High neutrophil count in girls and women with hyperinsulinaemic hyperandrogenism: normalization with metformin and flutamide overcomes the aggravation by oral contraception

Lourdes Ibáñez1,3, Adriana Maria Jaramillo1, Angela Ferrer1 and Francis de Zegher2

1 Endocrinology Unit, Hospital Sant Joan de Déu, University of Barcelona, Spain and 2 Department of Pediatrics, University of Leuven, Belgium

3 To whom correspondence should be addressed at: Endocrinology Unit, Hospital Sant Joan de Déu, University of Barcelona, Passeig de Sant Joan de Déu, 2, 08950 Esplugues, Barcelona, Spain. Email: libanez{at}hsjdbcn.org


    Abstract
 Top
 Abstract
 Introduction
 Study population and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: The endocrine hallmark of polycystic ovary syndrome (PCOS) is hyperinsulinaemic hyperandrogenism; another facet of PCOS is low-grade inflammation. METHODS: In adolescents and young women with hyperinsulinaemic hyperandrogenism (n=118; mean age 16 years, body mass index 22 kg/m2), we analysed whether the PCOS-associated rise in leukocyte count is already detectable at young age and, if so, whether such elevation is lowered by metformin, flutamide–metformin, oral contraception (OC), or their combination. RESULTS: Leukocyte count (x1000/mm3) in patients was high versus controls (7.5 ± 0.1 versus 6.4 ± 0.1; P < 0.001) due to a rise in neutrophils (4.2 ± 0.1 versus 3.0 ± 0.1; P < 0.001). Randomized studies at mean ages of 12.5 years (n=24) and 15.2 years (n=33) demonstrated normalizing effects of metformin (850 mg/day; P < 0.001) and, respectively, metformin plus flutamide (62.5 mg/day) on neutrophil counts; in young women (18.3 years; n=41), the neutrophil count rose further on OC monotherapy (P=0.003), but normalized on the same OC plus flutamide–metformin (P < 0.001 versus OC alone). CONCLUSIONS: (i) A high leukocyte count is already present in girls with hyperinsulinaemic hyperandrogenism, and this is due to a raised neutrophil count; (ii) this hyperneutrophilia is attenuated by metformin or flutamide–metformin, and is amplified by OC monotherapy; (iii) if these treatments are combined, the normalizing effect of flutamide–metformin overcomes the OC effect on neutrophil count.

Key words: flutamide/leukocytosis/metformin/oral contraception/ovarian hyperandrogenism


    Introduction
 Top
 Abstract
 Introduction
 Study population and methods
 Results
 Discussion
 Acknowledgements
 References
 
Polycystic ovarian syndrome (PCOS), a variable constellation of anovulatory hyperandrogenism with hyperinsulinaemia and/or dyslipidaemia, is the most frequent endocrine disorder of young women (Asunción et al., 2000Go; Dunaif and Thomas, 2000Go; Baumann and Rosenfield, 2002Go).

One of the multiple facets of PCOS is a state of low-grade inflammation, as judged by moderate elevations of circulating markers such as C-reactive protein (CRP), interleukin-6 (IL-6) and leukocyte count (Kelly et al., 2001Go; Morin-Papunen et al., 2003aGo; Boulman et al., 2004Go; Ibáñez et al., 2004aGo; Tarkun et al., 2004Go; Orio et al., 2005Go). This chronic, low-grade inflammatory state has been linked to the degree of insulin resistance and to the early development of atherosclerosis (Tarkun et al., 2004Go; Orio et al., 2005Go). Treatment effects on the leukocyte count are not available but, using CRP and IL-6 as markers, metformin has been shown to attenuate the pro-inflammatory state, while oral contraceptives (OC) seem to aggravate it, even if they contain cyproterone acetate or drospirenone as progestagen (Morin-Papunen et al., 2003aGo; Ibáñez et al., 2004aGo; Ibáñez and de Zegher, 2004aGo).

Flutamide–metformin plus ethinylestradiol–drospirenone has been developed as a combined therapy capable of reverting the hyperinsulinaemic hyperandrogenism, anovulation, dyslipidaemia, adipocytokine imbalance and body adiposity of PCOS toward normal, and that may therefore improve the long-term prognosis of women with PCOS (Ibáñez and de Zegher, 2003Go, 2004aGo; Ibáñez et al., 2003aGo); low-dose flutamide does not appear to be hepatotoxic (Ibáñez et al., 2005Go); both metformin and flutamide play a pivotal role in the efficacy (Ibáñez et al., 2002aGo, 2004bGo; Ibáñez and de Zegher, 2005Go) and, for safety reasons, this duo has been complemented by an OC, a drospirenone OC being abdominally more lipolytic than a gestodene OC (Ibáñez and de Zegher, 2004bGo). In the early stages of PCOS development (age 8–14 years), metformin in monotherapy lowers circulating IL-6 effectively, provided there is no associated obesity (Ibáñez et al., 2004aGo).

We have now analysed whether the PCOS-associated rise in leukocyte count, as reported in women aged ~25 years (Orio et al., 2005Go), is already detectable at a younger age and, if so, whether such elevation is lowered by treatment with metformin, flutamide–metformin, OC or a combination of these.


    Study population and methods
 Top
 Abstract
 Introduction
 Study population and methods
 Results
 Discussion
 Acknowledgements
 References
 
Study population and ethics
Prompted by the report of a raised leukocyte count in women with PCOS (Orio et al., 2005Go), we analysed the unpublished blood count results from our recent studies which explored new treatments in girls, adolescents and young women with established or incipient PCOS, but which focused primarily on other efficacy and safety indices. Incipient PCOS was defined as a condition in which girls already present the endocrine–metabolic abnormalities of PCOS, but do not yet present hirsutism (Ferriman–Gallwey score < 8) or menstrual disturbances (Ferriman and Gallwey, 1961Go; Ibáñez et al., 2002bGo, 2004cGo). Table I lists the baseline characteristics of the total study population (n=118). Table II displays the treatment options that were compared in randomized studies (Ibáñez and de Zegher, 2003Go, 2004aGo; Ibáñez et al., 2003Go, 2004cGo) including a total of 24 non-obese girls with incipient PCOS, and 94 non-obese adolescents or young women with PCOS.


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Table I. Baseline data in non-obese girls, adolescents and young women with hyperinsulinaemic hyperandrogenism, and in healthy controls matched for age, height and weight

 

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Table II. Treatment effects on leukocyte count were assessed in five randomized studies conducted in different phases of polycystic ovarian syndrome development

 
All studies were conducted in Barcelona, none being supported by the pharmaceutical industry. All studies were approved by the Institutional Review Board of Sant Joan University Hospital; informed consent was obtained from parents or young women, and assent from minors.

Each study population has been reported in detail (Ibáñez et al., 2003Go, 2004aGo,bGo,cGo; Ibáñez and de Zegher, 2003Go, 2004aGo, 2005Go).

Common inclusion criteria were: (i) post-menarcheal status; (ii) hyperinsulinaemia on a standard 2 h oral glucose tolerance test (oGTT), defined as peak serum insulin levels >150 mU/ml and/or mean serum insulin >84 mIU/ml (Ibáñez et al., 1997Go; Vidal-Puig and Moller, 1997Go); (iii) excessive 17-hydroxyprogesterone (17-OHP) response (>160 ng/dl) to GnRH agonist (leuprolide acetate, 500 mg s.c.; Abbott, Madrid, Spain) (Ibáñez et al., 2002bGo,cGo); (iv) body mass index (BMI) < 26 kg/m2.

Specific inclusion criteria for Study 1 [untreated versus metformin; Table II (Ibáñez et al., 2004cGo)] were: (i) menarche 6–12 months before study start; (ii) birthweight for gestational age below –1.5 SDS (this corresponds to a birthweight below ~2.7 kg in term Catalan girls); (iii) history of precocious pubarche [presence of pubic hair before the age of 8 years (Ibáñez et al., 2004aGo,cGo), because the combination of these criteria is known to confer a high risk for progression to full-spectrum PCOS in adolescence (Ibáñez et al., 1998Go, 2001aGo, 2004aGo,cGo).

Specific inclusion criteria for Studies 2 and 3 [untreated versus flutamide plus metformin; Table II (Ibáñez et al., 2003Go)], and Studies 4 and 5 [OC versus OC plus flutamide–metformin; Table II (Ibáñez et al., 2003Go, 2004aGo)]: (i) menarche ≥2 years before study start; (ii) hirsutism (Ferriman–Galwey score ≥8), amenorrhoea (no menses for >3 months), or oligomenorrhoea (intermenstrual phase of >45 days); (iii) hyperandrogenaemia {elevated serum androstenedione, total testosterone, or free androgen index [testosteronex100/sex hormone binding globulin (SHBG)]} (Ibáñez et al., 1997Go, 2002cGo).

Common exclusion criteria were: evidence for thyroid dysfunction, Cushing syndrome or hyperprolactinaemia; glucose intolerance (Expert Committe on the Diagnosis and Classification of Diabetes Mellitus, 1997Go), family or personal history of diabetes mellitus; late-onset congenital adrenal hyperplasia (New et al., 1983Go; Sakkal-Alkadour et al., 1996Go); use of medication known to affect gonadal or adrenal function, or carbohydrate–lipid metabolism; anaemia or serum electrolyte anomalies; smoking; abnormal results in screening tests for liver and kidney function; acute inflammatory processes, as identified by history or physical examination.

Specific exclusion criteria for Study 1 [untreated versus metformin] were: the presence of hirsutism or menstrual disturbances at start of study.

Study design
All studies were open-labelled and compared 3 month treatment effects between randomized groups. Table II lists the compared treatments, the medication doses, and the age groups.

Haemoglobin, leukocyte count and endocrine–metabolic assessment
Fasting blood glucose, haemoglobin (Hb) and leukocyte count were determined at 0–3 months, within 2 h after venipuncture; among the other assessed variables were serum insulin, lipid profile, SHBG, testosterone, and indices of hepatic and renal function.

Baseline assessments were performed in the follicular phase (day 3–7) or after 2 months of amenorrhoea. Haemoglobin, leukocyte count and hormonal levels were compared to healthy post-menarcheal females of similar age and BMI; this control group was composed of girls with short–normal stature (percentile range 10–25), and adolescents and young women seen for screening purposes at Barcelona Hospital. Control girls received no medication for ≥3 months, and presented no evidence of thyroid dysfunction, an acute inflammatory disorder, liver or kidney dysfunction.

Assays and statistics
Haemoglobin and leukocyte count were assessed by an automatic cell counter (ABX Pentra 120; ABX Diagnostics, Montpellier, France) calibrated with an ABX Minotrol 16 (ABX Diagnostics); the intra-assay coefficient of variation (CV) determined on five replicates of each leukocyte measurement was ≤1%. Glucose was measured by the glucose oxidase method. Immunoreactive insulin was assayed by IMX (Abbott, Santa Clara, CA, USA); intra- and inter-assay CV were 4.7 and 7.2%. Serum testosterone, androstenedione, 17-OHP and SHBG were assayed as described (Ibáñez and de Zegher 2003Go, 2004aGo; Ibáñez et al., 2003Go, 2004cGo). Samples for hormonal parameters were stored at –20°C until assay.

For uniformity, results are expressed as mean ± SEM. Two-sided t-tests (paired or unpaired, as appropriate) were used for statistical comparisons between groups; per variable, only one comparison was performed; significance level was set at P < 0.05.


    Results
 Top
 Abstract
 Introduction
 Study population and methods
 Results
 Discussion
 Acknowledgements
 References
 
Baseline
Table I shows that total leukocyte count was relatively high in girls with incipient PCOS and in adolescents or young women with non-obese PCOS. In this age range, the relatively elevated leukocyte count was attributable to the absolute neutrophil count, which was on average >1000 cells/mm3 above the norm. Lymphocytes, monocytes, basophils and eosinophils did not contribute significantly to the rise in leukocyte count.

Treatment
Table III summarizes the 3 month changes in neutrophil and lymphocyte counts observed after initiating randomized treatments. In none of the treatment groups were significant changes noted in monocyte, basophil or eosinophil counts (data not shown).


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Table III. Baseline values in neutrophil and lymphocyte counts, and changes (0–3 months) following randomized treatments in three phases of polycystic ovarian syndrome development (total n =118)

 
Study 1: untreated versus metformin in early post-menarche
The neutrophil count in metformin-treated girls with incipient PCOS (mean age 12.5 years) dropped by >1000 cells/mm3 within 3 months (P < 0.001 versus baseline; P < 0.001 versus untreated), whereas the normal lymphocyte count was not detectably affected by metformin therapy.

Studies 2 and 3: untreated versus flutamide–metformin in adolescence
The high neutrophil count in PCOS adolescents treated with flutamide–metformin (mean age 15.2 years) dropped by ~500 cells/mm3 within 3 months (P < 0.02 versus untreated girls), whereas the normal lymphocyte count was not detectably affected by this treatment.

Studies 4 and 5: OC versus (OC plus flutamide–metformin) in young women
Results with gestodene and drospirenone OC were similar, and were therefore pooled. The high neutrophil count in young PCOS women rose further with OC in monotherapy (P=0.003), but dropped on the same OC plus flutamide–metformin (P < 0.02 versus baseline; P < 0.001 versus OC alone). Figure 1 illustrates the additive effect of low-dose flutamide–metformin on the high neutrophil count in young and non-obese PCOS women starting on OC. The normal lymphocyte count did not change appreciably on either therapy.



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Figure 1. Changes (0–3 months) in the neutrophil count of 24 girls (mean age 12.5 years; body mass index 21 kg/m2) randomized to remain untreated or to receive metformin (850 mg/day), and of 41 young women (18 years; 22 kg/m2) randomized to receive oral contraception (OC) in monotherapy or in combination with low-dose flutamide (62.5 mg/day) and metformin (850 mg/day). The relatively high neutrophil count at baseline was lowered (P < 0.001) by metformin in girls, and by the addition of low-dose flutamide–metformin in young women starting on OC.

 

    Discussion
 Top
 Abstract
 Introduction
 Study population and methods
 Results
 Discussion
 Acknowledgements
 References
 
There are two major strategies in the therapy for non-obese PCOS: one uses an OC from one of four generations of estro-progestagens, which also induce a SHBG rise; the other uses insulin sensitization and/or androgen receptor blockade, for example, with generics as metformin and/or low-dose flutamide (Dunaif et al., 1996Go; Nestler and Jakubowicz, 1997Go; de Leo et al., 1998Go; Diamanti-Kandarakis et al., 1998Go, 2003Go; Ibáñez et al., 2000aGo,bGo, 2001bGo, 2002aGo, 2003Go, 2004aGo,bGo; Moghetti et al., 2000Go; Elter et al., 2002Go; Ibáñez and de Zegher, 2003Go, 2004aGo, bGo, 2005Go; Morin-Papunen et al., 2003bGo; Gambineri et al., 2004Go; Guido et al., 2004Go; Palep-Singh et al., 2004Go).

In girls with incipient PCOS and in adolescents and young women with PCOS, we studied the effects of some of these treatments on leukocyte count. The results indicate: (i) that a relatively high leukocyte count is already present in girls and adolescents with hyperinsulinaemic hyperandrogenism, and is at this young age essentially attributable to an augmented neutrophil count; (ii) that this relative hyperneutrophilia is attenuated by metformin or low-dose flutamide–metformin, and is further amplified by OC monotherapy; and (iii) that, if these treatments are combined, the normalizing effects of flutamide–metformin overcome the OC effects on neutrophil count. These results remain to be confirmed in larger studies, in other ethnic populations and in other age ranges; until our findings have been corroborated in breadth and over time, they may be viewed as being of chiefly pathophysiological interest, rather than of immediately therapeutic relevance.

The present findings align well with those of earlier studies that used CRP or IL-6 as markers of the chronic, low-grade inflammation in PCOS (Morin-Papunen et al., 2003aGo, Ibáñez and de Zegher 2003Go, 2004aGo, 2005Go; Ibáñez et al., 2002aGo, 2003Go, 2004bGo). Collectively, these studies disclose the anti-inflammatory and anti-adipose benefit of giving metformin to girls with incipient PCOS, and of adding low-dose flutamide–metformin to an OC in young women with PCOS (Table IV). The anti-inflammatory effects of metformin and flutamide may underpin, or ensue from, the widely normalizing effects that these compounds have on hyperinsulinaemic hyperandrogenism, dyslipidaemia, body adiposity, elevated CRP and hypo-adiponectinaemia in PCOS patients (Diamanti-Kandarakis et al., 1998Go, Diamanti-Kandarakis et al., 2003Go; Ibáñez et al., 2002aGo, 2003Go, 2004bGo; Morin-Papunen et al., 2003aGo; Ibáñez and de Zegher, 2003Go, 2004aGo, 2005Go). Recently, there has been a remarkable increment in the recognition of inter-linkages among low-grade inflammation, insulin resistance, fat excess and endothelial dysfunction in PCOS and other hyperinsulinaemic states (Libby, 2002Go; Fernández-Real and Ricart, 2003Go; Goldfine and Kahn, 2003Go; Weisberg et al., 2003Go; Xu et al., 2003Go; Dandona et al., 2004Go; Fonseca et al., 2004Go; Tarkun et al., 2004Go; Orio et al., 2005Go; Sjöholm and Nyström, 2005Go). Regardless of the precise mechanisms involved, our study strengthens the evidence that metformin and flutamide–metformin may be regarded as, respectively, prime and adjuvant therapies for girls and young women with incipient or overt PCOS.


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Table IV. Polycystic ovarian syndrome (PCOS) is characterized by a pro-inflammatory and pro-adipose state

 


    Acknowledgements
 Top
 Abstract
 Introduction
 Study population and methods
 Results
 Discussion
 Acknowledgements
 References
 
Supported by the Social Security Research Fund, Health Institute Carlos III, Spain (PI/021013). F.d.Z. is a Clinical Research Investigator of the Fund for Scientific Research, Flanders, Belgium.


    References
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 Abstract
 Introduction
 Study population and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on February 21, 2005; resubmitted on March 28, 2005; accepted on April 15, 2005.