1 Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology and Infertility, 2 Department of Internal Medicine, Division of Endocrinology, Faculty of Medicine, Hacettepe University and 3 Department of Obstetrics and Gynecology, Faculty of Medicine, Basikent University and 4 Department of Nuclear Medicine, Faculty of Medicine, Hacettepe University, Ankara, Turkey
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Abstract |
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Key words: insulin resistance/low-dose step-up protocol/metformin/polycystic ovary syndrome/rFSH
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Introduction |
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Clomiphene citrate is the primary agent used for ovulation induction in infertile patients with PCOS. Clomiphene citrate will induce ovulation in ~80% but will achieve pregnancy in only 35% of these patients (Macgregor et al., 1968). Exogenous gonadotrophins are traditionally employed to restore ovulation in clomiphene citrate-resistant cases. Low-dose step-up protocol is the treatment of choice and yields a high rate of monofollicular growth and ovulation (White et al., 1996
; Yarali et al., 1999
).
On the basis of the theory that insulin resistance and resultant hyperinsulinaemia impede ovulation and may be important contributors to the pathophysiology of PCOS, it may be postulated that insulin sensitizers might improve the abnormal endocrine milieu associated with PCOS, resulting in an increase in ovulatory cycles and pregnancy. Metformin is a biguanide that has been used for many years in Europe for the treatment of diabetes mellitus. Metformin has been reported to improve insulin resistance (Velazquez et al., 1994; Diamanti-Kandarakis et al., 1998
; Nestler et al., 1998
; Glueck et al., 1999
; Kolodziejczyk et al., 2000
; Moghetti et al., 2000
), decrease serum total testosterone (Pirwany et al., 1999
; Kolodziejczyk et al., 2000
) and free testosterone concentrations (Nestler et al., 1998
; Moghetti et al., 2000
), increase SHBG levels (Nestler and Jakubowicz, 1996
), improve menstrual cyclicity (Velazquez et al., 1997
; Diamanti-Kandarakis et al., 1998
; Morin-Papunen et al., 1998
) and documented ovulation (Nestler et al., 1998
; Glueck et al., 1999
) in patients with PCOS. However, in other studies, metformin had no beneficial effect on insulin resistance (Crave et al., 1995
; Acbay and Gundogdu, 1996
; Ehrmann et al., 1997
; Pirwany et al., 1999
) and various endocrine parameters (Crave et al., 1995
; Acbay and Gundogdu, 1996
; Ehrmann et al., 1997
).
There is a paucity of data on the use of metformin as an adjunct to ovulation induction in infertile patients with PCOS. Nestler et al. reported a significant increase in the number of ovulatory cycles in conjunction with clomiphene citrate compared with placebo (Nestler et al., 1998). De Leo et al., using the conventional protocol for exogenous gonadotrophin treatment in clomiphene citrate-resistant PCOS patients, reported an orderly follicular growth and a reduction in multifollicular development with co-administration of metformin (De Leo et al., 1999
). To our knowledge there are no data evaluating the effect of metformin on ovarian response when co-administered during exogenous gonadotrophin treatment using the low-dose step-up protocol.
The aim of this prospective randomized, placebo-controlled study is to analyse the impact of metformin on ovarian response when co-administered during a low-dose step-up protocol using recombinant (r)FSH.
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Materials and methods |
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Exclusion criteria included the presence of any infertility factor other than PCOS, the use of medications known to alter insulin secretion or action, endocrinopathies, including non-classic congenital adrenal hyperplasia due to 21-hydroxylase deficiency, Cushing's syndrome, hyperprolactinaemia or thyroid dysfunction. Patients with impaired glucose tolerance or type 2 diabetes were excluded. No patient was excluded on the basis of body mass index (BMI).
Informed consent was obtained from all patients and the Institutional Review Board approved the study.
Experimental protocol
At the time of entry into the study, all the women were in the equivalent of the follicular phase of the menstrual cycle and they had either spontaneous or progesterone-induced menses during the preceding week. At first visit, patients' weight, height, waist and hip circumferences were measured. The BMI [weight (kg)/height2 (m2)] and the waist:hip ratio (WHR) were calculated.
An i.v. cannula was inserted into a forearm or antecubital fossa vein between 08000900 h and blood samples were obtained for baseline serum concentrations of LH, FSH, testosterone, free testosterone, androstenedione, estradiol (E2), 17-OH progesterone, dehydroepiandrosterone sulphate (DHEAS) and leptin.
Oral glucose tolerance test
OGTT was performed after a 3 day, 300 g carbohydrate diet and 12 h overnight fasting. A 75 g oral glucose load was administered and blood samples were collected through an intravenous cannula for plasma glucose and insulin concentrations at 0, 30, 60, 90 and 120 min.
Frequently sampled i.v. glucose tolerance test (FSIGTT)
Intravenous cannulas were placed in both antecubital veins. A bolus of 50% glucose solution (0.3 gr/kg) was injected over 1 min at time zero. Regular human insulin (0.03 IU/kg Humulin®; Eli Lilly and Corp., IN, USA) was administered as an i.v. bolus at 20 min. Blood samples were collected at 15, 10, 5, 1, 2, 3, 4, 5, 6, 8, 10, 14, 19, 22, 25, 30, 50, 70, 100, 140 and 180 min for determination of plasma glucose and insulin concentrations.
Following the completion of baseline studies, 32 women were randomized to oral placebo (n = 16) or metformin (Glucophage Retard; Ilsan-Iltas Pharmaceuticals, Istanbul, Turkey) (n = 16) groups using computer-generated numbers. The study was double-blinded. The women took metformin or placebo alone for 6 weeks to allow metformin to exert its putative insulin-sensitizing effect before exogenous gonadotrophin treatment. The dose of metformin was 850 mg (two tablets daily). Patients were instructed not to modify their usual eating habits throughout the study.
Restoration of spontaneous ovulation was monitored by weekly serum progesterone levels and ovulation was assumed to have occurred when this was >5 ng/ml. Those who spontaneously ovulated on placebo or metformin did not undergo subsequent ovulation induction with rFSH.
The pretreatment studies were repeated after 6 weeks in all subjects excluding the two patients who spontaneously conceived. Metformin or placebo was continued during ovulation induction with rFSH until the day of HCG administration.
Ovulation induction with rFSH (Follitropin alpha, Gonal-F; AresSerono, Geneva, Switzerland) using the low-dose step-up protocol was performed as previously described (White et al., 1996; Yarali et al., 1999
). Treatment was commenced on day 35 of a spontaneous or progesterone-induced menstrual bleeding. The starting dose was 75 IU of FSH s.c. daily. The initial dose of 75 IU/day was maintained for up to 14 days unless follicle maturity was reached so that HCG would be administered. Ovarian response was initially monitored by serum E2 levels every 23 days. When serum E2 level was >100 pg/ml (conversion factor = 3.671; 367 pmol/l), monitoring was continued by daily transvaginal ultrasonography and serum E2 levels. If no ovarian response was noted after 14 days of 75 IU/day therapy, the daily dose was increased by half a vial (37.5 IU). Any further increment, if necessary, was made by half a vial (37.5 IU) at weekly intervals to a maximum of 187.5 IU/day. If a dominant follicle emerged, the dose of FSH (threshold dose) was maintained until the follicle reached a mean diameter of 17 mm. At that point, HCG (Profasi; Serono, Aubonne, Switzerland) at a dose of 10 000 IU was administered by i.m. injection. If there were more than three follicles of
15 mm in diameter, the cycle was cancelled due to the risk of multiple pregnancy and/or ovarian hyperstimulation syndrome (OHSS). If there was no ovarian response after 35 days of treatment, the cycle was cancelled. Blood was taken for measurement of progesterone 68 days after HCG administration and ovulation was assumed to have occurred when progesterone level was >5 ng/ml (conversion factor = 3.18; 15.9 nmol/l). A serum pregnancy test was performed 1315 days after administration of HCG.
Assay methods
Blood samples were centrifuged immediately, and serum was stored at 20°C until assayed. Plasma glucose was measured by the glucose oxidase technique (Boehringer, Mannheim, Germany). LH, FSH, E2, total testosterone and DHEAS were measured by chemiluminescent enzyme immunoassay (Immulite 2000; Diagnostic Products Corp., Los Angeles, CA, USA) with an average inter-assay coefficient of variation (CV) of 8% and intra-assay CV of 7%. Leptin was measured by using an immunoradiometric assay (Active Human Leptin IRMA, DSL-23100; Diagnostic Systems Laboratories Inc., Webster, TX, USA) with intra-assay and inter-assay CV of 4.9 and 6.6% respectively. Free testosterone, androstenedione and insulin were measured by radioimmunoassay (Diagnostic Systems Laboratories). The average inter-assay and intra-assay CV were 6.7 and 6.4% respectively. Plasma 17-OH progesterone was measured by radioimmunoassay (Immunotech, Marseille, France) with an intra-assay CV of 5.4% and inter-assay CV of 4.5%. To avoid inter-assay variation, all samples were analysed in duplicate in a single assay.
Data analysis
Using glucose and insulin data from the OGTT and FSIGTT, the following parameters were calculated: fasting glucose to insulin ratio [FG:I ratio (mg/10-4 IU)], glucose and insulin area under curves during OGTT (AUCglucose and AUCinsulin), the first phase insulin secretion [area in response to glucose: AIRg (µIU/ml min)] in response to glucose, calculated as the mean increment above basal of insulin values measured at 2, 3, 4, 5, 6, 8 and 10 min during FSIGTT, the insulin sensitivity index [SIx10-4 (min-1.pmol-1.l-1)] calculated using the minimal model analysis (MINMOD) program (Bergman et al., 1981). The SI represents the increase in net fractional glucose clearance rate per unit change in plasma insulin concentration after the i.v. glucose load.
Statistical analysis
SPSS for Windows for 9.0 (SPSS, Chicago, IL, USA) was used for statistical analysis. Student's t-test, MannWhitney U-test, 2-test, Fisher's exact test were used where appropriate. Areas under curves for glucose and insulin were calculated using the trapezoidal formula. Data are given as mean ± SD. P
0.05 was considered statistically significant.
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Results |
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One patient on placebo and six patients on metformin ovulated spontaneously (P < 0.05). In the metformin group, ovulation was noted at week 3 in five patients and at week 6 in one patient. The only ovulation with placebo was noted at week 6. All these seven patients did not undergo subsequent ovulation induction with rFSH. Two patients on metformin conceived spontaneously. Of these two patients, one has an ongoing pregnancy and the other had a miscarriage.
On entry into the study, the metformin and placebo groups did not differ with respect to the anthropometric variables (Table I). BMI and WHR remained stable over the course of the study in both groups.
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Hormonal and metabolic characteristics of study subjects
Responses during OGTT and IVGTT
By design, no subject had impaired glucose tolerance or type 2 diabetes. Before treatment with placebo or metformin for 6 weeks, all parameters of insulin resistance, including the baseline mean fasting insulin level, FG:I ratio, AUCinsulin, SI and AIRg values were comparable between the two groups (Table II). In neither group was there any change in mean fasting insulin, fasting glucose/insulin ratio, AUCglucose, AUCinsulin, SI and AIRg values after 6 weeks of treatment with placebo or metformin (Table II
). In the metformin group, AUCinsulin decreased from 50 040 ± 21 918 to 43 806 ± 17 064 pmol/l min, and AIRg decreased from 4016 ± 2484 to 3065 ± 1690 pmol/l min; however, these differences did not reach statistical significance.
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Data on ovulation induction with rFSH
The baseline characteristics of the placebo (n = 15) and metformin (n = 10) groups undergoing ovulation induction with rFSH, including the mean age, duration of infertility, all insulin resistance indices and endocrine parameters, were comparable (data not shown).
All parameters of ovarian response, including the mean duration of stimulation, total dose of rFSH used, E2 on the day of HCG and endometrial thickness on the day of HCG were comparable between the two groups (not significant) (Table III). Monofollicular development and ovulation rates were also comparable between the two groups (not significant). Two cycles were cancelled in the placebo group; one due to no ovarian response and the other due to development of more than three follicles of
15 mm diameter. One patient in the placebo group and three patients in the metformin group conceived. Of the three patients who conceived in the metformin group, two have an ongoing pregnancy and the remaining had a miscarriage. The only patient who conceived in the placebo group has an ongoing pregnancy.
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Discussion |
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Previous studies have reported conflicting data on the effect of metformin on insulin resistance and endocrine parameters in patients with PCOS. Although some studies reported an improvement in insulin resistance (Velazquez et al., 1994; Diamanti-Kandarakis et al., 1998
; Nestler et al., 1998
; Glueck et al., 1999
; Kolodziejczyk et al., 2000
; Moghetti et al., 2000
), others failed to observe any statistically significant effect (Crave et al., 1995
; Acbay and Gundogdu, 1996
; Ehrmann et al., 1997
; Pirwany et al., 1999
), in agreement with the present study. The effect of metformin on endocrine parameters has also been variable (Taylor, 2000
). The three studies reporting no effect of metformin on insulin resistance have not provided data on restoration of ovulation (Crave et al., 1995
; Acbay and Gundogdu, 1996
; Ehrmann et al., 1997
). However, in 15 obese patients with PCOS, despite no change in fasting insulin concentration, a significant improvement in spontaneous ovulation was reported with metformin (Pirwany et al., 1999
).
Several hypotheses have been proposed to explain why metformin successfully lowers insulin and androgen levels in some studies but not in others. These include variations in patient inclusion criteria, BMI, dosing and duration of metformin therapy, and genetic background. In the study in which metformin had no effect on hyperinsulinaemia and androgen excess (Ehrmann et al., 1997), the mean BMI was 39 kg/m2. However, BMI is not the sole predictor of success of metformin therapy, since the mean BMI of the current study and another negative study (Acbay and Gundogdu, 1996
) are ~27 and ~30 kg/m2 respectively. The variability of responses to comparable regimens of metformin in individual studies underscores the role of ethnic background and other factors contributing to a great diversity of clinical and hormonal profiles found among women with PCOS.
There is a lack of data on the effect of metformin therapy when used as an adjunct to ovulation induction in infertile patients with PCOS. To our knowledge, there is only one study evaluating the effect of pretreatment with metformin on exogenous gonadotrophin treatment in infertile women with clomiphene citrate-resistant PCOS (De Leo et al., 1999). Twenty women were randomized to two groups. The first group consisted of 10 patients treated with urinary (u)FSH alone for two cycles and then for 1 month with metformin, after which they underwent a third cycle of combined metformin and uFSH stimulation. The second group (n = 10) was treated with metformin for 1 month before undergoing ovulation induction with combined metformin and uFSH for one cycle. Metformin was given at a dose of 500 mg three times daily. Conventional protocol was employed for uFSH treatment. In the first group, two women conceived; hence eight women in the first group were treated with metformin before the final stimulation cycle with uFSH. Therefore, a total of 19 cycles with uFSH alone and 18 cycles with uFSH and metformin were performed and compared. The number of follicles >15 mm in diameter on the day of HCG administration, the percentage of cycles with HCG withheld because of excessive follicular development and plasma levels of E2 on the day of HCG administration were significantly lower in cycles performed after metformin treatment. Two pregnancies occurred in women treated with uFSH alone and three pregnancies occurred in those treated also with metformin. The authors concluded that co-administration of metformin led to an orderly induced ovulation with uFSH and lowered the risk of multifollicular development and hence OHSS. There are two drawbacks of this study. First, by design, crossing-over of the first group to the second group may create selection bias and should be avoided in studies with terminal events such as pregnancy (Daya, 1993
). Second, there is no data on the effect of metformin on insulin resistance and endocrine parameters. Of interest, no patient ovulated in response to metformin only.
Multiple follicular development resulting in high rates of multiple pregnancy and OHSS is the major drawback of conventional protocols. Low-dose step-up protocol is the treatment of choice in clomiphene citrate-resistant cases and yields a high rate of monofollicular growth and ovulation (White et al., 1996; Yarali et al., 1999
). The principle behind the low-dose step-up protocol is to find the threshold level of FSH that will lead to the development of a single pre-ovulatory follicle, thereby avoiding multiple pregnancy and OHSS. Dale et al. examined the impact of insulin resistance on the outcome of ovulation induction with the low-dose step-up protocol in 42 clomiphene citrate-resistant women with PCOS (Dale et al., 1998
). The insulin-resistant women required a higher gonadotrophin dose and a longer time to achieve follicular maturation. Cycle cancellation due to multifollicular development was significantly greater in the insulin-resistant cases. Although ovulation rates were similar between the insulin-resistant and non-insulin-resistant groups, the conception rate was significantly better in the non-insulin-resistant PCOS women.
To our knowledge, there are no data on the effect of co-administration of metformin during a low-dose step-up protocol using rFSH in clomiphene citrate-resistant PCOS patients. In contrast to the study by De Leo (De Leo et al., 1999), all parameters of ovarian response were comparable between the placebo and metformin groups in our study.
We should stress that our study is a pilot study, and a lack of difference in insulin sensitivity and ovulation induction parameters may result from Type 2 statistical error due to limited sample size and high variation. Further well-designed, powerful, prospective randomized studies are warranted to delineate the role of metformin for co-administration during low-dose step-up protocol using exogenous gonadotrophins.
We conclude that in clomiphene citrate-resistant PCOS women with normal glucose tolerance: (i) metformin may markedly restore spontaneous ovulation with no improvement in insulin resistance; and (ii) metformin has no significant effect on ovarian response during low-dose step-up protocol using rFSH.
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Acknowledgements |
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Notes |
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References |
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accepted on October 19, 2001.