Effects of metformin on inappropriate LH release in women with polycystic ovarian syndrome and insulin resistance

RBMOnline - Vol 12 No 6. 2006 669–683 Reproductive BioMedicine Online; www.rbmonline.com/Article/ www.rbmonline.com/Article/2243 on web 28 March 2006

Article Effects of metformin on inappropriate LH release in women with polycystic ovarian syndrome and insulin resistance Dr Alfredo Ulloa-Aguirre obtained his MD, DSc, and specialty degrees in Internal Medicine and Reproductive Endocrinology at the National University of Mexico. In 1980, as a Rockefeller Foundation post-doctoral fellow, he studied reproductive biology and endocrinology at the University of Pennsylvania. In 1996 and 2003 he was Visiting Professor at the Oregon National Primate Research Centre, where he is currently Collaborating Scientist. He leads the Research Unit in Reproductive Medicine at the Mexican Institute of Social Security, has published prolifically and received many awards. He is an editor of RBMOnline. His research focuses on gonadotrophins and gonadotrophin receptors, the GnRH receptor, and the neuroendocrine regulation of gonadotrophin secretion. Dr Alfredo Ulloa-Aguirre Alfredo Ulloa-Aguirre1,6, Lesly Portocarrero2, Teresa Zariñán1, Aleida Olivares3, Sebastián Carranza-Lira1, Johannes D Veldhuis4, Juan C López-Alvarenga5 1 Research Unit in Reproductive Medicine, Hospital de Ginecobstetricia ‘Luis Castelazo Ayala’, IMSS; 2Instituto Nacional de Neurología y Neurocirugía, SSA; 3Research Unit in Developmental Biology, Hospital de Especialidades, CMN SXXI, IMSS, México D.F.; 4Mayo Clinic, Rochester, MN; 5Department of Genetics, Southwest Foundation for Biomedical Research, San Antonio, TX, USA 6 Correspondence: Research Unit in Reproductive Medicine, IMSS, Apdo. Postal 99–065, Unidad Independencia, México D.F., C.P.10101, México. Tel/Fax: +52 55 56162278; e-mail: [email protected]

Abstract The role of hyperinsulinaemia in neuroendocrine abnormalities in polycystic ovarian syndrome (PCOS) is controversial. The present study applied frequent blood sampling to assess the response of LH to metformin treatment in insulin-resistant women with PCOS. Thirteen predominantly overweight women with PCOS were studied before and after treatment with 1.5 g/day metformin for 3 months. Serum LH and testosterone were measured every 10 min for 10 h; LH was measured for an additional 2 h after gonadotrophin-releasing hormone (GnRH) administration. LH pulses were characterized by cluster analysis, secretory LH episodes by a deconvolution procedure, and synchronicity of paired LH-testosterone concentrations by lag-specific cross-correlation. After treatment, basal LH concentrations, amplitude of LH pulses, LH secretory amplitude, response to exogenous GnRH, and basal testosterone concentrations significantly decreased in seven patients, whereas in the remaining women these parameters remained unaltered. Before treatment, decreased coordinate LH and testosterone release was manifested by all patients; metformin treatment led to re-establishment of the feed-back control of testosterone on LH secretory rates by −20 to 0 min. Treatment did not modify the glucose:insulin ratio or serum insulin concentrations. In conclusion, administration of metformin allowed the identification of two subsets of PCOS women in whom neuroendocrine abnormalities may improve independently of the presence of insulin resistance or hyperinsulinaemia. Keywords: insulin resistance, LH, metformin, polycystic ovarian syndrome, testosterone

Introduction Polycystic ovarian syndrome (PCOS) is one of the most common endocrine disorders in young women (Azziz et al., 2004). Although the diagnostic criteria for PCOS are still controversial, the diagnosis is generally based on the presence of reproductive manifestations, including peripubertal onset of oligo-ovulation or anovulation, clinical or biochemical hyperandrogenism and

reduced fertility (Ehrmann, 2005; Escobar-Morreale et al., 2005). A substantial (50–60%) proportion of PCOS patients show insulin resistance, which is more frequently observed in obese than in lean PCOS patients (Dunaif et al., 1989; Grulet et al., 1993; Dunaif 1997); the presence of insulin resistance in PCOS is of major significance, due to its association with an increased risk of type II diabetes mellitus, dyslipidaemia and cardiovascular disease (Dahlgren et al., 1992; Dunaif 1997; Ehrmann et al., 1999;

669

Article - LH release and metformin treatment in PCOS - A Ulloa-Aguirre et al. Legro et al., 2001; Paradisi et al., 2001; Ehrmann, 2005). The primary pathophysiological mechanisms underling this complex and heterogeneous disorder remain obscure. From a neuroendocrine perspective, a number of studies using frequent sampling over extended periods of time have documented a marked increase in mean serum LH concentrations related to augmented pulse amplitude and frequency in PCOS women, providing evidence for accelerated hypothalamic gonadotrophinreleasing hormone (GnRH) pulse generator output in this disorder (Yen et al.,., 1970; Rebar et al., 1976; Burger et al., 1985; Venturoli et al.,., 1988; Berga et al., 1993; Morales et al., 1996; Arroyo et al., 1997; Taylor et al., 1997). Concomitantly, several studies have also documented increased pituitary sensitivity to GnRH in PCOS women (Waldstricher et al., 1988; Cheung and Chang, 1995; Morales et al., 1996; Nestler and Jakubowicz, 1997), with both neuroendocrine abnormalities leading eventually to increased LH secretion as a key factor that contributes to overproduction of ovarian androgens. The role of hypothalamic−pituitary dysfunction in the pathophysiology of PCOS is emphasized by the attenuated ovarian steroid production following suppression of gonadotroph activity by GnRH analogue administration (Gilling-Smith et al., 1997). Several neuroendocrine abnormalities appear to contribute to the failure of integrative hypothalamo−pituitary−theca cells in PCOS, including disruption of the joint synchrony of LH and androgen secretion [which apparently emerges peripubertally in PCOS women (Veldhuis et al., 2001b)], low concentrations of progesterone output (presumably resulting from infrequent ovulatory cycles) (Buckler et al., 1988; Fiad et al., 1996), and impairment of negative feed-back mechanisms of ovarian steroids at the level of the hypothalamic GnRH pulse generator and/or the gonadotroph (Daniels and Berga, 1997; Pastor et al., 1998; Eagleson et al., 2003). The relationship between inappropriate LH secretion in PCOS women and insulin resistance in PCOS is unclear. In one study, the spontaneous and exogenous GnRH-stimulated LH secretion was unaltered by prolonged insulin infusion (Patel et al., 2003), and in others administration of metformin or pioglitazone (both insulin-sensitizing agents) did not modify the impairment of the hypothalamic GnRH pulse generator and/or the response to exogenous GnRH administration (Ehrman et al., 1997; Eagleson et al.,., 2003; Mehta et al., 2005). Further, in one of these studies (Eagleson et al., 2003), administration of metformin for 4 weeks increased serum LH concentrations, LH pulse amplitude and the response to exogenous GnRH administration. By contrast, several studies have documented a decrease in basal serum LH concentrations in both obese and non-obese PCOS women (Velazquez et al., 1994; Pirwany et al., 1999; Genazzani et al., 2004), as well as reduction in LH pulse amplitude under metformin treatment (Genazzani et al., 2004). Although it is known that insulin enhances GnRH actions in vitro (Adashi et al., 1981) and in vivo (López-Alvarenga et al., 2002), critical information is still scarce as to whether the pituitary sensitivity per se to endogenous and exogenous GnRH is altered by facilitating insulin action in PCOS patients.

670

To further examine the nature of neuroendocrine alterations in the reproductive axis, their relationship with insulin resistance, and their response to a widely used insulin sensitizer in women with PCOS, the following experimental strategy was applied: (i) patient selection was restricted to insulin-resistant PCOS patients; (ii) frequent sampling was carried out over extended periods of

time in basal conditions and after administration of metformin; (iii) cluster and multiparameter deconvolution analyses were performed to quantitate pulsatile LH secretion before (baseline) and after a low-dose pulse of exogenous GnRH; (iv) crosscorrelation analysis was used to investigate functional linkages between LH and testosterone; and (v) the in-vitro biological to immunological LH ratio was measured before and during metformin administration to provide an indirect indication of the occurrence of structural changes in the LH molecule in response to the insulin-sensitizing agent administered.

Materials and methods Subjects Patients with PCOS were selected from the outpatient clinic. The study was approved by the local human ethics committee. The initial criteria for eligibility included age between 18 and 40 years, presence of insulin resistance, clinical or biochemical hyperandrogenism, oligomenorrhoea (≤6 menstrual periods in the last year) or amenorrhoea, previous history of infertility, and polycystic ovaries by transvaginal ultrasound. Initially, 17 PCOS women who met the inclusion criteria agreed to participate in the study. Patients were 18–35 years-old (median, 25.7 years), exhibited normal [20.2−25 kg/m2 (median, 21.8 kg/m2, n = 4)] or increased [27.7−42.2 kg/m2 (median, 32.1 kg/m2), n = 13] body mass index (BMI), hirsutism, insulin resistance (glucose:insulin ratio <4.5), enlarged (>10 cm3 by transvaginal ultrasound) and polycystic ovaries, and had normal serum prolactin (<310 mIU/ ml) and 17α-hydroxyprogesterone (<7 nmol/l) concentrations. Screening laboratory tests of hepatic, renal, haematological, and thyroid function were normal. None had taken any medications over at least 3 months before the onset of the study, and none had diabetes mellitus. All patients were advised to use barrier contraception until the end of the study. Written informed consent was provided before participation. Four patients were eventually eliminated from the study because of pregnancy, surgery for a large ovarian cyst, refusal to continue in the study, and failure to attend on the 2nd sampling day (see below).

Experimental protocol Blood sampling was performed on two occasions, before and after metformin treatment. Oligomenorrhoeic women were studied 2–5 days after the onset of spontaneous menstrual bleeding. On each occasion, volunteers were admitted to the metabolic research ward at 0730 hours and an indwelling heparinized catheter was placed in an antecubital vein. Subjects remained recumbent and were provided light meals at 0900 hours and 1400 hours. Beginning at 0800 hours, blood samples were obtained every 10 min for 12 h. The first 10h segment was used to monitor endogenous GnRH-driven (spontaneous) LH secretion. After 10 h of blood sampling, a pulse of 100 ng/kg body weight GnRH (Serono de Mexico, SA de CV, Mexico) was injected in a rapid i.v. bolus. After the first admission, patients were given metformin (Roche-Syntex, México), 500 mg 3 times daily and then examined every month for compliance with all of their medication as assessed by pill counting. Subjects were readmitted after 12–14 weeks of metformin treatment, and blood sampled following the protocol

Article - LH release and metformin treatment in PCOS - A Ulloa-Aguirre et al. described above. Sera from both sampling days were stored frozen at –20ºC until assayed.

Assays The LH radioimmunoassay (RIA) employed 125I-radio-labelled LH-I3 (specific activity 70–90 μCi/μg protein), antiserum to human LH-2 (final dilution 1:800,000), and LER-907 as standard [1 mg LER-907 = 277 IU of the Second International Reference Preparation (IRP) of Human Menopausal Gonadotrophins] (National Hormone & Peptide Program, Torrance, CA, USA) (Castro-Fernández et al.,., 2002; López-Alvarenga et al., 2002). The detection threshold was 0.7 IU/l. Intra-assay and interassay coefficients of variation were 4.1−6.2%, and 6.1−11.3% over displacement ranges of 15–23, 45–59 and 75–84% respectively. Serum FSH concentrations were determined by RIA using the 2nd IRP-human menopausal gonadotrophin standard, as previously described (Zariñán et al., 2001). Testosterone concentrations were quantitated in each 10-h sample series by solid phase RIA (Diagnostic Products Corporation, Los Angeles, CA, USA). Serial samples from each individual were analysed in the same assay in duplicate. Steroid hormone-binding globulin (SHBG) was measured by immunofluorometry (Delfia, Wallac Oy, Turku, Finland). Oestradiol, progesterone, 17α-hydroxyprogesterone (17-OHP), androstenedione and dehydroepiandrosterone sulphate (DHEA-S) were measured in the first morning sample from each study day by solid-phase RIA (Diagnostic Products Corporation). Intra-assay coefficients of variation averaged <8% (FSH), <5% (oestradiol, androstenedione, progesterone, 17OHP, DHEA-S, and testosterone), and <3.5% (SHBG). Insulin and glucose concentrations were analysed using an enzymelinked immunosorbent assay and by the glucose-oxidase method (Aguilar-Salinas et al., 2002), respectively. Samples for FSH, steroid hormones, SHBG, glucose and insulin were analysed by duplicate in a single assay run. Basal concentrations (first morning sample) of all measurements were compared with values obtained in 23 eumenorrhoeic, ovulatory women who volunteered to participate in the study and who were sampled at 0800 hours. Nine women from this group were frequently sampled (every 10 min) over 2 h before receiving a low (100 ng/kg) GnRH dose and their LH concentration values served as reference to compare the immunoactive and bioactive LH response of the study group to exogenous GnRH administration.

In-vitro bioassay of LH In-vitro LH bioassay and RIA were applied separately to single baseline (10-h) and GnRH-stimulated (2-h) serum pools (brackets in Figure 1). Both assays employed highly purified human recombinant LH produced by Chinese hamster ovary cells (kindly provided by Organon International BV, Oss, The Netherlands) as standard for the corresponding reference curves. The LH bioassay monitors cAMP production by the human embryonic kidney-293 cell line stably transfected with the full-length human LH receptor cDNA (Castro-Fernández et al.,., 2000; López-Alvarenga et al., 2002). Total (intracellular plus extracellular) cAMP concentrations were determined by RIA after acetylation, as described earlier (Zambrano et al., 1999). To equalize volume, samples were diluted with sera collected from women treated with oral contraceptives, wherein LH immunoreactivity and bioactivity were undetectable. Each sample was assayed at three dilutions (12.5, 25 and 50 μl) in triplicate. The final concentration (vol/vol) of serum was <10%.

Assay sensitivity was 0.075 mIU (LER-907)/tube. Interassay and intra-assay coefficients of variation were <18 and <10% respectively, at the ED50.

Cluster analysis and deconvolution analysis of spontaneous and GnRHinduced LH release A conventional serum hormone concentration peak analysis method (cluster; Veldhuis and Johnson, 1986) was initially applied to analyse the pulsatile properties of spontaneous LH secretion. A threshold change corresponding to a t-statistic of 2.0 for both the peak up-stroke and peak down-stroke, with a nadir and peak cluster sizes of two and one points respectively, were designed to allow for an acceptable positive accuracy and sensitivity, as previously shown by in-vivo simulation analysis (Veldhuis and Johnson, 1986). Thereafter, deconvolution analysis was applied to quantitate the frequency, amplitude, and mass of LH secretory bursts from the 10-h (basal) and 2-h (post-GnRH) serum LH concentration time series (Veldhuis et al., 1987; Veldhuis and Johnson, 1992). To more accurately determine these parameters, a fixed two-component populational half-life value (15.6 min) was used in the program applied to analyse the data, as previously suggested (Keenan et al., 2000, 2004). Univariate approximate entropy (ApEn) was employed to quantitate pattern regularity of serial LH measurements (Pincus 1991; Veldhuis et al., 2001a). This statistical approach allows measurement of relative orderliness in time series data, with minimal dependence on mean pulse amplitude, interpulse baseline, or subthreshold sample uncertainty, and thus complements conventional pulse detection (Pincus et al., 1999; Veldhuis et al., 2001a,d). For each time series, a normalized ratio of observed-to-random ApEn was calculated as the mean ratio of observed to random ApEn values calculated by shuffling the original data series 1000 times (Veldhuis et al., 2001a). ApEn ratios below unit denote more orderly patterns of hormone release, and vice versa.

Cross-correlation analysis Cross-correlation analysis quantitates the strength of a linear relationship between successively time-lagged measurements in two paired and equally spaced time series (Veldhuis et al., 1994; Mulligan et al., 1997; Pincus et al., 1997). This procedure computes successive lag-specific Pearson’s correlation coefficients (rr values). Cross-correlation is applied for paired data values considered simultaneously (zero time lag) and at various time lags defined by multiples of the sampling interval (Veldhuis et al., 1994). In the present study, LH concentrations (time series A) were compared pairwise with those of testosterone (time series B) measured simultaneously (zero lag), later (positive lag) and earlier (negative lag). Error estimates of the cross-correlation r-values were propagated from the pooled intrasample variances, based on the series length and the number of lag units (k) considered (Mulligan et al., 1997). The overall statistical significance of group r values at any given time were appraised via the one-sample Kolmogorov−Smirnov statistic applied to the null hypothesis that the z-score distribution of r-values is random normal with zero mean and unit standard deviation (Veldhuis et al., 1994).

671

Article - LH release and metformin treatment in PCOS - A Ulloa-Aguirre et al.

Statistical analysis Paired Student’s t-test was applied to compare all measures before and after metformin treatment. One-way analysis of variance followed by the Bonferroni protected unpaired Student’s t-test were applied to compare basal concentrations of LH, FSH, steroid hormones, SHBG, testosterone/SHBG molar ratio, insulin, glucose:insulin ratio, and GnRH-stimulated LH response among different groups (responders versus non-responders or normal women versus PCOS patients), as appropriate; all measures were transformed logarithmically to equalize residual variance prior to analysis of variance (ANOVA). Areas under the LH curve (AULHC) in basal conditions (10-h sampling period) and in response to exogenous GnRH were calculated by the trapezoidal method. ΔLH was defined as the difference between the maximal exogenous GnRH-stimulated LH concentration and the mean LH concentration preceding the GnRH dose. Multivariate analysis of variance (MANOVA) was employed to analyse changes in basal testosterone and progesterone concentrations (first morning samples) over time, setting as a fixed variable the LH response to metformin treatment (response or no response in terms of LH decrease; see Results section). Basal testosterone and progesterone were log transformed and repeated measurements for both steroids were used in a nested model; post-hoc contrasts were made using the Fisher’s test. Values are reported as the mean ± SEM unless indicated. P < 0.05 was considered statistically significant.

Results Analysis of the individual serum LH concentration profiles sampled every 10 min over 10 h (spontaneous LH release) before and after metformin treatment allowed the identification of two distinct groups of PCOS women according to the LH response to administration of the insulin-sensitizing agent. For the purpose of the study, responders were defined a priori as those showing a decrease of ≥30% [which corresponds to twice the maximal interassay coefficient of variation accepted in the laboratory for the LH RIA employed (i.e. 15%)] in the AULHC in response to metformin administration. Based on this criterion, seven PCOS women were unambiguously identified as responders and six patients as non-responders (Figure 1 and Table 1). Both groups were similar in age and BMI; two women in each group were lean. Clinically, there was no improvement in cycle regularity in either group and all women remained oligomenorrhoeic during the treatment period. Pretreatment concentrations of glucose, insulin, LH, FSH, DHEA-S, progesterone and oestradiol were comparable in both groups, whereas the LH:FSH ratio, serum androgens, testosterone/SHBG index, and 17-OHP were significantly higher in the non-responsive group (all P < 0.05).

Spontaneous LH secretion

672

Figure 1 shows group mean (±SEM) serum LH concentration profiles sampled every 10 min over 10 h (spontaneous LH release). Cluster and deconvolution analyses were applied to evaluate integrated serum LH concentration responses to metformin treatment. PCOS women who responded to metformin administration according to the criteria described above, exhibited significant reductions in mean LH concentrations, nadir concentrations, valley concentrations and amplitude of serum LH pulses as disclosed by cluster analysis (all P <

0.05); no changes in these parameters were detected in the metformin-unresponsive group (Table 2). When the data were analysed by deconvolution analysis, women who responded to metformin showed a 40% reduction in the mass of LH secreted per burst and, hence, in the pulsatile LH secretion rate (Table 3). This was due to selective attenuation of LH secretory burst amplitude (i.e. a reduced maximal rate of LH secretion attained per burst). Administration of metformin was not associated with a significant alteration in LH pulse frequency as determined by either Cluster or deconvolution analyses. Figures 2 and 3 illustrate individual LH concentration and secretory profiles from each group. To estimate changes in the orderliness of LH release in PCOS women treated with metformin, ApEn was used. As shown in Table 2, the synchrony outflow of LH in the group of responders was statistically indistinguishable from that exhibited by the non-responsive group both before and after treatment. In fact, the ApEn ratio in both groups was similar to that previously reported in healthy young women sampled during the early follicular phase (Pincus et al., 1997).

GnRH-stimulated LH release Before metformin treatment, LH concentrations peaked 20 min and 25 min (median) after GnRH injection in the responder and non-responder groups, respectively; the time to achieve maximal GnRH-stimulated LH responses after treatment was also similar in both groups (30 min). At baseline, the exogenous GnRHstimulated LH secretory burst mass, absolute LH response (ΔLH), mean LH concentrations, and AULHC were comparable in both PCOS groups but higher than those exhibited by normal women. Women who responded to metformin administration showed a significant attenuation (>50%, P < 0.05) in all these measures, whereas in the non-responders they remained unaltered (Table 3 and Figure 1). The attenuation in GnRHstimulated LH response exhibited by the metformin responders was not due to differing baseline concentrations, because the per cent increments in LH response before and after treatment were significantly different (P < 0.05).

Bioactive LH concentrations and in-vitro bioactive:immunoreactive LH ratio Metformin treatment did not modify basal bioactive LH concentrations in the responders group, whereas in the nonresponsive women bioactive LH concentrations increased from 32 ± 6 to 42 ± 7 IU/l after metformin treatment (Table 4). In the responders group, basal B:I LH ratios before and after treatment remained unaltered, whereas in the group of non-responders this ratio decreased modestly from 1.9 ± 0.4 to 1.4 ± 0.3; this reduction in B:I LH ratio was apparently due to a significant increase in immunoreactive LH content within the corresponding serum pool (P < 0.05, Table 4). After GnRH administration, bioactive LH concentrations rose in both groups; under metformin treatment, the bioactive LH response to exogenous GnRH was attenuated in the group of responders but not in the metformin unresponsive group. Although the post-GnRH B:I LH ratio was higher in responders than in nonresponders both before and after treatment, these differences did not reach statistical significance. In both PCOS groups, bioactive LH and the B:I LH ratio during spontaneous (basal)

Article - LH release and metformin treatment in PCOS - A Ulloa-Aguirre et al. and exogenous GnRH-stimulated LH secretion were higher than those detected in serum pools of eumenorrhoeic women sampled during the early follicular phase of an ovulatory cycle.

Serum glucose, insulin and steroid hormone concentrations and crosscorrelation analysis of LH versus testosterone

to metformin treatment [P < 0.048 for groups of LH response (responsive and non-responsive groups) and P < 0.052 for time (pretreatment and post-treatment)]. Similar results were found for serum progesterone concentrations (MANOVA for LH groups, P < 0.087; for time, P < 0.029). Serum concentrations of androstenedione, progesterone, oestradiol and SHBG as well as the testosterone/SHBG molar index did not change in response to metformin in either PCOS group. There were slight, but statistically insignificant post-treatment reductions in serum 17-OHP concentrations in both groups.

Metformin administration did not provoke detectable changes in serum glucose or insulin concentrations; consequently, the glucose:insulin ratio remained unaltered in all but two patients (one from each group). A significant (P < 0.05) reduction in total serum testosterone concentrations (measured in the first morning sample and in samples collected over 10 h) was detected in the group of PCOS women who exhibited a significant decrease in both spontaneous and exogenous GnRHstimulated LH secretion in response to metformin administration (Table 1 and Figure 4A,B). Three patients from the nonresponder group presented a detectable (21−60%) reduction in serum testosterone concentrations in the first morning sample and five exhibited a modest, albeit not significant decrease in mean (10-h) testosterone concentrations. Nevertheless, multivariate analysis of variance revealed that in both groups basal testosterone concentrations (first morning sample) modestly declined over time independently of the LH response

Cross-correlation analysis was applied to evaluate bivariate (timelagged) linear relationships in hormone secretion. The results are summarized in Figure 5. No significant linkage between serum LH and testosterone concentrations was detected in basal conditions (before metformin treatment) when serial samples from PCOS women were analysed either separately (responders and non-responders) or as a single group. After treatment, serum LH and testosterone concentrations were negatively related at time lags of 0 and −20 min [i.e. LH rose (or fell) 0−20 min after testosterone concentration fell (or rose) reciprocally]. Although the power of the study was sufficient to demonstrate statistical significance of this negative feedback-like relationship only when all PCOS women were considered as a whole, the crosscorrelation profile exhibited by the PCOS women who responded to metformin (Figure 5B) strongly suggests that the significance of the detected relationship occurred at the expense of changes occurring in this latter group.

Figure 1. Immunoreactive LH concentrations determined in serum samples collected every 10 min for 10 h before, and for 2 h after, an i.v. pulse of exogenous gonadotrophin-releasing hormone (GnRH) (arrows) in women with polycystic ovarian syndrome (PCOS) before and after treatment with 1.5 g/day of metformin. Values are group mean (±SEM). (A) PCOS women who showed ≥30% decrease in the area under LH curve (AULHC) under metformin treatment; (B) PCOS women who did not respond to metformin treatment in terms of changes in the AULHC. The grey-shaded areas in (A) and (B) show the range of basal and exogenous GnRH-stimulated serum LH concentrations in nine normal women frequently sampled for 4 h. Aliquots of samples from each study period (basal and exogenous GnRH-stimulated, delineated by brackets above and below the concentration curves) were pooled and the resulting samples were analysed for LH immunoactivity and in-vitro bioactivity as described in Materials and methods.

673

Article - LH release and metformin treatment in PCOS - A Ulloa-Aguirre et al.

Table 1. Clinical, biochemical and hormonal data in women with polycystic ovarian syndrome, grouped according to their response to metformin treatment. Data from 23 normal women sampled during the early follicular phase of a menstrual cycle are also shown. Biochemical and hormonal values correspond to the first morning sample obtained on each study day.

Age (median and range) BMI (kg/m2) Glucose (mg/dl) Insulin (μIU/ml) G:I ratio LH (IU/l) FSH (IU/l) LH:FSH ratio AULHC (IU/l/10 h) Androstenedione (nmol/l) Testosterone (nmol/l) Mean testosterone (nmol/l/10 h) SHBG (nmol/l) Testosterone/SHBG indexd 17-OHP (nmol/l) DHEA-S (μmol/l) Progesterone (nmol/l) Oestradiol (pmol/l)

Responders (n = 7) Post-treatment Pretreatment

Non-responders (n = 6) Pretreatment Post-treatment

Normal women (n = 23)

27 (18–35) 34.2 ± 2.1 77.0 ± 4.0 29.7 ± 4.1a 3.0 ± 0.4a 19.0 ± 4.0a 12.0 ± 0.5 1.6 ± 0.4 10,447 ± 2575 9.7 ± 1.1 2.1 ± 0.2a 1.1 ± 0.1 35.8 ± 5.3a 6.6 ± 0.9a 3.9 ± 0.5 5.3 ± 0.6 2.1 ± 0.4 157 ± 18

25 (19–32) 33.0 ± 2.0 78.0 ± 7.3 34.00 ± 7.7a 2.7 ± 0.4a 27.0 ± 7.0a 10.0 ± 0.9 2.5 ± 0.5a,c 8792 ± 2660 15.1 ± 0.7a,c 3.3 ± 0.3a,c 2.0 ± 0.4c 19.4 ± 2.4a,c 19.8 ± 3.7a,c 6.2 ± 0.1a,c 6.7 ± 0.9 2.7 ± 0.3a 162 ± 26

27 (24–30) 22.4 ± 1.0 83.0 ± 1.5 9.7 ± 1.3 10.1 ± 0.9 11.0 ± 1 12 ± 2 0.92 ± 0.1 − 10.7 ± 0.7 1.4 ± 0.1 − 71.0 ± 8.1 2.5 ± 0.5 3.7 ± 0.4 5.9 ± 0.8 1.7 ± 0.1 118 ± 11

− 34.1 ± 2.3 80.0 ± 5.3 29.5 ± 5.9a 2.8 ± 0.4a 12.0 ± 2.0b 11.0 ± 1.2 1.2 ± 0.2 6447 ± 1430b 10.1 ± 0.5 1.4 ± 0.1b 0.8 ± 0.1b 32.4 ± 7.1a 5.5 ± 0.9a 3.3 ± 0.3 5.5 ± 0.8 1.3 ± 0.3 131 ± 14

− 32.2 ± 2.2 79.0 ± 5.1 35.0 ± 9.3a 3.7 ± 1.2a 26.0 ± 6.0a,c 11.0 ± 0.8 2.4 ± 0.4a,c 10,925 ± 2425c 17.2 ± 1.7a,c 2.9 ± 0.6a,c 1.8 ± 0.3c 24.2 ± 4.4a 18.8 ± 4.2a,c 5.3 ± 0.2a,c 7.2 ± 1.1 2.0 ± 0.2 197 ± 27a

P < 0.05 versus normal women. P < 0.05 versus pretreatment. P < 0.05 versus responders in the same study period. d Testosterone × 100/SHBG. BMI = 17-OHP = 17α-hydroxyprogesterone; AULHC = area under LH curve; body mass index; DHEA-S = dehydroepiandrosterone sulphate; G:I ratio = glucose: insulin ratio; SHBG = sex hormone binding globulin. a

b c

Table 2. Cluster and deconvolution analyses data, and approximate entropy (ApEn ratio) of LH in polycystic ovarian syndrome patients before and after treatment with 1.5 g of metformin per day for 3 months.

Cluster analysis No. of peaks/10 h Maximal peak height (IU/l) Incremental peak amplitude (IU/l) Peak area (IU/l) No. of valleys/10 h Mean valley levels (IU/l) Nadir concentrations (IU/L/10 h) Deconvolution analysis Peak number/10 h Pulse mass (IU/l) Pulsatile secretion rate (IU/l/10 h) Mean LH concentrations (IU/l/10 h) EpEn (1,35%) ratio P < 0.05 versus pretreatment. P < 0.05 versus responders.

a

b

674

Responders Pretreatment

Post-treatment

Non-responders Pretreatment Post-treatment

8±1 22 ± 5 7±1 212 ± 51 8±1 16 ± 4 15 ± 4

8±1 14 ± 3a 4 ± 1a 96 ± 27a 9±1 10 ± 2a 9 ± 2a

9±1 27 ± 6 8±2 181 ± 30 9±1 21 ± 5 18 ± 4

10 ± 1 29 ± 4b 8 ± 1b 183 ± 25 10 ± 1 23 ± 4b 20 ± 4b

9 13 ± 2 131 ± 25 19 ± 5 1.4 ± 0.1

10 8 ± 2a 78 ± 18a 11 ± 3a 1.3 ± 0.6

10 10 ± 2 103 ± 25 22 ± 5 1.2 ± 0.1

10 11 ± 1 116 ± 10 24 ± 4b 1.3 ± 0.1

Article - LH release and metformin treatment in PCOS - A Ulloa-Aguirre et al.

Table 3. Absolute LH response (ΔLH), mean LH concentrations, area under the LH curve, pulse mass, and % LH change over baseline concentrations in samples from exogenous gonadotrophin-releasing hormone-stimulated polycystic ovarian syndrome patients before and after treatment with 1.5 g of metformin per day for 3 months. Data from nine normal women frequently sampled (every 10 min for 4 h) during the early follicular phase of a menstrual cycle are also shown.

ΔLH (IU/l) Mean LH concentration (IU/l) AULHC (IU/l/2 h) Pulse mass (IU/l) Per cent LH change

Responders (n = 7) Pretreatment Post-treatment

Non-responders (n = 6) Pretreatment Post-treatment

Normal women (n = 9)

124 ± 44a 84 ± 33a 9199 ± 3585 118 ± 45 764 ± 153a

92 ± 33a 74 ± 25a 8782 ± 2915 93 ± 37 608 ± 159a

23 ± 4 25 ± 8 3995 ± 560 62 ± 19 331 ± 54

38 ± 9b 29 ± 7b 3625 ± 874b 41 ± 13b 445 ± 67b

177 ± 70a,c 90 ± 24a,c 10,925 ± 2657a,c 84 ± 30 623 ± 169a

P < 0.05 versus normal women. P < 0.05 versus pretreatment. c P < 0.05 versus responders in the same study period. a

b

Figure 2. Illustrative basal 10-hour profiles of serum immunoreactive LH concentrations in four women with polycystic ovarian syndrome, before and after metformin treatment. (A) and (B) show the patterns in two patients classified as responders; (C) and (D) depict the patterns from two non-responsive women. Asterisks denote the maximal peak value as disclosed by cluster analysis.

675

Article - LH release and metformin treatment in PCOS - A Ulloa-Aguirre et al.

Figure 3. Illustrative serum immunoreactive LH concentration profiles (A, C, E and G) and deconvolution-resolved LH secretory rates (B, D, F and H) in two patients with polycystic ovarian syndrome, before (left graphs) and after (right graphs) treatment with 1.5 g/day metformin. (A−D) show the patterns in a patient classified as responder; (E−H) depict the patterns from a non-responsive woman.

676

Article - LH release and metformin treatment in PCOS - A Ulloa-Aguirre et al.

Table 4. Immunoreactive and bioactive LH concentrations before (basal) and after gonadotrophin-releasing hormone (GnRH) administration in serum pools from women with polycystic ovarian syndrome before and after treatment with 1.5 g of metformin for 3 months. Data from nine normal women frequently sampled during the early follicular phase of a menstrual cycle are also shown.

Basal Immunoreactive LH (IU/l) Bioactive LH (IU/l) B:I ratio Post-GnRH Immunoreactive LH (IU/l) Bioactive LH (IL/l) B:I ratio

Responders (n = 7) Post-treatment Pretreatment

Non-responders (n = 6) Pretreatment Post-treatment

Normal women (n = 9)

14 ± 2a 29 ± 5a,d 2.2 ± 0.4d

12 ± 2a 27 ± 5a,d 2.2 ± 0.2d

18 ± 2a,d 32 ± 6a,d 1.9 ± 0.4d

42 ± 13b,c,d 42 ± 7a,b,c,d 1.4 ± 0.3

10 ± 1 10 ± 2 1.1 ± 0.1

34 ± 9 122 ± 16d 4.6 ± 1.8d

29 ± 9 68 ± 6c,d 2.9 ± 0.7d

47 ± 11d 82 ± 9d 1.8 ± 0.2d

56 ± 11b,c,d 114 ± 12b,c,d 2.2 ± 0.5d

22 ± 5 17 ± 3 0.85 ± 0.1

B:I ratio = bioactive:immunoreactive ratio. a P < 0.05 basal versus post-GnRH in the same group. b P < 0.05 versus responders in the same study period. c P < 0.05 versus pretreatment period in the same group. d P < 0.05 versus normal women.

Figure 4. Illustrative 10-h LH and testosterone (T) concentration profiles in four women with polycystic ovarian syndrome, before (black lines) and after (grey lines) treatment with 1.5 g/day metformin. (A) and (B) show the patterns in two patients classified as responders; (C) and (D) depict the patterns from two non-responder women.

677

Article - LH release and metformin treatment in PCOS - A Ulloa-Aguirre et al.

Figure 5. Median (± absolute range) cross-correlation coefficients (rr values; y-axis) plotted against various lag times (x-axis, time in min separating the successively correlated serum hormone concentrations) in 13 polycystic ovarian syndrome women (A), and patients classified as responders (B) or non-responders (C), before (left graphs) and after (right graphs) metformin treatment. Crosscorrelation analysis was applied to paired daytime serum concentration profiles of LH-testosterone. Symbols at various lags in A reflect the statistical significance of the group correlation coefficients at this lag (a, P = 0.021; b, P = 0.018).

678

Article - LH release and metformin treatment in PCOS - A Ulloa-Aguirre et al.

Discussion Polycystic ovarian syndrome is characterized by inappropriate pituitary gonadotrophin secretion, with increased release of LH and decreased concentrations of FSH, with the augmented LH release occurring at the expense of increases in both LH pulse frequency and amplitude (Yen et al.,., 1970; Rebar et al., 1976; Burger et al., 1985; Venturoli et al., 1988; Berga et al., 1993; Morales et al., 1996; Arroyo et al.,., 1997; Taylor et al., 1997; García-Rudaz et al., 1998). Although the precise mechanism(s) responsible for the enhanced LH secretion in PCOS is unclear, insulin resistance and compensatory hyperinsulinaemia, which are common in both lean and obese PCOS women, have been proposed as potential mechanisms for the gonadotrophin secretory abnormalities characteristic of this syndrome (Dahlgren et al., 1992; Dunaif, 1997; Ehrmann et al., 1999, 2005; Legro et al., 2001; Paradisi et al., 2001; Fleming, 2005). However, studies employing insulin-lowering agents to address this issue have yielded contradictory results. Some reports have found significant decrements in serum LH concentrations after treatment with insulin-sensitizing agents (Velazquez et al., 1994; Nestler and Jakubowicz, 1997; Pirwany et al., 1999; Kolodziejczyk et al., 2000; Genazzani et al., 2004), whereas others have been unable to document changes in either basal LH release or pituitary LH response to exogenous GnRH as a result of treatment (Ehrmann et al., 1997; Morin-Papunen et al., 1998a, 2000; Moghetti et al., 2000; Hung et al., 2001; Eagleson et al., 2003; Mehta et al., 2005). For example, Mehta and colleagues (2005) recently reported that administration of pioglitazone for 20 weeks to obese women with PCOS and hyperinsulinaemia did not modify mean LH concentrations, LH pulse frequency or amplitude, as well as the gonadotrophin response to exogenous GnRH, whereas in another study metformin administration over 6 months restored both LH spontaneous episodic secretion and ovarian function in non-obese, normoinsulinaemic PCOS patients (Genazzani et al., 2004). Although differences in BMI may explain these apparent discrepancies (Arantes et al., 2004), it has been shown that non-obese and obese PCOS patients do not represent distinct pathophysiological subsets of this disorder but rather a continuous spectrum of gonadotrophin abnormalities that vary with body fat (Morales et al., 1996; Taylor et al., 1997). In the PCOS women studied here, values for both LH pulse frequency and amplitude were uniformly increased when compared with those previously reported for normal women in the follicular phase of the menstrual cycle (Evans et al., 1992; South et al., 1993). In this population of predominantly overweight, insulin-resistant PCOS women, metformin administration for 3 months allowed the identification of two subsets of patients: one in which metformin was remarkably effective in decreasing both spontaneous (basal) and exogenous GnRH-stimulated LH secretion, and the other in which treatment was completely ineffective in modifying LH secretory abnormalities and increased pituitary responsiveness to exogenous GnRH. Further, in the metformin-responsive group the changes in neuroendocrine function occurred in the face of a persistently low glucose:insulin ratio, which is a useful measure of insulin sensitivity in women with PCOS (Parra et al., 1994; Legro et al., 1998). This latter finding [which should not be surprising considering the variable results on insulin resistance obtained with the use of this particular drug in lean and obese PCOS women (Ehrmann et al., 1997; Morin-Papunen et al.,

1998a, 2000; Pirwany et al., 1999; Moghetti et al., 2000; Hung et al., 2001; Arantes et al., 2004) indicates that the correction of the inappropriate LH secretion and responsiveness to exogenous GnRH exhibited by the responder group were not related to improvements in insulin resistance and/or hyperinsulinaemia. The present findings contrast with those reported by Eagleson and colleagues (2003), who rather observed a marked increase in mean LH concentrations and LH pulse amplitude following administration of metformin for 4 weeks in some, but not all the obese PCOS women studied. Although the reason for the latter contrast is not readily clear, plausible explanations may include differences in timing of study, degree of obesity and/or duration of treatment. In fact, Genazzani and colleagues (2004) recently found that administration of metformin for 6 months restored spontaneous LH secretion in non-obese, non-insulin-resistant PCOS women. At first sight, the disparity of LH responses to metformin treatment among the PCOS women studied may be disturbing. Nevertheless, it is worth noting that patients in the non-responsive group had higher LH:FSH ratios, a more marked increase in basal androstenedione, total testosterone, testosterone/SHBG index and 17-OHP concentrations, and lower SHBG concentrations than women in the responsive group. Further, women in the metformin-responsive group presented a modest yet significant (P < 0.05) reduction in serum testosterone concentrations after metformin exposure; this subset of PCOS women may therefore have had less hypothalamic and pituitary androgen exposure, allowing for improvement in mean LH concentrations and sensitivity to endogenous GnRH stimulation upon metformin treatment. In this vein, it was also interesting to find that metformin-responsive patients also showed a significant reduction (P < 0.05) in maximally GnRHstimulated LH release, which was not related to the reduction in pre-injection concentrations of the gonadotrophin since the per cent change from baseline also differed between the untreated and treated state. Given that the effects of metformin on the hypothalamic−pituitary function in the responsive PCOS group were not related to improvement in insulin resistance and compensatory hyperinsulinaemia or to weight loss, it seems reasonable to locate the hypothalamic-pituitary unit and/or the ovary as potential sites of action for metformin. At the hypothalamic−pituitary level, metformin might influence the action of a number of factors that directly or indirectly control the strength of the hypothalamic GnRH impulse and/or LH synthesis (Berga and Yenn, 1989; Morin-Papunen et al., 1998b; Spritzer et al., 2001; Veldhuis et al., 2001c; Ortega-González et al., 2005), and consequently attenuate GnRH-dependent pituitary feedforward signalling, LH release, and testosterone production. The possibility that the hypothalamic−pituitary unit may represent a target for metformin action is supported by a recent study in which administration of this drug to patients with primary hypothyroidism led to suppression of serum thyroid-stimulating hormone concentrations (Vigersky et al., 2005). The observation that metformin treatment did not modify the increased LH pulse frequency in the treatment-responsive group and that deconvolution analysis documented a marked (>50%) reduction in LH secretory burst mass after infusion of exogenous GnRH, specifically points towards the gonadotroph as a potential target for the effects of treatment on LH release, and additionally emphasizes the existence of an intrinsic (and autonomous) hypothalamic defect as a contributor for the inappropriate gonadotrophin secretion in PCOS. Alternatively,

679

Article - LH release and metformin treatment in PCOS - A Ulloa-Aguirre et al. metformin may exert actions directly in the ovary to attenuate testosterone biosynthesis, leading to reduced hypothalamic and/ or pituitary androgen exposure and correction of inappropriate LH secretion. In this scenario, the reduction of the androgenic substrate for the aromatase enzyme at both the ovarian and pituitary levels may additionally contribute to abrogate the existing exaggerated GnRH-stimulated release of LH (la Marca et al., 2002). This possibility is consistent with evidence derived from in-vitro and in-vivo studies that indicates that metformin may directly inhibit androgen production in human thecal cells (Attia et al., 2001) and attenuate human chorionic gonadotrophin-stimulated ovarian cytochrome P-450c17α activity (la Marca et al., 2000).

Huhtaniemi 1991) and/or the hyperinsulinaemia (Nagami et al., 1999), and thus may be an additional factor for the excessive ovarian androgen production in adult PCOS women. It is possible that the attenuation in testosterone levels observed in the responsive group, albeit sufficient for altering the secretory dynamics of LH, was not of the magnitude required to modify glycosylation of the gonadotrophin, which is an important determinant for both the plasma half-life and the potency of the gonadotrophin at the target cell level (Bergendah and Veldhuis, 2001). Alternatively, the persistent hyperinsulinaemia may have obscured the effect of the changes in steroid hormone milieu provoked by metformin on bioactive LH synthesis and/ or release.

Several studies have shown that both basal and GnRH stimulated LH release are unaffected by metformin treatment (Ehrmann et al., 1997; Eagleson et al., 2003). Consistent with these observations, it was found that a subset of predominantly obese women was completely unresponsive to treatment with this drug, despite presenting a clinical phenotype similar to the subgroup of patients who responded to treatment. Differences in regulators at and/or outside the reproductive axis, metformin clearance, and/or severity of the disease may account for the disparity in responses to metformin administration among PCOS women. In this vein, numerous genes involved in an array of biological functions that are differentially expressed in normal and PCOS ovaries have been recently identified (Diao et al., 2003; Wood et al., 2003; Escobar-Morreale et al., 2005), raising the possibility that several subsets of PCOS patients with differing altered gene expression may exist. Different molecular signatures among PCOS subgroups may reasonably explain the wide genetic, clinical and biochemical heterogeneity characteristic of this complex disease (Chang et al., 2005), and consequently the variety of responses to similar or identical therapeutic strategies.

In summary, in a subgroup of women with PCOS, administration of metformin for 3 months decreased both spontaneous and exogenous GnRH-stimulated LH secretion as well as basal testosterone concentrations, and allowed reestablishment of the feedback control of ovarian steroids on LH secretory dynamics, without altering LH pulse frequency and the biopotency of the gonadotrophin at the target cell level. Since the partial restoration of the inappropriate LH secretion resulting from metformin exposure occurred in the absence of detectable changes in insulin sensitivity, it is possible that the effects of this drug were exerted through direct or indirect actions at the pituitary gland, the theca cell compartment, or both. Further studies are required to more precisely identify the factor(s) subserving the response to metformin in PCOS as well as those subgroups of patients that may benefit from this particular agent.

A previous study in adolescent girls with PCOS documented a prominent disruption of the bihormonal joint synchrony between LH and testosterone (Veldhuis et al., 2001b) with loss of rapid feedforward coupling between LH and testosterone output. Several pathophysiological features of PCOS, including blunting of progestin-dependent negative feedback regulation of pulsatile LH secretion, excessive LH feedforward drive, and loss of feedback restraint manifested by the failure of high bioavailable androgen concentrations to repress LH hypersecretion, have been proposed to mediate altered feedback control in this disorder (Veldhuis et al., 2001b). The present study suggested that the decreased coordinate LH and testosterone release manifested by adolescent PCOS women persists in adulthood, and additionally that treatment with metformin led to re-establishment of a feedback control of testosterone (or its aromatization product) on LH secretory rates by −20 to 0 min. This finding suggests that the more autonomous (less feedbackdependent) LH secretion prevailing in PCOS may be partially corrected by this drug in a particular subgroup of patients.

680

In agreement with previous studies employing heterologous invitro bioassays (reviewed in Bergendah and Veldhuis, 2001), the homologous LH bioassay applied to the present study revealed elevated basal and GnRH-stimulated bioactive LH values in PCOS women, which were not modified by metformin exposure in either group. Highly bioactive LH in PCOS has been attributed to the altered sex steroid hormone milieu (Ding and

Acknowledgements The authors are indebted to the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK; Bethesda, MD, USA) and Dr AF Parlow (Harbor-UCLA Medical Centre, Torrance, CA, USA) for the human FSH and LH RIA reagents and the anti-cAMP antibody. This study was supported by grant IMSS-2002/170 from the Fondo para el Fomento de la Investigación, Instituto Mexicano del Seguro Social, México. Leslie Portocarrero is a graduate student supported by the Consejo Nacional de Ciencia y Tecnología (CONACyT), México. Dr Alfredo Ulloa-Aguirre is recipient of a Research Career Development Award from the Fundación IMSS, México.

References Adashi EY, Hsueh AJ, Yen SS 1981 Insulin enhancement of luteinizing hormone and follicle-stimulating hormone release by cultured pituitary cells. Endocrinology 108, 1441–1449. Aguilar-Salinas CA, Rojas R, Gómez-Pérez FJ et al. 2002 Prevalence and characteristics of early-onset type 2 diabetes in Mexico. American Journal of Medicine 113, 569–574. Arantes GA, Soares JM, Leme E et al. 2004 Nonobese women with polycystic ovary syndrome respond better than obese women to treatment with metformin. Fertility and Sterility 81, 355–360. Arroyo A, Laughlin GA, Morales AJ, Yen SSC 1997 Inappropriate gonadotropin secretion in polycystic ovary syndrome: influence of adiposity. Journal of Clinical Endocrinology and Metabolism 82, 3728–3733. Attia GR, Raine WE, Carr BC 2001 Metformin directly inhibits androgen production in human thecal cells. Fertility and Sterility 76, 517–524.

Article - LH release and metformin treatment in PCOS - A Ulloa-Aguirre et al. Azziz R, Woods KS, Reyna R et al. 2004 The prevalence and features of the polycystic ovary syndrome in an unselected population. Journal of Clinical Endocrinology and Metabolism 89, 2745– 2749. Berga SL, Yen SSC 1989 Opioidergic regulation of LH pulsatility in women with polycystic ovary syndrome. Clinical Endocrinology (Oxf) 30, 177–184. Berga SL, Guzick DS, Winters SJ 1993 Increased luteinizing hormone and α-subunit secretion in women with hyperandrogenic anovulation. Journal of Clinical Endocrinology and Metabolism 77, 895–901. Bergendah M, Veldhuis JD 2001 Is there a physiological role for gonadotorphin oligosaccharide heterogeneity in humans? III. Luteinizing hormone heterogeneity: a medical physiologist´s perspective. Human Reproduction 16, 1058–1064. Buckler HM, Phillips SE, Cameron IT et al. 1988 Vaginal progesterone administration before ovulation induction with exogenous gonadotropins in polycystic ovarian syndrome. Journal of Clinical Endocrinology and Metabolism 67, 300–306. Burger CW, Korsen T, Van Kessel H et al. 1985 Pulsatile luteinizing hormone patterns in the follicular phase of the menstrual cycle, polycystic ovarian disease and non-PCOD secondary amenorrhea. Journal of Clinical Endocrinology and Metabolism 61, 1126–1132. Castro-Fernández C, Olivares A, Söderlund D et al. 2000 A preponderance of circulating basic isoforms is associated with decreased plasma half-life and biological to immunological ratio of gonadotropin-releasing hormone-releasable luteinizing hormone in obese men. Journal of Clinical Endocrinology and Metabolism 85, 4603–4610. Chang WY, Knochenhauer ES, Bartolucci AA, Azziz R 2005 Phenotypic spectrum of polycystic ovary syndrome: clinical and biochemical characterization of the three major clinical subgroups. Fertility and Sterility 83, 1717–1723. Cheung AP, Chang RJ 1995 Pituitary responsiveness to gonadotrophin-releasing hormone agonist stimulation: a dose– response comparison of luteinizing hormone/follicle-stimulating hormone secretion in women with polycystic ovary syndrome and normal women. Human Reproduction 10, 1054–1059. Dahlgren E, Janson PO, Johansson S et al. 1992 Polycystic ovary syndrome and risk for myocardial infraction-evaluated from a risk factor model based on a prospective study of women. Acta Obstetrica et Gynecologica Scandinavica 71, 599–604. Daniels TL, Berga SL 1997 Resistance of gonadotropin releasing hormone drive to sex steroid-induced suppression in hyperandrogenic anovulation. Journal of Clinical Endocrinology and Metabolism 82, 4179–4183. Diao F-Y, Xu M, Hu Y et al. 2004 The molecular characteristics of polycystic ovary syndrome (PCOS) ovary defined by human ovary cDNA microarray. Journal of Molecular Endocrinology 33, 59–72. Ding YQ, Huhtaniemi I 1991 Preponderance of basic isoforms of serum luteinizing hormone (LH) is associated with high bio/ immuno ratio of LH in healthy women and women with polycystic ovarian disease. Human Reproduction 6, 346–350. Dunaif A 1997 Insulin resistance and the polycystic ovary syndrome: mechanisms and implications for pathogenesis. Endocrine Reviews 18, 774–800. Dunaif A, Segal KR, Futterweit W, Dobrjansky A 1989 Profound peripheral insulin resistance, independent of obesity, in polycystic ovary syndrome. Diabetes 38, 1165–1174. Eagleson CA, Bellows AB, Hu K et al. 2003 Obese patients with polycystic ovary syndrome: evidence that metformin does not restore sensitivity of the gonadotropin-releasing hormone pulse generator to inhibition by ovarian steroids. Journal of Clinical Endocrinology and Metabolism 88, 5158–5162. Ehrmann DA 2005 Polycystic ovary syndrome. New England Journal of Medicine 352, 1223–1236. Ehrmann DA, Barnes RB, Rosenfield RL et al. 1999 Prevalence of impaired glucose tolerance and diabetes in women with polycystic ovary syndrome. Diabetes Care 22, 141–146. Ehrmann DA, Cavaghan MK, Imperial J et al. 1997 Effects of metformin on insulin secretion, insulin action, and ovarian

steroidogenesis in women with polycystic ovary syndrome. Journal of Clinical Endocrinology and Metabolism 82, 524–530. Escobar-Morreale, HF, Luque-Ramírez M, San Millán JL 2005 The molecular-genetic basis of functional hyperandrogenism and the polycystic ovary syndrome. Endocrine Reviews 26, 251–282. Evans WS, Sollenberger MJ, Booth RA Jr et al. 1992 Contemporary aspects of discrete peak-detection algorithms. II. The paradigm of the luteinizing hormone pulse signal in women. Endocrine Reviews 13, 81–104. Fiad TM, Cunningham SK, McKenna TJ 1996 Role of progesterone deficiency in the development of luteinizing hormone and androgen abnormalities in polycystic ovary syndrome. European Journal of Endocrinology 135, 335. Fleming R 2005 Recruitment prior to ovarian stimulation: ways of improving follicular recruitment. Reproductive BioMedicine Online 10, 55–59. García-Rudaz MC, Ropelato G, Escobar ME et al. 1998 Augmented frequency and mass of LH discharged per burst are accompanied by marked disorderliness of LH secretion in adolescents with polycystic ovary syndrome. European Journal of Endocrinology 139, 621–630. Genazzani AD, Battaglia C, Malavasi B et al. 2004 Metformin administration modulates and restores luteinizing hormone spontaneous episodic secretion and ovarian function in nonobese patients with polycystic ovary syndrome. Fertility and Sterility 81, 114–119. Gilling-Smith C, Story H, Rogers V, Franks S 1997 Evidence for a primary abnormality of theca cell steroidogenesis in the polycystic ovary syndrome. Clinical Endocrinology (Oxford) 47, 93–99. Grulet H, Hecart AC, Delemer B et al. 1993 Roles of LH and insulin resistance in lean and obese polycystic ovary syndrome. Clinical Endocrinology (Oxford) 38, 621–626. Hung YNE, Ming SWA, Cung HP 2001 Effects of metformin on ovulation rate, hormonal and metabolic profiles in women with clomiphene-resistant polycystic ovaries: a randomized, doubleblinded placebo-controlled trial. Human Reproduction 16, 1625–1631. Keenan DM, Alexander SL, Irvine CHG et al. 2004 Reconstruction of in-vivo time-evolving neuroendocrine dose–response properties unveils admixed deterministic and stochastic elements in interglandular signaling. Proceedings of the National Academy of Sciences of the USA 101, 6740–6745. Keenan DM, Sun W, Veldhuis JD 2000 A stochastic biomathematical model of the male reproductive hormone system. SIAM Journal on Applied Mathematics 61, 934–965. Kolodziejczyk B, Duleba AJ, Spaczynski RZ, Pawelczyk L 2000 Metformin therapy decreases hyperandrogenism and hyperinsulinemia in women with polycystic ovary syndrome. Fertility and Sterility 73, 1149–1154. la Marca A, Morgante G, Palumbo M et al. 2002 Insulin-lowering treatment reduces aromatase activity in response to folliclestimulating hormone in women with polycystic ovary syndrome. Fertility and Sterility 78, 1234–1239. la Marca A, Obinchemti Egbe T, Morgante G et al. 2000 Metformin treatment reduces ovarian cytochrome P-450c17α response to human chorionic gonadotrophin in women with insulin resistancerelated polycystic ovary syndrome. Human Reproduction 15, 21–23. Legro RS, Kunselman AR, Dunaif A 2001 Prevalence and predictors of dyslipidemia in women with polycystic ovary syndrome. American Journal of Medicine 117, 607–613. Legro RS, Finegood D, Dunaif A 1998 A fasting glucose to insulin ratio is a useful measure of insulin sensitivity in women with polycystic ovary syndrome. Journal of Clinical Endocrinology and Metabolism 83, 2694–2698. López-Alvarenga JC, Zariñán T, Olivares A et al. 2002 Poorly controlled type I diabetes mellitus in young men selectively suppresses luteinizing hormone secretory burst mass. Journal of Clinical Endocrinology and Metabolism 87, 5507–5515. Mehta RV, Patel KS, Coffler MS et al. 2005 Luteinizing hormone secretion is not influenced by insulin infusion in women with

681

Article - LH release and metformin treatment in PCOS - A Ulloa-Aguirre et al.

682

polycystic ovary syndrome despite improved insulin sensitivity during pioglitazone treatment. Journal of Clinical Endocrinology and Metabolism 90, 2136–2141. Moghetti P, Castello R, Negri C et al. 2000 Metformin effects on clinical features, endocrine and metabolic profiles, and insulin sensitivity in polycystic ovary syndrome: a randomized, double blind, placebo-controlled 6-month trial, followed by open, longterm clinical evaluation. Journal of Clinical Endocrinology and Metabolism 85, 139–146. Morales AJ, Laughlin GA, Butzow T et al. 1996 Insulin, somatotropic, and luteinizing hormone axes in lean and obese women with polycystic ovary syndrome: common and distinct features. Journal of Clinical Endocrinology and Metabolism 81, 2854–2864. Morin-Papunen LC, Vauhkonen I, Koivunen RM et al. 2000 Endocrine and metabolic effects of metformin versus ethinyl estradiol−cyproterone acetate in obese women with polycystic ovary syndrome: a randomized study. Journal of Clinical Endocrinology and Metabolism 85, 3161–3168. Morin-Papunen LC, Koivunen RM, Ruokonen A, Martikainen HK 1998a Metformin therapy improves the menstrual pattern with minimal endocrine and metabolic effects in women with polycystic ovary syndrome. Fertility and Sterility 69, 691–696. Morin-Papunen LC, Koivunen RM, Tomás C et al. 1998b Decreased serum leptin concentrations during metformin therapy in obese women with polycystic ovary syndrome. Journal of Clinical Endocrinology and Metabolism 83, 2566–2568. Mulligan T, Iranmanesh A, Johnson ML et al. 1997 aging alters feedforward and feedback linkages between LH and testosterone in healthy men. American Journal of Physiology 42, R1407–R1413. Nagami M, Osuampke C, Kelver ME 1999 Increased bioactive luteinizing hormone levels and bio/immuno ratio in women with hyperthecosis of the ovaries: possible role of hyperinsulinemia. Journal of Clinical Endocrinology and Metabolism 84, 1685– 1689. Nestler JE, Jakubowicz DJ 1997 Lean women with polycystic ovary syndrome respond to insulin reduction with decreases in ovarian P450c17α activity and serum androgens. Journal of Clinical Endocrinology and Metabolism 82, 4075–4079. Ortega-González C, Cardoza L, Coutiño B et al. 2005 Insulin sensitizing drugs increase the endogenous dopaminergic tone in obese insulin-resistant women with polycystic ovary syndrome. Journal of Endocrinology 184, 233–239. Paradisi G, Steinberg HO, Hempfling A 2001 Polycystic ovary syndrome is associated with endothelial dysfunction. Circulation 103, 1410–1405. Parra A, Ramírez A, Espinoza de los Monteros A 1994 Fasting glucose/insulin ratio. An index to differentiate normo- from hyperinsulinemic women with polycystic ovary syndrome. Revista de Investigación Clínica 46, 363–368. Pastor CL, Griffin-Korg ML, Aloi JA et al. 1998 Polycystic ovary syndrome: evidence for reduced sensitivity of the gonadotropinreleasing hormone pulse generator to inhibition by estradiol and progesterone. Journal of Clinical Endocrinology and Metabolism 83, 582–590. Patel K, Coffler MS, Dahan MH et al. 2003 Increased luteinizing hormone secretion in women with polycystic ovary syndrome is unaltered by prolonged insulin infusion. Journal of Clinical Endocrinology and Metabolism 88, 5456–5461. Pincus SM 1991 Approximate entropy as a measure of system complexity. Proceedings of the National Academy of Sciences of the USA 88, 2297–2301. Pincus SM, Hartman ML, Roelfsema F et al. 1999 Hormone pulsatility discrimination via coarse and short time sampling. American Journal of Physiology 277, E948–E957. Pincus SM, Veldhuis JD, Mulligan T et al. 1997 Effects of age on the irregularity of LH and FSH serum concentrations in women and men. American Journal of Physiology 274, E898–E995. Pirwany IR, Yates RWS, Cameron IT, Fleming R 1999 Effects of the insulin sensitizing drug metformin on ovarian function, follicular growth and ovulation rate in obese women with oligoamenorrhoea. Human Reproduction 14, 2963–2968.

Rebar R, Judd HL, Yen SSC, Rakoff J et al. 1976 Characterization of the inappropriate gonadotropin secretion in polycystic ovary syndrome. Journal of Clinical Investigation 57, 1320–1329. South SA, Asplin CM, Carlsen EC et al. 1993 Alterations in luteinizing hormone secretory activity in women with insulindependent diabetes mellitus and secondary amenorrhea. Journal of Clinical Endocrinology and Metabolism 76, 1048–1053. Spritzer PM, Poy M, Wiltgen D et al. 2001 Leptin concentrations in hirsute women with polycystic ovary syndrome or idiopathic hirsutism: influence on LH and relationship with hormonal, metabolic, and anthropometric measurements. Human Reproduction 16, 1340–1346. Taylor AE, McCourt B, Martin KA et al. 1997 Determinants of abnormal gonadotropin secretion in clinically defined women with polycystic ovary syndrome. Journal of Clinical Endocrinology and Metabolism 82, 2248–2256. Velazquez EM, Mendoza S, Hamer T et al. 1994 Metformin therapy in polycystic ovary syndrome reduces hyperinsulinemia, insulin resistance, hyperandrogenemia, and systolic blood pressure, while facilitating normal menses and pregnancy. Metabolism 43, 647–654. Veldhuis JD, Johnson ML 1992 Deconvolution analysis of hormone data. Methods in Enzymology 210, 539–575. Veldhuis JD, Johnson ML 1986 Cluster analysis: a simple, versatile and robust algorithm for endocrine pulse detection. American Journal of Physiology 250, E486-E493. Veldhuis JD, Johnson ML, Veldhuis OL et al. 2001a Impact of pulsatility of the ensemble orderliness (approximate entropy) of neurohormone secretion. American Journal of Physiology 281, R1975-R1985. Veldhuis JD, Pincus SM, García-Rudaz M et al. 2001b Disruption of the joint synchrony of luteinizing hormone, testosterone, and androstenedione secretion in adolescents with polycystic ovarian syndrome. Journal of Clinical Endocrinology and Metabolism 86, 72–79. Veldhuis JD, Pincus SM, García-Rudaz MC et al. 2001c Disruption of the synchronous secretion of leptin, LH, and ovarian androgens in nonobese adolescents with the polycystic ovarian syndrome. Journal of Clinical Endocrinology and Metabolism 86, 3772– 3778. Veldhuis JD, Straume M, Iranmanesh A et al. 2001d Secretory process regularity monitors neuroendocrine feedback and feedforward signaling strength in humans. American Journal of Physiology 280, R721–R279. Veldhuis JD, Johnson ML, Faunt LM, 1994 Assessing temporal coupling between two, or among three or more, neuroendocrine pulse trains: cross-correlation analysis, simulation methods, and conditional probability testing. Methods in Neuroscience 20, 336–376. Veldhuis JD, Carlson ML, Johnson ML 1987 The pituitary gland secretes in bursts: appraising the nature of glandular secretory impulses by simultaneous multiple-parameter deconvolution of plasma hormone concentrations. Proceedings of the National Academy of Sciences of the USA 84, 7686–7690. Venturoli S, Porcu E, Fabbri R et al. 1988 Episodic pulsatile secretion of FSH, LH, prolactin, oestradiol, oestrone, and LH circadian variations in polycystic ovary syndrome. Clinical Endocrinology (Oxf) 28, 90–107. Vigersky RA, Filmore-Nassar A, Glass AR 2006 Thyrotropin suppression by metformin. Journal of Clinical Endocrinology and Metabolism 91, 225–227. Waldstreicher J, Santoro NF, Hall JE et al. 1988 Hyperfunction of the hypothalamic-pituitary axis in women with polycystic ovarian disease: indirect evidence for partial gonadotroph desensitization. Journal of Clinical Endocrinology and Metabolism 66, 165–172. Wood JR, Nelson VL, Ho C et al. 2003 The molecular phenotype of polycystic ovary syndrome (PCOS) theca cells and new candidate PCOS genes defined by microarray analysis. Journal of Biological Chemistry 278, 26380–26390. Yen SSC, Vela P, Rankin J 1970 Inappropriate secretion of folliclestimulating hormone and luteinizing hormone in polycystic ovarian

Article - LH release and metformin treatment in PCOS - A Ulloa-Aguirre et al. disease. Journal of Clinical Endocrinology and Metabolism 30, 435–442. Zambrano E, Zariñán T, Olivares A et al. 1999 Receptor binding activity and in vitro biological activity of the human FSH charge isoforms as disclosed by heterologous and homologous assay systems. Endocrine 10, 113–121. Zariñán T, Olivares A, Söderlund D et al. 2001 Changes in the

biological:immunological ratio of basal and GnRH-releasable FSH during the follicular, pre-ovulatory and luteal phases of the human menstrual cycle. Human Reproduction 16, 1611–1618. Received 10 January 2006; refereed 26 January 2006; accepted 20 February 2006.

683