect of AMH upon the expression of aromatase and LH receptors by cultured granulosa cells of rat and porcine immature ovaries. Endocrine 2:553–558
  16. Grossman MP, Nakajima ST, Fallat ME, Siow Y Mullerian-inhibiting substance inhibits cytochrome P450 aromatase activity in human granulosa lutein cell culture. Fertil Steril, in press
  17. Laven JSE, Imani B, Eijkemans MJC, Fauser BCJM 2002 New approach to polycystic ovary syndrome and other forms of anovulatory infertility. Obstet Gynecol Surv 57:755–767[CrossRef][Medline]
  18. Kevenaar ME, Themmen APN, Laven JSE, Sonntag B, Lie Fong S, Uitterlinden AG, de Jong FH, Pols HAP, Simoni M, Visser JA 2007 Anti-Mullerian hormone and anti-Mullerian hormone type II receptor polymorphisms are associated with follicular phase estradiol levels in normo-ovulatory women. Hum Reprod 22:1547–1554[Abstract/Free Full Text]
  19. Balen AH, Laven JSE, Tan SL, Dewailly D 2003 Ultrasound assessment of the polycystic ovary: international consensus definitions. Hum Reprod Update 9:505–514[Abstract/Free Full Text]
  20. van Santbrink EJP, Hop WCJ, Fauser BCJM 1997 Classification of normogonadotropic infertility: polycystic ovaries diagnosed by ultrasound versus endocrine characteristics of polycystic ovary syndrome. Fertil Steril 67:452–458[CrossRef][Medline]
  21. Kevenaar ME, Meerasahib MF, Kramer P, van de Lang-Born BM, de Jong FH, Groome NP, Themmen APN, Visser JA 2006 Serum Anti-Mullerian hormone levels reflect the size of the primordial follicle pool in mice. Endocrinology 147:3228–3234[CrossRef][Medline]
  22. Sodergard R, Backstrom T, Shanbhag V, Carstensen H 1982 Calculation of free and bound fractions of testosterone and estradiol-17β to human plasma proteins at body temperature. J Steroid Biochem 16:801–810[CrossRef][Medline]
  23. de Ronde W, van der Schouw YT, Pols HA, Gooren LJ, Muller M, Grobbee DE, de Jong FH 2006 Calculation of bioavailable and free testosterone in men: a comparison of 5 published algorithms. Clin Chem 52:1777–1784[Abstract/Free Full Text]
  24. Hofman A, Grobbee DE, de Jong PT, van den Ouweland FA 1991 Determinants of disease and disability in the elderly: the Rotterdam Elderly Study. Eur J Epidemiol 7:403–422[CrossRef][Medline]
  25. The International HapMap Consortium 2003 The International HapMap Project. Nature 426:789–796[CrossRef][Medline]
  26. Al-Qahtani A, Muttukrishna S, Appasamy M, Johns J, Cranfield M, Visser JA, Themmen APN, Groome NP 2005 Development of a sensitive enzyme immunoassay for anti-Mullerian hormone and the evaluation of potential clinical applications in males and females. Clin Endocrinol (Oxf) 63:267–273[CrossRef][Medline]
  27. Kananen K, Markkula M, Rainio E, Su JG, Hsueh AJ, Huhtaniemi IT 1995 Gonadal tumorigenesis in transgenic mice bearing the mouse inhibin {alpha}-subunit promoter/simian virus T-antigen fusion gene: characterization of ovarian tumors and establishment of gonadotropin-responsive granulosa cell lines. Mol Endocrinol 9:616–627[Abstract/Free Full Text]
  28. Zhang H, Vollmer M, De Geyter M, Litzistorf Y, Ladewig A, Durrenberger M, Guggenheim R, Miny P, Holzgreve W, De Geyter C 2000 Characterization of an immortalized human granulosa cell line (COV434). Mol Hum Reprod 6:146–153[Abstract/Free Full Text]
  29. van den Berg-Bakker CA, Hagemeijer A, Franken-Postma EM, Smit VT, Kuppen PJ, van Ravenswaay Claasen HH, Cornelisse CJ, Schrier PI 1993 Establishment and characterization of 7 ovarian carcinoma cell lines and one granulosa tumor cell line: growth features and cytogenetics. Int J Cancer 53:613–620[Medline]
  30. Visser JA, Olaso R, Verhoef-Post M, Kramer P, Themmen APN, Ingraham HA 2001 The serine/threonine transmembrane receptor ALK2 mediates Mullerian inhibiting substance signaling. Mol Endocrinol 15:936–945[Abstract/Free Full Text]
  31. Korchynskyi O, ten Dijke P 2002 Identification and functional characterization of distinct critically important bone morphogenetic protein-specific response elements in the Id1 promoter. J Biol Chem 277:4883–4891[Abstract/Free Full Text]
  32. Schneider S, Roessli D, Excoffier L 2000 Arlequin version 2.000: a software for population genetics data analysis. Geneva: Genetics and Biometry Laboratory, University of Geneva
  33. Barrett JC, Fry B, Maller J, Daly MJ 2005 Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics 21:263–265[Abstract/Free Full Text]
  34. di Clemente N, Wilson C, Faure E, Boussin L, Carmillo P, Tizard R, Picard JY, Vigier B, Josso N, Cate R 1994 Cloning, expression, and alternative splicing of the receptor for anti-Mullerian hormone. Mol Endocrinol 8:1006–1020[Abstract/Free Full Text]
  35. Nachtigal MW, Ingraham HA 1996 Bioactivation of Mullerian inhibiting substance during gonadal development by a kex2/subtilisin-like endoprotease. Proc Natl Acad Sci USA 93:7711–7716[Abstract/Free Full Text]
  36. Belville C, Van Vlijmen H, Ehrenfels C, Pepinsky B, Rezaie AR, Picard JY, Josso N, di Clemente N, Cate RL 2004 Mutations of the anti-mullerian hormone gene in patients with persistent mullerian duct syndrome: biosynthesis, secretion, and processing of the abnormal proteins and analysis using a three-dimensional model. Mol Endocrinol 18:708–721[Abstract/Free Full Text]
  37. Franks S, McCarthy MI, Hardy K 2006 Development of polycystic ovary syndrome: involvement of genetic and environmental factors. Int J Androl 29:278–285; discussion 286–290
  38. Abbott DH, Dumesic DA, Franks S 2002 Developmental origin of polycystic ovary syndrome—a hypothesis. J Endocrinol 174:1–5[Abstract]
  39. Stewart DR, Dombroski BA, Urbanek M, Ankener W, Ewens KG, Wood JR, Legro RS, Strauss 3rd JF, Dunaif A, Spielman RS 2006 Fine mapping of genetic susceptibility to polycystic ovary syndrome on chromosome 19p13.2 and tests for regulatory activity. J Clin Endocrinol Metab 91:4112–4117[Abstract/Free Full Text]
  40. Urbanek M, Woodroffe A, Ewens KG, Diamanti-Kandarakis E, Legro RS, Strauss 3rd JF, Dunaif A, Spielman RS 2005 Candidate gene region for polycystic ovary syndrome on chromosome 19p13.2. J Clin Endocrinol Metab 90:6623–6629[Abstract/Free Full Text]
  41. Wacholder S, Chanock S, Garcia-Closas M, El Ghormli L, Rothman N 2004 Assessing the probability that a positive report is false: an approach for molecular epidemiology studies. J Natl Cancer Inst 96:434–442[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Hum ReprodHome page
M. E. Kevenaar, A. P.N. Themmen, A. J. van Kerkwijk, O. Valkenburg, A. G. Uitterlinden, F. H. de Jong, J. S.E. Laven, and J. A. Visser
Variants in the ACVR1 gene are associated with AMH levels in women with polycystic ovary syndrome
Hum. Reprod., January 1, 2009; 24(1): 241 - 249.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kevenaar, M. E.
Right arrow Articles by Visser, J. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kevenaar, M. E.
Right arrow Articles by Visser, J. A.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*OMIM
*Protein*SNP
*UniGene
*Substance via MeSH
Related Collections
Right arrow Female Endocrinology

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals