Effects of metformin on adrenalsteroidogenesis in women with polycysticovary syndrome

Antonio la Marca, M.D.,* Giuseppe Morgante, M.D.,* Tiziana Paglia, B.Sc.,*Liliana Ciotta, M.D.,† Antonio Cianci, M.D.,† and Vincenzo De Leo, M.D.*

University of Siena, Siena, and University of Catania, Catania, Italy

Objective: To determine whether the administration of metformin, an insulin-sensitizing agent, is followedby changes in adrenal steroidogenesis in women with polycystic ovary syndrome (PCOS).

Design: Prospective trial.

Setting: Department of Obstetrics and Gynecology, University of Siena, Siena, Italy.

Patient(s): Fourteen women with PCOS.

Intervention(s): Blood samples were obtained before (215 and 0 minutes) and after (15, 30, 45, and 60minutes) the administration of ACTH (250mg). Metformin then was given at a dosage of 500 mg three timesa day for 30–32 days, at which time the pretreatment study was repeated.

Main Outcome Measure(s): The adrenal androgen responses to ACTH before and after treatment withmetformin.

Result(s): Ovulation occurred in two women (14%) in response to metformin treatment. A significantreduction in basal concentrations of free testosterone and a significant increase in concentrations of sexhormone-binding globulin were observed. The administration of metformin was associated with a significantreduction in the response of 17a-hydroxyprogesterone, testosterone, free testosterone, and androstenedione toACTH. The ratio of 17a-hydroxyprogesterone to progesterone, which indicates 17a-hydroxylase activity, andthe ratio of androstenedione to 17a-hydroxyprogesterone, which indicates 17,20-lyase activity, were signif-icantly lower after a month of metformin treatment, indicating a reduction in the activities of these enzymes.

Conclusion(s): The administration of metformin to unselected women with PCOS led to a reduction in theadrenal steroidogenesis response to ACTH. This finding supports the hypothesis that high insulin levelsassociated with PCOS may cause an increase in plasma levels of adrenal androgens. (Fertil Sterilt 1999;72:985–9. ©1999 by American Society for Reproductive Medicine.)

Key Words: PCOS, metformin, ACTH, adrenal, androgens

It is well known that high plasma concen-trations of androgens in patients with polycys-tic ovary syndrome (PCOS) may be due toincreased ovarian and adrenal steroid produc-tion. There is evidence that adrenal disorderssuch as Cushing’s syndrome and androgen-secreting tumors often are associated with mi-cropolycystic ovaries (1, 2). Dynamic tests alsohave identified many hyperandrogenic patientsin whom the plasma androgen concentrationshave a large adrenal component (3).

The association between PCOS-related hy-perandrogenemia and insulin resistance is welldocumented. Insulin resistance and the result-ing raised plasma levels of insulin are reportedto be responsible for high androgen concentra-

tions in patients with PCOS. There is evidencethat insulin stimulates ovarian estrogen, andro-gen, and progesterone secretion in vitro (4).Insulin inhibits hepatic secretion of sex hor-mone-binding globulin (SHBG), increasing theavailability of androgens (5). Insulin also mayinhibit hepatic synthesis of insulin-like growthfactor binding protein-1, leading to increasedavailability of insulin-like growth factor I inthe ovary (6).

Several years ago, Lanzone et al. (7) dem-onstrated that hyperinsulinemia in women withPCOS affects adrenal androgen production;they found that the response of androstenedi-one and 17a-hydroxyprogesterone (17-OHP)to ACTH was greater in hyperinsulinemic

Received March 30, 1999;revised and accepted June25, 1999.Reprint requests: VincenzoDe Leo, M.D., Departmentof Obstetrics andGynecology, University ofSiena, Policlinico LeScotte, Viale Bracci, 53100Siena, Italy (FAX: 39-0577-233464; E-mail: deleo@unisi.it).* Department of Obstetricsand Gynecology, Universityof Siena.† Department of Obstetricsand Gynecology, Universityof Catania.

HYPERANDROGENISMFERTILITY AND STERILITY tVOL. 72, NO. 6, DECEMBER 1999Copyright ©1999 American Society for Reproductive MedicinePublished by Elsevier Science Inc.Printed on acid-free paper in U.S.A.

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women with PCOS than in normoinsulinemic women withPCOS. There also is evidence from in vitro and in vivostudies that insulin potentiates the response of adrenal ste-roidogenesis to ACTH (8).

The aim of the present study was to determine whether theadministration of metformin, an insulin-sensitizingagent, isfollowed by changes in adrenal steroidogenesis in womenwith PCOS.

MATERIALS AND METHODS

SubjectsWe recruited 14 women with a mean (6SD) age of 246

3 years (range, 20–28 years). They all had PCOS diagnosedon the basis of oligomenorrhea (,6 menstrual periods inthe previous year) or amenorrhea and hyperandrogenemia.These irregular menstrual cycles had been present sincemenarche. Twelve of the women had a body mass index of.27. None of the women had hypothyroidism or hyperthy-roidism, Cushing’s syndrome, adrenal hyperplasia, or hyper-prolactinemia. They all had evidence of micropolycysticovaries on transvaginal ultrasound examination. None of thewomen were taking drugs or had any significant pathologiccondition. The study was approved by the institutional re-view board of the University of Siena. Written informedconsent was obtained from each subject.

Study DesignThe women were studied in the follicular phase of the

menstrual cycle, as determined by plasma concentrations ofprogesterone of,2 ng/mL. At 8 A.M., after overnight bedrest and fasting, an indwelling catheter was inserted into theantecubital vein and saline solution was infused slowlythroughout the ACTH test to keep the vein patent. Bloodsamples were obtained before (215 and 0 minutes) and after(15, 30, 45, and 60 minutes) the administration of ACTH(250 mg).

Metformin (Metforal; Guidotti, Milan, Italy) was given ata dosage of 500 mg three times a day for 30–32 days, atwhich time the pretreatment study was repeated. Spontane-ous ovulation was checked by obtaining blood samples 20days and 27 days after the initiation of metformin adminis-tration. Two women had serum progesterone values in thepostovulatory range and were excluded from the study.

AssaysPlasma ACTH, LH, FSH, E2, SHBG, cortisol, testoster-

one, androstenedione, and 17-OHP levels were measured bydouble-antibody RIA with use of Radim kits (Rome, Italy)for LH, FSH, cortisol, androstenedione, and DHEAS, DPCkits (Los Angeles, CA) for ACTH and 17-OHP, and Sorinkits (Saluggia, Italy) for testosterone, E2, and SHBG. Thesamples were assayed in duplicate at two dilutions. Allsamples from each subject were assayed together. Qualitycontrol pools at low, medium, and high hormone levels

were included in each assay. The intraassay and interassaycoefficients of variation did not exceed 10% and 15%,respectively.

Statistical AnalysisTo compare the response before and after metformin

therapy, basal hormone concentrations, maximum incre-ments (the maximum rise above baseline levels), and theareas under the curve (cumulative rise above baseline levels)were compared using Student’s two-tailedt test. The areasunder the curves were calculated by the trapezoidal methodand expressed in picograms per milliliter per time as appro-priate. The ratios of the areas of substrates to products beforeand after metformin administration were compared for dif-ferences in 17a-hydroxylase (17-OHP/progesterone) and17,20-lyase (androstenedione/17-OHP) activities.P,.05was considered statistically significant.

RESULTS

The administration of metformin did not lead to anychange in the body mass index of the women (Table 1).Complete data from 12 of the 14 women could be analyzed.Ovulation occurred in 2 women (14%) in response to met-formin therapy. After a month of therapy, a significant re-duction in basal concentrations of free testosterone (196 3pg/mL versus 136 3 pg/mL, P,.05) and asignificantincrease in concentrations of SHBG (43.96 8.2 nmol/Lversus 57.66 9.2 nmol/L, P,.05) wereobserved (Table1). Basal plasma concentrations of LH, androstenedione,

T A B L E 1

Clinical and hormonal data of 12 women with polycysticovary syndrome before and after metformin therapy.

Variable

Study group

Beforemetformin

therapy

Aftermetformin

therapy

BMI (kg/m2) 28.46 3.1 28.26 3.5LH level (mIU/mL) 11.86 1.5 11.26 1.6FSH level (mIU/mL) 6.46 0.5 6.26 0.7E2 level (pg/mL) 866 24 846 18T level (pg/mL) 3206 58 2946 48FT level (pg/mL) 196 3 136 3*Cortisol level (ng/mL) 2186 42 1986 65SHBG level (nmol/L) 43.96 8.2 57.66 9.2*17-OHP level (pg/mL) 1,3006 300 1,2006 350P level (pg/mL) 8206 150 7306 130DHEAS level (mg/mL) 2.26 0.4 2.16 0.4A level (pg/mL) 1,5506 320 1,3506 380

Note: Values are means6 SD. A 5 androstenedione; BMI5 body massindex; FT5 free testosterone; 17-OHP5 17a-hydroxyprogesterone; P5progesterone; SHBG5 sex hormone-binding globulin; T5 testosterone.* P,.05.

la Marca. Effects of metformin. Fertil Steril 1999.

986 la Marca et al. Metformin and adrenal steroidogenesis Vol. 72, No. 6, December 1999

testosterone, DHEAS, and 17-OHP decreased, but notsignificantly.

The administration of metformin was associated with asignificant reduction (as maximum increment and area underthe curve) in the response of 17-OHP, testosterone, freetestosterone, and androstenedione to ACTH (Figs. 1–4).There was no significant change in basal levels of cortisol orin the response of cortisol after a month of metformintherapy (data not shown).

The ratio of 17-OHP to progesterone, which indicates17a-hydroxylase activity, and the ratio of androstenedione to17-OHP, which indicates 17,20-lyase activity, were signifi-

cantly lower after a month of metformin therapy (36.16 6.8versus 24.36 5, P,.05, and0.596 0.11 versus 0.46 0.07,P,.05, respectively), indicating a reduction in the activitiesof these enzymes (Fig. 5).

DISCUSSION

Metformin, a biguanide, normally is used to treat nonin-sulin-dependent diabetes. Its multiple mechanisms of actioninclude inhibition of gluconeogenesis in the liver and stim-ulation of peripheral uptake of glucose (9, 10).

The findings of this study were that metformin causes a

F I G U R E 1

The response of 17-OHP to the administration of ACTH wassignificantly lower after metformin treatment (A) than beforemetformin treatment (B). -h- 5 before treatment; © 5 aftertreatment. *P,.05.

la Marca. Effects of metformin. Fertil Steril 1999.

F I G U R E 2

The response of androstenedione (A) to the administration ofACTH was significantly lower after metformin treatment (A)than before metformin treatment (B). -h- 5 before treatment;© 5 after treatment. *P,.05.

la Marca. Effects of metformin. Fertil Steril 1999.

F I G U R E 3

The response of testosterone (T) to the administration ofACTH was significantly lower after metformin treatment (A)than before metformin treatment (B). -h- 5 before treatment;© 5 after treatment. *P,.05.

la Marca. Effects of metformin. Fertil Steril 1999.

F I G U R E 4

The response of free testosterone (FT) to the administrationof ACTH was significantly lower after metformin treatment (A)than before metformin treatment (B). -h- 5 before treatment;© 5 after treatment. *P,.05.

la Marca. Effects of metformin. Fertil Steril 1999.

FERTILITY & STERILITY t 987

reduction in basal plasma concentrations of free testosteroneand in the adrenal secretion of androgens in response toACTH, as well as an increase in concentrations of SHBG, inwomen with PCOS. No changes in body mass index wereobserved.

Various recent studies on the effects of metformin inwomen with PCOS have produced contrasting results (11–14). Nestler and Jakubowicz (11) found a reduction in basalconcentrations of 17-OHP, testosterone, and free testoster-one and an increase in basal concentrations of SHBG after 8weeks of therapy (500 mg of metformin three times a day) inobese women with PCOS. They also found a reduction inconcentrations of 17-OHP, testosterone, free testosterone,DHEAS, and androstenedione and an increase in concentra-tions of SHBG in lean women with PCOS (12) after 4–6weeks of therapy (same dosage). Ehrmann et al. (13) founda reduction in concentrations of SHBG and total testosteroneand no change in concentrations of free testosterone after 12weeks of metformin therapy. Another study showed an im-provement in the menstrual pattern and fertility of womenwith PCOS after 4–6 weeks of therapy (14).

Our results are partly in agreement with previous find-ings. One month of metformin therapy produced ovulatorycycles in two women (14%). Our results suggest that themain effect of metformin was on the liver, with increasedsecretion of SHBG. The reduction in free testosterone pre-sumably was a secondary effect.

The reduced response of 17-OHP and androstenedione toACTH is compatible with a reduction in adrenal cytochromeP-450 activity. Metformin presumably brought about a re-duction in 17a-hydroxylase and 17,20-lyase activity by re-ducing insulin levels.

The observation that the adrenal response to ACTH wasmodified after metformin administration suggests that insu-lin could have a role in the regulation of adrenal steroido-genesis. It is unlikely that the drug has a direct effect onthese enzymes, but it is possible that the reduction of freetestosterone secondary to the increase in SHBG may havecaused a reduction in the adrenal response to ACTH. As werecently demonstrated, the administration of flutamide, anonsteroid antiandrogen, to women with PCOS induces asignificant reduction in the adrenal androgen response tocorticotropin-releasing hormone, probably by blocking thenormal enhancing effect of androgens on adrenal androgenproduction (15). It seems unlikely that metformin could havecaused a reduction in adrenal steroidogenesis merely as aconsequence of the reduction in free testosterone since wedid not find any correlation between the degree of reductionof free testosterone and the reduction in adrenal androgenresponses to ACTH (data not shown).

Nestler and Jakubowicz (16) hypothesized that by reduc-ing hyperinsulinemia, metformin led to a reduction in ovar-ian cytochrome P-450 17a-activity, as shown by a reducedresponse of 17-OHP to GnRH stimulation. Perhaps throughthe insulin-like growth factor system, insulin increases themessenger RNA and enzyme levels of the rate-determiningenzyme in steroidogenesis cholesterol side-chain cleavagecytochrome P-450, and reverses LH-induced down-regula-tion of the messenger RNA and 17a-hydroxylase/17,20-lyase cytochrome P-450, the rate-limiting enzyme in ratovarian steroidogenesis (17). Moreover, Moghetti et al. (8)demonstrated in vivo and in vitro that insulin infusion inhyperandrogenic women potentiates ACTH-stimulated adre-nal steroidogenesis and that the effect of insulin seems to beassociated with relative impairment of 17,20-lyase activity.

Hyperinsulinemic patients with PCOS have higher andro-stenedione and 17-OHP levels in response to ACTH thannormoinsulinemic patients with PCOS (7). We intentionallydid not screen the women for insulin resistance, so ourresults are applicable to unselected women with PCOS. Ourstudy demonstrated that the administration of metformin tounselected women with PCOS led to a reduction in theadrenal steroidogenesis response to ACTH. This findingprovides support for the hypothesis that high insulin levelsassociated with PCOS may cause an increase in plasmalevels of adrenal androgens.

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F I G U R E 5

The ratio of 17-OHP to progesterone (P), which indicates17a-hydroxylase activity (A), and the ratio of androstenedi-one (A) to 17-OHP, which indicates 17,20-lyase activity (B),were significantly lower after a month of metformin treatment.* P,.05.

la Marca. Effects of metformin. Fertil Steril 1999.

988 la Marca et al. Metformin and adrenal steroidogenesis Vol. 72, No. 6, December 1999

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