Abnormal Inhibin A and Inhibin B Secretion in Obese Women With and Without Insulin Resistance

Abstract

Aim. The present study aimed to establish inhibin A and B serum levels during the menstrual cycle of obese women, and its usefulness as an index of luteal-phase follicular development.

Materials and methods. Twenty-one obese patients (mean body mass index: 34.9 3.7 kg/m^sup 2^; range: 30.0-45.0 kg/m^sup 2^) were submitted to basal hormonal measurements and an oral glucose tolerance test after challenge with 75 g glucose. Progesterone and inhibin A and B levels were determined 5-7 days after the menstrual cycle and 7 days prior to expected menses.

Results. As expected, an increase in inhibin A and a decrease in inhibin B were observed when first-phase samples were compared with samples obtained after 15-20 days. On the other hand, the percentage variation of both inhibin A and B was at least four times smaller than the values for normal women described previously by other authors employing the same enzyme-linked immunosorbent assays. A small number of obese women presented ovulatory cycles characterized by progesterone concentration higher than 5.8 ng/ml. The percentage elevation (> 190%) of inhibin A in the second samples was in agreement with the progesterone levels, but it seemed to be more sensitive for the detection of follicle luteinization.

Conclusion. We conclude that obese women present less percentage variation of both inhibin A and B during the menstrual cycle, associated with a low frequency of ovulatory cycles. In obese women, the percentage increase of inhibin A can represent an additional marker to recognize follicle luteinization.

Keywords: Obesity, inhibin, anovulation, follicular luteinization, insulin resistance

Introduction

Excessive weight is a well-established public health disorder [1] associated with increased cardiovascular risk [2], especially in those patients with predominant visceral fat deposition [3,4]. Insulin resistance and the resultant hyperinsulinemia is the pivotal pathophysiological mechanism, determining a clinical profile characterized by visceral obesity, systemic arterial hypertension, dyslipidemia and a prothrombotic and pro-inflammatory state, also known as the metabolic syndrome [5,6].

Hyperinsulinemia stimulates the ovary directly, but is also able to potentiate the effects of gonadotropins, especially in the theca cells, with a resultant increase in androgen secretion [7]. Additionally, levels of bioactive free androgens are increased secondary to the reduction of liver sex hormone-binding globulin (SHBG) production [8]. Overall, the complete action of excessive insulin concentration includes an increase of insulin-like growth factor-I (IGF-I) production and the elevation of IGF-I receptor expression in the ovary [9]. In obese women, insulin resistance is significantly improved after weight loss [10].

Patients with polycystic ovary syndrome (PCOS) are frequently obese and have detectable insulin resistance and concomitant hyperandrogenism associated with abnormal inhibin secretion [11]. In PCOS patients, elevation of inhibin B concentration is usually detected and is possibly related to persistence of the follicular phase [12]. A decrease in inhibin A has also been described in obese women [13].

Study in normo-ovulatory women [14] confirms that inhibin B is produced mainly by small antral follicles. Its levels increase during the early to mid-follicular phases and decline during the late follicular phase. Inhibin A is produced predominantly by preovulatory follicles and serum concentrations are therefore high during the late follicular phase.

Our aim in the present study was to describe the inhibin A and inhibin B concentrations during the menstrual cycle of obese women, and to correlate inhibin levels to insulin resistance and the severity of obesity.

Materials and methods

Patients and samples

Twenty-one obese patients were prospectively evaluated at the Gynecology Unit and Endocrinology Unit of outpatient clinics of Santa Casa de So Paulo – Faculty of Medical Sciences. Patients reporting the use of compounds with antiandrogenic, hypoglycemic or anoretic actions or who had a history of menstrual disturbances (cycle length < 25 days or > 35 days) were excluded. Each patient signed written informed consent, approved by the institution’s ethical committee, before inclusion in the study. Chronological age of the patients ranged from 16 to 45 years. Obesity was defined as body mass index (BMI) > 30 kg/m^sup 2^. Abdominal fat predominance was represented by waist/hip ratio > 0.85. The patients were categorized by obesity degree into two groups: BMI < 35 kg/m^sup 2^ and BMI ≥ 35 kg/m^sup 2^.

Clinical and metabolic characteristics were obtained in basal conditions at the beginning of the study, before any diet or exercise prescription. A second sample was taken 7 days prior to expected menses, and progesterone concentration, representing a second phase sample. A glucose tolerance test was performed after oral challenge with 75 g of glucose. Two samples for inhibin determination were collected from each patient. The first corresponded to the first phase of the menstrual cycle and was obtained between 5 and 7 days after the beginning of a spontaneous menstrual episode. A second sample was taken 7 days prior to expected menses.

Assays

Hormones determined in the first sample included:

* Thyroid-stimulating hormone by immunoradiometric assay (IRMA) (TSH IKTS; Diagnostic Products Corporation, Los Angeles, CA, USA);

* Thyroxine (T^sub 4^) by radioimmunoassay (RIA) (DSL-3200 Active(TM) RIA; Diagnostic Systems Laboratories, Inc., Webster, TX, USA);

* Free T^sub 4^ by RIA (Gamma Coat ^sup 125^I-RIA Kit; DiaSorin, Inc., Stillwater, MN, USA);

* Estradiol by RIA (Coat-A-Coimt TKE; Diagnostic Products Corporation);

* SHBG by immunofluorometric assay (Delfia(TM); Perkin Elmer, Turku, Finland);

* IGF-I by IRMA (Active(TM) IRMA; Diagnostic Systems Laboratories, Inc.);

* Δ^sub 4^ androstenedione by RIA (DSL-3800 ACTIVE(TM) RIA; Diagnostic Systems Laboratories, Inc., Webster, TX, USA);

* Total testosterone by RIA (DSL-4100 ACTIVE(TM) RIA; Diagnostic Systems Laboratories, Inc., Webster, TX, USA);

* Free testosterone by RIA (DSL-4900 ACTIVE(TM) RIA; Diagnostic Systems Laboratories, Inc., Webster, TX, USA);

* Progesterone by RIA (Coat-A-Count TKPG; Diagnostic Products Corporation; and

* Insulin by RIA (TKIN; Diagnostic Products Corporation). The area under the curve (AUC) for insulin response was calculated by the trapezoidal method [15].

Inhibin A and inhibin B were measured in both samples by enzyme- linked immunosorbent assay (Serotec Ltd, Kidlington, UK), employing the method described by Groome and colleagues [16]. Determinations of inhibins were performed in duplicate using the same assay by one of the authors (C.A.L.) at the Molecular Medicine Laboratory Physiology Department. The intra-assay coefficient of variation was less than 15%.

Percentage inhibin variation (% ΔINH) was calculated as a relative increment using the equation: % ΔINH = 100(INH – INH1)/ INH 1, where INH1 and INH2 represent the inhibin concentration determined in samples obtained in the first and second phase of the menstrual cycle, respectively.

Statistical analysis

Statistical analyses were performed using the software SigmaStat for Windows, version 2.0.3 (SPSS Inc., Chicago, IL, USA). Non- parametric tests were used for both unpaired (Mann-Whitney) and paired (Wilcoxon) samples. Correlations between variables were first tested by establishing Pearson or Spearman coefficients and complemented by simple linear regression analysis. Significance was defined as p < 0.05.

Results

Twelve of the 21 obese women evaluated in this study presented mild obesity characterized by BMI < 35 kg/km^sup 2^. The remaining nine had BMI ≥ 35 kg/m^sup 2^, but only one had BMI > 40 kg/ km^sup 2^. Visceral obesity was detected in 19 patients (12 with BMI < 35 kg/m^sup 2^ and seven with BMI ≥ 35 kg/m^sup 2^).

The results of total cholesterol and its fractions, glucose and insulin concentrations are described in Table I. Insulin resistance, characterized by AUC for insulin after glucose overload > 183 mUI/ ml, was observed in seven patients (four with BMI < 35 kg/m^sup 2^ and three with BMI ≥ 35 kg/m^sup 2^).

Table I. Clinical characteristics and metabolic profile of obese women according to body mass index (BMI) category.

Table II. Basal hormonal values obtained during the first phase of the menstrual cycle in obese women, according to body mass index (BMI) category.

The hormonal concentrations determined in the patients during the first phase of the menstrual cycle are summarized in Table II. Hypothyroidism or hyperandrogenism was not detected in any patient, which is in agreement with the absence of clinical features related to these disorders. There were no differences in hormonal levels when obese women were classified according to BMI, suggesting that in these cases the severity of obesity did not influence basal hormonal concentrations. This is at least in part due to the selection of cases with isolated obesity not associated with hirsutism, severe acanthosis or overt diabetes mellitus. There was no correlation between BMI and the AUC for insulin after glucose overload.

Concentrations of inhibin A and B determined in the first- andsecond-phase samples are presented in Table III. The percentage variation (% ΔINH) of both inhibins was calculated as described above. As expected, there was a significant increase of inhibin A and a decrease of inhibin B and this was consequent to the presence of ovulation and luteinization of the dominant follicle observed in some patients. On the other hand, the percentage variation of inhibin A and B presented by the obese women was significantly reduced compared with that described previously by Groome and co- workers [16,17] employing the same assays for inhibin measurements. As a group, obese women showed a median increment of 72% for inhibin A from the first- to the second-phase sample, which is significantly smaller than the increment of at least 600% described for normal women. Inhibin B decreased by 45%, also significantly less than the 500% reduction observed in normal controls [18]. Overall, these findings suggest that a reduced frequency of ovulatory cycles is present in the obese patients evaluated in this study.

In agreement with these findings, we detected progesterone concentrations higher than 5.8 ng/ml in only nine of 21 (42.8%) obese women. When the progesterone levels were correlated to the percentage variation of inhibins (Figure 1), a significant correlation was observed for inhibin A (r = 0.61; p = 0.001). Only a trend for negative correlation was found for inhibin B (r = -0.32; p = 0.16). An increment greater than 190% in inhibin A was detected in seven of the nine patients with progesterone concentrations greater than 5.8 ng/ml, representing a cut-off value with 91% sensitivity and 78% specificity. Due to the wide variation, it was not possible to establish the cut-off value for inhibin B. There was no significant difference between BMI or AUC for insulin and the percentage variation of inhibin A or B, suggesting that the presence of obesity was sufficient to determine an abnormal ovulatory pattern independent of its severity.

Table III. Inhibin A and B values of obese woman, according to body mass index (BMI) category and phase of the menstrual cycle.

Figure 1. Correlation between progesterone level and the percentage variation of (A) inhibin A and (B) inhibin B in obese women. Percentage variation of inhibin A and B was calculated as described in the text. The dashed horizontal line (190%) corresponds to the percentage variation with the highest sensitivity for the detection of follicular luteinization. Progesterone concentrations were obtained 21-26 days after the beginning of the menstrual cycle. The dashed vertical line represents the minimal progesterone concentration (5.8 ng/ml) to consider follicular luteinization.

The levels of gonadotropins obtained in the first phase of the menstrual cycle were normal in all patients. Median values and range of variation of 2.5 (0.5-8.6) and 5.4 (1.6-12.0) were observed for luteinizing hormone and follicle-stimulating hormone, respectively. No significant differences were found when patients were divided according to BMI or AUC for insulin. There was no correlation between IGF-I concentration and patient’s BMI, with a median (range) of 0.63 (0.27-1.73) mU/ml. IGF-I values were also similar to those described previously for non-obese women. SHBG levels were significantly lower in the obese group with BMI ≥ 35 kg/m^sup 2^ (p = 0.041).

Discussion

In women, obesity may be one of the components of a complex metabolic syndrome or be part of PCOS. In both conditions, anovulation is a frequent associated abnormality. The impact of isolated obesity, with or without established insulin resistance, on the inhibin secretory pattern has not been well characterized. Obesity alone can potentially compromise menstrual cycle regularity and the rate of fertility.

In our study, which included women with varying degrees of obesity and predominant visceral fat distribution with or without insulin resistance, but with no other signs of PCOS or metabolic syndrome, a high frequency of anovulation was detected. This was confirmed both by the persistence of low progesterone concentrations and the reduced percentage increase of inhibin A, suggesting inadequate follicular luteinization. Our results are in agreement with those described by Cortet-Rudelli and associates [19]. The effects of obesity were observed even in the less severe cases and also in those with no insulin resistance or abnormal IGF-I levels, indicating that in these cases a central mechanism could be involved, determining abnormal gonadotropin secretion. An increase in peripheral and ovarian bioactivity of IGF-I is also possible, since obesity was previously associated with higher IGF-I bioavailability and action.

We conclude that, independently from its severity or association with insulin resistance, obesity is able to reduce the frequency of ovulation and concomitantly determine abnormal progesterone and inhibin secretion. Obese women present insufficient inhibin A increment and smaller inhibin B reduction, compatible with more frequent ovulatory failure than in normal individuals. Future studies are needed to determine whether inhibin measurements are more accurate than progesterone for identifying inadequate luteinization of the dominant follicle in obese patients.

Acknowledgement

We are grateful to the Support Center for Scientific Publications of Santa Casa de So Paulo – Faculty of Medical Sciences for the editorial assistance.

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ANTONIO PEDRO AUGE1, CARLOS ALBERTO LONGUI2,3, ROBERTO A. ALMEIDA PRADO1, MYLENE ROCHA NEVES3, ANDREA HIROTA4, NEWTON EDUARDO BUSSO1, OSMAR MONTE2,4, & JOS MENDES ALDRIGHI1

1 Department of Gynecology and Obstetrics, Endocrinology Gynecology Unit, Santa Casa de So Paulo – Faculty of Medical Sciences, So Paulo, Brazil, 2 Department of Pediatrics, Pediatric Endocrinology Unit, Santa Casa de So Paulo – Faculty of Medical Sciences, So Paulo, Brazil, 3 Department of Physiology, Molecular Medicine Unit, Santa Casa de So Paulo Faculty of Medical Sciences, So Paulo, Brazil, and 4 Department of Medicine, Endocrinology Unit, Santa Casa de So Paulo – Faculty of Medical Sciences, So Paulo, Brazil

Correspondence: A. P. Auge, Rua Jos Maria Lisboa, 207 apto. 91, CEP 01423-000 Jardim Paulista, So Paulo, SP, Brazil. Tel/Fax: 55 11 31684848. E-mail: pedroauge@terra.com.br

Copyright CRC Press Jul 2005