Early follicular anti-mullerian hormone as a predictor of ovarian response during ICSI cycles

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1 Middle East Fertility Society Journal (2010) 15, Middle East Fertility Society Middle East Fertility Society Journal ORIGINAL ARTICLE Early follicular anti-mullerian hormone as a predictor of ovarian response during ICSI cycles Ashraf Moawad a,c, *, Hanan Abd Elmawgood b, Mahmoud Shaeer c a Faculty of Medicine, Al-Azhar University, Egypt b Faculty of Pharmacy for Girls, Al-Azhar University, Egypt c IVF Unit, Enjab Hospital for Infertility, Sharjah, United Arab Emirates Received 20 June 2010; accepted 4 July 2010 Available online 31 July 2010 KEYWORDS Anti-mullerian hormone; Ovarian response; ICSI Abstract Objective: To evaluate the use of AMH in predicting the ovarian response in ICSI cycles compared to other markers of ovarian function. Design: Prospective study. Setting: IVF/ICSI Unit; Enjab Hospital for infertility, Gulf Medical College and Research Centre (UAE) and Biochemistry department, Faculty of Pharmacy for Girls, Al-Azhar University (Egypt). Subject(s): A total of 220 infertile women attending an ART program (ICSI) for the first time during the period from June 2007 to October 2009 who met the inclusion criteria were our subjects. On day-3 of the menstrual cycle, serum levels of AMH, FSH, LH, E2, and inhibin B were measured for each woman. Early antral follicles were evaluated by vaginal ultrasound. Thereafter, the patients were classified according to oocyte count into two groups; Good responders (those with P4 oocytes) and poor responders (those with <4 oocytes). Intervention(s): None. Main outcome measure(s): Comparison of day-3 serum AMH levels in both groups. Antral follicle count, basal FSH, LH, E2 and inhibin B were also compared. * Corresponding author at: IVF Unit, Enjab Hospital for Infertility, P.O. Box 31705, Sharjah, United Arab Emirates. address: drashrafmoawad@yahoo.com (A. Moawad) Ó 2010 Middle East Fertility Society. Production and Hosting by Elsevier B.V. All rights reserved. Peer-review under responsibility of Middle East Fertility Society. doi: /j.mefs Production and hosting by Elsevier

2 282 A. Moawad et al. Results: The serum level of AMH, AFC and FSH levels were significantly different in both groups. Parameters such as serum LH, E2 and inhibin B levels were not significantly different between the two groups. Ovarian response was significantly correlated with basal AMH levels, FSH levels and AFC. However, AMH levels were highly correlated with the number of retrieved oocytes (P < 0.001) than did AFC (P < 0.01) or FSH (P < 0.05) on day-3 of the cycle. Day-3 AMH was more sensitive and specific with higher predictivity for ovarian response than either day-3 AFC or day-3 FSH. Conclusion: Serum AMH levels may reflect ovarian response better than the usual hormone markers. Ó 2010 Middle East Fertility Society. Production and Hosting by Elsevier B.V. All rights reserved. 1. Introduction Careful evaluation of patients and proper treatment with right techniques are essential for the successful outcome of assisted reproduction. To obtain satisfactory results, it is necessary to assess ovarian reserve before planning treatment. Contemporary markers for ovarian reserve include age, basal folliclestimulating hormone (FSH) levels, and estradiol (E2) levels (1,2). Antral follicle count, serum inhibin B levels, ovarian volume, and vascular resistance have also been studied as markers of ovarian reserve (3 7). Provocative and dynamic tests such as a gonadotropin-releasing hormone (GnRH) agonist test and clomiphene citrate test have also been introduced recently, and more effective parameters are being sought (6,8). Ovarian reserve relates to both the quantity and the quality of the ovarian follicle pool. The number of primordial follicles that are left in the ovary at a given age is therefore an important indicator for ovarian reserve and dictates reproductive events, such as age at menopause (9). Although direct measurement of the primordial follicle pool is impossible, it has been shown that the number of antral follicles in the ovaries is proportionally related to the size of primordial follicle stock from which they were recruited (10). Therefore, the antral follicle count (AFC) is believed to represent the quantitative aspect of ovarian aging (7). Unfortunately, markers that may directly reflect oocyte quality are clearly lacking at the moment. Consequently, the age-related decrease in fertility cannot be determined through a direct test. Only through measurement of the quantity of the oocytes can information on the quality aspects of ovarian reserve be obtained (11,12). Ovarian response to ovarian hyperstimulation in IVF is another way in which the quantitative ovarian reserve may come to expression. Although poor response may be considered to be a sign of diminished ovarian reserve, it may also be caused by other factors, such as under-dosing in obesity or in certain FSH-receptor polymorphisms (13). Assessment of the true nature of a poor ovarian response may help to direct the management of the patient regarding treatment (11,14,15). Additionally, correct identification of poor responders, especially in older patients, before entering an IVF program is important, because it could help in proper management regarding gonadotropin dosing and denial of treatment. For this purpose, the tests of choice currently are the AFC or basal FSH, as was shown in a comparative review (6). Anti-mullerian hormone, a member of the transforming growth factor b family, is produced in the granulosa cells (16). The highest level of AMH expression is present in granulosa cells of secondary, pre-antral, and small antral follicles up to 6 mm in diameter (17), whereas in follicles growing into dominance, this expression ceases (18,19). Secreted from pre antral and early antral follicles, AMH regulates ovarian activity and follicular steroidogenesis. Animal studies have revealed that not only does AMH decrease aromatase activity of FSH-stimulated granulosa cells, but it also decreases the number of luteinizing hormone (LH) receptors, and regulates testosterone production in theca cells (20). Anti-mullerian hormone is barely detectable at birth and reaches the highest values after puberty, then decreases progressively with age and becomes undetectable at menopause (21,22). Serum AMH levels have been shown to strongly correlate with the number of antral follicles (23,24) and have appeared to be cycle independent (25,26). From several studies, AMH has emerged as a predictor of ovarian response to hyperstimulation (27,28) and possibly even of the chance of becoming pregnant after IVF (29). However, the question of whether serum AMH measurements on cycle day-3 reflect ovarian follicular status better than the usual hormonal parameters remained unanswered. Hence, the present investigation was designed to weigh the relationship between AMH levels and ovarian response against that between other putative markers of ovarian function and fertility potential, including antral follicle count and serum levels of FSH, LH, estradiol (E2) and inhibin B on cycle day Materials and methods 2.1. Patients This prospective study included a total of 220 women attending the intracytoplasmic sperm injection (ICSI) program of IVF Department (Enjab Hospital) for the first time between June 2007 and October In order to be included, the candidate had to meet the following criteria: regular ovulatory menstrual cycles, normal BMI (18 25 kg/m 2 ), no current hormone therapy, and no current or past diseases affecting ovaries or gonadotrophin or sex steroid secretion, clearance or excretion and adequate visualization of both ovaries at transvaginal ultrasound scanning. Women who had polycystic ovarian syndrome (30) or prior enrolled in this study were excluded. The study got approval from UAE ethical committee (UEC) and the informed consent of patients was documented before being included in the study Setting This study was conducted at private IVF/ICSI units (Enjab Hospital for infertility and Gulf Medical College and Research Centre, Sharjah, UAE) and Biochemistry department,

3 Early follicular anti-mullerian hormone as a predictor of ovarian response during ICSI cycles 283 Faculty of Pharmacy for Girls, Al-Azhar University (Cairo, Egypt) Hormonal assessment On day-3 of the menstrual cycle, each woman underwent blood sampling by venipuncture for measurement of serum levels of AMH, FSH, LH, E2 and inhibin B at approximately 10:00 am. Serum was separated from all blood samples and frozen in aliquots at 20 C until used for subsequent centralized analysis. Serum AMH levels were determined using an ultrasensitive enzyme-linked immunosorbent assay (ELISA) as described by (31). Serum levels of FSH, LH, E2 and inhibin B were determined using an automated multi-analysis system with chemiluminescence detection Ultrasound assessment Later in the morning, ovarian ultrasound scanning was performed using a MHz multi-frequency transvaginal probe (MEDISON SONOACE 8000 live, KOREA) by one operator (A.M.). The objective of the ultrasound examination was to evaluate the number and sizes of early antral follicles. The sum of all follicles measuring 2 10 mm in mean diameter in both ovaries was considered Stimulation protocol and ICSI procedures The women participating in this study followed a long GnRH agonist protocol that began with daily S.C. injections of 0.1 mg triptoreline (Ipsen pharma biotech, France) on day 21 of the pre-stimulation cycle. The GnRH agonist was continued until the day of HCG administration. E2 levels less than 50 pg/ml on cycle day-3 and an endometrial thickness less than 4 mm indicated down-regulation. Gonadotropin was administered daily by S.C. injection of follitropin beta (Puregon; Organon, Netherlands) in a dose of 250 IU/day started from the third day of the cycle for 5 days after which the level was adjusted according to the associated ovarian response in order to stimulate follicular development. The resultant ovarian response was monitored by transvaginal ultrasound and serum E2 levels. In cases of less than two growing follicles on cycle day 14, treatment cycle was canceled. When three or more follicles reached a maximum diameter of 18 mm, 10,000 IU of hcg (Pregnyl; Organon, Holland) was administered. Transvaginal oocyte retrieval was performed under general anesthesia by one operator (A.M.; who was blinded to the results of the hormone assays) h after hcg injection. The study group was divided into two subgroups according to the number of oocytes retrieved. Patients with an oocyte count of four or more were considered good responders and patients with less than four were considered poor responders. A maximum of three embryos were transferred under ultrasonic guide on day-3 after oocyte retrieval. Before transfer, the embryos were evaluated microscopically to ensure 6 8 cells in each embryo of grades 1 2. On the 12th 14th days of the transfer, a serum b-hcg test was performed to confirm pregnancy Data registration and statistical analysis The results were tabulated and statistically analyzed using a computer program SPSS (statistic a package for social science), version 15. The sample mean (X), standard deviation (SD), and standard error of the mean as well as the range were obtained for numerical variables. For non-numerical variables, the frequency, distribution and percentage were calculated. The student s (t) test was used to test the significance of the difference between the two independent means. The Chi square test (v 2 ) was used to test whether the distribution of a certain phenomenon among two or more groups was equal or not. Correlation coefficient (r) was used to find out a correlation between two parameters where (r) value will be either +1 (positive correlation), 0 (no correlation) or 1 (negative correlation). To evaluate significance of a test, we determined its sensitivity, specificity, +ve predictive value (PPV) and ve predictive value (NPV). The probability (P) value was calculated and a P-value <0.05 was considered statistically significant. 3. Results Two hundred and twenty seven patients undergoing ICSI and eligible for our criteria were started the treatment in this study. Four patients with very poor response (less than 2 growing follicles on cycle day 14) were excluded. Two hundred and twenty three patients were grouped on the day of ovum pick up. Patients with an oocyte count of four or more were considered good responders (group A = 174) and patients with less than four as poor responders (group B = 49). Three patients were again excluded (1 patient in group A and 2 patients in group B) because no embryos were available for transfer. Finally 220 patients were enrolled in this study. The flowchart of the patients included in the study is shown in Fig. 1. Good Responders N= 174 No ET N=1 Good Responders group N=173 Figure 1 Started the treatment N=227 OPU =223 Enrolled in the study N=220 < 2 growing follicles N=4 Poor Responders N=49 No ET N=2 Poor Responders Group N = 47 Flowchart of the patients included in this study.

4 284 A. Moawad et al. The patient features (Table 1) were comparable in both groups and has no significant difference regarding the mean age, duration of infertility, parity, and BMI. As shown in Table 2, AMH and AFC were significantly higher in good responders group (4.79 ± lg/l, and 8.71 ± 3.04, respectively) compared to poor responders group (1.67 ± 9.4 lg/l, and 4.34 ± 3.7, respectively) P -value <0.001 and <0.01, respectively. FSH was significantly lower in the good responders group (5.92 ± 2.17 miu/ml) compared to the poor responders group (8.36 ± 6.2 miu/ml) P-value <0.05. On the other hand, LH, E 2 and inhibin B levels did not show any statistically significant differences between the good responders groups (5.63 ± 4.61 miu/ml, ± 7.02 pg/ml and ± pg/ml, respectively) and poor responders group (6.87 ± 3.02 miu/ml, ± 8.64 pg/ml and ± pg/ml) P-value >0.05. As a methodology-based classification, the good responders group had a significantly higher mean regarding the number of follicles P18 mm on the day of hcg and retrieved oocyte count (10.71 ± 3.21 and 9.32 ± 3.23, respectively) compared to the poor responders group (5.76 ± 2.76 and 3.44 ± 0.73, respectively), P-value <0.01. However, there were no significant differences between the studied groups regarding percentage of MII oocyte, fertilization rate and number of transferred embryos (73.63 ± 14.47%, ± 14.54% and 3.36 ± 0.19, respectively) in good responders group vs. (78.96 ± 19.57%, ± 13.58% and 2.95 ± 0.26, respectively) poor responders group). Although, the pregnancy rate was higher in good responders group (31.21%) compared to the poor responders group (27.66%), no statistically significant difference was observed (Table 3). When we evaluated the relationship between retrieved oocyte counts and the parameters used to predict the ovarian response (Table 4), it was found that basal AMH levels, FSH levels and the number of antral follicles were statistically correlated. The number of oocytes retrieved was not correlated with basal LH, E2, and inhibin levels. Day-3 AMH levels correlated with age (r = 0.431; P < 0.01), antral follicle count (r = 0.412; P < 0.01) and retrieved oocyte count (r = 0.493; P < 0.001) but was not correlated with either chemical pregnancy or clinical pregnancy (r = 0.123; P > 0.05). On the other hand, day-3 FSH level was correlated only with day-3 E2 levels (r = 0.293; P < 0.05). To identify the best parameter and threshold values that would yield the optimal mix of false-positive and false-negative fractions for the prediction of the number of oocytes to be collected, we used logistic regression and ROC curves analyzes for day-3 AMH, FSH and the number of the antral follicles (Fig. 2). Among all the parameters for ROC curves, day-3 AMH level had the highest sensitivity and specificity values, and the curve referring to AMH was placed on the top (area under the curve [AUC]: 0.66), followed by antral follicles count (AUC: 0.58) and FSH (AUC: 0.47). Depending on the ROC curve for each significant parameter used, the cut-off value was selected (1.7 lg/l for AMH, 4 for AFC and 8 MIU/mL for FSH). Table 1 Subject characteristics in the studied groups. Good responders (No. 173) Poor responders (No. 47) P-Value Age (years) ± ± Infertility duration (years) ± ± Parity (No) ± ± BMI (kg/m 2 ) ± ± Table 2 Day-3 basal hormonal and ultrasonic data in the studied groups. Good responders (No. 173) Poor responders (No. 47) P-Value AMH (lg/l) 4.79 ± ± 9.4 <0.001 FSH (miu/ml) 5.92 ± ± 6.2 <0.05 LH (miu/ml) 5.63 ± ± E 2 (pg/ml) ± ± Inhibin B (pg/ml) ± ± Antral follicle count 8.71 ± ± 3.7 <0.01 Table 3 Ovarian stimulation outcomes in the studied groups. Good responders (No. 173) Poor responders (No. 47) P-Value Follicles No. P18 mm on day of hcg ± ± 2.76 <0.01 Retrieved oocytes count 9.32 ± ± 0.73 <0.01 MII oocyte (%) ± ± Fertilization rate ± ± Embryos transferred 3.36 ± ± Pregnancy rate 54/173 (31.21%) 13/47 (27.66%) >0.05

5 Early follicular anti-mullerian hormone as a predictor of ovarian response during ICSI cycles 285 Table 4 Correlation coefficients between the number of oocytes collected and the parameters investigated. Coefficient of correlation (r) Significance (P-value) Day-3 AMH <0.001 Day-3 FSH <0.05 Day-3 LH Day-3 E Day-3 inhibin B Antral follicle count <0.01 Figure 2 ROC curve for the parameters investigated (upper line for AMH, middle line for AFC and lower line for FSH). As shown in Table 5, day-3 AMH was more sensitive and specific than either day-3 AFC or day-3 FSH in prediction of poor ovarian response (92.9% and 95.5% vs. 84.2% and 91.8% in AFC and 82.9% and 90.2% in FSH, respectively). Also, day-3 AMH has higher predictivity for ovarian response (PPV and NPV) than either day-3 AFC or day-3 FSH (82.95% and 98.3% vs % and 96.5% in AFC and 61.7% and 96.5% in FSH, respectively). On the other hand, diagnostic accuracy of day-3 AMH in prediction of ovarian response was better than that of day-3 AFC or day-3 FSH (92.4% vs. 85.2% and 82.8%, respectively). 4. Discussion The present investigation was designed to evaluate the association between the serum AMH levels in early follicular phase and the number of retrieved oocytes, inspite of clinically similar serum LH, E2 and inhibin B concentrations on day-3 of the menstrual cycle. On the other hand, it was designed to weigh the relationship between AMH levels and ovarian response against that between other putative markers of ovarian function and fertility potential, including antral follicle count (which has been shown to be the best predictor of poor response after IVF) (6) and serum levels of FSH on cycle day-3. In the current study, the good responders group had significantly higher basal AMH (P < 0.001) and AFC (P < 0.01) and significantly lower FSH (P < 0.05) compared to the poor responders group which gave us an opportunity to underline the importance of AMH and its relation with the number of retrieved oocytes. On the other hand, LH, E 2 and inhibin B levels did not show any statistically significant differences between both the groups (P > 0.05). In agreement with the current study, Muttukrishna et al. (32) found that the basal AMH level was significantly lower in the canceled group and the baseline FSH were significantly higher in the canceled group compared to the completed cycle group. Another study showed that the levels of baseline FSH and E2, but not LH, were significantly lower in cycles resulting in a normal ovarian response as well as cycles resulting in clinical pregnancy (33). Van Rooij et al. (23) demonstrated that the baseline levels of FSH, but not E2, were higher in poor responders and that the AMH levels were lower in the poor responders compared to normal responders. It was also stated that a stricter FSH value (8 IU/mL) would be required for the group of patients with poor prognosis (1,2,33). Ficicioglu et al. (34) showed that the baseline levels of AMH were lower in poor responders compared to normal responders and both groups had similar mean baseline FSH levels (lower than 8 IU/mL). In the current study, basal AMH levels, FSH levels and the number of antral follicles were statistically correlated with the number of retrieved oocytes. However, AMH levels highly correlated with the number of retrieved oocytes (P < 0.001) than did AFC (P < 0.01) or FSH (P < 0.05) on day-3 of the cycle. Therefore, serum AMH is tightly related to ovarian response and retrieved oocytes count with a relationship that was remarkably more intense than those obtained with AFC and serum levels of FSH. On the other hand, the number of oocytes retrieved was not correlated with basal LH, E2, and inhibin levels (Table 4). This study also showed an association between AMH level and age (P < 0.01), antral follicle count (P < 0.01) and retrieved oocytes count (P < 0.001). Day-3 FSH level was correlated only with day-3 E2 levels (P < 0.05). Therefore, serum AMH levels may reflect the size of the antral follicle pool and hence, may provide a marker associated with the anticipated number of oocytes to be retrieved after controlled ovarian stimulation for IVF/ICSI. Table 5 Performance of anti-mullerian hormone and other significant parameters used in predicting ovarian response. Sensitivity Specificity PPV NPV Diagnostic accuracy (%) AMH threshold (1.7 lg/l) 39/42 (92.9%) 170/178 (95.5%) 39/47 (82.9%) 170/173 (98.3%) 92.4 AFC threshold (4) 32/38 (84.2%) 167/182 (91.8%) 32/47 (68.1%) 167/173 (96.5%) 85.2 FSH threshold (8 miu/ml) 29/35 (82.9%) 167/185 (90.2%) 29/47 (61.7%) 167/173 (96.5%) 82.8

6 286 A. Moawad et al. The age-related decrease in AMH levels (22) probably results from the relative follicular attrition that characterizes the decline of ovarian function, with a noticeable reduction in the number of early antral follicles (35). The negative relationship between day-3 serum AMH levels and the women s age seen in the present study supports this hypothesis. The reported correlation between serum AMH levels on cycle day-3 and the number of oocytes retrieved after COS (27) is in keeping with the relationship between AMH and the number of early antral follicles shown in the present study. Indeed, the number of early antral follicles is a putative predictor of COS outcome (36,37). The correlation between serum AMH levels and early antral follicle count confirms preliminary data reported by others (22). However, the latter mentioned authors observed higher coefficients of correlation for this relationship (0.71) than the present study (0.41). In agreement with this study, Seifer et al. (27) demonstrated that a higher day-3 serum AMH concentrations were associated with greater number of retrieved oocytes. Thus, the basal antral follicle count and basal AMH levels are good tools to use in counseling patients (18,23,27,34,38). Fanchin et al. (18) indicate not only that serum AMH levels are strongly correlated with ovarian follicular status during the early follicular phase, but also that this relationship is more strict than that obtained with other hormonal markers (inhibin B, E2, LH or FSH) of the follicular functioning. They concluded that serum AMH measurements on cycle day-3 are a better predictor of the number of early antral follicles than conventional hormone measurements. Also, Ficicioglu et al. (34) have found that there is an association between AMH levels, antral follicles count, and the number of retrieved oocytes. Recently, Razieh et al. (39) demonstrated that serum AMH levels were highly correlated with the number of antral follicles, HCG day follicles count, and the number of retrieved oocytes than did E2, FSH, or LH on day-3 of the cycle. In good responders, with increasing AMH levels, the antral follicle, the growing follicle, and oocyte retrieval counts would also increase. Against the findings of the current study, Bancsi et al. (40) found that antral follicle count is the best single indicator for ovarian response. The combination of antral follicle count with basal FSH and inhibin B levels improved prediction. Ficicioglu et al. (4) found serum inhibin B to be the determinant with the highest sensitivity among age, body mass index, basal FSH, E2, and antral follicle count. Similar to previous reports on ovarian reserve, we found that basal antral follicle count is correlated (but weakly than did AMH) with the number of retrieved oocytes during assisted reproduction cycles. Although antral follicle count was not as accurate as AMH in predicting success or failure, it correlated with current measures of ovarian response. However, accurate antral follicle counts depend on the clinician s experience and the technical properties of the ultrasound used. By contrast, AMH levels are obtained by objective measurements performed in laboratory medium and thus are free of interobserver variability and personal comments. According to RCO curve, the cut-off value for AMH was set at 1.7 lg/l and we found that AMH has a high sensitivity (92.9%) and specificity (95.5%) with a positive predictive value of 98.3%. In agreement with the current study, Ficicioglu et al. (34) used 0.25 pg/ml as a cut-off level for AMH and they found that AMH is the best indicator of ovarian reserve with a high sensitivity and specificity. Not only that, AMH had a higher predictability for the number of retrieved oocytes (PPV of 96%). Also, we used four antral follicles as a threshold basal count (Table 5) and we did observe that patients with fewer than four retrieved oocytes had lower day-3 AMH levels and fewer antral follicles. However, Frattarelli et al. (41) in their 289-patient series, attempted to find a threshold basal antral follicle count but found no absolute value. Although, the pregnancy rate was higher in the good responders group compared to poor responders group, no statistically significant difference was observed (Table 3). Also, day-3 AMH levels were not correlated with either chemical pregnancy or clinical pregnancy (Table 4). In agreement with this study, Deffieux and Antoine (42) demonstrated that day-3 AMH levels can predict the number of oocytes retrieved, but the AMH level cannot predict the likelihood of pregnancy. Also, Ficicioglu et al. (34) have found that the levels of AMH predict the number of oocytes with a positive predictive value of 96%, although it has little value for predicting pregnancy. However, Razieh et al. (39) demonstrated that a higher serum AMH level has a relation with chemical pregnancy outcome but it does not have any correlation with clinical pregnancy and miscarriage rates. Another study showed that AFC has a significant association with the number of oocytes retrieved and is predictive of clinical pregnancy (43). In conclusion, it appears that AMH serum levels are associated with ovarian response in ART cycles better than the usual hormone markers and can be used to serve as a novel marker for ovarian reserve. On the other hand, AMH level cannot predict the likelihood of pregnancy. However, further studies are needed to determine whether AMH can accurately predict the ART outcomes or not. References (1) Kwee J, Elting MW, Schats R, Bezemer PD, Lambalk CB, Schoemarker J. 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