Clinical Chemistry 55:3
519–526 (2009)
Endocrinology and Metabolism
Steroid Profiles in Ovarian Follicular Fluid from Regularly
Menstruating Women and Women after
Ovarian Stimulation
Mark M. Kushnir,1,2* Tord Naessen,3 Dmitrijus Kirilovas,3 Andrey Chaika,4 Jelena Nosenko,4
Iryna Mogilevkina,4 Alan L. Rockwood,1,5 Kjell Carlström,6 and Jonas Bergquist2
BACKGROUND: Information on the concentrations of
steroids in ovarian follicular fluid (FF) from regularly
menstruating (RM) women has been limited because
of the absence of methods for the simultaneous quantification of multiple steroids in small volumes of FF.
We studied steroid profiles in FF during the early follicular phase of the menstrual cycle and after ovarian
stimulation for in vitro fertilization (IVF), and compared concentrations with published values obtained
by immunoassay (IA).
METHODS:
We used liquid chromatography–tandem
mass spectrometry (LC-MS/MS) to measure 13 steroids in 40-␮L aliquots of FF samples from 21 RM
women and from 5 women after ovarian stimulation
for IVF. Relationships between concentrations of
steroids and their ratios (representations of the enzyme activities) were evaluated within and between
subgroups.
RESULTS:
The concentrations of testosterone (Te), androstenedione (A4), and estradiol (E2) measured by
LC-MS/MS were lower than those previously reported
in studies with IAs. In RM women, androgens were the
most abundant class of steroids, with A4 being the major constituent. The concentrations of 17-hydroxyprogesterone (17OHP), total androgens, and estrogens
were 200- to 1000-fold greater in FF than in serum.
Compared with RM women, FF samples from women
undergoing ovarian stimulation had significantly
higher concentrations of E2 (P ⫽ 0.021), pregnenolone
(P ⫽ 0.0022), 17OHP (P ⫽ 0.0007), and cortisol (F)
(P ⫽ 0.0016), and significantly higher ratios of F to
1
ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT;
Analytical Chemistry/Department of Physical and Analytical Chemistry, Uppsala University, Uppsala, Sweden; 3 Departments of Obstetrics and Gynecology,
Uppsala University Hospital, Uppsala, Sweden; 4 Department of Obstetrics,
Gynecology and Perinatology, Donetsk State Medical University, Donetsk,
Ukraine; 5 Department of Pathology, University of Utah, Salt Lake City, UT;
6
Division of Clinical Chemistry, Department of Laboratory Medicine, Karolinska
Institutet, University Hospital at Huddinge, Huddinge, Stockholm, Sweden.
* Address correspondence to this author at: ARUP Institute for Clinical and
Experimental Pathology, 500 Chipeta Way, Salt Lake City, UT 84108. Fax (801)
2
cortisone (P ⫽ 0.0006), E2 to estrone (P ⫽ 0.0008), and
E2 to Te (P ⫽ 0.0013).
CONCLUSIONS: The data provide the first MS-based concentration values for 13 steroids in ovarian FF from
RM women, from estrogen- and androgen-dominant
follicles, and from women after ovarian stimulation
for IVF.
© 2008 American Association for Clinical Chemistry
In women of fertile age, ovarian follicles are the main
source for a major fraction of the estrogens present in
the circulation. Although ovarian follicles also contribute circulating androgens, the adrenal cortex is the major source of the androgens in the circulation. Follicular steroids are secreted by granulosa, theca, and hilus
cells under the control of gonadotropins, and the hormonal microenvironment affects follicle development
and oocyte viability (1 ). Higher estradiol (E2)7 concentrations in follicular fluid (FF) are associated with
healthy mature follicles containing oocytes capable of
meiosis, whereas higher androgen concentrations are
indicative of atretic changes (1–5 ). With the introduction of in vitro fertilization (IVF), a number of studies
have analyzed FF from women undergoing ovarian
stimulation. The majority of these studies were undertaken to identify prognostic variables for the likelihood
of a successful implantation (6–8 ). Relatively few publications, however, have focused on the steroid hormones present in the FF of regularly menstruating
(RM) women and on the relationship between steroid
concentrations and follicular development (3–5,
9 –11 ).
584-5207; e-mail [email protected].
Received May 9, 2008; accepted October 24, 2008.
Previously published online at DOI: 10.1373/clinchem.2008.110262
7
Nonstandard abbreviations: E2, estradiol; FF, follicular fluid; IVF, in vitro fertilization; RM, regularly menstruating; IA, immunoassay; MS, mass spectrometry;
LC-MS/MS, liquid chromatography–tandem MS; EDF, estrogen-dominant follicle; ADF; androgen-dominant follicle; Te, testosterone; E1, estrone; E3, estriol;
Pregn, pregnenolone; 17OHPregn, 17-hydroxypregnenolone; 17OHP, 17hydroxyprogesterone; 11DC, 11-deoxycortisol; F, cortisol; E, cortisone; A4,
androstenedione; DHEA, dehydroepiandrosterone.
519
The information available on the steroids in FF
and their concentrations is conflicting (5, 9, 10 ). This
situation is due in part to the very limited volume of FF
that can be obtained from follicles of RM women and
the absence of methods for simultaneous quantitative
analysis of multiple analytes in small samples. Previous
studies have measured steroids in FF with immunoassays (IAs) (12–15 ), which are known to have a high
potential for cross-reactivity (16 –18 ), or with GC-MS
methods, which are more specific but require larger
sample aliquots (19 –21 ). Recent advances in biological
mass spectrometry (MS) have overcome the problems
associated with poor specificity in earlier methods and
have enabled simultaneous quantification of multiple
steroids. To our knowledge, no comprehensive published study has used liquid chromatography–tandem
MS (LC-MS/MS) methods for quantitative analysis of
multiple steroids in FF from RM women.
Our aims were to use high-sensitivity LC-MS/MS
methods to evaluate steroid profiles in FF samples from
RM women during the early follicular phase of menstrual cycle and from women after ovarian stimulation
for IVF, to compare the patterns of steroid distribution
in FF samples from estrogen-dominant follicles (EDFs)
and androgen-dominant follicles (ADFs), to compare
the distribution patterns of steroid concentrations in
serum and FF in RM women during the early follicular
phase of the menstrual cycle, and to compare the steroid concentrations in FF obtained in this study with
published values obtained by other methods.
Materials and Methods
STUDY PARTICIPANTS
Twenty-one RM women who responded to advertising
in the media were recruited at the Donetsk Regional
Center of Mother and Child Care, Donetsk, Ukraine.
The women entered the hospital for laparoscopic treatment of infertility, presumably caused by pelvic adhesions. All women had regular cycles, and pelvic ultrasound examinations revealed typical healthy ovaries.
The women were in good general health and had not
taken hormonal medications or oral contraceptives
during the 3 months before inclusion in the study. The
RM women had periods with intervals of 21–32 days
(Table 1). The ultrasound examinations showed typical ovarian images, with no signs of polycystic ovaries
or increased stromal density. Table 1 summarizes the
clinical and anthropometric characteristics of the
participants.
We also sampled FF from 5 Swedish patients
undergoing IVF treatment because of male-factor, tubalfactor, nonovarian endometriosis–related, or unexplained infertility. These patients underwent infertility
treatment at the Uppsala University Hospital, Uppsala,
520 Clinical Chemistry 55:3 (2009)
Table 1. Anthropometric and reproductive
characteristics of RM healthy women of
fertile age (n ⴝ 21).a
Variable
Age, years
28 (3.2)b
Height, cm
165 (6.2)
Weight, kg
64.8 (10.4)
Body mass index, kg/m2
23.9 (3.8)
Parity
Menstrual cycles during previous 12 months, n
Menstrual cycle day at FF sampling
Menstrual cycle length, days
Hirsutism indexd
Current smokers, n
2.1 (1.7)c
12
6 (4–7)
28 (21–32)
3 (1–8)
9
a
Data are presented as the mean (SD) or as the median (range), as
indicated.
b
Age range, 21–34 years.
c
Parity range, 1– 8.
d
Modified Ferriman–Gallwey scale.
Sweden. The treatment protocol for the IVF patients
consisted of pituitary down-regulation by means of the
gonadotropin-releasing hormone analog Buserelin
(Hoechst) and the “long” protocol initiated at the midluteal phase (900 ␮g/day, intranasal administration).
Recombinant follicle-stimulating hormone (Gonal-f威;
Serono) was injected subcutaneously (225 IU/day),
starting on cycle day 3. The dose was adjusted from
cycle day 7 as necessary. Human chorionic gonadotropin (10 000 IU Profasi威 HP; Serono) was administered
when one or more follicles had reached a diameter
⬎23 mm. Oocytes were retrieved transvaginally under
ultrasound guidance 36 h after human chorionic gonadotropin administration. All women participating in
the study were Caucasian. Informed consent was obtained from all of the women, and the ethics committees of Donetsk State Medical University and Uppsala
University approved the study protocol.
COLLECTION AND HANDLING OF FF SAMPLES
FF samples were obtained from the RM women during
laparoscopic adhesiolysis performed between day 4
and day 7 of the follicular phase of a cycle. FF samples
were aspirated from ovarian follicles (5– 8 mm in diameter), pooled for each study participant, and centrifuged. The samples were transferred and stored in
Cryovial tubes (SciMart) below ⫺20 °C. Follicle size
was measured by transvaginal ultrasonography during
laparoscopic adhesiolysis. For the women undergoing
ovarian stimulation for IVF, FF was aspirated from single follicles ⬎15 mm in diameter into Falcon tubes (BD
Steroid Profiles in Ovarian Follicular Fluid
Biosciences). The samples were transferred into SciMart Cryovial tubes and stored at ⫺70 °C until analysis. FF samples were packed in dry ice during transport
between the participating centers.
REAGENTS AND CALIBRATORS
Testosterone (Te), estrone (E1), 17␤-E2, 17␣-E2, estriol
(E3), pregnenolone (Pregn), 17-hydroxypregnenolone
(17OHPregn), 17-hydroxyprogesterone (17OHP), 11deoxycortisol (11DC), cortisol (F), cortisone (E), hydroxylamine, formic acid, trifluoroacetic acid, dansyl
chloride, and sodium carbonate were purchased from
Sigma–Aldrich. Androstenedione (A4), dehydroepiandrosterone (DHEA), dihydrotestosterone, and androstanedione were purchased from Steraloids. The internal standards were deuterium-labeled analogs of the
steroids: d3-Te, d3-Pregn, d2-11DC, d8-17OHP, d317OHPregn, d4-F, and d3-E were purchased from
Cambridge Isotope Laboratories; d4-E1, d3-E2, d3-E3
were purchased from C/D/N Isotopes. HPLC-grade
methanol, acetonitrile, and methyl-tert-butyl ether
were all obtained from VWR. All other chemicals were
of the highest purity commercially available.
LC-MS/MS METHODS
We used LC-MS/MS methods to measure steroid concentrations in FF. Estrogens were analyzed as dansyl
derivatives (22, 23 ), ketosteroids were analyzed as
oxime derivatives (24, 25 ), and F and E were analyzed
nonderivatized (26 ). The HPLC system consisted of
1200 Series HPLC pumps (Agilent Technologies), a 10port switching valve, a vacuum degasser, and an HTC
PAL autosampler (LEAP Technologies) equipped with a
fast-wash station. An API 4000 tandem mass spectrometer (Applied Biosystems/MDS Sciex) was used in the
positive-ion mode with a TurbolonSpray™ ion source.
Sample preparation, chromatographic-separation conditions, and mass transitions used in the methods are
outlined in Table 1 of the Data Supplement that accompanies the online version of this article at http://
www.clinchem.org/content/vol55/issue3. The quadrupoles Q1 and Q3 were tuned to unit resolution, and the
MS conditions were optimized for the maximum signal
intensity of each steroid. Two mass transitions were
monitored for each steroid and its internal standard.
The primary mass transitions were used to measure the
concentration of each steroid, and the specificity of the
analysis for each steroid in every sample was evaluated
by comparing the concentrations measured with the
primary and secondary mass transitions of each steroid
and its internal standard (27, 28 ). Data were analyzed
quantitatively with Analyst™ 1.4.2 software (Applied
Biosystems). We used 6 calibrators to generate calibration curves for every set of samples and included 3 QC
samples with every set of samples. Assay imprecision
was ⬍15% (see Table 1 in the online Data Supplement). No ion suppression was observed in the methods (22–26 ) during analyses of FF samples. Steroid recoveries from FF samples were between 94% and
125%, compared with serum samples. The methods
showed the same lower limits of quantification and upper limits of linearity for FF and serum samples.
STATISTICAL ANALYSIS
Unless otherwise stated, results were expressed as the
median and the range. The Mann–Whitney U-test was
used to compare groups, and P values ⬍0.05 were
considered statistically significant. Statistical analyses were performed with the JMP software package
(SAS Institute).
Results
The steroids measured in FF samples belong to 5
classes: pregnenolones, progestins, androgens, estrogens, and glucocorticoids (see Fig. 1 in the online Data
Supplement). Fig. 1 shows pie diagrams of the distributions of steroid concentrations in FF samples from
RM women and from women after ovarian stimulation
for IVF. A4 was the predominant steroid (46.8%) in
RM women, followed by 17OHP and Pregn, whereas
17OHP was the predominant steroid (54.0%) in
women undergoing ovarian stimulation, followed by
E2 and Pregn (Fig. 1). Compared with RM women, FF
samples from women undergoing ovarian stimulation
had significantly higher concentrations of E2, Pregn,
17OHP, and F; significantly higher E2/E1 ratios (indicative of 17␤-hydroxysteroid oxidoreductase activity),
E2/Te ratios [indicative of CYP19 (aromatase) activity], and F/E ratios (indicative of 11␤-hydroxysteroid
oxidoreductase activity); and significantly lower concentrations of 17OHPregn, 11DC, E, DHEA, A4, and
Te (Table 2).
We also evaluated the distribution patterns of steroid concentrations in ADFs (n ⫽ 13) and EDFs (n ⫽
8) in RM women. ADFs were defined as follicles with
E2/Te ratios ⬍4, and EDFs were defined as those with
E2/Te ratios ⬎4 (4 ). The medians and central 90%
intervals (i.e., 5th –95th percentile) for the E2/Te ratio
were 0.63 (0.1–2.5) and 14.1 (4.0 –73.0) in the ADF and
EDF groups, respectively. Fig. 2 shows pie diagrams of
the distributions of median steroid concentrations in
the ADF and EDF groups. Table 3 presents results for
steroids with significant differences between ADFs and
EDFs. A4 was the predominant steroid (56.4%) in
ADFs, followed by 17OHP and DHEA. A4 was also the
predominant steroid in EDFs (30.8%), followed by
17OHP and E2 (Fig. 2). Compared with ADFs, EDFs
had significantly higher E2 concentrations, signifiClinical Chemistry 55:3 (2009) 521
Fig. 1. Distribution of median concentrations of steroids in FF from RM women during the early follicular phase
(left) and from women after ovarian stimulation (right).
Table 2. Concentrations of steroids in FF samples,
as measured by LC-MS/MS.a
RM women
(n ⴝ 21)
Women with
ovarian
stimulation for
IVF (n ⴝ 5)
Pregn, ␮g/L
52 (16–89)
110 (74–460)
0.0022
17OHPregn, ␮g/L
32 (4.4–60)
3.0 (1.5–6.9)
0.0016
Variable
P
17OHP, ␮g/L
180 (65–310)
520 (380–1400)
0.0007
11DC, ␮g/L
4.1 (1.8–6.6)
1.5 (0.6–3.2)
0.0063
F, ␮g/L
17 (3.9–38)
53 (38–64)
0.0016
E, ␮g/L
32 (19–47)
12 (7.7–27)
0.0047
86 (34–190)
2.7 (1.7–4.3)
0.0006
420 (200–830)
6.8 (2.9–67)
0.0006
DHEA, ␮g/L
A4, ␮g/L
Te, ␮g/L
18 (6.2–43)
0.3 (0.01–2.7)
0.0006
Androstanedione,
␮g/L
2.0 (0.6–6.2)
0.5 (0.3–2.5)
0.029
9.2 (6.39–72.7)
0.0006
Androgens, total, 534 (252–997)
␮g/L
E1, ␮g/L
34 (3.3–140)
24 (12–36)
0.416
E2, ␮g/L
31 (2.6–302)b
235 (119–389)
0.021
E3, ␮g/L
0.47 (0.1–2.3)b
0.86 (0.8–2.0)
0.182
260 (132–426)
0.047
Estrogens, total,
␮g/L
b
77 (11–388)
F/E ratio
0.55 (0.14–1.19) 4.33 (2.27–5.55)
E2/E1 ratio
0.66 (0.15–3.51)b 9.71 (8.30–13.38) 0.0008
E2/Te ratio
1.5 (0.12–42)b
628 (85–4050)
a
0.0006
0.0013
Data are presented as the median and central 90% of the distribution (i.e.,
5th–95th percentile).
b
One result was excluded as an outlier (with the Mahalanobis test).
522 Clinical Chemistry 55:3 (2009)
cantly higher E2/E1 ratios, and significantly lower A4
and Te concentrations (Table 3).
We evaluated the relationships between the concentrations of the steroids involved in the pathway (see
Fig. 1 in the online Data Supplement) and the associations between the concentrations of the steroids within
the ADF and EDF subgroups. In ADFs, the 17OHPregn
concentration was associated with the DHEA concentration (r ⫽ 0.95; P ⫽ 0.001), the concentration of
17OHP was associated with concentrations of E1 (r ⫽
0.88; P ⫽ 0.001), Te (r ⫽ 0.86; P ⫽ 0.001), and androstanedione (r ⫽ 0.98, P ⫽ 0.002), and the A4 concentration was significantly associated with the Te concentration (r ⫽ 0.85; P ⫽ 0.001). In EDFs, the 17OHP
concentration was positively associated with the concentrations of 11DC (r ⫽ 0.96; P ⫽ 0.002) and E3
(r ⫽ 0.88; P ⫽ 0.003) and was negatively associated
with the F/11DC ratio (indicative of CYP11 activity;
r ⫽ ⫺0.87; P ⫽ 0.004). The 17OHPregn concentration
was positively associated with the DHEA concentration
(r ⫽ 0.99; P ⫽ 0.001), whereas the E2 concentration was
positively associated with the E3 concentration (r ⫽ 0.92;
P ⫽ 0.001) and negatively associated with the F/11DC
ratio (CYP11 activity; r ⫽ ⫺0.93; P ⫽ 0.001). The A4/
17OHP ratio (CYP17 activity) was negatively associated
with the E2/Te ratio (CYP19 activity; r ⫽ ⫺0.89; P ⫽
0.002); the F/11DC ratio (CYP11 activity) was negatively
associated with the 17OHP concentration.
The concentrations of steroids in FF samples from
women after ovarian stimulation measured in this
study with LC-MS/MS methods were compared (Table
4) with values reported in 3 previous studies that used
Steroid Profiles in Ovarian Follicular Fluid
Fig. 2. Distribution of median concentrations of steroids in FF from ADFs (left) and EDFs (right) of RM women. EDFs,
follicles with an E2/Te ratio >4; ADFs, follicles with an E2/Te ratio <4 [Klein et al. (4 )].
IA methods (13–15 ) and 1 study that used liquid chromatography followed by spectrophotometric detection
(12 ). Lower concentrations for DHEA, A4, E2, and F
and considerably lower Te concentrations were observed in the present study than were obtained with
these other techniques (Table 4).
Discussion
During last decade, tandem MS has become the
method of choice for analyzing endogenous steroids
(18–27 ). To our knowledge, there have been no published LC-MS/MS– based measurements of steroids in
ovarian FF samples from RM women and from women
after ovarian stimulation for IVF. In our study, LCMS/MS methods produced lower concentrations for
Table 3. Variables showing significant differences
between FF samples from ADFs and EDFs in
RM women.a
ADF (n ⴝ 13)
EDF (n ⴝ 8)
A4, ␮g/L
590 (330–890)
300 (180–410)**
Te, ␮g/L
25 (15–54)
7.5 (6.0–21)**
E2, ␮g/L
14 (2.0–43)
190 (33–490)***
Variable
E1, ␮g/L
E2/E1 ratio
a
22 (3.3–97.1)
0.42 (0.15–2.44)
83 (15.5–139.9)*
2.16 (0.81–6.64)**
Data are presented as the median and central 90% of the distribution (i.e.,
5th–95th percentile). *P ⬍ 0.05; **P ⬍ 0.01; ***P ⬍ 0.001.
all androgens, especially for Te, than those obtained
with other techniques (Table 4) (12–15 ). E2, A4, and
17OHP concentrations in FF samples from RM women
were comparable to previously published values obtained with IAs (29 –31 ). In FF samples from women
undergoing ovarian stimulation for IVF, the concentrations of DHEA, E1, and E2 obtained with LCMS/MS and IA methods were comparable (Table 4).
The good agreement might be due to the relatively low
concentrations of possible cross-reacting compounds,
i.e., other 3␤-hydroxy-5-ene steroids and estrogens
(Table 2). On the other hand, large differences were
found for the 3-oxo-4-ene steroids (A4, Te, and F),
with IA values being much higher than those obtained
with LC-MS/MS. The large differences were likely due
to cross-reactivity with other 3-oxo-4-ene steroids in
IAs, notably 17OHP and progesterone, which are present
at very high concentrations in the FF of women undergoing ovarian stimulation (Table 2). The comparable concentrations of 17OHP and E obtained with LC-MS/MS
and liquid chromatography–spectrophotometry (12 )
may be because both methods used chromatographic
separation; however, A4 concentrations measured by liquid chromatography–spectrophotometry were affected
by interference, likely caused by the similar chromatographic retentions of A4 and other androgens (25 ).
In previous reports, we described our LC-MS/MS–
based measurements of steroid concentrations in serum samples from women of reproductive age (22–
26 ). Compared with serum, the concentrations of
17OHP, androgens, and estrogens were 200- to 1000Clinical Chemistry 55:3 (2009) 523
Table 4. Reported median concentrations of steroids in FF samples collected at oocyte retrieval from women
undergoing ovarian stimulation.a
Present
study
Follicular diameter, mm
Method
17OHP, ␮g/L
a
⬎15
LC-MS/MS
520
De Sutter et al.
(12 )
Andersen
(13 )
Bergh et al.
(14 )
Smitz et al.
(15 )
⬎12
⬎12
IA
IA
IA
460
—
—
—
NAb
LC-Spectro
DHEA, ␮g/L
2.7
—
—
A4, ␮g/L
6.8
19.3
Te, ␮g/L
0.3
—
NA
4.8
—
14.1
18.6
14.6
2.9
5.5
4.4
E1, ␮g/L
24
—
—
29
—
E2, ␮g/L
240
390
594
373
431
F, ␮g/L
53
—
—
—
188
E, ␮g/L
12
—
—
—
18
NA, data not available; LC-Spectro, liquid chromatography–spectrophotometry.
fold greater in FF during the early follicular phase of the
menstrual cycle. This finding is consistent with the fact
that ovarian follicles are the major site of biosynthesis
for 17OHP and estrogens and an important site of androgen biosynthesis in women of reproductive age
(32 ). The significant differences between RM women
and women undergoing ovarian stimulation for IVF
with respect to steroid concentrations in FF samples
(Table 2) clearly reflect the effect of increased gonadotropin stimulation and androgen consumption for increased estrogen biosynthesis in women undergoing
ovarian stimulation. Furthermore, the finding might
reflect an increasingly reductive environment, as reflected by increased E2/E1 and F/E ratios. The occurrence of 17␤-hydroxysteroid oxidoreductase in the
ovaries is very well known, and the presence of 11␤hydroxysteroid oxidoreductase has also been demonstrated (33 ). The increased concentrations of Pregn
and 17OHP in women undergoing ovarian stimulation
for IVF may also reflect a generally increased stimulation of follicular steroidogenesis. The concentrations
of F and E in FF samples were within the reference
limits for serum (26 ). This result suggests that glucocorticoids are likely distributed to ovarian follicles by
transport from the peripheral circulation, because
11␤-hydroxylase has never been demonstrated in the
ovaries; however, one cannot exclude ovarian production of 11DC by 21-hydroxylation from 17OHP, because 21-hydroxylase is present not only in the adrenal
cortex but also in a number of other tissues, including
the ovaries (34 ). The low lower limits of quantification
for nonovarian steroids was important in this study
because it allowed measurement of low concentrations of steroids that have not previously been reported for FF.
524 Clinical Chemistry 55:3 (2009)
We observed substantial differences between
ADFs and EDFs in Te, E2, E1, and A4 concentrations
and in the E1/E2 ratio (Table 4). In addition, ADFs
and EDFs differ in the associations between the concentrations of many of the steroid intermediates of
the pathway; these associations are indicative of the
relative enzyme activities in the ADF and EDF groups
(35 ). Differentiation of follicles based on the prevalence of androgens or estrogens and evaluation of
the corresponding enzyme activities could be useful
for determining the mechanisms of selection of the
dominant follicle.
The methods used in this study (22–27 ) allowed
the quantification of 13 steroids from 40 ␮L of FF. To
analyze these steroids with IA-based methods would
require several milliliters of FF, which is an unrealistic
sample size for follicles during the early follicular stage
of the menstrual cycle. In addition, there are some pitfalls associated with use of IAs for analyzing FF samples. Compared with serum, FF has substantially
higher concentrations of some steroids, and the differences in concentration may cause cross-reactivity that
is not observed with serum samples (for which IAs are
typically validated). Another problem is the need to
dilute FF samples to reduce steroid concentrations to
concentration ranges measurable by the IA. The characteristics of the diluents may alter the equilibrium
with binding proteins and with conjugated forms of the
steroids, thereby affecting the observed concentrations. These problems are not relevant to MS-based
methods. For these reasons and because the tandem
MS methods used in this study have been extensively
validated (22–26 ) and shown to be free of interference
[i.e., analysis specificity was evaluated in every sample
by monitoring multiple fragments of each steroid
Steroid Profiles in Ovarian Follicular Fluid
(27 )], we believe that the results presented in this study
are a truer representation of the concentrations of steroids in FF than the IA-based results of earlier studies.
Although our study provides the first MS measurements of concentrations of steroids in FF during
the early follicular phase and after ovarian stimulation
for IVF, it has some limitations. The RM women,
whose samples were used in the study, underwent laparoscopic operations for presumed pelvic adhesions. All
individuals included in the study were women of European descent, so generalization to other populations
should be done with caution.
In conclusion, our data provide the first MS-based
concentrations for multiple steroids in ovarian FF samples from RM women and from women after ovarian
stimulation. The ability to accurately analyze multiple
steroids from minute samples of FF, together with calculated product–precursor ratios of steroids and their metabolites (indicative of enzyme activities), is crucial for a
better understanding of typical ovarian physiology, the
menstrual cycle, anovulation, and the effects of different
ovarian stimulation regimens used in IVF programs.
Author Contributions: All authors confirmed they have contributed to
the intellectual content of this paper and have met the following 3 re-
quirements: (a) significant contributions to the conception and design,
acquisition of data, or analysis and interpretation of data; (b) drafting
or revising the article for intellectual content; and (c) final approval of
the published article.
Authors’ Disclosures of Potential Conflicts of Interest: Upon
manuscript submission, all authors completed the Disclosures of Potential Conflict of Interest form. Potential conflicts of interest:
Employment or Leadership: A.L. Rockwood, University of Utah.
Consultant or Advisory Role: A.L. Rockwood, ARUP Laboratories.
Stock Ownership: None declared.
Honoraria: None declared.
Research Funding: J. Bergquist, Family Planning Foundation, Uppsala, Sweden, by grants 629-2002-6821 and 621-2005-5379 from the
Swedish Research Council; M.M. Kushnir, ARUP Institute for Clinical and Experimental Pathology; A.L. Rockwood, ARUP Institute
for Clinical and Experimental Pathology.
Expert Testimony: None declared.
Role of Sponsor: The funding organizations played no role in the
design of study, choice of enrolled patients, review and interpretation
of data, or preparation or approval of manuscript.
Acknowledgments: We thank A. Wayne Meikle (Departments of
Medicine and Pathology, University of Utah, Salt Lake City, Utah)
for discussions and suggestions, and we thank Drs. Svetlana Korniyenko and Anna Yakovets (Donetsk State Medical University,
Donetsk, Ukraine) for assistance with sample collection. We are
grateful to all of the women who participated in the study.
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