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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2005-2142
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The Journal of Clinical Endocrinology & Metabolism Vol. 91, No. 6 2327-2333
Copyright © 2006 by The Endocrine Society

Follicle Luteinization in Hyperandrogenic Follicles of Polycystic Ovary Syndrome Patients Undergoing Gonadotropin Therapy for in Vitro Fertilization

Shu C. Foong, David H. Abbott, Michael A. Zschunke, Timothy G. Lesnick, Jennifer L. Phy and Daniel A. Dumesic

Departments of Obstetrics and Gynecology (S.C.F., J.L.P.), Biostatistics (T.G.L.), and Experimental Pathology (M.A.Z.), Mayo Clinic, Rochester, Minnesota 55905; National Primate Research Center (D.H.A., D.A.D.), University of Wisconsin, Madison, Wisconsin 53715; Department of Obstetrics and Gynecology (D.H.A.), University of Wisconsin, Madison, Wisconsin 53792; and Reproductive Medicine and Infertility Associates (D.A.D.), Woodbury, Minnesota 55125

Address all correspondence and requests for reprints to: Daniel A. Dumesic, M.D., Reproductive Medicine and Infertility Associates, 2101 Woodwinds Drive, Woodbury, Minnesota 55125. E-mail: danieldumesic{at}aol.com.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Context: Polycystic ovary syndrome (PCOS) is a reproductive disorder of ovarian hyperandrogenism and insulin resistance characterized by abnormal luteinization of small follicles. After exposure to GnRH analog/FSH stimulation for in vitro fertilization (IVF), however, it is unclear whether such PCOS follicles remain abnormally luteinized during the resumption of oocyte maturation in vivo.

Objective: The aim of this study was to determine whether PCOS follicles exposed to GnRH analog/FSH stimulation for IVF show abnormal luteinization.

Design: This study was a prospective cohort.

Setting: The setting was an institutional practice.

Patients: Eleven PCOS and 30 normoandrogenic ovulatory women were included.

Intervention(s): All subjects received GnRH analog/FSH therapy after basal serum hormone determinations.

Main Outcome Measure(s): Follicle fluid aspirated at oocyte retrieval from the first follicle of each ovary was assayed for gonadotropins, steroids, insulin, and glucose. LH receptor mRNA expression was determined in granulosa cells of the same follicle.

Results: In PCOS patients with basal hyperandrogenemia and hyperinsulinemia, total oocyte number was increased and follicle diameter was decreased, despite normal maximal serum estradiol levels. Within PCOS follicles, progesterone levels were reduced (P < 0.01), despite comparable bioactive LH and insulin levels and granulosa cell LH receptor mRNA expression; estradiol levels were normal, despite diminished FSH availability (P < 0.004). Elevated androstenedione (P < 0.01), testosterone (P < 0.001), and glucose (P < 0.01) levels also occurred. In PCOS follicles containing mature oocytes, however, elevated androgen levels were accompanied by both normal progesterone concentrations and a normal inverse relationship between glucose depletion and lactate accumulation.

Conclusion: Hyperandrogenic follicles with mature oocytes from PCOS women receiving GnRH analog/recombinant human FSH therapy for IVF show sufficient glucose utilization for normal luteinization.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
POLYCYSTIC OVARY SYNDROME (PCOS) is a common reproductive syndrome of women characterized by arrested development of small follicles, ovarian hyperandrogenism, and hyperinsulinemia from insulin resistance. Such small PCOS follicles also are prematurely advanced in development, as evidenced by cell-culture studies showing that granulosa cells from such follicles prematurely respond to LH with progesterone (P4) hypersecretion and overexpress LH receptors (1, 2). This premature granulosa cell luteinization in small PCOS follicles appears to represent insulin-induced amplification of LH action, with in vitro studies showing insulin-enhanced P4 responsiveness of granulosa cells to LH (3, 4).

Nevertheless, P4 levels may (5) or may not (6, 7, 8) be elevated in small PCOS follicles and also may rise abnormally as PCOS follicles undergo terminal differentiation (9, 10), suggesting that factors other than insulin influence intrafollicular P4 production. Exposure of cultured human granulosa-luteal cells to high concentrations of aromatizable androgens, for example, impairs P4 production in vitro, with testosterone (T) (10–6 mol/liter) reducing human chorionic gonadotropin (hCG)-stimulated P4 secretion (11) and with androstenedione (A4) (10–5 mol/liter) also diminishing insulin-induced P4 responsiveness to LH (12). Moreover, the nonaromatizable androgen dihydrotestosterone (DHT) decreases FSH-induced P4 secretion by porcine cumulus oocyte complexes (13) and inhibits rat granulosa cell proliferation (14), raising the possibility that androgen excess within the PCOS follicle impairs P4 production during its terminal differentiation.

Alternatively, glycolysis is the metabolic process by which glucose is progressively converted to lactate by granulosa cells as energy substrate for terminal differentiation and oocyte maturation (15, 16). Glycolysis by human granulosa cells is gonadotropin dependent (17), with glucose depletion and lactate accumulation in the follicle as it increases in size (17, 18). In PCOS granulosa-luteal cells exposed to insulin in vitro, glucose uptake and lactate production are decreased (19, 20), whereas glycogen synthesis is diminished (21), possibly implicating impaired glucose metabolism with abnormal luteinization in the terminally differentiated PCOS follicle.

This study examines whether follicles from PCOS patients undergoing GnRH analog/recombinant human (rh) FSH therapy for in vitro fertilization (IVF) demonstrate abnormal luteinization and, if so, whether it is associated with hyperandrogenism and/or impaired glycolysis.


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

After approval by the Mayo Institutional Review Board, 30 normoandrogenic ovulatory women and 11 PCOS patients undergoing gonadotropin therapy for IVF were recruited. Normoandrogenic ovulatory women were receiving assisted reproduction for nonovarian indications (male factor, n = 18; endometriosis, n = 4; tubal factor, n = 6, and combined tubal and male-factor infertility, n = 2), whereas PCOS patients had previously received unsuccessful attempts for conception with ovulation induction or had male-factor infertility requiring assisted reproduction. All women signed informed consent before study participation.

General inclusion criteria for all study participants included age less than 38 yr, normal serum prolactin levels, and normal thyroid function studies. No woman had galactorrhea, endometriomas, or ovarian cysts greater than 18 mm in diameter.

All normoandrogenic ovulatory women had regular menstrual cycles occurring every 21–35 d, luteal serum P4 values [>3 ng/ml (SI conversion, 3.18 nmol/liter)], absence of hirsutism (modified Ferriman-Gallwey score <8), and normal midfollicular serum androgen levels, as described previously (22). None had polycystic ovaries by transvaginal ultrasound (TVUS) (23). Six women in the normoandrogenic ovulatory group were obese [body mass index (BMI) ≥ 30 kg/m2] (24).

All PCOS patients had intermenstrual intervals more than 35 d with hirsutism (i.e. modified Ferriman-Gallwey score ≥8) and/or total or free serum T levels more than 2 SD above the mean for normoandrogenic ovulatory women in our patient population (25). No PCOS patient had evidence of late-onset 21-hydroxylase deficiency [serum 17-hydroxyprogesterone (17-OHP4) >2.0 ng/ml] or adrenal virilizing tumor. All PCOS patients had at least one ovary fulfilling the sonographic criteria of polycystic morphology (23) by TVUS. Four PCOS patients were obese.

Baseline blood sampling

Blood sampling for FSH, LH, dehydroepiandrosterone sulfate (DHEAS), A4, total T, and SHBG was performed in PCOS patients during a period of amenorrhea and in normoandrogenic ovulatory women between cycle d 5–10 of the menstrual cycle preceding IVF. On the same day, blood sampling for glucose and insulin was performed under fasting conditions and was repeated at 30-min intervals during a 75-g, 2-hr oral glucose tolerance test.

Gonadotropin stimulation for IVF and oocyte retrieval

Normoandrogenic ovulatory women began leuprolide acetate (Lupron; TAP Pharmaceuticals, Lake Forest, IL) therapy on menstrual cycle d 21 to induce pituitary down-regulation. Anovulatory PCOS patients began leuprolide acetate after initial treatment with medroxyprogesterone acetate, 10 mg orally for 10 d. In both study groups, leuprolide acetate was initiated at a dose of 1.0 mg sc each day until pituitary down-regulation was determined [e.g. no ovarian cysts >18 mm in diameter and serum estradiol (E2) <50 pg/ml]. The leuprolide acetate dose was then reduced to 0.5 mg daily until the day of hCG administration.

After pituitary down-regulation, blood sampling for gonadotropin determinations was repeated, and treatment with rhFSH (Gonal-F; Serono Laboratories, Rockland, MA) was initiated sc with a starting dose of 225 IU daily for the first 3 d of stimulation. Thereafter, daily dosing was increased or decreased as clinically indicated. Serial E2 levels and two-dimensional TVUS follicle measurements were performed until at least two dominant follicles reached at least 18 mm in diameter and serum E2 levels reached approximately 300 pg/ml per dominant follicle. hCG (10,000 IU, im) was then administered, followed by transvaginal oocyte retrieval 36 h later.

At oocyte retrieval, follicular fluid was aspirated from the first follicle of each ovary, which was selected by size (at least 15 mm in diameter) and accessibility. After initial aspiration of follicular fluid, the collection tube was changed, and the same follicle was flushed with media until the oocyte was retrieved, if possible. The same procedure was repeated on the contralateral ovary, and fluid uncontaminated by blood from each of the two follicles was individually assayed for hormone determinations (normal follicles, n = 55; PCOS follicles, n = 22; see Statistical analysis). One oocyte was collected at random from either of the two follicles for an independent study. Of 30 oocytes recovered from 30 selected follicles in normoandrogenic ovulatory women, 25 were metaphase II, two were metaphase I, one was germinal vesicle stage, one was atretic, and one was indeterminate. Of 11 oocytes recovered from 11 selected follicles in PCOS patients, nine were metaphase II and two were germinal vesicle stage.

Preparation of mural granulosa and cumulus cells

Immediately after obtaining follicular fluid and flush media from the first follicle of each ovary, mural granulosa cells were washed in Dulbecco’s PBS (Sigma, St. Louis, MO) supplemented with 5 mg/ml human serum albumin (HSA) (Irvine Scientific, Santa Ana, CA) and were prepared as described previously (22). Cumulus cells were separated from the oocyte using 0.1% hyaluronidase (Sigma) in human tubal fluid-HEPES-buffered medium (Irvine Scientific) with 10% serum substitute supplement (Irvine Scientific) at 37 C for approximately 3 min. Both mural granulosa and cumulus cells were suspended in 1.0 ml PBS-HSA, were centrifuged separately using an upper isolate 47.5% (Irvine Scientific) gradient, and were counted by a hemocytometer, after which they were stored in 1.5 ml Eppendorf tubes in TRIzol (Invitrogen, Carlsbad, CA). Samples were snap frozen in liquid nitrogen and were stored at –70 C for later RNA extraction using the TRIzol protocol (Invitrogen), followed by treatment with deoxyribonuclease (DNA-free; Ambion, Austin, TX).

RT and quantitative real-time PCR

RT was performed using TaqMan Reverse Transcription Reagents (PE Biosystems, Foster City, CA) as described previously (22). Quantitative real-time PCR was performed using primer and probe sequences specific for LH receptor and 28S rRNA (Applied Biosystems, Foster City, CA) using published sequences (26) to analyze mural granulosa and cumulus cells from one follicle per subject. Standard curve dilutions were established using quantified DNA templates (single-stranded oligonucleotides; Integrated DNA Technologies, Coralville, IA) that corresponded to each gene amplicon. Using standard curve dilutions loaded within each 96-well optical plate, copy numbers of LH receptor and 28S rRNA were determined using the ABI PRISM 7700 sequence detection system (Applied Biosystems). Expression of LH receptor mRNA was normalized by dividing the mean copy number of the gene of interest by the mean copy number of 28S within each sample. Data are expressed per 108 28S mRNA.

Follicular fluid sampling on the day of oocyte retrieval

Follicular fluid was transferred to a clean Falcon centrifuge tube, was centrifuged at 1800 x g for 5 min to pellet follicular debris, and then was stored in 2.0 ml cryovials (Sarstedt, Newton, NC) at –70 C. Follicular fluid samples were transported on dry ice to the National Primate Research Center at the University of Wisconsin (Madison, WI) for steroid, insulin, glucose, and lactate determinations, with correction for total protein concentration. There was no detectable insulin (<0.1 µIU/ml) present in any media used for cell preparation (human tubal fluid-HEPES-buffered medium with 10% serum substitute supplement and PBS-HSA) (22).

Hormone assays

Baseline serum FSH, LH, high-sensitivity T, SHBG, and DHEAS were measured by chemiluminescent immunoassay, serum A4 by RIA, serum insulin by immunoenzymatic assay, and serum glucose using the hexokinase reagent from Roche Molecular Biochemicals (Indianapolis, IN) at the Immunochemical Core Laboratory of the Mayo General Clinical Research Center (Rochester, MN), as described previously (22). Free T was calculated using the ratio of total T to SHBG (27).

Follicular fluid FSH, E2, A4, 17-OHP4, DHEA, and insulin as well as serum FSH at pituitary down-regulation were measured by RIA in the National Primate Research Center Hormone Assay Services Laboratory (22, 28, 29). The intraassay coefficients of variation (CVs) were as follows: FSH, 3.6%; E2, 5.7%; A4, 4.9%; 17-OHP4, 9.0%; DHEA, 10.8%; and insulin, 4.6%. The interassay CVs were as follows: FSH, 6.9%; E2, 18.6%; A4, 17.2%; 17-OHP4, 18.7; DHEA, 12.9%; and insulin, 7.9%. Serum and follicular fluid bioactive (bio) LH was measured by the mouse Leydig cell bioassay using the rhLH-RP1 reference preparation (22, 28, 29) to accurately reflect the follicle bio components of LH and after hCG injection, combined bio components of LH and CG. The intraassay and interassay CVs for bioLH were 13.5 and 28.2%, respectively. P4, T, and DHT were measured by an enzyme immunoassay. The intraassay CVs were as follows: P4, 3.6%; T, 1.8%; and DHT, 3.2%. The interassay CVs were as follows: P4, 20.0%; T, 19.0%; and DHT, 18.3%. Glucose was measured by the glucose oxidase method. The intraassay and interassay CVs for glucose were 2.9 and 4.0%, respectively. The intraassay and interassay CVs for lactate were 1.8 and 3.2%, respectively.

The total protein in follicular fluid aspirates was measured by the Biuret Reaction in a bovine serum albumin protein assay reagent (Pierce, Rockford, IL), and follicular fluid hormone values were adjusted for protein content (per milligram of bovine serum albumin) to quantitatively reflect the volume of follicular fluid present, as described previously (22, 28, 29).

Statistical analysis

Basal serum hormone determinations and patient/IVF cycle characteristics were compared between normoandrogenic ovulatory women and PCOS patients using Student’s t test. Logarithmic transformations were performed when necessary to meet assumptions in regression modeling. Regression models with estimation by generalized estimating equations (30) were used to compare intrafollicular hormone levels between normoandrogenic ovulatory women and PCOS patients, while adjusting for intrasubject correlations due to more than one follicle per patient (22, 28). Hormone levels in follicles containing a metaphase II (mature) oocyte were compared between normoandrogenic ovulatory women and POCS patients using Student’s t test because only one such follicle per individual was studied. Student’s t test also was used to compare LH receptor mRNA expression by granulosa cell type. Linear regression analysis was used to compare intrafollicular glucose levels with respective steroid and lactate levels. Values are expressed as mean ± SD; P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Patient and IVF cycle characteristics

Baseline characteristics of a subset of our normoandrogenic ovulatory women and PCOS patients have been described previously (22). There were no significant differences between female groups in age, BMI or serum FSH, DHEAS, and fasting serum glucose levels (Table 1Go). Serum LH, T, free T, and A4 concentrations were higher in PCOS patients than normoandrogenic ovulatory women, as were fasting serum insulin and 2-h postprandial glucose and insulin levels.


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  		TABLE 1. Characteristics of normoandrogenic ovulatory women and PCOS patients undergoing GnRH analog/rhFSH therapy for IVF

 
After pituitary desensitization, serum FSH and bioLH/CG concentrations immediately preceding rhFSH therapy did not differ between female groups (FSH, P = 0.9; bioLH/CG, P = 0.4). The amount of rhFSH administered and the duration of rhFSH treatment were similar in normoandrogenic ovulatory women and PCOS patients, causing comparable maximum serum E2 levels on the day of hCG administration (Table 2Go). Despite the same amount of gonadotropin therapy, however, total number of oocytes retrieved was greater, whereas average follicle diameter was smaller, in PCOS patients vs. normoandrogenic ovulatory women.


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  		TABLE 2. IVF cycle characteristics of normoandrogenic ovulatory women and PCOS patients undergoing GnRH analog/rhFSH therapy for IVF

 
Intrafollicular hormone concentrations

Intrafollicular FSH levels were significantly lower in PCOS patients than in normoandrogenic ovulatory women (P < 0.004), without a female group difference in intrafollicular bioLH/CG concentrations (Table 3Go). Follicular fluid P4 levels in PCOS patients also were significantly reduced (P < 0.005), whereas A4 and T concentrations were significantly elevated compared with normoandrogenic ovulatory women (A4, P < 0.006; T, P < 0.001). Otherwise, follicular fluid 17-OHP4, DHEA, DHT, and E2 concentrations were similar between the two female groups. Despite comparable amounts of insulin in follicles of the two female groups, however, intrafollicular glucose levels were significantly elevated in PCOS follicles (P < 0.01).


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  		TABLE 3. Intrafollicular hormone levels of 30 normoandrogenic ovulatory women and 11 PCOS patients undergoing GnRH analog/rhFSH therapy for IVF

 
Because oocyte maturity is positively correlated with intrafollicular P4 production (10) and glucose utilization (16), steroid and metabolic measurements also were examined in the subset of normal follicles (n = 25) and PCOS follicles (n = 9) containing a mature oocyte. Age, BMI, amount of rhFSH administered, duration of rhFSH therapy and maximum serum E2 levels on the day of hCG administration were similar between these 25 normoandrogenic ovulatory women (age, 30.8 ± 2.5 yr; BMI, 26.2 ± 6.7 kg/m2; rhFSH administered, 2173 ± 649 IU; rhFSH duration, 10.0 ± 1.4 d; maximum serum E2 level, 1915 ± 770 pg/ml) and nine PCOS patients (age, 30.6 ± 4.2 yr, P = 0.8; BMI, 31.0 ± 11.4 kg/m2, P = 0.1; rhFSH administered, 2158 ± 1128 IU, P = 0.9; rhFSH duration, 10.4 ± 2.5 d, P = 0.5; maximum serum E2 level, 2487 ± 1010 pg/ml, P = 0.1).

Intrafollicular androgen levels were higher in these nine PCOS patients (T, 44.6 ± 28.1 pg/mg; A4, 0.3 ± 0.3 ng/mg) vs. these 25 normoandrogenic ovulatory women (T, 26.2 ± 11.3 pg/mg, P < 0.009; A4, 0.2 ± 0.1 ng/mg, P < 0.08). P4, insulin, and glucose levels in PCOS follicles with mature oocytes (P4, 159.1 ± 76.5 ng/mg; insulin, 0.19 ± 0.21 µU/mg; glucose, 12.1 ± 2.2 µg/mg) were comparable with normal follicles matched for oocyte maturity (P4, 214.3 ± 102.4 ng/mg, P = 0.2; insulin, 0.12 ± 0.04 µU/mg, P = 0.2; glucose, 10.7 ± 3.4 µg/mg, P = 0.2), with all other intrafollicular steroid concentrations also being similar between female groups (data not shown).

In follicles with mature oocytes, significant negative correlations between intrafollicular glucose and lactate existed in normoandrogenic ovulatory women (Y = –1.2X + 7.2; R2 = 0.55; P < 0.001) and PCOS patients (Y = –1.4X + 8.3; R2 = 0.72; P < 0.004) (Fig. 1Go). The slopes of the linear regression lines for intrafollicular glucose and lactate concentrations were comparable in normoandrogenic ovulatory women and PCOS patients, with both slopes less than –2.0 [the molar ratio of lactate produced per glucose used by anaerobic glycolysis (17)]. There also was a significant negative correlation between intrafollicular glucose and P4 levels in normoandrogenic ovulatory women (Y = –14.9X + 81.6; R2 = 0.29; P < 0.007) and PCOS patients (Y = –10.4X + 62.8; R2 = 0.55; P < 0.025), with similar slopes of regression lines for both female groups. There were no significant correlations between intrafollicular glucose concentrations and any other steroid levels (data not shown).


Figure 1
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  		FIG. 1. Linear regression lines for glucose vs. lactate (A, B) and P4 (C, D) concentrations in follicles with mature oocytes from normoandrogenic ovulatory women (A, C; n = 25) and PCOS patients (B, D; n = 9). Significant and similar negative correlations between intrafollicular glucose and lactate levels existed in both female groups (normoandrogenic ovulatory women, Y = –1.2X + 7.2, R2 = 0.55, P < 0.001; PCOS patients, Y = –1.4X + 8.3, R2 = 0.72, P < 0.004), with both slopes less than –2.0 (the molar ratio of lactate produced per glucose used by anaerobic glycolysis). Significant and similar negative correlations between intrafollicular glucose and P4 levels also existed in both female groups (normoandrogenic ovulatory women, Y = –14.9X + 81.6, R2 = 0.29, P < 0.007; PCOS patients, Y = –10.4X + 62.8, R2 = 0.55, P < 0.025).

 
Granulosa cell LH receptor mRNA expression

In 26 of 30 follicles from normoandrogenic ovulatory women and in 8 of 11 follicles from PCOS patients, there were sufficient cumulus cells and mural granulosa cells for LH receptor mRNA determinations. The back-transformed mean levels and 95% confidence intervals (per 108 28S mRNA) of LH receptor mRNA expression in cumulus cells [135,520 (96,480–190,360)] and in mural granulosa cells [165,990 (106,950–257,610)] of normoandrogenic ovulatory women were comparable with those in cumulus cells [252,390 (77,640–820,540); P = 0.1] and in mural granulosa cells [251,690 (120,240–526,850); P = 0.3] of PCOS patients, respectively. Also, in 21 of 25 normal follicles and in 6 of 9 PCOS follicles with mature oocytes and sufficient granulosa cells for mRNA analysis, the back-transformed mean levels and 95% confidence intervals (per 108 28S mRNA) of LH receptor mRNA expression in cumulus cells and in mural granulosa cells were similar between female groups [cumulus cells: normoandrogenic ovulatory, 145,010 [95,300–220,630] vs. PCOS, 148,900 (35,130–631,090), P = 0.9; mural granulosa cells: normoandrogenic ovulatory, 151,760 (97,520–236,180) vs. PCOS, 163,390 (78,650–339,430), P = 0.9].


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
Small antral follicles of PCOS patients prematurely advance in development. Granulosa cells from small PCOS follicles show premature luteinization through their ability to hypersecrete P4 in response to LH and to overexpress LH receptors in vitro (1, 2). Such premature granulosa cell luteinization in PCOS patients has been associated with hyperinsulinemia because insulin acts through its own receptor to amplify FSH-induced granulosa cell differentiation (3, 4, 31). Specifically, in vitro studies show that insulin enhances FSH-induced P4 responsiveness of human granulosa cells to LH (3) and upregulates murine granulosa cell LH receptor expression (32), although P4 levels in small PCOS follicles may (5) or may not (6, 7) be elevated.

Without considering the positive association between oocyte maturity and intrafollicular P4 production (10), P4 levels were reduced in PCOS follicles exposed to GnRH analog/rhFSH therapy, confirming the attenuated P4 rise within the PCOS follicle as it increases in size (9). Importantly, the degree of pituitary down-regulation before gonadotropin therapy was similar in the two female groups, as were granulosa cell LH receptor mRNA expression and intrafollicular bioLH activity 36 h after hCG administration; intrafollicular insulin levels and BMI also were comparable in normal women and PCOS patients, supporting our previous findings that the amount of insulin in follicles of women is positively correlated with adiposity (22). Controlling for the amounts of intrafollicular bioLH and insulin in proximity to their own granulosa cell receptors, our data do not show exaggerated luteinization in terminally differentiated follicles of PCOS patients receiving GnRH analog/rhFSH therapy.

Instead, such PCOS follicles had reduced P4 levels with elevated androgen concentrations. Elevated intrafollicular T levels in our PCOS patients and other such individuals (10) support in vitro studies of PCOS theca cells showing increased androgen biosynthesis and augmented expression of several steroidogenic enzymes, including cytochrome P450 cholesterol side-chain cleavage, 17{alpha}-hydroxylase/17-20 lyase (P450c17), and 3ß-hydroxysteroid dehydrogenase (33, 34). Elevated levels of aromatizable androgens in our PCOS follicles vs. normal follicles, however, were less than those required in other studies to reduce hCG-stimulated P4 production (11) or to diminish insulin-induced P4 responsiveness to LH (12) in cultured human granulosa-luteal cells.

Moreover, our PCOS patients had normal intrafollicular DHT and E2 levels, showing that their follicles had undergone transition from 5{alpha}-reductase to aromatase activity during gonadotropin therapy (35, 36). In fact, E2 production by these PCOS follicles was maintained with reduced FSH availability, corresponding with exaggerated E2 responsiveness of cultured PCOS granulosa cells to FSH (7, 37). Normal DHT levels in our PCOS follicles exposed to FSH did not affect intrafollicular steroidogenesis, although elevated 5{alpha}-reductase activity in small PCOS follicles suppresses granulosa cell E2 secretion (35, 38), whereas DHT in animals impairs gonadotropin-stimulated E2 secretion (39), decreases FSH-induced P4 secretion by cultured cumulus oocyte complexes (13), and inhibits granulosa cell proliferation in vitro (14).

Also governing follicle luteinization, glucose is used by granulosa cells as energy substrate for terminal differentiation and oocyte maturation (15, 16). This gonadotropin- dependent process causes glucose depletion and lactate accumulation in the follicle as it increases in size (17, 18), with anaerobic glycolysis during oocyte maturation producing two lactate molecules for every glucose molecule metabolized (18). Controlling for oocyte maturation, normal and PCOS follicles had both comparable sizes and similar negative slopes of the regression lines for intrafollicular glucose and lactate concentrations, suggesting the same degree of anaerobic glycolysis, with a lesser degree of glucose metabolized through other pathways. Such normal and PCOS follicles also had similar negative slopes of the regression lines for intrafollicular glucose and P4 concentrations, along with comparable P4 concentrations, consistent with glucose utilization for follicle luteinization (15) and normal insulin-stimulated P4 release by cultured PCOS granulosa-luteal cells (20).

When hormone data from nonobese normoandrogenic ovulatory women (n = 24) and PCOS patients (n = 7) were analyzed independently, androgen levels remained significantly elevated (A4, P < 0.004; T, P < 0.0002), whereas FSH levels remained significantly reduced (P < 0.0001) in PCOS follicles, despite similar insulin levels between PCOS and normal follicles (P = 0.5). In these nonobese individuals, PCOS follicles and normal follicles had similar glucose (P = 0.5) and P4 (P = 0.2) levels after controlling for follicle diameter and oocyte maturity (i.e. metaphase II oocytes only). Although an adverse effect of combined obesity and PCOS on terminal follicle luteinization may be undetectable in this study because of its limited statistical power, hyperandrogenic follicles from nonobese PCOS patients undergo appropriate terminal differentiation with normal insulin exposure.

In conclusion, hyperandrogenic follicles with mature oocytes from PCOS women receiving GnRH analog/rhFSH therapy for IVF show sufficient glucose utilization for normal luteinization, which should aid oocyte developmental competence, although other effects of androgen excess on the human oocyte remain to be explored.


   Acknowledgments
 
We thank Rebekah R. Herrmann for her contribution toward the preparation of the figures in this manuscript. All human studies were approved by the Mayo Institutional Review Board.


   Footnotes
 
This work was supported by National Institutes of Health Grants U01 HD044650, R01 RR 13635, Mayo Clinical Research Grant 2123-01, Mayo Grant M01-RR-00585, and P51 RR 000167 (to the National Primate Research Center, University of Wisconsin, Madison, and Serono Pharmaceuticals). This work was partially supported by National Institutes of Health as part of the National Institute of Child Health and Human Development National Cooperative Program on Female Health and Egg Quality under cooperative agreement U01 HD044650.

The authors have nothing to declare.

First Published Online March 21, 2006

Abbreviations: A4, Androstenedione; bio, bioactive; BMI, body mass index; CG, chorionic gonadotropin; CV, coefficient of variation; DHEAS, dehydroepiandrosterone sulfate; DHT, dihydrotestosterone; E2, estradiol; HSA, human serum albumin; IVF, in vitro fertilization; P4, progesterone; PCOS, polycystic ovary syndrome; rh, recombinant human; T, testosterone; TVUS, transvaginal ultrasound.

Received September 27, 2005.

Accepted March 15, 2006.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

  1. Willis D, Watson H, Mason H, Galea R, Brincat M, Franks S 1998 Premature response to LH of granulosa cells from anovulatory women with polycystic ovaries: relevance to mechanism of anovulation. J Clin Endocrinol Metab 83:3984–3991[Abstract/Free Full Text]
  2. Jakimiuk AJ, Weitsman SR, Navab A, Magoffin DA 2001 Luteinizing hormone receptor, steroidogenesis acute regulatory protein, and steroidogenic enzyme messenger ribonucleic acids are overproduced in thecal and granulosa cells from polycystic ovaries. J Clin Metab Endocrinol 86:1318–1323
  3. Willis D, Mason H, Gilling-Smith C, Franks S 1996 Modulation by insulin of follicle-stimulating hormone and luteinizing hormone actions in human granulosa cells of normal and polycystic ovaries. J Clin Endocrinol Metab 81:302–309[Abstract]
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