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The Decrease in Luteinizing Hormone Secretion in Response to Weight Reduction Is Inversely Related to the Severity of Insulin Resistance in Overweight Women¹

The Family Federation of Finland (T.L.B., O.H., R.S., D.A.); and Department of Medicine, Divisions of Internal Medicine (M.L.) and Obstetrics and Gynecology (R.K., M.S.), Helsinki University, FIN-00100 Helsinki, Finland

Address all correspondence and requests for reprints to: Tarja L. Bützow, M.D., The Family Federation of Finland, Kalevankatu 16 A, FIN-00100 Helsinki, Finland. E-mail: tarja.butzow{at}vaestoliitto.fi

	Abstract

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Controversial effects of weight reduction on gonadotropin secretion in obesity have been reported. As a result of pulsatility, single serum samples or frequent sampling studies are somewhat limited with regard to monitoring LH and FSH concentrations. We studied follicular phase nocturnal urinary (nu) LH and FSH secretion and glucose metabolism (150-min euglycemic hyperinsulinemic clamp) during 1 menstrual cycle/30-day period before and after weight reduction in 10 severely overweight infertility patients (age, 29 ± 3.1 yr; body mass index, 37.1 ± 3.3 kg/m²; ±SEM). A 6-week very low calorie diet was followed by a 4-week normocaloric period. The urinary LH and FSH results reported represent samples taken 12 to 2 days before the LH surge, or 10 consecutive samples in the case of amenorrhea.

We observed a decrease of 8% (P < 0.001) in percent body fat mass and a 5% (P < 0.005) reduction in waist to hip ratio. Mean nu-LH decreased by 45% [6.06 ± 1.05 (±SEM) to 3.22 ± 0.71 IU/L], whereas mean nu-FSH remained unchanged. Insulin-stimulated glucose uptake increased by 41% (P < 0.01), which was accounted for by a significant increase in nonoxidative glucose disposal (P = 0.003). Serum sex hormone-binding globulin concentrations increased by 39% (P < 0.01), and insulin-like growth factor (IGF)-binding protein-1 (IGFBP-1) levels increased by 46% (P < 0.05). Fasting serum insulin concentrations decreased by 38%, those of leptin by 37%, those of androstenedione by 32%, those of testosterone by 20% (all P < 0.01), and those of dehydroepiandrosterone sulfate by 13% (P < 0.05). The percent change in nu-LH correlated negatively with glucose uptake (r = -0.76; P < 0.01) and the increase in serum sex hormone-binding globulin (r = -0.85; P < 0.005) and positively with the percent change in waist to hip ratio (r = 0.79; P < 0.01). The absolute nu-LH levels after weight reduction correlated significantly with fasting insulin concentrations (r = 0.88; P < 0.001) and negatively with glucose uptake (r = -0.67; P < 0.05). No significant relationships were found between absolute levels or changes in nu-LH concentrations and leptin, IGF-I, IGFBP-3, or IGFBP-1 concentrations. Our findings suggest that weight reduction with a very low calorie diet results in a decrease in nu-LH concentrations, a reduction in the LH/FSH ratio, and FSH predominance favoring folliculogenesis. The decrease in LH concentrations is inversely related to the severity of insulin resistance. It is possible that the decrease in LH secretion with weight reduction is more dependent on the absolute levels of insulin sensitivity than on the degree of general adiposity.

	Introduction

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THE ROLE OF obesity in menstrual disorders as well as the beneficial effect of weight reduction on ovulatory function in overweight anovulatory women were described as early as 5 decades ago (1, 2). As overweight is an increasing problem for fertility (3), weight reduction has been incorporated as a part of infertility treatment (4). Hence, it has become crucial to gain understanding of the physiological links between weight reduction and reproductive hormones. Overweight is associated with a number of metabolic and endocrine aberrations that seem to interact with the mechanisms behind disrupted folliculogenesis and anovulation. Substantial evidence suggests that the metabolic-endocrine cascade resulting in anovulation is linked to insulin resistance and hyperinsulinemia. Altered insulin metabolism leads to decreased concentrations of circulating sex hormone-binding globulin (SHBG) (5, 6, 7, 8, 9), hyperandrogenemia, increased biological action of androgens, and changes in the functionality of the insulin-like growth factor (IGF) system at both endocrine and paracrine levels (10, 11, 12). Recently, leptin, a nutrition- and adiposity-dependent protein with a potential to modulate the gonadotropin axis in several mammals, has been proposed as a possible link between energy metabolism and ovarian function. Its role in human reproductive physiology, however, is not completely understood (13, 14).

Hypersecretion of LH and an increased LH/FSH ratio unfavorable for folliculogenesis are frequently found in obese infertility patients (15). An association between acute energy depletion or food intake and LH secretory dynamics is reported in normal women, athletes, and men (16, 17, 18, 19). Clinical data on the effects of weight reduction on longer term gonadotropin function in overweight women are limited. Although no change in LH pulsatility after weight reduction was demonstrated in a study by Guzick et al. (20), recent studies suggest that obesity and gonadotropin function are related (21) and that weight reduction has an effect on gonadotropin secretion (22).

Against such a background it is important to study gonadotropin secretory dynamics. The accuracy of a single LH measurement is limited as a result of LH pulsatility, whereas studies involving frequent sampling monitor a relatively short period of time. Highly sensitive time-resolved immunofluorometric assays (IFMAs) for LH and FSH have been used, giving comparable results in serum and urine in children (23, 24). Daily urinary LH and FSH assayed in the first morning urine allow the accumulation of information over long periods of time. We therefore set out to study whether weight reduction is followed by changes in urinary LH and/or FSH concentrations, and how these may be related to changes in insulin, glucose uptake (euglycemic hyperinsulinemic clamp), body composition, and the concentrations of leptin, steroid hormones, IGF-I, and IGF-binding proteins (IGFBPs).

	Materials and Methods

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Ten young (age, 29 ± 3.1 yr), severely overweight [body mass index (BMI), 37.1 ± 3.3 kg/m²] hyperandrogenic but otherwise healthy women seeking help because of infertility and with no recent history of dieting or weight loss were recruited from the infertility clinic. The women were selected according to the following criteria: age, 20–35 yr; BMI, more than 30; waist to hip ratio (WHR), more than 0.9; anovulation or oligoamenorrhea; a typical finding of polycystic ovaries on vaginal ultrasound exhibiting enlarged volume and increased number (>20) of visible subcortical small follicles; androstenedione levels of 10 nmol/L or more or testosterone levels of 2.7 nmol/L or more; primary or secondary sterility or more than 2 yr duration; no binge eating (The Bulimic Investigatory Test Edinburg questionnaire, <20 points); normal serum concentrations of TSH, PRL, and dehydroepiandrosterone sulfate (DHEAS); normal tubal patency, no endometriosis or fibroids; no coronary, liver, kidney, or thyroid diseases; and no contraindications for a very low calorie diet (VLCD). The experimental protocol was approved by the ethics committees of the Family Federation of Finland and the University of Helsinki, Department of Medicine, Division of Internal Medicine. All participants received detailed verbal and written descriptions of the study and signed an informed consent document.

Study design

A intervention study was performed using the patients as their own controls. A 6-week VLCD (650 kcal/day; Dietta Mini, Semper-MediFood OY, Helsinki, Finland) was used, comprising five meals per day (130 kcal/meal). The patients were allowed coffee, noncaloric beverages, and trace amounts of vegetables (100 kcal/day). Smoking was prohibited. Weight loss and daily VLCD consumption were carefully monitored in weekly group meetings held by a trained nurse. The diet was followed by a 4-week recovery period when no further weight loss was allowed, and the subjects returned to an energy-balanced normocaloric diet. One menstrual cycle (or a 30-day period in cases of amenorrhea) was studied before (period 1) and after (period 2) weight reduction. Sampling in period 2 started from the onset of bleeding after the 4-week normocaloric period or immediately after it in cases of amenorrhea. Patients were followed by vaginal ultrasonography and serum estradiol and progesterone measurements one or two times per week to verify the correct timing of sampling. Baseline serum hormone concentrations were those in samples taken in the early follicular phase on cycle days 3–6 in conditions when estradiol concentrations were less than 0.20 nmol/L and progesterone concentrations less than 3.5 nmol/L, and no dominant follicles were found in vaginal ultrasonography.

Procedures before and after weight reduction

In periods 1 and 2, in the early follicular phase, the patients underwent a 75-g, 3-h oral glucose tolerance test (OGTT), followed a few days later by a 150-min euglycemic hyperinsulinemic clamp (45 mU/m²·min) in combination with indirect calorimetry (25, 26). In this study insulin-stimulated glucose uptake is expressed per kg fat-free mass. Body composition was assessed using a bioelectrical impedance method (27). Anthropometric measures included weight (kilograms), height (centimeters), waist circumference at the umbilical level, and hip circumference at the widest point at symphysis (centimeters). All such measures were taken by a single trained nurse. A 3-day food record and a depression questionnaire were completed. Patients collected their first morning urine daily for 30 days or over one menstrual cycle before and after weight reduction. The urinary LH and FSH results reported represent means of samples collected during the follicular phase (12 to 2 days before the LH surge or 10 consecutive samples in cases of amenorrhea). All samples were taken after an overnight fast.

Assays

Daily urinary samples were stored at 4 C, as described by Demir et al. (23). All samples from a single cycle were analyzed in one assay. Serum samples were stored at -18 C until assayed. All measurements were carried out in duplicate. Nocturnal urinary (nu) LH and nu-FSH were determined using highly sensitive, time-resolved IFMAs (Delfia, Wallac, Inc.). For LH determination, LH subunit-specific kits (LHSpec) were used. The intraassay coefficient of variation (CV) for LH in both serum and urine was less than 5% at 4.0 IU/L (serum) and at four different levels ranging from 1.5–95.0 IU/L in urine. For FSH, the intraassay CV was 3% at 12 IU/L in serum and 8% at 8.5 IU/L in urine. A detailed validation of the urinary gonadotropin assay has been described by Demir et al (23). Plasma glucose concentrations measured during the OGTT and hyperinsulinemic clamp were determined by the glucose oxidase method using an automatic analyzer (Beckman Instruments, Inc./Hybritech, Palo Alto, CA), and those of insulin were determined by a double antibody RIA (Phadeseph Insulin RIA, Pharmacia Biotech, Uppsala, Sweden) with a sensitivity of 2 mU/L, an intraassay CV less than 3%, and an interassay CV less than 10%. Well established RIAs were used for androstenedione, DHEAS, and testosterone. Uric acid was measured using the Roche uric acid peroxidase-antiperoxidase test, with a Hitachi 911 automatic analyzer (Hialeah, FL). SHBG concentrations were determined using immunoenzymometric assay (Medix Biochemica, Kauniainen, Finland), with a sensitivity of 2 nmol/L and intra- and interassay CVs of 3.0% and 3.9%, respectively. Serum leptin concentrations were measured by using a commercial RIA (Linco Research, Inc., St. Charles, MO) with a sensitivity of 0.4 ng/mL and intra- and interassay CVs of 2.7–9.9% and 9.1–7.4% at levels of 4–20 ng/mL (28). Concentrations of IGF-I were measured by RIA using a commercial kit (INCSTAR Corp., Stillwater, MN), and those of IGFBP-1 and -3 were measured by IFMAs using methods described previously (10, 29).

Statistical analyses

Statistical analyses were performed using a BMDP statistical software package (BMDP Statistical Software, Los Angeles, CA). The paired t test was used for comparisons before and after VLCD intervention. Correlations between variables were tested by calculating Pearson’s correlation coefficient. P < 0.05 was considered significant. Results are given as the mean ± SEM.

	Results

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A 6-week VLCD followed by a 4-week normocaloric period resulted in a significant 11% weight reduction [BMI, 36.1 ± 1.4 to 32.0 ± 1.1 kg/m² (P < 0.01), percent body fat mass (%BFM), 39.2 ± 0.9% to 36.0 ± 1.0% (P < 0.01)] and a small reduction in fat-free body mass (61 ± 2.0 to 57 ± 1.8 kg; P < 0.01). WHR also decreased (0.96 ± 0.03 to 0.91 ± 0.02; P < 0.01).

A 45% decrease in transverse mean follicular phase nu-LH was observed between periods 1 and 2 (Fig. 1; 6.06 ± 1.05 to 3.22 ± 0.71 IU/L; P < 0.05), whereas mean nu-FSH remained relatively constant (7.09 ± 1.06 and 7.83 ± 1.06 IU/L; P = NS). The nu-LH/FSH ratio therefore decreased by 45% (0.97 ± 0.2 to 0.49 ± 0.11; P < 0.05).

View larger version (19K): [in this window] [in a new window]   Figure 1. Mean nu-LH and nu-FSH concentrations and the LH/FSH ratio before and after weight reduction based on transverse means of 10 consecutive morning urine samples collected over the follicular phase.

Figure 2 illustrates the correlation between changes in nu-LH and changes in WHR, SHBG concentrations, the absolute level of glucose uptake before VLCD, and the correlation between nu-LH and fasting insulin concentrations after VLCD. The percent change in nu-LH concentrations correlated negatively with glucose uptake in period 2 (r = -0.69; P < 0.05) and positively with uric acid (period 2: r = 0.82; P < 0.005), androstenedione (period 1: r = 0.8; P < 0.01; period 2: r = 0.85; P < 0.005), and testosterone (period 1: r = 0.77; P < 0.01; period 2: r = 0.87; P < 0.001) concentrations. Period 2 absolute nu-LH mean concentrations correlated negatively with glucose uptake (r = -0.67; P < 0.05) and positively with fasting serum insulin (Fig. 2b) and the absolute change in %BFM (r = 0.77; P < 0.01).

View larger version (18K): [in this window] [in a new window]   Figure 2. Pearson’s correlation between percent change in nu-LH and glucose uptake/fat-free mass in period 1 (A), nu-LH levels and fasting insulin in period 2 (B), percent change in nu-LH and absolute change in SHBG (C), and percent change in WHR (D).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study weight reduction by means of a very low caloric diet resulted in a significant reduction in nu-LH secretion during the follicular phase. The decrease in nu-LH was inversely associated with the degree of insulin resistance and correlated with the reduction of WHR and the reversal of hyperandrogenism. The magnitude of the nu-LH decrease did not, however, correlate with BMI or percent body fat mass, or their changes.

Modulation of pulsatile GnRH secretion is under the influence of a variety of neurotransmitters, hormones, and growth factors, including adipose tissue and gastrointestinal hormonal feedback in response to fuel homeostasis and intermediary metabolism of carbohydrates, fat, and proteins. The relationship between LH secretion and energy availability in women has been studied mainly in conditions of acute energy restriction (16, 17, 18, 19). In such conditions energy restriction results in a profound decrease in IGF-I concentrations (17, 30). As the present study was not performed under conditions of acute energy restriction, the stability of IGF-I levels was observed before and after weight reduction. The minute decrease in fat-free mass together with constant levels of IGF-I also demonstrate the adequacy of protein supplementation, as protein depletion has been shown to reduce IGF-I concentrations. In weight reduction studies, compliance with weight intervention is critical. In the present study the diet was closely followed. The patients lost weight in the range of 6–17 kg (6.3–14.4%) and lost fat (5.3–11.6%BFM), and they all completed the study. The VLCD method was found to be safe and effective to achieve metabolically significant weight reduction in infertility patients.

Previous weight reduction studies have characterized LH secretion using either a 24-h transverse mean of frequent sampling or monitoring serum levels in one to three single samples taken at 20- to 30-min intervals (19, 20, 22). Even 24-h frequent sampling, however, is too short a period when LH secretion should be monitored over several days. We therefore used a method established in pediatric endocrine units (23). Our study provides an approximation of LH secretion based on daily first morning urinary LH measurements over 10 days in the follicular phase. The LH levels represent a longer follow-up than has been reported in previous weight reduction studies.

Although visceral obesity, infertility, and hypersecretion of LH coincide, the relationship between LH and insulin resistance is not well established. In the present study weight reduction as an intervention was aimed at changing insulin-stimulated glucose metabolism and insulin resistance, and a clear 41% response was achieved. The mean concentrations of LH decreased as a result of this intervention and in relation to the degree of insulin resistance as measured both before and after weight reduction, suggesting a possible physiological relationship between insulin resistance and LH secretion. Our findings of correlations between nu-LH change and LH concentrations with insulin resistance, hyperinsulinemia, and change in abdominal obesity as a result of weight reduction are therefore of significance. In our study LH reduction was inversely related to the degree of insulin resistance both before and after weight intervention. The reduction in LH concentrations was not correlated with variables of adiposity such as BMI, leptin, or %BFM. This suggests that the change in adipose tissue mass would not primarily predict the change in nu-LH. The reduction of abdominal fat, the magnitude of the increase in SHBG concentrations, and the concentrations of androgens were all significantly related to the reduction of nu-LH. These finding together with the negative relationship between LH reduction and glucose uptake support the primary, inhibitory role of insulin resistance in the reduction of LH concentrations with weight reduction. Leenen et al. found that an abundance of visceral fat measured by magnetic resonance imaging was significantly associated with diminished levels of SHBG. With weight reduction, the loss of visceral fat led to rises in SHBG concentrations (31). Furthermore, a reduction of abdominal fat has been found to be critical to achieve restoration of reproductive potential (22). Our findings concerning the decrease in LH secretion are in line with these observations.

Taken together, in this study weight reduction by means of a very low calorie diet resulted in a decrease in follicular phase LH concentrations, a decrease in the LH/FSH ratio, and FSH predominance favoring folliculogenesis. The decrease in LH concentrations was inversely related to the degree of insulin resistance. It is therefore possible that the decrease in LH secretion with weight reduction is more dependent on the absolute levels of insulin sensitivity than on the degree of general adiposity. Further studies are needed to define the optimal treatment of insulin resistance and the role of weight reduction for an abdominally obese, hyperandrogenic, severely insulin-resistant female.

	Acknowledgments

 
The LH and FSH kits were generously provided by Wallac, Inc. (Turku, Finland), and the very low calorie diets were supplied by Semper-MediFood (Helsinki, Finland). We thank Celtrix Pharmaceuticals, Inc. (Santa Clara, CA), for the generous gift of recombinant IGFBP-3 used for generation of IGFBP-3 antibodies. We are grateful to Ms. Anne Kaljunen for secretarial assistance, to Dr. Helena Korpelainen, Ph.D., for her assistance with the statistics, and to Dr. Nicholas Bolton for reviewing the language. The help of Ms. Ulla Väinämö, Ms. Seija Heikkilä, Dr. Aila Rissanen, Dr. Liisa Valsta, Dr. Tiina Lampisjärvi, Dr. Tiinamaija Tuomi, and Dr. And Demir is appreciated.

	Footnotes

 
¹ This work was supported by a Yrjö Similä Grant from the Finnish Cultural Foundation (to T.L.B.), the Helsinki University Central Hospital Research Fund (to M.L. and R.K.), and the Federation of the Finnish Life and Pension Insurance Companies (to M.S.).

² Former head of the Infertility Clinic of the Family Federation of Finland. Current address: Karolinska Institute, Department of Obstetrics and Gynecology, Huddinge Hospital, S-14186 Huddinge, Sweden.

Received December 17, 1999.

Revised May 22, 2000.

Accepted June 15, 2000.

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