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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2006-2719
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The Journal of Clinical Endocrinology & Metabolism Vol. 92, No. 8 3285-3291
Copyright © 2007 by The Endocrine Society

Lower Levels of Urinary 2-Hydroxyestrogens in Polycystic Ovary Syndrome

Sana Salih, Xia Xu, Timothy D. Veenstra, Antoni J. Duleba, Hala Fouad, Manubai Nagamani and Ayman Al-Hendy

Department of Obstetrics and Gynecology (S.S., H.F., M.N., A.A.-H.), University of Texas Medical Branch, Galveston, Texas 77555; Laboratory of Proteomics and Analytical Technologies (X.X., T.D.V.), SAIC-Frederick, Inc., Frederick, Maryland 21702; and Department of Obstetrics and Gynecology (A.J.D.), Yale University, New Haven, Connecticut 06511

Address all correspondence and requests for reprints to: Ayman Al-Hendy, M.D., Ph.D., F.R.C.S.C., F.A.C.O.G., Department of Obstetrics and Gynecology, University of Texas Medical Branch, 301 University Boulevard, Galveston, Texas 77555-0587. E-mail: ayalhend{at}utmb.edu.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Context: Women with polycystic ovary syndrome (PCOS) have anovulation due to arrested follicular maturation. The substrate (2-hydroxyestrogen) and product (2-methoxyestrogen) of catechol-O-methyl transferase (COMT) have been shown to modulate proliferation and angiogenesis of granulosa cells.

Objective: The objective of the study was to evaluate COMT ovarian expression as well as the production of estrogen metabolites (2-hydroxyestrogen and 2-methoxyestrogen) in subjects with PCOS.

Design: Immunohistochemistry was used to assess COMT expression in ovarian tissues. Urinary levels of 10 different estrogens and estrogen metabolites were measured using enzyme-labeled immunoassays and/or liquid chromatography with tandem mass spectrometry.

Setting: The study was conducted at a tertiary university referral center.

Patients and Other Participants: Ovarian tissues were obtained from six control subjects and six subjects with PCOS. Fasting first-void urinary samples were collected from 49 subjects with PCOS and 36 healthy control subjects.

Main Outcome Measure(s): COMT protein expression in ovarian tissues was measured. Urinary levels of 2-hydroxyestrogen and 2-methoxyestrogen levels in PCOS patients were also measured.

Results: Whereas immunohistochemistry showed that COMT was expressed in ovaries from control and PCOS subjects, its expression was significantly higher in ovaries from subjects with PCOS, in both the follicular structures and ovarian stroma. The urinary 2-hydroxyestrogen level was significantly lower in subjects with PCOS, compared with normal controls (P = 0.009). Additionally, urinary 2-hydroxyestrogen levels negatively correlated with serum insulin levels in subjects with PCOS (r = –0.333, P =0 .031).

Conclusions: Urinary 2-hydroxyestrogen is decreased in subjects with PCOS, which could be due in part to increased ovarian expression of COMT. Further studies are needed to ascertain the role of estrogen metabolism in PCOS before this information can be used in clinical settings.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
POLYCYSTIC OVARY SYNDROME (PCOS), the most common endocrinopathy, affects 4–10% of reproductive-age women (1) and is characterized by hyperandrogenism and ovulatory dysfunction (2). Anovulation, the hallmark of PCOS, is caused by arrested follicular maturation and protracted granulosa cell proliferation, the mechanism of which is currently unknown. Insulin resistance and hyperandrogenism have been associated with PCOS (2). The ovaries are the main site for estrogen production. Estrogen is metabolized by oxidative and conjugative reactions generating metabolites with distinct biologic activity (3, 4). Estrogen is aromatically hydroxylated by the cytochrome P450 1A1 and 1B1 group of enzymes to produce either 16-hydoxyestrogens or catecholestrogens (2-hydroxyestrogen and 4-hydroxyestrogen) (5). The main catechol estrogen, 2-hydroxyestrogen, functions as an antiestrogen (6) in several estrogen-dependent systems such as the breast (7) and uterus (8). In extrahepatic tissues, catechol estrogens are detoxified primarily by O-methylation catalyzed by catechol-O-methyl transferase (COMT) to produce methoxyestrogens. 2-Methoxyestrogen, the COMT-methylated product of 2-hydroxyestrogen, is more lipophilic and has a longer half-life, compared with its substrate. 2-Methoxyestrogen seems to possess bimodal cellular effects because it induces cell proliferation at low doses. At higher doses, however, it is one of the most potent apoptotic and antiangiogenic steroid compounds known to man (9). The abundance of 2-hydroxyestrogen and 2-methoxyestrogen in any given tissue is determined mainly by the relative expression and activity of COMT. The balance between these two estrogen metabolites is central in determining estrogen bioavailability and estrogenic cellular effects (10).

Perturbed estrogen metabolism has been associated with a variety of gynecological pathologies (11). We recently described an association between the high-activity COMTVal/Val genotype and uterine leiomyomata (8). In the ovary, 2-hydroxyestrogen has been shown to enhance the action of the FSH by enhancing hormone-stimulated cAMP production (12). Previous studies have shown that 2-methoxyestrogen inhibits ovarian angiogenesis and granulosa cell proliferation (12, 13, 14). Therefore, we hypothesize that increased expression and activity of COMT in ovarian follicles leads to a decrease in cellular levels of 2-hydroxyestrogen and a corresponding increase in local production of 2-methoxyestrogen. This increased expression and activity of COMT may contribute to the pathogenesis of follicular arrest and anovulation in PCOS. In this work, we report on COMT expression in ovarian tissues from control subjects and subjects with PCOS. We also compare urinary levels of 2-hydroxyestrogen and 2-methoxyestrogen, as well as several estrogens and estrogen metabolites, in subjects with PCOS and matched healthy controls. Understanding the role of perturbed estrogen metabolism in PCOS pathogenesis should lead to the development of new therapeutic treatments for this complicated syndrome.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Human subjects

The study was approved by the Institutional Review Board of the University of Texas Medical Branch (Galveston, TX), and informed consent was obtained from each subject. This study was conducted in accordance with the guidelines in the Declaration of Helsinki. We recruited 49 subjects with PCOS and 36 healthy controls with normal ovulatory cycles for the study. Attempts were made to match the two groups in all aspects including age, body mass index (BMI), race, and hormonal profile. Both subjects with PCOS and control subjects were not on any hormonal treatment for at least 3 months before inclusion in the study. All of the study subjects were nonsmokers. All control subjects had regular menstrual cycles and proven fertility. Samples (peripheral blood and urine) were collected from control subjects in the early proliferative phase of the menstrual cycle to avoid the preovulatory or luteal phase estradiol rise.

The diagnosis of PCOS was established according to the National Institutes of Health/National Institute of Child Health and Human Development criteria (15). Subjects with PCOS were diagnosed by the presence of anovulation accompanied by clinical or biochemical hyperandrogenism, after exclusion of adrenal enzyme deficiencies, Cushing’s syndrome, and ovarian or adrenal tumors that could present in a similar fashion. Anovulation was established from menstrual history of oligomenorrhea (cycle length > 45 d) or amenorrhea (cycle length > 6 months). Clinical hyperandrogenism was defined as the presence of hirsutism, acne, or signs of virilization (temporal balding or clitoral enlargement). Hirsutism was assessed by Ferriman-Gallwey-Lorenzo scores; patients with a score greater than 6 were considered hirsute (16). All patients with PCOS had hirsutism. Subjects with possible ovarian tumor [testosterone > 200 ng/dl (57.68 nmol/liter)] and neoclassical congenital adrenal hyperplasia [17-hydroxyprogesterone levels > 2 ng/ml (0.66 nmol/liter)] were excluded from the study. All subjects had polycystic ovaries on pelvic ultrasound examination (15) as well as normal TSH-stimulating hormone and prolactin levels. Height and weight were measured, and BMI was calculated for all subjects and controls. We measured estradiol and total testosterone, and we performed a standard oral glucose tolerance test with glucose and insulin levels measured at baseline and 1, 2, and 3 h after oral glucose administration. The areas under the curve for insulin and glucose were calculated using the trapezoidal method. Control subjects had normal ovulatory cycles as shown by cycle d 21 progesterone greater than 8 ng/ml (2.515 nmol/liter).

Immunohistochemistry

Ovarian COMT expression was assessed in six subjects with PCOS and six age-, BMI-, and ethnicity-matched healthy controls with normal ovulatory cycles using immunohistochemistry analysis with COMT-specific polyclonal antibodies. Normal human ovarian tissue specimens were collected from patients undergoing oophorectomy as part of a total abdominal hysterectomy for abnormal uterine bleeding. Tissue was collected from the subjects with PCOS and stored after laparoscopic ovarian wedge resections performed for the treatment of infertility in women with PCOS. Immunohistochemistry was performed as previously described (17). Primary anti-COMT polyclonal antibody (a kind gift from Dr. Tenure, Orion Corp., Orion-Faros, Research Center, Helsinki, Finland) was applied to sections at a 1:250 dilution.

Clinical sample collection

Venous blood and first-void urine samples (after overnight fasting) were collected from study subjects. Blood samples from healthy controls were collected in the first week of their menstrual cycle to match the hormonal milieu of subjects with PCOS and avoid the preovulatory estrogen rise. Serum was separated and stored at –80 C until use. Urine was collected in a sterile container with 50 mg of L-ascorbic acid and frozen immediately at –20 C. Ascorbic acid was added to the urine to prevent autooxidation of 2-hydroxyestrogen to the corresponding 2,3-O-quinone (18).

Hormonal assays

Insulin and 17-hydroxyprogesterone levels were measured with a specific double-antibody RIA using 125I-labeled hormones, whereas total testosterone, dehydroepiandrosterone sulfate, SHBG, and LH levels were measured by coated-tube RIA using kits obtained from Diagnostic Systems Laboratories (Webster, TX) following the manufacturer’s instructions. Plasma glucose levels were measured using the glucose oxidase technique, and samples were run in duplicate. High- and low-level controls were run with each assay, and the results of the assay were accepted only if the controls were within the expected range. Intraassay variation ranged from 1.5 to 2.5%, and interassay variation ranged from 3.8 to 7.4%.

ELISA urinary test

Urinary levels of 2-hydroxyestrogen and 16{alpha}-hydroxyestrogen were measured using ELISA in 49 subjects with PCOS and 36 healthy control subjects. Urinary estrogen metabolites were measured using the Estramet urinary estrogen metabolite kit (Immuna Care Corp., Blue Bell, PA) according to the manufacturer’s instructions (19). Unextracted urine samples were brought to room temperature and centrifuged to precipitate any sediment; 10 ml of urine were used for each assay. Based on initial assays using the Estramet kit, all samples from subjects were diluted 1:3 with diluents supplied by the manufacturer. Kinetic analysis was performed at 405 nM, and readings were taken every minute for 2 min using a Thermomax microplate reader (Molecular Devices, Sunnyvale, CA). All samples, standards, and controls were assayed in triplicate. Urinary creatinine was assayed in duplicate using a commercially available colorimetric assay (Ortho-Clinical Diagnostics, Rochester, NY) and was used for normalization. Results for the concentration of each metabolite were reported as nanograms per milliliter per milligram of creatinine. Samples from each patient were assayed in a single assay to eliminate interassay variation.

HPLC/electrospray ionization-tandem mass spectrometry analysis

To further investigate the role of estrogen metabolism and COMT activity in PCOS, we measured 10 urinary endogenous estrogens and estrogen metabolites in 49 subjects with PCOS and 36 healthy control subjects by liquid chromatography/electrospray ionization-tandem mass spectrometry operating in a selected reaction monitoring mode as described previously (20). The liquid chromatography/tandem mass spectrometry assay was performed on coded samples where the operator was blinded to the clinical assignment of the sample donor.

Statistical analysis

Statistical analyses of the urine data were performed using the SAS 9.2.1 computer program (SAS Institute, Cary, NC). All laboratory experiments were performed in triplicate. Student’s t test was used for comparing levels between subjects with PCOS and control subjects. The relationships between the measured variables were assessed by Pearson correlation coefficient and stratified analyses. The Wilcoxon rank sum test was used to confirm the results. The skewness statistics, as well as the median and interquartile ranges, were tested for the data. Data were presented as mean ± SD, and differences were considered significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
All of the women in the PCOS group had either oligomenorrhea (cycle length > 45 d) or amenorrhea (cycle length > 6 months) and hirsutism. Subjects with PCOS tended to be younger, be more obese, have lower gravidity, and have higher serum total testosterone levels than controls (Table 1Go). Subjects with PCOS had significantly increased fasting insulin levels, compared with control women (Table 1Go), whereas glucose levels were normal, indicating insulin resistance. Serum estradiol levels were not significantly different between subjects with PCOS and matched controls at the time of sample collection (Table 1Go), suggesting that the two cohorts were well matched regarding menstrual cycle phase or in vivo estrogen hormonal milieu.


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  		TABLE 1. Study subjects demographics and serum hormone levels

 
Immunohistochemistry was used to determine the expression and localization of COMT in control and polycystic human ovaries. There was clear cytoplasmic COMT immunostaining in the granulosa cells, theca interna cells, and stromal cell layer (Fig. 1AGo). The cytoplasmic immunoreactivity for COMT in the control ovaries was observed in the granulosa cells layer surrounding the oocytes, in the ovarian stromal cells, and in the theca interna cell layer surrounding the growing follicles (Fig. 1Go, B–F, control). The intensity of the COMT immunoreactivity was markedly increased in polycystic ovaries, compared with control ovaries (Fig. 1Go, B–F).


Figure 1
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  		FIG. 1. A, COMT immunohistochemistry in a mature follicle in a normal ovary. The immunoreactivity of the human ovarian tissue for COMT polyclonal antibody was evaluated. There is clear cytoplasmic COMT immunostaining in the granulosa and theca interna cells and stromal cell layer. B, Immunohistochemical reactivity of COMT in normal vs. polycystic ovaries. The intensity of COMT expression in the ovarian stroma is shown (x100). C, Intense COMT immunoreactivity is evident in the PCOS ovarian stroma and wall of a cystic follicle. D, Primordial ovarian follicles in the PCOS ovary demonstrate intense COMT immunoreactivity in both the granulosa (arrow) and stromal cells, compared with normal primordial follicle (x400). E, Weak COMT immunoreactivity in a multilaminar primary follicle in the cytoplasm of the granulosa cells and in the surrounding stromal cells in a normal ovary. F, Section in the wall of a rare secondary ovarian follicle in PCOS. Note the intense reaction in the granulosa (arrowheads), theca interna (arrows), and stromal cells, compared with normal ovarian follicle (x200).

 
As an indirect assessment of COMT activity in subjects with PCOS, we measured estrogen metabolites that constitute either a substrate or a product of the COMT enzyme. We measured 10 different estrogens and estrogen metabolites in urine samples from 49 subjects with PCOS and 36 healthy control women by ELISA and liquid chromatography/tandem mass spectrometry-2 (Tables 2Go and 3Go). Urinary 16{alpha}-hydroxyestrone levels were used as internal controls, and no statistical difference was seen between subjects and controls (Tables 2Go and 3Go). Furthermore, the PCOS and control samples did not significantly differ in urinary estrone, estradiol, or estriol levels (Table 2Go). This was in agreement with the findings of similar serum estradiol levels between the two groups (Table 1Go). When assayed by the ELISA technique, there were markedly reduced urinary 2-hydroxyestrogen levels in subjects with PCOS when compared with normal controls (Table 3Go). This result was further confirmed by liquid chromatography/tandem mass spectrometry, which showed significantly reduced levels of 2-hydroxyestrone, 2-hydroxyestradiol, and total 2-hydroxyestrogen in the urine of subjects with PCOS, compared with controls. The urinary 2-methoxyestrogen levels were not significantly different between subjects with PCOS and healthy controls (Table 2Go).


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  		TABLE 2. Urinary estrogens and estrogen metabolites in PCOS vs. control (liquid chromatography/tandem mass spectrometry)

 

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  		TABLE 3. Urinary 2-hydroxyestrogen and 16-hydroxyestrogen in PCOS vs. control (ELISA)

 
In subjects with PCOS, there was a significant positive correlation between serum estradiol levels and urinary 2-hydroxyestrogen levels. There was negative correlation between urinary 2-hydroxyestrogen and insulin, as well as BMI. BMI was the only parameter that showed a statistically significant negative association with 2-hydroxyestrogen in the control group (Table 4Go).


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  		TABLE 4. Correlation between urinary 2-hydroxyestrogen levels and other variables in PCOS and control groups

 
We stratified analysis of the data by BMI classes to further analyze the effect of BMI on urinary 2-hydroxyestradiol levels (21). Because the sample sizes for low weight and normal weight are too small in PCOS (supplemental Table 1, published as supplemental data on The Endocrine Society’s Journals Online Web site at http://jcem.endojournals.org), the BMI was grouped into two categories (nonobese vs. obese) in the stratified analysis. We conducted stratified analyses on variables that were found significantly different between subjects and controls by BMI status (Table 5Go). There was a significant difference between subjects with PCOS and controls in 2-hydroxyestradiol levels in the obese stratum of BMI status. It is important to note that the SD in Tables 2Go, 3Go, and 5Go was large and sometimes close to the mean, suggesting a skewed distribution. The skewness statistics showed that the majority of the data were significantly skewed (supplemental Tables 2–4, published as supplemental data on The Endocrine Society’s Journals Online Web site at http://jcem.endojournals.org). Given that qualitative skewwness was significant, the median and interquartile ranges of the data were calculated and presented in the tables (Tables 2–4GoGoGo, supplemental data). Furthermore, the Wilcoxon rank sum test was used to compare variables between PCOS and control. The significant levels from both parametric (Tables 2Go, 3Go, and 5Go) and nonparametric tests (Tables 2–4GoGoGo, supplemental data) were very similar, further confirming the results. This is probably due to the relatively large sample size of the study subjects (49 for PCOS and 36 for control).


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  		TABLE 5. BMI stratified analysis between PCOS and control women.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
Here we report for the first time that COMT is expressed in human ovaries. Only one other recent report (22) has demonstrated COMT expression in a human luteinized granulosa cell line. Furthermore, immunoreactive COMT was overexpressed in PCOS ovaries, compared with normal ovaries. COMT substrate (2-hydroxyestrone and 2-hydroxyestradiol) levels were significantly lower in urine samples from subjects with PCOS, compared with controls when tested by two independent techniques (ELISA and liquid chromatography/tandem mass spectrometry). The urinary levels of 2-methoxyestrogen, the COMT-mediated methylated product of 2-hydroxyestrogen, were not significantly elevated in subjects with PCOS. Additionally, the urinary 16{alpha}-hydroxylation pathway metabolites were unchanged between subjects with PCOS and matched controls. Because estrogen hydroxylation toward the 16{alpha} or C2 pathway uses the same intermediate, our finding suggests that the decrease in urinary 2-hydroxyestrogen levels in subjects with PCOS is a specific step in the pathway that seems to be altered. Furthermore, we have provided preliminary evidence that COMT is overexpressed in ovarian tissues from subjects with PCOS, thus raising the possibility that the increased levels of 2-hydroxyestrogen are at least partially due to enhanced COMT-mediated methylation rather than diminished production. Furthermore, serum estradiol levels and urinary levels of estradiol, estrone, and estriol were similar between subjects with PCOS and controls at the time of sample collection; this suggests that the observed difference in 2-hydroxyestrogen levels is highly specific and does not simply represent an overall decrease in estrogen.

Increased COMT expression in the ovary is expected to lead to a decrease in the level of 2-hydroxyestrogen and an increase in the level of in situ 2-methoxyestrogen. We measured these two levels and other estrogen metabolites levels in urine samples in lieu of measuring the ovarian levels because of the obvious difficulty in collecting fresh ovarian samples from subjects with PCOS. Using this method, we found that urinary levels of 2-hydroxyestrogen are significantly lower in women with PCOS. Interestingly, there was a significant positive correlation between urinary 2-hydroxyestrogen and serum estradiol levels in subjects with PCOS. More importantly, urinary 2-hydroxyestrogen levels negatively correlated with serum insulin levels, as well as BMI, in subjects with PCOS. The higher serum insulin levels in subjects with PCOS corresponded with lower urinary 2-hydroxyestrogen levels. There is a clinical correlation between higher serum insulin levels and the severity of the PCOS phenotype, which may suggest that urinary 2-hydroxyestrogen levels would also correlate with PCOS severity, with the lowest levels occurring in more severe cases. This correlation may not simply be a statistical phenomenon but also a mechanistic one. It is currently unclear whether the lower concentrations of 2-hydroxyestrogen directly participate in the pathogenesis of PCOS or if they are simply a surrogate marker for an aberrant pathway. Several reports have demonstrated that 2-hydroxyestrogen does function as an antiestrogen in the breast (7, 23). We reported a similar finding in the uterus (8). 2-Hydroxyestrogen is synthesized in ovarian follicles and is present in follicular fluid; it has effects that are discrete from and thus potentially additive to the actions of other trophic hormones such as gonadotropin, androgens, estradiol, catecholamines, and GH (12, 24). 2-Hydroxyestrogen stimulates steroidogenesis of the ovary (12). Catecholestrogens were effective stimulators of progesterone secretion in vitro by rats and porcine granulosa cells (24). Additionally, 2-hydroxyestrogen acts as antiestrogen and enhances the action of FSHs by augmenting hormone-stimulated cAMP production in the ovary (12). Therefore, it is conceivable that a 2-hydroxyestrogen deficiency would perturb FSH-mediated granulosa cell responses and interfere with normal follicular development.

Decreased urinary 2-hydroxyestrogen levels in subjects with PCOS may be a consequence of altered activities of COMT in the ovary or other estrogen metabolizing enzymes elsewhere in the body. We were unable to quantitatively assess COMT protein and/or RNA expression in ovarian tissues from subjects with PCOS because we do not have access to such samples. We have, however, recently demonstrated that selective COMT inhibitor Ro 41–0960 induced a dose-dependent increase in granulosa cell proliferation as well as P450 side chain cleavage transcription (25). We have also recently shown that COMT inhibitor treatment of rats decreased physiological follicular apoptosis (assessed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling test) in rat ovaries, which suggests that COMT is the central step in this pathway (26). Our hypothesis is supported by the fact that COMT is the limiting step in the rate of accumulation of catecholestrogens (10). Catecholestrogens have the highest affinity for the COMT enzyme, and hydroxylation of estrogen to catecholestrogen is followed immediately by COMT catalyzed O-methylation. Furthermore, the O-methylated products are the major urinary estrogen metabolites. Most importantly, the efficiency of the O-methylation is reflected in the extremely short half-life of the catecholestrogens (27). On the other hand, the 2-hydroxylation of estrogen to catecholestrogens in the liver and other peripheral tissues is mediated by different isoforms of cytochrome P450 with overlapping functions. There are large interindividual variations in cytochrome P450 enzyme levels (up to 20-fold) caused by genetics and environmental factors such as drug administration, tobacco smoking, alcohol ingestion, and dietary composition (28). Finally, in a recent comprehensive microarray analysis of 33,000 genes between long-term culture theca cells from subjects with PCOS, compared with healthy controls, Wood et al. (29) found no detectable difference in various estrogen-metabolizing P450 enzymes.

Higher ovarian COMT would also lead to increasing levels of 2-methoxyestrogen in the ovarian milieu. Even though there was slightly elevated urinary 2-methoxyestrogen in subjects with PCOS, it did not achieve statistical significance. Caution should be exercised when correlating levels in urine to the concentration of 2-methoxyestrogen in the ovarian microenvironment because these metabolites are actively and selectively secreted by the renal system (30). In addition, 2-methoxyestrogens are lipophilic and have a very high affinity for the sex hormone-binding protein, which may affect their excretion pattern (31). There is also the possibility that the power of this study may not have been sufficient for the difference to reach statistical significance. The follicle is the site of 2-methoxyestrogen production in the ovary, with concentrations of 30 nmol/liter (10.5 ng/ml) in humans, and 0.82 µM (247 ng/ml) in mare follicles (32, 33). 2-Methoxyestrogen is lipophilic and may concentrate in cell membranes or other cellular compartments at even higher concentrations. It has been shown to inhibit proliferation and angiogenesis of ovarian follicle, and it is capable of inducing granulosa cell atresia in culture and triggering follicular apoptosis in mice ovaries (14, 24).

In this study, we measure estrogen metabolites in first-void urinary specimens. Others have used 24-h urine collection or a fixed time period of collection (34). Levels of 2-hydroxyestrogen in first-void urine were found to be representative of a 24-h collection (35). Age, weight, smoking status, or CYP1A1 genotype did not appear to have an effect on the urinary 2-hydroxyestrogen/creatinine levels (36). This is in partial agreement with our data that found no correlation between age and urinary 2-hydroxyestrogen levels in our PCOS cohort (Table 4Go). Weight, however, negatively correlated with 2-hydroxyestrogen levels in our study. This is possibly due to differences in the genetic and demographic backgrounds of the population from which the data were collected in the two studies. Obese patients were overrepresented in our study; therefore, the association between obesity and 2-hydroxyestrogen was more evident. The role of obesity in the pathogenesis of PCOS and its contribution to the perturbed estrogen metabolism in PCOS is unsettled (37). Our findings do not address this issue because we have not looked at the quantitative expression of COMT in adipose tissue. As indicated earlier, however, in this study, there was no difference in mean plasma estradiol level (Table 1Go) or urinary estrogen (estradiol, estrone, and estriol) levels (Table 2Go) between the PCOS and control groups.

In conclusion, we have reported here for the first time that there are significantly lower levels of urinary 2-hydroxyestrogen in subjects with PCOS when compared with normal controls. We have also demonstrated higher expression of immunoreactive COMT in PCOS ovaries, compared with normal ovaries. This finding may provide some insight into the pathophysiology of PCOS as well as propose novel therapeutic targets. Further work will be needed to elucidate the significance of perturbed estrogen metabolism and the role of different estrogen receptors in the pathogenesis of PCOS.


   Acknowledgments
 
We appreciate the technical assistance of Ye Wang and the statistical help of Dr. Alai Tan. We confirm that all individuals acknowledged for contributions to the study have explicitly agreed to this designation.


   Footnotes
 
This work was supported by Grants HD046228, HD40207, and Contract NO1-CO-12400 from the National Cancer Institute, National Institutes of Health. The study sponsors had no role in the study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit the paper. T.D.V. acknowledges that the content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does the mention of trade names, commercial products, or organizations imply endorsement by the U.S. government.

Disclosure Statement: The authors have nothing to disclose.

First Published Online May 29, 2007

Abbreviations: BMI, Body mass index; COMT, catechol-O-methyl transferase; PCOS, polycystic ovary syndrome.

Received December 8, 2006.

Accepted May 23, 2007.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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