A prospective, comparative analysis of anti-Müllerian hormone, inhibin-B, and three-dimensional ultrasound determinants of ovarian reserve in the prediction of poor response to controlled

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A prospective, comparative analysis of anti-M€ullerianhormone, inhibin-B, and three-dimensional ultrasounddeterminants of ovarian reserve in the prediction ofpoor response to controlled ovarian stimulationKannamannadiar Jayaprakasan, M.R.C.O.G., Bruce Campbell, Ph.D., James Hopkisson, M.D.,Ian Johnson, D.M., 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, Nottinghamshire, United Kingdom

Objective: To compare three-dimensional ultrasound parameters, antral follicle count (AFC), ovarian volume, andovarian vascularity indices with anti-M€ullerian hormone (AMH) and other conventional endocrine markers for theprediction of poor response to controlled ovarian hyperstimulation (COH) during assisted reproduction treatment(ART).Design: Prospective study.Setting: University-based assisted conception unit.Patient(s): One hundred thirty-five women undergoing the first cycle of ART.Intervention(s): Transvaginal three-dimensional ultrasound assessment and venipuncture in the early follicularphase of the menstrual cycle immediately before ART.Main Outcome Measure(s): Poor ovarian response and nonconception.Result(s): Antral follicle count (Exp(B): 0.65) and AMH (Exp(B): 0.13) were the most significant predictors ofpoor ovarian response on multiple regression analysis and their predictive accuracy was similar, with an area underthe curve (AUC) of 0.935 and 0.905, respectively. The AFC and AMH, as a combined test, did not significantlyimprove the level of prediction (AUC ¼ 0.946). The sensitivity and specificity for prediction of poor ovarian re-sponse were 93% and 88% for AFC and 100% and 73% for AMH at an optimum cutoff values of %10 and %0.99ng/mL, respectively. Age (Exp(B): 1.191) was the only significant predictor of nonconception, although its predic-tive accuracy was also low (AUC ¼ 0.674).Conclusion(s): The AFC and AMH are the most significant predictors of poor response to ovarian stimulation dur-ing ART. The AMH and AFC, either alone or in combination, demonstrate a similar predictive power but are notpredictive of nonconception, which is dependent on the woman’s age. (Fertil Steril� 2010;93:855–64. �2010 byAmerican Society for Reproductive Medicine.)

Key Words: In vitro fertilization, three-dimensional (3D) ultrasound, ovarian reserve, antral follicle count, anti-M€ullerian hormone

The response to controlled ovarian hyperstimulation (COH)during assisted reproduction treatment (ART) is highly vari-able, even among women of similar ages (1). This undoubt-edly reflects the intersubject variation in ovarian reserve,which is primarily determined by the size of the primordialfollicular pool at birth and the rate of its decline during repro-ductive life, both of which are genetically determined (2, 3).Age alone, therefore, does not reflect the reproductive poten-tial, or ovarian reserve, of a woman (4).

eptember 17, 2008; revised October 16, 2008; accepted Octo-

008; published online November 30, 2008.

thing to disclose. B.C. has nothing to disclose. J.H. has nothing

se. I.J. has nothing to disclose. N.R.-F. has nothing to disclose.

assays undertaken in this study were supported by an investi-

l grant from Merck-Serono, Feltham, Middlesex, United King-

quests: Kannamannadiar Jayaprakasan, M.R.C.O.G., NUR-

Floor, East Block, Queen’s Medical Centre, Nottingham, Not-

shire, United Kingdom NG7 2UH (FAX: 44-115-82-30651;

[email protected]).

2/10/$36.0016/j.fertnstert.2008.10.042 Copyright ª2010 American S

Pretreatment assessment of ovarian reserve allows identifi-cation of women who are likely to have low, intermediate, orexaggerated response to COH and facilitates appropriate pre-treatment counseling and modification of an individual’streatment protocol in an attempt to maximize their potentialresponse (5). All of the tests that have been described to eval-uate ovarian reserve aim to quantify the ovarian follicular co-hort. Although the ultrasound assessment of the antral folliclecount (AFC) and ovarian volume provide direct measuresof ‘‘selectable’’ and gonadotropin-dependant follicles, endo-crine markers, such as anti-M€ullerian hormone (AMH), in-hibin-B, E2, and FSH indirectly reflect the early growingfollicle population (6). Although the majority of these testsoffer some prediction of poor ovarian response (7), AMHand AFC have repeatedly been found to offer the highest pre-dictive value (8–10). The AFC, measured using conventionaltwo-dimensional ultrasound, appears to perform equally aswell as AMH. A recent study by our group has shown thatthe three-dimensional (3D) assessment of AFC is more reli-able and more valid because it allows off-line analysis of

Fertility and Sterility� Vol. 93, No. 3, February 2010 855ociety for Reproductive Medicine, Published by Elsevier Inc.

ultrasound data and provides more spatial information thanreal-time, two-dimensional ultrasound (11). Three-dimen-sional ultrasound also allows an objective assessment of ovar-ian volume and total blood flow to the ovary (12, 13), both ofwhich have been suggested as important determinants ofovarian reserve (14–17). Further evaluation is needed to de-termine whether 3D measures of ovarian reserve offer anyclinical advantage over the clinical and endocrine markersof ovarian reserve. At present no studies have comparedAMH, inhibin-B, and other conventional endocrine markersof ovarian reserve to those derivable from 3D ultrasound data.

Studies have shown that a small, but significant, proportion(2%–30%) of women undergoing COH experience poor re-sponse (8) for which there is no universally accepted defini-tion. Poor response is generally considered to have occurredif the cycle is cancelled due to an inadequate response tostimulation or when three or less oocytes are retrieved ategg collection as four oocytes are generally required toachieve two embryos, given an average fertilization rate of50%–60% (18). Therefore, poor ovarian response, definedin this way, is associated with a significantly reduced concep-tion and live birth rates. In this study we aim to test the hy-pothesis that 3D ultrasonographic markers of ovarianreserve are better predictors of poor response to COH duringART than conventional clinical and endocrine measures. Wespecifically looked at the prediction of poor response in par-ticipants considered to be potential normal responders, ex-cluding women with polycystic ovaries (PCO) who are atrisk of exaggerated ovarian response and ovarian hyperstim-ulation syndrome (OHSS) (19).

MATERIALS AND METHODS

Experimental Design

The study was performed at the University of Nottingham’sAssisted Conception Unit (NURTURE: Nottingham Univer-sity Research and Treatment Unit in Reproduction) betweenSeptember 2006 and April 2008. We prospectively recruited150 consecutive participants aged less than 41 years with reg-ular menstrual cycles of 21–35 days duration. All of them hadan early follicular phase FSH level of less than 12 IU/L andwere undergoing their first cycle of ART. They underwent ve-nipuncture and a baseline pretreatment 3D ultrasound assess-ment in the early follicular phase (days 2–4) of thespontaneous menstrual cycle before starting treatment withGnRH agonists in the luteal phase of the same cycle. Partic-ipants were excluded if they had a history of ovarian surgeryor were found to have PCOs as defined by Rotterdam PCOSconsensus workshop group (20) or an ovarian cyst or folliclemeasuring 20 mm or more in diameter. The study was ap-proved by the National Health Service (NHS) research ethicscommittee and the Nottingham University NHS Trust Re-search and Development (R & D) department. Informed,written consent was obtained before the enrollment of eachsubject. The study was conducted in accordance with the eth-ical principles that have their origin in the Declaration of Hel-sinki, 1996, the principles of Good Clinical Practice, and the

856 Jayaprakasan et al. AMH and 3D US markers of ovarian

Department of Health Research Governance Framework forHealth and Social Care, 2005.

Data Acquisition

All participants had a transvaginal scan performed by a singleinvestigator (K.J.) using a Voluson Expert 730 (GE MedicalSystems, Kretz, Austria) and a four-dimensional 5- to 9-MHz transvaginal probe. Participants were scanned withtheir legs supported by stirrups in a modified Lloyd Daviesposition to limit discomfort and ensure free manipulationof the transvaginal transducer. Our technique for the acquisi-tion of 3D volumetric and power Doppler data has been de-scribed in detail (13), but briefly this included an initialtwo-dimensional ultrasound assessment of the pelvis to ex-clude any obvious pathology before the application of a re-gion of interest over the ovary that defined the volume tobe acquired. An automated mechanical sweep of this regionthrough 90� was then undertaken using the slow sweepmode and the resultant multiplanar display examined to en-sure that the entire ovary had been captured. Power Dopplerwas then applied using predefined settings, which offer thebest compromise between small ovarian vessel detectionand artifact (21). The volume mode was entered once an ad-equate power Doppler signal had been obtained and the resul-tant truncated sector defining the area of interest moved andadjusted to ensure that a complete ovarian volume was ob-tained. Two volume acquisitions for each ovary, one withgray scale and the other with power Doppler information,were obtained. The data were subsequently transferred toa personal computer by a universal serial bus device withoutany data compression.

Data Measurement

All measurements were made on a personal computer using4D View (version 7.0; GE Medical Systems, Kretz, Austria)by a single investigator (K.J.). The 3D gray scale ovarian vol-ume dataset was initially displayed in the multiplanar viewand the total number of antral follicles measuring 2–10 mmin diameter were counted as previously described (22).Briefly, this involved measurement of largest follicle in twoplanes to ensure this was 10 mm or smaller and counted allthereafter. All follicles measuring more than 10 mm were ex-cluded. Virtual Organ Computer-aided AnaLysis (VOCAL;GE Medical Systems) was used to measure ovarian volumethrough the delineation of the ovarian cortex in the B (trans-verse image) plane as the volume was rotated 180� through 9�

rotation steps (12). Quantification of power Doppler informa-tion within the resultant 3D ovarian model was performed us-ing ‘‘histogram facility’’ and three indices of vascularity weregenerated: the vascular index represents the ratio of powerDoppler information within the total dataset relative to bothcolor and gray information, the flow index reflects themean power Doppler signal intensity, and the vascularizationflow index, which is a combination of the other two indices(23, 24).

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The 3D technique has two components, volume acquisitionand off-line data assessment, and the intraobserver and inter-observer reproducibility of these components has been estab-lished for the measurement of AFC, ovarian volume, andovarian vascularity (12, 13, 25, 26). In this study, two mea-surements of each variable were made for each dataset andthe mean value used for analysis. The mean intraclass corre-lation coefficient and 95% confidence interval (CI) were0.983 (0.968–0.992) and 0.989 (0.960–0.997) for measure-ment of the number of antral follicles and ovarian volume, re-spectively, within the observer, which are indicative of a highdegree of measurement reliability. Measurement of the vascu-lar indices also showed a high degree of intraobserver reliabil-ity returning mean intraclass correlation coefficients (95% CI)of 0.982 (0.974–0.0.991), 0.985 (0.977–0.993), and 0.983(0.976–0.990) for vascular index, flow index, and vasculariza-tion flow index measurements, respectively.

Inhibin-B and Hormonal Assays

Blood samples were collected from each subject into twoplain tubes. All samples were immediately centrifuged toseparate the serum. One tube was used for the FSH, LH,and E2 assays, which were performed within 2–3 hours aftervenipuncture or within 24 hours in which case the serum wasstored at 2�C until assayed. The other serum sample was fro-zen at -20�C and stored until sufficient samples were avail-able for the inhibin-B and anti-M€ullerian hormone (AMH)assays, which were performed in batches. All of the assayswere performed in duplicate and the mean value, whichwas used for statistical analysis, is presented.

The FSH, LH, and E2 levels were measured using the mi-croparticle enzyme immunoassay method on an AxSYMauto-analyzer (AxSYM; Abbott Laboratories, Abbott Park,IL). The lowest detection limit and the intra-assay and inter-assay coefficients of variation for FSH were 0.37 IU/L,<5%,and<5%, respectively. The lowest detection limit and the in-tra-assay and interassay coefficients of variation for LH were0.5 IU/L, <7%, and <8%, respectively. The lowest detectionlimit and the intra-assay and interassay coefficients of varia-tion for E2 were 8 pmol/L, 2.9%–11%, and 4.8%–15.2%,respectively.

Measurement of serum AMH levels was performed usingthe MIS/AMH enzyme-linked immunosorbent assay kit(Diagnostic System Lab, Webster, TX). The lowest detectionlimit and the intra-assay and interassay coefficients of varia-tion were 0.006 ng/mL, <5%, and <8%, respectively. In-hibin-B was measured using the inhibin-B ELISA kit(Diagnostic System Lab). The lowest detection limit andthe intra-assay and interassay coefficients of variation were7 pg/mL, <6%, and <8%, respectively.

Treatment Protocol

All participants underwent IVF treatment using a standardlong protocol. This involved down-regulation with GnRHagonists (500 mg/day of buserelin [Suprefact; Aventis

Fertility and Sterility�

Pharma, Kent, United Kingdom] or 800 mg/day of nafarelin[Synarel; Pharmacia, Milton Keynes, United Kingdom])started in the midluteal phase of the menstrual cycle 7 daysbefore the expected date of menstruation. Two weeks later,after confirmation of ovarian suppression through demonstra-tion of an endometrial thickness of less than 5 mm and noovarian activity on ultrasound scan in association with anE2 level less than 200 pmol/L, ovarian stimulation was com-menced using recombinant FSH (Gonal-F; Serono Pharma-ceuticals Ltd., Feltham, United Kingdom). Participantsreceived a daily dose of 225 IU or 300 IU according to localprotocol and this dose was kept constant throughout the dura-tion of stimulation. The ovarian response was monitoreddaily by serial transvaginal ultrasound and serum E2 mea-surements. Choriogonadotropin alfa (6,500 IU of Ovitrelle;Serono Pharmaceuticals) was administered when therewere at least three follicles measuring 18 mm or more in di-ameter and transvaginal, ultrasound-guided oocyte retrievalperformed 36 hours later. Participants who did not developat least three follicles measuring 18 mm or more in diameterafter 14 days of recombinant FSH treatment were advised todiscontinue treatment (cycle cancellation) or convert to IUItreatment, dependent on other clinical factors including thetubal status and the seminal fluid quality according to theguidelines proposed by the National Institute of ClinicalExcellence (27). However, treatment was continued, andoocyte recovery performed, if the couple preferred this optionprovided there were at least two follicles measuring 18 mm ormore. One or two embryos were replaced according to thewishes of the couple and the number of embryos available.Luteal support using progestogen pessaries (Cyclogest; ShirePharmaceuticals Ltd., Basingstoke, Hants, United Kingdom)was provided from the day of embryo transfer and the level ofserum hCG was measured 16 days later to determine the out-come. If the test was positive, as defined by a hCG level morethan 50 IU/L, a transvaginal ultrasound was arranged 2 weekslater to confirm the viability of the pregnancy. A repeat ultra-sound scan at 12 weeks of gestation ensured that the preg-nancy remained clinically viable.

Statistical Analysis

The primary outcome measure was poor ovarian response,which was defined as the retrieval of three or less oocytesor cycle cancellation (18). Poor responders were then com-pared with the rest of the study group who were considered‘‘normal responders.’’

Statistical Package for the Social Sciences (version 14.0;SPSS, Chicago, IL) was used for statistical analysis. The dis-tribution of the data was checked for normality using a normalprobability plot. The unpaired t-test or Mann-Whitney U test,which were applied to normally distributed and skewed data,respectively, were used to examine for significant differencesin each variable between poor responders and normalresponders. A P value of less than .05 was considered statisti-cally significant. Linear regression analysis was used to eval-uate the value of age, body mass index (BMI), smoking, FSH,

857

LH, E2, inhibin-B, AMH, and each ultrasound parameter forthe prediction of the number of oocytes retrieved. Logistic re-gression analysis was then applied to evaluate the effect of thesame variables on the prediction of poor response and noncon-ception. Receiver operating characteristic (ROC) curve anal-ysis was performed to quantify the ability of any significantpredictors to discriminate between poor responders and nor-mal responders and between pregnant and nonpregnant par-ticipants. Areas under the ROC curves (AUCROC) werecompared using the MEDCALC software package (version9.2.0; MedCalc Software, Mariakerke, Belgium) (28).

Participants Included for Analysis

Of the 150 participants who met the inclusion criteria, 5 ofthese participants were excluded as they had PCO accordingto the Rotterdam criteria. Eight additional participants wereexcluded as they had ovarian follicles or cysts measuringmore than 20 mm in diameter and data were incomplete intwo participants. Therefore, the final study group comprised135 participants.

RESULTS

The mean age and mean FSH level of this group was 33.5 �3.5 years (24–40 years) and 7.1 � 1.8 IU/L (2.95–11.96 IU/L), respectively. Their mean BMI was 24.3 � 3.4 kg/m2

(20–35 kg/m2). The participants had a variety of causative fac-tors for their subfertility including tubal disease (32 partici-pants; 23.7%), endometriosis (9 participants; 6.7%), malefactor infertility (49 participants; 36.3%), combined factors(3 participants; 2.2%), and unexplained subfertility (42 partic-ipants; 31.1%).

TABLE 1Comparison of baseline clinical, endocrine, and ultraresponder groups.

Parameters Normal responder

Age (y) 33.3 � 3.6 (24–Body mass index (kg/m2) 24.4 � 3.4 (20–Basal FSH level (IU/L) 7 � 1.8 (2.9Basal LH level (IU/L) 5.5 � 3.0 (1.3FSH:LH ratio 1.5 � 0.8 (0.2Basal E2 level (pmol/L) 160.7 � 56.0 (42Inhibin-B (pg/mL) 51.6 � 28.7 (7–Anti-M€ullerian hormone (ng/mL) 1.48 � 0.75 (0.Total antral follicle count 15.7 � 4.3 (5–2Mean ovarian volume (cm3) 6.6 � 1.9 (2.5Mean vascular index (%) 7.1 � 4.0 (1.3Mean flow index (0–100) 35.8 � 4.8 (23.Mean vascular flow index (0–100) 2.7 � 1.7 (0.3Mean echogenicity (0–100) 37.0 � 5.6 (21.

Note: Values presented as mean � SD (range).

Jayaprakasan. AMH and 3D US markers of ovarian reserve. Fertil Steril 2010.

858 Jayaprakasan et al. AMH and 3D US markers of ovarian

Treatment was cancelled before the administration of hCGin six participants. In five participants (3.7%) treatment wascancelled as there was an inadequate response to ovarian stim-ulation, but in the other woman (0.7%) it was cancelled as shewas considered at an increased risk of the development of se-vere OHSS due to an unexpected, exaggerated multifollicularresponse, associated with an E2 level of R35,000 pmol/L, onthe 12th day of stimulation. An additional four participants(3%) experienced failed fertilization and three (2.2%) hadfailed cleavage. One subject developed severe OHSS soon af-ter oocyte retrieval and was subsequently cancelled to reducethe risk of exacerbation of the disease through the administra-tion of luteal support or in the event of pregnancy. Overall, 121participants had embryo transfer, therefore with replacementof a single embryo in 12 participants and two embryos in theremaining 109 participants. Sixty-one pregnancies resulted,giving a conception rate of 45.2% per cycles initiated and50.4% per embryo transfer. Three of these were later con-firmed as biochemical pregnancies, as there was no evidenceof a pregnancy on ultrasound 2 weeks later at 6 weeks’ gesta-tion. One subject had an ectopic pregnancy. Four pregnanciesmiscarried during the next 6 weeks, resulting in an ongoingpregnancy rate (PR) of 39.3% per cycles initiated (53/135)and 43.8% per embryo transfer (53/121). Five ongoing preg-nancies were twins, giving an implantation rate of 25.2% (53/230) per embryo transfer.

Overall 15 participants (11.1%) demonstrated poor ovarianresponse; 10 participants had %3 oocytes retrieved and 5participants were cancelled due to inadequate follicularresponse. Clinically, although BMI was similar in bothgroups (Table 1), the poor responders were significantly olderthan the normal responders (35.7 � 1.9 vs. 33.3 � 3.6 years;

sound characteristics between normal and poor

s (n [ 120) Poor responders (n [ 15) P value

40) 35.7 � 1.9 (33–39) < .0535) 24.0 � 2.9 (20–30) .605–11.96) 8.3 � 1.5 (4.8–10.0) < .01–28.3) 5.3 � 1.9 (2.0–9.0) .71–5.5) 1.8 � 0.8 (0.9–4.2) .22–373) 182.2 � 77.6 (76–357) .31164) 58.7 � 62.6 (7–264.9) .4519–4.31) 0.58 � 0.28 (0.12–0.99) < .0012) 8.6 � 1.9 (5–12) < .001–9.9) 5.3 � 1.5 (3.5–8.2) < .01–25.5) 6.6 � 4.0 (0.6–13.0) .700–47.0) 35.2 � 5.3 (25.9–46.6) .68–10.9) 2.5 � 1.6 (0.2–4.9) .752–53.4) 39.8 � 4.8 (30.1–49.0) .22

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TABLE 2Univariate and multivariate analysis of baseline markers of ovarian reserve for the prediction of thenumber of oocytes retrieved at egg collection during assisted reproduction treatment.

ParametersRegression coefficient (mean

and 95% CI) P value R2

Age �0.148 (�0.341, 0.046) .133

0.470

Basal FSH �0.062 (�0.442, 0.325) .753Basal E2 �0.008 (�0.019, 0.003) .156Anti-M€ullerian hormone 2.353 (1.041, 3.665) < .001Antral follicle count 0.481 (0.230, 0.733) < .002Mean ovarian volume 0.028 (�0.375, 0.431) .892Mean vascular index 0.161 (�0.701, 1.023) .713Mean vascular flow index �0.209 (�2.209, 1.791) .156

Note: Significant predictors on univariate analysis are included in the multiple linear regression analysis model. Body massindex, basal LH, inhibin-B, ovarian flow index, and ovarian echogenicity were not predictive on univariate analysis.

CI ¼ confidence interval.

Jayaprakasan. AMH and 3D US markers of ovarian reserve. Fertil Steril 2010.

TABLE 3Multivariate regression analysis of basalmarkers of ovarian reserve for the predictionof poor response during assisted reproductiontreatment.

Parameters Odds ratio 95% CI P value

Age 1.172 0.841–1.633 .35Basal FSH 1.156 0.793–1.685 .45Anti-M€ullerian

hormone0.130 0.018–0.934 < .05

Antral folliclecount

0.650 0.445–0.948 < .05

Mean ovarianvolume

1.029 0.627–1.688 .91

Note: Significant predictors on univariate analysis areincluded in the multiple logistic regression analysismodel. Body mass index, basal LH, E2, inhibin-B,ovarian vascular indices (vascular index, flow index,and vascular flow index), and ovarian echogenicitywere not predictive on univariate analysis.

CI ¼ confidence interval.

Jayaprakasan. AMH and 3D US markers of ovarian reserve. Fertil Steril 2010.

P<.05). Poor responders had significantly lower AMH levels(P<.001) and significantly higher FSH levels (P<.01) thanthe normal responders. The poor responders also had a lowertotal AFC (P<.001) and smaller ovaries (P<.05). The otherendocrine (basal LH, E2, and inhibin-B) and ultrasoundmarkers (ovarian vascularity and perfusion) were similar inboth groups. The number of oocytes retrieved was signifi-cantly lower (P<.001) in the poor responders comparedwith the rest of the group (1.8 � 1.4 vs. 11.7 � 5.2), as wasthe number of embryos transferred (0.7 � 0.9 vs. 1.8 �0.5; P<.001) and the on-going PRs despite a comparablestarting dose (270.0 � 38.0 vs. 258.8 � 37.5 IU; P¼.27)and total dose of gonadotropin (2,870.0 � 579.9 vs.2.855.2 � 608.6 IU; P¼.88) and duration of ovarian stimula-tion (10.5 � 1.4 vs. 10.9 � 1.3 days; P¼.17) between thegroups.

Univariate and multivariate linear regression analysis of thevariables studied for the prediction of the number of oocytesretrieved are depicted in Table 2. Although several of the pa-rameters were predictive on univariate analysis, AFC andAMH were the only significant predictors on multivariateanalysis. The number of oocytes retrieved can be estimated us-ing the regression equation: Y ¼ -1.476 þ 0.594 � AFC þ2.378 � AMH (100 R2 ¼ 44.4%). Similar results were seenfor prediction of poor ovarian response with several of themeasures being predictive on univariate but not multivariatelogistic regression analysis, which again revealed AFC andAMH as the only significant predictors (Table 3). The ROCcurve analysis (Fig. 1) showed that AFC and AMH wereequally predictive of poor ovarian response, as demonstratedby a similar AUC (0.935 and 0.905, respectively; P¼.34).Evaluation of AFC and AMH as a combined test did not sig-nificantly improve the level of prediction (AUC ¼ 0.946).

The sensitivity, specificity, positive and negative predictivevalue, positive likelihood ratio, and post-test probability for

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the prediction of poor ovarian response at different cutofflevels for AFC and AMH are shown in Tables 4 and 5, respec-tively. Although the optimum cutoff levels, as indicated bythe highest sum of sensitivity and specificity, were %10and %0.99 ng/mL for AFC and AMH, respectively, thepost-test probability was highest at cutoff levels of %8 and%0.59 ng/mL. When both AFC and AMH are considered to-gether with their respective optimum cutoff levels of %10and %0.99 ng/mL, the sensitivity, specificity, positive

859

FIGURE 1

The receiver operating characteristic curve analysisof antral follicle count, anti-M€ullerian hormone(AMH), and other conventional markers of ovarianreserve for the prediction of poor response tocontrolled ovarian hyperstimulation during assistedreproduction treatment (ART).

Jayaprakasan. AMH and 3D US markers of ovarian reserve. Fertil Steril 2010.

likelihood ratio, and post-test probability could be marginallyimproved to 0.93%, 0.92%, 11.2%, and 58.3%, respectively.

Multiple logistic regression analysis of all of the clinical,hormonal, and ultrasound parameters demonstrated that age(Exp(B): 1.191, 95% CI 1.050–1.343; P<.01) was the only

TABLE 4Performance of antral follicle count at different cutoduring assisted reproduction treatment.

Antral folliclecount cutoff levels(total count)

Subjects thisapplied to (n) Sensitivity

%7 4 0.13%8 13 0.47%9 15 0.53%10a 29 0.93%11 40 1.00

Note: The shift from the pretest probability (11.1%) to the posfollicle count is shown.

PPV ¼ positive predictive value; NPV ¼ negative predictive vaa Optimum cutoff level.

Jayaprakasan. AMH and 3D US markers of ovarian reserve. Fertil Steril 2010.

860 Jayaprakasan et al. AMH and 3D US markers of ovarian

significant predictor of nonconception, defined as the absenceof an on-going pregnancy. However, the discriminative abil-ity of age for the prediction of nonconception was low, as in-dicated by an AUC of only 0.674 on ROC curve analysis. TheAFC and AMH, the most significant predictors of poor ovar-ian response, were not predictive of nonconception.

DISCUSSION

This is the first study to compare the predictive value of ovar-ian parameters measured using 3D ultrasound with that ofAMH, inhibin-B, and other conventional clinical and endo-crine markers in determining the response to ovarian stimula-tion as measured by the number of oocytes retrieved and therate of poor ovarian response and outcome in terms of clinicalPR after ART. The data in this study indicate that a pretreat-ment AFC, measured using 3D ultrasound, and AMH are themost significant predictors of the number of oocytes retrievedand of poor ovarian response. When compared with eachother, AFC and AMH appear equally predictive of poor re-sponse. However, none of the markers studied were predic-tive of nonconception, with the exception of the woman’sage, although its ability to predict an unsuccessful treatmentcycle appears to be low.

Our findings are in agreement with previous studies exam-ining the ability of AMH and AFC to predict both the numberof oocytes retrieved and the chance of poor ovarian response(9, 10, 29, 30). However, our work differs from theses studiesin that the follicle counts were made using 3D ultrasound,which has been shown to provide more reliable and validmeasurements (11). Still, AMH demonstrated an equivalentpredictive ability as that of AFC in our study, indicatingthat AMH has the potential to replace FSH as the widelyused test of ovarian reserve, but with a significantly betterpredictive accuracy. Despite the low number of poor re-sponders in this study, our findings of an equivalent anda high degree of predictive ability of both the AFC and

ff levels as a predictor of poor ovarian response

Specificity PPV NPV DLRPost-test

probability

0.98 0.45 0.90 9.00 52.9%0.95 0.54 0.93 9.33 53.8%0.93 0.49 0.94 8.00 50.0%0.88 0.49 0.99 7.47 48.3%0.79 0.37 1.00 4.80 37.5%

t-test probability of poor response according to the antral

lue; þLR ¼ positive likelihood ratio.

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TABLE 5Performance of anti-M€ullerian hormone at different cutoff levels as a predictor of poor ovarianresponse during assisted reproduction treatment.

Anti-M€ullerian hormonecutoff levels (ng/mL)

Subjects thisapplied to (n) Sensitivity Specificity PPV NPV DLR

Post-testprobability

%0.59 17 0.53 0.93 0.49 0.94 7.11 47.0%%0.7 21 0.60 0.90 0.43 0.95 6.00 42.8%%0.8 28 0.73 0.86 0.39 0.96 5.18 39.3%%0.9 37 0.87 0.80 0.37 0.98 4.33 35.1%%0.99a 47 1.00 0.73 0.32 1.00 3.75 31.9%

Note: The shift from the pretest probability (11.1%) to the post-test probability of poor response according to the absolutelevel of anti-M€ullerian hormone is shown.

PPV ¼ positive predictive value; NPV ¼ negative predictive value; þLR ¼ positive likelihood ratio.a Optimum cutoff level.

Jayaprakasan. AMH and 3D US markers of ovarian reserve. Fertil Steril 2010.

AMH for poor response are still likely to be valid as they werealso the most significant predictors of the number of oocytesretrieved.

Ovarian aging is known to influence the ovarian responseto stimulation, as measured by the oocyte yield at retrieval,and is characterized by the progressive depletion of the pri-mordial follicular cohort (6). The AFC is positively corre-lated with the primordial follicular population (31) and isa significant predictor of poor ovarian response. This studysuggests that the AFC provides an optimum sensitivity andspecificity of 0.93 and 0.88, respectively, and a post-test prob-ability of 48.3% at a cutoff value of 10 or less. The specificityand post-test probability can be improved to 0.95 and 53.8%,respectively, by reducing the cutoff level to 8 or less, but thisis at the expense of a significant decrease in the sensitivity ofAFC to 0.54. The antral follicles visualized on conventionalultrasound measure 2 mm or more in diameter. These folli-cles, if functionally viable, are mostly gonadotropin respon-sive and selectable for further growth and developmentthrough to the preovulatory stage, therefore once the ‘‘FSHthreshold’’ is surpassed during COH. Therefore, the AFC isa direct marker of the recruitable follicular cohort. Con-versely, AMH, a member of transforming growth factor(TGF)-b family, is predominantly secreted by the granulosacells (GC) of preantral and small antral follicles of up to 4mm in diameter (32, 33), most of which are not evident on ul-trasound. Although AMH appears to be positively related toultrasonographically detectable antral follicles as demon-strated by a significant correlation (r ¼ 0.62; P<.001) be-tween AMH and AFC in this study, it is measuringa different group of follicles and may provide additional in-formation to that derivable from ultrasound. However, therewas no obvious value to using AFC and AMH as a combinedtest for the prediction of poor ovarian response, which wasonly marginally improved in this study. In addition, the abil-ity to discriminate poor responders from normal respondersas a combined test (AUC¼ 0.946), as assessed by ROC curve

Fertility and Sterility�

analysis, was not significantly different from that as individ-ual tests (AUC for AFC and AMH¼ 0.935 and 0.905, respec-tively). Considering the expense of these tests and the lack ofany significant improvement in predictive ability with a com-bined approach, it would seem unnecessary to perform bothinvestigations for the pretreatment prediction of ovarianresponse.

Although ovarian volume was predictive of both the num-ber of oocytes retrieved and poor response on univariate anal-ysis, as reported in other studies (17, 34), its predictive abilitywas less than that of AFC and AMH. The superior predictivepower of AFC over ovarian volume in this study is in agree-ment with the majority of studies (15, 35, 36) and a recentmeta-analysis (37). Although the AFC is a direct measureof the ‘‘selectable’’ follicle population, ovarian volume isan indirect indicator of the size of the follicle cohort and isnot only influenced by the number of follicles but also theirsize. It has been suggested that the larger antral follicle pop-ulation (7–10 mm) remains relatively constant despite a sig-nificant decrease in the number of smaller follicles (2–6 mm)as the age of the woman increases (38), possibly secondary toadvanced follicular growth and early selection of the domi-nant follicle due to elevated FSH levels associated with theloss of negative feedback that occurs as ovarian reserve de-clines (39). The size of the follicles may compensate forthe decrease in total AFC in women with advanced ovarianaging, and this may explain why ovarian volume is less pre-dictive of ovarian response than AFC in all the reported com-parative studies. Ovarian vascularity, the other 3D ultrasoundovarian parameter studied, was not predictive of poor ovarianresponse in both univariate and multivariate analysis and wasless predictive of the number of oocytes retrieved than AFC.This is in agreement with all of the reported comparativestudies that have used 3D ultrasound for blood flow assess-ment (15, 40–42). Any reduction in ovarian blood flow maybe a late phenomenon, however, as 3D indices of vascularityare similar in women aged %30, 31–35, and 36–40 years, but

861

significantly lower in those aged more than 40 years (43).This suggests that ovarian stromal blood flow is likely to bea late marker of poor ovarian response to COH.

In this study, neither inhibin-B nor any of the other con-ventional markers proved to be predictive of ovarian re-sponse. Inhibin-B and E2 are produced by the GCs of earlyantral follicles and therefore, reflect the size of the growingfollicular cohort (44–46). However, the levels of inhibin-Band E2 are regulated through pituitary FSH secretion (47)and the negative feedback loops within the hypothalamic–pi-tuitary–ovarian axis, which means that the levels of thesemarkers are inter-related and dependent on each other andnot simply the number and the size of the growing follicles.The lack of a significant predictive power of age and FSH inthis study may have been biased by the selection of partici-pants, who were expected to have normal ovarian responsebased on their age (<41 years) and FSH levels (<12 IU/L)(18, 48). However, poor response was demonstrated in a sig-nificant proportion (11.1%) of our study participants who arerepresentative of the majority of patients undergoing IVFworldwide (49). Although poor responders demonstratecompromised IVF outcome regardless of the woman’s age(4), the sensitivity and the negative predictive value of a nor-mal FSH level is relatively low, with many women consid-ered to have a normal ovarian reserve based on thesevalues alone, responding poorly to ovarian stimulation(50). Furthermore, women more than 40 years of age are ex-pected to have less success because of reduced oocyte qual-ity, as indicated by lower implantation rates even when thefollicular response is deemed adequate and an adequate num-ber of embryos are available for transfer (51). We used ageand FSH, therefore, to specifically select a group of partici-pants expected to respond normally to ovarian stimulation asthis allowed us to focus on the newer predictors of ovarianreserve.

The AMH as a test of ovarian reserve has many potentialadvantages over AFC and other conventional markers. Itcan be measured on any day of the menstrual cycle as itsserum levels do not vary significantly during the menstrualcycles (52–54) and a single measurement is predictive ofovarian response during ART (55). However, a wide rangeof serum AMH concentrations have been described anda reliable cutoff level has yet to be defined. There is nointernational assay standard for AMH measurement, whichmay explain the discordance between different studies andmakes comparison between laboratories difficult (56). Esti-mation of the total number of antral follicles is easy to per-form and can be undertaken during a routine ultrasoundexamination, which is an integral part of the pretreatmentassessment of women undergoing ART. It can be performedbefore treatment is started or after down-regulation hasbeen confirmed before ovarian stimulation with a similaraccuracy for the prediction of poor ovarian response (57).There is a legitimate argument, therefore, that the AFCshould be considered the first choice test for the assessmentof ovarian reserve until a uniform cutoff level of AMH has

862 Jayaprakasan et al. AMH and 3D US markers of ovarian

been agreed and a widely available, standardized assaytechnique is developed. The AFC assessment is operator de-pendent (52, 58), but this can be improved through the ac-quisition of 3D data (11) and the subsequent off-lineassessment of these data, which has been shown to improvemeasurement reliability even among inexperienced ob-servers (25). This does not validate the routine use of 3Dultrasound in clinical practice, and experienced observerscan use real-time two-dimensional ultrasound to reliablydetermine the total AFC (11, 59). The additional ultrasoundparameters derivable through 3D imaging, namely ovarianvolume and vascularity (13, 60), were not significant pre-dictors in the multivariate analysis.

Ovarian reserve tests have limited value in the predictionof nonconception and their routine use in clinical practicehas been questioned (7). Pregnancy may occur even at ex-treme cutoffs for an abnormal test result and therefore, IVFtreatment cannot be denied based on these tests, especiallyin participants who are seeking first cycle of treatment (6).The results of this study concur with this viewpoint as agewas the only significant predictor of nonconception andeven then, its ability to discriminate those participants whoconceived from those that did not, was poor (AUC ¼0.674) and of insufficient power to warrant withholding treat-ment from certain women. However, identification of partic-ipants who are likely to respond poorly during IVF treatmentis clinically relevant as the couple could be counseled accord-ingly and made aware that they have an increased chance ofcycle cancellation and a significantly lower chance of suc-cess, allowing them to make an informed decision. Such pre-diction may also allow clinicians to formulate individualizedtreatment protocols to improve or at least maximize ovarianresponse. The current level of evidence, based on a singlerandomized controlled trial in a small population (n ¼ 52),suggests no advantage to the use of higher starting doses ofgonadotropin in predicted poor responders (61), and suchan approach may increase the number of abnormal embryos(62). An accurate predictor will at least allow the develop-ment of further studies in an attempt to improve the outcomefor such couples who, regardless of the impressions and pre-dictions of the medical profession, often remain determinedto have at least one attempt at ART. We should counselthem accordingly, but not stop our attempts to maximize theirchances.

In conclusion, pretreatment AFC and AMH are the mostsignificant predictors of the number of oocytes retrievedand of poor ovarian response to ovarian stimulation duringART. Although AFC and AMH, either alone or in combina-tion, demonstrate a similar predictive power, 3D ultrasono-graphic ovarian parameters, such as ovarian volume,vascularity, and perfusion, do not appear to have any signif-icant value in the prediction of poor ovarian response.None of the markers studied, including AFC and AMH, aresignificant predictors of nonconception with the exceptionof the woman’s age, although the predictive ability is insuffi-ciently low to warrant modification of treatment.

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Acknowledgments: The authors thank George Bouliotis (Trent Research &

Development Support Unit, the University of Nottingham) for his assistance

with the statistical analysis and Catherine Pincott-Allen (School of Human

Development, University of Nottingham) for her assistance with the hor-

monal assays.

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