Lille II; and b Laboratory of Endocrinology, C.H.R.U. and Universite de Lille II, Lille, France

Transcription

1 Increased anti-m ullerian hormone and decreased FSH levels in follicular fluid obtained in women with polycystic ovaries at the time of follicle puncture for in vitro fertilization Virginie Desforges-Bullet, M.D., a Cecile Gallo, M.D., a Catherine Lefebvre, M.D., a Pascal Pigny, Ph.D., b Didier Dewailly, M.D., a and Sophie Catteau-Jonard, Ph.D. a a Department of Endocrine Gynaecology and Reproductive Medicine, H^opital Jeanne de Flandre, C.H.R.U. and Universite de Lille II; and b Laboratory of Endocrinology, C.H.R.U. and Universite de Lille II, Lille, France Objective: To confirm the increased levels of anti-m ullerian hormone (AMH) in preovulatory follicles from patients with polycystic ovary syndrome (PCOS) and to study the role of other hormones involved in folliculogenesis in this increased secretion. Design: Prospective study. Setting: University hospital in France. Patient(s): Twenty-two patients with PCOS and 20 controls undergoing IVF. Intervention: On the day of oocyte retrieval, follicular fluid (FF) from one small follicle (8 13 mm) (SF) and one large follicle (16-23 mm) (LF) was collected in each patient. Main Outcome Measure(s): Per-follicle AMH, FSH, estradiol, androstenedione, hcg, and progesterone levels, and pregnancy rate. Result(s): In FF from both SF and LF of PCOS patients, AMH level was significantly increased, and FSH level was significantly decreased when compared with controls. Both hormone levels were negatively and significantly related in controls but not in PCOS. The AMH levels from SF and LF were significantly lower in patients who began a pregnancy. Conclusion(s): Our findings suggest that the granulosa cells from polycystic ovaries continue to produce elevated levels of AMH, possibly because of impaired access of FSH to follicles. Such an excess in FF AMH may have harmful consequences on oocyte quality and final maturation through unknown mechanisms. (Fertil Steril Ò 2010;94: Ó2010 by American Society for Reproductive Medicine.) Key Words: Anti-M ullerian hormone, M ullerian inhibiting substance, polycystic ovary syndrome, FSH, follicular fluid Anti-M ullerian hormone (AMH), a member of the transforming growth factor-b superfamily, is known to be secreted by the granulosa cells (GCs) of growing follicles from the primary to the large antral follicle stage (1). It has been suggested that AMH may be involved in the initiation of primordial follicle growth in mice (2) and in the cyclic recruitment of growing follicles (3). Consequently, AMH could be involved in the abnormal folliculogenesis in polycystic ovary syndrome (PCOS), which affects 5% 10% of women of reproductive age worldwide and is the most common cause of oligoanovulation, infertility, and hyperandrogenism (4). The pathophysiology of the ovulatory disorder remains unclear, but two abnormalities in folliculogenesis Received December 23, 2008; revised February 24, 2009; accepted March 2, 2009; published online April 9, V.D-B. has nothing to disclose. C.G. has nothing to disclose. C.L. has nothing to disclose. P.P. has nothing to disclose. D.D. has nothing to disclose. S.C-J. has nothing to disclose. Reprint requests: Didier Dewailly, M.D., Department of Endocrine Gynaecology and Reproductive Medicine, H^opital Jeanne de Flandre, C.H.R.U., Lille, France (TEL: ; FAX: ; ddewailly@chru-lille.fr). have been proposed (5). Polycystic ovaries have an abnormally rich pool of growing follicles from classes 1 5 (%5 mm) (6) and a disturbance in the selection and subsequent maturation of a dominant follicle. Studies have shown that serum AMH levels are increased in women with PCOS compared with normoovulatory women, corresponding to the follicle excess seen on ultrasonographic examination (7, 8). However, some authors argued that the increased serum AMH is due to increased production per GC, suggesting an intrinsic GC dysregulation in PCOS. Assays in follicular fluid from small follicles (SFs) (9) and in cell-conditioned media from cultured GC in vitro (10) support this last hypothesis. Not only is AMH expression increased, but it also might be protracted in polycystic ovary (PCO) follicles. Immunocytochemistry of unstimulated follicles from normal women shows that AMH expression is nearly undetectable in follicles larger than 8 mm, where it seemed restricted to the GC of the cumulus (1). However, Fallat et al. (11) reported a significantly higher AMH level in the follicular fluid (FF) and serum of patients with PCOS undergoing IVF than in patients undergoing IVF for 198 Fertility and Sterility â Vol. 94, No. 1, June /$36.00 Copyright ª2010 American Society for Reproductive Medicine, Published by Elsevier Inc. doi: /j.fertnstert

2 endometriosis or pelvic adhesions. Similarly, Dumesic et al. (12) found elevated FFAMH levels in patients with PCOS vs. those with normoandrogenic ovulation on the day of oocyte retrieval. The higher AMH levels in FF from patients with PCOS suggest an increased capacity of mature GC from PCO to produce AMH. Consistent with this hypothesis, our recent data using real time quantitative polymerase chain reaction indicated an increased AMH gene expression in GC obtained the day of oocyte retrieval in patients with PCOS undergoing IVF when compared with controls (13). The reasons for this persistant, increased AMH production in mature follicles could be a global GC upregulation, because we also showed higher levels of androgen receptor and FSH receptor mrnas in mature follicles from PCOS patients, in close relationship with AMH mrnas (13). To extend the previous data from Fallat et al. (11) and Dumesic et al. (12) and to better understand the AMH dysregulation in PCOS, we searched in this study for relationships between hcg, FSH, AMH, and androgens in FF. We focused our attention on FSH that has been shown to exert an inhibitory effect on expression and action of AMH in adult rat ovaries (14). Follicular fluid from SFs and large follicles (LFs) was collected and examined separately to determine whether the degree of follicular maturation influences the data. MATERIALS AND METHODS Subjects This prospective study included 42 women aged years referred for IVF: 22 patients with PCOS and 20 patients with normal ovulatory function. Patients were selected from a group undergoing IVF for treatment of tubal and/or male infertility. The study required no modification of our routine IVF protocols. All patients gave informed consent before their inclusion in this study. This study was approved by the institutional Review Board of the University Hospital of Lille. Patients in the PCOS group were diagnosed according to the Rotterdam criteria (15). The diagnosis was based on the association of at least two of three following criteria: [1] ovulatory disturbance (oligomenorrhea or amenorrhea); [2] hyperandrogenism, as defined either clinically by hirsutism (modified Ferriman and Gallway score > 6), severe acne or seborrhoea, or biologically by a testosterone serum level greater than 0.6 ng/ml and/or androstenedione (A) level greater than 2.2 ng/ml; and [3] more than 12 follicles in the 2- to 9-mm range in each ovary on ultrasound examination and/or an ovarian volume greater than 10 ml. The control population was referred to our department for IVF because of tubal and/or male infertility. Exclusion criteria were a history of menstrual disturbances (i.e., cycle length either <25 days or >35 days), hirsutism, abnormal serum level of prolactin or androgens (i.e., serum testosterone above 0.6 ng/ml), decreased ovarian reserve (i.e., FSH >12 IU/L and/or AMH < 8 pmol/l), and PCO on ultrasonograpic examination (15). Blood Hormone Samples Serum FSH, AMH, and A levels were measured during the early follicular phase (2nd to 5th day of the menstrual cycle). In patients with PCOS, the last menstrual period was either spontaneous or induced by the administration of didrogesterone (10 mg/d for 7 days). Follicle-stimulating hormone level was measured with a Cobas analyser (Roche Diagnostics, Meylan, France) using two site electrochemiluminescence immunoassays. Serum AMH levels were assessed using the second generation enzyme immunoassay AMH-EIA (ref A16507) provided by Beckman Coulter Immunotech (Villepinte, France), as described previously (16). Androstenedione levels were determined using a radioimmunoassay provided by Diagnostic Systems Laboratories (Villepinte, France) according to the manufacturer s instructions. Ovarian Stimulation Pituitary desensitization with the GnRH agonist triptorelin (0.1 mg/d; Decapeptyl; Ferring, Malmo, Sweden) was initiated during the luteal phase before IVF treatment or, for the patients with dysovulation, on the first day of menses. Follicle growth was stimulated after 12 days of desensitization with SC injection of recombinant follicle-stimulating hormone (rec FSH; Puregon; Organon Laboratories, Saint-Denis, France) with doses ranging IU/d. Doses were based on our multiparameter calculation of the initial dose and adjusted accordingly after ovarian monitoring. Follicle growth was monitored by serum estradiol (E 2 ) levels and transvaginal ultrasound. Ovulation was induced with hcg (5,000 IU; Serono) when at least three follicles greater than 16 mm in diameter were detected on ultrasound examination and the leading follicle of the three reached mm in diameter. Oocyte retrieval was performed 36 hours later under transvaginal ultrasound guidance. Collection of Follicular Fluid Follicle fluid from one SF (8 13 mm) and one LF (16 23 mm) was collected in separate tubes. Follicle size was determined by ultrasound examination immediately before retrieval. The nature of oocyte retrieval required the use of the same aspirating needle in each ovary. Thus, to avoid mixing the fluid from SFs and LFs, the FF samples were aspirated in a certain order. Follicular fluid from one LF was aspirated first. The FF from two SFs was then aspirated, but the fluid was not used for the study. Finally, FF from another SF was aspirated for use in the study. Oocytes were isolated and FFs were centrifuged at 350 g for 5 minutes to remove red blood cells. The supernatants were then stored at 80 C for hormonal assays. Hormone Measurements in Follicular Fluid Estradiol, progesterone (P), A, FSH, hcg and AMH levels were assessed in FF using the same immunoassays used on Fertility and Sterility â 199

3 blood samples (see previous discussion for FSH, A, AMH). The E 2 and P levels were measured using a competitive chemiluminescent immunoassay on an automated multianalysis system (Architect; Abbott Diagnostics Division, Rungis, France). For P, the linearity was up to 40 ng/ml vs pg/ ml for E 2. The hcg level was measured on the Cobas analyser (Roche Diagnostics, Meylan, France) using two site electrochemiluminescence immunoassays. For E 2, P, and A measurements, the FF was diluted to 1:500, 1:800, or 1:50, respectively, with the specific diluting agent provided by the manufacturer (E 2, P) or in the zero standard (A). For AMH, most of the FF samples were tested undiluted. In case of concentrations greater than 125 pmol/l, the AMH FF levels were reassessed on 1:10 diluted samples in the reaction buffer provided by the manufacturer. Statistics Because values from any parameter were not normally distributed, all comparisons between PCOS and controls were performed using the nonparametric Mann-Whitney U test. The Wilcoxon test was used for paired comparisons between small and large follicles. Univariate analysis of correlations between intrafollicular levels of different hormones was performed with the nonparametric Spearman s test. Multiple linear regression on log-transformed values was performed to search for a possible confounding effect of age and/or rec FSH doses. All statistical procedures were run on SPSS 11.5 (SPSS Inc., Chicago, IL). Statistical significance was set at P % RESULTS Population Clinical and endocrine parameters in normoovulatory controls and in patients with PCOS are summarized in Table 1. Mean age was significantly higher in controls (P¼0.013), as was the mean cumulative rec FSH dose received by each patient (P¼0.0001). Serum AMH and A levels and basal antral follicle count in the early follicular phase were significantly higher in the PCOS group (P¼0.008, P¼0.012, and P < , respectively). These differences remained significant after controlling for age. There was no significant difference for body mass index, basal FSH serum levels, number of retrieved oocytes, number of embryos, pregnancy rate, and aspirated follicle sizes. Follicular Fluid Hormone Levels in PCOS and Controls In the PCOS group, the mean AMH level was significantly increased in FF obtained from both SFs and LFs compared with controls (Fig. 1; Table 1). More than 50% of the patients had an FF AMH level greater than the 95th percentile of controls. Conversely, the mean FSH level was significantly decreased in both SFs and LFs compared with controls (Fig. 2; Table 1). More than 50% of the patients had an FF FSH level less than the 5th percentile of controls. These differences remained significant after controlling for age and the total dose of rec FSH. Mean A, hcg, E 2, and P levels from both SFs and LFs did not differ from the PCOS and control groups (Table 1). Mean AMH level was significantly higher in SFs than in LFs in both PCOS and control groups (PCOS, P < and P¼0.007, respectively; controls, P¼0.005 and P¼0.001, respectively; Table 1). Conversely, mean FSH and P levels were significantly higher in LFs than in SFs in both PCOS and control groups (PCOS, P¼0.005 and P < , respectively; controls, P¼0.002 and P < , respectively). Mean E 2 and A levels were not significantly different between SFs and LFs in either the PCOS or control groups (Table 1). Relationships between Intrafollicular Hormone Levels By univariate analysis of data from PCOS follicles, AMH and E 2 levels tended to be negatively related in SFs only (Spearman s Rho: r ¼ 0.42, P¼0.05). The AMH had no significant relationship with other hormone levels. By univariate analysis of data from control follicles, AMH did not correlate to E 2, but it was negatively and significantly correlated to FSH levels in both SFs and LFs (Spearman s Rho: r ¼ 0.66, P¼0.001; and r ¼ 0.64, P¼0.002). Follicle-stmulating hormone was also negatively and significantly correlated to E 2 in both SFs and LFs (r ¼ 0.56, P ¼0.01; r ¼ 0.51, P¼0.02). Androstenedione levels were positively and significantly related to E 2 in LFs exclusively (r ¼ 0.66, P¼0.01). In both groups, FF FSH levels in SFs and LFs were positively correlated to the total dose of rec FSH received by each patient (r ¼ 0.62, P < ; r ¼ 0.66, P < , respectively). However, introducing this parameter in a multiple linear regression analysis did not modify the data from univariate analysis. Relationship between Intrafollicular AMH Levels and Pregnancy In the whole group, the mean SF and LF AMH mean contents were significantly lower in the group of patients who began a pregnancy (n ¼ 16) than in those who did not (n ¼24) (22.0 [ ] vs [ ] and 18.2 [ ] vs [ ] pmol/l, P¼0.018 and P¼0.023, respectively). Separate analysis in each group did not reach statistical significance. The other intrafollicular hormone mean levels were not different between the patients who began a pregnancy and those who did not. DISCUSSION An increased production of AMH has been previously reported in GC cultures and in FF from unstimulated SFs (4 10 mm) in women with PCOS (9, 10). Such an elevation is expected during early follicle stages when AMH production is physiologically at its highest level (1). In keeping 200 Desforges-Bullet et al. AMH in follicles from patients with PCOS Vol. 94, No. 1, June 2010

4 TABLE 1 Median values with 10th 90th percentiles (in parentheses) of the tested variables in normoovulatory controls and in patients with PCOS. Controls PCOS patients P value Number Age (y) 33.3 ( ) 29.5 ( ) Body mass 22.4 ( ) 23.8 ( ) 0.58 index (kg/m 2 ) Serum FSH (IU/L) 7.3 ( ) 5.9 (2.8 9) 0.06 Serum AMH (pmol/l) 19.6 ( ) 44.7 ( ) Serum A (ng/ml) 1.28 ( ) 1.87 ( ) Antral follicle count 8 (4.5 12) 21.8 ( ) < total rec FSH (IU) 2,138 (1,073 4,792) 1,300 (758 3,052) < Retrieved oocytes 12 (4 20) 13.5 ( ) 0.45 Inseminated oocytes 12 (4 18) 11.5 (3 22) 0.86 Number of embryos 7 (3 11) 7 (1.2 15) 0.23 Pregnancy rate (%) 50% 32% 0.12 SF size (mm) 11 (9 13) 11 (8 12.5) 0.98 LF size (mm) 20 ( ) 18 ( ) SF AMH (pmol/l) 22 (5.6 86) a 70 ( ) a < LF AMH (pmol/l) 18.2 ( ) 36.2 ( ) SF FSH (IU/L) 4.6 ( ) a 2.1 ( ) a < LF FSH (IU/L) 4.8 ( ) 2.4 (0.7 5) < SF E 2 (ng/ml) 425 (96.5 1,074) 382 ( ,381) 0.67 LF E 2 (ng/ml) 434 ( ) 343 ( ) 0.16 SF P (ng/ml) 8,925 (1,791 13,469) a 6,372 (1,643 15,598) a 0.18 LF P (ng/ml) 14,355 (6,179 17,528) 12,915 (7,316 20,742) 0.94 SF A (ng/ml) 60.2 ( ) 73.7 ( ) 0.38 LF A (ng/ml) 71 ( ) 64 ( ) 0.08 SF hcg (IU/L) 36.2 ( ) a 41 ( ) a 0.53 LF hcg (IU/L) 28.1( ) 36.2 ( ) 0.28 a Significantly different from LF. Desforges-Bullet. AMH in follicles from patients with PCOS. Fertil Steril with this physiologic concept, our study also demonstrated higher FF AMH levels in SFs than in LFs. However, an exaggerated AMH production in women with PCOS is still present in mature follicles. Our recent study using real time quantitative polymerase chain reaction and western blot indicated an AMH gene expression in GCs harvested from stimulated preovulatory follicles of women undergoing IVF (13). These data argue for a sustained AMH production in stimulated preovulatory follicles and rules out the theory that increased AMH levels in FF from these follicles reflect only prior hypersecretion. Several explanations can be postulated to understand the exaggerated AMH production in women with PCOS at a late follicle stage. First, PCOS follicles may exhibit an abnormal AMH response to the hcg used to trigger ovulation. Previous studies have reported that cultured GCs from patients with PCOS increased their AMH production in response to LH, compared with controls (10). However, the present study demonstrated no relationship between FF AMH and hcg levels in either the PCOS or control groups, suggesting that hcg did not influence AMH production, contradicting our previous hypothesis (13). Second, it has been suggested that the increased production of AMH may be due to elevated concentrations of androgens that are characteristically found in patients with PCOS (13, 17). Previous studies have indicated a positive relationship between serum AMH and circulating androgen levels in PCOS patients (11, 18, 19), suggesting that androgens may exert a stimulatory effect on AMH production. However, the FF A levels in our PCOS group did not differ from the control group and no correlation was found between A and AMH levels. In keeping with this finding, Yding Andersen et al. (20) and Dumesic et al. (21) recently reported that FF concentrations of AMH were unrelated to FF A and testosterone levels in respectively unstimulated and stimulated follicles from patients without PCOS patients. Also, Das et al. (9) did not observe any statistically significant relationship between FF AMH and serum free Fertility and Sterility â 201

5 FIGURE 1 Box-and-whisker plots showing the intrafollicular AMH levels in SFs and LFs from patients with PCOS and in SFs and LFs from controls. Horizontal small bars represent the 10th 90th percentile range, and the boxes indicate the 25th 75th percentile range. The horizontal line in each box corresponds to the median. FIGURE 2 Box-and-whisker plots showing the intrafollicular FSH levels in SFs and LFs from patients with PCOS and in SFs and LFs from controls. Horizontal small bars represent the 10th 90th percentile range, and the boxes indicate the 25th 75th percentile range. The horizontal line in each box corresponds to the median. Desforges-Bullet. AMH in follicles from patients with PCOS. Fertil Steril testosterone levels in small follicles from unstimulated PCOS patients. Last, the increased AMH levels in FF from PCOS follicles may be due to the lack of available and/or active FSH. Our study is the first to relate FF AMH and FSH levels in patients with PCOS. Lower rec FSH cumulative doses received by our patients with PCOS could explain their lower FF FSH levels than in control FFs, because both parameters were strongly correlated. However, the FSH level in PCOS follicles still remained significantly lower even after controlling for rec FSH cumulative dose, suggesting a partial insufficiency of endogenous FSH. We also found that the mean basal serum level in PCOS patients was approximately 20% lower than in controls, yet this difference was only close to statistical significance and not as marked as the decrease in FF FSH (approximately 50% lower than in controls). This raises the possibility that, in addition to the moderate serum FSH insufficiency, some unknown factor(s) in PCO may render FSH less available at the follicular level. Similar findings have been reported by Foong et al. (22), who also theorized that Desforges-Bullet. AMH in follicles from patients with PCOS. Fertil Steril there is a reduced availability of FSH to the follicle. Although the exact cause of this phenomenon is unknown, the lack of available FSH may explain the exaggerated FF AMH levels. Indeed, FSH has been shown to exert an inhibitory effect on expression and action of AMH in adult rat ovaries (14). Similarly, several studies in women have reported a negative relationship between serum FSH and AMH levels in the early follicular phase of nonstimulated (7, 23 25) and FSH stimulated (16) cycles. A recent study also found that FF AMH levels negatively correlated with FF FSH concentrations in stimulated follicles from normoovulatory patients (21). That in the present study such a strong negative correlation was found only in the control population reinforces our previous hypothesis that FSH does not properly exert its negative effect on AMH secretion in women with PCOS (16). However, we must recognize that it is speculative and that the series is too small for a firm conclusion. One might argue that such a defect in FSH action would also lead to lower follicular E 2 levels in PCOS. However, 202 Desforges-Bullet et al. AMH in follicles from patients with PCOS Vol. 94, No. 1, June 2010

6 this was not the case in our study. In agreement, Foong et al. (22) reported similar FF E 2 levels between normal women and those with PCOS. They reasoned that, despite lower FF FSH levels, the E 2 levels were similar because mature follicles in patients with PCOS were more sensitive to FSH. This conclusion agrees with in vitro data (26 28) and with our observation of higher FSH-R gene expression in such mature follicles from women with PCOS compared with controls (13). However, this hypothesis does not fit with the lack of correlation between FF FSH and FF E 2 in our patients with PCOS nor with the negative correlation between these parameters in our control women. Obviously, more data are needed to better understand the relationship between FSH, FSH-R and E 2 in mature follicles. Of note, the administration of hcg 36 hours before FF collection represses aromatase expression and subsequently E 2 production, making it difficult to obtain informative data with this approach (29). Finally, the negative relationship between FF AMH levels and pregnancy rate demonstrated in our study raises the hypothesis of a negative link between AMH and final oocyte maturation. Consistent with this hypothesis, Cupisti et al. (30) reported an inverse correlation between FF AMH and the maturation and developmental potential of oocytes during COH. In contrast, Fanchin et al. (31) and Takahashi et al. (32) suggested that higher FF AMH levels were associated with better rates of fertilization, embryo implantation, and pregnancy. This discrepancy could be explained by differences in the studied populations, and further experimental data are necessary to confirm and explain these results. In conclusion, although COH facilitates the maturation of follicles in patients with PCOS, these follicles continue to present abnormalities that have been previously described in unstimulated smaller follicles. In particular, they continue to produce elevated levels of AMH, possibly because of impaired access of FSH to follicles. Such an excess in FF AMH may have harmful consequences on oocyte quality and final maturation through unknown mechanisms. However, future research must be performed to clarify the relationship between AMH and FSH, and the potential role of AMH on oocyte quality. Acknowledgments: The authors thank Vanessa Vanderpool for her contributions to the English translation. REFERENCES 1. Weenen C, Laven JS, Von Bergh AR, Cranfield M, Groome NP, Visser JA, et al. Anti-Mullerian hormone expression pattern in the human ovary: potential implications for initial and cyclic follicle recruitment. Mol Hum Reprod 2004;10: Durlinger AL, Gruijters MJ, Kramer P, Karels B, Ingraham HA, Nachtigal MW, et al. Anti-Mullerian hormone inhibits initiation of primordial follicle growth in the mouse ovary. Endocrinology 2002;143: Durlinger AL, Gruijters MJ, Kramer P, Karels B, Kumar TR, Matzuk MM, et al. Anti-Mullerian hormone attenuates the effects of FSH on follicle development in the mouse ovary. Endocrinology 2001;142: Norman RJ, Dewailly D, Legro RS, Hickey TE. Polycystic ovary syndrome. Lancet 2007;370: Jonard S, Dewailly D. The follicular excess in polycystic ovaries, due to intra-ovarian hyperandrogenism, may be the main culprit for the follicular arrest. Hum Reprod Update 2004;10: Hughesdon PE. Morphology and morphogenesis of the Stein-Leventhal ovary and of so-called hyperthecosis. Obstet Gynecol Surv 1982;37: Pigny P, Merlen E, Robert Y, Cortet-Rudelli C, Decanter C, Jonard S, et al. Elevated serum level of anti-mullerian hormone in patients with polycystic ovary syndrome: relationship to the ovarian follicle excess and to the follicular arrest. J Clin Endocrinol Metab 2003;88: Laven JS, Mulders AG, Visser JA, Themmen AP, De Jong FH, Fauser BC. Anti-Mullerian hormone serum concentrations in normoovulatory and anovulatory women of reproductive age. J Clin Endocrinol Metab 2004;89: Das M, Gillott DJ, Saridogan E, Djahanbakhch O. Anti-Mullerian hormone is increased in follicular fluid from unstimulated ovaries in women with polycystic ovary syndrome. Hum Reprod 2008;23: Pellatt L, Hanna L, Brincat M, Galea R, Brain H, Whitehead S, et al. Granulosa cell production of anti-mullerian hormone is increased in polycystic ovaries. J Clin Endocrinol Metab 2007;92: Fallat ME, Siow Y, Marra M, Cook C, Carrillo A. Mullerian-inhibiting substance in follicular fluid and serum: a comparison of patients with tubal factor infertility, polycystic ovary syndrome, and endometriosis. Fertil Steril 1997;67: Dumesic DA, Lesnick TG, Booher J, Wong A, Ball GD, Abbott DH. Intrafollicular anti-m ullerian hormone (AMH) levels positively correlate with follicle sensitivity to follicle stimulating hormone (FSH) and are elevated in polycystic ovary syndrome (PCOS). Fertil Steril 2007;88(Suppl 1):S Catteau-Jonard S, Jamin SP, Leclerc A, Gonzales J, Dewailly D, di Clemente N. Anti-Mullerian hormone, its receptor, FSH receptor, and androgen receptor genes are overexpressed by granulosa cells from stimulated follicles in women with polycystic ovary syndrome. J Clin Endocrinol Metab 2008;93: Baarends WM, Uilenbroek JT, Kramer P, Hoogerbrugge JW, van Leeuwen EC, Themmen AP, et al. Anti-mullerian hormone and anti-mullerian hormone type II receptor messenger ribonucleic acid expression in rat ovaries during postnatal development, the estrous cycle, and gonadotropin-induced follicle growth. Endocrinology 1995;136: Revised 2003 consensus on diagnostic criteria and long-term health risks related to polycystic ovary syndrome (PCOS). Hum Reprod 2004;19: Catteau-Jonard S, Pigny P, Reyss AC, Decanter C, Poncelet E, Dewailly D. Changes in serum anti-mullerian hormone level during low-dose recombinant follicular-stimulating hormone therapy for anovulation in polycystic ovary syndrome. J Clin Endocrinol Metab 2007;92: Andersen CY, Lossl K. Increased intrafollicular androgen levels affect human granulosa cell secretion of anti-mullerian hormone and inhibin- B. Fertil Steril 2008;89: Pigny P, Jonard S, Robert Y, Dewailly D. Serum anti-mullerian hormone as a surrogate for antral follicle count for definition of the polycystic ovary syndrome. J Clin Endocrinol Metab 2006;91: Eldar-Geva T, Margalioth EJ, Gal M, Ben-Chetrit A, Algur N, Zylber- Haran E, et al. Serum anti-mullerian hormone levels during controlled ovarian hyperstimulation in women with polycystic ovaries with and without hyperandrogenism. Hum Reprod 2005;20: Yding Andersen C, Rosendahl M, Byskov AG. Concentration of anti- Mullerian hormone and inhibin-b in relation to steroids and age in follicular fluid from small antral human follicles. J Clin Endocrinol Metab 2008;93: Dumesic DA, Lesnick TG, Stassart JP, Ball GD, Wong A, Abbott DH. Intrafollicular antimullerian hormone levels predict follicle responsiveness to follicle-stimulating hormone (FSH) in normoandrogenic ovulatory women undergoing gonadotropin releasing-hormone analog/ recombinant human FSH therapy for in vitro fertilization and embryo transfer. Fertil Steril 2008, in press. Fertility and Sterility â 203

7 22. Foong SC, Abbott DH, Zschunke MA, Lesnick TG, Phy JL, Dumesic DA. Follicle luteinization in hyperandrogenic follicles of polycystic ovary syndrome patients undergoing gonadotropin therapy for in vitro fertilization. J Clin Endocrinol Metab 2006;91: Seifer DB, MacLaughlin DT, Christian BP, Feng B, Shelden RM. Early follicular serum mullerian-inhibiting substance levels are associated with ovarian response during assisted reproductive technology cycles. Fertil Steril 2002;77: van Rooij IA, Broekmans FJ, te Velde ER, Fauser BC, Bancsi LF, de Jong FH, et al. Serum anti-mullerian hormone levels: a novel measure of ovarian reserve. Hum Reprod 2002;17: Fanchin R, Schonauer LM, Righini C, Guibourdenche J, Frydman R, Taieb J. Serum anti-mullerian hormone is more strongly related to ovarian follicular status than serum inhibin B, estradiol, FSH and LH on day 3. Hum Reprod 2003;18: Erickson GF, Magoffin DA, Garzo VG, Cheung AP, Chang RJ. Granulosa cells of polycystic ovaries: are they normal or abnormal? Hum Reprod 1992;7: Mason HD, Willis DS, Beard RW, Winston RM, Margara R, Franks S. Estradiol production by granulosa cells of normal and polycystic ovaries: relationship to menstrual cycle history and concentrations of gonadotropins and sex steroids in follicular fluid. J Clin Endocrinol Metab 1994;79: Almahbobi G, Anderiesz C, Hutchinson P, McFarlane JR, Wood C, Trounson AO. Functional integrity of granulosa cells from polycystic ovaries. Clin Endocrinol (Oxf) 1996;44: Voss AK, Fortune JE. Levels of messenger ribonucleic acid for cholesterol side-chain cleavage cytochrome P-450 and 3 beta-hydroxysteroid dehydrogenase in bovine preovulatory follicles decrease after the luteinizing hormone surge. Endocrinology 1993;132: Cupisti S, Dittrich R, Mueller A, Strick R, Stiegler E, Binder H, et al. Correlations between anti-mullerian hormone, inhibin B, and activin A in follicular fluid in IVF/ICSI patients for assessing the maturation and developmental potential of oocytes. Eur J Med Res 2007;12: Fanchin R, Mendez Lozano DH, Frydman N, Gougeon A, di Clemente N, Frydman R, et al. Anti-Mullerian hormone concentrations in the follicular fluid of the preovulatory follicle are predictive of the implantation potential of the ensuing embryo obtained by in vitro fertilization. J Clin Endocrinol Metab 2007;92: Takahashi C, Fujito A, Kazuka M, Sugiyama R, Ito H, Isaka K. Anti- Mullerian hormone substance from follicular fluid is positively associated with success in oocyte fertilization during in vitro fertilization. Fertil Steril 2008;89: Desforges-Bullet et al. AMH in follicles from patients with PCOS Vol. 94, No. 1, June 2010