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Department of Endocrine Gynaecology and Reproductive Medicine (S.C.-J., D.D.), Hôpital Jeanne de Flandre, Centre Hospitalier Régional Universitaire, and Faculty of Medicine of Lille, Université de Lille II, 59037 Lille, France; Institut National de la Santé et de la Recherche Médicale, Unit 782 (S.P.J., A.L., J.G., N.d.C.), Clamart F-92140, France; and University of Paris-Sud, Unit Mixed of Research S0782 (S.P.J., A.L., J.G., N.d.C.), Clamart F-92140, France
Address all correspondence and requests for reprints to: Dewailly Didier, Department of Endocrine Gynaecology and Reproductive Medicine, Hôpital Jeanne de Flandre, C.H.R.U., 59037 Lille, France. E-mail: ddewailly{at}chru-lille.fr.
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Abstract |
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Design: The aim of this prospective study was to analyze the expression of these genes at the last stages of follicular maturation in GCs from 17 patients with PCOS and 15 controls undergoing controlled ovarian hyperstimulation during a cycle with in vitro fertilization.
Materials and Methods: On the day of oocyte retrieval, follicular fluids were collected from small follicles (SF; 8–13 mm) and large follicles (17–22 mm) in separate tubes. Total RNAs and proteins were extracted from GCs. Reverse transcription was performed and quantification of gene expression levels was achieved by real-time quantitative PCR.
Results: AMH and FSHR mRNA levels were significantly higher in PCOS than in controls in GCs from both SF and large follicles. Likewise, AR and AMH receptor II mRNA levels in GCs from SF were significantly higher in PCOS compared with controls. In both PCOS patients and controls, AMH and AR mRNA levels correlated strongly, positively, and independently to FSHR mRNA levels.
Conclusion: Using quantitative RT-PCR, AMH, AMH receptor II, FSHR, and AR genes were shown to be overexpressed by GCs from stimulated follicles of women with PCOS undergoing controlled ovarian hyperstimulation. This could be the sign of a maturation defect or may reflect hyperandrogenism.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Whether such abnormalities persist in the dominant follicles obtained under controlled ovarian hyperstimulation for in vitro fertilization (IVF) is unknown. If so, this could explain in part the impaired oocyte quality that has been reported in PCOS during IVF (13, 14). We therefore chose to study GCs from small and large follicles (SF and LF, respectively) collected during oocyte retrieval in patients undergoing controlled ovarian hyperstimulation with IVF. SF and LF were examined separately to point differences between mature and immature dominant follicles. The aim of this study was therefore to perform a functional analysis of GC and compare the expression of AMH, FSH receptor (FSHR), and AR genes by GCs from mature and immature dominant follicles in PCOS patients and controls. AMH type II receptor (AMHR-II) expression was also studied as a marker of AMH bioactivity. LH receptor expression was not studied because it is suppressed by human chorionic gonadotropin (hCG) administration (15, 16).
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Subjects and Methods |
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This prospective study included 17 patients with PCOS and 15 women with normal ovulatory function undergoing IVF for treatment of tubal and/or male infertility. Women were between 20 and 40 yr old. The study required no modification of our routine IVF protocol.
According to the Rotterdam criteria (17), the diagnosis of PCOS was based on the association of at least two of three following criteria: 1) ovulatory disturbance, mainly oligomenorrhea or amenorrhea; 2) hyperandrogenism, as defined clinically by hirsutism (modified Ferriman and Gallway score >6), severe acne/seborrhea, and/or biologically by a testosterone serum level greater than 0.6 ng/ml and/or 
-4-androstenedione greater than 2.2 ng/ml; and 3) more than 12 follicles in the 2- to 9-mm range in each ovary at ultrasonography and/or an ovarian volume higher 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 d or >35 d), hirsutism, abnormal serum level of prolactin or androgens (i.e. serum testosterone above 0.6 ng/ml), and PCO at ultrasonography.
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.
Ovarian stimulation
Pituitary desensitization was started with the GnRH agonist triptorelin 0.1 mg/day (Decapeptyl; Ferring, Malmö, Sweden) during the luteal phase before IVF treatment or the first day of menses for the patients with dysovulation. Follicle growth was stimulated after 12 d of desensitization by injecting recombinant FSH (rFSH; Puregon; Organon Laboratories, Saint-Denis, France) at 150–250 IU/d. Follicle growth was monitored according to serum estradiol levels and transvaginal ultrasound. Human chorionic gonadotropin (Merck Serono, Geneva, Switzerland), 5000 IU, was administered when the leading follicle reached 18–20 mm in diameter together with at least three follicles greater than 16 mm detected by ultrasonography. Oocyte retrieval was performed 36 h later under transvaginal ultrasound guidance.
Collection of GCs
Follicle size was determined immediately before retrieval under ultrasound, and follicular fluids (FF) from SF (8–13 mm) and LF (17–22 mm) were collected in separate tubes. The size of SF and LF was chosen approximately similar to a previous study of Fanchin et al. (18). The 8- to 13-mm follicles (SF) are intermediate dominant follicles lastly recruited. SF and LF were examined separately to point differences between mature and immature dominant follicles. To avoid mixing fluids of SF vs. LF from one ovary in the same aspirating needle, FF of LF were aspired first. Then two SF were aspired but their FF were not used for the study. Lastly, FF of the other SF were aspired. Oocytes were isolated and follicular fluids from SF and LF were pooled separately and centrifuged through a one-step density Percoll gradient [(vol/vol) phosphate-buffered saline/Percoll] at 350 x g for 15 min to remove red blood cells. Luteinized GCs were collected at the interface, washed with PBS, and stored in lysis buffer at –20 C until mRNAs and proteins analysis.
For each patient, GC from either SF or LF were pooled to extract enough RNA to allow their quantification and to study the expression of several genes.
RNA extraction and reverse transcription
Total RNAs were extracted from GCs using the NucleoSpin RNA/protein kit (Macherey-Nagel, Düren, Germany). For each patient and each category of follicle, reverse transcription was performed in a total volume of 20 µl with the first-strand cDNA synthesis kit for RT-PCR (Roche Diagnostics, Indianapolis, IN) using 0.37 µg RNA, avian myeloblastosis virus reverse transcriptase, and random primers p(dN)6 as recommended by the manufacturer.
Quantitative real-time PCR
Evaluation of gene expression levels was achieved by real-time quantitative PCR kinetics using the SYBR Green I chemistry. Real-time PCR was performed with 5 µl of appropriate diluted cDNA, 500 nM of forward and reverse specific primers for human AMH, AMHR-II, FSHR, AR (Table 1
), 3 mM MgCl2, and 1x LightCycler FastStart reaction mix SYBR Green I (Roche Diagnostics) in a LightCycler (Roche Diagnostics). The PCR protocol used an initial denaturing step at 95 C for 10 min followed by 45 cycles at 95 C for 10 sec, annealing temperature (Table 1) for 10 sec, 72 C for 2–10 sec (Table 1) with a transition rate of 20 C/sec. Crossing point values were acquired using the second derivative maximum method of the LightCycler software 3.3 (Roche). The specificity of the desired product was documented with the analysis of the melting curve. The melting curve was achieved by first cooling samples to 60 C at a transition rate of 20 C/sec after 30 sec of incubation, and a slow-heating step at a rate of 0.1 C/sec until a maximum temperature of 95 C. The mix was next cooled at 40 C for 1 min, at a transition rate of 20 C/sec. Quantification of gene expression was based on a standard curve for each target gene with known amounts of testicular cDNAs and was included in each LightCycler real-time PCR experiment. Relative gene expression was calculated as a ratio of each target gene concentration to housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) concentration.
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Proteins were purified according to the total RNA and protein isolation kit (Macherey-Nagel). After loading cell lysates on the Nucleospin RNA/protein column, flow-throughs were precipitated with an equal volume of protein precipitator. After a 10-min incubation and a centrifugation for 5 min at 11000 rpm, protein pellets were washed with 50% ethanol and resuspended in 50 µl of protein loading buffer. Proteins were then dissolved during 3 min at 98 C and centrifuged for 1 min at 11000 rpm to remove insoluble material. Protein concentrations were determined using the BCA protein assay kit (Pierce Chemical Co., Rockford, IL). Ten micrograms of proteins were loaded on 7.5% SDS-PAGE (Bio-Rad Laboratories, Hercules, CA) under reducing conditions [3% (vol/vol) 2β-mercaptoethanol] and transferred onto a Protran BA85 nitrocellulose membrane (Schleicher & Schuell, Keene, NH) as previously described (19). AMH was detected using an anti-AMH monoclonal antibody specific of the N-terminal domain of AMH (20) (monoclonal antibody 10.6) at 1 µg/ml and horseradish peroxidase-labeled antimouse IgG antibody at 1:5000 (Jackson ImmunoResearch Laboratories, Bar Harbor, ME). Bands were visualized with the ECL Plus detection reagent (Amersham Pharmacia Biotech, Piscataway, NJ).
Statistics
Because values from any parameter were not normally distributed, all comparisons between PCOS and controls were performed using the nonparametric Mann-Whitney test. Wilcoxon test was used for paired comparisons between SF and LF.
Univariate analysis of correlations between expression of the different genes was performed with the nonparametric Spearman test. Multiple linear regression was then performed to test these different correlations using log-transformed values.
All statistical procedures were run on SPSS 11.5 (SPSS Inc., Chicago, IL). P 
0.05 was considered statistically significant.
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Results |
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Main clinical parameters in normoovulatory controls and patients with PCOS are summarized in Table 2. No difference between the two populations was found for the age, the total dose of rFSH received by each patient and the number of LF aspired. Mean body mass index and number of SF aspired were significantly higher in the PCOS group.
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AMH expression determined by quantitative RT-PCR was about 3-fold higher in PCOS patients than controls (P = 0.016 and P = 0.008, respectively) (Table 3) in both SF and LF. AMH mRNAs in SF tended to be higher than in LF in patients with PCOS (P = 0.08) and controls (P = 0.25) (Table 3 and Fig. 1A).
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). The 70-kDa band corresponds to noncleaved AMH and the 56-kDa band to the N-terminal domain of AMH. In LF, no difference was found between PCOS and controls (Fig. 2).
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In GCs from both SF and LF, FSHR mRNA level was significantly higher in PCOS patients than controls (P = 0.004 and P = 0.007, respectively). FSHR expression was significantly higher in SF than LF, both in PCOS and controls (P = 0.019 and P = 0.048, respectively) (Table 3
and Fig. 1B).
AR and AMHR-II gene expression in GCs
In GCs from SF only, AR and AMHR-II mRNA levels were significantly higher in PCOS patients than controls (P = 0.007 and P = 0.024, respectively). In PCOS patients only, AR (and not AMHR-II) expression was significantly higher in SF than LF (P = 0.049) (Table 3 and Fig. 1, C and D).
Relationships between the expression of the different genes
Using a Spearman test, no correlation was found between the expression of the different genes in LF from controls. AMH and FSHR mRNA levels correlated strongly and positively in both SF (Spearman’s Rho = 0.644, P = 0.013) and LF (Table 4) from PCOS patients and SF from controls (R = 0.776, P = 0.003). AR and FSHR mRNA levels were also significantly and positively correlated in both SF (R = 0.510, P = 0.037) and LF (Table 4) from PCOS patients and in SF from controls (R = 0.591, P = 0.026). In PCOS patients, AMHR-II expression significantly and positively correlated with AR expression in SF (R = 0.672, P = 0.003) and LF and with AMH and FSHR messengers in LF (Table 4). In SF from controls, there was no correlation between the expression of AMHR-II and those of AR, AMH, and FSHR. Because of the numerous univariate correlations (Table 4), multiple linear regression was performed on the data from LF in PCOS patients. When FSHR expression was introduced as the dependent variable, its relationships with AMH and AR mRNAs persisted in an independent manner, whereas AMHR-II was rejected by the model.
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Discussion |
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Second, both AMH mRNA and protein levels were higher in GCs from PCOS than from controls (Figs. 1A and 2). These results are in agreement with Pellatt et al. (12), who reported that in vitro, GCs from PCOS patients secrete more AMH than GCs from controls. Serum levels of AMH are 2- to 3-fold higher in women with PCOS, compared with ovulatory women with normal ovaries (11, 23, 24). We previously hypothesized that AMH excess in PCOS was due only to the increased number of small antral follicles, each follicle presumably secreting the same amount of AMH than in a normal ovary (11). Conversely, the present study as well as the data of Pellatt et al. (12) argue for an increased expression of AMH per GC in PCOS. Consequently, the high levels of serum AMH in PCOS are likely a combination of an increased number of small follicles and an increased production of AMH per cell. Given that GCs from each category of follicle were pooled, it was not possible to determine whether all or only some of the follicles from PCOS expressed more AMH.
In our study, the increased expression of AMH by GCs from PCOS was associated with higher AR, FSHR, and AMHR-II mRNA levels. The presence of AR at advanced stages of maturation was in agreement with the immunocytochemical data from Horie et al. (25). However, using quantitative immunocytochemistry, Hillier et al. (26) reported that in the primate (common marmoset) ovary, ARs were most abundant in healthy preantral and early antral follicles, being scarce or absent in preovulatory follicles. To our knowledge, this is the first time that an expression of the AR gene is reported in GCs from human stimulated preovulatory follicles. The higher expression of AR in SF than LF in PCOS is in agreement with the decline of AR expression during follicle maturation (26), thus indicating that AR synthesis in PCOS follicles is still under physiological regulation. The absence of such a decrease in controls may be explained by lower levels of AR mRNAs in follicles from normal women. The higher expression of AR in PCOS follicles than controls follicles is in agreement with Weil et al (7), who reported an increased AR expression in GCs of testosterone-treated monkeys from the preantral to the large antral follicle stage. This suggests that hyperandrogenism stimulates AR expression. A relationship between intrafollicular androgen concentration and its receptor level should be studied to confirm this hypothesis in human follicles.
In our study FSHR expression was significantly higher in PCOS patients than controls in GCs from both SF and LF. This is in agreement with previous studies showing that GCs from PCOS cells express high amounts of FSHRs and are highly responsive to this hormone in culture (8, 27). This excess of FSHR mRNAs in PCOS patients is perhaps an amplification of a physiological phenomenon. Indeed, in controls, FSHRs were also expressed in SF and LF but at lower levels. Second, in the normal ovary, the total number of FSHR was about 2-fold higher at midfollicular phase than at the other periods (28). Moreover, other authors reported that human GCs in the late follicular phase bound less FSH than GCs in the midfollicular phase (29). In agreement, in our study, the amount of FSHR mRNAs was significantly lower in LF than SF from both PCOS patients and controls, although cells were terminally differentiated by hCG administration.
Whether these two phenomenons (i.e. excessive AR and FSHR expression) may reflect exaggeration and/or abnormal persistence of physiological properties is further supported by the tight relationship we observed between FSHR and AR expression. Both were strongly and positively correlated in SF from PCOS patients and controls and in LF from PCOS patients. This correlation persisted after controlling for confounding factors by multiple linear regression. In keeping with this result, Weil et al. (30) reported a highly significant positive correlation between FSHR and AR mRNA levels in GCs from testosterone-treated or control primate follicles. Hyperandrogenism is likely the culprit because androgen treatment significantly increased FSHR mRNAs in GCs (by 
50–100%, depending on follicle size) (30). So we hypothesize that FSHR expression is increased by androgens through the increase of AR mRNAs. In our study, FSHR expression was also strongly and positively correlated with the amount of AMH mRNAs both in SF and LF from PCOS patients and in SF from controls. This strong relationship persisted after multiple linear regression analysis. Therefore, we hypothesize that AMH, FSHR, and AR excessive expression in PCOS patients are linked and are all a consequence of hyperandrogenism. However, androgen assays in FF and further experimental data are necessary to confirm this hypothesis, in particular the relationship between hyperandrogenism and the AMH increase. Indeed, a relationship between androgens and AMH in follicular fluid was established in a first study (31) but not a second one (32).
Our data suggest that AMH is probably not just residual to an anterior secretion but still has an action in the final phase of follicle maturation. Indeed, in our study, the expression pattern of AMH and its receptor was found similar. This is in agreement with previous studies in rodents reporting that AMHR-II expression colocalizes with AMH mRNAs in GCs of the postnatal ovary (33). The AMH action on the follicle is probably more important in PCOS patients compared with controls, and the question then arises about its possible impact on oocyte quality that is known to be poor in PCOS patients (13, 14). In support of this assumption, a lower oocyte quality has been reported in patients with high serum AMH levels (34).
In this study, we have shown that the expression of AMH, AR, FSHR, and AMHR-II was higher in follicles from PCOS patients compared with controls, under rFSH and hCG stimulation. This may reflect an intrinsic GC dysregulation, in which LH (or hCG) and hyperandrogenism could be involved. The higher levels of AMH, AR, FSHR, and AMHR-II mRNAs in PCOS and their tendency to be lower in dominant mature follicles compared with immature follicles strengthen the hypothesis of a delayed GC maturation in PCOS patients (35, 36). Finally, despite an apparent normal development under rFSH, PCOS dominant follicles retain some of their basal abnormalities possibly impacting on oocyte quality. However, further data on the relationships between hCG, AMH, and androgens in FF are necessary to elucidate the pathophysiological relevance of such findings.
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Footnotes |
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Disclosure Statement: The authors have nothing to disclose.
First Published Online August 12, 2008
Abbreviations: AMH, Anti-Müllerian hormone; AMHR-II, AMH type II receptor; AR, androgen receptor; FF, follicular fluid; FSHR, FSH receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GC, granulosa cell; hCG, human chorionic gonadotropin; IVF, in vitro fertilization; LF, large follicles; PCOS, polycystic ovary syndrome; rFSH, recombinant FSH; SF, small follicles.
Received June 6, 2008.
Accepted August 5, 2008.
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References |
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-hydroxylase and steroidogenic acute regulatory protein gene promoters in normal and polycystic ovary syndrome theca cells. J Clin Endocrinol Metab 85:2304–2311This article has been cited by other articles:
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  E. Diamanti-Kandarakis, A. Piouka, S. Livadas, C. Piperi, I. Katsikis, A. G Papavassiliou, and D. Panidis Anti-mullerian hormone is associated with advanced glycosylated end products in lean women with polycystic ovary syndrome Eur. J. Endocrinol., May 1, 2009; 160(5): 847 - 853. [Abstract] [Full Text] [PDF]
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 H. H Ortega, N. R Salvetti, and V. Padmanabhan Developmental programming: prenatal androgen excess disrupts ovarian steroid receptor balance Reproduction, May 1, 2009; 137(5): 865 - 877. [Abstract] [Full Text] [PDF]
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