COMPARING AMH, AFC AND FSH FOR PREDICTING HIGH OVARIAN RESPONSE IN WOMEN UNDERGOING ANTAGONIST PROTOCOL

COMPARING AMH, AFC AND FSH FOR PREDICTING HIGH OVARIAN RESPONSE IN WOMEN UNDERGOING ANTAGONIST PROTOCOL Nguyen Xuan Hoi1, Nguyen Manh Ha2 1 National Obstetrics and Gynecology Hospital, 2Hanoi Medical Unviversity The aim of this study was to assess the predictive values of AFC, AMH and FSH in predicting high ovarian response during in - vitro fertilization (IVF). We recruited 600 IVF patients who were receiving GnRH antagonist therapy and recombinant FSH for ovarian stimulation. High ovarian response during IVF was defined as > 15 oocytes retrieved. AMH, FSH and AFC levels were assessed on cycle day 2. We found that the AMH threshold value for high ovarian response was 4.04 ng/ml with a sensitivity of 73% and a specificity of 61%. The AFC threshold value for high ovarian response was 10.5, with a sensitivity of 78.7% and a specificity of 52%. The FSH threshold value for high ovarian response was 6.14 (IU/L) with a sensitivity of 53.2% and a specificity of. The area under the curve (AUC) of AMH, AFC and FSH were 71%, 65%, 62.7%, respectively. Conclusions: AMH was the best marker for predicting high ovarian response during IVF, followed by AFC and FSH. Keywords: AMH, FSH, AFC, high ovarian response, GnRH antagonist I. INTRODUCTION individualization of the ovarian stimulation A high ovarian response to ovarian stimula- treatment regimen and to counsel patients tion during in-vitro fertilization (IVF) has been about the risk of OHSS. Factors used to pre- associated with increased cancellation rates, dict ovarian stimulation include markers of compromised pregnancies, and live birth rates ovarian reserve such as follicle stimulating [1]. A high ovarian response also increases hormone (FSH) and antral follicle count (AFC). the risk for development of ovarian hyperstimulation syndrome (OHSS). OHSS is an excessive response to ovarian stimulation, characterized by increased vascular permeability and ovarian enlargement. Moderate and severe forms of OHSS may occur in 3% to 10% of all IVF cycles and the incidence may reach 25% among women undergoing IVF treatment [2]. Thus, early identification of potential high responders is necessary to enable Recently, anti - Müllerian hormone (AMH) has been used as a reliable indicator of ovarian reserve [3; 4]. Determining an AMH threshold is important in order to identify women who are at risk of high ovarian response and OHSS [5]. Some studies have shown that AMH is an accurate biomarker for predicting OHSS [6; 7]. Others have compared the predictive values of AMH, AFC and FSH for ovarian response. In controlled ovarian hyperstimulation, AFC has been found to be a Corresponding author: Nguyen Xuan Hoi, National Obstetrics and Gynecology Hospital E-mail: doctorhoi@gmail.com Received: 20 October 2016 Accepted: 10 December 2016 better predictor of ovarian response than AMH [8; 9]. However, the predictive values of AMH, FSH and AFC in IVF women undergoing the antagonist protocol are not fully understood. 57

This study was designed to assess the there were 2 follicles of 18 mm. Oocyte predictive values of AFC, AMH, and FSH in retrieval was conducted 36 hours after hcg predicting high ovarian response during IVF. administration.the criteria for ovarian response was based on the number of oocytes retrie- II. SUBJECTS AND METHODS ved [10]. High ovarian response was defined as more than 15 oocytes retrieved. 1. Subjects Female members of infertile couples Measurement of AFC, AMH, and FSH undergoing IVF antagonist treatment at the To determine AMH and FSH levels, eligible National Assisted Reproductive Technology subjects had 3 ml of blood drawn on day 2 of Center were eligible to participate in this study. their menstrual cycle and just prior to FSH The research was conducted at the National stimulation. Serum separation was done within Assisted Reproductive Technology Center in one hour after blood collection. Serum was Vietnam. All patients in this study met the stored at 20 C and then transferred to selection criteria and voluntarily agreed to testing laboratories within 24 hours after blood participate. sampling. Serum AMH levels were determined using the AMH Gen II assay (Beckman Inclusion criteria Coulter, Texas, USA; lowest detection limit Patients between the ages of 18-45 cur- 0.08 ng/ml) and the FSH level was deter- rently receiving ovarian stimulation with a mined using the electrochemiluminescence gonadotropin - releasing hormone (GnRH) method (Roche, Mannheim, Germany; assay antagonist protocol and recombinant FSH at sensitivity 0.100 miu/ml). To determine AFC the National Assisted Reproductive Technolo- levels, eligible subjects underwent transvagi- gy Center were included in the study. nal 2-dimensional ultrasounds (7.5MHz, Aloka, Japan) on day 2 of their cycle. Total AFC level Exclusion criteria was measured by including all follicles of 2 Patients who had undergone other stimula- 10 mm in both ovaries. tion regimens, such as the long protocol and the agonist protocol, or who had participated 3. Research ethics in egg donation, were ineligible to participate. Research subjects were informed about the goals of the study and voluntarily agreed 2. Methods to participate. All personal information was be This prospective study was conducted at the National Hospital of Obstetrics and Gynecology in Vietnam from October 2014 to June kept confidential. The study protocol was approved by National Hospital of Obstetrics and Gynecology. 2015. The study included 600 IVF patients receiving the GnRH antagonist protocol with III. RESULTS recombinant FSH. The starting dose of recombinant FSH was based on patient age, AMH level, and AFC level. Human chorionic gonadotrophin (hcg) was administered when 58 1. Patient characteristics and ovarian stimulation outcomes 600 patients were eligible to participate in

the study. Demographic and clinical data, in- was 12.09 ± 5.74. Te lowest number of folli- cluding basal AFC, AMH, and FSH, were as cles was 3 follicles, while the highest number follows: of follicles was 30 follicles. Finally, oocytes The average age of participants was 31.7 ± retrieved per trigger averaged to 13.21 ± 6.66, 5.2, with the group of 30-34 year olds with a range from 0-30 oocytes. accounting for 42% of the patients. The Results of ovarian response youngest patient was 18 years old and the oldest was 45 years old. 54.7% of patients had primary infertility, while 45.3% had secondary infertility. The average duration of infertility was 5.0 ± 3.2 years, with 52.2% having less than 5 years of Poor response accounted for 4.7% of participants (28 patients in total), normal response accounted for 62.3% of participants (374 patients in total), and high response accounted for 33% of participants (198 patients in total). infertility. 33 patients had an infertility duration of more than 10 years. 44.2% of patients had unexplained infertility. Characteristics of AFC, AMH, basal FSH, and E2 2. Comparing the predictive value of AMH, AFC, and FSH for predicting high ovarian response Our data showed that an AFC threshold of The lowest AFC value was 1, the highest 8 had a sensitivity of 78.7% and a specificity of AFC value was 30 and the average AFC value 52% for predicting high ovarian response. The was 13.0 ± 10.8. In terms of AMH level, the AFC value was highly correlated with the num- lowest AMH level was 0.2, while the highest ber of oocytes retrieved that reflex ovarian AMH level was 23.6 and the average AMH reserve, with a correlation coefficient of r = level was 4.57 ± 3.25. 0.34 (p < 0.001). AFC had a weak correlation The lowest basal FSH level was 0.09, the highest FSH level was 15.00 and the average FSH level was 5.97 ± 4.56. with high ovarian response (r = 0.167, p < 0.05), indicating their poor value as indicator for high ovarian response. Finally, the lowest E2 level was 1.54, the In terms of FSH, our study found that the highest E2 level was 174.00 and the average FSH threshold to predict high ovarian re- E2 level was 36.22 ± 19.00. sponse was 6.14 (IU/L), with 53.2% sensitivity Ovarian stimulation and cycle outcomes and specificity. The average number of total rfsh doses We could not determine the predictive was 1971,2 ± 753,4 IU, with the lowest dose at value of E2, since there were no statistical 400 IU and the highest dose at 6750 IU. differences between E2 concentration among Duration of ovarian stimulation was 9.84 ± the 3 groups. We also found no correlation 1.16 days. The shortest duration of ovarian between the concentration of E2 and the num- stimulation was eight days, while the longest ber of oocytes retrieved. was 15 days. The average number of follicles 14mm * The predictive value of AMH for high ovarian response 59

Table 1. The predictive value of AMH for high ovarian response High ovarian response (> 15 oocytes retrieved) Threshold value Sensitivity Specificity 3.125 85% 45% 3.49 81% 51% 3.54 80% 52% 3.62 78% 54% 3.87 73% 58% 3.95 73% 59% 4.04 73% 61% 4.12 72% 61% 4.21 71% 61% 4.25 69% 62% AMH (ng/ml) The AMH threshold to predict high ovarian response was 4.04 ng/ml, with 73% sensitivity and 61% specificity. The predictive value of AFC for high ovarian response Table 2. The predictive value of AFC for high ovarian response High ovarian response (> 15 oocytes retrieved) Threshold value Sensitivity Specificity 5.5 96.4% 14.1% 6.5 92.9% 18.6% 7.5 88.3% 25% 8.5 85.3% 34.1% 9.5 82.8% 42.4% 10.5 78.7% 52.0% 11.5 70.7% 56.3% 12.5 61.6% 61.6% 13.5 50.0% 68.6% 14.5 44.4% 72.9% AFC 60

An AFC threshold of 8 had a sensitivity of 78.7% and a specificity of 52% for predicting high ovarian response. The predictive value of FSH for high ovarian response Table 3. The predictive value of FSH with high ovarian response High ovarian response (> 15 oocytes retrieved) FSH (IU/L) Threshold value Sensitivity Specificity 6.07 56.0% 69.1% 6.09 55.8% 69.2% 6.10 53.5% 72.2% 6.12 53.2% 72.2% 6.14 53.2% 6.15 53.0% 6.17 52.7% 6.18 52.0% 72,7% 6.19 51.7% The FSH threshold to predict high ovarian response was 6.14 (IU/L), with 53.2% sensitivity and specificity. Comparing the predictive value of AMH, AFC and FSH for predicting high ovarian response Table 4. AMH, AFC and FSH thresholds to predict high ovarian response High ovarian response (> 15 oocytes retrieved) Threshold value Sensitivity Specificity AUC AMH (ng/ml) 4.04 73% 61% 71% AFC 10.5 78.7% 52.0% 65% FSH (IU/L) 6.14 53.2% 62.7% AMH had the best predictive value in determining which women would have high ovarian response, followed by AFC and finally FSH, as demonstrated by each measurement s sensitivity and specificity. 61

Figure 1. Receiver operating characteristics (ROC) curves for AMH, FSH and AFC in predicting high ovarian response Multivariate analysis for predictive factors of high ovarian response Table 5. Multivariate analysis for predictive factors of high ovarian response High ovarian response (n = 198 patients) Predictive factors P OR CI 95% AMH 4.04 (n = 307) < 4.04 (n = 293) < 0.001 2.69 1.80-4.00 AFC 10.5 (n = 345) < 10.5 (n = 255) < 0.001 2.67 1.76-4.07 FSH 6.14 (n = 332) > 6.14 (n = 268) < 0.001 2.11 1.42-3.14 The adjusted odds ratio (OR) of having a high ovarian response based on AMH 4.04 ng/ml was 2.69, as compared with AMH < 4.04 ng/ml (95% CI, p < 0.001). Conversely, the OR of having a high ovarian response based on AFC 10.5 was 2.67, as compared with AFC < 10.5 (95% CI, p < 0.001). Finally, the OR of having a high ovarian response based on FSH 6,14 IU/l was 2.11, as compared with FSH > 6.14 IU/l (95% CI, p < 0.001). IV. DISCUSSION Our results showed that AMH and AFC are larger than the area under the curve for AFC good predictors of high ovarian response in (AUC = 65%). AMH is more highly correlated women undergoing the GnRH antagonist to the number of oocytes retrieved at pick - up protocol. AMH appears to be a superior (r = 0.338) than AFC (r = 0.167). We found no predictor to AFC, since we found that the area correlation between FSH and E2 and the under the curve for AMH (AUC = 71%) was number of oocytes retrieved, indicating that 62

these factors are not predictors of high ovarian as a predictive marker for ovarian response. response. These results are in agreement with They concluded that AMH is the most reliable previous studies [11; 12]. marker Recent studies have suggested that the use of AMH as a marker of ovarian response has clinical advantages when assessing ovarian reserve. A meta-analysis with data from more than 20 studies concluded that AMH was a more accurate and robust biomarker of ovarian response in IVF than of ovarian reserve [16]. Moreover, AMH has a number of obvious clinical advantages, since AMH levels vary less across different menstrual cycles, within one menstrual cycle, during a pregnancy period, and when undergoing GnRH agonist treatment [13]. This variation is often seen with other ovarian biomarkers [13]. AMH can be assessed at any time point during the FSH, LH, E2 and inhibin B [13]. menstrual cycle, whereas AFC and other Our findings are in agreement with previous studies which found that the combination of AFC and AMH enhances prediction of ovarian response. However, there are limited data and conflicting results in the literature with regards to comparing AMH and AFC to predict the number of oocytes retrieved. Ficicioglu et al revealed that the level of AMH, as an indicator of ovarian reserve, is more sensitive and specific than AFC, with an AUC for AMH of 92% and for AFC of 78% [12]. On the contrary, Mutlu et al measured basal levels of AMH, FSH and AFC in 192 patients prior to IVF treatment and demonstrated that AFC is better than AMH at predicting poor ovarian response [14]. The AUC values from this study were 93%, 86% and 75% for AFC, AMH, FSH, respectively, indicating that in our study, AFC was better at predicting poor ovarian response. Similarly, Kwee et al found the AUC for AFC and AMH to be 93% and 85%, respectively, biomarkers have to be measured at the start of the menstrual cycle. AFC can be used as a prognostic indicator of ovarian response in patients with a history of ovarian surgery, or in patients with endometriosis in the ovaries. So far, AMH has been found to be a useful, convenient, and promising marker to assess ovarian reserve and to predict ovarian response. The real value of the above information lies in its ability to help predict a female patient's required dose of rfsh. In our study, the target for ovarian stimulation was set at 7 15 oocytes at retrieval. Seven or more oocytes are considered to give a reasonable chance ( 25%) of pregnancy, and the risk of developing moderate/severe ovarian hyperstimulation syndrome (OHSS) is low in patients with 15 oocytes. Severe OHSS was most frequent in patients with high ovarian reserve and who were given high rfsh doses. In contrast, in patients with low ovarian reserve and who demonstrating that AFC seemed to perform were treated with low or medium doses of slightly rfsh, fewer or no oocytes were retrieved, better than AMH for predicting hyperresponse [15]. cycles were cancelled, and the proportion of Recently, Fleming et al reviewed the cur- oocytes retrieved below the stimulation target rent evidence evaluating individualized ovarian was higher. In these patients, high doses of stimulation protocols using AMH concentration rfsh may be appropriate. 63

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