Diagnosis of polycystic ovaries by three-dimensional

transvaginal ultrasound
Michael C. Allemand, M.D.,a Ian S. Tummon, M.D.,a Jennifer L. Phy, D.O.,a,b
Shu C. Foong, M.D.,a Daniel A. Dumesic, M.D.,a,c and Donna R. Session, M.D.a,d
a

Mayo Clinic College of Medicine, Rochester, Minnesota; b The Centre for Reproductive Medicine, Lubbock, Texas;
Reproductive Medicine and Infertility Associates, Woodbury, Minnesota; and d Department of Obstetrics and Gynecology,
Emory University, Atlanta, Georgia
c

Objective: To study diagnostic thresholds for polycystic ovary (PCO).

Design: Retrospective cohort study.
Setting: Academic hospital.
Patient(s): Normoandrogenic ovulatory women and patients with polycystic ovary syndrome (PCOS).
Intervention(s): Two-dimensional (2D) and three-dimensional (3D) transvaginal ultrasound.
Main Outcome Measure(s): The mean follicle number per ovary (FNPO) of both ovaries and the maximum
number follicles in a single sonographic plane (FSSP) of either ovary were determined using 3D transvaginal
ultrasound. Ovarian volume was determined using 2D transvaginal ultrasound.
Result(s): Twenty-nine normoandrogenic ovulatory women were compared with 10 patients with PCOS.
Diagnostic thresholds for PCO with 100% specificity as determined by receiver operator characteristic (ROC)
curves were 20 for mean FNPO, 10 for maximum FSSP, and 13 cm3 for ovarian volume. Both 2D and 3D
transvaginal ultrasound were highly accurate in the diagnosis of PCO as determined by areas under the curve
(AUC) that were 90% for all three measures.
Conclusion(s): Mean FNPO and maximum FSSP by 3D transvaginal ultrasound have comparable high accuracy
for diagnosis of PCO. The diagnostic threshold with 100% specificity for mean FNPO is 20, which is greater
than suggested by the Rotterdam Consensus Workshop in 2003. Use of the consensus standard, consequently,
may result in overdiagnosis of PCO. A threshold of 20 mean FNPO using 3D transvaginal ultrasound may be
appropriate to minimize false-positive diagnoses of PCO. (Fertil Steril 2006;85:214 9. 2006 by American
Society for Reproductive Medicine.)
Key Words: Diagnosis, threshold, accuracy, polycystic ovary, three-dimensional, transvaginal ultrasound, twodimensional

Polycystic ovary syndrome (PCOS) is a heterogeneous disorder

characterized by chronic anovulation, hyperandrogenism, and
polycystic ovaries (PCO) (1, 2). This heterogeneity has led to
the ongoing challenge of establishing accurate diagnostic criteria. Following the 1990 NIH Conference on PCOS (3), diagnostic criteria included hyperandrogenism or hyperandrogenemia, oligo-ovulation, and exclusion of other endocrinopathies.
In a landmark simultaneous publication in Fertility and Sterility
(1) and Human Reproduction (2), the Rotterdam Consensus
Workshop on PCOS in 2003 changed diagnostic guidelines by
adding the ultrasonographic criteria of a mean follicle number
per ovary (FNPO) of both ovaries 12 or ovarian volume 10
mL to hyperandrogenism and oligo-ovulation (1). Two of the
three criteria must be present to diagnose PCOS (1).
Establishing a standardized definition of PCO by ultrasound has been difficult, generating numerous publications
(4 14). The original ultrasound definition of PCO, described
Received February 16, 2005; revised and accepted July 8, 2005.
Presented at the 60th Annual Meeting of the American Society for Reproductive Medicine, Philadelphia, Pennsylvania, October 2004.
Reprint requests: Michael C. Allemand, M.D., Mayo Clinic, 200 First
Street SW, Rochester, Minnesota 55905 (FAX: 507-284-1774; E-mail:
allemand.michael@mayo.edu).

214

by Adams et al. in 1985, is the presence of 10 or more

follicles, measuring 2 8 mm in diameter, arranged peripherally around a dense core of stroma or scattered throughout
an increased amount of stroma (5). This definition, performed using abdominal two-dimensional (2D) ultrasound to
count the maximum number of follicles in a single sonographic plane (FSSP), has remained the most widely used in
clinical practice. Since 1990, other studies have reported the
use of ovarian volume as a marker for PCO, and a volume
10 cm3 is now included in the Rotterdam criteria as an
accepted ultrasonographic marker for the diagnosis of PCOS
(1, 8, 10, 11, 15). Other ultrasonographic criteria for PCO
proposed in recent years, which attempt to quantify the
amount of increased ovarian stroma, have met with less
widespread acceptance (12, 15).
The addition of ultrasound criteria to the recommendations for PCOS diagnosis represents a fundamental change,
and emphasizes the need for identifying the most accurate
method for the identification of PCO.
The goals of this study were to use transvaginal ultrasound
to determine diagnostic thresholds for PCO and to quantify
the accuracy of these thresholds.

Fertility and Sterility Vol. 85, No. 1, January 2006

Copyright 2006 American Society for Reproductive Medicine, Published by Elsevier Inc.

0015-0282/06/$32.00
doi:10.1016/j.fertnstert.2005.07.1279

MATERIALS AND METHODS

The Mayo Institutional Review Board approved this retrospective cohort study (772-04), in which normoandrogenic
ovulatory women were compared with PCOS patients. Subjects had given consent for their records to be reviewed
according to Minnesota law (MN Statute Section 144.335).
Normoandrogenic ovulatory women with a history of
male factor or tubal factor infertility, who were undergoing
preparations for IVF treatment, were recruited between July
2001 and May 2002. All women had ovulatory menstrual
cycles of 2135 days with luteal serum progesterone 3.0
ng/mL, normal physical examination confirming lack of
hirsutism with a modified Ferriman-Gallwey score 7 (16),
no active thyroid disease, galactorrhea, 21-hydroxylase deficiency, or diabetes mellitus (2-hour postprandial glucose
200 mg/dL). Two women had undergone unilateral oophorectomy for benign disease (i.e., benign cystic teratoma and
endometrioma).
The PCOS patients with a history of anovulatory infertility and undergoing preparations for IVF were recruited between May 2002 and February 2004. All PCOS patients had
chronic anovulation and hirsutism or biochemical hyperandrogenism, excluding specific ovarian, adrenal, and pituitary
disorders (17). Chronic anovulation was defined as amenorrhea of 3 months duration or oligomenorrhea (i.e., intermenstrual intervals greater than 35 days) with adequately
timed serum progesterone levels 3.0 ng/mL (18, 19). Hirsutism was defined as a modified Ferriman-Gallwey score
7 (16). Biochemical hyperandrogenism was defined as
serum testosterone, non-sex hormone-binding globulinbound testosterone, or dihydrotestosterone greater than two
SD above the mean for nonhirsute, ovulatory women (18,
19). No PCOS patient had a history of ovarian surgery or had
any taken any medications known to affect ovarian function
over the preceding two months.
Two-dimensional transvaginal ultrasound was performed in normoandrogenic and PCOS patients on cycle
day 5 of the follicular phase of the menstrual cycle and
during a period of amenorrhea, respectively, to assess
mean ovarian volume of both ovaries and to rule out
follicles that were 10 mm in size. Two-dimensional
transvaginal ultrasound was used to calculate ovarian
volume because of its ease of attainment and reliability as
compared with other techniques. A single ultrasound machine was used for all studies (HDI 5000; Advanced
Technology Laboratories Ultrasound, Inc., Bothell, WA).
The maximum longitudinal, anteroposterior, and transverse diameters were determined by freehand in manually
obtained consecutive 2D images in real time. A 4 8 MHz
vaginal probe (C8-4v, Advanced Technology Laboratories Ultrasound, Inc.) was used to determine follicle size
and ovarian volume using the formula for a prolate ellipsoid (0.5237 D1 D2 D3, with D1, D2, and D3
representing the maximum longitudinal, anteroposterior,
and transverse diameters) (20, 21).
Fertility and Sterility

Following 2D transvaginal ultrasound, three-dimensional

(3D) transvaginal ultrasound was performed with the same
ultrasound machine (Advanced Technology Laboratories Ultrasound, Inc.) and stored on CD-ROM to determine the
mean FNPO of both ovaries as well as the maximum FSSP
of either ovary. Two to four independent observers, blinded
to one anothers results, analyzed the stored images. Follicles were counted in transparent reconstructed volumes using 3D imaging software (Advanced 3DI version 1.0; Vital
Images, Inc., Minnetonka, MN).
On the day after transvaginal ultrasound, serum glucose,
dehydroepiandrosterone sulfate (DHEAS), and total testosterone were measured after a 12-hour fast as previously
described (20). Serum testosterone, DHEAS, progesterone,
and glucose were measured as previously described at the
Immunochemical Core Laboratory of the Mayo General
Clinical Research Center (20).
General patient characteristics and ovarian volume
were compared between normoandrogenic, ovulatory
women and PCOS patients using t-tests. Ferriman-Galwey
scores, FSSP, and FNPO were compared between both
female groups using Mann-Whitney U tests. Intraclass
coefficients of correlation were calculated to analyze interobserver variation (n 2) for FSSP and FNPO estimates. Receiver operator characteristic (ROC) curves
(Medcalc 7.2; Mariakerke, Belgium) were used to examine whether mean FNPO, maximum FSSP, and ovarian
volume were related to the dependent variable, with PCO
considered as a binary outcome (22).
Results are expressed as mean SD with a 95% confidence interval (CI); P.05 was considered significant.
RESULTS
A total of 39 patients (29 normoandrogenic and 10 PCOS)
were studied. Their general characteristics are presented in
Table 1. The PCOS patients had higher body mass indexes
(BMIs), higher modified Ferriman-Gallwey scores (reflecting clinical hirsutism), and lower serum progesterone (reflecting anovulation), as compared with normoandrogenic
women. There were no differences in age, DHEAS, and total
testosterone between both female groups.
Ultrasound differences between both female groups are
given in Table 2. The mean FNPO of both ovaries was
greater in PCOS patients (29.8 11.5 [95% CI 21.6 38.1])
than in normoandrogenic women (9.5 3.1 [95% CI 8.2
10.6], P.0001). Similarly, the average maximum FSSP of
either ovary was greater in PCOS patients (13.3 3.2 [95%
CI 11.0 15.6]) than in normoandrogenic women (6.4 1.4
[95% CI 5.8 6.9)], P.0001). The mean ovarian volume for
PCOS patients was 13.6 3.5 cm3 (95% CI 11.0 16.1) and
7.3 2.1 cm3 (95% CI 6.5 8.1) for normoandrogenic
women (P.0001).
Diagnostic accuracy of both FNPO and maximum FSSP
was high, as judged by ROC areas under curve (AUC) that
215

TABLE 1
General patient characteristics.
Variablea
Age (y)
BMI (kg/m2)
DHEAS (ug/mL)
Total testosterone (nmol/L)
Modified Ferriman-Gallwey
Cycle day 21 P4 (ng/mL)
Fasting glucose (mg/dL)

Normoandrogenic
(n 29)

PCOS
(n 10)

30.9 3.5
24.0 5.5
1.0 0.3
39.0 10.6
1.0 1.1
13.4 6.3
93.6 5.6

31.2 3.9
32.2 10.8
1.6 1.0
48.9 22.6
13.8 3.9
1.3 1.4
89.8 9.1

P
.82
.004
.22
.26
.001
.001
.33

Note: PCOS polycystic ovary syndrome; BMI body mass index; DHEAS dehydroepiandrosterone sulfate.
a
Values are means SD.
Allemand. Diagnosis of PCO by 3D transvaginal ultrasound. Fertil Steril 2006.

were 90% for both measures (22). A mean FNPO 20.1

predicted PCO with 100% specificity and 70% sensitivity
(AUC 98.7%). As depicted in Figure 1, a mean FNPO 20.1
resulted in no false positive diagnoses of PCO among normoandrogenic women, while resulting in false negatives in 2
of the 10 PCOS patients in our study. A maximum FSSP of
10.0 predicted PCO with 100% specificity and 90% sensitivity (AUC 99.0%).
Diagnostic accuracy of ovarian volume as judged by ROC
also was high (22). An ovarian volume of 13.0 cm3 predicted PCO with a specificity of 100% and a sensitivity of
50% (AUC 94.8%).
Intraclass coefficients of correlation between observers for
maximum FSSP and mean FNPO were 0.72 and 0.82, indicating strong and excellent correlation, respectively.
DISCUSSION
The goals of the present study were to use transvaginal
ultrasound to determine diagnostic thresholds for PCO and

to quantify the accuracy of these thresholds. Three-dimensional transvaginal ultrasound was used to measure mean
FNPO of both ovaries and maximum FSSP of either ovary,
while 2D ultrasound was used to measure ovarian volume. A
comparison was made between well-characterized, normoandrogenic, ovulatory subjects vs. PCOS patients. Diagnostic thresholds with 100% specificity for PCO were 20
for mean FNPO, 10 for maximum FSSP, and 13 cm3 for
ovarian volume. Mean FNPO, maximum FSSP, and ovarian
volume all showed high accuracy as demonstrated by AUCs
90% for all three measures (22).
Three-dimensional imaging improves spatial awareness
(23), stores information for later use, and gives an improved
record of anatomy (24). Three-dimensional transvaginal ultrasound, however, gives quantitatively different information
than 2D imaging when counting ovarian antral follicles (25).
A mean difference of 1.6 antral follicles was observed comparing 3D vs. 2D transvaginal ultrasound; this difference
was especially noted in the higher range of counts (25).
Applying this correction factor to our 3D data would corre-

TABLE 2
Ultrasound differences between normoandrogenic and PCOS patients.
Variablea

Normoandrogenic
(n 29)

PCOS
(n 10)

Mean FNPOb
Maximum FSSPc
Ovarian volume (cm3)

9.5 3.1 (8.210.6)

6.4 1.4 (5.86.9)
7.3 2.1 (6.58.1)

29.8 11.5 (21.638.1)

13.3 3.2 (11.015.6)
13.6 3.5 (11.016.1)

.0001
.0001
.0001

Note: PCOS polycystic ovary syndrome; FNPO follicle number per ovary; FSSP follicles in a single sonographic
plane; CI confidence interval.
a
Values are mean SD and 95% CI.
b
Values are mean of both ovaries.
c
Values are maximum of either ovary.
Allemand. Diagnosis of PCO by 3D transvaginal ultrasound. Fertil Steril 2006.

216

Allemand et al.

Diagnosis of PCO by 3D transvaginal ultrasound

Vol. 85, No. 1, January 2006

FIGURE 1
Dot-plot of individual results of mean follicle number per ovary (FNPO) in normoandrogenic subjects and
PCOS patients. Horizontal line at FNPO 20.1 designates 100% specificity and 70% sensitivity for
diagnosis of PCO.

Allemand. Diagnosis of PCO by 3D transvaginal ultrasound. Fertil Steril 2006.

late to a threshold mean FNPO of 18 using 2D transvaginal

ultrasound for the diagnosis of PCO. Although speculative,
such a correction acknowledges the expectation that one
would find more follicles using a 3D technique, and allows
for comparison with published data using a 2D approach.
The 2003 Rotterdam Consensus Workshop defines ultrasonographic criteria for PCO as the: Presence of 12 or more
follicles in each ovary measuring 29 mm in diameter,
and/or increased ovarian volume (10 mL) (1). Only one
ovary fitting this definition is sufficient to define PCO (1).
The literature cited to support this definition includes Pache
et al., van Santbrink et al., and Jonard et al. (8, 10, 13).
Pache et al. studied 29 normoovulatory women and 52
PCOS patients using 2D transvaginal ultrasound to determine cutoff levels for the size and number of ovarian follicles, ovarian echogenicity, and ovarian volume to discriminate between the two groups (8). They found differences
between the groups in the median values of all measures
(P.001). The authors concluded that due to overlap in the
distribution of the data, only a combination of mean follicular size (median value 3.8 mm in PCOS vs. 5.1 mm in
controls) and mean ovarian volume (median 9.8 mL in
PCOS vs. 5.9 mL in controls) had the necessary power to
Fertility and Sterility

discriminate between the two groups, with a sensitivity of

92% and specificity of 97% (8).
van Santbrink et al. used 2D transvaginal ultrasound to
correlate mean FNPO, ovarian volume, and ovarian stromal
echogenicity to FSH, LH, testosterone, and androstenedione
levels in 48 control subjects with regular menstrual cycles
vs. 330 oligo- or amenorrheic patients with a history of
infertility (10). Polycystic ovaries were defined as an ovarian
volume 10.8 mL or an elevated FNPO 10 (10). The goal
of the study was not to use ultrasound to define diagnostic
criteria for PCO, but to correlate gonadotropin and hormone
levels to ultrasound findings in two populations of women.
The criteria used in the study to determine if an ovary
appeared polycystic (i.e., ovarian volume 10.8 or FNPO
10) was arbitrary, making interpretation of the hormonal
correlations difficult. Additionally, the oligo- or amenorrheic
population in this study was not well characterized by current diagnostic criteria for PCOS, making comparison to
more recent studies a challenge.
Jonard et al. in 2003 compared 112 normal controls to 214
well-characterized PCOS patients using 2D transvaginal ultrasound to determine diagnostic thresholds for PCO by
FNPO (13). When all follicles 29 mm in size were consid217

ered together, PCOS patients were found to have a mean

FNPO higher than that of the control subjects: 15.5 [range
10 27.5] vs. 6.0 [range 4.510]; P.0001 (13). They found
that a threshold mean FNPO of 12 follicles that were 29
mm offered the best discrimination between PCOS patients
and normal controls, with a sensitivity of 99% and a specificity of 75%. Although subgroup analysis did find an increased number of follicles in the 25-mm size range in
PCOS patients, clinical applicability is limited in attempting
to stratify follicles by size in the range of 25 mm. In the
end, this is not necessary because their data demonstrates the
mean FNPO of follicles 29 mm in size provided the best
clinical assessment of PCO status (13).
Our data conflict with those of Jonard et al. and the
recommendations of the Rotterdam PCOS Consensus Workshop in that our threshold mean FNPO is substantially
higher. A number of potential reasons could cause this
discrepancy. Our study utilized 3D transvaginal ultrasound
rather than 2D as in the three articles cited by the consensus
statement (8, 10, 13). This could certainly be expected to
result in more follicles being identified, although as discussed previously, even with attempts to correct for this
difference in technique, our recommended threshold remains
higher than current guidelines.
In addition, the images used to establish the 3D data for
this study were recorded on CD-ROM and reviewed later by
multiple observers. Recorded images might be expected to
result in the identification of more follicles than real-time
interpretation, as demonstrated by Jonard et al., because the
observer has more time to carefully examine the images (10).
However, the use of multiple observers for each measure
should minimize the tendency of any one observer to overestimate the number of follicles in an ovary. As we have
shown with analysis of intraclass coefficients of variation,
there was strong to excellent correlation between all observers, indicating that there was not a tendency to overestimate
follicles by any one observer, and a single individual could
reliably perform the same analysis.
In the present study, both normoandrogenic and PCOS
patients were carefully characterized. Normoandrogenic patients likely truly represent the healthy normal population
and the PCOS patients likely truly have PCOS. Additionally,
the results of this study agree with prior literature regarding
a maximum FSSP of 10 as well as a mean ovarian volume of
10.2 mL (if best compromise between sensitivity and specificity is used) as diagnostic thresholds for PCO, lending
validity to our recommended mean FNPO threshold of 20
using 3D transvaginal ultrasonography.
Data from normandrogenic women and PCOS patients
were collected at different times; however, the same ultrasound techniques and the same 3D software were used in
both groups.
Although the study sample size is small, with standard
assumptions of 0.05 and power 0.90, it is sufficient
218

Allemand et al.

to detect differences of eight follicles in mean FNPO, three

follicles in maximum FSSP, and 3 cm3 in ovarian volume
between normoandrogenic women and PCOS patients.
There is a fundamental difference between counting
FSSP)and determining FNPO. If one accepts that 10 follicles in a single plane represents PCO, it is likely that more
follicles are within that ovary outside the single plane of
ultrasound examination, thus the recommendation that a
mean FNPO 12 be used as the diagnostic cutoff for PCO
with 2D transvaginal ultrasound is questionable (1).
Use of the consensus standard of a mean FNPO 12 may
result in overdiagnosis of PCO (1, 2). The diagnostic threshold for PCO with 100% specificity for mean FNPO determined by 3D transvaginal ultrasound is 20, which is higher
than that suggested by the Rotterdam consensus using 2D
transvaginal ultrasound (1, 2). Given the data suggesting
fewer follicles will be identified using 2D as compared with
3D transvaginal ultrasound, a mean FNPO 18 may be an
appropriate diagnostic threshold for PCO when using this
technique.
Acknowledgment: This work was supported by the National Institutes of
Health Grant U01 HD044650-02 as well as a grant by Serono Pharmaceuticals.

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