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Center for Research in Reproduction (S.C., C.R.M., C.A.E., J.C.M.) and Division of Endocrinology, Department of Internal Medicine (S.C., C.R.M., C.A.E., J.C.M.), University of Virginia Health System, Charlottesville, Virginia 22908; and Division of Reproductive Endocrinology, Department of Reproductive Medicine, University of California-San Diego (R.Y.Y., R.J.C.), La Jolla, California 92093
Address all correspondence and requests for reprints to: Dr. Sandhya Chhabra, Center for Research in Reproduction, Box 800391, University of Virginia Health System, Charlottesville, Virginia 22908. E-mail: skc6u{at}virginia.edu.
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Abstract |
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Introduction |
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Mean plasma LH concentrations, LH pulse frequency, and LH pulse amplitude are increased in PCOS compared with normally cycling women in the early to midfollicular phase. Administration of estrogen (E2) and progesterone (P) can decrease pulsatile LH secretion in PCOS (4, 5, 6), and the attenuation of GnRH pulse frequency is associated with subsequent selective FSH secretion with follicular maturation (5). Data in humans (7) have shown that more rapid GnRH frequencies are associated with increased LH and decreased FSH secretion and rodent studies have indicated increased expression of the LHß gene (8). These findings suggest that the increased GnRH pulse frequency in PCOS increases LH synthesis and secretion, leading to persistent LH stimulation of the ovaries, increased ovarian androgen secretion, and impaired follicular maturation.
Adult women with PCOS and hyperandrogenemic (HA) adolescents both demonstrate abnormalities of ovulation and abnormally elevated LH pulsatility (4, 9). Most women with PCOS have menstrual irregularities dating back to menarche. Venturoli et al. (10) showed that these women have abnormalities in LH secretion as early as before menarche, and detailed studies (11, 12, 13) have confirmed this observation. Data suggest that adult women with PCOS are relatively insensitive to P inhibition of LH (GnRH) pulse secretion (14, 15), an abnormality that can be reversed by blockade of androgen action (16). It is unclear, however, when this insensitivity to P develops, or if this abnormality of steroid feedback is present in HA adolescents.
We hypothesized that adult PCOS and adolescent HA are due in part to dysregulation of the hypothalamic-pituitary-ovarian axis. Adults with PCOS demonstrate increased mean LH concentration and increased LH pulse frequency, the latter reflecting a persistently rapid GnRH pulse frequency (approximately one pulse per hour) (17). Adolescent girls with hyperandrogenemia also demonstrate increased LH pulse frequency compared with age-matched controls (9, 12, 13). As a consequence of elevated LH, relatively low levels of FSH, and increased plasma testosterone (T) levels, follicular maturation and ovulation are impaired. To evaluate this further, we studied HA adolescents and normal controls (NC) by measuring LH pulse frequency before and after a 7-d course of exogenous E2 and P to produce midluteal levels.
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Subjects and Methods |
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Normal and HA adolescents were recruited from the pediatric and adult endocrinology clinics and by advertisement. Fourteen girls were classified as HA based on the presence of clinical evidence of hyperandrogenemia, including hirsutism (excess facial hair and/or male escutcheon) and either oligo- or amenorrhea (cycle length > 35 d). Nine adolescents with no evidence of hyperandrogenism served as controls.
The human investigation committee and the General Clinical Research Center (GCRC) advisory committee of the University of Virginia Health System approved this study. Informed assent and consent were obtained from all study volunteers and their parents, respectively. Subjects came to the GCRC for an out-patient screening exam. A medical history and physical exam were performed. Blood was drawn at 0800 h for complete blood count, chemistry and liver panels, prolactin, LH, FSH, total T, 17-hydroprogesterone, dehydroepiandrosterone sulfate (DHEA-S), and TSH. With the exception of total T and LH in HA subjects, all laboratory values were within normal limits.
Normally cycling subjects were initially admitted to the GCRC between cycle d 8 and 10. HA subjects were admitted at least 60 d after the last episode of menstrual bleeding, except for one HA subject who was studied on d 12 because she reported vaginal bleeding every 35–45 d. Subjects were admitted to the GCRC at 1700 h (d 0). An iv line was placed in the forearm vein on arrival, and pregnancy and anemia were excluded in all participants with measurements of human chorionic gonadotropin-ß and hemoglobin, respectively. Blood samples were collected between 1900 and 0700 h. Samples were taken every 10 min for LH and FSH and every 2 h for E2, P, and T. Lights were extinguished at 2300 h to facilitate sleep. An additional fasting sample was obtained between 0300 and 0600 h for androstenedione, insulin, SHBG, and estrone (E1) determinations. GnRH (25 ng/kg) was administered at 0600 h (after 11 h of sampling) to determine LH responsiveness to GnRH stimulation in 15 of the 23 subjects. This procedure was discontinued when GnRH became unavailable at our institution. All subjects who received GnRH had an appropriate LH response. The subjects were offered meals at standard GCRC meal times.
Starting on the day of GCRC discharge, subjects began oral estradiol (Estrace, Warner Chilcott, Rockaway, NJ; 0.5–1.0 mg daily). Subjects also began oral P suspension (25–100 mg at 0700, 1500, and 2300 h). Estradiol and P doses were based on subject age and body mass index (BMI). To achieve mean plasma concentrations over the range of 2–8 ng/ml for 7 d, the oral P suspension was formulated by the research pharmacy using micronized P (PharmaTek, Huntington, NY) (18). Each subject was instructed to eat a small snack with the micronized P to improve the consistency of absorption. Subjects were also given iron supplements (ferrous gluconate, 325 mg daily or twice daily depending on BMI). On study d 3 and 5, subjects had blood E2 and P levels measured 2 h after the 1500 h P dose to ensure compliance with these medications. The second GCRC admission on d 7 was identical with the d 0 study, except that E2 and P were continued. After completion of sampling, subjects discontinued oral E2 and P and were given iron supplements for 1 month. After completion of the study, HA subjects were given the option of being seen by a pediatric endocrinologist for additional treatment if they had not already established care with a physician.
Hormonal measurements
All samples from each individual were analyzed in duplicate in the same assay for each hormone. All samples were analyzed at the University of Virginia Center for Research and Reproduction Ligand Core Laboratory. LH and FSH were measured using chemiluminescence (Diagnostic Products Corp., Los Angeles, CA), with assay sensitivities of 0.1 and 0.05 IU/liter. Intraassay coefficients of variation (CVs) were 4.1% and 5.0%, and interassay CVs were 5.3% and 6.9%, respectively. E2, P, and T were measured by RIA (Diagnostic Systems Laboratories, Inc., Webster, TX); assay sensitivities were 1.5 pg/ml, 0.05 ng/ml, and 5 ng/ml, respectively; intraassay CVs were 6.0%, 6.7%, and 9.0%, respectively; and interassay CVs were 15.1%, 7.5%, and 11.4%, respectively. E1 was measured by RIA (Diagnostic Systems Laboratories, Inc.; sensitivity, 15 pg/ml; intraassay CV, 8.8%; interassay CV, 13.8%). SHBG was measured using chemiluminescence (Diagnostic Products Corp.; sensitivity, 0.2 nmol/ml; intraassay CV, 3.5%; interassay CV, 3.8%). Free T was calculated from total T and SHBG using the following formula: FT = T – (N)(FT)/(KT)(SHBG) – (KT)(T) + (N)(KT)(FT), where FT is the free T concentration (picomoles per liter), KT is the association constant of SHBG (1.0 x 109) for T, T is the total T concentration (nanograms per deciliter), SHBG is the SHBG concentration (nanomoles per liter), and N = (KA)(CA) + 1, where KA is the association constant of albumin (3.6 x 104) for T, and CA is the concentration of albumin (assumed to be 4.3 g/dl) (19, 20).
Data and statistical analysis
All data are presented as the mean ± SEM unless otherwise indicated. LH pulses were identified using the computer algorithm Cluster 7, with parameters of threshold change corresponding to a t statistic of 2.45 for both up- and downstrokes. Missing LH values represented less than 0.5% of the total and were not replaced. Based on previous studies, pulses defined by the Cluster program were accepted if the increment in LH was greater than 0.25 IU/liter for pulses with a peak value less than 1 IU/liter, greater than 0.5 IU/liter for pulses with a peak value between 1 and 5 IU/liter, and greater than 1 IU/liter for pulses with a peak value greater than 5 IU/liter (21).
All hypothesis tests were two-sided and were conducted at the 0.05 level of significance. Given the small sample of subjects studied, we employed nonparametric statistical tests, and exact tests were routinely performed. Two-sample exact Wilcoxon tests were used for comparisons between NC and HA girls. The apparent sensitivity of the GnRH pulse generator to suppression by P was assessed as 1) the slope defined by the reduction in LH pulses per 11 h vs. the d 7 P concentration, and 2) the slope defined by the percent reduction in LH pulses per 11 h vs. the d 7 P concentration (see Fig. 1
). Exact Wilcoxon rank-sum tests were used to compare these slopes between HA and NC as well as to compare adolescent females with historical data for adult counterparts.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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. Three HA subjects had elevated mean second admission P concentrations, are not included in this table, and were excluded from subsequent statistical analysis. These three subjects suppressed LH pulses from a mean of 8.0 ± 1.1 to 3.3 ± 0.3 pulses/11 h, with an average P level of 25.3 ± 4.5 ng/ml. Data from these subjects confirm previous findings that P levels in excess of 15 ng/ml will suppress LH pulsatility in all adult women, including those with PCOS (14).
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Results before and after 7 d of oral E2 and P in NC and HA girls are shown in Table 2 and Fig. 1
. On d 0, LH pulse frequency was higher in the HA group (9.1 ± 0.4 vs. 7.0 ± 0.1; P < 0.01); thus, analysis of pulse frequency reduction on d 7 is shown as both absolute change and percent change (d 0–7). On d 7, both NC and HA subjects achieved similar E2, E1, and P [7.8 ± 1.3 (24.7 ± 4.2 nmol/liter) vs. 7.9 ± 0.8 ng/ml (25.0 ± 2.6 nmol/liter)] levels.
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, data for adolescent subjects are plotted on the background of normal and PCOS adult women (14). Comparison of adolescent NC and HA groups showed that the slopes of the absolute reduction in LH pulses were not different (–0.60 ± 0.14 vs. –0.36 ± 0.11), but NC showed greater suppressibility when expressed as the percent reduction (–0.09 ± 0.02 vs. –0.04 ± 0.01; P < 0.05). The latter in part reflects the complete suppression of LH pulsatility in two NC subjects, one of whom was early pubertal (Tanner stage 1). On review of the raw data, there appeared to be two distinct groups of HA subjects, those in whom LH pulsatility was suppressed similarly to NCs and those in whom LH pulses were not suppressed. Based on this observation, the data were reanalyzed to evaluate these three groups (NC, HA P-suppressible, and HA P-nonsuppressible). Comparison of the slopes of LH pulse reduction vs. P showed that five HA subjects suppressed similar to NC subjects when expressed as the absolute reduction in LH pulses (HA P-suppressible, –0.69 ± 0.12; NC, –0.60 ± 0.14), and four HA suppressed similar to NC subjects when the percent reduction was used (HA P-suppressible, –0.08 ± 0.01; NC, –0.09 ± 0.02). The HA P-nonsuppressible subjects (slopes, –0.08 ± 0.03 and –0.014 ± 0.006 for LH pulse reduction and percent reduction, respectively) were different from both the HA P-suppressible group and NC when either reduction or percent reduction was used for analysis (P < 0.05 and P < 0.005, respectively).
The two groups of HA subjects responded differently despite achieving similar levels of E2 and P on d 7 [E2, 113.4 ± 14.8 (416 ± 54 pmol/liter) and 137.8 ± 31.5 pg/ml (506 ± 115 pmol/liter); P, 8.2 ± 1.5 ng/ml (25.9 ± 4.8 nmol/liter) and 7.5 ± 0.5 ng/ml (23.9 ± 1.5 nmol/liter) for the HA P-nonsuppressible and HA P-suppressible, respectively]. P levels determined on d 3 and 5 of the study confirmed medication compliance, with peak values approximately 3-fold greater than the average d 7 P levels for each subject. The two groups were also similar in regard to age (17.1 ± 0.6 and 15.4 ± 0.7 yr), years post menarche (4.3 ± 0.8 and 3.0 ± 0.3 yr), BMI (33.2 ± 3.6 and 35.8 ± 2.8 kg/m2), and BMI for age percentile (85.1 ± 12.0 and 97.8 ± 1.0). HA P-nonsuppressible and HA P-suppressible groups were also similar in baseline biochemistry: total T, 45.7 ± 7.5 (1.6 ± 0.3 nmol/liter) and 53.1 ± 4.4 (1.8 ± 0.2 nmol/liter) ng/dl; SHBG, 19.8 ± 5.6 and 10.2 ± 2.7 nmol/liter; free T, 43.4 ± 10.2 and 56.1 ± 4.8 pmol/liter; DHEA-S, 145 ± 38 (3.9 ± 1.0 µmol/liter) and 88.5 ± 21.3 (2.4 ± 0.6 µmol/liter) µg/dl; androstenedione, 1.63 ± 0.14 (5.7 ± 0.5 nmol/liter) and 2.09 ± 0.33 (7.3 ± 1.2 nmol/liter) ng/ml; and fasting insulin, 31.2 ± 3.3 (224 ± 23 pmol/liter) and 17.7 ± 2.7 (127 ± 19 pmol/liter) mIU/ml. Of interest, however, all the HA P-suppressible individuals were of Hispanic descent, whereas HA P-nonsuppressible subjects consisted of four Caucasians and two African-Americans (P < 0.01, Hispanic vs. non-Hispanic). Therefore, it appears that there is a subset of adolescent individuals who phenotypically are hyperandrogenemic, but, despite similar levels of plasma T, physiologically behave like their normal counterparts.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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In previous studies we showed that elevated plasma T impaired P inhibition of the GnRH pulse generator in women with PCOS and could be reversed by treatment with the androgen receptor antagonist, flutamide (16). Thus, we explored whether hyperandrogenemia exerts similar effects on hypothalamic sensitivity to E2 and P in adolescent girls. Our results indicate that some HA adolescent girls are similar to adults with PCOS in that the GnRH pulse generator is less sensitive to suppression by physiological concentrations of E2 and P, whereas other HA girls show normal LH frequency suppression with luteal levels of P. In efforts to determine why these two groups of HA adolescents behaved differently, we reviewed all clinical and biochemical data. There were no differences in levels of T or androgens, degree of obesity, or age. However, HA subjects who suppressed were fewer years after menarche and were all of Hispanic descent.
The HA P-suppressible subjects were also younger (3.0 ± 0.3 vs. 4.3 ± 0.8 yr after menarche for HA P-nonsuppressible subjects). Although this difference did not achieve significance, it suggests that the duration of exposure to androgen excess may have been shorter in the HA P-suppressible group. Van Hooff et al. (28) found that both LH and androgen concentrations increased in the first 6 yr after menarche in girls with regular menstrual cycles, irregular menstrual cycles, and oligomenorrhea. These data indicate that LH and T levels increase with gynecological age regardless of menstrual history, but comparison among these groups of girls showed that those with oligomenorrhea had significantly higher LH and T concentrations throughout the 6 yr, even at younger gynecological ages. Taken together, these findings support the concept that duration of androgen exposure could influence responses of the GnRH pulse generator to physiological levels of estradiol and P.
Interestingly, all of the HA adolescents who suppressed with physiological E2 and P administration were of Hispanic descent. These girls behaved similarly to their normal counterparts, suggesting that certain ethnicities could be protected from the hypothalamic effects of hyperandrogenemia. Several large, unbiased population studies indicate that there is no difference in the prevalence of PCOS among various ethnic groups within the United States (29, 30), although some smaller studies have demonstrated a higher incidence of PCOS in certain populations (31, 32), but to date there are no data showing that differences in response to physiological GnRH suppression exist among various ethnic groups. Other studies have shown that women of different ethnic backgrounds were more insulin resistant than weight-matched Caucasian women with PCOS (31, 33) despite similar androgen levels. The findings in our small sample support the possibility that there are as yet unidentified factors that may modify the effects of hyperandrogenemnia in individuals from different ethnic groups. Confirmation of this suggestion awaits the study of larger numbers of hyperandrogenic adolescents with different genetic backgrounds. Nonetheless, the present data show that the sensitivity of the GnRH pulse generator to P inhibition is not related simply to the degree of hyperandrogenemia; rather, it may reflect genetic factors.
In conclusion, the results indicate that abnormalities of hypothalamic function (i.e. suppression of GnRH pulse secretion by ovarian steroids) are present in some hyperandrogenemic girls during adolescence. Thus, the impaired ability of progestins to suppress GnRH secretion in adults with PCOS may have its origins in the early stages of pubertal maturation. However, not all adolescents with elevated T levels exhibit this defect, perhaps reflecting duration of exposure or currently unrecognized genetic differences.
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Acknowledgments |
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Footnotes |
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First Published Online February 22, 2005
Abbreviations: BMI, Body mass index; CV, coefficient of variation; DHEA-S, dehydroepiandrosterone sulfate; E1, estrone; E2, estrogen; HA, hyperandrogenemic; NC, normal control; P, progesterone; PCOS, polycystic ovarian syndrome; T, testosterone.
Received December 3, 2004.
Accepted February 11, 2005.
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(T3) are two NC subjects who were at earlier stages of puberty (Tanner breast stages 1 and 3, respectively). To convert P values to nanomoles per liter, multiply by 3.18.