Scholarly article on topic 'Anti-Müllerian hormone in polycystic ovary syndrome and normo-ovulatory women: Correlation with clinical, hormonal and ultrasonographic parameters'

Anti-Müllerian hormone in polycystic ovary syndrome and normo-ovulatory women: Correlation with clinical, hormonal and ultrasonographic parameters Academic research paper on "Veterinary science"

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{"Anti-Müllerian hormone" / "Polycystic ovary syndrome" / Hyperandrogenism}

Abstract of research paper on Veterinary science, author of scientific article — Adel F. Begawy, Akmal N. El-Mazny, Nermeen A. Abou-Salem, Nagwa E. El-Taweel

Abstract Background Although the ultimate pathogenesis of polycystic ovary syndrome (PCOS) remains obscure, the distinctive feature is failure of follicular maturation resulting in anovulation and accumulation of preantral and small antral follicles which contribute significantly to the production of anti-Müllerian hormone (AMH). Objectives To compare serum AMH levels between PCOS and normo-ovulatory women; and to investigate whether AMH correlates to clinical, hormonal and ultrasonographic parameters in both groups. Design Comparative observational cross-sectional study. Setting Department of Obstetrics and Gynecology, Kasr El-Aini Teaching Hospital, Faculty of Medicine, Cairo University. Subjects Thirty-five women with PCOS according to the Rotterdam consensus; and 35 normo-ovulatory-matched controls with male, tubal or unexplained infertility. Methods Serum levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), testosterone, androstenedione, estradiol, fasting insulin and AMH were measured in the early follicular phase (day 3–4) of natural cycle or progestin-induced withdrawal bleeding (in PCOS); together with transvaginal sonography for detection of the number of small follicles (<10mm) and calculation of ovarian volume. Main outcome measures Correlation between AMH and clinical, hormonal and ultrasonographic parameters in both groups. Results AMH was significantly higher in the PCOS group. In the whole group of patients and in each group separately, AMH was positively correlated to LH, LH/FSH, number of follicles <10mm and ovarian volume; and negatively correlated to FSH. No correlation was found between AMH and age, BMI, estradiol or fasting insulin. Testosterone and androstenedione were positively correlated to AMH in the PCOS group exclusively (r=0.557; P=0.001 and r=0.451; P=0.007, respectively). Multiple regression analysis demonstrated that testosterone was the only determinant for AMH level (r=0.485; P<0.001). Conclusions Hyperandrogenism is associated with increased AMH secretion in PCOS patients, possibly due to increased number of small antral follicles. Assessment of AMH levels before and after the treatment of hyperandrogenism should be recommended in the plan of management of PCOS.

Academic research paper on topic "Anti-Müllerian hormone in polycystic ovary syndrome and normo-ovulatory women: Correlation with clinical, hormonal and ultrasonographic parameters"

Middle East Fertility Society Journal (2010) 15, 253-258

Middle East Fertility Society Middle East Fertility Society Journal

www.mefsjournal.com www.sciencedirect.com

ORIGINAL ARTICLE

Anti-Miillerian hormone in polycystic ovary syndrome and normo-ovulatory women: Correlation with clinical, hormonal and ultrasonographic parameters

Adel F. Begawy a *, Akmal N. El-Mazny a, Nermeen A. Abou-Salem a, Nagwa E. El-Taweel b

a Department of Obstetrics and Gynecology, Faculty of Medicine, Cairo University, Egypt b Department of Clinical and Chemical Pathology, Faculty of Medicine, Cairo University, Egypt

Received 3 January 2010; accepted 22 February 2010 Available online 22 September 2010

KEYWORDS

Anti-Müllerian hormone; Polycystic ovary syndrome; Hyperandrogenism

Abstract Background: Although the ultimate pathogenesis of polycystic ovary syndrome (PCOS) remains obscure, the distinctive feature is failure of follicular maturation resulting in anovulation and accumulation of preantral and small antral follicles which contribute significantly to the production of anti-Mullerian hormone (AMH).

Objectives: To compare serum AMH levels between PCOS and normo-ovulatory women; and to investigate whether AMH correlates to clinical, hormonal and ultrasonographic parameters in both groups.

Design: Comparative observational cross-sectional study.

Setting: Department of Obstetrics and Gynecology, Kasr El-Aini Teaching Hospital, Faculty of Medicine, Cairo University.

Subjects: Thirty-five women with PCOS according to the Rotterdam consensus; and 35 normo-ovulatory-matched controls with male, tubal or unexplained infertility.

Methods: Serum levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), testosterone, androstenedione, estradiol, fasting insulin and AMH were measured in the early follicular phase (day 3-4) of natural cycle or progestin-induced withdrawal bleeding (in PCOS); together with

Corresponding author. E-mail address: adel_faroük1@yahoo.com (A.F. Begawy).

1110-5690 © 2010 Middle East Fertility Society. Production and Hosting by Elsevier B.V. All rights reserved.

Peer-review under responsibility of Middle East Fertility Society. doi:10.1016/j.mefs.2010.08.005

transvaginal sonography for detection of the number of small follicles (<10 mm) and calculation of ovarian volume.

Main outcome measures: Correlation between AMH and clinical, hormonal and ultrasonographic parameters in both groups.

Results: AMH was significantly higher in the PCOS group. In the whole group of patients and in each group separately, AMH was positively correlated to LH, LH/FSH, number of follicles <10 mm and ovarian volume; and negatively correlated to FSH. No correlation was found between AMH and age, BMI, estradiol or fasting insulin. Testosterone and androstenedione were positively correlated to AMH in the PCOS group exclusively (r = 0.557; P = 0.001 and r = 0.451; P = 0.007, respectively). Multiple regression analysis demonstrated that testosterone was the only determinant for AMH level (r = 0.485; P < 0.001).

Conclusions: Hyperandrogenism is associated with increased AMH secretion in PCOS patients, possibly due to increased number of small antral follicles. Assessment of AMH levels before and after the treatment of hyperandrogenism should be recommended in the plan of management of PCOS.

© 2010 Middle East Fertility Society. Production and Hosting by Elsevier B.V. All rights reserved.

1. Introduction

Polycystic ovary syndrome (PCOS) is the most common endocrine disorder among women in the reproductive age. It is characterized by anovulation manifested as oligomenorrhea or amenorrhea, elevated levels of androgens and luteinizing hormone (LH), and polycystic ovaries by ultrasound. PCOS also encompasses a broad spectrum of clinical, hormonal and ultrasonographic characteristics (1).

The dimeric glycoprotein anti-Mullerian hormone (AMH) is a member of the transforming growth factor-B superfamily. It has been mainly studied for its regulatory role in male sex differentiation. It is produced by the Sertoli cells of the fetal testis, and induces the regression of the Miillerian ducts (2,3). In females, AMH is produced only after birth by granulosa cells from preantral and small antral follicles (4,5). AMH has an inhibitory effect on the primordial follicle recruitment as well as on the responsiveness of growing follicles to follicle-stimulating hormone (FSH) (6,7). This ovary-specific expression pattern in granulosa cells of growing non selected follicles makes AMH an ideal marker for ''ovarian reserve'' (8). Several studies have reported a correlation between serum AMH levels and the number of small antral follicles (9-12).

Although the ultimate pathogenesis of PCOS remains obscure, the distinctive feature is failure of follicular maturation, despite initial recruitment, resulting in anovulation and accumulation of preantral and small antral follicles, which contribute significantly to the production of AMH (11,12). AMH also inhibits aromatase activity, suggesting that AMH contributes to the severity of PCOS (13). The objectives of the present study were to compare serum AMH levels between PCOS and normo-ovulatory women; and to investigate whether AMH correlates to clinical, hormonal and ultrasonographic parameters in both groups.

2. Subjects and methods

This comparative observational cross-sectional study was conducted at the Department of Obstetrics and Gynecology, Kasr El-Aini Teaching Hospital, Faculty of Medicine, Cairo University between June and November 2009. The study was approved by the local ethical committee and a total of 70

patients were prospectively enrolled in the study, after taking their informed consent. The inclusion criteria were: age between 20 and 35 years, both ovaries present, no previous ovarian operation, adequate visualization of ovaries on transvaginal sonography, and no current hormone therapy.

The PCOS group consisted of 35 women diagnosed according to the Rotterdam consensus (14) based on the association of at least two of the three following criteria: (1) Oligo- and/or anovulation (<6 menstrual periods/year); (2) hyperandroge-nism, as defined either by hirsutism (Ferriman-Gallwey score >8), or minor signs such as acne or seborrhea, and/or testosterone >3 nmol/l and/or androstenedione >12 nmol/l; and (3) ultrasound criterion of PCO (at least 12 follicles 2-9 mm in diameter per ovary, and/or increased ovarian volume of at least 10 mm3 (15)).

The control group consisted of 35 healthy women with male, tubal or unexplained infertility. They had regular ovula-tory cycles (25-35 days), no endocrine abnormalities (normal prolactin, basal FSH and estradiol, and no hyperandroge-nism), and normal ultrasonic ovarian morphology. The control women were matched with PCOS women for age (±2 years) and body mass index, BMI (±10%).

Transvaginal sonography was performed for detection of the number of small follicles (<10 mm), and calculation of ovarian volume using the formula of ellipsoid (p/6 x length x width x height).

Blood sampling for hormone measurement was performed in the early follicular phase (day 3-4 after the last menstrual period) both in PCOS and control women. Each participant was subjected to withdrawal of 6 mL venous blood after 810 h fasting on a plain tube and centrifuged after clotting. Serum samples were separated and stored at —70 0C until assayed. Serum LH (Cat. No. L2KLH2) (16), FSH (Cat. No. L2KFS2) (17) and androstenedione (Cat. No. L5KAO2) (18) levels were measured using two-site chemiluminescent immunometric assay, on Immulite 2000 analyzer DPC® (Diagnostic Product Corporation, Los Angeles, CA, USA). Serum testosterone (19), and estradiol (20) levels were measured using electro-chemiluminescence immunoassay, on Elecsys immunoassay analyzer (Roche Diagnostics GmbH, D-68298 Mannheim). Fasting serum insulin (21) levels were measured using radioimmunoassay (RIA) (ANOGEN 2355 Derry Road East, Unit 23, Mississauga, Ontario CANADA L5S 1V6). Serum AMH (22)

levels were measured using Enzyme-Linked Immunosorbent Assay (ELISA), using kits supplied by DSL® (Diagnostic System Laboratories Inc., Texas, USA).

2.1. Testosterone assay

Fifty microliters of sample were incubated with a testosterone-specific biotinylated antibody and a testosterone derivative labeled with a ruthenium complex. Application of the voltage to the electrode then induces chemiluminescent emission which was measured by a photomultiplier. Results were determined via a calibration curve which was instrument-specifically generated by 2-point calibration and a master curve provided via the reagent barcode.

2.2. Estradiol assay

Thirty-five microliters of the sample were incubated with an estradiol-specific biotinylated antibody, an immunocomplex was formed, the amount of which was dependent upon the analyte concentration in the sample, then induces chemilumi-nescent emission which was measured by a photomultiplier. Results were determined via a calibration curve which was instrument-specifically generated by 2-point calibration and a master curve provided via the reagent barcode.

2.3. Insulin assay

ANOGEN Insulin test based on radioimmunoassay, an analytical technique in which radio-labeled Insulin competes with unlabeled Insulin for binding sites on anti-Insulin immobilized to the inside wall of the tube. Standards of known Insulin concentrations were run concurrently with the samples being assayed and a standard curve was plotted. The unknown Insulin concentration in each sample was calculated from this curve.

2.4. AMH assay

It was a competitive enzyme immunoassay. The immunoplate in this kit was pre-coated with secondary antibody and the nonspecific binding sites were blocked. The secondary antibody can bind to the Fc fragment of the primary antibody (peptide antibody) whose Fab fragment will be competitively bound by both biotinylated peptide and peptide standard or target peptide in the sample. The biotinylated peptide was able to interact with streptavidin-horseradish peroxidase (SA-HRP) which catalyzes the substrate solution composed of 3,3',5,5'-tetramethylbenzidine (TMB) and hydrogen perox-idase to produce a blue-coloured solution. The enzyme substrate reaction was stopped by hydrogen chloride (HCl) and the solution turns to yellow. The intensity of the yellow colour was directly proportional to the amount of biotinylated peptide-SA-HRP complex but inversely proportional to the amount of the peptide in standard solutions or samples. This was due to the competitive binding of biotinylated peptide and the peptide in standard solutions or samples to the pep-tide antibody (primary antibody). A standard curve of a pep-tide with known concentration can be established accordingly. The peptide with unknown concentrations in samples can be determined by the extrapolation of the standard curve.

Statistical analysis was performed using Student's t-test and the Mann-Whitney U-test as appropriate. Correlations between AMH and the various parameters were evaluated using Pearson moment correlation coefficient (r). Multiple regression analysis was used to evaluate the preferential effect of the different studied variables on AMH level. A P-value 60.05 was considered statistically significant. All statistical procedures were run on SPSS® (Statistical Package for the Social Science; SPSS Inc., Chicago, IL, USA) for MS Windows®.

3. Results

Table 1 presents the clinical, hormonal and ultrasonographic data of the PCOS and control groups. The mean FSH and estradiol were not significantly different between the two groups. The mean LH, LH/FSH, testosterone, androstenedi-one, fasting insulin, AMH, number of follicles <10 mm and ovarian volume were significantly higher in the PCOS group.

Table 2 presents the correlation between AMH and clinical, hormonal and ultrasonographic parameters in the two groups

Table 1 Clinical, hormonal and ultrasonographic data of the PCOS and control groups. Results are expressed as mean ± SD.

PCOS (n = 35) Control P Sig.

(n = 35)

Age (years) 27.5 ± 4.1 28.6 ± 4.6 0.295 NS

BMI (kg/m2) 26.1 ± 4.9 25.9 ± 3.7 0.848 NS

LH (IU/l) 8.9 ± 4.4 4.7 ± 2.3 <0.001 S

FSH (IU/l) 5.6 ± 1.7 6.1 ± 1.8 0.236 NS

LH/FSH 1.9 ± 0.9 0.8 ± 0.4 <0.001 S

Testosterone (nmol/l) 2.3 ± 0.8 1.5 ± 0.6 <0.001 S

Androstenedione 9.2 ± 4.3 6.6 ± 1.8 0.002 S

(nmol/l)

Estradiol (pmol/l) 110.1 ± 56.i 6 118.4 ± 58.1 0.547 NS

Fasting insulin (mlU/l) 8.1 ± 4.3 4.6 ± 2.4 <0.001 S

AMH (pmol/l) 42.6 ± 23.8 16.1 ± 7.5 <0.001 S

No. of follicles <10 mm 21.3 ± 7.3 7.5 ± 3.4 <0.001 S

Ovarian volume (mm3) 28.7 ± 6.7 7.8 ± 1.6 <0.001 S

NS = non-significant and S = significant.

Table 2 Correlation between AMH and clinical, hormonal and ultrasonographic parameters in the whole group of patients (n = 70).

r P Sig.

Age -0.205 0.089 NS

BMI -0.130 0.283 NS

LH 0.281 0.018 S

FSH -0.358 0.002 S

LH/FSH 0.426 <0.001 S

Testosterone 0.472 <0.001 S

Androstenedione 0.371 0.002 S

Estradiol -0.095 0.434 NS

Fasting insulin -0.114 0.347 NS

No. of follicles <10 mm 0.627 <0.001 S

Ovarian volume 0.478 <0.001 S

NS = non-significant and S = significant.

Table 3 Correlation between AMH and clinical, hormonal and ultrasonographic parameters in the PCOS and control groups.

PCOS (n = 35) Control (n = 35)

r P Sig. r P Sig.

Age -0.137 0.433 NS -0.098 0.575 NS

BMI -0.086 0.623 NS -0.195 0.262 NS

LH 0.336 0.048 S 0.341 0.045 S

FSH -0.347 0.041 S -0.356 0.036 S

LH/FSH 0.421 0.012 S 0.398 0.036 S

Testosterone 0.557 0.001 S 0.199 0.504 NS

Androstenedione 0.451 0.007 S 0.227 0.379 NS

Estradiol -0.085 0.627 NS -0.074 0.673 NS

Fasting insulin -0.062 0.723 NS -0.179 0.607 NS

No. of follicles <10 mm 0.625 <0.001 S 0.475 0.008 S

Ovarian volume 0.436 0.009 S 0.369 0.029 S

NS = non-significant and S = significant.

together. Significant positive correlations were found between AMH, and LH, LH/FSH, testosterone, androstenedione, number of follicles <10 mm and ovarian volume; while a significant negative correlation was found between AMH and FSH. No correlation was found between AMH and age, BMI, estradiol or fasting insulin.

Table 3 presents the correlation between AMH and clinical, hormonal and ultrasonographic parameters in the PCOS and control groups separately. The above correlations were almost similar except for testosterone and androstenedione which were positively correlated to AMH in the PCOS group exclusively (r = 0.557; P = 0.001 and r = 0.451; P = 0.007, respectively).

Multiple regression analysis was performed in the PCOS group including AMH as the dependant variable, and LH, FSH, LH/FSH, testosterone, androstenedione, number of follicles <10 mm and ovarian volume as independent variables. Testosterone was the only determinant for AMH level (r = 0.485; P < 0.001), whereas other parameters were no longer significantly related.

4. Discussion

The results of the present study have shown higher serum AMH levels in PCOS group than in controls (Table 1); and a significant positive correlation between AMH and number of follicles <10 mm in the whole group of patients (Table 2) and in each group separately (Table 3), which is in line with the finding that serum AMH levels reflect the number of small antral follicles demonstrated in several studies (9-12,23-30).

Pallet et al. (28) demonstrated an exponential fall in AMH concentration follicular fluid as the follicle size increased. The highest concentration was found in fluid from small follicles while present in very low amounts in those follicles above 9 mm in diameter which was particularly apparent once the follicle reached 10 mm in diameter, i.e. the size at which follicle selection occurs.

Our findings regarding LH and LH/FSH (Tables 2 and 3) are comparable with the results of previous studies (11,26,31). However, Pigny et al. (12) found no relationship between AMH and LH and LH/FSH in PCOS and controls.

The results of the present study revealed no significant correlations between AMH and age, BMI, estradiol or fasting

insulin (Tables 2 and 3). This is in agreement with Pigny et al. (12). However, Nardo et al. (30) indicated that AMH is generally decreased with chronological age and increased with insulin; and Chen et al. (29) found that AMH had a significant negative association with both BMI and insulin resistance.

In the present study, significant positive correlations were found between AMH and serum testosterone and androstene-dione in the PCOS group exclusively (Table 3). These findings are in accordance with the results of previous studies (11,12,25,26,29), and add to the existing evidence for small ovarian follicles in the production of both AMH and andro-gens. However, Nardo et al. (30) indicated that AMH is similarly related to androgens in women with and without PCOS.

Pigny et al. (12) suggested that the increase in AMH serum levels in PCOS is a consequence of androgen-induced excess in small antral follicles and that each follicle produces normal amount of AMH. However, Pallet et al. (28) found that raised serum AMH in PCOS is a reflection of both an increase in production per cell and the increase in follicle number since they used cells from size-matched follicles in patients and controls plated at the same density.

It could be also speculated that since AMH inhibits FSH-induced aromatase activity in cultured mouse (32) and human (33) granulosa cells, it may also be responsible for the reduced aromatase activity in PCO granulosa cells (34) and contributes to the elevated androgen levels. Moreover, Criso-sto et al. (35) proposed that AMH expression is modulated by androgens in bovine granulosa cells from small follicles; suggesting that androgens, by inhibiting AMH expression, may promote follicle recruitment, increasing the early growing follicular pool.

Multiple regression analysis demonstrated that testosterone was the only determinant for AMH level in the PCOS group (r = 0.485; P < 0.001). This is in contrast with Pigny et al. (12) where only the number of 2-5 mm follicles, but not andro-gens, was significantly related to AMH. However, Eldar-Geva et al. (26) revealed that both the number of small follicles and serum androgens were correlated to AMH.

In conclusion, the present study has demonstrated higher serum AMH levels in PCOS group than in controls; and significant positive correlations between AMH and testosterone and androstenedione in the PCOS group exclusively. Furthermore,

multiple regression analysis demonstrated that testosterone was the only determinant for AMH level in the PCOS group. It could be speculated that hyperandrogenism may contribute to increased number of small antral follicles leading to increased AMH secretion in PCOS patients. Assessment of AMH levels before and after the treatment of hyperandroge-nism should be recommended in the plan of management of PCOS.

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