A prospective, comparative analysis of anti-M

ullerian
hormone, inhibin-B, and three-dimensional ultrasound
determinants of ovarian reserve in the prediction of
poor response to controlled ovarian stimulation
Kannamannadiar Jayaprakasan, M.R.C.O.G., Bruce Campbell, Ph.D., James Hopkisson, M.D.,
Ian Johnson, D.M., and Nick Raine-Fenning, Ph.D.
NottinghamUniversity 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, and
ovarian vascularity indices with anti-Mullerian hormone (AMH) and other conventional endocrine markers for the
prediction 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-ve women undergoing the rst cycle of ART.
Intervention(s): Transvaginal three-dimensional ultrasound assessment and venipuncture in the early follicular
phase 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 signicant predictors of
poor ovarian response on multiple regression analysis and their predictive accuracy was similar, with an area under
the curve (AUC) of 0.935 and 0.905, respectively. The AFC and AMH, as a combined test, did not signicantly
improve the level of prediction (AUC 0.946). The sensitivity and specicity 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 %0.99
ng/mL, respectively. Age (Exp(B): 1.191) was the only signicant predictor of nonconception, although its predic-
tive accuracy was also low (AUC 0.674).
Conclusion(s): The AFC and AMH are the most signicant 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 not
predictive of nonconception, which is dependent on the womans age. (Fertil Steril

2010;93:85564. 2010 by
American Society for Reproductive Medicine.)
Key Words: In vitro fertilization, three-dimensional (3D) ultrasound, ovarian reserve, antral follicle count, anti-
Mullerian 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 reects the intersubject variation in ovarian reserve,
which is primarily determined by the size of the primordial
follicular 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 reect the reproductive poten-
tial, or ovarian reserve, of a woman (4).
Pretreatment assessment of ovarian reserve allows identi-
cation of women who are likely to have low, intermediate, or
exaggerated response to COH and facilitates appropriate pre-
treatment counseling and modication of an individuals
treatment protocol in an attempt to maximize their potential
response (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 follicle
count (AFC) and ovarian volume provide direct measures
of selectable and gonadotropin-dependant follicles, endo-
crine markers, such as anti-Mullerian hormone (AMH), in-
hibin-B, E
2
, and FSH indirectly reect the early growing
follicle population (6). Although the majority of these tests
offer some prediction of poor ovarian response (7), AMH
and AFC have repeatedly been found to offer the highest pre-
dictive value (810). The AFC, measured using conventional
two-dimensional ultrasound, appears to perform equally as
well as AMH. A recent study by our group has shown that
the three-dimensional (3D) assessment of AFC is more reli-
able and more valid because it allows off-line analysis of
Received September 17, 2008; revised October 16, 2008; accepted Octo-
ber 17, 2008; published online November 30, 2008.
K.J. has nothing to disclose. B.C. has nothing to disclose. J.H. has nothing
to disclose. I.J. has nothing to disclose. N.R.-F. has nothing to disclose.
Endocrine assays undertaken in this study were supported by an investi-
gational grant from Merck-Serono, Feltham, Middlesex, United King-
dom.
Reprint requests: Kannamannadiar Jayaprakasan, M.R.C.O.G., NUR-
TURE, B Floor, East Block, Queens Medical Centre, Nottingham, Not-
tinghamshire, United Kingdom NG7 2UH (FAX: 44-115-82-30651;
E-mail: jayaprakasan@hotmail.co.uk).
0015-0282/10/$36.00 Fertility and Sterility

Vol. 93, No. 3, February 2010

855
doi:10.1016/j.fertnstert.2008.10.042 Copyright 2010 American Society for Reproductive Medicine, Published by Elsevier Inc.
ultrasound data and provides more spatial information than
real-time, two-dimensional ultrasound (11). Three-dimen-
sional ultrasound also allows an objective assessment of ovar-
ian volume and total blood ow to the ovary (12, 13), both of
which have been suggested as important determinants of
ovarian reserve (1417). Further evaluation is needed to de-
termine whether 3D measures of ovarian reserve offer any
clinical advantage over the clinical and endocrine markers
of ovarian reserve. At present no studies have compared
AMH, inhibin-B, and other conventional endocrine markers
of ovarian reserve to those derivable from3Dultrasound data.
Studies have shown that a small, but signicant, proportion
(2%30%) of women undergoing COH experience poor re-
sponse (8) for which there is no universally accepted deni-
tion. Poor response is generally considered to have occurred
if the cycle is cancelled due to an inadequate response to
stimulation or when three or less oocytes are retrieved at
egg collection as four oocytes are generally required to
achieve two embryos, given an average fertilization rate of
50%60% (18). Therefore, poor ovarian response, dened
in this way, is associated with a signicantly reduced concep-
tion and live birth rates. In this study we aim to test the hy-
pothesis that 3D ultrasonographic markers of ovarian
reserve are better predictors of poor response to COH during
ART than conventional clinical and endocrine measures. We
specically 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 at
risk of exaggerated ovarian response and ovarian hyperstim-
ulation syndrome (OHSS) (19).
MATERIALS AND METHODS
Experimental Design
The study was performed at the University of Nottinghams
Assisted Conception Unit (NURTURE: Nottingham Univer-
sity Research and Treatment Unit in Reproduction) between
September 2006 and April 2008. We prospectively recruited
150 consecutive participants aged less than 41 years with reg-
ular menstrual cycles of 2135 days duration. All of themhad
an early follicular phase FSH level of less than 12 IU/L and
were undergoing their rst cycle of ART. They underwent ve-
nipuncture and a baseline pretreatment 3D ultrasound assess-
ment in the early follicular phase (days 24) of the
spontaneous menstrual cycle before starting treatment with
GnRH agonists in the luteal phase of the same cycle. Partic-
ipants were excluded if they had a history of ovarian surgery
or were found to have PCOs as dened by Rotterdam PCOS
consensus workshop group (20) or an ovarian cyst or follicle
measuring 20 mm or more in diameter. The study was ap-
proved by the National Health Service (NHS) research ethics
committee and the Nottingham University NHS Trust Re-
search and Development (R & D) department. Informed,
written consent was obtained before the enrollment of each
subject. 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
Department of Health Research Governance Framework for
Health and Social Care, 2005.
Data Acquisition
All participants had a transvaginal scan performed by a single
investigator (K.J.) using a Voluson Expert 730 (GE Medical
Systems, Kretz, Austria) and a four-dimensional 5- to 9-
MHz transvaginal probe. Participants were scanned with
their legs supported by stirrups in a modied Lloyd Davies
position to limit discomfort and ensure free manipulation
of the transvaginal transducer. Our technique for the acquisi-
tion of 3D volumetric and power Doppler data has been de-
scribed in detail (13), but briey this included an initial
two-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 dened the volume to
be acquired. An automated mechanical sweep of this region
through 90

was then undertaken using the slow sweep

mode and the resultant multiplanar display examined to en-
sure that the entire ovary had been captured. Power Doppler
was then applied using predened settings, which offer the
best compromise between small ovarian vessel detection
and artifact (21). The volume mode was entered once an ad-
equate power Doppler signal had been obtained and the resul-
tant truncated sector dening the area of interest moved and
adjusted to ensure that a complete ovarian volume was ob-
tained. Two volume acquisitions for each ovary, one with
gray scale and the other with power Doppler information,
were obtained. The data were subsequently transferred to
a personal computer by a universal serial bus device without
any data compression.
Data Measurement
All measurements were made on a personal computer using
4D 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 view
and the total number of antral follicles measuring 210 mm
in diameter were counted as previously described (22).
Briey, this involved measurement of largest follicle in two
planes to ensure this was 10 mm or smaller and counted all
thereafter. All follicles measuring more than 10 mm were ex-
cluded. Virtual Organ Computer-aided AnaLysis (VOCAL;
GE Medical Systems) was used to measure ovarian volume
through the delineation of the ovarian cortex in the B (trans-
verse image) plane as the volume was rotated 180

through 9

rotation steps (12). Quantication of power Doppler informa-

tion within the resultant 3D ovarian model was performed us-
ing histogramfacility and three indices of vascularity were
generated: the vascular index represents the ratio of power
Doppler information within the total dataset relative to both
color and gray information, the ow index reects the
mean power Doppler signal intensity, and the vascularization
ow index, which is a combination of the other two indices
(23, 24).
856
Jayaprakasan et al. AMH and 3D US markers of ovarian reserve Vol. 93, No. 3, February 2010
The 3Dtechnique has two components, volume acquisition
and 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, and
ovarian vascularity (12, 13, 25, 26). In this study, two mea-
surements of each variable were made for each dataset and
the mean value used for analysis. The mean intraclass corre-
lation coefcient and 95% condence interval (CI) were
0.983 (0.9680.992) and 0.989 (0.9600.997) for measure-
ment of the number of antral follicles and ovarian volume, re-
spectively, within the observer, which are indicative of a high
degree of measurement reliability. Measurement of the vascu-
lar indices also showed a high degree of intraobserver reliabil-
ity returning meanintraclass correlation coefcients (95%CI)
of 0.982 (0.9740.0.991), 0.985 (0.9770.993), and 0.983
(0.9760.990) for vascular index, owindex, and vasculariza-
tion ow index measurements, respectively.
Inhibin-B and Hormonal Assays
Blood samples were collected from each subject into two
plain tubes. All samples were immediately centrifuged to
separate the serum. One tube was used for the FSH, LH,
and E
2
assays, which were performed within 23 hours after
venipuncture or within 24 hours in which case the serum was
stored at 2

C until assayed. The other serum sample was fro-

zen at -20

C and stored until sufcient samples were avail-

able for the inhibin-B and anti-Mullerian hormone (AMH)
assays, which were performed in batches. All of the assays
were performed in duplicate and the mean value, which
was used for statistical analysis, is presented.
The FSH, LH, and E
2
levels were measured using the mi-
croparticle enzyme immunoassay method on an AxSYM
auto-analyzer (AxSYM; Abbott Laboratories, Abbott Park,
IL). The lowest detection limit and the intra-assay and inter-
assay coefcients of variation for FSH were 0.37 IU/L, <5%,
and <5%, respectively. The lowest detection limit and the in-
tra-assay and interassay coefcients of variation for LH were
0.5 IU/L, <7%, and <8%, respectively. The lowest detection
limit and the intra-assay and interassay coefcients of varia-
tion for E
2
were 8 pmol/L, 2.9%11%, and 4.8%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-assay and interassay coefcients 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 and
the intra-assay and interassay coefcients of variation were
7 pg/mL, <6%, and <8%, respectively.
Treatment Protocol
All participants underwent IVF treatment using a standard
long protocol. This involved down-regulation with GnRH
agonists (500 mg/day of buserelin [Suprefact; Aventis
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 days
before the expected date of menstruation. Two weeks later,
after conrmation of ovarian suppression through demonstra-
tion of an endometrial thickness of less than 5 mm and no
ovarian activity on ultrasound scan in association with an
E
2
level less than 200 pmol/L, ovarian stimulation was com-
menced using recombinant FSH (Gonal-F; Serono Pharma-
ceuticals Ltd., Feltham, United Kingdom). Participants
received a daily dose of 225 IU or 300 IU according to local
protocol and this dose was kept constant throughout the dura-
tion of stimulation. The ovarian response was monitored
daily by serial transvaginal ultrasound and serum E
2
mea-
surements. Choriogonadotropin alfa (6,500 IU of Ovitrelle;
Serono Pharmaceuticals) was administered when there
were at least three follicles measuring 18 mm or more in di-
ameter and transvaginal, ultrasound-guided oocyte retrieval
performed 36 hours later. Participants who did not develop
at least three follicles measuring 18 mm or more in diameter
after 14 days of recombinant FSH treatment were advised to
discontinue treatment (cycle cancellation) or convert to IUI
treatment, dependent on other clinical factors including the
tubal status and the seminal uid quality according to the
guidelines proposed by the National Institute of Clinical
Excellence (27). However, treatment was continued, and
oocyte recovery performed, if the couple preferred this option
provided there were at least two follicles measuring 18 mmor
more. One or two embryos were replaced according to the
wishes of the couple and the number of embryos available.
Luteal support using progestogen pessaries (Cyclogest; Shire
Pharmaceuticals Ltd., Basingstoke, Hants, United Kingdom)
was provided fromthe day of embryo transfer and the level of
serum hCG was measured 16 days later to determine the out-
come. If the test was positive, as dened by a hCG level more
than 50 IU/L, a transvaginal ultrasound was arranged 2 weeks
later to conrm 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 dened as the retrieval of three or less oocytes
or 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 normal
probability 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 signicant differences
in each variable between poor responders and normal
responders. A P value of less than .05 was considered statisti-
cally signicant. Linear regression analysis was used to eval-
uate the value of age, body mass index (BMI), smoking, FSH,
Fertility and Sterility

857
LH, E
2
, inhibin-B, AMH, and each ultrasound parameter for
the prediction of the number of oocytes retrieved. Logistic re-
gression analysis was then applied to evaluate the effect of the
same 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 signicant
predictors to discriminate between poor responders and nor-
mal responders and between pregnant and nonpregnant par-
ticipants. Areas under the ROC curves (AUC
ROC
) were
compared using the MEDCALC software package (version
9.2.0; MedCalc Software, Mariakerke, Belgium) (28).
Participants Included for Analysis
Of the 150 participants who met the inclusion criteria, 5 of
these participants were excluded as they had PCO according
to the Rotterdam criteria. Eight additional participants were
excluded as they had ovarian follicles or cysts measuring
more than 20 mm in diameter and data were incomplete in
two participants. Therefore, the nal study group comprised
135 participants.
RESULTS
The mean age and mean FSH level of this group was 33.5
3.5 years (2440 years) and 7.1 1.8 IU/L (2.9511.96 IU/
L), respectively. Their mean BMI was 24.3 3.4 kg/m
2
(2035 kg/m
2
). 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%), male
factor infertility (49 participants; 36.3%), combined factors
(3 participants; 2.2%), and unexplained subfertility (42 partic-
ipants; 31.1%).
Treatment was cancelled before the administration of hCG
in six participants. In ve participants (3.7%) treatment was
cancelled as there was an inadequate response to ovarian stim-
ulation, but in the other woman (0.7%) it was cancelled as she
was considered at an increased risk of the development of se-
vere OHSS due to an unexpected, exaggerated multifollicular
response, associated with an E
2
level of R35,000 pmol/L, on
the 12th day of stimulation. An additional four participants
(3%) experienced failed fertilization and three (2.2%) had
failed cleavage. One subject developed severe OHSS soon af-
ter oocyte retrieval and was subsequently cancelled to reduce
the risk of exacerbation of the disease through the administra-
tion of luteal support or in the event of pregnancy. Overall, 121
participants had embryo transfer, therefore with replacement
of a single embryo in 12 participants and two embryos in the
remaining 109 participants. Sixty-one pregnancies resulted,
giving a conception rate of 45.2% per cycles initiated and
50.4% per embryo transfer. Three of these were later con-
rmed as biochemical pregnancies, as there was no evidence
of a pregnancy on ultrasound 2 weeks later at 6 weeks gesta-
tion. One subject had an ectopic pregnancy. Four pregnancies
miscarried during the next 6 weeks, resulting in an ongoing
pregnancy 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 ovarian
response; 10 participants had %3 oocytes retrieved and 5
participants were cancelled due to inadequate follicular
response. Clinically, although BMI was similar in both
groups (Table 1), the poor responders were signicantly older
than the normal responders (35.7 1.9 vs. 33.3 3.6 years;
TABLE 1
Comparison of baseline clinical, endocrine, and ultrasound characteristics between normal and poor
responder groups.
Parameters Normal responders (n [120) Poor responders (n [15) P value
Age (y) 33.3 3.6 (2440) 35.7 1.9 (3339) <.05
Body mass index (kg/m
2
) 24.4 3.4 (2035) 24.0 2.9 (2030) .60
Basal FSH level (IU/L) 7 1.8 (2.9511.96) 8.3 1.5 (4.810.0) <.01
Basal LH level (IU/L) 5.5 3.0 (1.328.3) 5.3 1.9 (2.09.0) .71
FSH:LH ratio 1.5 0.8 (0.25.5) 1.8 0.8 (0.94.2) .22
Basal E
2
level (pmol/L) 160.7 56.0 (42373) 182.2 77.6 (76357) .31
Inhibin-B (pg/mL) 51.6 28.7 (7164) 58.7 62.6 (7264.9) .45
Anti-M ullerian hormone (ng/mL) 1.48 0.75 (0.194.31) 0.58 0.28 (0.120.99) <.001
Total antral follicle count 15.7 4.3 (522) 8.6 1.9 (512) <.001
Mean ovarian volume (cm
3
) 6.6 1.9 (2.59.9) 5.3 1.5 (3.58.2) <.01
Mean vascular index (%) 7.1 4.0 (1.325.5) 6.6 4.0 (0.613.0) .70
Mean ow index (0100) 35.8 4.8 (23.047.0) 35.2 5.3 (25.946.6) .68
Mean vascular ow index (0100) 2.7 1.7 (0.310.9) 2.5 1.6 (0.24.9) .75
Mean echogenicity (0100) 37.0 5.6 (21.253.4) 39.8 4.8 (30.149.0) .22
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 reserve Vol. 93, No. 3, February 2010
P<.05). Poor responders had signicantly lower AMH levels
(P<.001) and signicantly higher FSH levels (P<.01) than
the normal responders. The poor responders also had a lower
total AFC (P<.001) and smaller ovaries (P<.05). The other
endocrine (basal LH, E
2
, and inhibin-B) and ultrasound
markers (ovarian vascularity and perfusion) were similar in
both groups. The number of oocytes retrieved was signi-
cantly lower (P<.001) in the poor responders compared
with the rest of the group (1.8 1.4 vs. 11.7 5.2), as was
the number of embryos transferred (0.7 0.9 vs. 1.8
0.5; P<.001) and the on-going PRs despite a comparable
starting dose (270.0 38.0 vs. 258.8 37.5 IU; P.27)
and total dose of gonadotropin (2,870.0 579.9 vs.
2.855.2 608.6 IU; P.88) and duration of ovarian stimula-
tion (10.5 1.4 vs. 10.9 1.3 days; P.17) between the
groups.
Univariate and multivariate linear regression analysis of the
variables studied for the prediction of the number of oocytes
retrieved are depicted in Table 2. Although several of the pa-
rameters were predictive on univariate analysis, AFC and
AMH were the only signicant predictors on multivariate
analysis. The number of oocytes retrieved can be estimatedus-
ing the regression equation: Y -1.476 0.594 AFC
2.378 AMH (100 R
2
44.4%). Similar results were seen
for prediction of poor ovarian response with several of the
measures being predictive on univariate but not multivariate
logistic regression analysis, which again revealed AFC and
AMH as the only signicant predictors (Table 3). The ROC
curve analysis (Fig. 1) showed that AFC and AMH were
equally predictive of poor ovarian response, as demonstrated
by a similar AUC (0.935 and 0.905, respectively; P.34).
Evaluation of AFC and AMH as a combined test did not sig-
nicantly improve the level of prediction (AUC 0.946).
The sensitivity, specicity, positive and negative predictive
value, positive likelihood ratio, and post-test probability for
the prediction of poor ovarian response at different cutoff
levels for AFCand AMHare shown in Tables 4 and 5, respec-
tively. Although the optimum cutoff levels, as indicated by
the highest sum of sensitivity and specicity, were %10
and %0.99 ng/mL for AFC and AMH, respectively, the
post-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 %10
and %0.99 ng/mL, the sensitivity, specicity, positive
TABLE 2
Univariate and multivariate analysis of baseline markers of ovarian reserve for the prediction of the
number of oocytes retrieved at egg collection during assisted reproduction treatment.
Parameters
Regression coefcient (mean
and 95% CI) P value R
2
Age 0.148 (0.341, 0.046) .133
0.470
Basal FSH 0.062 (0.442, 0.325) .753
Basal E
2
0.008 (0.019, 0.003) .156
Anti-M ullerian hormone 2.353 (1.041, 3.665) <.001
Antral follicle count 0.481 (0.230, 0.733) <.002
Mean ovarian volume 0.028 (0.375, 0.431) .892
Mean vascular index 0.161 (0.701, 1.023) .713
Mean vascular ow index 0.209 (2.209, 1.791) .156
Note: Signicant predictors on univariate analysis are included in the multiple linear regression analysis model. Body mass
index, basal LH, inhibin-B, ovarian ow index, and ovarian echogenicity were not predictive on univariate analysis.
CI condence interval.
Jayaprakasan. AMH and 3D US markers of ovarian reserve. Fertil Steril 2010.
TABLE 3
Multivariate regression analysis of basal
markers of ovarian reserve for the prediction
of poor response during assistedreproduction
treatment.
Parameters Odds ratio 95% CI P value
Age 1.172 0.8411.633 .35
Basal FSH 1.156 0.7931.685 .45
Anti-M ullerian
hormone
0.130 0.0180.934 <.05
Antral follicle
count
0.650 0.4450.948 <.05
Mean ovarian
volume
1.029 0.6271.688 .91
Note: Signicant predictors on univariate analysis are
included in the multiple logistic regression analysis
model. Body mass index, basal LH, E
2
, inhibin-B,
ovarian vascular indices (vascular index, ow index,
and vascular ow index), and ovarian echogenicity
were not predictive on univariate analysis.
CI condence interval.
Jayaprakasan. AMH and 3D US markers of ovarian reserve. Fertil Steril 2010.
Fertility and Sterility

859
likelihood ratio, and post-test probability could be marginally
improved 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 1.0501.343; P<.01) was the only
signicant predictor of nonconception, dened as the absence
of 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. The
AFC and AMH, the most signicant predictors of poor ovar-
ian response, were not predictive of nonconception.
DISCUSSION
This is the rst study to compare the predictive value of ovar-
ian parameters measured using 3D ultrasound with that of
AMH, 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 the
rate of poor ovarian response and outcome in terms of clinical
PR after ART. The data in this study indicate that a pretreat-
ment AFC, measured using 3D ultrasound, and AMH are the
most signicant predictors of the number of oocytes retrieved
and of poor ovarian response. When compared with each
other, 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 womans
age, although its ability to predict an unsuccessful treatment
cycle appears to be low.
Our ndings are in agreement with previous studies exam-
ining the ability of AMH and AFC to predict both the number
of oocytes retrieved and the chance of poor ovarian response
(9, 10, 29, 30). However, our work differs from theses studies
in that the follicle counts were made using 3D ultrasound,
which has been shown to provide more reliable and valid
measurements (11). Still, AMH demonstrated an equivalent
predictive ability as that of AFC in our study, indicating
that AMH has the potential to replace FSH as the widely
used test of ovarian reserve, but with a signicantly better
predictive accuracy. Despite the low number of poor re-
sponders in this study, our ndings of an equivalent and
a high degree of predictive ability of both the AFC and
FIGURE 1
The receiver operating characteristic curve analysis
of antral follicle count, anti-M ullerian hormone
(AMH), and other conventional markers of ovarian
reserve for the prediction of poor response to
controlled ovarian hyperstimulation during assisted
reproduction treatment (ART).
Jayaprakasan. AMH and 3D US markers of ovarian reserve. Fertil Steril 2010.
TABLE 4
Performance of antral follicle count at different cutoff levels as a predictor of poor ovarian response
during assisted reproduction treatment.
Antral follicle
count cutoff levels
(total count)
Subjects this
applied to (n) Sensitivity Specicity PPV NPV DLR
Post-test
probability
%7 4 0.13 0.98 0.45 0.90 9.00 52.9%
%8 13 0.47 0.95 0.54 0.93 9.33 53.8%
%9 15 0.53 0.93 0.49 0.94 8.00 50.0%
%10
a
29 0.93 0.88 0.49 0.99 7.47 48.3%
%11 40 1.00 0.79 0.37 1.00 4.80 37.5%
Note: The shift from the pretest probability (11.1%) to the post-test probability of poor response according to the antral
follicle count is shown.
PPV positive predictive value; NPV negative predictive value; LR positive likelihood ratio.
a
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 reserve Vol. 93, No. 3, February 2010
AMHfor poor response are still likely to be valid as they were
also the most signicant predictors of the number of oocytes
retrieved.
Ovarian aging is known to inuence the ovarian response
to 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 is
a signicant predictor of poor ovarian response. This study
suggests that the AFC provides an optimum sensitivity and
specicity 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 specicity
and post-test probability can be improved to 0.95 and 53.8%,
respectively, by reducing the cutoff level to 8 or less, but this
is at the expense of a signicant decrease in the sensitivity of
AFC to 0.54. The antral follicles visualized on conventional
ultrasound measure 2 mm or more in diameter. These folli-
cles, if functionally viable, are mostly gonadotropin respon-
sive and selectable for further growth and development
through to the preovulatory stage, therefore once the FSH
threshold is surpassed during COH. Therefore, the AFC is
a direct marker of the recruitable follicular cohort. Con-
versely, AMH, a member of transforming growth factor
(TGF)-b family, is predominantly secreted by the granulosa
cells (GC) of preantral and small antral follicles of up to 4
mm in diameter (32, 33), most of which are not evident on ul-
trasound. Although AMH appears to be positively related to
ultrasonographically detectable antral follicles as demon-
strated by a signicant correlation (r 0.62; P<.001) be-
tween AMH and AFC in this study, it is measuring
a different group of follicles and may provide additional in-
formation to that derivable from ultrasound. However, there
was no obvious value to using AFC and AMH as a combined
test for the prediction of poor ovarian response, which was
only marginally improved in this study. In addition, the abil-
ity to discriminate poor responders from normal responders
as a combined test (AUC 0.946), as assessed by ROC curve
analysis, was not signicantly different from that as individ-
ual tests (AUCfor AFCand AMH0.935 and 0.905, respec-
tively). Considering the expense of these tests and the lack of
any signicant improvement in predictive ability with a com-
bined approach, it would seem unnecessary to perform both
investigations for the pretreatment prediction of ovarian
response.
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 ability
was less than that of AFC and AMH. The superior predictive
power of AFC over ovarian volume in this study is in agree-
ment with the majority of studies (15, 35, 36) and a recent
meta-analysis (37). Although the AFC is a direct measure
of the selectable follicle population, ovarian volume is
an indirect indicator of the size of the follicle cohort and is
not only inuenced by the number of follicles but also their
size. It has been suggested that the larger antral follicle pop-
ulation (710 mm) remains relatively constant despite a sig-
nicant decrease in the number of smaller follicles (26 mm)
as the age of the woman increases (38), possibly secondary to
advanced follicular growth and early selection of the domi-
nant follicle due to elevated FSH levels associated with the
loss of negative feedback that occurs as ovarian reserve de-
clines (39). The size of the follicles may compensate for
the decrease in total AFC in women with advanced ovarian
aging, 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 ultrasound
ovarian parameter studied, was not predictive of poor ovarian
response in both univariate and multivariate analysis and was
less predictive of the number of oocytes retrieved than AFC.
This is in agreement with all of the reported comparative
studies that have used 3D ultrasound for blood ow assess-
ment (15, 4042). Any reduction in ovarian blood ow may
be a late phenomenon, however, as 3D indices of vascularity
are similar in women aged %30, 3135, and 3640 years, but
TABLE 5
Performance of anti-M ullerian hormone at different cutoff levels as a predictor of poor ovarian
response during assisted reproduction treatment.
Anti-M ullerian hormone
cutoff levels (ng/mL)
Subjects this
applied to (n) Sensitivity Specicity PPV NPV DLR
Post-test
probability
%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%
%0.99
a
47 1.00 0.73 0.32 1.00 3.75 31.9%
Note: The shift fromthe pretest probability (11.1%) to the post-test probability of poor response according to the absolute
level of anti-M ullerian hormone is shown.
PPV positive predictive value; NPV negative predictive value; LR positive likelihood ratio.
a
Optimum cutoff level.
Jayaprakasan. AMH and 3D US markers of ovarian reserve. Fertil Steril 2010.
Fertility and Sterility

861
signicantly lower in those aged more than 40 years (43).
This suggests that ovarian stromal blood ow is likely to be
a 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 E
2
are produced by the GCs of early
antral follicles and therefore, reect the size of the growing
follicular cohort (4446). However, the levels of inhibin-B
and E
2
are regulated through pituitary FSH secretion (47)
and the negative feedback loops within the hypothalamicpi-
tuitaryovarian axis, which means that the levels of these
markers are inter-related and dependent on each other and
not simply the number and the size of the growing follicles.
The lack of a signicant predictive power of age and FSH in
this study may have been biased by the selection of partici-
pants, who were expected to have normal ovarian response
based on their age (<41 years) and FSH levels (<12 IU/L)
(18, 48). However, poor response was demonstrated in a sig-
nicant proportion (11.1%) of our study participants who are
representative of the majority of patients undergoing IVF
worldwide (49). Although poor responders demonstrate
compromised IVF outcome regardless of the womans 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 these
values 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 the
follicular response is deemed adequate and an adequate num-
ber of embryos are available for transfer (51). We used age
and FSH, therefore, to specically select a group of partici-
pants expected to respond normally to ovarian stimulation as
this allowed us to focus on the newer predictors of ovarian
reserve.
The AMH as a test of ovarian reserve has many potential
advantages over AFC and other conventional markers. It
can be measured on any day of the menstrual cycle as its
serum levels do not vary signicantly during the menstrual
cycles (5254) and a single measurement is predictive of
ovarian response during ART (55). However, a wide range
of serum AMH concentrations have been described and
a reliable cutoff level has yet to be dened. There is no
international assay standard for AMH measurement, which
may explain the discordance between different studies and
makes comparison between laboratories difcult (56). Esti-
mation of the total number of antral follicles is easy to per-
form and can be undertaken during a routine ultrasound
examination, which is an integral part of the pretreatment
assessment of women undergoing ART. It can be performed
before treatment is started or after down-regulation has
been conrmed before ovarian stimulation with a similar
accuracy for the prediction of poor ovarian response (57).
There is a legitimate argument, therefore, that the AFC
should be considered the rst choice test for the assessment
of ovarian reserve until a uniform cutoff level of AMH has
been agreed and a widely available, standardized assay
technique 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-line
assessment of these data, which has been shown to improve
measurement reliability even among inexperienced ob-
servers (25). This does not validate the routine use of 3D
ultrasound in clinical practice, and experienced observers
can use real-time two-dimensional ultrasound to reliably
determine the total AFC (11, 59). The additional ultrasound
parameters derivable through 3D imaging, namely ovarian
volume and vascularity (13, 60), were not signicant pre-
dictors in the multivariate analysis.
Ovarian reserve tests have limited value in the prediction
of nonconception and their routine use in clinical practice
has been questioned (7). Pregnancy may occur even at ex-
treme cutoffs for an abnormal test result and therefore, IVF
treatment cannot be denied based on these tests, especially
in participants who are seeking rst cycle of treatment (6).
The results of this study concur with this viewpoint as age
was the only signicant predictor of nonconception and
even then, its ability to discriminate those participants who
conceived from those that did not, was poor (AUC
0.674) and of insufcient power to warrant withholding treat-
ment from certain women. However, identication of partic-
ipants who are likely to respond poorly during IVF treatment
is clinically relevant as the couple could be counseled accord-
ingly and made aware that they have an increased chance of
cycle cancellation and a signicantly lower chance of suc-
cess, allowing them to make an informed decision. Such pre-
diction may also allow clinicians to formulate individualized
treatment protocols to improve or at least maximize ovarian
response. The current level of evidence, based on a single
randomized controlled trial in a small population (n 52),
suggests no advantage to the use of higher starting doses of
gonadotropin in predicted poor responders (61), and such
an 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 outcome
for such couples who, regardless of the impressions and pre-
dictions of the medical profession, often remain determined
to have at least one attempt at ART. We should counsel
themaccordingly, but not stop our attempts to maximize their
chances.
In conclusion, pretreatment AFC and AMH are the most
signicant predictors of the number of oocytes retrieved
and of poor ovarian response to ovarian stimulation during
ART. 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, are
signicant predictors of nonconception with the exception
of the womans age, although the predictive ability is insuf-
ciently low to warrant modication of treatment.
862
Jayaprakasan et al. AMH and 3D US markers of ovarian reserve Vol. 93, No. 3, February 2010
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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