By Genazzani, Alessandro D; Lanzoni, Chiara; Ricchieri, Federica; Baraldi, Enrica; Et al
Background. Polycystic ovary syndrome (PCOS) is a common endocrine disease that is frequently observed to be related to increased insulin resistance independent of body weight. The use of insulin-sensitizer compounds, such as metformin, permits great improvement of such metabolic abnormality, restoring ovarian function and gonadal steroid synthesis and reducing insulin resistance.
Aim. On this basis we aimed to evaluate a group of non-obese amenorrheic PCOS patients before and after 6 months of metformin administration (500 mg orally twice daily) to better understand upon which basis of clinical and endocrine parameters metformin administration might be chosen as a putative therapeutic tool.
Method. A group of non-obese PCOS patients (n = 42) was enrolled after informed consent. They underwent an oral glucose tolerance test for insulin, glucose and C-peptide levels and provided blood samples for determination of plasma levels of luteinizing hormone (LH), follicle-stimulating hormone, prolactin, estradiol, androstenedione, 17-hydroxyprogesterone, insulin, cortisol and testosterone levels on two occasions: before and on day 7 of the first menstrual cycle occurring after the 5th month of treatment.
Results. Plasma LH, estradiol, insulin and C-peptide were decreased significantly by metformin treatment in the entire group of PCOS patients. When subdividing PCOS patients according to insulin sensitivity (i.e. hyper- and normoinsulinemic subjects), a greater rate of positive endocrine changes was observed in hyperinsulinemic patients and the highest rate was observed in hyperinsulinemic hyperandrogenic subjects. Menstrual cyclicity was recovered in all patients under treatment.
Conclusions. Our data show that metformin modulates ovarian function and greatly affects LH secretion through reduction of the hyperandrogenic condition. The highest rate of endocrine changes was observed in the hyperinsulinemic hyperandrogenic non-obese PCOS patients. Our study demonstrates that metformin administration is more appropriate in hyperinsulinemic hyperandrogenic non-obese PCOS patients.
Keywords: Polycystic ovary syndrome, hyperandrogenism, hyperinsulinemia, metformin cloridate, menstrual cyclicity, insulin sensitivity
Hyperandrogenism, anovulation and metabolic disturbances are typical findings in obese but also in non-obese patients with polycystic ovary syndrome (PCOS) [1-4]. Indeed, recently insulin resistance/ hyperinsulinemia was indicated as one of the relevant causal factors in inducing the endocrine disorders that are associated with PCOS [5-7], independent of body weight . In vitro, insulin stimulates androgen synthesis in theca cells  and decreases secretion of sex hormone-binding globulin from the liver , increasing the availability of free androgens.
In addition to other therapeutic strategies (i.e. oral contraceptives, antiandrogen compounds), such findings have induced the use of specific treatments intended to reduce the hyperinsulinemic condition and, when present, the excess of weight. It is well known that weight loss is effective in restoring menstrual cyclicity and/or ovulation in overweight PCOS patients thanks to the reduction in fat mass and mainly the increase in tissue insulin sensitivity . It is of great relevance to point out that insulin resistance is recognized as a major risk factor for type 2 diabetes [10,11].
Many studies have demonstrated the efficacy of insulin- sensitizing compounds, such as metformin, in reducing PCOS- associated hyperinsulinemia and in correcting most of the endocrine and metabolic abnormalities found in women with PCOS [1,2,10,12]. Among the available compounds, metformin, a water-soluble oral biguanide, enhances insulin sensitivity in liver, where it inhibits hepatic glucose production, and in muscle tissue, where it improves glucose uptake and utilization [8,12], with minimal side-effects such hypoglycemia  and with no direct effect on insulin secretion by the pancreatic β-cells . The metformin- induced decrease in hyperinsulinemia underlies the significant modifications in ovarian response to gonadotropin stimulation [3,4,7,12,15-18], but, to date, the unsolved question is what kind of PCOS patients would certainly benefit from metformin administration. Obesity is a key factor in inducing hyperinsulinemia in PCOS patients and these patients improve greatly following administration of insulin-sensitizing compounds; however, a great many (though not all) normal-weight PCOS patients have also been demonstrated to benefit from such administration even in the absence of weight excess .
To try to elucidate which parameters might be proposed as the basis for administering an insulin-sensitizing compound such as metformin or not, a group of non-obese PCOS patients were studied before and after 6 months of metformin cloridrate treatment.
Material and methods
Subjects and study protocol
Forty-two women aged 18-28 years with PCOS were recruited for this study. These patients were selected among a population attending the Gynecological Endocrinology Center at the University of Modena and Reggio Emilia, Italy, according to the following criteria: (1) presence of micropolycystic ovaries at ultrasound; (2) hirsutism and/or acne, from grade mild to severe; (3) oligomenorrhea or amenorrhea; (4) absence of enzymatic adrenal deficiency and/or other endocrine disease; (5) normal prolactin level (range 5-25 ng/ ml); (6) no hormonal treatment for at least 6 months before the study; (7) body mass index (BMI)
All patients were from 5 to 15% above their ideal body weight and the mean BMI was 22.7+1.2 kg/m^sup 2^ ( standard error of the mean, SEM). The ratio of glucose to insulin was computed at baseline, to estimate the sensitivity to insulin [19,20]. Such ratio was >4.5, within the normal range in all subjects. Thirty-two patients were amenorrheic and ten oligomenorrheic (menstrual cycle every 50 days or more).
Patients were treated with metformin (Metforal; Guidotti, Pisa, Italy) 500 mg orally twice daily, 20 min before lunch and dinner, for 6 months.
Endocrine parameters were determined before and after treatment. At baseline, amenorrheic patients were studied on a random cycle day while oligomenorrheic patients were studied on day 7 of the menstrual cycle. The post-treatment endocrine parameters were determined on day 7 of the first menstrual cycle occurring after the 5th month of treatment.
On these occasions, all patients underwent an oral glucose tolerance test (OGTT) for insulin, glucose and C-peptide determinations, sampling 15 min before and 30, 60, 90, 120 and 240 min after the oral consumption of 100 g glucose. Time +90 min was considered indicative of the maximal response to the oral glucose load. Insulin sensitivity was computed was computed as glucose/ insulin ratio, since this has been shown to be a good index of insulin sensitivity in women with PCOS [19-21].
At the same time plasma levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), prolactin, estradiol, androstenedione, 17-hydroxyprogesterone (17OHP), insulin, cortisol and testosterone were determined. All collected samples were immediately centrifuged and plasma was stored at -20C until assayed.
Vaginal ultrasound was performed and the Ferriman-Gallway score determined before and after 6 months of treatment. Each patient kept a diary of their menstrual cyclicity, reporting the date of menstrual occurrence.
The study protocol was approved by the Human Investigation Committee of the University of Modena and Reggio Emilia, Italy.
All samples from each subject were assayed in duplicate in the same assay. Plasma LH and FSH concentrations were determined using a previously described immunofluorimetric assay [22,23]. The sensitivity of the assay, expressed as the minimal detectable dose, was 0.1 IU/ml. The cross-reactivities with free α- and β- subunits of LH, FSH and thyroid-stimulating hormone were less than 2% . Intra-assay and inter-assay coefficients of variation were 4.9 and 7.4%, respectively.
Plasma estradiol, 17OHP, androstenedione, cortisol and testosterone were determined by radioimmunoassay (Radim; Pomezia, Rome, Italy) as described previously . Based on two quality control samples the average within- and between-assay coefficients of variation were 4.0 and 9.7%, respectively.
Plasma insulin was determined using an immunoradiometric assay (Biosource Europa SA, Nivelles, Belgium). Based on two quality control samples the average within- and between-assay coefficients of variation were respectively 4.5 and 11.7%.
Plasma C-peptide concentrations were determined using a chemiluminescence assay (Immulite One; Diagnostic Products Corporation, Los Angeles, CA, USA). The average within- and between- assay coefficients of variation were 4.5 and 8.2%, respectively, based on two quality control samples.
We tested the data for significant differences between groups, after one-way analysis of variance, using Student’s t test for paired or unpaired data, as appropriate. Data are expressed as mean SEM.
Table I shows the endocrine pattern of all subjects before and during the 6th mont\h of metformin administration. These data show that metformin administration in non-obese PCOS subjects significantly modified plasma LH, testosterone and insulin levels, as well as LH/FSH ratio, while no changes were observed for androstenedione and 17OHP levels in plasma. Interestingly, the glucose/insulin ratio was perfectly normal (above the cut-off of 4.5) both before and under metformin administration. Considering results of the OGTT, the mean insulin response after 90 min was significantly reduced under metformin administration (Figure 1).
Table I. Endocrine parameters of all PCOS patients (n = 42) before and under metformin administration.
To gain a better understanding of these results, all patients were divided into two groups according to their insulin response in the OGTT: hyperinsulinemic (n = 27) and non-hyperinsulinemic (n = 15) PCOS patients. According to our laboratory, a hyperinsulinemic response to a glucose load occurs when insulin level is reported to be > 45 U/ml in at least two consecutive blood samples during the OGTT. When patients were considered according to the insulin response to the OGTT a completely different setting was outlined (Table II): hyperinsulinemic PCOS subjects showed a greater response to metformin administration than non-hyperinsulinemic (i.e. normoinsulinemic) PCOS subjects. The former had significant reductions in plasma LH, estradiol, testosterone, C-peptide and insulin levels as well as LH/FSH ratio after metformin administration. Conversely, normoinsulinemic subjects showed just the reduction of plasma LH level and LH/FSH ratio, with no change of any other endocrine parameter evaluated (Table II). Insulin response to the OGTT was also markedly different during metformin administration, being reduced only in hyperinsulinemic subjects (Figure 2). However, it must be pointed out that hyperinsulinemic patients remained far from being similar to normoinsulinemic subjects in terms of insulin response to glucose load, also under metformin administration.
With the purpose of obtaining insight into the hyperinsulinemic patients, they were subdivided according to their plasma androstenedione levels at baseline. Our laboratory considers hyperandrogenism as present when plasma androstenedione level is >3 ng/100 ml . The endocrine parameters of the hyperandrogenic (n = 15) and normoandrogenic (n = 12) hyperinsulinemic patients are reported in Table III. According to such subdivision, metformin administration induced significant changes in endocrine parameters in the hyperinsulinemic hyperandrogenic PCOS patients. As expected, there was a significant reduction of LH, insulin, C-peptide and LH/ FSH ratio, and also of plasma testosterone, androstenedione and 17OHP levels. Non-hyperandrogenic hyperinsulinemic PCOS patients showed only the reduction of insulin, C-peptide and estradiol levels in plasma. It is interesting to notice that these hyperandrogenic subjects showed also higher plasma LH and insulin levels than normoandrogenic subjects. These latter patients showed normal LH/ FSH ratio and no change in plasma levels of LH under metformin administration (Table III).
Figure 1. Insulin response to oral glucose tolerance test at time 0 and +90 min in patients with polycystic ovary syndrome (PCOS): [white square], all PCOS patients at baseline; [black square], all PCOS patients under metformin. Data means with standard error of the mean represented by vertical bars. After 6 months of metformin administration, mean insulin response to oral glucose load was significantly reduced over the whole group of PCOS patients: *p
Table II. Endocrine parameters of PCOS subjects divided according to insulin response during the OGTT.
When patients were considered according to their menstrual cyclicity, it was found that all amenorrheic subjects recovered the occurrence of menstrual cycles (Table IV), with normal cycles in 81% of the subjects. If patients were considered according to the presence of hyperinsulinism or its association with hyperandrogenism, it was revealed that all amenorrheic subjects recovered menstrual cyclicity (eumenorrhea or oligomenorrhea); however, no great differences were observed among the groups (Table V) although menstrual cycles occurred more frequently in hyperinsulinemic than in normoinsulinemic PCOS subjects.
None of the 42 patients studied reported a significant change in BMI during the study period. The Ferriman-Gallway score was significantly reduced in all patients (before: 23.1 1.4, under metformin: 13.80.9, p
Figure 2. Insulin response to oral glucose tolerance test at time 0 and +90 in hyper- and normoinsulimenic patients with polycystic ovary syndrome (PCOS): [white square], hyperinsulinemic PCOS patients at baseline; [black square], hyperinsulinemic PCOS patients under metformin; [black square], normoinsulinemic PCOS patients at baseline; [black square], normoinsulinemic PCOS patients under metformin. Data means with standard error of the mean represented by vertical bars. After 6 months of metformin administration, mean insulin response to oral glucose load was significantly reduced only in hyperinsulinemic subjects: *p
Table III. Endocrine parameters of hyperinsulinemic and non- hyperinsulinemic PCOS patients before and under metformin administration.
Table IV. Change in menstrual cycle frequency of PCOS women (n; = 42) during metformin administration.
The present study demonstrated that metformin induces significant positive endocrine changes in non-obese PCOS patients, restoring menstrual cyclicity in a great percentage of cases, and that the concomitant presence of hyperinsulinism and hyperandrogenism can be considered a clinical index to select patients to treat using metformin.
There is a great abundance of data demonstrating that metformin can be used to treat several disturbances typical of PCOS, including menstrual dysfunctions, hyperandrogenism, hyperinsulinism, insulin resistance, and infertility related to anovulation. It is well known that such positive effects are mediated through the reduction of insulin resistance and plasma insulin levels, which determines the reduction of circulating androgens [19,25-27]. Although to date metformin administration has been demonstrated to be effective both in obese and non-obese PCOS patients [9,10,19,25,28,29], obese PCOS patients have been demonstrated to be those in whom metformin has the greater chance to be effective since the treatment deeply affects body weight through the great improvement of metabolic pathways. Indeed, metformin-induced weight loss or weight loss due to dieting has been demonstrated to decrease insulin resistance, improve all symptoms of PCOS, and restore menstrual cyclicity and ovulation , thus supporting that excess of body fat is one of the main effectors of hyperinsulinemia and insulin resistance, as well as of all the endocrine abnormalities linked to them.
Despite the lack of excess of weight, non-obese PCOS patients have been reported as achieving optimal improvement of endocrine and menstrual disturbances when undergoing metformin administration [19,25]. Our data confirm previous reports, i.e. that metformin administration reduces LH, insulin and LH/FSH ratio with little or no apparent effect on plasma androgen levels , although other authors demonstrated a great efficacy [16,19,21,25]. Such discrepancy can be easily explained by the fact that most of these studies probably enrolled a mixture of PCOS patients with different (low or normal) insulin sensitivities or used quite different metformin dosages. For this reason in our study we tested the insulin response to oral glucose load (i.e. OGTT) of our patients and then analyzed them subdivided into groups on the basis of their insulin response.
Using such criteria we distinguished normo- and hyperinsulinemic PCOS patients. Although the two groups were not different before treatment, after metformin administration LH and LH/FSH ratio decreased in both groups but only the hyperinsulinemic patients showed significant reductions in plasma C-peptide, insulin and testosterone levels. These data are in agreement with previous reports showing that in-non obese PCOS patients the presence of normoinsulinemia might hide an exaggerated response to glucose load (i.e. hyperinsulinism)  and only the documentation of such abnormal response seems to indicate the higher level of eligibility to metformin administration. In addition, it is of interest to notice that the reduction of C-peptide is typical only in hyperinsulinemic subjects, thus confirming that metformin administration induces reduced synthesis of proinsulin and its cleavage to insulin.
Table V. Change in menstrual cycle frequency of PCOS women (n = 42) during metformin administration according to endocrine parameters.
Since hyperandrogenism is one of the endocrine characteristics of PCOS patients , to elucidate the issue further we divided the hyperinsulinemic subjects according to their plasma androstenedione levels at baseline. Such subdivision permitted us to outline that 15 out of 27 (55%) hyperinsulinemic PCOS patients were hyperandrogenic (i.e. plasma androstenedione > 3 ng/ml) and that among hyperinsulinemic PCOS subjects only those with the hyperandrogenic state showed elevated LH plasma levels and LH/FSH ratio > 2.5. Interestingly, only these patients were shown to benefit fully from the metformin administration since our data demonstrated the reduction of insulin, C-peptide, testosterone and androstenedione levels in plasma as well as LH and LH/FSH ratio. Conversely, non- hyperandrogenic hyperinsulinemic subjects showed only the reduction of plasma estradiol, C-peptide and insulin levels.
These observation\s confirm previous, extensively reported results  : that the increase of plasma LH levels frequently observed in PCOS patients is not related to the presence of hyperinsulinemia but to the presence of high androgen plasma levels. However, our data confirm the hypothesis that not every woman with insulin resistance and hyperinsulinemia develops hyperandrogenism and thus support the hypothesis that there are two subpopulations of PCOS patients, one with absence of ovarian hypersensitivity to insulin and one with a greater ovarian hypersensitivity to insulin, as recently proposed .
Menstrual cyclicity was severely compromised in our PCOS patients since there was a high rate of oligomenorrhea and a small percentage of amenorrhea. Nevertheless menstrual cyclicity was restored in all subjects under study. Indeed, all amenorrheic subjects recovered the occurrence of menses, although three out of ten became oligomenorrheic. Conversely, 26 out of 32 oligomenorrheic patients become eumenorrheic and only six did not show any change in their oligomenorrhea. When considering all patients according to the presence of hyperinsulinsm and/or hyperandrogenism, we observed that there was a trend to a higher rate of normalization of menstrual cyclicity in hyperinsulinemic subjects. However, our data support the fact that metformin administration induced the restoration of menstrual cyclicity by acting on all endocrine abnormalities (i.e. hyperinsulinsm, reduced insulin sensitivity, hyperandrogenism, high LH) independendy of the prevalence of each. Such results are in agreement with previous reports that metformin restores menstrual cyclicity in most PCOS patients [19,33], although our data show a higher success rate in terms of menstrual cyclicity since 70% of amenorrheic and 81% of oligomenorrheic patients under study became eumenorrheic after 6 months of metformin administration, taking in account that all amenorrheic subjects recovered menstrual cyclicity.
In conclusion, our study demonstrated that metformin can be proposed as a therapeutic strategy in non-obese PCOS patients since it is effective in restoring an appropriate endocrine balance that induces the occurrence of ovarian activity and menstrual bleeding. Moreover, our data clearly show that at the same time metformin decreases abnormal LH, LH/FSH ratio, testosterone and androstenedione levels only when such abnormalities are coupled to hyperinsulinemia. This observation permits us to infer that some but probably not all nonobese PCOS patients have a greater responsiveness to insulin-stimulated androgen synthesis from ovary and these PCOS patients are those who might achieve greater improvement than others from metformin administration. From a practical point of view our data suggest that hyperandrogenic hyperinsulinemic non-obese PCOS patients might gain more benefit from metformin than others.
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ALESSANDRO D. GENAZZANI1, CHIARA LANZONI1, FEDERICA RICCHIERI1, ENRICA BARALDI2, ELENA CASAROSA3, & VALERIO M. JASONNI1
1 Department of Obstetrics and Gynecology, University of Modena and Reggio Emilia, Modena, Italy, 2 Laboratory of Endocrinology, Azienda Policlinico, Modena, Italy, and 3 Department of Obstetrics and Gynecology, University of Pisa, Pisa, Italy
(Received 28 December 2006; revised 8 January 2007; accepted 10 January 2007)
Corresponden\ce: A. D. Genazzani, Gynecological Endocrinology Unit, Department of Obstetrics and Gynecology, University of Modena and Reggio Emilia, Via del Pozzo 71, 41100 Modena, Italy. Tel: +39 59 4222278. Fax: +39 59 4224394. E-mail: [email protected]
Copyright Taylor & Francis Ltd. Mar 2007
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