Hum. Reprod. Advance Access originally published online on October 27, 2005
Human Reproduction 2006 21(2):457-465; doi:10.1093/humrep/dei351
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Uterine effects of metformin administration in anovulatory women with polycystic ovary syndrome
1 Department of Obstetrics & Gynecology, University ‘Magna Graecia’ of Catanzaro, 2 Department of Molecular & Clinical Endocrinology and Oncology, 3 Department of Clinical and Experimental Medicine, 4 Department of Obstetrics & Gynecology, University ‘Federico II’ of Naples and 5 Department of Endocrinology, University of Palermo, Italy
6 To whom correspondence should be addressed at: Department of Gynecology & Obstetrics, University ‘Magna Graecia’ of Catanzaro, Via M. Greco vico XI, 88100 Catanzaro, Italy. E-mail: stefanopalomba{at}tin.it
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Abstract |
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BACKGROUND: Metformin has been shown to improve fertility in anovulatory patients with polycystic ovary syndrome (PCOS), inducing not only a high ovulation and pregnancy rate but also reducing the incidence of miscarriages. The aim of the present study was to evaluate the uterine effects of metformin in patients with PCOS who ovulated under metformin. METHODS: Thirty-seven non-obese primary infertile anovulatory patients with PCOS and another 30 age- and body mass index-matched healthy women (control group) were studied. PCOS patients were treated with metformin (850 mg twice daily) for 6 months, whereas the control group did not receive any treatment. In these PCOS patients who ovulated whilst under metformin treatment (PCOS group) and in controls, uterine, sub-endometrial and endometrial blood flow, and endometrial thickness and pattern were evaluated using serial ultrasonographic assessments. RESULTS: Before treatment, uterine, sub-endometrial and endometrial blood flows were significantly lower in patients with PCOS than in the control group. All indexes of uterine vascularization were significantly improved in the PCOS group with metformin treatment and were not different from the controls. Nor was any difference in endometrial thickness and pattern detected between PCOS and control groups. After grouping the data of PCOS patients who ovulated under metformin for cycles with favourable/unfavourable reproductive outcome, no difference in any parameter was observed. CONCLUSIONS: Metformin improves all surrogate markers of endometrial receptivity in PCOS patients, without difference between patients who had favourable or unfavourable reproductive outcome.
Key words: anovulation/endometrium/metformin/PCOS/ultrasound
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Introduction |
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Several approaches have been proposed to treat the ovarian dysfunction in women with polycystic ovary syndrome (PCOS) (Palomba et al., 2004a
). Metformin, an oral biguanide used in type-2 diabetes mellitus, has been shown to be an effective treatment for anovulatory PCOS patients not only in terms of menstrual cyclicity but also of fertility (Lord et al., 2003; Kashyap et al., 2004; Palomba et al., 2004b, 2005a).
Recent randomized controlled trials (RCT) have shown that anovulatory women with PCOS who have restored ovulation after metformin treatment exhibited a surprisingly high reproductive potential, with lower-than-expected rate of spontaneous miscarriage (Palomba et al., 2004b, 2005a). The reasons for these findings have not been determined but it is possible to hypothesize a beneficial action of the drug on endometrial development/receptivity to blastocyst implantation and/or on the quality of the oocytes/embryos. However, currently the relative impact of endometrial versus embryonic factors on implantation in human reproduction is not completely clear (Schwartz et al., 1997).
The primary function of the endometrium is to prepare for and to sustain the nidation of the embryo. Despite the ideal method of predicting uterine receptivity not having yet been established, the histological analysis of an endometrial biopsy specimen has been proposed (Noyes and Haman, 1953) and successively widely accepted as standard procedure for endometrial dating. Moreover, this last procedure is invasive and cannot be used in treatment cycles. On the other hand, ultrasound examination has been widely applied to study the endometrial receptivity in spontaneous and/or stimulated menstrual cycles (Pierson, 2003). Specifically, the assessment of uterine artery velocimetry and of the endometrial characteristics, such as thickness, pattern and vascularity, has been proposed (Pierson, 2003). Furthermore, these parameters seem to be only a surrogate of endometrial receptivity because controversial results have been reported with particular regard to their predictive value for conception (Friedler et al., 1996; Tropea et al., 2004).
Based on these considerations, the present study was carried out in order to evaluate the uterine effects, if any, of metformin in women with PCOS using several surrogate ultrasonographic parameters of uterine receptivity.
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Materials and methods |
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Patients
The patients consisted of 50 non-obese primary infertile anovulatory women with PCOS initially enrolled in a wider RCT (Palomba et al., 2005a
). The diagnosis of PCOS was made according to the National Institutes of Health (NIH) criteria (Zawadzki and Dunaif, 1992). Exclusion criteria for all subjects included: age <20 or >34 years; body mass index (BMI) >30 and <18 kg/m2; neoplastic, metabolic (including glucose intolerance), hepatic, and cardiovascular disorder or other concurrent medical illness; hypothyroidism, hyperprolactinaemia, Cushing’s syndrome, and non-classical congenital adrenal hyperplasia; current or previous (within the last 6 months) use of oral contraceptives, glucocorticoids, antiandrogens, ovulation induction agents, antidiabetic and anti-obesity drugs or other hormonal drugs. Other exclusion criteria were: no uterine bleeding after progesterone challenge test, organic pelvic diseases, previous pelvic surgery, suspected peritoneal factor infertility, tubal or male factor infertility. We excluded also women who were intended to start a diet or a specific programme of physical activity. All subjects had a normal physical activity and none drank alcoholic beverages.
Controls
Controls consisted of healthy volunteer females with regular menstrual cycles and at least one spontaneous previous pregnancy ended in the birth of a healthy baby.
Their healthy state was determined by medical history, physical and pelvic examination, and complete blood chemistry. The normal menstrual cycle was diagnosed after a 3 month pre-study period. During this period, all healthy women recorded on a daily diary the characteristics of their menses. A normal menstrual cycle was defined as cyclic uterine bleedings with duration of 4–5 days and a frequency of 26–32 days. The quantity of blood flow was defined as ‘normal’ subjectively by each woman and objectively using a serum haemoglobin assay. At entry, the normal ovulatory state was confirmed by serial transvaginal sonography (TVS) and plasma progesterone levels assayed during the mid-luteal phase of the cycle (7 days before the expected menses). Women with clinical and/or biochemical hyperandrogenism or with polycystic ovaries (PCO) were excluded from the control group.
Thirty-seven controls were carefully matched one-to-one with the 37 cases (see below) for age and BMI. Specifically, a control was considered age- and BMI-matched with a case when the difference in chronological age and in BMI was 
12 months or 0.5 kg/m2 respectively.
Ethics
The procedures used were in accordance with the guidelines of the Helsinki Declaration on human experimentation. The study was approved by the Institutional Review Board of the University ‘Magna Graecia’ of Catanzaro. The purpose of the protocol was carefully explained to each woman, and written consent was obtained from them before beginning the study.
Protocol and treatment
At study entry, all subjects underwent venous blood withdrawal to evaluate a complete hormonal assay and serum glucose and insulin levels at baseline and after oral glucose tolerance test (OGTT). The fasting glucose:insulin ratio (GIR) (mg/10–4 IU), the area under curve (AUC) for glucose and insulin, and the AUCglucose:AUCinsulin ratio, and the free androgen index (FAI) [testosterone (nmol/l)/sex hormone-binding globulin (SHBG, nmol/1)x100] were also calculated.
At baseline, in each subject the same operator calculated the modified Ferriman–Gallwey score, evaluated the patients’ daily physical activity, job, and daily activities using a well-validated semi-quantitative questionnaire, (Palomba et al., 2002), performed a TVS and assessed the anthropometric measurements [including height, weight, BMI and waist:hip ratio (WHR)].
PCOS patients were treated with metformin (850 mg twice daily). The patients were instructed to take the tablets with meals and to have sexual intercourse every 2 days for four times as suggested by serial TVS (mean diameter >18 mm of the leading follicle). The duration of the treatment was 6 months. Patients who conceived during the study suspended treatment and were followed throughout pregnancy.
All subjects were instructed to follow their usual diet and physical activity.
During the study, the ovulation, pregnancy, abortion, and live-birth rates were evaluated in each woman. These data have been already reported elsewhere (Palomba et al., 2005a).
Each subject underwent serial TVS examinations by the same experienced operator (T.R.) and the following parameters were assessed: uterine and ovarian dimensions and blood flows, follicular/corpus luteum dimensions and vascularity; endometrial thickness, pattern, and vascularity. Furthermore, in the present study, only the evaluations of the uterine parameters in the PCOS patients who ovulated under metfomin will be described, while the data regarding the effect of metformin on ultrasonographic parameters of ovarian function will be reported elsewhere.
To assess the relationship, if any, between uterine effects of metformin administration and reproductive outcomes, the data were analysed considering for each patient all ovulatory cycles induced by metformin treatment until pregnancy was achieved, and categorizing retrospectively the cycles according to different outcome, i.e. ovulation alone and early pregnancy loss (group A) or ongoing pregnancy (group B).
Ultrasonographic and Doppler assessments
Serial TVS examinations were performed with the use of an ultrasonic scanner (Aplio; Toshiba Medical Systems, Rome, Italy) equipped with a 7.5 MHz vaginal probe between 08:00 and 11:00 in order to avoid the effects of circadian rhythms on uterine blood flow (Zaidi et al., 1995a) and before or after spontaneous uterine contractions (Dastidar and Dastidar, 2003). Subjects rested for 
15 min before TVS examination and completely emptied the bladder to minimize any external effects on blood flow. Just before TVS examination, systolic and diastolic blood pressures and heart rate were taken, and, in the cases of abnormal values, the examination was delayed until these parameters were normalized.
In the PCOS group, scans were performed before and throughout treatment. Specifically, they were obtained before treatment on days 3, 14 and 21 after the onset of progesterone-induced menses during a pre-study period, whereas during treatment the following schedule was adopted: the first assessment in early follicular phase (day 3 after the onset of progesterone-induced menses), every 3 days beginning 7th day after treatment started and daily when a follicle achieved 16 mm, and finally 7 days before expected menses. Ovulation was retrospectively defined with the observation of follicular dimension decrease and of liquid in the cul-de-sac, and confirmed by plasma progesterone assay.
In the control group, scans were obtained during three different visits (day 3 of the menstrual cycle, and 14 and 7 days before the expected menses) of the same cycle.
Uterine arteries were studied by using a colour Doppler system, whereas endometrial and sub-endometrial flows were evaluated with the use of a power Doppler system.
The bias due to inter-observer error was avoided because the ultrasonographic assessments were performed by a single operator. The intra-observed error was evaluated in five out of 30 healthy controls by three consecutive measurements at 15 min intervals and calculated using analysis of variance (ANOVA). The intra-observer coefficients of variation for the endometrial thickness and for uterine, sub-endometrial and endometrial blood flow were respectively 4, 8, 10 and 10%.
Endometrial thickness
The endometrium was measured by longitudinal scans including the whole endometrium at the point of its maximum thickness. Both endometrial outer limits were included in the measurement, assuming that the amount of intracavity fluid was not significant. In all cases, a mean of three measurements was considered.
Endometrial echogenicity pattern
In the peri-ovulatory and mid-luteal phases, the endometrial pattern was subjectively evaluated by comparison with the echogenicity of the surrounding myometrium.
In the peri-ovulatory phase, it was described as ‘homogeneous pattern’ or as ‘triple-line pattern’. The ‘homogeneous pattern’ was defined as a single hyperechoic, hypoechoic or isoechoic layer with absent or poorly defined central echogenic line, whereas the ‘triple-line pattern’ was defined as a multilayered endometrium consisting of a prominent outer and inner hyperechoic line and inner hypoechoic regions (Gonen and Casper, 1990a).
In the mid-luteal phase the endometrial echogenicity pattern was classified in four grades: grade 1, hypoechoic pattern with a minimal hyperechoic component (basalis) and a distinct lumen; grade 2, 
50% hypoechoic and 50% hyperechoic pattern with a distinct lumen; grade 3, predominantly hyperechoic pattern with a minimal central hypoechoic area and a distinct lumen; grade 4, a totally hyperechoic pattern. The grade 4 pattern was considered satisfactory for a normal luteal phase and for this reason the mid-luteal phase endometrial echogenicity patterns were categorized as ‘high-grade pattern’, corresponding to grade 4, and as ‘low-grade pattern’, corresponding to grades 1–3 (Doherty et al., 1993).
Pulsed Doppler measurements
Colour flow images of the ascending main branch of the uterine arteries on the right and left side of the cervico-uterine junction at the level of the internal os in a longitudinal plane before it entered the uterus were visualized and studied by positioning a sample volume over the area of maximum colour and by activating the pulsed Doppler. The mean of three consecutive waveforms of good quality was analysed. For each patient, the blood flow impedance of both sides was calculated.
A power flow map was then superimposed on the sub-endometrial and endometrial regions. The Doppler flow indices of vessels seen at the sub-endometrial and endometrial regions were studied at random on the areas of maximum colour intensity, and the lowest values for resistance to flow were recorded.
Blood flow impedance was evaluated and expressed as pulsatility index [PI = (peak systolic velocity – peak diastolic velocity)/mean velocity] and resistance index [RI = (peak systolic velocity – peak diastolic velocity)/peak systolic velocity]. These two parameters were calculated electronically by the machine.
A high-pass filter of 125 Hz was used. The spatial peak temporal average intensity of ultrasound for B-mode and Doppler examinations was <100 mW/cm2, which is within the safety limits recommended by the Bioeffects Committee of the American Institute of Ultrasound in Medicine (Barnett et al., 2000).
Computerized analysis of sub-endometrial and endometrial vascularity
Images (from uterine fundus to the cervico-uterine junction at the level of the internal os in a longitudinal plane) with colour signals obtained by power Doppler were recorded for each patient and acquired using an advanced digital camera, and then processed.
The presence of sub-endometrial and endometrial vascularity was noted, and their detection rates were calculated in each group as number of positive examinations for sub-endometrial and endometrial vascularization divided by the total number of power Doppler assessments performed.
The extension of vascularity was successively studied using advanced image analysis software (Image-Pro Plus 4.5; Media Cybernetics Inc., Silver Spring, MD, USA). Specifically, a single operator, blinded for image allocation, quantified the percentage of positive areas (% pixels) to power Doppler. The selected fields comprised sub-endometrial and endometrial areas. Endometrial borders were set as the outer limits of the hyperechogenic myometrium–endometrium interface, whereas sub-endometrial areas were considered arbitrarily 1 cm around the endometrial borders.
Statistical analysis
Data were expressed as mean ± SD and analysed using the intention-to-treat (ITT) method.
The Kolmogorov–Smirnov statistic with a Lilliefors significance level was used for testing normality for continuous variables that were analysed with the paired and the unpaired t-test, and with the repeated measures analysis of variance (ANOVA) with Bonferroni test for the post hoc analysis, as appropriate. For categorical variables Pearson’s 
2-test and Fisher’s exact test were applied as needed. P 0.05 was considered significant. The Statistics Package for Social Science (SPSS 13.0.1, December 2004; SPSS Inc., Chicago, IL, USA) was used for statistical analyses.
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Results |
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The data were obtained on a population of 37 women with PCOS and 30 control subjects. In fact, in the PCOS group a total of 13 patients was excluded for the following reasons: missed first follow-up visit (three patients), precocious treatment suspension (one patient), significant reduction in body weight after the first months of treatment (one patient), inadequate visualization of the endometrium at TVS for retroverted uterus (two patients), grossly irregular ultrasonographic appearance of the myometrium (one patient), refusal to undergo the complete ultrasonographic assessment (five patients). On the other hand, in the control group seven healthy women missed the first visit because they refused to undergo serial and intensive ultrasonographic assessments.
In Table I are shown the clinical, hormonal and metabolic parameters detected in PCOS and control groups. The two groups were well-matched for age and BMI.
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All PCOS patients had PCO at TVS examination (Balen et al., 2003). Modified Ferriman–Gallwey score, LH, 17-OHP, testosterone, androstenedione, FAI, fasting insulin, and AUCinsulin were significantly (P < 0.05) higher in the PCOS group than in control group (Table I). On the contrary, SHBG, GIR, AUCglucose:AUCinsulin ratio were significantly (P < 0.05) lower in the PCOS group than in the control group (Table I).
The metformin treatment was generally well tolerated and a detailed description of the data regarding the safety has already been shown (Palomba et al., 2005a).
All PCOS patients were ovulating under metformin administration. After grouping the PCOS patients who ovulated under metformin for favourable (group A) or unfavourable (group B) reproductive outcomes, no difference in any clinical, hormonal or metabolic parameter was detected (Table II).
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Endometrial thickness and pattern
At baseline, the endometrial thickness assessed in the early follicular phase was not different between the PCOS and control groups (Table III). No difference in endometrial thickness was observed between PCOS women who ovulated under metformin treatment and control subjects in any period of the cycle (Table III).
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As expected, at baseline no anovulatory PCOS patient showed a triple-line and a high-grade endometrial pattern (Table III).
At the peri-ovulatory phase assessment, the percentage of cycles with triple-line endometrial pattern was not significantly different between women with PCOS who ovulated under metformin treatment and healthy controls (Table III). At the mid-luteal phase assessment, the percentage of cycles with high-grade pattern was again not significantly different between PCOS women who ovulated under metformin treatment and healthy controls (Table III).
After grouping the ovulatory cycles of the PCOS group for reproductive outcome, no difference between groups A and B was observed in endometrial thickness, and in percentage of cycles with triple-line and high-grade endometrial patterns in peri-ovulatory and mid-luteal phases, respectively (Table IV).
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Uterine artery blood flow
No difference between RI/PI values evaluated on the right and left was observed in PCOS, before and during treatment, and in the control group (data not shown). At baseline, RI/PI values were significantly (P < 0.05) higher in PCOS than in the control group and no variation within PCOS group was detected (Table III).
PI and RI values were significantly (P < 0.05) improved during metformin administration in ovulatory PCOS patients and not different from those detected in the controls (Table III).
After grouping the ovulatory cycles of the women with PCOS for reproductive outcomes, no difference was observed between groups A and B in uterine artery impedance (Table IV).
Sub-endometrial and endometrial vascularity
At baseline, the detection rate for sub-endometrial and endometrial blood flow was significantly (P < 0.05) lower in PCOS patients than in controls (Table III).
The detection rate for sub-endometrial and endometrial vascularity was significantly (P < 0.05) increased in PCOS women who ovulated under metformin treatment and not different from those detected in the control subjects
At baseline the extension of vascularity was significantly (P < 0.05) lower in the PCOS group than in the control group, whereas under treatment a significant increase was detected in the PCOS group. The extension of vascularity was not different between PCOS patients who ovulated under metformin and controls (Table III).
Before treatment, RI/PI values on endometrial and sub-endometrial vessels were significantly (P< 0.05) higher in the PCOS group than in the control group (Table III). A significant (P< 0.05) improvement from baseline was observed in PCOS patients who ovulated under metformin administration (Table III). No difference in RI/PI on sub-endometrial and endometrial vessels was again observed between the PCOS group and the control group (Table III).
After grouping the ovulatory cycles of PCOS group for reproductive outcome, no difference was observed between groups A and B in the detection rate, neither in sub-endometrial and endometrial blood flows nor in percentage of positive areas for sub-endometrial and endometrial vessels nor in RI/PI (Table IV).
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Discussion |
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In a recent RCT, clomiphene citrate and metformin administration induced a similar correction of the ovulatory function in infertile PCOS women, whereas a higher improvement of fertility and reproductive outcome was detected after the latter treatment (Palomba et al., 2005a
). According to these findings we have hypothesized a different and specific effect of metformin on uterine receptivity, and to test this hypothesis the current study was performed.
Our study confirms (Battaglia et al., 1995, 1996, 1997; Aleem and Predanic, 1996; Ajossa et al., 1999) that anovulatory PCOS patients have an alteration in uterine vascularization. In fact, PI and RI values, two measures of blood impedance inversely related to blood flow, were not only unchanged throughout the cycle (Vrtacnik-Bokal and Meden-Vrtovec, 1998) but also significantly higher in comparison with healthy controls.
Elevated impedance to blood flow in the uterine arteries appears to be detrimental for endometrial receptivity (Goswamy et al., 1988; Kurjak et al., 1991; Steer et al., 1992, 1994; Cacciatore et al., 1996; Tekay et al., 1996). A high PI detected in the uterine arteries at the time of implantation predicts a poor chance of conception in spontaneous and gonadotrophin-stimulated transfer cycles, suggesting that uterine perfusion may be a pivotal factor for a favourable reproductive outcome (Steer et al., 1992; Cacciatore et al., 1996; Tekay et al., 1996). During IVF cycles, the implantation rate is decreased when uterine artery PI is >3.3–3.5 at the time of HCG administration (Dickey, 1997). The reduction in uterine perfusion seems to be associated with a lower reproductive probability not only in patients affected by PCOS, but also in patients with unexplained recurrent pregnancy loss (Habara et al., 2002).
Even though changes in uterine arterial resistance might reflect uterine receptivity, a more accurate and sensible method seems to be the evaluation of sub-endometrial vascularity (Zaidi et al., 1995b; Tohma et al., 1997; Basir et al., 2001; Chien et al., 2002). Chien et al. (2002) showed that the pregnancy and implantation rates were related to sub-endometrial flow and to vascular penetration, whereas no relationship was detected with PI/RI. Our data showed a significant decrease in sub-endometrial and endometrial vascularity in PCOS women, assessed as RI/PI indices and as number and/or areas of coloured spots at computerized analysis of the image. In the current study, a normalization of these parameters was observed in PCOS women who ovulated under metformin treatment, confirming the data obtained by Jakubowicz et al. (2001). In this last study, in fact, metformin improved endometrial vascular penetration and decreased the impedance of uterine spiral arteries in PCOS women treated with clomiphene citrate (Jakubowicz et al., 2001). In fact, vascular penetration, studied using the method described by Zaidi et al. (1995b), showed increases in zones 3 (vessels penetrating the inner hypoechogenic area) and 4 (vessels penetrating the layer surrounding the uterine cavity), zones corresponding to the endometrial layers, as examined in the current study.
Metformin could act directly or indirectly on uterine vascularity. In this regard, it seems that insulin resistance did not play a key role in reducing uterine perfusion in PCOS (Ajossa et al., 2002). Conversely, it is possible to suppose that metformin acts on uterine perfusion, reducing androgen levels (Nestler and Jakubowicz, 1996) and thus their vasoconstrictive effect on vascular tissues (Fischer and Swain, 1977; Nestler and Jakubowicz, 1996; Ajossa et al., 2002).
Our previous data (Orio et al., 2004) have shown that in PCOS women there are increased concentrations of endothelin 1 (ET-1), a peptide synthesized in the vascular endothelial cells with vasoconstrictive action, and that metformin administration improves this alteration (Orio et al., 2005). The beneficial effect of metformin on serum ET-1 levels could be another indirect mechanism by which this drug acts on uterine vascularity. In fact, ET-1 is expressed in human endometrium (Economos et al., 1992) and inversely related to plasma estradiol levels (Polderman et al., 1993; Ylikorkala et al., 1995; Cacciatore et al., 1997), suggesting that ET-1 and estradiol could operate in concert, modulating endometrial blood flow.
Endometrial thickness has been considered a marker for uterine receptivity and for pregnancy prediction with different results (Gonen and Casper, 1990b; Dickey et al., 1993; Dickey and Holtkamp, 1996; Habara et al., 2002). Certainly, patients with a thick endometrium have a pregnancy rate higher in comparison with those having a thin endometrium, and a minimum endometrial thickness is necessary for an effective endometrial receptivity and, thus, for the occurrence of pregnancy (Yaman et al., 2000). In this regard, our findings demonstrated no effect of metformin on endometrial thickness in comparison with healthy women. These data are consistent with those also observed by Jakubowicz et al. (2001) and in another of our sub-analyses (Palomba et al., 2005b). To the contrary, Kocak et al. (2002) demonstrated a significant effect of metformin on endometrial thickness in PCOS women treated with clomiphene citrate.
In the current study, PCOS women who ovulated under metformin treatment showed a triple-line endometrial pattern in a percentage of cases similar to those observed in healthy controls. As already suggested by Hock et al. (1997), endometrial pattern is another critical factor influencing the chance of pregnancy in cycles of ovulation induction plus timed intercourse and a triple-line pattern is associated with a significantly higher pregnancy rate. When the endometrial echogenicity was studied using objective methods, e.g. computer-assisted measurements, a significant relationship was also observed between this parameter and implantation and pregnancy rates in IVF cycles, confirming that this parameter can be useful to predict endometrial receptivity (Fanchin et al., 2000).
A similar percentage of high-grade pattern in the mid-luteal phase was also observed in ovulating PCOS patients under metformin treatment and in healthy controls. The percentage of high-grade endometrial pattern detected in the current study was similar to those obtained by Triwitayakorn et al. (2002) and by Nakamura et al. (1997) in healthy women.
In the endometrium of the PCOS women there is an altered modulation of the expression of the endometrial androgen receptors (AR). Contrary to healthy women, an elevation in the endometrial expression of AR during the late luteal phase has been detected in PCOS patients, and this elevation seems to be strongly related to 
v
3 integrin, a well-characterized biomarker of endometrial receptivity (Apparao et al., 2002). The AR activation seems to have a negative impact on the expression of a homeobox gene, i.e. HOXA10, a gene implicated in the endometrial plasticity during menstrual cycles and essential for endometrial receptivity (Taylor, 2000). HOXA10, in fact, is significantly and inversely related to testosterone, but is not regulated by insulin (Cermik et al., 2003). Elevated levels of androgens, as observed in PCOS women, may have a detrimental effect on endometrial function via HOXA10 modulation (Cermik et al., 2003).
Based on these considerations, the beneficial actions of metfomin on the reproductive outcomes could be due to an indirect effect of metformin on serum androgen levels (Nestler and Jakubowicz, 1996). In this regard, Jakubowicz et al. (2001) showed that metformin increased the mid-luteal phase concentrations of serum glycodelin and insulin-like growth factor I (IGF-I), two putative biomarkers of endometrial receptivity, of 3- and 4-fold respectively, and that this effect was related to the decrease in androgen levels. However, a direct effect of metformin on endometrium via insulin action cannot be excluded. Insulin stimulates glucose oxidation activity in the late luteal phase in human endometrium, suggesting its involvement in the metabolic activities of endometrial tissue (Hackl, 1973; Truchan et al., 1987). In addition, insulin receptors are present at the endometrial level, reaching their maximal expression in the secretory phase, further supporting the hypothesis that insulin directly influences endometrial growth through its mitogenic and metabolic effects (Straus, 1984; Strowitzki et al., 1993). Finally, glucose transporter 1 (GLUT 1), a non-insulin-regulated transponder, is expressed at endometrial levels in animals and humans (Korgun et al., 2001; Strowitzki et al., 2001), indicating the importance of glucose transportation in the endometrium (Jansson et al., 1993; Barros et al., 1995; Strowitzki et al., 2001). At this level, another insulin-dependent transporter, GLUT 4, is also expressed, and its expression seems to be regulated by body weight and insulin (Mioni et al., 2004). Specifically, a recent experimental study (Mioni et al., 2004) has shown that GLUT 4 expression is impaired in PCOS patients, suggesting that in these subjects the insulin resistance and the hyperinsulinaemia induce an inadequate GLUT 4 expression and, thus, a reduced glucose supply. The consequences of this alteration could be responsible for the impaired endometrial receptivity and metformin could be effective in restoring endometrial functionality (Mioni et al., 2004).
Unfortunately, the present study cannot suggest specifically any putative mechanism by which metformin acts on uterine receptivity. In fact, the hormonal and metabolic evaluations were performed only at baseline in order to improve the compliance to the protocol study, and to reduce the drop-outs and the costs (Palomba et al., 2005a). In addition, no significant correlation was detected between our ultrasonographic data and hormonal/metabolic and/or demographic parameters assessed at study entry. Furthermore, these findings were probably due to a bias selection because our study population was composed of a well-selected and very homogeneous patient population.
Another important finding of our study was the absence of any differences in any parameters evaluated between cycles termed in a favourable or unfavourable reproductive outcome. Despite these data suggesting that no parameters evaluated in the current study are predictive for ongoing pregnancy, the lack of differences in endometrial receptivity marker between ovulating patients with favourable and unfavourable reproductive outcome could also be due to the low number of subjects of the latter group.
In conclusion, our results demonstrate that metformin administration acts on the uterus improving several surrogate parameters of endometrial receptivity. To date, it is very difficult to suggest a specific and unique action of metformin. At the same time, it is possible to maintain that metformin could act on the reproductive apparatus in different and various ways, i.e. reduction of hyperandrogenaemia and insulin resistance. Further data are necessary to explain the exact mechanism by which metformin exerts its beneficial effects on the uterus.
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Acknowledgements |
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The authors thank all the Gynecological Units of the Calabria and Campania Regions that have given their help in the subjects’ enrolment.
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References |
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Submitted on August 1, 2005; resubmitted on September 12, 2005; accepted on September 20, 2005.
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