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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2006-1582
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The Journal of Clinical Endocrinology & Metabolism Vol. 92, No. 1 240-245
Copyright © 2007 by The Endocrine Society

Granulosa Cell Production of Anti-Müllerian Hormone Is Increased in Polycystic Ovaries

Laura Pellatt, Laurine Hanna, Mark Brincat, Ray Galea, Henrietta Brain, Saffron Whitehead and Helen Mason

Clinical Development Sciences (L.P., H.M.), Graduate Entry Programme (L.H.), and Basic Medical Sciences (H.B., S.W., H.M.), St. George’s University of London, London SW17 0RE, United Kingdom; and St. Luke’s Hospital (M.B., R.G.), University of Malta Medical School, Msida MDA 06, Malta

Address all correspondence and requests for reprints to: Ms. Laura Pellatt, Clinical Developmental Sciences, St. George’s University of London, Cranmer Terrace, London SW17 0RE, United Kingdom.


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Context: There has been renewed interest in anti-Müllerian hormone (AMH) because of its role in the ovary. Data on its actions are sparse, but it appears to inhibit follicle growth. Interestingly, serum AMH is two to three times higher in women with polycystic ovary (PCO) syndrome than women with normal ovaries.

Objective: We examined the production of AMH by cells from a range of follicle sizes from normal ovaries and compared this with production by ovulatory and anovulatory (anov) PCOs.

Design: Granulosa cells (GCs) and theca and follicular fluid (ff) were isolated from intact follicles. Cells were cultured for 48 h ± FSH or LH, and AMH was measured in ff and cell-conditioned media (CM).

Results: AMH levels in ff and GC-CM ranged from 42 to 2240 and 0.025 to 1.7 ng/ml, respectively, and were low or undetectable in ff and GC-CM from follicles greater than 9 mm, luteinized cells, and theca and stroma. The mean level of AMH was four times higher in GC-CM from ovulatory PCOs [mean (range) 1.56 (0.025–7)] and 75 times higher from anovPCO [21.4 (17.2–43 ng/ml)] than normal ovaries [0.37 (0.025–1.7)]. Neither LH nor FSH had an effect on AMH production by GCs from normal ovaries, but in cells from PCOs, FSH significantly decreased AMH, and in contrast, LH increased AMH.

Conclusions: The reduction of AMH in follicles greater than 9 mm from normal ovaries appears to be an important requirement for the selection of the dominant follicle. AMH production per GC was 75 times higher in anovPCOs, compared with normal ovaries. This increase in AMH may contribute to failure of follicle growth and ovulation seen in polycystic ovary syndrome.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 References
 
ANTI-MÜLLERIAN HORMONE (AMH), also known as Müllerian-inhibiting substance, is a member of the TGFß superfamily. Secreted by the Sertoli cells of the testis from 8 wk gestation until puberty, its classical action is to regress the Müllerian ducts during male fetal development (1). AMH is now coming under renewed scrutiny, however, because of its possible role in the ovary, in which it is expressed postnatally until menopause and is located in the granulosa cells (2). In both rodents and humans, its expression was absent from nongrowing primordial follicles, was highest in small growing follicles of the preantral and antral stages, and was again lost in luteinized and atretic follicles (3, 4). Human follicles of the size after selection of the dominant follicle have not been studied.

This renewed scrutiny of AMH is due to a series of interesting publications. Several studies have demonstrated that serum AMH levels in women decline with advancing age (5, 6), and AMH has therefore been proposed as a marker of ovarian reserve (7). Indeed, an over-the-counter kit is now available in the United Kingdom in which AMH is one of a triad of hormones used to calculate ovarian reserve index. The relationship between the number of follicles in the ovary and serum AMH has also led to the demonstration that AMH is a good predictor of poor response to in vitro fertilization treatment (8).

Knowledge of the actual function of AMH in the ovary is rather limited, and much of the information that we do have has been extrapolated from studies of AMH null mice (9, 10, 11). These mice had an increase in the number of follicle-initiating growth, which was reflected in an increase in the number of growing follicles of all stages. This overgrowth of follicles resulted in premature cessation of cyclicity in these animals (9). Cultured ovaries from neonatal mouse cultures with AMH contained 40% fewer growing follicles (11). Interestingly, AMH also reduced FSH-stimulated follicle growth in isolated follicle cultures (10). Taken together, these data suggest that AMH retards follicle growth and reduces sensitivity to FSH. Studies in which ovarian cells were exposed to AMH protein supported these findings in that cell proliferation, steroidogenesis, and induction of LH receptors, all required for follicle progression, were inhibited (12, 13).

A second major reason for the resurgence of interest in AMH is the fact that serum levels in women with polycystic ovary syndrome (PCOS) are 2- to 3-fold higher than in ovulatory women with normal ovaries (14, 15, 16). PCOS affects 5–20% of women of reproductive age (17) and is the primary cause of anovulatory infertility (18). It is characterized by polycystic ovaries (PCOs) with hyperandrogenism, obesity, elevated LH, and insulin resistance (19). As an exclusively granulosa cell product, it was assumed that the rise in serum AMH in PCOS was a consequence of the increased number of follicles in these ovaries. Given that AMH appears to be an inhibitor of follicle growth, however, we were interested to see whether AMH production per cell was raised in PCOS.

We examined the production of AMH by cells from the normal ovary and compared this with AMH production by ovulatory and anovulatory PCOs. Given the likelihood of AMH production being modulated by gonadotropins, we also examined the effect of LH and FSH on granulosa cell AMH production.


   Patients and Methods
Top
 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 References
 
Ovarian dissection

Informed written consent was obtained from women undergoing transabdominal hysterectomy with bilateral oophorectomy for benign gynecological conditions at St. George’s Hospital (London, UK), Hope Hospital (Manchester, UK), and St. Luke’s Hospital (Malta). Ethical approval was granted by the relevant committee in each institution. Details of menstrual history (cycle length and duration) and medication were obtained before surgery. The ovaries removed from each patient were seen by a pathologist before a portion of each was taken to the laboratory for dissection. Morphology and ovulatory status were assigned as previously published and were based on ovarian size, follicle sizes and numbers, the presence of a dominant follicle or corpus luteum, and the amount and density of stroma as determined by dissection in conjunction with patient history (20). The timing of surgery was random. Patient details and follicle numbers isolated and used in each experiment are shown in Table 1Go.


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  		TABLE 1. Details of patients whose follicles were included in the study

 
Follicles were isolated from the surrounding stroma under sterile conditions using binocular microscopy as previously described (21). Accurate measurement of follicle diameter was determined with calipers before the follicular fluid was aspirated. A cross-wise incision into the follicle was then made to expose the fragile granulosa cell layer, which was flushed away from the theca with a stream of Medium 199 (M199; Invitrogen Ltd., Paisley, Scotland, UK) and collected. Finally the theca cell layer was carefully peeled off and digested in an enzyme cocktail for 30 min at 37 C with gentle agitation (22). The granulosa or theca cells were cultured in M199 supplemented with penicillin/streptomycin, L-glutamine, and amphotericin B (all purchased from Invitrogen) pooled from either a number of follicles from a single patient or individual follicles, based on the number of follicles and cells and the experimental protocol. Only cells from those follicles visually designated healthy were used. A healthy follicle is well vascularized and contains the expected number of granulosa cells on subsequent counting (23).

Granulosa cells were plated at 5 x 104 and theca cells at 2.5 x105 cells/well. Pieces of stroma were removed from normal ovaries and 10- to 50-mg pieces cultured in 24-well plates with 1 ml M199. Cells and tissue were incubated at 37 C in 95% air-5% CO2 in a humidified incubator.

Granulosa-luteal cell (GLC) preparation

GLCs were isolated and prepared from follicular aspirates collected from women undergoing in vitro fertilization treatment, with ethical approval and informed written consent. Isolation of GLCs was performed as previously described (22). Cells were plated out at 5 x104 cells/well in a 96-well plate with 200 µl of serum (5%) supplemented medium for 48 h, after which the medium was removed and replaced with experimental medium as detailed below.

Protocol

Experimentation on nonluteinized cells was performed on the day of dissection. To assess AMH production with increasing follicle size in normal ovaries, granulosa cells were counted using Trypan blue exclusion and plated out at a density of 5 x 104 in 96-well plates in 200 µl of supplemented M199 for 48 h. The cells from each follicle were cultured separately except those obtained from patients 4 and 6 in which cells from two follicles of similar size were pooled (4 + 6 mm and 13 + 15 mm). After removal of serum-supplemented medium, GLCs were also incubated in serum-free medium for 48 h.

For studies of comparative AMH production among normal, ovulatory (ov) PCOs, or anovulatory (anov) PCOs, cells were pooled from follicles sized 2–10 mm from a single patient and incubated as above. Cells from eight normal, nine ovPCOs, and six anovPCOs were cultured. To investigate the effect of FSH, cells from normal ovaries or PCOs were pooled from 2- to 10-mm follicles and incubated with or without purified human FSH (5 ng/ml; Endocrine Services, Bideford on Avon, UK) with testosterone (5 x 10–7 M) added as an aromatase substrate for 48 h. Estradiol was measured in the same medium as a marker of response to FSH. Each experiment was performed in a minimum of triplicate wells, and four experiments were performed in normal ovaries and eight in PCOs. To investigate the effects of LH, cells from individual or pooled follicles greater than 10 mm (i.e. after acquisition of LH receptors) from five separate patients with normal ovaries and four with ovPCOs were incubated with 5 ng/ml LH for 48 h. Theca cells and pieces of stroma were incubated in serum-free media without treatment for 48 h. All reagents used were purchased from Sigma (Poole, Dorset, UK) unless otherwise stated. AMH was measured in the cell-conditioned media using a serum-based ELISA (Diagnostic Systems Laboratories, Oxon, UK) (23). Initially we validated this ELISA for measurement of AMH in cell-conditioned medium, samples diluted in parallel with serum-based standards; there was good recovery and there was no effect of repeat freeze/thaw cycles.

Analyses

Mean AMH levels in follicles from normal ovaries sized 2–10 mm were compared with levels in follicles greater than 10 mm using unpaired Student’s t test. Comparisons among normal ovaries, ovPCOs, and anovPCOs were performed using Kruskal-Wallis with Tukey-Kramer post hoc test. Only follicles sized 2–10 mm from normal ovaries were included in this latter comparison to avoid bias caused by the increased number of small follicles from PCOs. Due to the wide variation in absolute AMH levels between experiments, the results from the LH- and FSH-treated granulosa cells were calculated as a percentage of the control with the control set at 100%. Results were compared with Mann-Whitney test; P ≤ 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 References
 
AMH production and levels in granulosa cells, theca, stroma, follicular fluid, and GLCs from normal ovaries

AMH was measured in follicular fluid from 14 follicles between 3 and 12 mm in diameter. The concentration ranged from 42 to 2240 ng/ml (Fig. 1Go) with levels being highest in fluid from small follicles and being present in very low amounts in those follicles above 9 mm. Granulosa cells were isolated from 17 follicles ranging in size from 4 to 19 mm from 13 women with normal ovaries (Fig. 1Go). The concentration of AMH produced by the cultured cells ranged from 0.025 to 1.7 ng/ml, with the highest level again being made by cells from the smallest follicles. Cells from follicles of greater than 10 mm produced AMH at or below the detection limit of the assay (0.025 ng/ml) as did GLCs, theca, and stroma (data not shown).


Figure 1
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  		FIG. 1. Left, AMH concentration in follicular fluid from a range of individual follicles isolated from women with regular ovulatory cycles and normal ovaries. Note the loss of AMH in fluid from follicles greater than 10 mm. Right, AMH concentration (nanograms per milliliter per 50,000 cells) in granulosa cell-conditioned medium from a range of follicle sizes from normal ovaries. AMH fell exponentially as the follicle size increased and again granulosa cells from follicles greater than 10 mm produced low or undetectable levels of AMH.

 
Comparison of AMH production in granulosa cells from normal ovaries, ovPCOs, and anovPCOs

The mean (range) number of follicles isolated from portions of ovPCOs and anovPCOs was 13 (6–20) and 29 (18–40), respectively, and cells from all follicles from 2 to 10 mm were pooled. Follicle size distribution among the three groups did not differ significantly. Levels of AMH produced by granulosa cells from 2- to 10-mm follicles from normal ovaries ranged from undetectable to 1.7 ng/ml, with a mean of 0.37 ng/ml. There was a greater range of AMH production by cultured cells from ovPCOs, with levels ranging up to 7 ng/ml (mean 1.56), with the highest concentration again being in medium from cells from 3- to 5-mm follicles. Medium conditioned by cells from anovPCOs contained remarkably high levels of AMH with a range of 17.22 to 42.96 ng/ml and a mean of 27.4. This mean is 16 times higher than the highest level from normal ovaries and 75 times higher than the mean normal level. AMH production by granulosa cells from normal ovaries, ovPCOs, and anovPCOs were significantly different from each other (P = 0.0005, Kruskal-Wallis) (Fig. 2Go).


Figure 2
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  		FIG. 2. AMH concentration in granulosa cell-conditioned medium from normal ovaries (n = 8), ovulatory (n = 9), and anovulatory (n = 6) PCOs. AMH production was significantly different between normal ovaries, ovPCOs, and anovPCOs (P < 0.001). The mean concentration in cells from anovPCOs was 75 times higher than the mean for normal ovaries.

 
Effect of FSH and LH on AMH production

FSH had no effect on AMH production by granulosa cells from normal ovaries (Fig. 3Go). In contrast, FSH treatment of granulosa cells from PCOs significantly reduced AMH with the mean percentage production falling by 30% (P = 0.008, Mann-Whitney). Estradiol production in response to FSH was significantly increased in the same experiments in each case. Incubation of cells from dominant follicles with LH increased AMH by a modest 28% but caused levels to rise by 4-fold in cells from polycystic ovaries (Fig. 3Go).


Figure 3
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  		FIG. 3. AMH production in response to gonadotropin: white bars, control; gray bars, FSH or LH (5 ng/ml). There was no significant difference in production of AMH after FSH treatment in granulosa cells from normal ovaries (n = 4); however, FSH significantly reduced AMH production by granulosa cells from PCOs (n = 8), compared with controls (P = 0.008). There was a slight but not significant increase in AMH in response to LH (P = 0.06) in cells from follicles greater than 10 mm from normal ovaries (n = 5), but a mean 4-fold increase in cells from PCOs (n = 4, P = 0.06).

 

   Discussion
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
References
 
This is the first report of AMH concentrations in a large size range of follicles from normal unstimulated human ovaries; it demonstrates an exponential fall as the follicle size increased. This was seen in both follicular fluid and in granulosa cell-conditioned medium and was particularly apparent once the follicle reached 10 mm in diameter, i.e. the size at which follicle selection occurs. Production by cells collected after human chorionic gonadotropin (hCG) was largely undetectable. This suggests that the decrease in AMH is important for the dominant follicle to be selected and reinforces the suggestion that AMH inhibits follicle growth. Serum AMH levels in women with normal ovaries range between 1.2 and 2.4 ng/ml (25, 26, 27). AMH levels in follicular fluid in this study were between 100 and 1000 times higher, emphasizing local granulosa cell production and follicular fluid accumulation.

We have also demonstrated that the levels of AMH in media conditioned by granulosa cells from anovPCOs are on average 75 times higher than in media of cells from normal ovaries. Because cells from sized-matched follicles in all three groups were plated at the same density, the AMH production in anovPCOs is significantly higher per granulosa cell. Serum levels of AMH have been shown to be 2- to 3-fold higher in women with PCOS, compared with ovulatory women with normal ovaries (14, 15), and there are more follicles in anovPCOs, compared with normal ovaries (28). The raised serum AMH in PCOS is therefore a reflection of both an increase in production per cell and the increase in follicle number. Although the levels produced by cells from both ovPCOs and anovPCOs were higher than normal, the latter were considerably higher than the former. We have shown that AMH has virtually disappeared from follicles proceeding beyond selection in normal ovaries, signifying that the high levels in anovPCOs may indeed be sufficient to inhibit folliculogenesis. One possible criticism of our data is that the mean age of the patients in our PCOS group was lower than that of our controls, and it has clearly been shown that serum AMH declines with age. The latter, however, gives an integrated measure that reflects the total number of antral follicles in both ovaries. In contrast, this study was performed to compare production per granulosa cell, i.e. to determine production independently of ovarian reserve. We did not find any relationship between age and AMH production per cell; however, this relationship would be masked by differences in follicle size.

These results appear to be at variance with those studies comparing the relationship between the number of small follicles and serum AMH in normal ovaries and PCOs in which it was shown that the slope of the regression line did not differ between the two morphologies (16). The conclusion reached by these authors was that each follicle from a PCO was secreting the same amount of AMH as one from a normal ovary. Closer analysis of the data obtained by ultrasound does, however, suggest some possible reasons for the difference. The relationship between follicle number and AMH only held for 2- to 5-mm follicles and was lost in those of 6–9 mm, and although size matched between groups, our pools do contain significant numbers of follicles of 6–9 mm. Second, the patients in the ultrasonographic studies were a mixed group with patients with amenorrhea, oligomenorrhea, and regular cycles. It is stated that the patients with anovulation did have higher levels, but the correlation with AMH was not analyzed separately. One other possibility is that size-matched follicles from polycystic ovaries contain significantly fewer granulosa cells than those from normal ovaries, and so the increase per cell is partly negated. This has not been our experience; however, we have not made a systematic analysis of granulosa cell number in these follicles. Given our finding that AMH was so greatly increased per cell in PCOs, these differences are still difficult to reconcile and remain largely unexplained.

The cause of raised AMH production in PCOS is unknown; however, clinical studies point to androgens as a likely candidate. There was a correlation between serum androgen and AMH in patients with PCOS (16, 29, 30), and in particular, levels in women undergoing in vitro fertilization were higher when the patient had hyperandrogenism and PCOs than in women with PCOs and normal androgens (31). Interestingly, androgens tend to be higher in anovulatory women with PCOS than in their ovulatory counterparts (32), and androgen secretion is higher per theca cell in anovulatory women than ovulatory (33). Raised androgens could therefore not only explain the increased AMH production in PCOs but could also explain why we found higher levels in anovPCOs than ovPCOs.

We were rather surprised by the findings of our experiment with FSH. Although FSH has been shown to inhibit AMH production in rat fetal Sertoli cells (34), it had no effect in the equivalent human cells (35). To our knowledge there has been only one study investigating the effects of FSH on granulosa cell AMH production. In this study, FSH had no effect on AMH mRNA expression in luteinized cells, and the protein was not measured (35). We also found no effect on AMH production in cells from normal ovaries, which seems surprising, given the apparent ability of AMH to inhibit FSH responsiveness. In contrast, AMH was reduced by FSH in cells from PCOs. The difference in response is hard to explain, although cells from PCOS have been shown to be hypersensitive to FSH in other regards, i.e. in terms of estradiol production (20) and in response to induction of ovulation and controlled ovarian hyperstimulation (36). It is tempting to speculate that the FSH given as a treatment for induction of ovulation might inhibit local production of AMH as one of its mechanisms of action. Cells from normal ovaries also produced very little response to LH, whereas this gonadotropin stimulated AMH production 4-fold in cells from PCOs. The differential response is again hard to explain, although it has previously been suggested that granulosa cells from PCOS acquire LH responsiveness at a smaller size than those from normal ovaries (37). The cells in this study, however, were all from follicles that were already greater than 10 mm, i.e. they all should have had LH receptors. Many women with PCOS have raised serum levels of LH, and these data indicate that this may further increase ovarian AMH production.

To conclude, this report is the first to show that levels of AMH are on average 75 times higher in granulosa cells from anovPCOs, compared with levels in normal ovaries, and that this increase is per cell and not simply due to an increase in the number of follicles. It is important to bear in mind that these raised levels could be misleading if used as a marker of ovarian reserve. The overproduction of AMH could have important implications for the mechanism of anovulation in these women. The finding of AMH type II receptor (AMHRII) on granulosa and theca cells (38) makes the determination of the actions of AMH in the human ovary an important next step.


   Acknowledgments
 
We thank George Hammett (Diagnostic Systems Laboratories) for the AMH ELISA kits and the patients and staff of the Lister Assisted Conception Unit and The Assisted Reproduction and Gynaecology Centre, London, United Kingdom.


   Footnotes
 
This study was supported by a Wellcome Trust grant (to H.M. and S.W.).

The authors have nothing to disclose.

First Published Online October 24, 2006

Abbreviations: AMH, Anti-Müllerian hormone; anov, anovulatory; GLC, granulosa-luteal cell; M199, Medium 199; ov, ovulatory; PCO, polycystic ovary; PCOS, PCO syndrome.

Received July 20, 2006.

Accepted October 13, 2006.


   References
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

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