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Clinical significance of serum concentration of anti-Müllerian hormone in obese women with polycystic ovary syndrome
RBMOnline - Vol 15. No 5. 2007 495-499 Reproductive BioMedicine Online; www.rbmonline.com/Article/2954 on web 25 September 2007
!RTICLE #LINICAL SIGNIlCANCE OF SERUM CONCENTRATION OF ANTI -àLLERIAN HORMONE IN OBESE WOMEN WITH POLYCYSTIC OVARY SYNDROME 3AMIR %L (ALAWATY IS AN OBSTETRICIAN AND GYNECOLOGIST IN THE 7OMENS 3PECIALIZED (OSPITAL +ING &AHD -EDICAL #ITY 2IYADH 3AUDI !RABIA WHERE A HIGH PROPORTION OF THE PATIENT POPULATION SEEKS TREATMENT FOR INFERTILITY AND ENDOCRINE RELATED DISEASES (E IS A &ELLOW OF THE 2OYAL #OLLEGE OF 3URGEONS OF %DINBURGH AND HE COMPLETED HIS 0H$ IN 4HE .ETHERLANDS
$R 3AMIR %L (ALAWATY 3AMIR %L (ALAWATY !HMED 2IZK -ANAL +AMAL -ONA !BOUL(ASSAN (EBA !L 3AWAH /LFAT .OAH (ESHAM !L )NANY $EPARTMENT OF /BSTETRICS 'YNECOLOGY #AIRO 5NIVERSITY $EPARTMENT OF /BSTETRICS 'YNECOLOGY "ENHA 5NIVERSITY $EPARTMENT OF #HEMICAL 0ATHOLOGY #AIRO 5NIVERSITY #ORRESPONDENCE -OUSTAPHA (ASSANIN 3T -ANIAL #AIRO %GYPT E MAIL KAAINIH YAHOOCOM
!BSTRACT In the human ovary, expression of anti-Müllerian hormone (AMH) is detected primarily in granulosa cells of preantral and small antral follicles. The aim of this study was to compare serum AMH measurements in obese women with polycystic ovary syndrome (PCOS) with those in obese normo-ovulatory women and to evaluate the role of AMH as a predictor of ovulation induction by clomiphene citrate compared to FSH. Sixty-eight obese women with PCOS were compared to 17 normoovulatory obese women. All women had a body mass index greater than 30 kg/m2. Women with PCOS received clomiphene citrate (150 mg/day) for 5 days starting from day 3 of cycle and were subdivided into responsive and non-responsive groups. There was a significant difference in AMH concentration between women with PCOS and the control group (P < 0.05) and also between women with PCOS who responded to clomiphene citrate and those who did not (P < 0.01). A value of 1.2 ng/ ml AMH could be used to predict response to clomiphene citrate in obese women with PCOS (sensitivity 71%, specificity 65.7%). AMH production increases in women with PCOS compared to controls. AMH measurement could also be useful in the prediction of ovarian response to clomiphene citrate. Keywords: anti-Müllerian hormone, clomiphene citrate, obesity, ovulation induction, polycystic ovary syndrome
)NTRODUCTION Polycystic ovary syndrome (PCOS) is one of the most common causes of anovulatory infertility, affecting 4–7% of women (Pasquali and Gambineri, 2006). Obesity may either be a trigger for developing the syndrome when inherited predisposition exists, or an independent causative factor by inducing reduced sensitivity to insulin (Ciaraldi et al., 1997; Sozen and Arici, 2000). Clomiphene citrate represents a convenient and safe first choice treatment and is widely available for induction of ovulation in women with PCOS. (Imani et al., 2002). Currently, patients who do not respond to clomiphene citrate are identified only after 3–6 months of treatment (Ghobadi et al., 2007). If such
patients could be distinguished earlier, time could be saved by changing clomiphene citrate-resistant anovulatory patients to alternative treatments. Anti-Müllerian hormone (AMH) could be used to fulfill this target. AMH is a dimeric glycoprotein made up of two monomers attached to each other by disulphide bonds. It belongs to the transforming growth factor β superfamily, which acts on tissue growth and differentiation (Lee et al., 1996). Sertoli cells in the male produce AMH, which induces the degeneration of the Müllerian ducts and provides for the normal formation of the male genital system. Sertoli cell secretion of AMH continues throughout life. Concentrations drop following puberty,
© 2007 Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB3 8DB, UK
Article - Anti-Müllerian hormone in obese women with PCOS - S El-Halawaty et al.
decreasing slowly to a low post-puberty value. In females, it is produced by the granulosa cells of early developing follicles. After puberty and the onset of menstrual cycles, serum AMH concentration decreases progressively until it becomes undetectable at around the time of menopause (Lee et al., 1996; Seifer et al., 2002). As AMH is largely expressed throughout folliculogenesis, serum concentrations of AMH may represent both the quantity and quality of the ovarian follicle pool. FSH at day 3 of the cycle is a commonly used diagnostic blood test to help in prediction of ovarian reserve (Johnson et al., 2006) but it does not seem adequate on its own to enable prediction of response to ovarian stimulation (Kwee et al., 2006). In addition, available data in the medical literature has demonstrated that obesity is an important parameter that negatively affects the response to ovarian stimulation with clomiphene citrate (Kousta et al., 1997; Imani et al., 1998; Douchi et al., 2004). Accordingly, both FSH and AMH were compared regarding their value in prediction of ovarian response to clomiphene citrate. An additional aim was to compare serum AMH measurement in obese women with PCOS to obese normo-ovulatory women.
-ATERIALS AND METHODS This was a prospective controlled clinical trial in which obese subfertile women with PCOS, n = 68 and body mass index (BMI) greater than 30 kg/m2, were compared to obese normo-ovulatory women who comprised the control group (n = 17). Participants were recruited from the Outpatient Clinic of Gynecology of Kasr El-Aini Cairo University Hospital and Benha University Hospital. The study was conducted between March 2006 and November 2006 after ethical committee approval. A full history was taken and clinical examination was performed. BMI was calculated as weight (kg)/height (m)2. Only women with anovulation as a cause of infertility were included. The clinical criteria were oligomenorrhoea (menstrual interval longer than 6 weeks) or amenorrhoea (no menstrual loss for 6 months) dating from menarche, and/or hirsutism. The main aim of this study was to measure AMH and FSH for prediction of response to clomiphene citrate and, as Rotterdam criteria require two out of three parameters, clinical hyperandrogenism was also a focus. At the same time, biochemical markers were assayed as increased LH:FSH ratio and normal FSH concentrations. The ultrasound criteria were enlarged ovaries with an increased stroma and more than 10 subcapsular follicles of 3–8 mm diameter. Women having bilateral tubal block, organic uterine or ovarian pathology and women whose husbands were found to be infertile on semen analysis were excluded. Other excluded conditions were hypo/hyperthyroidism, hyperprolactinaemia and Cushing’s syndrome as detected by history, examination and investigations.
All women with PCOS received clomiphene citrate (Clomid; Global Napi Pharmaceuticals under license of Hoechst, Marion Roussel, France) 50 mg/8 h from the third day of the cycle and for 5 days. Transvaginal ultrasonographic serial folliculometry to check ovulation was performed from day 8 until day of HCG
administration. Human chorionic gonadotropin 10,000 IU (HCG; Pregnyl, NV Organon International, The Netherlands) was administered via intramuscular single injection when the leading follicle reached 18 mm in diameter.
3AMPLE COLLECTION In the early follicular phase (day 3), fasting blood samples were collected from all women for the measurement of serum insulin, glucose, LH, FSH and AMH. Those who responded to treatment were subjected to oestradiol assay on the day of HCG administration and progesterone assay on day 22 of the cycle. Blood samples were taken from all women (6 ml) and collected in vaccutainer tubes (4 ml in plain tubes and 2 ml in tubes with sodium fluoride). All samples were then centrifuged at 2000 g for 15 min. Serum and plasma were separated and stored at –20 °C until assayed. The concentrations of FSH, LH, oestradiol, progesterone and fasting insulin were assayed using reagents supplied by DPC (Diagnostic Products Corporation, Los Angeles, USA) by enzyme chemiluminescence immunoassay using the Immulite system. Progesterone concentration above 10 ng/ml was considered to indicate an appropriate ovulatory response. Fasting blood glucose was measured on an automated autoanalyzer (Hitachi 917) using reagents supplied by Roche (Roche Diagnostics GmbH, D-68298 Mannheim, Germany). Serum AMH was determined by quantitative sandwich enzyme-linked immunosorbent assay (ELISA) (Teixeira et al., 2001) using kits supplied by DSL, Cat No. 14400 (Diagnostic Systems Laboratories, Inc., Webster, Texas, USA). Insulin resistance (IR) was calculated using the Homeostasis Assessment Model (HOMA) as follows: HOMA IR = fasting insulinaemia (μIU/ml) × fasting glucose (mmol/l)/22.5 (Matthews et al., 1985).
3TATISTICAL EVALUATION Data are presented as mean ± SD. For statistical analysis, comparison between different parameters in the present study was performed using Student’s t-test for comparing parametric data. For comparing non-parametric data, chi-squared tests and Mann–Whitney tests were performed. Pearson’s correlation co-efficient was calculated to evaluate the relation of AMH to number of follicles. Sensitivity and specificity were calculated to estimate the best cut-off value using receiver operating characteristic (ROC) curve. All statistical calculations were performed using the computer programmes Arcus Quickstat Biomedical version l.0 and Microsoft Excel version 2000.
2ESULTS Sixty-eight obese women having PCOS were compared to 17 normo-ovulatory obese women (Table 1). There was a significant difference in AMH (P = 0.0254) and LH (P = 0.0167) between both groups. HOMA IR was apparently higher in women with PCOS
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Article - Anti-Müllerian hormone in obese women with PCOS - S El-Halawaty et al.
but it did not reach statistical significance. Other demographic criteria did not show statistical difference (Table 1). Women with PCOS were further subdivided into two groups based on transvaginal ultrasonography results and progesterone concentrations after ovulation induction with clomiphene citrate: Group I, 17 women who responded to clomiphene citrate and Group II, 51 women who did not respond to clomiphene citrate. There were no significant differences between the groups in age, infertility duration, BMI, hormonal profile and fasting blood glucose concentration. However, there was a highly significant difference in AMH between the groups (P = 0.0081, Table 2). In the present study, the ROC curve showed a cut-off value of 1.2 ng/ml AMH with area under curve more than 0.71, sensitivity 71% and specificity 65.7% to predict response to clomiphene citrate in obese women with PCOS (Figure 1). There appeared to be a weak (not significant) correlation between AMH concentration and the number of follicles >18 mm observed by ultrasound on the day of HCG administration (Figure 2, correlation coefficient (r) = –0.18)
Figure 1. Area under receiver operating characteristic (ROC) curve with a cut-off value for anti-Müllerian hormone.
Table 1. Comparison between women with polycystic ovary syndrome and normo-ovulatory women. Parameter
Control group (n = 17)
PCOS group (n = 68)
P-value
Age (years) Body weight (kg) Height (m) BMI (kg/m2) LH (mIU/ml) FSH (mIU/ml) AMH (ng/ml) Fasting glucose (mg/dl) Fasting insulin (μIU/ml) HOMA IR
27.65 ± 5.70 98.4 ± 14.21 1.63 ± 0.05 36.4 ± 5.67 4.1 ± 2.6 4.53 ± 2.03 1.95 ± 1.8 86.5 ± 17.61 9.3 ± 5.52 2.36 ± 1.67
28.21 ± 4.8 97.8 ± 14.61 1.61 ± 0.05 36.7 ± 5.75 10.2 ± 3.52 4.53 ± 1.58 3.31 ± 3.14 90.1 ± 24.39 12.8 ± 9.14 4.18 ± 3.4
NS NS NS NS 0.0167 NS 0.0254 NS NS NS
All values are mean ± standard deviation; AMH = anti-Müllerian hormone; BMI = body mass index; HOMA IR = Homeostasis Assessment Model insulin resistance; NS = not statistically significant; PCOS = polycystic ovary syndrome.
Table 2. Comparison between women with PCOS who responded to clomiphene citrate and those who did not respond. Parameter
Responsive cases (n = 17)
Non-responsive cases (n = 51)
P-value
Age (years) Duration of infertility (years) Body weight (kg) Height (m) BMI (kg/m2) LH (mIU/ml) FSH (mIU/ml) Fasting glucose (mg/dl) Fasting insulin (μIU/ml) HOMA IR AMH (ng/ml) Oestradiol (pg/ml) Follicles >18 mm on day of HCG (n) Endometrial thickness (mm) Progesterone (ng/ml)
28.82 ± 3.80 4.71 ± 2.66 101.94 ± 9.85 1.64 ± 0.06 38.25 ± 4.77 10.38 ± 2.08 5.94 ± 4.22 85.29 ± 17.61 10.95 ± 10.52 2.36 ±1.67 4.38 ± 3.30 181.06 ± 60.22 1.65 ± 0.49 6.35 ± 0.70 11.76 ± 2.02
29.55 ± 4.60 5.39 ± 3.12 95.93 ± 14.61 1.64 ± 0.05 35.58 ± 5.75 10.03 ± 2.41 4.53 ± 1.58 88.18 ± 24.39 8.63 ± 5.14 1.89 ± 1.33 1.89 ± 1.33 Not measureda 0 5.54 ± 0.74 1.93 ± 1.42
NS NS NS NS NS NS NS NS NS NS 0.0081 – – 0.0004 <0.0001
All values are mean ± standard deviation; AMH = anti-Müllerian hormone, BMI = body mass index; HCG = human chorionic gonadotrophin; HOMA IR = Homeostasis Assessment Model insulin resistance; NS = not statistically significant. a Oestradiol was not measured as no ovarian response was detected by ultrasound.
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Article - Anti-Müllerian hormone in obese women with PCOS - S El-Halawaty et al.
predicts ovarian response to clomiphene citrate with sensitivity 71% and specificity 65.7%. More studies are needed to test which value would be most useful in clinical practice. The present study provides an addition to the available evidence supporting an interrelationship between obesity and PCOS. There is evidence, however, to suggest that adipose tissue plays an important role in the development and maintenance of PCOS pathology. For example, a close correlation has been shown between adiposity and symptom severity in women with PCOS (Barber et al., 2006).
Figure 2. Correlation between anti-Müllerian hormone concentration and number of follicles observed on ultrasound in responsive women.
$ISCUSSION Fifty per cent of all women with PCOS are obese, and the underlying pathogenetic mechanisms appear to involve insulin resistance and hyperinsulinaemia, the magnitude of which is greater in obese than in non-obese women with PCOS (Salehi et al., 2004). In the present study, all women had PCOS and all were obese with BMI >30 kg/m2. AMH production was found to be increased in obese women with PCOS compared to obese controls. This finding was consistent with other reports in the medical literature (Cook et al., 2000; Laven et al., 2004). There was also a significant difference (P = 0.0081) in AMH concentration between women with PCOS who responded to clomiphene citrate and those who did not. The comparisons between both groups were based upon their response to ovulation induction, which is a clinically oriented outcome. It should be noted that FSH concentration was not significantly different between those women who responded and those who did not. Keeping in mind that AMH concentration was statistically different, it may be assumed that AMH is a better predictor for response to ovulation induction by clomiphene citrate than FSH in obese women with PCOS. In addition, AMH has another advantage over FSH in that it can be measured on any day of the cycle not only on day 3. However, it should be noted that measurement of AMH is still expensive and kits may not be available locally, while the measurement of FSH is widely available in all the laboratories. There was a non-significant weak correlation between AMH concentration and the number of follicles observed by ultrasound (Figure 2). This could be attributed to the mild nature of ovarian stimulation via clomiphene citrate, allowing growth of only one or two follicles.
The data was further analysed to try to detect the best cut-off value for AMH by ROC curve; in the era of evidence-based medicine, the sensitivity and specificity of a diagnostic tool have limited use but should be supplemented with cut-off value. In the present study, it was found that a cut-off of 1.2 ng/ml
In the current study, there was no significant difference in fasting insulin concentrations between women with PCOS who were responsive to clomiphene citrate and those who were non-responsive, although there was a trend towards higher values in those who were responsive. HOMA IR was apparently higher in women with PCOS than the control group, although this difference did not reach statistical significance. It has been shown by other investigators that women with PCOS with insulin resistance are more likely to be obese and resistant to clomiphene citrate (Mor et al., 2004). It has also been demonstrated that in women with PCOS, and therefore with an excessive follicle pool, short-term reduction of insulinpromoted LH action by metformin treatment failed to influence follicle responses to exogenous FSH (Fleming, 2005). In conclusion, AMH production increases in women with PCOS compared to controls. If kits are available, AMH measurement could be useful in the prediction of ovarian response to clomiphene citrate in obese anovulatory women.
2EFERENCES Barber TM, McCarthy MI, Wass JA, Franks S 2006 Obesity and polycystic ovary syndrome. Clinical Endocrinology 65, 137–145. Ciaraldi TP, Morales AJ, Hickman MG et al. 1997 Cellular insulin resistance in adipocytes from obese polycystic ovary syndrome subjects involves adenosine modulation of insulin sensitivity. Journal of Clinical Endocrinology and Metabolism 82, 1421– 1425. Cook CL, Siow Y, Taylor S, Fallat ME 2000 Serum Müllerian inhibiting substance levels during normal menstrual cycles. Fertility and Sterility 73, 859–861. Douchi T, Oki T, Yamasaki H et al. 2004 Body fat patterning in polycystic ovary syndrome women as a predictor of the response to clomiphene. Acta Obstetricia et Gynecologica Scandinavica 83, 838–841. Fleming R 2005 Recruitment prior to ovarian stimulation: ways of improving follicular recruitment. Reproductive BioMedicine Online 10 (Suppl. 3), 55–59. Ghobadi C, Nguyen TH, Lennard MS et al. 2007 Evaluation of an existing nomogram for predicting the response to clomiphene citrate. Fertility and Sterility 87, 597–602. Imani B, Eijkemans MJ, te Velde ER et al. 2002 A nomogram to predict the probability of live birth after clomiphene citrate induction of ovulation in normogonadotropic oligoamenorrheic infertility. Fertility and Sterility 77, 91–97. Imani B, Eijkemans MJ, te Velde ER et al. 1998 Predictors of patients remaining anovulatory during clomiphene citrate induction of ovulation in normogonadotropic oligomenorrheic infertility. Journal of Clinical Endocrinology and Metabolism 83, 2361– 2365. Johnson NP, Bagrie EM, Coomarasamy A et al. 2006 Ovarian reserve tests for predicting fertility outcomes for assisted reproductive technology: the International Systematic Collaboration of Ovarian
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Article - Anti-Müllerian hormone in obese women with PCOS - S El-Halawaty et al.
Reserve Evaluation protocol for a systematic review of ovarian reserve test accuracy. BJOG 113, 1472–1480. Kousta E, White DM, Franks S 1997 Modern use of clomiphene citrate in induction of ovulation. Human Reproduction Update 3, 359–365. Kwee J, Schats R, McDonnell J et al. 2006 The clomiphene citrate challenge test versus the exogenous follicle-stimulating hormone ovarian reserve test as a single test for identification of low responders and hyperresponders to in-vitro fertilization. Fertility and Sterility 85, 1714–1722. Laven JS, Mulders AG, Visser JA et al. 2004 Anti-Müllerian hormone serum concentrations in normoovulatory and anovulatory women of reproductive age. Journal of Clinical Endocrinology and Metabolism 89, 318–323. Lee MM, Donahoe PK, Hasegawa T et al. 1996 Müllerian inhibiting substance in humans: normal levels from infancy to adulthood. Journal of Clinical Endocrinology and Metabolism 81, 571–576. Matthews DR, Rudenski AS, Naylor BA et al. 1985 Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 28, 412–419. Mor E, Zograbyan A, Saadat P et al. 2004 The insulin resistant subphenotype of polycystic ovary syndrome: clinical parameters and pathogenesis. American Journal of Obstetrics and Gynecology 190, 1654–1660.
Pasquali R, Gambineri A 2006 Polycystic ovary syndrome: a multifaceted disease from adolescence to adult age. Annals of the New York Academy of Sciences 1092, 158–174. Salehi M, Bravo-Vera R, Sheikh A et al. 2004 Pathogenesis of polycystic ovary syndrome: what is the role of obesity? Metabolism 53, 358–376. Seifer DB, MacLaughlin DT, Christian BP et al. 2002 Early follicular serum Müllerian inhibiting substance levels are associated with ovarian response during assisted reproductive technology cycles. Fertility and Sterility 77, 468–471. Sozen I, Arici A 2000 Hyperinsulinism and its interaction with hyperandrogenism in polycystic ovary syndrome. Obstetrical and Gynecological Survey 55, 321–328. Teixeira J, Maheswaran S, Donahoe PK 2001 Müllerian inhibiting substance: an instructive developmental hormone with diagnostic and possible therapeutic applications. Endocrine Reviews 22, 657–674. Declaration: The authors report no financial or commercial conflicts of interest. Received 23 May 2007; refereed 13 June 2007; accepted 15 August 2007.
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!RTICLE #LINICAL SIGNIlCANCE OF SERUM CONCENTRATION OF ANTI -àLLERIAN HORMONE IN OBESE WOMEN WITH POLYCYSTIC OVARY SYNDROME 3AMIR %L (ALAWATY IS AN OBSTETRICIAN AND GYNECOLOGIST IN THE 7OMENS 3PECIALIZED (OSPITAL +ING &AHD -EDICAL #ITY 2IYADH 3AUDI !RABIA WHERE A HIGH PROPORTION OF THE PATIENT POPULATION SEEKS TREATMENT FOR INFERTILITY AND ENDOCRINE RELATED DISEASES (E IS A &ELLOW OF THE 2OYAL #OLLEGE OF 3URGEONS OF %DINBURGH AND HE COMPLETED HIS 0H$ IN 4HE .ETHERLANDS
$R 3AMIR %L (ALAWATY 3AMIR %L (ALAWATY !HMED 2IZK -ANAL +AMAL -ONA !BOUL(ASSAN (EBA !L 3AWAH /LFAT .OAH (ESHAM !L )NANY $EPARTMENT OF /BSTETRICS 'YNECOLOGY #AIRO 5NIVERSITY $EPARTMENT OF /BSTETRICS 'YNECOLOGY "ENHA 5NIVERSITY $EPARTMENT OF #HEMICAL 0ATHOLOGY #AIRO 5NIVERSITY #ORRESPONDENCE -OUSTAPHA (ASSANIN 3T -ANIAL #AIRO %GYPT E MAIL KAAINIH YAHOOCOM
!BSTRACT In the human ovary, expression of anti-Müllerian hormone (AMH) is detected primarily in granulosa cells of preantral and small antral follicles. The aim of this study was to compare serum AMH measurements in obese women with polycystic ovary syndrome (PCOS) with those in obese normo-ovulatory women and to evaluate the role of AMH as a predictor of ovulation induction by clomiphene citrate compared to FSH. Sixty-eight obese women with PCOS were compared to 17 normoovulatory obese women. All women had a body mass index greater than 30 kg/m2. Women with PCOS received clomiphene citrate (150 mg/day) for 5 days starting from day 3 of cycle and were subdivided into responsive and non-responsive groups. There was a significant difference in AMH concentration between women with PCOS and the control group (P < 0.05) and also between women with PCOS who responded to clomiphene citrate and those who did not (P < 0.01). A value of 1.2 ng/ ml AMH could be used to predict response to clomiphene citrate in obese women with PCOS (sensitivity 71%, specificity 65.7%). AMH production increases in women with PCOS compared to controls. AMH measurement could also be useful in the prediction of ovarian response to clomiphene citrate. Keywords: anti-Müllerian hormone, clomiphene citrate, obesity, ovulation induction, polycystic ovary syndrome
)NTRODUCTION Polycystic ovary syndrome (PCOS) is one of the most common causes of anovulatory infertility, affecting 4–7% of women (Pasquali and Gambineri, 2006). Obesity may either be a trigger for developing the syndrome when inherited predisposition exists, or an independent causative factor by inducing reduced sensitivity to insulin (Ciaraldi et al., 1997; Sozen and Arici, 2000). Clomiphene citrate represents a convenient and safe first choice treatment and is widely available for induction of ovulation in women with PCOS. (Imani et al., 2002). Currently, patients who do not respond to clomiphene citrate are identified only after 3–6 months of treatment (Ghobadi et al., 2007). If such
patients could be distinguished earlier, time could be saved by changing clomiphene citrate-resistant anovulatory patients to alternative treatments. Anti-Müllerian hormone (AMH) could be used to fulfill this target. AMH is a dimeric glycoprotein made up of two monomers attached to each other by disulphide bonds. It belongs to the transforming growth factor β superfamily, which acts on tissue growth and differentiation (Lee et al., 1996). Sertoli cells in the male produce AMH, which induces the degeneration of the Müllerian ducts and provides for the normal formation of the male genital system. Sertoli cell secretion of AMH continues throughout life. Concentrations drop following puberty,
© 2007 Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB3 8DB, UK
Article - Anti-Müllerian hormone in obese women with PCOS - S El-Halawaty et al.
decreasing slowly to a low post-puberty value. In females, it is produced by the granulosa cells of early developing follicles. After puberty and the onset of menstrual cycles, serum AMH concentration decreases progressively until it becomes undetectable at around the time of menopause (Lee et al., 1996; Seifer et al., 2002). As AMH is largely expressed throughout folliculogenesis, serum concentrations of AMH may represent both the quantity and quality of the ovarian follicle pool. FSH at day 3 of the cycle is a commonly used diagnostic blood test to help in prediction of ovarian reserve (Johnson et al., 2006) but it does not seem adequate on its own to enable prediction of response to ovarian stimulation (Kwee et al., 2006). In addition, available data in the medical literature has demonstrated that obesity is an important parameter that negatively affects the response to ovarian stimulation with clomiphene citrate (Kousta et al., 1997; Imani et al., 1998; Douchi et al., 2004). Accordingly, both FSH and AMH were compared regarding their value in prediction of ovarian response to clomiphene citrate. An additional aim was to compare serum AMH measurement in obese women with PCOS to obese normo-ovulatory women.
-ATERIALS AND METHODS This was a prospective controlled clinical trial in which obese subfertile women with PCOS, n = 68 and body mass index (BMI) greater than 30 kg/m2, were compared to obese normo-ovulatory women who comprised the control group (n = 17). Participants were recruited from the Outpatient Clinic of Gynecology of Kasr El-Aini Cairo University Hospital and Benha University Hospital. The study was conducted between March 2006 and November 2006 after ethical committee approval. A full history was taken and clinical examination was performed. BMI was calculated as weight (kg)/height (m)2. Only women with anovulation as a cause of infertility were included. The clinical criteria were oligomenorrhoea (menstrual interval longer than 6 weeks) or amenorrhoea (no menstrual loss for 6 months) dating from menarche, and/or hirsutism. The main aim of this study was to measure AMH and FSH for prediction of response to clomiphene citrate and, as Rotterdam criteria require two out of three parameters, clinical hyperandrogenism was also a focus. At the same time, biochemical markers were assayed as increased LH:FSH ratio and normal FSH concentrations. The ultrasound criteria were enlarged ovaries with an increased stroma and more than 10 subcapsular follicles of 3–8 mm diameter. Women having bilateral tubal block, organic uterine or ovarian pathology and women whose husbands were found to be infertile on semen analysis were excluded. Other excluded conditions were hypo/hyperthyroidism, hyperprolactinaemia and Cushing’s syndrome as detected by history, examination and investigations.
All women with PCOS received clomiphene citrate (Clomid; Global Napi Pharmaceuticals under license of Hoechst, Marion Roussel, France) 50 mg/8 h from the third day of the cycle and for 5 days. Transvaginal ultrasonographic serial folliculometry to check ovulation was performed from day 8 until day of HCG
administration. Human chorionic gonadotropin 10,000 IU (HCG; Pregnyl, NV Organon International, The Netherlands) was administered via intramuscular single injection when the leading follicle reached 18 mm in diameter.
3AMPLE COLLECTION In the early follicular phase (day 3), fasting blood samples were collected from all women for the measurement of serum insulin, glucose, LH, FSH and AMH. Those who responded to treatment were subjected to oestradiol assay on the day of HCG administration and progesterone assay on day 22 of the cycle. Blood samples were taken from all women (6 ml) and collected in vaccutainer tubes (4 ml in plain tubes and 2 ml in tubes with sodium fluoride). All samples were then centrifuged at 2000 g for 15 min. Serum and plasma were separated and stored at –20 °C until assayed. The concentrations of FSH, LH, oestradiol, progesterone and fasting insulin were assayed using reagents supplied by DPC (Diagnostic Products Corporation, Los Angeles, USA) by enzyme chemiluminescence immunoassay using the Immulite system. Progesterone concentration above 10 ng/ml was considered to indicate an appropriate ovulatory response. Fasting blood glucose was measured on an automated autoanalyzer (Hitachi 917) using reagents supplied by Roche (Roche Diagnostics GmbH, D-68298 Mannheim, Germany). Serum AMH was determined by quantitative sandwich enzyme-linked immunosorbent assay (ELISA) (Teixeira et al., 2001) using kits supplied by DSL, Cat No. 14400 (Diagnostic Systems Laboratories, Inc., Webster, Texas, USA). Insulin resistance (IR) was calculated using the Homeostasis Assessment Model (HOMA) as follows: HOMA IR = fasting insulinaemia (μIU/ml) × fasting glucose (mmol/l)/22.5 (Matthews et al., 1985).
3TATISTICAL EVALUATION Data are presented as mean ± SD. For statistical analysis, comparison between different parameters in the present study was performed using Student’s t-test for comparing parametric data. For comparing non-parametric data, chi-squared tests and Mann–Whitney tests were performed. Pearson’s correlation co-efficient was calculated to evaluate the relation of AMH to number of follicles. Sensitivity and specificity were calculated to estimate the best cut-off value using receiver operating characteristic (ROC) curve. All statistical calculations were performed using the computer programmes Arcus Quickstat Biomedical version l.0 and Microsoft Excel version 2000.
2ESULTS Sixty-eight obese women having PCOS were compared to 17 normo-ovulatory obese women (Table 1). There was a significant difference in AMH (P = 0.0254) and LH (P = 0.0167) between both groups. HOMA IR was apparently higher in women with PCOS
RBMOnline®
Article - Anti-Müllerian hormone in obese women with PCOS - S El-Halawaty et al.
but it did not reach statistical significance. Other demographic criteria did not show statistical difference (Table 1). Women with PCOS were further subdivided into two groups based on transvaginal ultrasonography results and progesterone concentrations after ovulation induction with clomiphene citrate: Group I, 17 women who responded to clomiphene citrate and Group II, 51 women who did not respond to clomiphene citrate. There were no significant differences between the groups in age, infertility duration, BMI, hormonal profile and fasting blood glucose concentration. However, there was a highly significant difference in AMH between the groups (P = 0.0081, Table 2). In the present study, the ROC curve showed a cut-off value of 1.2 ng/ml AMH with area under curve more than 0.71, sensitivity 71% and specificity 65.7% to predict response to clomiphene citrate in obese women with PCOS (Figure 1). There appeared to be a weak (not significant) correlation between AMH concentration and the number of follicles >18 mm observed by ultrasound on the day of HCG administration (Figure 2, correlation coefficient (r) = –0.18)
Figure 1. Area under receiver operating characteristic (ROC) curve with a cut-off value for anti-Müllerian hormone.
Table 1. Comparison between women with polycystic ovary syndrome and normo-ovulatory women. Parameter
Control group (n = 17)
PCOS group (n = 68)
P-value
Age (years) Body weight (kg) Height (m) BMI (kg/m2) LH (mIU/ml) FSH (mIU/ml) AMH (ng/ml) Fasting glucose (mg/dl) Fasting insulin (μIU/ml) HOMA IR
27.65 ± 5.70 98.4 ± 14.21 1.63 ± 0.05 36.4 ± 5.67 4.1 ± 2.6 4.53 ± 2.03 1.95 ± 1.8 86.5 ± 17.61 9.3 ± 5.52 2.36 ± 1.67
28.21 ± 4.8 97.8 ± 14.61 1.61 ± 0.05 36.7 ± 5.75 10.2 ± 3.52 4.53 ± 1.58 3.31 ± 3.14 90.1 ± 24.39 12.8 ± 9.14 4.18 ± 3.4
NS NS NS NS 0.0167 NS 0.0254 NS NS NS
All values are mean ± standard deviation; AMH = anti-Müllerian hormone; BMI = body mass index; HOMA IR = Homeostasis Assessment Model insulin resistance; NS = not statistically significant; PCOS = polycystic ovary syndrome.
Table 2. Comparison between women with PCOS who responded to clomiphene citrate and those who did not respond. Parameter
Responsive cases (n = 17)
Non-responsive cases (n = 51)
P-value
Age (years) Duration of infertility (years) Body weight (kg) Height (m) BMI (kg/m2) LH (mIU/ml) FSH (mIU/ml) Fasting glucose (mg/dl) Fasting insulin (μIU/ml) HOMA IR AMH (ng/ml) Oestradiol (pg/ml) Follicles >18 mm on day of HCG (n) Endometrial thickness (mm) Progesterone (ng/ml)
28.82 ± 3.80 4.71 ± 2.66 101.94 ± 9.85 1.64 ± 0.06 38.25 ± 4.77 10.38 ± 2.08 5.94 ± 4.22 85.29 ± 17.61 10.95 ± 10.52 2.36 ±1.67 4.38 ± 3.30 181.06 ± 60.22 1.65 ± 0.49 6.35 ± 0.70 11.76 ± 2.02
29.55 ± 4.60 5.39 ± 3.12 95.93 ± 14.61 1.64 ± 0.05 35.58 ± 5.75 10.03 ± 2.41 4.53 ± 1.58 88.18 ± 24.39 8.63 ± 5.14 1.89 ± 1.33 1.89 ± 1.33 Not measureda 0 5.54 ± 0.74 1.93 ± 1.42
NS NS NS NS NS NS NS NS NS NS 0.0081 – – 0.0004 <0.0001
All values are mean ± standard deviation; AMH = anti-Müllerian hormone, BMI = body mass index; HCG = human chorionic gonadotrophin; HOMA IR = Homeostasis Assessment Model insulin resistance; NS = not statistically significant. a Oestradiol was not measured as no ovarian response was detected by ultrasound.
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Article - Anti-Müllerian hormone in obese women with PCOS - S El-Halawaty et al.
predicts ovarian response to clomiphene citrate with sensitivity 71% and specificity 65.7%. More studies are needed to test which value would be most useful in clinical practice. The present study provides an addition to the available evidence supporting an interrelationship between obesity and PCOS. There is evidence, however, to suggest that adipose tissue plays an important role in the development and maintenance of PCOS pathology. For example, a close correlation has been shown between adiposity and symptom severity in women with PCOS (Barber et al., 2006).
Figure 2. Correlation between anti-Müllerian hormone concentration and number of follicles observed on ultrasound in responsive women.
$ISCUSSION Fifty per cent of all women with PCOS are obese, and the underlying pathogenetic mechanisms appear to involve insulin resistance and hyperinsulinaemia, the magnitude of which is greater in obese than in non-obese women with PCOS (Salehi et al., 2004). In the present study, all women had PCOS and all were obese with BMI >30 kg/m2. AMH production was found to be increased in obese women with PCOS compared to obese controls. This finding was consistent with other reports in the medical literature (Cook et al., 2000; Laven et al., 2004). There was also a significant difference (P = 0.0081) in AMH concentration between women with PCOS who responded to clomiphene citrate and those who did not. The comparisons between both groups were based upon their response to ovulation induction, which is a clinically oriented outcome. It should be noted that FSH concentration was not significantly different between those women who responded and those who did not. Keeping in mind that AMH concentration was statistically different, it may be assumed that AMH is a better predictor for response to ovulation induction by clomiphene citrate than FSH in obese women with PCOS. In addition, AMH has another advantage over FSH in that it can be measured on any day of the cycle not only on day 3. However, it should be noted that measurement of AMH is still expensive and kits may not be available locally, while the measurement of FSH is widely available in all the laboratories. There was a non-significant weak correlation between AMH concentration and the number of follicles observed by ultrasound (Figure 2). This could be attributed to the mild nature of ovarian stimulation via clomiphene citrate, allowing growth of only one or two follicles.
The data was further analysed to try to detect the best cut-off value for AMH by ROC curve; in the era of evidence-based medicine, the sensitivity and specificity of a diagnostic tool have limited use but should be supplemented with cut-off value. In the present study, it was found that a cut-off of 1.2 ng/ml
In the current study, there was no significant difference in fasting insulin concentrations between women with PCOS who were responsive to clomiphene citrate and those who were non-responsive, although there was a trend towards higher values in those who were responsive. HOMA IR was apparently higher in women with PCOS than the control group, although this difference did not reach statistical significance. It has been shown by other investigators that women with PCOS with insulin resistance are more likely to be obese and resistant to clomiphene citrate (Mor et al., 2004). It has also been demonstrated that in women with PCOS, and therefore with an excessive follicle pool, short-term reduction of insulinpromoted LH action by metformin treatment failed to influence follicle responses to exogenous FSH (Fleming, 2005). In conclusion, AMH production increases in women with PCOS compared to controls. If kits are available, AMH measurement could be useful in the prediction of ovarian response to clomiphene citrate in obese anovulatory women.
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