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Department of Internal Medicine (M.E.K., P.K., B.M.N.v.d.L.-B., F.H.d.J., A.P.N.T., J.A.V.), Erasmus MC, 3000 DR Rotterdam, The Netherlands; and School of Biological and Molecular Sciences (M.F.M., N.P.G.), Oxford Brookes University, Headington, Oxford OX3 0BP, United Kingdom
Address all correspondence and requests for reprints to: Jenny A. Visser, Ph.D., Department of Internal Medicine, Room Ee532, Erasmus MC, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands. E-mail: j.visser{at}erasmusmc.nl.
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Abstract |
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Introduction |
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Assessment of the ovarian reserve is important in the infertility clinic, where ovarian aging is characterized by decreased ovarian responsiveness to exogenous gonadotropin administration and poor pregnancy outcome. Currently, early follicular phase serum levels of FSH, inhibin B, and estradiol (E2) are measured to assess the ovarian reserve in women. Upon ovarian aging, serum levels of inhibin B and E2 decline and subsequently FSH levels rise (9). However, these markers constitute the classical hypothalamus-pituitary-gonadal feedback loop and, therefore, are not independent of each other. Furthermore, changes in serum levels of FSH, inhibin B, and E2 occur relatively late in the reproductive aging process (10). Therefore, in addition to these hormones, the number of antral follicles [antral follicle count (AFC)] is determined by ultrasonography, because the AFC gives a better prediction of the ovarian reserve (11). Recently, measurement of serum anti-Müllerian hormone (AMH) levels has been added to the panel of markers for ovarian aging (12, 13, 14).
AMH, also known as Müllerian-inhibiting substance, is expressed in granulosa cells of growing follicles (15, 16, 17, 18, 19). Detailed studies in rodents have shown that AMH expression is flanked by two major regulatory steps of folliculogenesis, i.e. initial follicle recruitment and cyclic selection for dominance (20, 21). AMH expression starts in the granulosa cells of primary follicles, directly after differentiation from flattened pregranulosa cells of primordial follicles. Highest expression is observed in granulosa cells of preantral and small antral follicles, whereas expression is absent during the FSH-dependent final stages of follicle growth (16, 17, 19, 21, 22). In the human ovary, AMH is expressed in a similar pattern, with expression first appearing in granulosa cells of primary follicles and being strongest in preantral and small antral follicles. AMH expression disappears in follicles of increasing size and is lost in large antral follicles, where weak staining only remains present in the granulosa cells of the cumulus (23).
This specific expression pattern of AMH in growing nonselected follicles has lead us and others to study whether serum AMH levels are indicative for the number of growing follicles (reviewed in Ref. 24). Indeed, in women, serum AMH levels decline with increasing age and changes in serum AMH levels were apparent before changes in other serum markers of ovarian aging, such as FSH and inhibin B, were present. Furthermore, AMH levels correlated strongly with the AFC (12, 13). In contrast with other serum markers, AMH levels remain relative constant during the menstrual cycle (25, 26). Furthermore, studies suggest that serum AMH levels are not influenced by the gonadotropic status, and only reflect the follicle population (13, 27, 28).
Despite accumulating data on the use of AMH serum levels as a marker for ovarian reserve, the relationship between AMH levels and the size of the primordial follicle pool has not been studied directly, because direct assessment of the size of this pool is not possible in women.
With the development of a mouse AMH ELISA, we have addressed this question in aging mice. In this study, we show that, similar to women, serum AMH levels decline with increasing age in mice. The decline in AMH levels correlates with the decrease in the number of growing follicles with aging, and most importantly, with the size of the primordial follicle pool. These findings show that serum AMH levels reflect the quantitative aspect of ovarian reserve in rodents.
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Materials and Methods |
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Two-site immunoassay development
To identify an optimum pair of antibodies to allow sensitive detection of AMH, every antibody was tested as capture antibody in combination with all other biotinylated detection antibodies. Ten 96-well microplates (Nunc Maxisorb; SLS, Nottingham, UK) were coated with each of the different antibodies (raised against human AMH, also reacting with rat AMH) at 2 µg/ml in 0.05 M bicarbonate buffer (pH 9.4) and incubated at 4 C overnight, before incubation with a blocking buffer containing 0.5% (wt/vol) casein (Mast Group Ltd., Bootle, UK; Ref. M2052) and 6% (wt/vol) sucrose in PBS for 1 h. Next, plates were emptied, dried, and stored in aluminum pouches with desiccant. Recombinant human AMH, recombinant rat AMH, and various mammalian sera were diluted in high-performance ELISA (HPE) immunoassay buffer (Mast Group Ltd.; Ref. M1940), and 50 µl/well was added to duplicate wells. Sera were diluted at 1:5 in HPE immunoassay buffer, 50 µl/well of each sample was added to duplicate wells on the plate, and plates were shaken at room temperature for 2 h. After washing with PBS/Tween (0.005% Tween 20) three to five times, 50 µl/well of each of the 10 biotinylated monoclonal antibodies was added at a concentration of 0.3 µg/ml diluted in 1% (wt/vol) casein buffer and incubated at room temperature for 1 h. Plates were again washed five times with PBS/Tween and incubated for 30 min with poly HRP-streptavidin conjugate (Mast Group Ltd.; Ref. M2051), added at a 1:10000 dilution in 1% (wt/vol) casein buffer. After a thorough wash with PBS/Tween followed by deionized water, the plates were developed with the tetramethylbenzidine substrate (Insight Biotechnology International, Wembley, UK). The best combination of detector and capture antibodies to allow detection of AMH in mouse serum was selected. This was F2B/7A for detection and F2B/12H for capture. This combination also provided highly sensitive assays for rat and human AMH. The assay is available through DSL Inc. (DSL-10-14400).
Animals
C57BL/6J wild-type mice were obtained from the Animal Facility of the Erasmus Medical Center (Rotterdam, The Netherlands) and were kept under standard animal housing conditions in accordance with the National Institutes of Health guidelines for the Care and Use of Experimental Animals. Animals were killed at various ages ranging from 4–18 months of age, when possible at estrus. For each age group, ovaries from eight to 10 mice were collected. To determine the day of the cycle, daily vaginal smears were taken for a period of at least 2 wk and examined as described previously (20). Mice were killed at 1400 h by decapitation, blood samples were collected, and serum was isolated as described previously (20). Serum samples were stored at –20 C until assayed for AMH. Ovaries were removed and fixed overnight in Bouin’s fluid at room temperature. For histological examination of the follicle population, fixed ovaries were embedded in paraffin, and after routine histological procedures, 8-µm sections were mounted on slides and stained with hematoxylin and eosin.
Follicle counting
Follicle count was performed as described previously (20), using one ovary per animal. Follicles were classified based on their mean diameter, which was determined by measuring two perpendicular diameters in the section in which the nucleolus of the oocyte was present. Primordial follicles (diameter <20 µm) were counted in every 10th section in all mice, whereas growing follicles were counted in every fifth section in four randomly selected mice per age group.
Immunohistochemistry
For immunohistochemical staining, sections were mounted on 3-aminopropyltriethoxysilane (Sigma-Aldrich Chemie BV, Zwijndrecht, The Netherlands)-coated slides. After deparaffinization, sections were quenched for 20 min in 3% H2O2/methanol solution to block endogenous peroxidase activity, washed with water, and transferred to PBS. Sections were subjected to heat-induced antigen retrieval for 3 x 5 min at 700 W in 0.01 M citric acid buffer, pH 6.0 (Merck, Darmstadt, Germany), in a microwave oven, cooled down to room temperature, rinsed in PBS, subsequently incubated with a biotinylated AMH mouse monoclonal antibody (antibody 5/6A, MCA2246; Serotec; Ref. 23), and diluted 1:100 at 4 C overnight followed by a wash step with PBS. Next, sections were incubated for 30 min at room temperature with streptavidin-biotin-peroxidase complex (ABC; diluted 1:200 in PBS; Dako, Glostrup, Denmark) and washed three times with PBS, and the peroxidase activity was developed with 0.07% 3,3'-diaminobenzidine tetrahydrochloride (Sigma-Aldrich). Finally, all sections were counterstained with hematoxylin.
Quantitative analysis of the AMH staining intensity in four to five follicles per age group was performed using the ImageJ software 1.35p (National Institutes of Health, Bethesda, MD; http://rsb.info.nih.gov/ij/).
To biotinylate the 5/6A antibody, the antibody was concentrated to approximately 1.5 mg/ml and dialyzed against 0.1 M NaHCO3 with several changes overnight. Sulfo-NHS-LC-Biotin (Pierce, Perbio Sciences Nederland B.V., Etten-Leur, The Netherlands) was dissolved in water to give a 2-mg/ml solution, and 50 µl were added to 1 ml of antibody (at 1 mg/ml) and incubated for 2 h at 4 C. The biotinylation reaction was stopped by adding 0.1 ml of 1 M NH4Cl. Next, the solution was dialyzed against PBS with two changes over 2 d. A 0.1% sodium azide solution was prepared, and the biotinylated antibody was stored at 4 C.
AMH ELISA procedure
A standard line was included for each plate. Human AMH standards made in working strength HPE buffer and mouse serum samples diluted 1:41 in HPE buffer were added to duplicate wells (50 µl/well) on F2B12/H antibody-coated microplates and incubated for 2 h at room temperature. After washing with PBS/Tween (300 µl/well three to five times), 50 µl of biotinylated AMH monoclonal antibody F2B7/A was added at a 1:3000 dilution in 1% (wt/vol) casein buffer. After 1 h incubation at room temperature, the plate was washed five times with PBS/Tween (300 µl/well). Next, the wells were incubated for 30 min at room temperature with the poly HRP conjugate (50 µl/well) at a 1:20,000 dilution in 1% (wt/vol) casein buffer. After washing with PBS/Tween (300 µl/well, five to seven times), followed by washing with deionized water, tetramethylbenzidine substrate (100 µl/well) was added. After 10 min of incubation at room temperature in the dark, the chromogenic reaction was stopped by adding 100 µl of 6% (vol/vol) phosphoric acid to each well, and absorbances were read at 450 nm with a reference wavelength set at 655 nm using a micro plate reader (Bio-Rad, Hemel Hempstead, UK).
Statistical analysis
Data were analyzed with SSPS11 (SPSS Inc., Chicago, IL) and expressed as mean ± SEM. Data were evaluated for statistical differences by one-way ANOVA, followed by Duncan’s new multiple range test. Differences were considered significant at P < 0.05. The correlations between different parameters were assessed using Spearman’s correlation coefficients.
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Results |
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Western blot analysis of human and rat recombinant AMH showed that the F2B/7A antibody recognizes epitopes in the pro-region under both reducing (Fig. 1
) and nonreducing (results not shown) conditions, whereas antibody F2B/12H recognizes epitopes in the mature region of AMH under nonreducing conditions only (Fig. 1). Based on the recognition of both regions, the assay is expected to measure total AMH.
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Assay range, sensitivity, and specificity
The range of the AMH standards used in this assay was from 5–0.037 ng/ml (Fig. 2). The detection limit, defined as the mean of the absorbance of the blank replicates + 2 SDs, was 6.3 pg/ml. The mean interassay and intraassay coefficients of variation were less than 10% and less than 5%, respectively.
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). In addition, six serum samples were spiked with a known amount of recombinant rat AMH. The average percentage recovery of recombinant AMH from mouse serum samples was 103.9 ± 3.0%.
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). In contrast, the number of growing follicles did not decline in mice of 4–8 months of age, and their decline only became evident in older mice, with numbers in mice of 10–14 months of age being significantly different from those in mice of 16–18 months of age (P < 0.05) (Fig. 4B).
Spearman’s correlation coefficient showed that the numbers of growing follicles correlated strongly with the numbers of primordial follicles in the same animal (r = 0.93, P < 0.0001) (Fig. 4C).
AMH expression in aging mice
The decrease in serum AMH with increasing age might be caused by a decline in AMH expression per follicle. Therefore, we performed immunohistochemistry to determine the expression pattern of AMH in ovaries of 4- to 16-month-old mice (Fig. 5). At 4 months of age, AMH staining was found in granulosa cells of small growing follicles up to the small antral stage. Weak staining was observed in primary follicles with a single layer of granulosa cells. Strongest expression was observed in large preantral follicles with several layers of granulosa cells, whereas expression decreased in small antral follicles. AMH expression was absent in large antral follicles. Ovaries of aging (8 and 12 months old) and aged (16 months old) mice showed a similar expression pattern, with AMH expression being strongest in the large preantral follicles. Furthermore, the AMH staining intensity of similar sized follicles in aging and aged mice appeared to be similar to those of 4-month-old mice (Fig. 5). Indeed, quantitative analysis of the immunolabeling using ImageJ software showed no difference in the staining intensities (results not shown).
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). In addition, AMH levels correlated strongly with the numbers of primordial follicles (r = 0.83, P < 0.0001) (Fig. 6B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, serum levels of AMH have been shown to be a promising marker for ovarian aging in women (reviewed in Ref. 24). In this study, we show that, similar to the situation in women, AMH levels decrease in mice of increasing age. This decline in serum AMH is not reflected by a similar change in AMH expression level. Using immunohistochemical analysis, we show that the expression level of AMH within growing follicles remains similar with increasing age, indicating that the expression of AMH is independent of other aging markers such as FSH and inhibin B. Furthermore, the constant AMH expression per follicle suggests that the decrease in serum AMH levels in aging mice directly reflects the decrease in number of AMH-expressing follicles. Indeed, serum AMH levels correlated strongly with the number of growing follicles in this study. This finding is in agreement with results obtained in women in whom serum AMH levels correlated with AFC (12, 14).
It has been suggested that the size of the primordial follicle pool is reflected by the number of growing follicles, in both women and mice (11, 32). This suggestion was confirmed in the current study in which we observed a high correlation between numbers of growing and primordial follicles. Moreover, we observed that serum levels of AMH were strongly correlated with the number of primordial follicles. Both AMH levels and the number of primordial follicles declined with increasing age. However, whereas size of the primordial follicle pool decreased from the age of 4 months onward, AMH levels did not change initially. Similarly, the number of growing follicles remained constant during the early reproductive period in mice, which explains the constant levels of AMH during this period. This suggests that compensatory mechanisms are present to maintain the number of growing follicles, and therefore, serum AMH levels, at a constant level despite a declining primordial follicle pool. It has been suggested that protection from oocyte degeneration and atresia may play a role in the preservation of the growing follicle pool (32). It is also possible that relatively more follicles are recruited from the declining primordial follicle pool to establish a constant number of growing follicles. However, in unilateral ovariectomized mice and rats, in which the primordial follicle pool is reduced by 50%, an increased recruitment was only observed immediately after ovariectomy to establish a normal number of ovulations. In the long-term, a reduction of the number of atretic antral follicles appears to be the main mechanism to obtain the appropriate number of preovulatory follicles (33, 34). Interestingly, these compensatory ovulations are absent in aged unilateral ovariectomized animals as they show an earlier onset of irregular cyclicity and infertility than control mice (35, 36).
Although little is known about the mechanisms that regulate follicle recruitment and early follicle growth, it is likely that both stimulatory and inhibitory signals are involved. These may be factors produced by both oocyte and granulosa cells acting in an autocrine and paracrine fashion. AMH has been identified as one of the factors that inhibit follicle recruitment (20). However, despite the increased AMH levels relative to the size of the primordial follicle pool, primordial follicle recruitment, as reflected by the number of remaining follicles, is not decelerated during early reproductive life. Thus, our present results on serum levels of AMH suggest that AMH is not a dominant factor in the regulation of follicle numbers in aging mice. However, a strong conclusion may not be drawn from this data, because serum AMH levels may not give a proper reflection of the intraovarian action of AMH.
From 8 months of age onward, both serum AMH levels and the number of growing follicles showed a steady decline, suggesting that after the size of the primordial pool has reached a certain threshold level, the compensatory mechanisms to maintain the pool of growing follicles at a fixed number may no longer be sufficient. Interestingly, toward the end of reproductive life, the depletion of the primordial follicle pool slowed down, whereas the gradual decline in the number of growing follicles did not change. This suggests that, during early reproductive life, the use of primordial follicles is relatively wasteful, whereas, at the end of reproductive life, primordial follicles are used more efficiently.
In conclusion, our results indicate that serum AMH is an excellent marker for the size of the primordial follicle pool and, therefore, for ovarian aging in mice. Our results also imply that AMH levels may only have predictive value when changes in fertility are already apparent because changes in serum levels were only evident in mice that already displayed cycle irregularities.
Similarly, also in women, serum AMH levels are predictive when changes in fertility are present. In women, cycle irregularities are preceded by a decline in fertility (2), and indeed changes in serum AMH levels are present before cycle irregularities occur (12, 37). Studies indicate that AMH is an equally good predictor as AFC for the decline in fertility (38). However, when after adjusting for age, only serum AMH levels remains predictive for cycle irregularities (37). Nevertheless, more studies are necessary to determine the relationship between serum AMH levels and status of fertility, preferably in a prospective study. Nevertheless, studies in women have shown that, currently, serum AMH levels are the earliest marker for ovarian aging, and give a better prediction than serum levels of FSH or inhibin B (12, 37, 38), which may be particularly important for women at risk of early ovarian aging, such as survivors of childhood cancer.
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Acknowledgments |
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Footnotes |
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First Published Online March 23, 2006
Abbreviations: AFC, Antral follicle count; AMH, anti-Müllerian hormone; E2, estradiol; HPE, high-performance ELISA.
Received December 14, 2005.
Accepted for publication March 13, 2006.
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