Hormonal Assays. MATERIALS AND METHODS Experimental Design. Treatment Protocol. Data Acquisition. Data Analysis. Statistical Analysis

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1 The cohort of antral follicles measuring 2 6 mm reflects the quantitative status of ovarian reserve as assessed by serum levels of anti-m ullerian hormone and response to controlled ovarian stimulation Kannamannadiar Jayaprakasan, Ph.D., Shilpa Deb, Milhan Batcha, M.D., James Hopkisson, M.D., Ian Johnson, D.M., Bruce Campbell, Ph.D., and Nick Raine-Fenning, Ph.D. Nottingham University Research and Treatment Unit in Reproduction (NURTURE), Division of Human Development, School of Clinical Sciences, University of Nottingham, Nottingham, United Kingdom Objective: To evaluate the relationship between serum anti-m ullerian hormone (AMH) and antral follicle size, and to ascertain which cohort of antral follicles is most predictive of the response to controlled ovarian stimulation during assisted reproduction treatment (ART). Design: Prospective study. Setting: University-based Assisted Conception Unit. Patient(s): One hundred thirteen women undergoing first cycle of ART. Intervention(s): Transvaginal 3D-ultrasound assessment and venipuncture in the early-follicular phase of the menstrual cycle. Main Outcome Measure(s): Serum AMH levels, number of mature oocytes retrieved and poor ovarian response. Result(s): The antral follicle cohorts measuring 2 to 3 mm, >3 to 4 mm, >4 to 5 mm, and >5 to 6 mm were most significantly correlated with AMH (r ¼.30,.27,.30, and.41, respectively) and the number of mature oocytes retrieved (r ¼.28,.23,.29, and.34, respectively). Although these follicle cohorts of 2-6 mm were significant predictors of the number of mature oocytes retrieved on regression analysis, their discriminative ability (area under the curve [AUC]: 0.829) for the prediction of poor ovarian response was similar to total counts made using cohorts of 2 to 4 mm, 2 to 5 mm, 2 to 8 mm, and 2 to 10 mm (AUCs: 0.794, 0.812, 0.852, and 0.826, respectively). Conclusion(s): The number of antral follicles measuring 2 to 6 mm is most reflective of the quantitative ovarian reserve. However, the ability of this group of antral follicles to predict poor ovarian response appears similar to that of the follicular cohorts of 2 to 4 mm, 2 to 5 mm, 2 to 8 mm, and 2 to 10 mm. (Fertil Steril Ò 2010;94: Ó2010 by American Society for Reproductive Medicine.) Key Words: In vitro fertilization, three-dimensional (3D) ultrasound, ovarian reserve, automated antral follicle count, anti-m ullerian hormone, sono-avc The outcome of assisted reproduction treatment (ART) can be predicted by estimating a woman s ovarian reserve, and a number of noninvasive tests incorporating clinical, endocrinologic, and ultrasonographic measures have been devised. Among these, the total antral follicle count (AFC) and serum levels of anti-m ullerian hormone (AMH) are the most predictive, direct tests of ovarian reserve screening (1 4). Although these two markers appear equally predictive of ovarian response during ART (4, 5), AFC is commonly the ovarian reserve test of choice because of the inherent availability Received June 26, 2009; revised October 8, 2009; accepted October 13, 2009; published online November 19, K.J. has nothing to disclose. S.D. has nothing to disclose. M.B. has nothing to disclose. J.H. has nothing to disclose. I.J. has nothing to disclose. B.C. has nothing to disclose. N.R.-F. has nothing to disclose. This article was presented orally at the 25th annual meeting of the European Society of Human Reproduction and Embryology (ESHRE) held at Amsterdam, the Netherlands (June 28 to July 1, 2009). Reprint requests: Kannamannadiar Jayaprakasan, Ph.D., MRCOG, NURTURE, B Floor, East Block, Queen s Medical Centre, Nottingham, Nottinghamshire, UK NG7 2UH (FAX: þ ; k.jayaprakasan@nottingham.ac.uk). of ultrasound machines within clinics and the relative ease with which follicle counts can be performed. The lack of an international assay standard for AMH measurements has limited its widespread use (6), although the recent development of commercial ultrasensitive sandwich ELISA assays and standard reference preparations seem to have addressed this issue (7). There is no consensus, however, on which follicles should be included in the total AFC (2), and the current evidence base includes studies using follicles measuring 2 to 5 mm (8, 9), 2 to 6 mm (10, 11), 2 to 8 mm (12), or2 to 10 mm (13, 14) in diameter. Examination of antral follicles grouped into cohorts by their size, particularly the smaller ones, may improve the predictive value of AFCs. Manual measurement of the number and diameter of each and every antral follicle, especially the smaller ones, is labor intensive and not straightforward. There is a definite variation in AFCs alone both within and between observers that is likely to be much more profound when the size of the follicle is also included in the assay. Three-dimensional (3D) ultrasound allows the user to acquire an ovarian volume, which can be analyzed off-line at a later time in a virtual real-time manner using one of several viewing options, all of which have been shown to improve the reliability of AFCs and, hypothetically, their validity in terms of predicting the number /$36.00 Fertility and Sterility â Vol. 94, No. 5, October doi: /j.fertnstert Copyright ª2010 American Society for Reproductive Medicine, Published by Elsevier Inc.

2 of oocytes likely to be retrieved during ART (15 17). Recently introduced software, Sono-Automatic Volume Calculation (sonoavc: General Electric Medical Systems, Zipf, Austria), allows semiautomated assessment of both the number and size of any fluid-filled hypoechoic structure within an acquired 3D volume (18, 19). Recent work has shown sono-avc with postprocessing provides a high degree of intra- and interobserver reliability for AFCs, and is superior to both conventional two-dimensional (2D) and manual 3D ultrasound techniques (20). The primary objective of this study was to evaluate the relationship between serum levels of AMH and antral follicle size, and to ascertain which cohort of antral follicles is most predictive of the response to controlled ovarian stimulation as determined by the number of mature oocytes retrieved during ART. The secondary objective was to examine the relationship between the cohorts of antral follicles stratified by size and other endocrinologic ovarian reserve markers. MATERIALS AND METHODS Experimental Design In this prospective study we aimed to recruit subjects, under the age of 41 years with regular menstrual cycles of between 21 to 35 days duration and an early follicular phase FSH level below 12 IU/L, and who were undergoing their first cycle of ART. Subjects were excluded if they had a history of ovarian surgery or were found to have an ovarian cyst or follicle measuring 20 mm or more in diameter. The study was performed at the University of Nottingham s Assisted Conception Unit (NURTURE: Nottingham University Research and Treatment Unit in Reproduction) between May 2007 and December The participants underwent venepuncture and a baseline, pretreatment 3D ultrasound assessment in the early follicular phase (days 2 to 4) of a spontaneous menstrual cycle before starting treatment. The study was approved by the Nottingham Research Ethics Committee and the hospital s Research and Development Department. Informed, written consent was obtained before the enrollment of each subject. Data Acquisition All participants had a transvaginal scan performed by a single investigator (K.J.) using a Voluson Expert 730 (GE Medical Systems, Zipf, Austria) and a four-dimensional (4D), 5- to 9-MHz transvaginal probe. Our technique for the acquisition of 3D volumetric data has been described in detail (21), but briefly, this included an initial 2D ultrasound assessment of the pelvis to exclude any obvious pathology before the application of a region of interest over the ovary, which defined the volume to be acquired. An automated mechanical sweep of this region through 90 was then undertaken using the slowest sweep mode (the high-quality setting), which provides a dataset with the highest resolution possible, and the resultant multiplanar display examined to ensure that the entire ovary had been captured. A volume dataset was acquired for each ovary, and these data were stored and then transferred to a personal computer via a digital video disc without any data compression. Data Analysis All measurements were made on a personal computer using 4D View (version 7.0; GE Medical Systems). The 3D gray-scale ovarian volume dataset was initially displayed in the multiplanar view and the number and size of each antral follicle calculated with sono-avc as previously described (22). Briefly, the image quality of the datasets was adjusted and optimized, in terms of magnification, and the render mode entered to generate a volume of interest within the dataset. The volume of interest was adjusted to exclude as much extraovarian information as possible, and to ensure that the whole ovary was included in the analysis. The threshold settings, which assign transparency associated with fluid to opaque voxels, were maintained for all datasets. Once the volume of interest had been correctly positioned, sonoavc was implemented. The follicles automatically identified by the software were displayed, with a specific color given to each individual follicle, and shown together with their dimensions and relative sizes (Fig. 1). Preliminary work had shown that this initial automated assessment of the ovary missed many follicles, both within stimulated and unstimulated ovaries, which were readily evident subjectively to any observer and that postprocessing is required for almost all datasets (20). The missed follicles are easily included in the analysis by manually clicking on them and each additional antral follicle identified in this way is given a new color and displayed together with those follicles that were originally automatically identified (23). The follicles were subdivided into cohorts according to their mean diameter calculated automatically by sonoavc: <2 mm, 2 to 3 mm, >3 to 4 mm, >4 to 5 mm, >5 to 6 mm, >6to7 mm, >7 to 8 mm, >8 to 9 mm, and >9 to 10 mm. There was a good inter- and intraobserver reproducibility for the measurement of antral follicle cohort of varying sizes with mean intra-class correlation coefficient (95% confidence interval) ranging from ( ) to ( ). Hormonal Assays FSH and estradiol levels were measured using the Microparticle Enzyme Immunoassay method on an AxSYM autoanalyzer (Abbott Laboratories, Abbott Park, IL). The lowest detection limit and the intra- and interassay coefficients of variation for FSH were 0.37 IU/L, <5%, and <5%, respectively. The lowest detection limit and the intra- and interassay coefficients of variation for estradiol were 8 pmol/l, 2.9% to 11%, and 4.8% to 15.2% respectively. Measurement of serum AMH levels was performed using the MIS/AMH enzyme-linked immunosorbent assay kit (Diagnostic System Lab, Webster, TX). The lowest detection limit and the intra- and interassay coefficients of variation were ng/ml, <5%, and <8%, respectively. The lowest AMH level detected in the current study population was 0.19 ng/ml. Inhibin B was measured using the Inhibin B enzyme-linked immunosorbent assay kit (Diagnostic System Lab). The lowest detection limit and the intra- and interassay coefficients of variation were 7 pg/ml, <6%, and <8%, respectively. Treatment Protocol All participants underwent IVF/intracytoplasmic injection (ICSI) treatment using a standard long protocol based on the unit s standardized operating protocol (SOP). This involved inducing pituitary down-regulation through the administration of gonadotrophin-releasing hormone (GnRH) agonists (500 mg/day of Buserelin; Suprefact, Aventis Pharma, Kent, UK, or 800 mg/day of Nafarelin; Synarel, Pharmacia, Milton Keynes, UK) started in the midluteal phase of the menstrual cycle. Two weeks later, following confirmation of ovarian suppression through demonstration of an endometrial thickness of <5 mm and no ovarian activity on ultrasound in association with an estradiol level below 200 pmol/l, ovarian stimulation was commenced using recombinant FSH (Gonal-F; Merck-Serono, Feltham, UK). Participants received a daily dose of 225 IU or 300 IU according to the SOP, which is based on the patient s age, and this dose was kept constant throughout the duration of stimulation. The ovarian response was monitored daily by serial transvaginal ultrasound and serum estradiol measurements. Choriogonadotropin alfa (6500 IU of Ovitrelle; Merck-Serono, Feltham, UK) was administered when there were at least three follicles measuring 18 mm or more in diameter and transvaginal, ultrasound-guided oocyte retrieval performed 36 hours later. Participants that did not develop at least three follicles measuring 18 mm or more in diameter after 14 days of rfsh treatment were advised to discontinue treatment (cycle cancellation) or convert to intrauterine insemination treatment dependent on other clinical factors including their tubal status and their partner s semen quality (24). Meiotic maturity of the retrieved oocytes was assessed after denudation for subjects having ICSI and 18 to 20 hours postinsemination for subjects having IVF. Statistical Analysis The main outcome measures were early follicular phase serum levels of AMH and the number of mature oocytes retrieved. The other outcome measures included serum levels of FSH, estradiol, and inhibin B. The Statistical Package for the Social Sciences (version 15.0; SPSS, Chicago, IL) was used for statistical analysis. The distribution of the data 1776 Jayaprakasan et al. Antral follicle size and ovarian reserve Vol. 94, No. 5, October 2010

3 FIGURE 1 Automated analysis of antral follicles using the Sono-Automatic Volume Calculation technique. was checked for normality using a normal probability plot. The relationship of each follicular cohort, stratified according to the mean diameter of the follicles, with the endocrine markers and the number of mature oocytes retrieved was evaluated using Spearman s correlation coefficient (r). Linear regression analysis was used to assess the value of each antral follicle cohort for the prediction of the number of mature oocytes retrieved at egg collection. Logistic regression analysis and receiver operating characteristic (ROC) curve analysis were performed to evaluate the ability of each antral follicle cohort to predict poor ovarian response, defined by cycle cancellation or the retrieval of less than four oocytes at the time of egg collection (25). AP-value of below.05 was considered statistically significant. RESULTS A total of 120 subjects were recruited for the study. Analysis was performed on 113 subjects after excluding 7 subjects who were found to have ovarian cysts or follicles measuring R20 mm on their baseline scan. The mean (SD; range) age, body mass index and basal FSH were 33.7 (3.6; 24 40) years, 24.2 (3.4; 20 35) kg/m 2 and 7.2 (1.9; ) IU/L, respectively. The mean (SD; range) duration of subfertility was 45.6 (32.6; 6 240) months. The mean (SD; range) number of antral follicles overall and within each follicular cohort and the serum levels of the various endocrine ovarian reserve markers are given in Table 1. There was no significant correlation between the total AFC, defined as the number of antral follicles visualized on ultrasound measuring up to 10 mm in diameter, and age (r ¼.13) or body mass index (r ¼.1). Although the total AFC was significantly correlated with serum AMH levels (r ¼.43; P<.001), there was no relationship between the total number of follicles and inhibin-b (r ¼.18), FSH (r ¼.10) or estradiol (r ¼.06). Significant correlations did exist, Fertility and Sterility â 1777

4 TABLE 1 Baseline ovarian reserve characteristics of the study population (n [ 113). Variables Mean ± SD Range Antral follicle cohorts (n) <2 mm to 3 mm >3 to 4 mm >4 to 5 mm >5 to 6 mm >6 to 7 mm >7 to 8 mm >8 to 9 mm >9 to 10 mm Total antral follicle counts (n) 2 to 6 mm to 8 mm to 10 mm FSH (IU/L) Estradiol (pmol/l) Inhibin B (pg/ml) AMH (ng/ml) AMH (pmol/l) a Note: AMH ¼ anti-m ullerian hormone. a Estimated values in pmol/l using the conversion factor 1 ng/ml ¼ 7.14 pmol/l. however, between the different antral follicle cohorts, stratified according to their size, and the clinical and endocrine markers of ovarian reserve (Table 2). The antral follicle cohorts measuring 2 to 3 mm, >3 to 4 mm, >4 to 5 mm, and >5 to 6 mm demonstrated the most significant degrees of correlation with AMH (r ¼.30,.27,.30, and.41, respectively; P<.01). Although the cohorts of follicles measuring >6 to 7 mm and >7 to 8 mm demonstrated less correlation with these markers (r ¼.20 and.24, respectively), but remained statistically significant (P<.05), there was such no relationship between AMH and follicles measuring <2 mm or >8 mm. Table 3 illustrates the relationship between the follicle cohorts and the markers of response to ovarian stimulation. Similar to the relationship with AMH, follicles measuring 2 to 3 mm, >3 to 4 mm, >4 to 5 mm, and >5 to 6 mm demonstrated the highest degree of correlation with the number of mature oocytes retrieved (r ¼.28,.23,.29, and.34, respectively; P<.05) (Table 3). Linear regression analysis confirmed that these follicular cohorts were significant predictors of the number of mature oocytes retrieved: R2 to 3 mm (P<.01), >3 to 4 mm (P<.05), >4 to 5 mm (P<.001), and >5 to 6 mm(p<.001). There was no significant relationship between number of mature oocytes and follicles measuring <2 mm or >6 mm and these follicular cohorts were not predictive of the number of mature oocytes retrieved on regression analysis. In view of the significant correlation between the follicle cohort measuring 2 to 6 mm and serum AMH and ovarian response, as defined by the number of mature oocytes retrieved, the predictive ability of this cohort of antral follicles was compared with that of total follicle counts made using cohorts of 2 to 4 mm, 2 to 5 mm, 2 to 8 mm, and 2 to 10 mm for the prediction of poor ovarian response (Table 4). Although of all these follicular cohorts proved to be significant predictors poor ovarian response on logistic regression analysis, their predictive ability was comparable as indicated by similar area under the curves (AUCs). DISCUSSION This is the first study to examine the relationship between antral follicle cohorts, stratified according to the size of each follicle, and AMH, the endocrine marker of ovarian reserve that correlates best with the response to gonadotrophins given to achieve controlled ovarian stimulation during IVF/ICSI treatment. The data in this study indicate that the number of antral follicles measuring 2 to 6 mm is most closely related to serum AMH levels, and is the best predictor of ovarian response as assessed by the number of mature oocytes retrieved at egg collection. This suggests that this follicular cohort is most reflective of the quantitative status of ovarian reserve. TABLE 2 Correlation between antral follicle cohorts of varying size with clinical and endocrine ovarian reserve markers (Spearman s correlation coefficient). Antral follicle size (mm) Age BMI AMH FSH Inhibin-B Estradiol < 2mm a 2 to 3 mm a 0.28 b 0.20 a >3 to4mm b >4 to5mm b a 0.09 >5 to6mm b >6 to7mm a 0.21 a >7 to8mm a 0.28 b >8 to 9 mm >9 to 10 mm a Note: AMH ¼ anti-m ullerian hormone; BMI ¼ body mass index. a P<.05. b P< Jayaprakasan et al. Antral follicle size and ovarian reserve Vol. 94, No. 5, October 2010

5 TABLE 3 Correlation between antral follicle cohorts of varying size and ovarian response to controlled ovarian stimulation (Spearman s correlation coefficient). Antral follicle size (mm) Peak Estradiol Total follicles aspirated Total eggs retrieved Mature eggs retrieved < 2 mm to 3 mm 0.31 b 0.26 b 0.27 b 0.24 b >3 to 4 mm a 0.20 a 0.23 a >4 to 5 mm 0.27 b 0.36 b 0.30 b 0.29 b >5 to 6 mm 0.29 b 0.39 b 0.36 b 0.34 b >6 to 7 mm a 0.20 a 0.18 >7 to 8 mm 0.22 a 0.21 a 0.22 a 0.18 >8 to 9 mm >9 to10mm a P<.05. b P<.01. Correlation is a fairly weak statistical test to derive any truly clinical relevant information from but was the only way to assess these data. The results given in Table 2 show several significant correlations but the relationship between the smaller follicles, namely, those measuring 2 to 3 mm, >3 to 4 mm, >4 to 5 mm, and >5 to 6 mm, and AMH in particular is more marked and readily evident than with the other measures of ovarian reserve. This significant relationship between the number of follicles within each of the smaller cohorts and serum AMH is supported by immunohistochemical work on human ovarian sections, which evaluated AMH expression in follicles of different size and at different degrees of development (26). Weenen et al. Showed AMH expression was strongest in preantral and small antral follicles measuring up to 4 to 6 mm and that expression then declines with increasing follicle size and disappears completely in follicles measuring >8 mm. The results are supported by quantitative data derived from analysis of follicular fluid AMH concentrations in small antral follicles of the ovaries of women undergoing surgical oophorectomy (27). Serum AMH levels reflect the size of the gonadotrophin-responsive follicle cohort, and this explains the performance of AMH as a quantitative predictor of ovarian reserve as assessed by ovarian response during IVF/ ICSI treatment (1, 3, 4, 28 30). TABLE 4 Univariate regression analysis of antral follicle cohorts of varying sizes for the prediction of poor ovarian response defined as cycle cancellation or retrieval of %3 oocytes. Antral follicle cohort size (mm) Odds ratio (mean and 95% CI) P value AUC 2 to ( ) < to ( ) < to ( ) to ( ) to ( ) Note: AUC ¼ area under the curve; CI ¼ confidence interval. Our data show that the smaller follicular cohorts, measuring 2 to 3 mm, >3 to 4 mm, >4 to 5 mm, and >5 to 6 mm, not only correlate with serum AMH levels but also with the number of mature oocytes retrieved following controlled ovarian stimulation. Conversely, the number of larger antral follicles measuring >6 to 10 mm demonstrates a weak correlation with AMH and ovarian response. This supports the hypothesis that the smaller antral follicle cohort represents the functional quantitative ovarian reserve as put forward by Haadsma et al. (31), who noted that age was associated with a steady decline in the number of follicles measuring 2 to 6 mm, whereas the number of follicles measuring 7 to 10 mm remained constant. When we compared normal and poor responders, as defined by their ovarian response to gonadotrophin stimulation, there was a significant difference in their total number of follicles measuring 2 to 6 mm ( vs ; P<.001) but not in the number of follicles measuring >6 to 10 mm ( vs ; P¼.11). It is likely that most of these larger antral follicles are already atretic, as has been shown in certain animal studies (32), and are not therefore truly reflective of functional ovarian reserve. These findings suggest that the total number of follicles measuring 2 to 6 mm is the most appropriate group to be considered in a total AFC rather than the other reported cohorts of 2 to 5 mm, 2 to 8 mm, and 2 to 10 mm, and that this may improve the ability of AFCs to predict poor ovarian response during IVF/ICSI treatment. The discriminative ability of the total AFC, however, appeared similar regardless of which cohort was included in the total AFC, as indicated by similar AUCs on ROC curve analysis (Table 4). This is in agreement with Khairy et al. (33), who concluded that the performance of AFCs for the prediction of poor ovarian response is not affected by differences in the follicle size cutoffs (2 5 mm, 2 8 mm, or 2 10 mm) used in its definition. This is possibly because of the fact that the total follicle count is largely defined by the number of smaller antral follicles measuring 2 to 6 mm anyway as these follicles accounted for 76% and 70% of the total number of antral follicles measuring 2 to 8 mm and 2 to 10 mm, respectively, in our study. In this study, neither inhibin-b nor any of the other conventional markers showed any significant correlation with AFC, unlike in other studies (34). Although inhibin-b and estradiol are produced by the granulosa cells of early antral follicles and therefore reflect the size of growing follicular cohort (35 37), the levels of inhibin- Fertility and Sterility â 1779

6 B and estradiol are regulated through pituitary FSH secretion (38) and the negative feedback loops within the hypothalamic pituitary ovarian axis, which means the levels of these markers are interrelated and dependent on each other and not simply the number and the size of growing follicles. Withholding IVF treatment based on the currently available tests of ovarian reserve, including AFCs, is not considered reasonable because of their inadequate ability to predict nonconception (3). Recent work by our group applying similar methodology to that described in this study has shown that the number of antral follicles measuring 2 to 4 mm may be an independent predictor of clinical pregnancy following IVF treatment (22). Although this work is limited through inclusion of women under 41 years of age, it does raise the possibility of redefining clinical cutoff levels for the total AFC for the prediction of cancellation or nonconception cycles. Future studies should be performed in larger populations and involve multiple centers to help derive these cutoff levels. In conclusion, this study has demonstrated that the number of antral follicles measuring 2 to 6 mm is most reflective of the quantitative status of ovarian reserve as indicated by the relationship of this follicular cohort with serum AMH levels and ovarian response to gonadotrophin stimulation during IVF/ICSI treatment. However, the ability of this group of antral follicles to predict poor ovarian response appears similar to that of the follicular cohorts currently used clinically. REFERENCES 1. 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: Hendriks DJ, Mol BW, Bancsi LF, Te Velde ER, Broekmans FJ. 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