Insulin and Messenger Ribonucleic Acid Expression of Insulin ...

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The Journal of Clinical Endocrinology & Metabolism 89(7):3561–3566 Copyright © 2004 by The Endocrine Society doi: 10.1210/jc.2003-031888

Insulin and Messenger Ribonucleic Acid Expression of Insulin Receptor Isoforms in Ovarian Follicles from Nonhirsute Ovulatory Women and Polycystic Ovary Syndrome Patients JENNIFER L. PHY, CHERYL A. CONOVER, DAVID H. ABBOTT, MICHAEL A. ZSCHUNKE, DAVID L. WALKER, DONNA R. SESSION, IAN S. TUMMON, ALAN R. THORNHILL, TIMOTHY G. LESNICK, AND DANIEL A. DUMESIC Departments of Obstetrics and Gynecology (J.L.P., D.L.W., D.R.S., I.S.T., A.R.T., D.A.D.), Internal Medicine (C.A.C., D.R.S., D.A.D.), Experimental Pathology (M.A.Z.), and Biostatistics (T.G.L.), Mayo Clinic, Rochester, Minnesota 55905; and Wisconsin Primate Research Center (D.H.A., D.A.D.) and Department of Obstetrics and Gynecology (D.H.A.), University of Wisconsin, Madison, Wisconsin 53792 Insulin action is mediated by two insulin receptor (IR) isoforms, differing in mitogenic and metabolic function. IR isoform expression might occur in human granulosa cells and could be altered in polycystic ovary syndrome (PCOS) from hyperinsulinemia. To determine the relationship between granulosa cell IR isoform expression and follicular fluid insulin concentration in individual follicles, 18 normal women and seven PCOS patients receiving gonadotropins for in vitro fertilization were studied. Glucose tolerance testing was performed before pituitary desensitization, and fasting serum insulin was measured at oocyte retrieval. Granulosa cells and fluid aspirated from the first follicle were used to determine IR isoform mRNA expression and insulin concentration, re-

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NSULIN PLAYS AN important role in regulating the response of human granulosa cells to gonadotropins. Exposure of cultured human granulosa cells to insulin stimulates sex steroid secretion and augments LH-induced progesterone release (1). Insulin action is mediated through its own receptor, which first appears in granulosa cells of preantral follicles, from immunohistochemical analysis and increases thereafter with follicle growth (2, 3). Interactions between granulosa cell insulin receptors and insulin present in the follicular fluid undoubtedly determine insulin action on the developing human follicle and its enclosed oocyte. An exaggerated response of granulosa cells to gonadotropins occurs in patients with polycystic ovary syndrome (PCOS), a reproductive disorder characterized by ovarian hyperandrogenism, chronic anovulation, and hyperinsulinemia (4, 5). Cultured granulosa cells from PCOS follicles

Abbreviations: AUC, Area under the curve; BMI, body mass index; CV, coefficient of variation; DHEAS, dehydroepiandrosterone sulfate; HSA, human serum albumin; IR, insulin receptor; IR-A, IR isoform A mediating primarily mitogenic signaling; IR-B, IR isoform B predominating in metabolic tissues; IVF, in vitro fertilization; OGTT, oral glucose tolerance test; PCOS, polycystic ovary syndrome; TVUS, transvaginal ultrasound. JCEM is published monthly by The Endocrine Society (http://www. endo-society.org), the foremost professional society serving the endocrine community.

spectively. IR isoform A mRNA expression was greater than that of IR isoform B expression in normal mural granulosa and cumulus cells, without a cell type effect. Intrafollicular insulin levels increased with adiposity and serum insulin levels at oocyte-retrieval but did not predict IR mRNA expression. Total IR mRNA expression, but not intrafollicular insulin levels, was elevated in PCOS patients, whereas intrafollicular insulin levels were increased in women with impaired glucose tolerance. Granulosa cell IR heterogeneity, together with adiposity-dependent intrafollicular insulin availability, introduces a novel mechanism by which insulin may affect granulosa cell function within the follicle. (J Clin Endocrinol Metab 89: 3561–3566, 2004)

hypersecrete estradiol in response to FSH (6). Granulosa cells of PCOS patients also overexpress LH receptors and acquire LH responsiveness at an earlier stage of development than those of normal follicles (7, 8). Moreover, follicle development during gonadotropin therapy for in vitro fertilization (IVF) is exaggerated in PCOS (9 –12) and is attenuated with concomitant metformin therapy (13). Putative mechanisms for excess insulin action on granulosa cells in PCOS include hyperinsulinemia from nonovarian insulin resistance (14 – 16), dysregulation of insulin receptor expression (16), and abnormal postinsulin receptor signaling mechanisms (5, 17). The insulin receptor (IR) exists as two separate isoforms that result from alternative splicing of exon 11 and differ by the absence or presence of 12 amino acids (18). Insulin receptor isoform A (IR-A) lacks the additional amino acids, mediates primarily mitogenic signaling and predominates in central nervous system, hemopoietic, fetal, and some tumor cells (19 –21). Insulin receptor isoform B (IR-B) contains the additional amino acids and predominates in metabolic tissues such as liver, muscle, and adipose (18). It is unknown whether insulin receptor heterogeneity exists in granulosa cells of women undergoing gonadotropin stimulation for IVF and, if so, whether its expression pattern is altered in PCOS. Using quantitative real-time RT-PCR, this study examined IR isoform expression in mural granulosa and cumulus cells from individual follicles of nonhirsute ovulatory women and

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Phy et al. • Insulin and Human Ovarian Follicles

PCOS patients undergoing gonadotropin stimulation for IVF. It also determines insulin levels within the fluid of the same follicle. The first aim of the study was to determine whether mRNA expression of IR-A and IR-B exists in human granulosa cells and, if so, whether the pattern of expression is altered by PCOS. Its second aim was to determine whether IR-A and IR-B mRNA expression in these granulosa cells is affected by intrafollicular insulin levels within the same follicle. Subjects and Methods Experimental subjects Following approval by the Mayo Institutional Review Board, 18 nonhirsute ovulatory women [age 31.1 ⫾ 2.6 yr; body mass index (BMI) 25.0 ⫾ 5.8 kg/m2 mean ⫾ sd] and seven PCOS patients (age 30.9 ⫾ 4.5 yr; BMI 30.9 ⫾ 10.2 kg/m2 mean ⫾ sd) undergoing gonadotropin therapy for IVF were recruited. Patients in the nonhirsute ovulatory (i.e. normal) group were receiving assisted reproduction for nonovarian indications, such as male or tubal factor infertility. Patients in the PCOS group had previously received unsuccessful attempts for conception with ovulation induction or had male factor infertility requiring assisted reproduction. All women provided signed informed consent before participating in the study. 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 nonhirsute ovulatory women had regular menstrual cycles occurring every 21–35 d, luteal serum progesterone values (⬎3 ng/ml), absence of hirsutism (modified Ferriman-Gallwey score ⬍ 8) (22) and normal midfollicular serum androgen levels (Table 1) (23). One patient had a unilateral oophorectomy for a benign indication. None had polycystic ovaries by transvaginal ultrasound (TVUS), defined as 10 or more follicles in one sonographic plane within or surrounding abundant stroma (24). Three women in the nonhirsute ovulatory group were obese (BMI ⬎ 30 kg/m2) (25). All PCOS patients had intermenstrual intervals greater than 35 days with either hirsutism (i.e. modified Ferriman-Gallwey score ⱖ 8) (22) and/or total or free serum testosterone levels greater than 2 sd above the mean for nonhirsute ovulatory women in our patient population (23). No PCOS patient had evidence of late-onset 21-hydroxylase deficiency (i.e. screening basal serum 17-hydroxyprogesterone level ⬎ 2.0 ng/ml) or adrenal virilizing tumor. All PCOS patients had at least one ovary

fulfilling the sonographic criteria of polycystic morphology (24) by TVUS. Three PCOS patients were obese.

Baseline blood sampling Blood sampling for immunoactive FSH, dehydroepiandrosterone sulfate (DHEAS), androstenedione, total testosterone, SHBG, and bioactive LH was performed in PCOS patients during a period of amenorrhea and in nonhirsute 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-h oral glucose tolerance test (OGTT).

Gonadotropin stimulation for IVF and oocyte retrieval Nonhirsute 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. 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 larger than 18 mm in diameter and serum estradiol ⬍ 50 pg/ml). The leuprolide acetate dose was then reduced to 0.5 mg daily until the day of human chorionic gonadotropin administration. After pituitary down-regulation, treatment with recombinant human FSH (Gonal-F, Serono Laboratories, Madrid, Spain) was administered 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 estradiol levels and two-dimensional TVUS follicle measurements were performed until at least two dominant follicles reached 18 mm or more in diameter and serum estradiol levels reached approximately 300 pg/ml per dominant follicle. Human chorionic gonadotropin (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. Follicle size was designated based on a 70% chance of obtaining a metaphase II oocyte from a follicle greater than 15 mm in diameter (26). After initial aspiration of follicular fluid uncontaminated by blood, the collection tube was changed and the same follicle was flushed with media until the oocyte was retrieved, if possible. Of 16 oocytes recovered from 18 follicles of nonhirsute ovulatory women, 14 were metaphase II, one was metaphase I, and one was atretic. Of six oocytes recovered from seven follicles of PCOS patients, five were metaphase II, and one was germinal vesicle stage.

TABLE 1. Patient characteristics Parametera

Nonhirsute ovulatory (n ⫽ 18)

PCOS (n ⫽ 7)

P

Age (yr) BMI (kg/m2) FSH (IU/liter) LH (IU/liter) DHEAS (␮mol/liter) Androstenedione (nmol/liter) Total testosterone (nmol/liter) Free testosterone index Fasting glucose (mmol/liter) Fasting insulin (pmol/liter) Glucose/insulin ratio (mg/10⫺4 U) 2-h prandial glucose (mmol/liter) 2-h prandial insulin (pmol/liter) AUC glucose release (2-h mmol/liter䡠min) AUC insulin release (2-h pmol/liter䡠min)

31.1 ⫾ 2.6 25.0 ⫾ 5.8 5.3 ⫾ 1.4 3.8 ⫾ 0.8 3.2 ⫾ 1.3 5.1 ⫾ 2.3 1.3 ⫾ 0.3 0.03 ⫾ 0.02 5.0 ⫾ 0.4 38.6 ⫾ 32.3 26.1 ⫾ 19.3 6.5 ⫾ 1.4 242.8 ⫾ 195.7 845.8 ⫾ 163.4 31,060.9 ⫾ 19,677.3

30.9 ⫾ 4.5 30.9 ⫾ 10.2 5.3 ⫾ 1.5 7.2 ⫾ 3.0 3.1 ⫾ 2.6 9.6 ⫾ 3.4 2.0 ⫾ 0.8 0.09 ⫾ 0.06 5.2 ⫾ 0.8 97.3 ⫾ 61.0 11.2 ⫾ 9.0 8.9 ⫾ 2.9 1,158.2 ⫾ 1,044.9 1,108.0 ⫾ 334.2 93,873.6 ⫾ 78,010.1

0.89 0.08 0.83 0.006 0.36b 0.01 0.01 0.002 0.65 0.03 0.03 0.003 0.01 0.05 0.04

Conversion factors: FSH, LH, IU/liter ⫽ mIU/ml; DHEAS, ␮mol/liter ⫻ 0.0027 ⫽ ng/ml; androstenedione, nmol/liter/3.49 ⫽ ng/ml; testosterone, nmol/liter/0.035 ⫽ ng/dl; glucose, mmol/liter/0.056 ⫽ mg/dl; insulin, pmol/liter/7.175 ⫽ ␮U/ml. a All values are represented as means ⫾ SD. b The lower laboratory testing limit for DHEAS was 0.8 ␮mol/liter. Values below this threshold were coded three ways: 0 ␮mol/liter, 0.8 ␮mol/liter, and undetectable. There was no significant difference by coding method between nonhirsute ovulatory women (n ⫽ 17) and PCOS patients (n ⫽ 7).


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Preparation of mural granulosa and cumulus cells

Hormone assays

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 Chemical Co., St. Louis, MO) supplemented with 5 mg/ml human serum albumin (HSA) (Irvine Scientific, Santa Ana, CA) under microscopic visualization using a 5-in. glass pipette attached to a 5-cc syringe. After repetitive rinsing, mural granulosa cells were disaggregated by pipetting in Chymotrypsin (Sigma), 2.5 mg in 1 ml PBS, at room temperature for approximately 2 min. The cell suspension was then diluted in 10 ml PBS-HSA and centrifuged at 600 g for 10 min. Cumulus cells were separated from the oocyte using 0.1% hyaluronidase (Sigma-Aldrich, Inc., St. Louis, MO) in human tubal fluid-HEPES-buffered medium (Irvine Scientific, Santa Ana, CA) with 10% Serum Substitute Supplement (Irvine Scientific) at 37 C for approximately 3 min per clinical denuding protocol. Both mural granulosa and cumulus cells were suspended in 1.0 ml PBS-HSA and centrifuged separately using an upper isolate 47.5% (Irvine Scientific) gradient to remove red blood cells and debris. Mural granulosa and cumulus cells were counted by hemocytometer and were stored in 1.5 ml Eppendorf tubes in TRIzol (Invitrogen, Life Technologies, Carlsbad, CA). Samples were snap frozen in liquid nitrogen and stored at ⫺70 C for later RNA extraction following the TRIzol protocol (Invitrogen). RNA was treated with deoxyribonuclease (DNA-free, Ambion, Inc., Austin, TX). To rule out the presence of insulin in culture media, human tubal fluid-HEPES-buffered medium with 10% serum substitute supplement and PBS-HSA medium used for mural granulosa and cumulus cell preparation were analyzed by a one-step immunoenzymatic assay (ACCESS ultrasensitive insulin, Beckman Coulter, Inc., Fullerton, CA). There was no detectable insulin (⬍0.1 ␮IU/ml) in either media.

Baseline serum FSH, LH, high-sensitivity testosterone, SHBG, and DHEAS were measured by chemiluminescent immunoassay; serum androstenedione 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). The interassay coefficients of variation (CVs) were: FSH, 6.0%; LH, 8.0%; testosterone, 4.4%; SHBG, 5.0%; DHEAS, 10.0%; androstenedione, 4.8%; insulin, 3.9%; and glucose, 1.3%. The intraassay CVs were: FSH, 5.6%; LH, 4.7%; testosterone, 4.2%; SHBG, 4.5%; DHEAS, 6.9%; androstenedione, 2.7%; insulin, 2.0%; and glucose, 0.9%. Serum insulin concentrations from day of oocyte retrieval and follicular fluid insulin were measured by RIA at the WNPRC with inter- and intraassay CVs of 4.6 and 2.4%, respectively. Free testosterone index was calculated using the ratio of total testosterone/SHBG (nanomoles/liter) (28).

RT and real-time PCR Reverse transcription was performed using TaqMan reverse transcription reagents (PE Biosystems, Foster City, CA) following the manufacturer’s instructions. Real-time quantitative PCR was performed using primer and probe sequences specific for IR-A, IR-B, and 28S rRNA (Applied Biosystems) using published sequences (27) to analyze mural granulosa and cumulus cells from one follicle per subject. In all real-time quantitative PCR, 5.0 ␮l cDNA prepared from each mural granulosa and cumulus cell sample were used for each reaction and loaded in triplicate into the same 96-well optical plate for detecting the gene of interest (i.e. IR-A, IR-B, and 28S). Standard curves dilutions were established using quantified DNA templates (single-stranded oligonucleotides, Integrated DNA Technologies, Inc., Coralville, IA) that corresponded to each gene amplicon. Using standard curve dilutions (i.e. IR-A and IR-B range 103 to 107 copies; 28S range 105 to 109 copies) loaded within each 96-well optical plate, copy numbers of IR-A and IR-B and 28S rRNA were determined using the ABI PRISM 7700 sequence detection system (Applied Biosystems). Optical plates contained samples from PCOS patients matched with samples from nonhirsute ovulatory women. Expression of IR-A and IR-B mRNA was normalized by dividing the mean copy number of the gene of interest by the mean copy number of 28S within each sample.

Follicular fluid and serum sampling on day of oocyte retrieval On the day of oocyte retrieval, blood sampling for determination of fasting serum insulin level was performed before iv sedation. Follicular fluid samples were obtained from the first follicle aspirated from each ovary. Follicular fluid was transferred to a clean Falcon centrifuge tube and centrifuged at 1800 ⫻ g for 5 min to pellet follicular debris. Total follicular fluid volume was determined by autopipette, and the follicular fluid was stored in 2.0-ml cryovials (Sarstedt, Inc. Newton, NC) at ⫺70 C. Serum and follicular fluid samples were transported on dry ice to the Wisconsin National Primate Research Center (WNPRC), University of Wisconsin (Madison, WI) for insulin determination, with correction for total protein concentration to quantitatively reflect the volume of follicular fluid present.

Statistical analysis Patient age and BMI (recorded on day of baseline blood sampling) were normally distributed and compared between groups using the Student t test. Hormonal measurements were expressed as mean ⫾ sd, and comparisons were made using the Wilcoxon rank sum test due to non-Gaussian distribution. Insulin response to OGTT was calculated by area under the curve (AUC) estimation using the trapezoidal rule. Log transformations were performed to meet assumptions in regression modeling. Linear regression analyses were used to estimate associations between IR-A or IR-B mRNA expressions and intrafollicular insulin levels from the same follicle. Linear regression analysis also was used to compare intrafollicular insulin levels with BMI, fasting, and prandial serum glucose and insulin levels before gonadotropin therapy and fasting serum insulin level on day of oocyte retrieval. Multiple linear regression analysis was used to compare IR-A and IR-B mRNA expression by granulosa cell type. Generalized estimating equations were used to adjust for intrafollicular insulin correlations between both follicles in each patient. P ⬍ 0.05 was considered significant.

Results

There were no significant differences in age or BMI between the nonhirsute ovulatory women and PCOS patients. Baseline serum LH, androstenedione, total testosterone, free testosterone index, and fasting serum insulin concentrations were higher in PCOS patients than nonhirsute ovulatory women (Table 1). Two-hour postprandial serum glucose and insulin levels and postprandial serum insulin levels by AUC measurement were greater in PCOS patients than nonhirsute ovulatory women. Impaired glucose tolerance (2-h OGTT glucose ⱖ140 mg/dl) (29) was detected in four PCOS patients (three of whom were obese) and two obese nonhirsute ovulatory women. Nonhirsute ovulatory women

In nonhirsute ovulatory women, the back-transformed mean levels of IR-A mRNA expression in cumulus cells (21878 per 108 28S mRNA) and mural granulosa cells (16218 per 108 28S mRNA) were similar (cell type effect, P ⬎ 0.05), as were the back-transformed mean levels of IR-B mRNA expression in both cell types (cumulus cells, 2042 per 108 28S; mural granulosa, 3311 per 108 28S mRNA)(cell type effect, P ⬎ 0.05) (Fig. 1A). mRNA expression of IR-A was greater than that of IR-B, adjusting for both cell types (P ⬍ 0.0001), with approximately 85% of total IR mRNA from all granulosa cells combined representing IR-A. There was a positive correlation between mural granulosa and cumulus cells from

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FIG. 1. Quantitative real-time RT-PCR of IR isoform mRNA expression in mural granulosa (MG) and cumulus cells (CC) from nonhirsute ovulatory women (A) and PCOS patients (B) undergoing gonadotropin therapy for IVF. Box plots summarize distribution of points with ends at the 25th and 75th quartiles. The line across the middle of each box identifies the median sample value. Whiskers extend to outermost data points that fall within distances computed. In both female types, IR-A was the predominant mRNA expressed in the two cell types combined, with total IR mRNA expression being significantly greater in PCOS patients than nonhirsute ovulatory women (P ⫽ 0.01). Conversion, log 10 ⫽ 1.0 factor.

granulosa, 8318 per 108 28S mRNA) (cell type effect, P ⬎ 0.05) (Fig. 1B). IR-A mRNA expression in both cell types combined was greater than that of IR-B (P ⬍ 0.0001), with 78% of total IR mRNA representing the IR-A isoform. Positive associations existed between mural granulosa and cumulus cells of the same follicle for mRNA expression of IR-A (R2 ⫽ 0.73, P ⬍ 0.0001) and of IR-B (R2 ⫽ 0.87, P ⬍ 0.0001). The level of total insulin receptor mRNA expression (i.e. IR-A and IR-B mRNA expressions combined) in cells from PCOS patients was approximately 2-fold greater than that of cells from normal women (P ⫽ 0.01), adjusting for both cell types. Intrafollicular insulin levels in PCOS patients were positively associated with BMI (P ⫽ 0.01), baseline fasting serum insulin (P ⬍ 0.0001), and postprandial serum insulin levels (2-h, P ⬍ 0.0001; AUC, P ⬍ 0.0001) before gonadotropin stimulation. Intrafollicular insulin levels also were positively associated with fasting serum insulin levels at the time of oocyte retrieval (P ⫽ 0.0005). Fasting serum insulin levels at the time of oocyte retrieval were higher in PCOS patients than in nonhirsute ovulatory women (P ⫽ 0.03); follicular fluid insulin concentrations in both female groups, however, were similar (P ⫽ 0.7). Consequently the mean follicular fluid/serum insulin ratio in PCOS patients (0.35, 0.22– 0.47 95% confidence interval) was lower than that of nonhirsute ovulatory women (P ⫽ 0.02). The follicular fluid insulin concentration in one PCOS ovary was comparable with that of the contralateral ovary (within-subject correlation ⫽ 0.71). The amount of intrafollicular insulin did not correlate with IR mRNA levels in mural granulosa (IR-A: R2 ⫽ 0.1, P ⫽ 0.4; IR-B: R2 ⫽ 0.2, P ⫽ 0.3) or cumulus cells (IR-A: R2 ⫽ 0.07, P ⫽ 0.6; IR-B: R2 ⫽ 0.3, P ⫽ 0.2) from the same follicle. Patients with glucose intolerance

the same follicle for mRNA expressions of IR-A (R ⫽ 0.73) and IR-B (R2 ⫽ 0.85). Intrafollicular insulin levels in nonhirsute ovulatory women were positively associated with BMI (P ⫽ 0.01) and fasting serum insulin levels on the day of oocyte retrieval (P ⬍ 0.0001). They were not associated with fasting serum insulin levels (P ⫽ 0.1) or postprandial serum insulin levels (2-h, P ⫽ 0.2; AUC, P ⫽ 0.2) before gonadotropin therapy. In each nonhirsute ovulatory woman, the insulin concentration in the follicle of one ovary was comparable with that in the follicle of the contralateral ovary (within-subject correlation ⫽ 0.69), with a mean follicular fluid to serum insulin ratio of 0.87 (95% confidence interval, 0.51–1.23). The amount of insulin in the follicle was not associated with the level of IR mRNA expression in mural granulosa cells (IR-A: R2 ⫽ 0.002, P ⫽ 0.9; IR-B: R2 ⫽ 0.05, P ⫽ 0.4) or cumulus cells (IR-A: R2 ⫽ 2.8e-8, P ⫽ 1.0; IR-B: R2 ⫽ 0.06, P ⫽ 0.4) from the same follicle. 2

PCOS patients

The back-transformed mean levels of IR-A mRNA expression in cumulus cells (40738 per 108 28S mRNA) and mural granulosa cells (25704 per 108 28S mRNA) of PCOS patients were comparable (cell type effect, P ⬎ 0.05). Both cell types also had similar back-transformed mean levels of IR-B mRNA expression (cumulus cells, 5495 per 108 28S; mural

In nonhirsute ovulatory women and PCOS patients combined, there was no difference in IR isoform mRNA expression based on the presence or absence of impaired glucose tolerance (mural granulosa: IR-A, P ⫽ 0.4; IR-B, P ⫽ 0.7; cumulus cells: IR-A, P ⫽ 0.6; IR-B, P ⫽ 0.6). Follicular fluid insulin concentrations, however, were elevated in women with impaired glucose tolerance (P ⫽ 0.01) (Fig. 2). Discussion

Insulin acts through its own receptor to modulate the response of granulosa cells to gonadotropins (1, 2). The present study demonstrates that insulin receptor heterogeneity occurs in mural granulosa and cumulus cells of women receiving gonadotropin therapy for IVF. IR-A is the predominant insulin receptor isoform in all granulosa cells and may affect their proliferation because IR-A has been shown to mediate mitogenic signaling in other cells (20, 30). The presence of IR-A mRNA in human granulosa cells also offers a novel mechanism by which insulin might regulate the responsiveness of these cells to gonadotropins, independent of IR-B-mediated metabolic effects on other insulin-sensitive target tissues (18, 19). Total insulin receptor mRNA expression (i.e. IR-A and IR-B mRNA expressions combined) in all granulosa cells was significantly greater in PCOS patients than in nonhirsute ovulatory women, whereas female type differences in


FIG. 2. Follicular fluid insulin levels in nonhirsute ovulatory women and PCOS patients combined, based on the presence (n ⫽ 6) or absence (n ⫽ 19) of impaired glucose tolerance. Box plots summarize distribution of points with ends at the 25th and 75th quartiles. The line across the middle of each box identifies the median sample value. Whiskers extend to outermost data points that fall within distances computed. Follicular fluid insulin concentrations were significantly elevated in women with impaired glucose tolerance (P ⫽ 0.01).

mRNA expression of individual insulin receptor isoforms were not apparent. Increased total insulin receptor mRNA expression in our PCOS patients differs from the downregulation of insulin receptor protein and the diminished insulin-induced glucose incorporation observed in primary cultures of granulosa cells from PCOS patients undergoing IVF (16, 31). Increased total insulin receptor mRNA expression in granulosa cells of our PCOS patients might represent differences in our study conditions; resistance of this cell type to insulin, similar to that previously reported in cells of these individuals (17, 31); or differential regulation of insulin receptor mRNA expression by other factors, such as IGFs (32, 33). Such increased total IR mRNA expression in granulosa cells may enhance IR/IGF-I receptor hybridization in PCOS because insulin receptor isoforms hybridize with each other and with the IGF-I receptor to form heterotetramers that bind IGFs (34). This mechanism might be responsible for the exaggerated IGF-I-induced DNA synthesis in cultured granulosa cells from PCOS patients undergoing IVF (31) and the increased ovarian IGF-I binding in rodents with experimental hyperinsulinemia (35). The primary determinant in predicting the insulin level in follicular fluid was adiposity, not PCOS. In other words, the amount of insulin in follicular fluid was positively correlated with BMI and fasting serum insulin levels at oocyte retrieval; intrafollicular insulin levels in nonhirsute ovulatory women and PCOS patients were similar. Elevated intrafollicular insulin concentration from adiposity might impair follicle development because anovulatory PCOS patients have a greater BMI than their ovulatory sisters, despite both siblings having ovarian hyperandrogenism (36). Moreover, ovulatory frequency is inversely related to body weight in prenatally androgenized female rhesus monkeys with the PCOS-like phenotype of chronic anovulation, ovarian hyperandrogenism, and hyperinsulinemia from insulin resistance (37). Adiposity-related effects on intrafollicular insulin concentration also might harm the oocyte because exposure of murine cumulus-oocyte complexes to insulin causes LH receptor up-regulation and reduced blastocyst development (38). Moreover, premature follicle luteinization and impaired

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oocyte competence accompany relative insulin excess in prenatally androgenized female rhesus monkeys undergoing gonadotropin therapy for IVF (39). In this regard, increased intrafollicular insulin concentrations were present in our women with impaired glucose tolerance, most of whom were obese, and might be responsible for the reduced fecundity previously reported in such individuals (40). Despite both female groups having the same amounts of insulin in the follicle, serum insulin levels at oocyte retrieval were significantly elevated in PCOS patients along with a slightly (but not significantly) higher BMI. The reduced mean follicle fluid/serum insulin ratio in PCOS patients suggests that transport of insulin from the circulation into the follicle may be impaired, although insulin concentrations in the blood may also be affected by increased production and/or decreased clearance. Ovaries from PCOS patients have reduced mRNA levels of both pro␣1(IV) collagen, a basement membrane component, and tissue inhibitor of metalloproteinase-3, an enzyme inhibitor that binds extracellular matrix and regulates molecular and cellular movement across the basement membrane (41, 42). Therefore, the amount of circulating insulin gaining access to the oocyte via the follicle may be influenced by proteolytic enzymes and enzyme inhibitors that affect its basement membrane. Our study required the aspiration of large FSH-primed follicles with a cumulus-oocyte complex to quantify IR mRNA expression in cumulus cells and mural granulosa cells adjacent and peripheral to the oocyte, respectively. Therefore, our results do not address the pathophysiology of premature follicle differentiation in PCOS because granulosa cells from IVF patients have already undergone terminal differentiation. Nevertheless, our finding of IR heterogeneity in mural granulosa and cumulus cells of periovulatory follicles introduces a novel mechanism by which insulin may affect granulosa cell function. One also might speculate that insulin action on granulosa cells of PCOS patients is governed by the influence of increased total IR mRNA expression on both insulin and IGF signaling and the effect of obesity on intrafollicular insulin concentration. Acknowledgments The authors acknowledge Rebekah R. Herrmann (Research Study Coordinator, Mayo Clinic, Rochester, MN) for her recruitment of patients and her many outstanding contributions to this study and Kimberly R. Kalli (Division of Endocrinology and Metabolism, Endocrine Research Unit, Mayo Clinic, Rochester, MN) for her scientific support and guidance. The authors also thank Assay Services of the National Primate Research Center (University of Wisconsin-Madison) for assistance with serum insulin and follicular fluid assays. Received October 30, 2003. Accepted March 28, 2004. Address all correspondence and requests for reprints to: Jennifer L. Phy, D.O., Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, Mayo Clinic, 200 First Street SW, Rochester, Minnesota 55905. E-mail: [email protected]. This work was supported by National Institutes of Health Grant U01 HD044650-01, Grant 5P51 RR 000167 to the National Primate Research Center, University of Wisconsin-Madison, Mayo Clinical Research Grant 2123-01, Mayo Grant M01-RR-00585 and Serono Pharmaceuticals.

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