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Institute of Reproductive and Developmental Biology (S.A.S., K.H., P.D.S.-B., L.J.W., S.F.), Wolfson and Weston Research Centre for Family Health, Imperial College London, Hammersmith Hospital, London W12 0NN, United Kingdom; Department of Mathematics (J.S.), Imperial College London, London SW7 2AZ, United Kingdom; Histopathology Department (A.M.F.), Division of Investigative Sciences, Imperial College London, St. Mary’s Hospital, London W2 1PG, United Kingdom; Department of Internal Medicine (A.P.N.T., J.A.V.), Erasmus Medical Center, 3015 GD Rotterdam, The Netherlands; and School of Biological and Molecular Sciences (N.P.G.), Oxford Brookes University, Headington Campus, Oxford OX3 0BP, United Kingdom
Address all correspondence and requests for reprints to: Kate Hardy, Institute of Reproductive and Developmental Biology, Wolfson and Weston Research Centre for Family Health, Imperial College London, Hammersmith Hospital, London W12 0NN, United Kingdom. E-mail: k.hardy{at}imperial.ac.uk.
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Abstract |
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 Top Abstract IntroductionPatients and MethodsResultsDiscussionReferences |
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Objective: The objective of this study is to investigate whether AMH is involved in accelerating the transition of follicles from primordial to primary stages in polycystic ovaries.
Design: This study compares AMH expression in archive tissue from normal and polycystic ovaries.
Setting: This is a laboratory-based study.
Patients: Ovarian tissue from seven normoovulatory women and 16 women with polycystic ovaries (five of whom were anovulatory) was used in this study. Ovaries were classified by histology and with reference to menstrual cycle history and ultrasound.
Main Outcome Measure: Presence and intensity of AMH expression in 1403 follicles was the main outcome measure.
Results: AMH was observed from the primordial stage onward. AMH immunostaining was observed in significantly fewer primordial (P = 0.007) and transitional follicles (P = 0.001) in ovaries from anovulatory women with polycystic ovaries compared with women with regular cycles and either normal or polycystic ovaries. AMH-negative follicles had fewer pregranulosa cells in the largest cross-section of the follicle at both the primordial (median, four and six for AMH-negative and -positive follicles, respectively; P < 0.0001) and transitional stages (median six and nine; P < 0.0007) in normal tissue, and fewer at the transitional stage (median, seven and 11; P < 0.0001) in tissue from anovulatory women with polycystic ovaries. This suggests that AMH expression is associated with granulosa cell mitosis.
Conclusions: These findings indicate a relative deficiency of AMH in primordial and transitional follicles in ovaries from anovulatory women with polycystic ovaries. This may contribute to disordered early follicle development in polycystic ovary syndrome.
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Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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AMH, also known as müllerian inhibiting substance, is a member of the TGFß superfamily and is a dimeric glycoprotein (5, 6). During male development, AMH is responsible for regression of müllerian duct derivatives (7). In the male, AMH expression is confined to the Sertoli cells of the testes during fetal and postnatal life, with levels declining as the germ cells enter meiosis (8, 9). In contrast, in the ovary, AMH is expressed only postnatally, in granulosa cells of growing follicles. AMH mRNA and protein have been detected in the cytoplasm of granulosa cells of follicles which have initiated growth (i.e. in follicles ranging from primary through to antral stages) in rats (8, 10, 11), mice (9, 12), sheep (13), and humans (14). Expression is strong in preantral and small antral follicles but reduced in larger antral and preovulatory follicles. Expression is not observed at the primordial stage or in atretic follicles. In the human ovary, AMH protein expression in granulosa cells has been demonstrated from 36 wk gestation through to menopause, at all stages of folliculogenesis except for primordial follicles (14, 15). AMH expression, observed from the primary stage, peaks at the large preantral and small antral stages (
4 mm) and declines in larger antral follicles (14).
Evidence for a role of AMH in control of follicular dynamics has arisen from recent mouse experiments. In AMH-null mice, the females are fertile with normal litter size but have an earlier depletion of the follicle pool and, hence, a shorter reproductive lifespan (4). The ovaries of these mice have more preantral and small antral follicles and fewer primordial follicles than their wild-type littermates. The increased progression of follicle development in the absence of AMH implies that AMH exerts a negative feedback effect on follicle growth. Further studies in which early postnatal mouse ovaries were cultured for 2–4 d in the presence of AMH showed a decreased proportion of growing follicles (12), supporting the proposed inhibitory role of AMH.
The finding of an increased proportion of growing follicles, and a reciprocal decrease in primordial follicles in both polycystic ovaries (3) and the AMH-null mouse (4) prompted us to examine the expression of AMH in polycystic ovaries. The aim of this study was to compare AMH protein expression, by immunohistochemistry, in ovaries from women with polycystic ovaries to that in ovary samples from women with normal ovarian morphology.
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Patients and Methods |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Whole ovary tissue samples (formalin fixed and paraffin embedded), from patients who had undergone oophorectomy for nonmalignant gynecological disorders between 1993 and 2000, were obtained from the histopathology tissue bank at St. Mary’s Hospital NHS Trust (London, UK). Ethical approval for the use of this tissue was obtained from the Local Research Ethics Committee, and the tissue had been removed with informed surgical consent. Tissue had been formalin fixed, dehydrated in ethanol, and embedded in paraffin wax blocks.
The samples were anonymous, retaining only essential clinical details: age of the patient, body mass index (BMI), menstrual history, and ultrasound assessment of ovarian morphology. For each tissue sample examined, histological classification of the ovary as normal or polycystic was made initially without reference to clinical data, as described below. Because most of the tissue samples were obtained from women who had undergone oophorectomy for intractable pelvic pain, preoperative endocrine data were not generally available.
Morphological classification
A single survey section was cut from each ovary. Sections were stained with hematoxylin and eosin and analyzed independently by two operators, a consultant histopathologist (A.M.F.) and a clinical fellow (L.J.W.). Tissue was classified into three groups as follows: 1) normal ovarian morphology with evidence of ovulation [i.e. corpora lutea and/or corpora albicantia (16, 17); Fig. 1
], 2) polycystic ovaries with evidence of ovulation (i.e. corpora lutea and/or corpora albicantia) (ovPCO), and 3) polycystic ovaries without evidence of ovulation (anovPCO). Samples were classified as PCO if the section contained 10 or more follicles of greater than 2 mm in diameter, had either increased density (or area) of stroma, and/or had a thickened tunica (17, 18) (Fig. 1).
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). Polycystic ovaries were defined on ultrasound by the presence of 10 or more follicles greater than 2 mm in diameter, increased stromal area, and/or ovarian volume greater than 9 ml (1). Menstrual history was particularly important in confirming the distinction between ovulatory and anovulatory women with PCO. Using these criteria, all five women in the anovulatory PCO group fit the European Society of Human Reproduction and Embryology/American Society for Reproductive Medicine Rotterdam consensus criteria for the diagnosis of PCOS (19). None of the normal ovary group and only one of the ovulatory PCO group (Table 1, subject N) was hirsute. Two of the anovulatory PCO group had hirsutism as a major presenting feature (Table 1, subjects T and X). A serum testosterone was available for subject T and was elevated at 3.6 nmol/liter. There were no significant differences between the three groups in terms of age or BMI (Kruskal Wallis). Ovarian volume was significantly greater in both PCO groups than in the normal ovary group (Table 1).
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Chemicals were from VWR International Ltd. (Lutterworth, UK) unless otherwise stated. Ovaries were serially sectioned at 5 µm, mounted on poly-L-lysine slides, and one section was used for immunohistochemistry. Staining of all slides was carried out simultaneously in a single session.
Slides were dewaxed in two changes of Histoclear (5 min each; National Diagnostics, East Riding, UK) and rehydrated through an alcohol series (BDH Analar, 100%, 100%, 70%, 2 min each; VWR International Ltd.). Antigen was retrieved with citrate buffer (0.01 mol/liter, pH 6, boiling for 20 min), and exogenous peroxidases were blocked with 0.3% hydrogen peroxide in methanol (30 min). Nonspecific antibody-binding was reduced by 20 min incubation with 20% (vol/vol) normal goat serum in PBS (Invitrogen Life Technologies, Paisley, UK) supplemented with 4% (wt/vol) BSA (Sigma-Aldrich, Poole, UK). Sections were incubated with a mouse antihuman AMH antibody, clone 5/6 [provided by N.P.G. and characterized in Weenen et al. (14); 3.75 µg/ml in 20% normal goat serum and 4% BSA] overnight at 4 C. A secondary antibody, goat antimouse (1 in 200 for 30 min; DakoCytomation Ltd., Ely, UK) was used. Labeling was visualized with a peroxidase-conjugated avidin biotin complex (60 min; Vector Laboratories, Peterborough, UK) and 3,3'-diaminobenzidine tetrahydrochloride (10 min in the dark; Zymed Laboratories, Cambridge, UK). Sections were counterstained for 2 min with Erlich’s hematoxylin to stain cell nuclei, dehydrated in alcohol (70%, 100%, 100%; 2 min each) and mounted in DPX.
As a negative control, the primary antibody was omitted. In addition, incubation with antibody was performed in the presence of an equivalent mass (or 5- or 10-fold excess) of recombinant AMH peptide ppg31 (prepared by N.P.G.). A 5-fold excess of AMH completely negated AMH immunostaining in both granulosa cells and oocytes (Fig. 2).
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Coded anonymized slides were examined on an E600 microscope (NikonUK Ltd., Kingston-upon-Thames, UK). Using a x60 objective for small preantral follicles and lower magnification objectives for larger follicles, high resolution images of every follicle in the entire section were captured by a DXM 1200 digital camera (Nikon) using the Lucia image analysis program (Nikon) and saved in TIFF format. The presence or absence of staining was independently assessed by two investigators (S.A.S. and P.D.S.-B.), each of whom was blinded to the clinical, ultrasound, and gross morphological classification.
AMH staining was assessed only in healthy follicles. Atretic follicles with pyknotic granulosa cells or oocytes were excluded from analysis. Healthy follicles were categorized according to developmental stage, and presence or absence of AMH staining was noted. The stages of follicle development were classified as 1) primordial, with one layer of flattened pregranulosa cells; 2) transitional, in which at least one, but not all, granulosa cell were cuboidal; 3) primary, with one complete layer of cuboidal granulosa cells; 4) secondary, with two to four layers of granulosa cells and beginnings of a thecal layer; 5) preantral, with more than five layers of granulosa cells and a well-defined thecal layer; and 6) antral (Fig. 2
, D–I). The intensity of staining was determined on screen, using a printed example of each follicle for relative comparison, and was classified according to the following criteria: –, no staining; +, very little staining; ++, moderate staining, and +++, intense staining (Fig. 2, D and J–L). To calculate the mean staining intensity of AMH, absence of staining was considered as 0, + = 1, ++ = 2, and +++ = 3.
Because not all early preantral follicles stained positive for AMH, we went on to determine whether the presence of AMH labeling in granulosa cells was associated with oocyte growth or granulosa cell division. The high-resolution images of every follicle were examined. In follicles where the oocyte nucleus was visible, and hence where the section could be considered to represent the largest cross-section of the follicle (20), the number of granulosa cells surrounding the oocyte was counted. In addition, an estimate of oocyte diameter was obtained by measuring, in two perpendicular dimensions, the inside diameter of the layer of granulosa cells. Formalin fixation does not preserve oocyte morphology well, precluding direct measurement of the oocyte itself.
Statistical analysis
Statistical analysis of AMH staining was carried out using Stata 8 for the Macintosh (Stata Corporation, College Station, TX). Mean proportions of stained follicles at each stage of development and in each type of ovary were estimated and compared using logistic regression (blogit command). This was also used to calculate and compare the coefficients for the trends in such proportions with stage. Confidence intervals and P values for comparisons between proportions in different groups were computed using robust SE with clustering by patients. This takes account of possible within-patient correlation in AMH staining.
Mean staining levels in each group (stage of development and ovary type) and their linear trends were analyzed using ordinary regression (regress command), again using robust SE with clustering by patients. In effect, this gives a more flexible and robust ANOVA type analysis.
The numbers of granulosa cells in the largest cross-section of AMH-positive and -negative follicles were not normally distributed and, therefore, were compared using nonparametric analysis (Mann-Whitney test, GraphPad InStat version 3.0a for Macintosh; GraphPad Software Inc., San Diego, CA). Oocyte diameter in AMH-positive and -negative follicles was compared in a similar manner.
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Results |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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). Staining was most pronounced in granulosa cells (Fig. 2B) and was confined to the cytoplasm (Fig. 2C). Staining was also present to a lesser degree in adjacent theca (Fig. 2, C and M). Oocyte morphology was poorly preserved in these formalin-fixed sections, but AMH immunostaining was present in almost all oocytes.
A total of 1403 follicles were analyzed (Table 2); AMH staining was detected in 1120 of these and was present in the majority of primary follicles but was less prevalent in primordial and transitional follicles (Fig. 2, E–I). Almost all follicles with two or more layers of granulosa cells (i.e. secondary follicles onward) showed positive AMH staining. The remaining 283 follicles were unstained and were most abundant at the early stages (primordial, transitional, and primary) (Fig. 2D and Table 2).
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and Table 2). In the normal ovary group, the majority of follicles were stained from the primordial stage onward, and there was no significant positive trend (between the primordial and the secondary stages) in the proportion of follicles stained (P = 0.21). However, in the two PCO groups, there was a significant positive trend in the proportion of AMH-positive follicles (ovPCO, P < 0.0001; anovPCO, P = 0.006). Between the primordial and secondary stages, the positive trend (combining trend and intercept with the y-axis) for anovPCO was significantly different from both normal (P = 0.002) and ovPCO (P < 0.0001) (Fig. 3A). By the secondary stage, nearly all follicles in each group were AMH positive.
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) and those with mild (+, Fig. 2J), moderate (++, Fig. 2K), and strong (+++, Fig. 2L) staining. The mean intensity of AMH staining increased significantly with advancing follicular stage for all three types of ovary (normal, P = 0.011; ovPCO, P < 0.0001; anov PCO, P < 0.0001) (Fig. 3B). The positive trend for increasing staining intensity in ovaries from anovulatory women with PCO was significantly different from those for women with regular cycles and normal (P = 0.003) or polycystic (P = 0.003) ovaries. At the primordial and transitional stages of follicles development, the mean staining intensity was significantly lower in the anovPCO group than the normal or ovulatory PCO groups (Fig. 3B). Staining levels in primary, secondary, and antral follicles were similar in the three types of ovary.
The number of granulosa cells surrounding the oocyte in the largest cross-section of the early preantral follicle varied, ranging from three to 12 for primordial and from three to 20 for transitional follicles (Fig. 4). In normal ovary, oocytes in AMH-negative follicles were surrounded by significantly fewer granulosa cells in the largest cross-section than oocytes in AMH-positive follicles (Fig. 4), at both primordial (P < 0.0001) and transitional (P = 0.0007) stages. In ovarian tissue from anovulatory women with PCO, a similar pattern was seen in transitional follicles (P < 0.0001), but not in primordial follicles.
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), but no larger than similar follicles in normal ovarian tissue.
AMH staining in granulosa cells was most intense adjacent to the oocyte (Fig. 2, B, F, H, and I). Immunostaining was also noted in the majority of oocytes. This staining could be inhibited in the presence of excess AMH peptide (Fig. 2P), but the specificity of such staining in the oocyte is open to question. In some growing follicles with intense AMH staining, AMH peptide was also detected in the surrounding theca and stroma cells (Fig. 2, B, C, M, and N).
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Discussion |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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). This study supports the hypothesis that reduced local exposure to AMH will result in a higher proportion of primordial follicles initiating growth in the polycystic compared with the normal ovary. By the primary stage, the majority of follicles in all types of ovary stained positive for AMH, and, by the secondary stage, close to 100% in each group were AMH positive. These data are similar to those reported previously by Weenen et al. (14). In the current study, nearly all antral follicles were AMH positive. Studies of circulating concentrations of AMH have shown higher levels in women with PCOS than in age-matched controls (21, 22, 23). This phenomenon almost certainly reflects the increased number of antral follicles in polycystic ovaries (23). It may seem paradoxical to propose that AMH deficiency contributes to disordered folliculogenesis in PCOS, but it is conceivable that the local concentration of AMH in the vicinity of the primordial pool is the principal determinant of its effect on follicle initiation. Primordial and early growing follicles lack a direct blood supply (24) and local paracrine signals must play an important part in regulating initiation of follicle growth. Synthesis of AMH is confined to granulosa cells (9), but in some growing follicles we observed what appeared to be diffusion of AMH protein beyond the granulosa cell into adjacent theca or stroma. This is consistent with the view that AMH, produced by growing follicles, exerts a local (paracrine) effect on nearby primordial (resting) follicles.
There was no association between oocyte growth and AMH expression in granulosa cells of primordial and transitional follicles in normal women. This is not surprising, as significant oocyte growth is not thought to occur before the primary stage (20) and our data confirm this. However, there was an association between AMH expression and the number of granulosa cells in the largest cross section of the follicle, suggesting that AMH expression is activated in proliferating granulosa cells. There is direct evidence that even squamous granulosa cells undergo a low rate of cell division, as demonstrated by proliferating cell nuclear antigen immunostaining of granulosa cells in occasional primordial follicles from a variety of species including human (25), macaque (25), baboon (26), and cow (27), with more widespread labeling in sheep (28). Furthermore, examination of small preantral follicles in rats exposed continuously to [3H]thymidine for up to 7 d showed labeling of squamous granulosa cells, and it was concluded that a proportion of these follicles were growing at a very slow rate (29). This, coupled with the variable number of granulosa cells in primordial follicles seen in this study, suggests that there is some granulosa cell proliferation before transformation from the flattened to the cuboidal granulosa cell phenotype (i.e. formation of transitional and primary follicles). An alternative explanation is that the follicles in which AMH is expressed were originally endowed (during follicle formation) with a larger number of granulosa cells and that these follicles are more likely to start growing. We conclude that AMH expression is associated with increased granulosa cell division or endowment at the primordial stage and can precede the transition to primary follicles.
In previous studies of mouse (12) and human (14) ovary, AMH protein was detected in follicles from the primary stage onward, as the granulosa cells become cuboidal. In this study, however, AMH protein was also detected in varying proportions of primordial and transitional follicles, depending on the type of ovary. The same tissue fixation, primary antibody (5/6A) and immunocytochemistry protocol were used in both this study and the previous analysis (14) of human tissue but the microscope and image analysis system used in the current study may have enhanced resolution capable of detecting immunoreactive peptide in thin pregranulosa cells of primordial follicles (Fig. 2E).
In conclusion, these observations are consistent with the hypothesis that, in the ovarian cortex, AMH deficiency contributes to disordered early follicle development in PCOS. An alternative interpretation is that reduced AMH expression is a marker of the abnormal early folliculogenesis, the primary cause of which remains unknown.
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Footnotes |
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Present address for A.M.F.: Department of Histopathology, Royal Free and University College Medical School, University College London, London WC1E 6BT, United Kingdom.
First Published Online July 19, 2005
Abbreviations: AMH, Anti-müllerian hormone; anovPCO, polycystic ovaries without evidence of ovulation; BMI, body mass index; ovPCO, polycystic ovaries with evidence of ovulation; PCO, polycystic ovary; PCOS, PCO syndrome.
Received April 26, 2005.
Accepted July 8, 2005.
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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), ovPCO (•), and anovPCO (
). Secondary+ includes all preantral follicles with two or more layers of granulosa cells. A, The proportions of follicles staining positive for AMH in the three types of ovary were significantly different at the primordial (P = 0.0015) and transitional (P = 0.0017) stages. Significantly fewer primordial and transitional follicles in anovulatory PCO samples were AMH positive compared with normal (a, P = 0.003) and ovulatory PCO (b, P = 0.0021) samples, respectively. B, The scoring system for calculating mean staining intensity was as follows: no staining = 0, + = 1, ++ = 2, and +++ = 3. The mean intensity of staining was significantly lower for anovulatory PCO at primordial (a, compared with normal and ovPCO, P = 0.003) and transitional (b, compared with normal, P = 0.04 and ovulatory PCO, P = 0.004) stages. No significant difference in staining intensity between the three groups at primary, secondary, and antral stages.
) and -positive follicles (
) in normal (F and H) and anovulatory polycystic (G and I) ovaries. In normal ovaries, 146 primordial and 146 transitional follicles were analyzed; in anovulatory PCO ovaries, 17 primordial and 62 transitional follicles were analyzed. Values are median ± 95% confidence intervals of the median. AMH-positive significantly different from AMH-negative: *, P = 0.014; **, P = 0.0007; ***, P < 0.0001.