MATERIALS AND METHODS Patients and Hormonal Treatment

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1 Levels of the epidermal growth factor-like peptide amphiregulin in follicular fluid reflect the mode of triggering ovulation: a comparison between gonadotrophin-releasing hormone agonist and urinary human chorionic gonadotrophin Peter Humaidan, M.D., a Lars Grabow Westergaard, M.D., D.M.Sc., b Anne Lis Mikkelsen, M.D., D.M.Sc., c Misao Fukuda, M.D., d and Claus Yding Andersen, D.M.Sc. e a Fertility Clinic, Skive Regional Hospital, Skive, Denmark; b Fertility Clinic, Department of Obstetrics and Gynecology, Odense University Hospital, Odense, Denmark; c Fertility Clinic, Holbæk Regional Hospital, Holbæk, Denmark; d M&K Health Institute, Ako, Japan; e Laboratory of Reproductive Biology, Section 5712, University Hospital of Copenhagen, Copenhagen, Denmark Objective: To detect differences in follicular fluid (FF) levels of amphiregulin (AR), depending on mode of triggering final oocyte maturation. Design: Prospective randomized trial. Setting: Three IVF units. Patient(s): Ninety-six patients undergoing IVF intracytoplasmic sperm injection. Intervention(s): Ovulation triggered with either urinary hcg or GnRH agonist (GnRH-a). Controls: 15 FF samples from small antral follicles (3 9 mm) and 12 FF samples from natural cycle. Main Outcome Measure(s): Follicular fluid concentration of AR, P 4,E 2, vascular endothelial growth factor, and inhibin B. Result(s): Significantly lower levels of AR were found in FF from the GnRH-a group versus the hcg group, versus ng/ml. In FF from natural cycles, levels of AR were significantly higher than those of GnRHa triggering but significantly lower than those of urinary hcg triggering. In small antral follicles only 5 out of 15 follicles contained measurable amounts of AR. When urinary hcg and GnRH-a triggering were compared, FF P 4 was significantly higher after urinary hcg triggering, whereas no difference was seen regarding E 2, vascular endothelial growth factor, and inhibin B. A total of 14% more metaphase II oocytes and 11% more transferable embryos were obtained after GnRH-a triggering. Conclusion(s): This study suggests that oocyte competence is linked to granulosa cell AR secretion. (Fertil Steril Ò 2011;95: Ó2011 by American Society for Reproductive Medicine.) Key Words: Amphiregulin, VEGF, GnRH agonist, GnRH antagonist, in vitro fertilization, triggering of ovulation The pivotal role of the midcycle surge of gonadotrophins for inducing ovulation and resumption of meiosis is well recognized. However, the physiologic processes by which FSH and LH secure the release of a fertilizable oocyte at ovulation involve a complicated network of regulatory substances. Animal studies have shown that members of the epidermal growth factor (EGF)-like family act as mediators of LH action on the preovulatory follicle (1, 2). On stimulation with LH-like activity mural granulosa cells of preovulatory follicles respond with a rapid and massive synthesis of EGF-like peptides, that is, amphiregulin (AR), epiregulin, and betacellulin (1 5). Especially AR and epiregulin have been shown to be potent mediators of oocyte maturation by stimulating EGF receptors in the cumulus cells (3, 6). Recently, isolated murine cumulus enclosed oocytes have been shown dose-dependently to respond to Received December 11, 2010; revised January 26, 2011; accepted February 7, 2011; published online March 5, P.H. has nothing to disclose. L.G.W. has nothing to disclose. A.L.M. has nothing to disclose. M.F. has nothing to disclose. C.Y.A. has nothing to disclose. Reprint requests: Peter Humaidan, M.D., Fertility Clinic, Skive Regional Hospital, Resenvej 25, 7800 Skive, Denmark ( peter. humaidan@viborg.rm.dk). AR by resuming meiosis (7). In humans, AR becomes expressed abundantly in preovulatory follicles on exposure to the midcycle surge of gonadotrophins or hcg, and the concentration of AR has been shown to correlate inversely with the fertilization rate (8). During ovarian hyperstimulation, the use of GnRH antagonist protocols for the prevention of a premature LH surge allows ovulation to be induced with a bolus of GnRH agonist (GnRH-a) as an alternative to hcg (9 11). In the pituitary GnRH-a displaces the GnRH antagonist, which activates the GnRH receptor, resulting in a surge of gonadotrophins (flare-up), similar to the natural midcycle surge of gonadotrophins. The surge of FSH elicited simultaneously with LH potentially could have a positive impact on oocyte maturation, LH receptor formation in granulosa cells, and cumulus expansion (12 16). The aim of the present study was to explore possible differences in follicular fluid (FF) hormones after triggering of final oocyte maturation with either GnRH-a or hcg, focusing on AR. MATERIALS AND METHODS Patients and Hormonal Treatment Follicular fluid was obtained from 96 patients with normal gonadotrophin levels undergoing IVF intracytoplasmic sperm injection (ICSI) and 2034 Fertility and Sterility â Vol. 95, No. 6, May /$36.00 Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc. doi: /j.fertnstert

2 TABLE 1 Follicle-stimulating hormone consumption, GnRH antagonist consumption, and E 2 levels during follicular phase in GnRH-a versus urinary hcg groups. Total GnRH-a Urinary hcg Started cycles, total FSH stimulation days, median (range) 8 (6 12) 8.5 (6 10) 8 (6 12) FSH consumption, IU (mean SEM) 1, , , First antagonist day, SD median (range) 6 (5 10) 6 (5 9) 7 (5 10) Antagonist consumption, mg (mean SEM) Se-E 2 (pmol/l), S Se-E 2 (pmol/l), S7 2, , , Se-E 2 (pmol/l), hcg day 4, , , Se-E 2 (pmol/l), OPU day 2, , , Note: OPU ¼ oocyte pickup; Se ¼ serum; S2 ¼ stimulation day 2; S7 ¼ stimulation day 7. participating in a prospective randomized, three-center study. Details on patient characteristics and hormonal treatment given have been published previously (17). In brief, patients received ovarian stimulation followed by cotreatment with a standard GnRH antagonist protocol; when at least two follicles had reached a size of 17 mm, patients were assigned randomly into two groups: the GnRH-a group or the hcg group. The GnRH-a group received final oocyte maturation with a single bolus of 0.5 mg buserelin SC (Suprefact; Hoechst, Hørsholm, Denmark). The hcg group received 10,000 IU of hcg SC for final oocyte maturation. Oocyte pickup was performed 34 hours later in both groups. The oocyte maturity was assessed in patients undergoing ICSI. The study was approved by the Ethics Committee of Viborg County, project number MCH, ClinicalTrials.gov NCT Follicular Fluid Sampling Two follicular samples without contamination of flushing medium, from the first follicle aspirated on each side, were collected. To eliminate granulosa cells the fluid was centrifuged at 500 g. A total of 146 FF samples were included; of these 73 FF samples were obtained from the GnRH-a group and 73 from the hcg group. Fifteen FF samples from small antral follicles (3 9 mm) and 12 preovulatory FF samples from natural cycles, aspirated after the midcycle surge but before ovulation, served as controls. Samples were stored at 20 C. All measurements were performed on individual follicles, and the results are given as the mean of measurements of individual follicles. Hormone Measurements Amphiregulin was measured with use of a commercially available sandwich ELISA kit (DY262; R&D Systems Europe, Abingdon, United Kingdom). According to the manufacturer the assay does not show any crossreactivity toward EGF, EGF receptor, heparin-binding EGF, or transforming growth factor a at a concentration of 50 ng/ml. The assay standard curve covered a concentration range from 1.5 to 1,000 pg/ml, and FF samples were diluted 1:500 with use of the reagent diluent to be covered by the standard curve. Samples were diluted parallel to the standard curve, and the interassay variation of a sample containing 59.8 ng/ml was 6.9% (N ¼ 10). Vascular endothelial growth factor (VEGF) was measured with use of a commercially available sandwich ELISA kit (DY293b; R&D Systems Europe). According to the manufacturer the assay does not show any cross-reactivity toward VEGF-C and VEGF-D at a concentration of 50 ng/ ml. The assay standard curve covered a concentration range from 2 to 2,000 pg/ml, and FF samples were diluted 1:10 to be covered by the standard curve. The interassay variation of a sample containing 1,536 pg/ml was 14% (N ¼ 28). Inhibin B was measured with use of a specific ELISA kit according to the manufacturer s instruction (Oxford Bio-innovation kit; Biotech-IgG, Copenhagen, Denmark). Before measurement, irrespective of whether samples derived from small antral or preovulatory follicles, all FF samples were diluted 1:100 or 1:500 in serum obtained from a pool of five postmenopausal women (with no inhibin B activity). The FF samples were pretreated with sodium dodecyl sulfate, heated, and exposed to hydrogen peroxide before they were applied to the wells of the plate and incubated overnight at room temperature. Subsequently, the plates were washed and incubated with detection antibody for 3 hours at room temperature. Substrate solution was applied and incubated for 1 hour. The amplifier solution was added, and the plates were read with an ELISA reader at 490 nm with its reference at 620 nm (coefficient of variation <7%). Estradiol and P were measured, with use of commercially available RIA kits (DSL and DSL-4300; Diagnostics System Laboratories, Webster, TX). Samples for both assays were diluted to 1:1,000 in steroid-free serum before measurement. Statistical Methods Comparison of outcomes between the two groups was performed by Student s t-test for parametric data and by Fisher s exact test for nonparametric data. The program used for analysis was SPSS 15 (SPSS, Inc., Chicago, IL). A P value <.05 was considered to be statistically significant. RESULTS Patient Characteristics and Stimulation No significant differences regarding demographic data were found. The total exogenous FSH consumption, total dose of GnRH antagonist, duration of stimulation, and E 2 levels during the follicular phase did not differ between the GnRH-a and hcg groups (Table 1). The distribution of IVF and ICSI cycles performed did not differ between the two groups. Follicular Fluid Contents of Hormones After Gonadotrophinreleasing Hormone Agonist and Human Chorionic Gonadotrophin Triggering and in the Natural Cycle Significantly lower levels of AR were found in FF from the GnRH-a group as compared with the hcg group, versus ng/ml (mean SD) (P¼.003). In preovulatory FF from natural menstrual cycles, levels of AR were ng/ml, a level significantly higher than that of GnRH-a triggering but significantly lower than that of hcg triggering (P<.001) (Table 2). No difference was seen regarding VEGF, 1, pg/ml versus 1, pg/ml, between the GnRH-a and hcg groups, respectively. However, the VEGF level of the natural cycle preovulatory FF, 2,248 Fertility and Sterility â 2035

3 TABLE 2 Follicular fluid concentration of VEGF and AR in small antral follicles and preovulatory natural cycle follicles versus stimulated follicles after GnRH-a and urinary hcg triggering. Small antral follicles Preovulatory follicles, natural cycle GnRH-a Urinary hcg FF aspirates (n) VEGF (pg/ml) a 2, b 1, c 1, c AR (ng/ml) a b d e Note: Values are expressed as mean SD. a Significantly different from the preovulatory follicles natural cycle, GnRH-a, and urinary hgg groups, P<.001. b Significantly different from the small antral follicles, GnRH-a, and urinary hgg groups, P<.001. c Significantly different from the preovulatory follicles natural cycle and small antral follicles groups, P<.001. d Significantly different from the small antral follicles, preovulatory follicles natural cycle, and urinary hgg groups, P¼.003. e Significantly different from the small antral follicles, preovulatory follicles natural cycle, and GnRH-a groups, P¼ pg/ml (mean SD), was significantly higher than that of both GnRH-a and hcg triggering (P<.001). In FF from small antral follicles only 5 out of 15 contained measurable amounts of AR ( ng/ml) and 6 of 15 VEGF ( pg/ml) (mean SD) (Table 2). Follicular fluid P concentration was significantly higher after hcg triggering (P¼.0004) than after triggering with GnRH-a. However, mean concentrations of FF E 2 and inhibin B did not differ between the GnRH-a and hcg groups, versus ng/ ml (Table 3). Oocytes and Transferable Embryos A significantly higher fertilization rate was seen after triggering with hcg as compared with GnRH-a in ICSI cycles (98% vs. 85%) (P¼.013). However, a non statistically significant difference in fertilization rate in favor of GnRH-a triggering in IVF cycles was seen (P¼.08) (Table 4). Assessment of the maturity of ICSI oocytes revealed significantly more MII oocytes after GnRH-a triggering as compared with hcg triggering (92% vs. 78%, respectively) (P¼.017). Moreover, significantly more transferable embryos (11% of oocytes retrieved) (IVF and ICSI) were present after GnRH-a triggering (51% vs. 40%, respectively) (P¼.005). DISCUSSION To the best of our knowledge this is the first study to explore possible differences in the intrafollicular levels of AR, after ovulation induction with either GnRH-a or hcg as compared with the natural surge of gonadotrophins. The study shows that the mode of triggering ovulation significantly affects the synthesis of AR. Thus, a significantly higher FF concentration of AR was found after hcg triggering as compared with triggering with GnRH-a and the natural surge of gonadotrophins. In contrast, a significantly higher concentration of VEGF was found in FF from natural cycle preovulatory follicles as compared with GnRH-a and hcg triggering. Only approximately one third of all human small antral follicles contained measurable levels of AR and VEGF. Interestingly, this study showed that significantly more metaphase II (MII) oocytes and more transferable embryos were obtained when GnRH-a was used for final oocyte maturation as compared with hcg. However, the fact that a significantly higher fertilization rate was seen with hcg as compared with GnRH-a in ICSI cycles is not readily explainable, and we are currently investigating whether this also is observed in a larger material. The collective bolus of LH-like activity released as a result of ovulation induction with GnRH-a is reduced significantly as compared with that of hcg (18, 19). The reduction in LH-like activity appears to induce a significantly higher proportion of oocytes to resume meiosis and reach the MII stage, suggesting that the process of ovulation induction may be optimized after GnRH-a triggering when compared with the standard use of hcg. Taken together, our study suggests that follicular synthesis of AR induced by ovulation induction has a significant impact on the oocyte maturation rate, as opposed to VEGF, suggesting that more optimal conditions for oocyte maturation might be achieved through the design of alternative methods for triggering ovulation. TABLE 3 Follicular fluid concentration of E 2,P 4, VEGF, inhibin B, and AR after GnRH-a versus urinary hcg triggering. Total GnRH-a Urinary hcg P value (t-test) OPU (n) FF aspirates (n) E 2 (ng/ml) NS P 4 (ng/ml) 13, , , Inhibin B (ng/ml) NS VEGF (pg/ml) 1, , , NS AR (ng/ml) Note: Values are expressed as mean SD. NS ¼ not significant; OPU ¼ oocyte pickup Humaidan et al. Amphiregulin and ovulation trigger Vol. 95, No. 6, May 2011

4 TABLE 4 Oocyte maturation, fertilization, and embryo development after GnRH-a versus urinary hcg triggering. Total GnRH-a Urinary hcg P value (Fisher s exact test, two-tailed) IVF Oocytes (n) Fertilized, no. (%) 471 (69) 237 (72) 234 (66).08 IVF: no. of embryos (% of fertilized) 405 (86) 210 (89) 195 (83) NS ICSI Oocytes (n) MII, no. (%) 124 (86) 72 (92) 52 (78).017 No. fertilized of MII (%) 112 (90) 61 (85) 51 (98).013 ICSI: no. of embryos (% of fertilized) 107 (96) 57 (93) 50 (98) NS IVF þ ICSI No. transferrable (% of oocytes retrieved) 309 (45) 167 (51) 142 (40).005 No. transferrable per cycle (mean SEM) NS No. transferred per cycle (mean SEM) NS Note: NS ¼ not significant. Intrafollicular protein levels of AR have been reported to peak at approximately 16 to 24 hours after ovulation induction (20). The present study demonstrated that hardly any AR was present before ovulation induction and that, interestingly, even after 35 hours, at the time of oocyte retrieval, there were still very high levels of AR: one to two orders of magnitude higher than before ovulation induction. In addition, the present study was intended to explore whether levels of AR reflected oocyte competence in a clinical setting where oocytes are retrieved approximately 35 hours after ovulation induction. As a bolus of hcg may represent a stronger signal for ovulation induction compared with that of the natural cycle and GnRH-a trigger, the synthesis of AR could be altered in a way leading to a reduced rate of MII oocytes. In the present study, the FF AR concentration in the GnRHa group was significantly lower as compared with that in the hcg group. At the same time, 14% more MII oocytes and 11% more transferable embryos were seen in the GnRH-a group. These findings confirm a recent trial by Inoue et al. (8), measuring three different EGF receptor ligands: EGF, transforming growth factor a, and AR, in FF obtained from patients undergoing IVF treatment. The authors found that AR was the most abundant EGF receptor ligand expressed in FF and that the concentration of AR in FF was 3,000 times as high as that in serum. Importantly, the AR concentration in FF was correlated significantly negatively with the fertilization rate. Moreover, a trend for an inverse correlation was found for AR and embryo quality. Finally, a significant inverse correlation between AR and FF exogenous hcg was found in the group of patients who conceived (8), which could indicate a down-regulation of AR synthesis because of the prolonged action of hcg. The finding of the present study that the highest AR concentration was found in FF of patients triggered with hcg compared with GnRH-a and the natural surge of gonadotrophins, combined with the fact that the MII rate and embryo quality in the GnRH-a group was significantly higher than that of the hcg group, could indicate a ceiling level of AR, above which oocyte competence is affected negatively. However, it also could reflect differences in kinetics of release or homeostasis of AR (21). Interestingly, more MII oocytes and good available embryos after GnRH-a triggering also were reported in a recent study in women with breast cancer (22). However, an increase in the number of MII oocytes and transferable embryos was not reported in a recent randomized controlled trial comparing GnRH-a triggering with hcg triggering in oocyte donors (23), a fact that could rely on differences in the type and dose of GnRH-a used. Vascular endothelial growth factor plays an important role for the vascularization of the follicle, and thus the regulation of the intrafollicular oxygen level and FF levels of VEGF directly correlated with perifollicular blood flow in patients undergoing IVF (24). A significantly higher FF concentration of VEGF has been reported in oocytes failing to fertilize, and high FF VEGF levels also have been associated with poor embryo quality and low pregnancy rates in IVF (25 27). In the present study, the concentration of VEGF in small antral follicles was very low, as only 6 out of 15 follicles contained measurable VEGF. In contrast, VEGF concentrations were significantly higher in both natural cycle preovulatory follicles and follicles after GnRH-a and hcg triggering, showing a clear up-regulation of VEGF with growth of the follicle and after the LH LH-like surge. Interestingly, the VEGF concentration in natural cycle FF was approximately twice as high as after GnRH-a and hcg triggering, a finding that is not readily explainable. Interestingly, no difference was seen in FF VEGF concentration between hcg and GnRH-a triggering. This is contrasted by the findings of Cerrillo et al. (28), who reported a significantly higher FF VEGF concentration after hcg triggering as compared with GnRH-a triggering. However, different types and doses of GnRH-a were used to trigger final oocyte maturation in the two trials: triptorelin (0.2 mg) in the study by Cerrillo et al. (28) versus buserelin 0.5 mg in the present study. Moreover, different assays for analysis of VEGF were used. A significantly lower FF concentration of P was found after GnRH-a triggering, whereas no difference was seen regarding E 2 and inhibin B. These results confirm those of a previous study (29), reporting a 25% lower FF P concentration after GnRHa triggering and no difference in E 2 and inhibin B, and the finding is also in line with the results of an earlier study (30), reflecting that P output is primarily LH activity dependent (31). Fertility and Sterility â 2037

5 In conclusion, the difference in MII rate and the significantly different levels of AR related to method of ovulation induction used (i.e., either GnRH-a or hcg), in combination with the differences in fertilization rate and embryo development, suggest that oocyte competence is linked to AR secretion. Although the present study does not provide a causative link between AR and oocyte competence, the results indicate that the process of ovulation induction might be further optimized and that the time has come to reconsider the standard use of hcg for ovulation trigger in assisted reproductive technologies. Acknowledgments: The authors thank the nurses and laboratory technicians of the participating clinics for their assistance during this study. Moreover, the excellent technical assistance by Ms. Marjo Westerdahl and Ms. Tine Roed is gratefully acknowledged. REFERENCES 1. Park JY, Su YQ, Ariga M, Law E, Jin SL, Conti M. 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Differential regulation of inhibin A and inhibin B by luteinizing hormone, follicle-stimulating hormone, and stage of follicle development. J Clin Endocrinol Metab 2001;86: Humaidan et al. Amphiregulin and ovulation trigger Vol. 95, No. 6, May 2011