Adipocyte insulin action following ovulation in polycystic ovarian syndrome

Philippa J.Marsden1,4, Alison P. Murdoch2 and Roy Taylor3

1 Department of Obstetrics and Gynaecology, Sunderland Royal Hospital, Kayll Road, Sunderland SR4 7TP, 2 Centre for Reproductive Medicine, Royal Victoria Infirmary, Queen Victoria Road, Newcastle upon Tyne NE1 4LP and 3 Department of Medicine, University of Newcastle upon Tyne, UK


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The role of anovulation and insulin resistance in the pathogenesis of polycystic ovarian syndrome (PCOS) remains to be determined. The aim of this study was to investigate whether the metabolic abnormality of insulin resistance in PCOS reflects, rather than causes, the ovarian dysfunction. Eight subjects with classical PCOS were studied on two occasions. Adipocyte insulin sensitivity together with hormonal and metabolic changes were investigated in patients with PCOS following prolonged amenorrhoea and then again in the early follicular phase after ovulation. Insulin receptor binding in amenorrhoeic subjects with PCOS was low at 0.78 ± 0.08% and this increased to 1.18 ± 0.19% after an ovulatory cycle (P < 0.05). Maximal insulin stimulated 3-O-methylglucose uptake was 0.70 ± 0.14 during amenorrhoea and increased to 1.08 ± 0.25 pmol/10 cm2 cell membrane (P < 0.05). Plasma testosterone fell (4.0 ± 0.4 to 2.3 ± 0.2 nmol/l; P < 0.001), luteinizing hormone fell (17.6 ± 2.3 to 6.7 ± 0.8 IU/l; P < 0.001) but plasma insulin concentrations remained unchanged following ovulation (14.6 ± 1.9 and 15.7 ± 3.8 pmol/l during amenorrhoea and after ovulation respectively). The results of this study suggest that chronic anovulation per se appears to modify the factors contributing to cellular insulin resistance seen in PCOS.

Key words: insulin action/ovulation/polycystic ovarian syndrome


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The pathogenesis of polycystic ovarian syndrome (PCOS) remains to be defined but appears to be complex and multifactorial, genetic factors (Hague et al., 1988Go; Franks et al., 1996Go) and obesity modifying the presentation of the syndrome (Ciampelli et al., 1997Go). Insulin resistance is thought to play an important role in the aetiology of PCOS. In-vivo and in-vitro studies have shown that in women with PCOS, the sensitivity of insulin to glucose metabolism is subnormal and modest hyperinsulinaemia prevails (Chang et al., 1980Go; Dunaif et al., 1989Go; Ciaraldi et al., 1992Go; Marsden et al., 1994Go) but the possibility that the reduction in insulin sensitivity is secondary to chronic anovulation rather than a primary phenomenon is still to be clarified.

Insulin and ovarian function appear to be intrinsically linked. Patients with syndromes of severe insulin resistance with acanthosis nigricans are usually hirsute and invariably have large polycystic ovaries suggesting that insulin resistance may cause ovarian dysfunction and alter ovarian morphology (Poretsky and Kalin, 1987Go). Strong evidence for a physiological link between insulin action and ovarian function has come from in-vitro studies. Insulin receptors have been demonstrated on all types of ovarian cells (Poretsky et al., 1985Go) and insulin has been shown to augment oestradiol production from granulosa cells and progesterone production from theca cells (Barbieri et al., 1984Go; Poretsky and Kalin, 1987Go). However, in-vivo studies attempting to address whether insulin resistance is the primary pathology or secondary to hyperandrogenaemia in PCOS give conflicting results. Suppression of androgens by the combined oral contraceptive pill or gonadotrophin releasing hormone (GnRH) analogue failed to alter insulin sensitivity (Lanzone et al., 1990Go; Korytkowski et al., 1995Go) whereas more recent studies of patients with PCOS showed an increased insulin sensitivity as androgens were suppressed with anti-androgen treatment (cyproterone acetate and GnRH analogue) (Dahlgren et al., 1998Go). Therefore it is unclear whether insulin resistance is the major aetiological factor in PCOS or secondary to other features associated with the syndrome. These studies were designed to investigate the interaction between chronic anovulation and insulin resistance in PCOS.

Steroid hormones per se are known to affect insulin sensitivity. For example clinical conditions such as pregnancy, where oestrogen and progesterone concentrations are markedly raised, have a substantial effect on carbohydrate, lipid and intermediary metabolism as well as altering insulin sensitivity (Ryan et al., 1985Go; Stanley et al., 1998Go). Artificially raised concentrations of the sex steroid hormones oestrogen and progesterone found in women taking the combined oral contraceptive pill also affect glucose tolerance and produce a degree of insulin resistance (Kasdorf and Kalkhoff, 1988Go). In addition in-vitro studies have recently shown a reduction in insulin receptor binding in the luteal phase of the menstrual cycle (Marsden et al., 1996Go).

Previous observations of women with PCOS have suggested that other associated endocrine abnormalities, such as hyperprolactinaemia (Murdoch et al., 1986Go), raised gonadotrophins and hyperandrogenaemia, improve after ovulation (Baird et al., 1977Go; Blankstein et al., 1987Go). In-vivo studies of insulin action showed more pronounced abnormalities in amenorrhoeic compared to oligomenorrhoeic women (Kustin et al., 1987Go) and more recently in-vivo studies have demonstrated a firm association between reduced insulin sensitivity and anovulatory cycles (Sharp et al., 1991Go; Rittmaster et al., 1993Go; Robinson et al., 1993Go). However, it is still unclear whether the reduction in insulin sensitivity is an association with, a reflection of or a causative factor in chronic anovulation in PCOS. There have been no in-vitro studies investigating whether insulin resistance in PCOS is secondary to chronic anovulation or part of the primary pathology.

The present study was therefore designed to investigate the hypothesis that the metabolic abnormality of insulin resistance in PCOS reflects rather than causes the ovarian dysfunction. Adipocyte insulin sensitivity was studied in patients with PCOS following prolonged amenorrhoea and then again after ovulation.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Subjects
Eight subjects with PCOS were studied. The subjects were recruited from the gynaecological/endocrine clinic and all presented with amenorrhoea requesting infertility investigations and treatment. All subjects were in good health and were not taking the oral contraceptive pill or any other medication. PCOS was defined by clinical features, endocrinological abnormalities and ovarian ultrasound. Since subtle metabolic abnormalities were sought, it was essential to set strict criteria for entry into the trial and a group of patients with carefully defined classical PCOS was studied.

Clinical features included oligomenorrhoea or amenorrhoea dating from the menarche. All subjects were hirsute with a score of >10 using the Ferriman/Gallwey score, a score of >10 being found in only 1.2% of the adult female population (Ferriman and Gallwey, 1961Go). None of the subjects had acanthosis nigricans. Endocrinological features included an increased luteinizing hormone (LH) concentration (>6 IU/l, normal range 1–6 IU/l), normal follicle stimulating hormone (FSH) concentration, increased testosterone concentration (>3 nmol/l, normal range 0.9–3.2 nmol/l) and increased androstenedione concentration (>10 nmol/l, normal range 1–12 nmol/l). Thyroid function and prolactin secretion were normal. Cushing's syndrome and congenital adrenal hyperplasia were excluded. Clinical and endocrinological parameters are shown in Table IGo.


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Table I. Clinical data from patients with polycystic ovarian syndrome studied during amenorrhoea and following an ovulatory cycle
 
Vaginal ultrasound was performed in all subjects and polycystic ovaries were diagnosed if the ovaries were enlarged with a thickened abnormal stroma and more than 10 cysts of 2–8 mm diameter, arranged peripherally around a dense core of stroma or scattered throughout an increased amount of stroma or both (Swanson et al., 1981Go; Adams et al., 1986Go).

The subjects were studied on two occasions, firstly during a period of amenorrhoea and then again following an ovulatory cycle. Subjects were amenorrhoeic for at least 8 weeks prior to the first study (mean 25 weeks, range 12–80 weeks). Ovarian status was assessed by measuring serum progesterone and oestradiol 1 week prior to and 1 week following the study as well as the study day itself. Vaginal ultrasound was performed on the study day to exclude follicular development. Ovulation induction using clomiphene citrate (50–100 mg for 5 days; Hoechst Marion Roussel Ltd, Uxbridge, Middx, UK) was commenced 1 week following the first study and progesterone estimations were performed in the mid-luteal phase of the menstrual cycle to confirm ovulation. If pregnancy did not ensue the second study was performed in the early follicular phase (day 2 to day 5) of the next menstrual cycle. No patient developed ovarian hyperstimulation syndrome. Circulating oestradiol concentrations in the ovulatory cycle preceding the biopsy in all patients were not significantly different from control subjects during an ovulatory cycle.

Study protocol
The study was approved by the Joint Ethics Committee of Newcastle upon Tyne District Health Authority. Written informed consent was obtained from each subject before study.

Methods
Subjects were studied twice, firstly during a period of amenorrhoea and then again after an ovulatory cycle. On both occasions, the studies were performed between 8 a.m. and 9 a.m. after an overnight fast. Venous blood was taken for oestradiol, LH, FSH, testosterone, androstenedione, fasting glucose and fasting insulin estimation prior to commencing the in-vitro studies of insulin sensitivity. Using an aseptic technique an incision was made (1–2 cm) just below the pubic hair line after subcutaneous infiltration with 1% lignocaine. Between 3 and 6 g of subcutaneous fat was obtained from the lower abdominal wall using dissecting forceps and a scalpel with a no. 11 blade. The adipose tissue was then transported to the laboratory in glucose/saline (5 mmol/l glucose/154 mmol/l NaCl) with HEPES 10 mmol at 37°C. The adipocyte isolation techniques described by Pedersen et al. (1981) were used. The adipose tissue was finely chopped and incubated for 90 min at 37°C in a HEPES buffer (pH 7.4), containing human serum albumin (25 g/l) and collagenase (0.5 g/l). The isolated adipocytes were then washed with a HEPES buffer, containing human serum albumin (50 g/l) as previously described (Taylor et al., 1985Go). Tissues and cells were suspended in a HEPES buffer containing the following substances (concentrations in mol/l): NaCl 135, KCl 4.8, MgSO4 1.7, CaCl2 0.5, NaH2PO4 0.2, Na2HPO4 1.0. The pH was adjusted to 7.4 at 37°C. Human serum albumin was present at 50 mg/ml and glucose at 5 mmol except for the glucose transport experiments where it was essential that glucose-free buffer was used. Cell number and total cell surface area/incubation tube were derived by measuring the diameter of 100 cells and calculating individual cell volume and cell surface area. The mean cell volume and surface area were calculated from the known lipocrit of the cell suspension (Pedersen et al., 1981Go). The adipose tissue biopsies were performed in the early follicular phase following the ovulatory cycle.

Glucose transport
Glucose uptake was measured using the method described by Pedersen and Gliemann (1981). Glucose uptake was initiated with direct injection of 14 µl of 3-O-methylglucose (final concentration of 43.3 µmol/l) and the reaction was stopped at 5 s by adding 3 ml 154 mmol/l NaCl containing 0.3 mmol/l of phloretin and 0.2% v/v of ethanol. Silicone oil was layered on the surface and the tubes were spun within 2 min at 3000 g for 90 s. The cell pellets were harvested with a disposable plastic pipette tip and placed in a vial containing 5 ml of scintillation fluid. The trapped extracellular radioactivity was measured by adding 3 ml of saline/phloretin solution before the addition of the glucose and this value was subtracted from all observed values. Glucose transport was expressed as pmol 3-O-methylglucose/10 cm2 cell membrane.

Adipocyte insulin binding
Insulin binding to adipocytes was measured as previously described (Pedersen et al., 1981Go) using A14-labelled mono-[125I]insulin (final concentration of 0.7–10 pmol/l) and insulin (final concentration 1 pmol/l to 100 000pmol/l) in duplicate incubation. Specific binding was calculated by subtracting the binding observed in the presence of 1.3x10–5 mol/l insulin from the total binding for each insulin concentration. The mean non-specific binding was 5.9% of total cell bound insulin. Binding was expressed as percentage specific binding per 10 cm2 adipocyte surface area. Previous work in our laboratory has demonstrated specific insulin binding to adipocytes from a group of normal women to be 1.78 ± 0.18% (Marsden et al., 1994Go).

Lipolysis inhibition
Lipolysis was measured by incubating 250 µl of 10% cell suspension with either 250 µl of buffer (basal rate) or 200 µl buffer and 50 µl noradrenaline (stimulated rate) or 150 µl buffer, 50 µl noradrenaline and varying concentrations of insulin (10–14 to 10–10mol/l) for 90 min at 37°C in a shaking waterbath. The medium did not contain caffeine as previously described (Pedersen and Hjollund, 1982Go) and this is likely to account for the observed exquisite sensitivity to insulin. The incubation was terminated with 2 ml of silicone oil and the tubes were centrifuged at 3000 g for 5 min. The silicone oil and the cells were then aspirated with a glass pipette and the infranatant was then stored at –40°C for subsequent assay of glycerol concentration. For glycerol analysis a perchloric acid (PCA) extract was prepared and glycerol was measured using an enzymatic fluorometric continuous-flow assay (Lloyd et al., 1978Go). Lipolysis was expressed as nmol glycerol released by 105 cells/90 min. Results were further calculated as a percentage of maximum stimulated rate of lipolysis, 0% representing basal lipolysis.

Materials
Chemicals: Purified human serum albumin was obtained from Hoechst Behring UK Ltd (now Hoechst Marion Roussel Ltd) (free of growth factors including insulin-like growth factor-I and insulin). Collagenase from Clostridium histolyticum (batch no. C-6885), phloretin, noradrenaline all from Sigma, London, UK. Crystalline porcine insulin and [125I]mono-iodoinsulin with the labelled iodine in tyrosine A14 were both from Novo Nordisk A/S, Bagsvaerd, Denmark. 3-O-Methyl-D-[U–14C]glucose (specific activity of 126 mCi/mmol) was obtained from Amersham International PLC, Amersham, Bucks, UK and silicone oil 200/50 from Dow Corning Corp, Poole, UK.

Statistics
Statistical analyses were performed using the Student's paired t-test or Wilcoxon signed-rank sum test as appropriate. Linear regression analysis was performed to assess correlation between variables. All results are expressed as mean ± SEM unless otherwise indicated.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Plasma hormone and biochemical data from patients with PCOS during amenorrhoea and after an ovulatory cycle is shown in Table IIGo. Maximum insulin receptor binding and maximum insulin action for individual patients with PCOS during amenorrhoea and after an ovulatory cycle is shown in Table IIIGo.


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Table II. Plasma hormone dataa from patients with PCOS studied during amenorrhoea and following an ovulatory cycle
 

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Table III. Maximum insulin receptor binding and maximum insulin action in patients with polycystic ovarian syndrome studied during amenorrhoea and following an ovulatory cycle
 
Adipocyte insulin binding
The insulin binding displacement curve is shown in Figure 1Go. Maximum specific binding was 0.78 ± 0.08% in PCOS during amenorrhoea and 1.18 ± 0.19% following ovulation per 10 cm2 cell membrane. This difference represents a 51% increase (P < 0.05). Insulin receptor binding at 10–10 mol/l was also significantly different (P < 0.01) (0.49 ± 0.07 and 0.74 ± 0.09 in PCOS during amenorrhoea and after ovulation respectively). Expression of data as cell number rather than surface area did not affect the conclusions. Receptor affinity as assessed by half maximum displacement by insulin was not significantly different between the two groups (ED50 170 ± 18 pmol versus 293 ± 72 pmol during amenorrhoea and following ovulation respectively).



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Figure 1. Specific insulin receptor binding to adipocytes at increasing concentrations of total insulin during amenorrhoea (filled circles) and after ovulation (open circles) in polycystic ovarian syndrome. Insulin receptor binding is expressed as percentage specific binding/10 cm2 adipocyte surface area.

 
Adipocyte glucose uptake
The dose–response curve for 3-O-methylglucose transport is shown in Figure 2Go. The basal rates of 3-0-methylglucose uptake were 0.47 ± 0.13 and 0.45 ± 0.09 pmol/10 cm2 cell membrane in PCOS during amenorrhoea and following ovulation respectively and were not significantly different between the two groups. Maximally insulin stimulated rates of 3-O-methylglucose transport were significantly increased after ovulation (0.70 ± 0.14 and 1.08 ± 0.25 pmol/10 cm2 membrane in PCOS during amenorrhoea and following ovulation respectively: P < 0.05). Half maximal insulin stimulation was observed at 351 ± 134 and 614 ± 388 pmol in PCOS during amenorrhoea and following ovulation respectively and was not signficantly different. Expression of data as cell number rather than surface area did not affect the conclusions.



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Figure 2. Dose–response curve for 3-O-methylglucose transport rate in adipocytes during amenorrhoea (filled circles) and after ovulation (open circles) in polycystic ovarian syndrome (PCOS). Glucose uptake is expressed as pmol 3-O-methylglucose/10 cm2 cell membrane. It should be noted that the maximal glucose transport in each patient did not necessarily occur at the highest insulin concentration. Therefore the maximal glucose transport result quoted in the text (0.70 ± 0.14 and 1.08 ± 0.25 pmol/10 cm2 membrane in PCOS during amenorrhoea and following ovulation respectively), which is for each patient, is not the same as that shown in the figure where all points of insulin concentration are plotted.

 
Lipolysis inhibition
Absolute rates of noradrenaline-stimulated lipolysis were 168.8 ± 38.9 and 249.9 ± 51.3 nmol glycerol released/105 cells/90 min for PCOS subjects during amenorrhoea and following ovulation respectively. The maximum percentage lipolysis inhibition observed was 31.9 ± 9.9% in PCOS during amenorrhoea and 30.1 ± 5.9% following an ovulatory cycle. Further increasing concentrations of insulin resulted in the expected paradoxical stimulation of lipolysis which was similar in the two groups.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This study demonstrates that the reduction in insulin receptor binding and insulin sensitivity seen in PCOS ameliorates after ovulation. The hormonal and metabolic changes associated with PCOS were also shown to normalize following ovulation.

Fasting insulin concentrations have been shown to be significantly higher in anovulatory women with PCOS compared with ovulatory subjects despite similar body mass index (BMI) (Sharp et al., 1991Go) and recently in-vivo studies have confirmed that insulin resistance appears to be more severe in patients with PCOS who are amenorrhoeic (Rittmaster et al., 1993Go; Robinson et al., 1993Go). However, there have been no longitudinal studies investigating the relationship of anovulation and insulin resistance in PCOS and there have been no in-vitro studies investigating the relationship of anovulation and insulin resistance in PCOS. The present study combined both these aspects aiming to establish whether the abnormalities in insulin sensitivity in PCOS are part of the primary pathology or secondary to chronic anovulation. In-vitro studies of insulin receptor binding and insulin action were performed in amenorrhoeic subjects with PCOS and then again after ovulation. Thus by using subjects as their own controls the study aimed to provide a more accurate assessment of whether the insulin resistance seen in PCOS ameliorated after ovulating rather than just comparing amenorrhoeic and regularly menstruating women with PCOS.

There have been few in-vitro studies using target cells to assess insulin sensitivity in PCOS. However, a profound post-receptor defect in insulin action has been clearly demonstrated with abnormalities of both lipolysis (Marsden et al., 1994Go; Ek et al., 1997Go) and glucose transport (Ciaraldi et al., 1992Go; Dunaif et al., 1992Go; Marsden et al., 1994Go). The abnormality of glucose transport has been shown to be possibly related to abnormalities of specific glucose transporters (Rosenbaum et al., 1993Go) although a recent study has suggested that the abnormality may be at an early step of insulin signalling that is common to both glucose transport and lipolysis (Ciaraldi et al., 1997Go). Conflicting results for insulin receptor binding to adipocytes in PCOS have been reported. We demonstrated a profound reduction in insulin receptor binding in a group of lean and obese patients with classical PCOS (Marsden et al., 1994Go). The two other studies (Ciaraldi et al., 1992Go; Dunaif et al., 1992Go) did not find a decrease in insulin receptor binding in patients with PCOS although both groups also failed to demonstrate the expected reduction in insulin receptor binding associated with obesity per se, thereby questioning their insulin receptor binding results.

The studies presented demonstrate again a marked reduction in insulin receptor binding in amenorrhoeic subjects with PCOS compared to that seen in normal women in the follicular phase of the menstrual cycle (Marsden et al., 1996Go). However, there was a 51% increase in insulin receptor binding after an ovulatory cycle which appeared to be related to an increase in receptor number as opposed to an increase in receptor affinity. There was also an increase in glucose transport in adipose tissue in subjects with PCOS following an ovulatory cycle. With respect to lipolysis inhibition two of the three subjects with a profound reduction in the ability to inhibit lipolysis achieved normal values of lipolysis inhibition after ovulation whereas three of the subjects appeared to have a normal ability to inhibit lipolysis whilst amenorrhoeic. These are small numbers and it may be that a larger group of women with PCOS over several cycles needs to be studied in order to detect differences in lipolysis inhibition.

There are several possible explanations of the association of insulin resistance with anovulation. Firstly, a reduction in insulin sensitivity could be secondary to chronic anovulation as suggested by normalization of other hormonal parameters after ovulation, the present study and recent in-vivo studies of insulin resistance in PCOS (Rittmaster et al., 1993Go). However, observations that patients with syndromes of severe insulin resistance due to genetic abnormalities in insulin receptors have large polycystic ovaries suggest that this is unlikely to be the only mechanism for insulin resistance in PCOS. Secondly a primary metabolic disorder of insulin action within the ovary itself could cause lack of selection of a dominant follicle resulting in chronic anovulation. Recent studies have shown that excessive serine phosphorylation of the insulin receptor leading to modulation of aromatase (P450c17) activity (the hormone essential for development of a dominant follicle and the prime regulator of androgen biosynthesis) could be the primary cause for insulin resistance in a significant proportion of women with PCOS (Dunaif, 1997Go). Thirdly there may be an additive effect between insulin resistance, obesity and anovulation in PCOS. The role of obesity in the pathogenesis of insulin resistance in PCOS remains to be determined although recent studies have suggested that obesity may be the predominant cause for a reduction in insulin sensitivity in obese PCOS whereas hyperinsulinaemia per se may be a primary feature of lean patients with PCOS (Ciampelli et al., 1997Go). However, the finding in the present study of increased insulin sensitivity after just one ovulatory cycle suggest that anovulation per se may also have a role in the aetiology of insulin resistance in PCOS or at least be an aggravating factor.

Significant differences in insulin receptor binding between the luteal and follicular phases have been demonstrated within the same normal ovulatory cycle (Marsden et al., 1996Go) demonstrating that variations in insulin receptor binding can occur within a relatively short space of time. Therefore the significant increase in binding after just one ovulatory cycle was not unexpected. However, the amount of insulin receptor binding after one ovulatory cycle in the present study still fell short of insulin receptor binding seen in subjects without PCOS studied in the follicular phase of the menstrual cycle (specific insulin receptor binding per 10 cm2 cell surface: 1.85 ± 0.14%; Marsden et al., 1996). This may be because a longer period of regular ovulatory cycles is required to correct the profound reduction in insulin receptor binding to values expected in the follicular phase of an ovulatory cycle in normal subjects.

Clinical and biochemical abnormalities in PCOS have been shown to be more pronounced in amenorrhoeic patients with PCOS compared to women with PCOS who have regular menstrual cycles (Zhang et al., 1984Go; Kustin et al., 1987Go). Chronic anovulation has been observed as having an aggravating affect on biochemical abnormalities in women with PCOS since normalization of hyperprolactinaemia (Murdoch et al., 1986Go), hyperandrogenaemia (Blankstein et al., 1987Go) and raised gonadotrophin concentrations (Baird et al., 1977Go) after ovulation in PCOS has been demonstrated. However, it is not clear whether these observations are secondary to resumption of ovulation or related to amelioration in obesity. Obese women with PCOS are more likely to demonstrate clinical and biochemical abnormalities and an improvement in endocrine function (androgens and fasting insulin) and clinical features (resumption of menstrual cycles and improvement in hirsutism) after weight loss has indeed been demonstrated (Kiddy et al., 1992Go). In the natural state of the disease it is often difficult to establish whether obesity precedes anovulation causing deterioration in clinical features and biochemical abnormalities or vice versa. However, normalization of biochemical abnormalities (plasma testosterone and LH concentrations) seen in PCOS has been observed after ovulation following ovarian wedge resection (Judd et al., 1976Go) and to a similar extent following ovarian electrocautery or down-regulation with a long-acting luteinizing hormone-releasing agonist (Gadir et al., 1990Go) suggesting that anovulation may have a role in the aetiology of the biochemical abnormalities in PCOS independent of obesity. The data from the present study show that androgen and gonadotrophin concentrations are lower following just one ovulatory cycle in PCOS. These findings suggest that the marked biochemical abnormalities seen in anovulatory PCOS are likely to be secondary to chronic anovulation.

Women with PCOS have normal or elevated oestradiol concentrations compared to control subjects despite being amenorrhoeic (Fox et al., 1991Go). This is thought to be due to a combination of increased secretion of oestradiol from the ovary (individual follicles producing low concentrations of oestradiol and oestrone but an overall increase from numerous follicles) and peripheral aromatization of circulating androgens in adipose tissue. The present study shows that oestradiol concentrations were significantly lower following ovulation. This decrease in oestradiol after just one ovulatory cycle associated with a marked decrease in androgens suggests that extraglandular peripheral conversion of androgens to oestadiol is the predominant cause for oestradiol concentrations in PCOS being higher than would be expected in a state of chronic anovulation.

Ovulation did not appear to reduce fasting insulin concentrations in this study. There is poor correlation between fasting insulin concentrations and insulin sensitivity and therefore the finding that fasting insulin concentrations did not fall is not entirely surprising. Fasting basal insulin concentrations have demonstrated hyperinsulinaemia in 30% of lean patients with PCOS (Conway, 1990Go) whereas the i.v. glucose tolerance test demonstrated a higher prevalence of insulin resistance in PCOS of 63% (Falcone et al., 1992Go). Euglycaemic clamp studies have demonstrated a similar incidence of reduced insulin sensitivity in PCOS (Dunaif et al., 1992Go). This study was designed to investigate whether a clearly identifiable in-vitro abnormality of insulin sensitivity in a group of patients with classical PCOS ameliorated after ovulation, rather than to determine the reaction of plasma insulin concentrations to ovulation, which is a weaker assessment of insulin sensitivity.

In summary, the results of this study suggest that chronic anovulation per se does modify the degree of cellular insulin resistance seen in PCOS as well as the biochemical abnormalities but is unlikely to be the major aetiological factor in insulin resistance in PCOS. The extent to which genetically determined abnormalities of insulin action pre-date the established syndrome remain to be established. However, the present study clearly indicates a partial reversibility of cellular insulin resistance in PCOS.


    Notes
 
4 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Adams, J., Polson, D.W. and Franks, S. (1986) Prevalence of polycystic ovaries in women with anovulation and idiopathic hirsutism. Br. Med. J., 293, 355–359.[ISI][Medline]

Baird, D.T., Corker, C.S., Davidson, D.W. et al. (1977) Pituitary–ovarian relationships in polycystic ovary syndrome. J. Clin. Endocrinol. Metab., 45, 798–809.[Abstract]

Barbieri, R.L., Makris, A. and Ryan, K.J. (1984) Insulin stimulates androgen accumulation in incubations of human ovarian stroma and theca. Obstet. Gynaecol., 64, 73S–80S.[Medline]

Blankstein, J., Rabinovici, J., Goldenberg, M. et al. (1987) Changing pituitary reactivity to follicle stimulating hormone and luteinising hormone releasing hormone after induced ovulatory cycles and after anovulation in patients with polycystic ovarian disease. J. Clin. Endocrinol. Metab., 65, 1164–1167.[Abstract]

Chang, R.J., Nakamura, R.M., Judd, H.L. et al. (1980) Insulin resistance in non-obese patients with polycystic ovarian disease. J. Clin. Endocrinol. Metab., 57, 356–359.[Abstract]

Ciampelli, M, Fulghescu, A.M., Cucinelli, F. et al. (1997) Heterogeneity in beta cell activity, hepatic insulin clearance and peripheral insulin sensitivity in women with polycystic ovary syndrome. Hum. Reprod., 12, 1897–1901.[Abstract]

Ciaraldi, T.P., El-Roeiy, A., Madar, Z. et al. (1992) Cellular mechanisms of insulin resistance in polycystic ovarian syndrome. J. Clin. Endocrinol. Metab., 75, 577–583.[Abstract]

Ciaraldi, T.P., Morales, A.J., Hickman, M.G. et al. (1997) Cellular insulin resistance in adipocytes from obese polycystic ovary syndrome subjects involves adenosine modulation of insulin sensitivity. J. Clin. Endocrinol. Metab., 82, 1421–1425.[Abstract/Free Full Text]

Conway, G.S. (1990) Insulin resistance and polycystic ovary syndrome. Contemp. Rev. Obstet. Gynaecol., 2, 4–39.

Dahlgren E., Landin K., Krotkiewski M. et al. (1998) Effects of two antiandrogen treatments on hirsutism and insulin sensitivity in women with polycystic ovary syndrome. Hum. Reprod., 13, 2706–2711[Abstract/Free Full Text]

Dunaif, A. (1997) Insulin resistance and the polycystic ovary syndrome: mechanism and implications for pathogenesis. Endocr. Rev., 18, 774–800.[Abstract/Free Full Text]

Dunaif, A., Segal, K.R., Futterweit, W. et al. (1989) Profound peripheral insulin resistance independent of obesity, in polycystic ovary syndrome. Diabetes, 38, 1165–1174.[Abstract]

Dunaif, A,, Segal, K.R., Shelley, D.R. et al. (1992) Evidence for distinctive and intrinsic defects in insulin action in polycystic ovary syndrome. Diabetes, 41, 1257–1266.[Abstract]

Ek, I., Arner, P., Bergqvist, A., et al. (1997) Impaired adipocyte lipolysis in nonobese women with the polycystic ovary syndrome: a possible link to insulin resistance. J. Clin. Endocrinol. Metab., 82, 1147–1153.[Abstract/Free Full Text]

Falcone, T., Little, A.B. and Morris, D. (1992) Impaired glucose effectiveness in patients with polycystic ovary syndrome. Hum. Reprod., 7, 922–925.[Abstract]

Ferriman, D. and Gallwey, J. (1961) Clinical assessment of body hair growth in women. J. Clin. Endocrinol. Metab., 21, 1440–1445.[ISI]

Fox, R., Corrigan, E., Thomas, P.G. et al. (1991) Oestrogen and androgen states in oligo-amenorrhoeic women with polycystic ovaries. Br. J. Obstet. Gynaecol., 98, 294–299.[ISI][Medline]

Franks, F., White, D., Gilling-Smith, C. et al. (1996) Hypersecretion of androgens by polycystic ovaries: the role of genetic factors in the regulation of cytochrome p450c17 alpha. Clin. Endocrinol. Metab., 10, 193–203.

Gadir, A.A., Khatim, M.S., Mowafi, R.S. et al. (1990) Hormonal changes in patients with polycystic ovarian disease after ovarian electrocautery or pituitary desensitisation. Clin. Endocrinol., 32, 749–754.[ISI][Medline]

Hague, W.M., Adams, J., Reeders, S.T. et al. (1988) Familial polycystic ovaries: a genetic disease? Clin. Endocrinol., 29, 593–605.[ISI][Medline]

Judd, H.L., Rigg, L.A., Anderson, D.C. et al. (1976) The effects of ovarian wedge resection on circulating gonadotrophin and ovarian steroid levels in patients with polycystic ovary syndrome. J. Clin. Endocrinol. Metab., 43, 347–355.[Abstract]

Kasdorf, G. and Kalkhoff, R.K. (1988) Prospective studies of insulin sensitivity in normal women receiving oral contraceptive agents. J. Clin. Endocrinol. Metab., 66, 846–852.[Abstract]

Kiddy, D., Hamilton-Fairley, D., Bush, A. et al. (1992) Improvement in endocrine and ovarian function during dietary treatment of obese women with polycystic ovary syndrome. Clin. Endocrinol., 36, 105–111.[ISI][Medline]

Korytkowski, M.T., Mokan, M., Horwitz, M.J. et al. (1995) Metabolic effects of oral contraceptives in women with polycystic ovary syndrome. J. Clin. Endocrinol. Metab., 80, 3327–3334.[Abstract]

Kustin, J., Kazer, R.R., Hoffman, D.I. et al. (1987) Insulin resistance and abnormal ovarian responses to human chorionic gonadotrophin in chronically anovulatory women. Am. J. Obstet. Gynaecol., 157, 1468–1473.[ISI][Medline]

Lanzone, A., Fulghescu, A.M., Andreani, C.L. et al. (1990) Insulin secretion in polycystic ovarian disease: effect of ovarian suppression by GnRH agonist. Hum. Reprod., 5, 143–149.[Abstract]

Lloyd, B., Burrin, J., Smythe, P. et al. (1978) Enzymatic fluorometric continuous-flow assays for blood glucose, lactate, pyruvate, alanine, glycerol and hydroxybutyrate. Clin. Chem., 34, 1724–1729.

Marsden, P.J., Murdoch, A. and Taylor, R. (1994) Severe impairment of insulin action in adipocytes from amenorrhoeic subjects with polycystic ovary syndrome. Metabolism, 43, 1536–1542[ISI][Medline]

Marsden, P.J., Murdoch, A. and Taylor, R. (1996) Adipocyte insulin action during the normal menstrual cycle. Hum. Reprod., 11, 968–974.[Abstract]

Murdoch, A.P., Dunlop, W. and Kendall-Taylor, P. (1986) Studies of prolactin secretion in polycystic ovary syndrome. Clin. Endocrinol., 24, 165–175.[ISI][Medline]

Pedersen, O. and Gliemann, J. (1981) Hexose transport in human adipocytes: factors influencing the response to insulin and kinetics of methylglucose and glucose transport. Diabetologia, 20, 630–635.[ISI][Medline]

Pedersen, O. and Hjollund, E. (1982) Insulin receptor binding to fat and blood cells and insulin action in fat cells from insulin dependent diabetics. Diabetologia, 31, 706–715.

Pedersen, O., Hjollund, E., Beck-Neilsen, H. et al. (1981) Insulin receptor binding and receptor-mediated insulin degradation in human adipocytes. Diabetologia, 20, 636–641.[ISI][Medline]

Poretsky, L. and Kalin, M.F. (1987) The gonadotrophic function of insulin. Endocr. Rev., 8, 132–141.[Abstract]

Poretsky, L., Grigorescu, F., Siebel, M. et al. (1985) Distribution and characterisation of insulin and insulin-like growth factor I receptors in normal human ovary. J. Clin. Endocrinol. Metab., 61, 728–734.[Abstract]

Rittmaster, R.S., Deshwal, N. and Lehman, L. (1993) The role of adrenal hyperandrogenism, insulin resistance and obesity in the pathogenesis of polycystic ovarian syndrome. J. Clin. Endocrinol. Metab., 76, 1295–1300.[Abstract]

Robinson, S., Kiddy, D., Gelding, S.V. et al. (1993) The relationship of insulin insensitivity to menstrual pattern in women with hyperandrogenism and polycystic ovaries. Clin. Endocrinol., 39, 351–355.[ISI][Medline]

Rosenbaum, D., Haber, R.S. and Dunaif, A. (1993) Insulin resistance in polycystic ovary syndrome: decreased expression of GLUT-4 glucose transporters in adipocytes. Am. J. Physiol., 264, E197–202.[Abstract/Free Full Text]

Ryan, E.A., O'Sullivan, M.J. and Sykler, J.S. (1985) Insulin action during pregnancy. Studies with the euglycaemic clamp technique. Diabetes., 34, 380–389.[Abstract]

Sharp, P.S., Kiddy, D.S., Reed, M.J. et al. (1991) Correlation of plasma insulin and insulin-like growth factor-I with indices of androgen transport and metabolism in women with polycystic ovary syndrome. Clin. Endocrinol., 35, 253–257.[ISI][Medline]

Stanley, K., Fraser, R. and Bruce, C. (1998) Physiological changes in insulin resistance in human pregnancy: longitudinal study with the hyperinsulinaemic euglycaemic clamp technique. Br. J. Obstet. Gynaecol., 105, 756–759.[ISI][Medline]

Swanson, M., Sauerbrei, E.E. and Cooperberg, P.L. (1981) Medical implications of ultrasonically detected polycystic ovaries. J. Clin. Ultrasound, 9, 219–222.[ISI][Medline]

Taylor, R., McCulloch, A.J., Zeuzem, S. et al. (1985) Insulin secretion, adipocyte insulin binding and insulin sensitivity in thyrotoxicosis. Acta Endocrinol., 109, 96–103.[Medline]

Zhang, Y., Stern, B. and Rebar, R. (1984) Endocrine comparison of obese menstruating and amenorrhoeic women. J. Clin. Endocrinol. Metab., 58, 1077–1083.[Abstract]

Submitted on October 23, 1998; accepted on May 28, 1999.