D-chiro-inositol in clinical practice: A perspective from The Experts Group on Inositol in Basic and Clinical Research (EGOI).

BACKGROUND: D-chiro-inositol is a natural molecule that, in association with its well-studied isomer myo-inositol, may play a role in treating various metabolic and gynecological disorders. OBJECTIVES: This perspective seeks to explore the mechanisms and functions of D-chiro-inositol, laying the foundations to discuss its use in clinical practice, across dysmetabolism, obesity, and hormonal dysregulation. METHODS: A narrative review of all the relevant papers known to the authors was conducted. OUTCOME: D-chiro-inositol acts through a variety of mechanisms, acting as an insulin sensitizer, inhibiting the transcription of aromatase, in addition to modulating white adipose tissue/brown adipose tissue trans differentiation. These different modes of action have potential applications in a variety of therapeutic fields including: PCOS, dysmetabolism, obesity, hypoestrogenic/hyperandrogenic disorders, and bone health. CONCLUSIONS: D-chiro-inositol mode of action has been studied in detail in recent years, resulting in a clear differentiation between D-chiro-inositol and its isomer myo-inositol. The insulin sensitizing activities of D-chiro-inositol are well understood; however, its potential applications in other fields, in particular obesity and hyperestrogenic/hypoandrogenic disorders in men and women, represent promising avenues of research that require further clinical study.

D-Chiro-Inositol and Myo-Inositol Induce WAT/BAT Trans-Differentiation in Two Different Human Adipocyte Models (SGBS and LiSa-2).

White adipose tissue/brown adipose tissue trans-differentiation is one of the main study targets for therapies against obesity and metabolic diseases. In recent years, numerous molecules able to induce such trans-differentiation have been identified; however, their effect in obesity therapies has not been as expected. In the present study, we investigated whether myo-inositol and its stereoisomer D-chiro-inositol could be involved in the browning of white adipose tissue. Our preliminary results clearly indicate that both, at 60 µM concentration, induce the upregulation of uncoupling protein 1 mRNA expression, the main brown adipose tissue marker, and increase mitochondrial copy number as well as oxygen consumption ratio. These changes demonstrate an activation of cell metabolism. Therefore, our results show that human differentiated adipocytes (SGBS and LiSa-2), assume the features typical of brown adipose tissue after both treatments. Furthermore, in the cell lines examined, we proved that D-chiro-inositol and myo-Inositol induce an increase in the expression of estrogen receptor mRNAs, suggesting a possible modulation by these isomers. We also found an increase in the mRNA of peroxisome proliferator-activated receptor gamma, a very important target in lipid metabolism and metabolic diseases. Our results open new opportunities for the use of inositols in therapeutic strategies to counteract obesity and its metabolic complications.

An innovative approach to polycystic ovary syndrome.

Myo-inositol and D-chiro-inositol are insulin sensitising agents. In the ovary, myo-inositol acts as second messenger of Follicle Stimulating Hormone (FSH). Both molecules were administered to Polycystic Ovary Syndrome (PCOS) women. The gynaecologist Vittorio Unfer was the first to give specific value to myo-inositol for the treatment of PCOS: this important innovation opened new ways of research to identify efficient therapies based on myo-inositol alone or with low doses of D-chiro-inositol. Significant successes were also gained using myo-inositol in treating male and female infertility. Unfer's researches allowed to identify "the D-Chiro-Inositol Paradox in the Ovary" and the best myo-inositol/D-chiro-inositol ratio (40:1) for the treatment of PCOS. Furthermore, his studies allowed to improve the inositol's efficacy using alpha-lactalbumin. As shown in this review, the main stages of Unfer's scientific career have been closely intertwined with important phases of the recent pharmacological research about the topic.

Inositols: From Established Knowledge to Novel Approaches.

Myo-inositol (myo-Ins) and D-chiro-inositol (D-chiro-Ins) are natural compounds involved in many biological pathways. Since the discovery of their involvement in endocrine signal transduction, myo-Ins and D-chiro-Ins supplementation has contributed to clinical approaches in ameliorating many gynecological and endocrinological diseases. Currently both myo-Ins and D-chiro-Ins are well-tolerated, effective alternative candidates to the classical insulin sensitizers, and are useful treatments in preventing and treating metabolic and reproductive disorders such as polycystic ovary syndrome (PCOS), gestational diabetes mellitus (GDM), and male fertility disturbances, like sperm abnormalities. Moreover, besides metabolic activity, myo-Ins and D-chiro-Ins deeply influence steroidogenesis, regulating the pools of androgens and estrogens, likely in opposite ways. Given the complexity of inositol-related mechanisms of action, many of their beneficial effects are still under scrutiny. Therefore, continuing research aims to discover new emerging roles and mechanisms that can allow clinicians to tailor inositol therapy and to use it in other medical areas, hitherto unexplored. The present paper outlines the established evidence on inositols and updates on recent research, namely concerning D-chiro-Ins involvement into steroidogenesis. In particular, D-chiro-Ins mediates insulin-induced testosterone biosynthesis from ovarian thecal cells and directly affects synthesis of estrogens by modulating the expression of the aromatase enzyme. Ovaries, as well as other organs and tissues, are characterized by a specific ratio of myo-Ins to D-chiro-Ins, which ensures their healthy state and proper functionality. Altered inositol ratios may account for pathological conditions, causing an imbalance in sex hormones. Such situations usually occur in association with medical conditions, such as PCOS, or as a consequence of some pharmacological treatments. Based on the physiological role of inositols and the pathological implications of altered myo-Ins to D-chiro-Ins ratios, inositol therapy may be designed with two different aims: (1) restoring the inositol physiological ratio; (2) altering the ratio in a controlled way to achieve specific effects.

D-chiro-inositol, an aromatase down-modulator, increases androgens and reduces estrogens in male volunteers: a pilot study.

BACKGROUND: Androgen deficiency affects men in the adulthood, causing several harmful effects at the reproductive and behavioural levels. Since aromatase is an enzyme that catalyses the conversion of androgens to estrogens, and it is responsible for an adequate balance of both sex hormones in males and females, the administration of molecules acting as down modulators may contribute to restore an abnormal enzymatic activity. A prospective pilot study was carried out to investigate the effect of D-chiro-inositol, a putative aromatase down-modulator, on serum levels of testosterone, estradiol, estrone, dehydroepiandrosterone and epiandrosterone from a group of adult male volunteers. Glucose, insulin, follicle-stimulating hormone, luteinizing hormone, inhibin B, D-chiro-inositol and myo-inositol serum levels were also measured. RESULTS: Male volunteers were selected according to age and body mass index. Subjects with altered glycemia and/or hormonal status, due to advanced age or abnormal weight, were enrolled in the study. Each of the 10 volunteers enrolled took oral D-chiro-inositol (1 g/day) for 1 month. Serum assays of selected markers were performed at baseline (control) and after treatment. D-chiro-inositol administration was associated to reduced serum levels of estrone (- 85.0%) and estradiol (- 14.4%), and increased serum levels of testosterone (+ 23.4%) and dehydroepiandrosterone (+ 13.8%). In addition, epiandrosterone levels were higher (+39%) after treatment. On the other hand, follicle-stimulating hormone, luteinizing hormone and inhibin B did not change. A trend toward a decrease of glycemia, insulinemia and Homeostatic Model Assessment index was observed after D-chiro-inositol treatment, although differences did not reach statistical significance. D-chiro-inositol treatment did not cause any noticeable adverse effect. CONCLUSIONS: Increased androgens and decreased estrogens seem to confirm that D-chiro-inositol acts as an aromatase down-modulator, but with a still unknown mechanism of action. This pilot study opens up new perspectives of research and therapeutic applications for D-chiro-inositol at different dosages and length of treatment. Authorization number 005/2020 released by the Local Ethics Committee of Alma Res Fertility Center, Rome. TRIAL REGISTRATION NUMBER: NCT04615767 (registry: ClinicalTrials.gov) Date of registration: November 3, 2020.

RéSUMé: CONTEXTE: L'insuffisance en androgènes affecte les hommes à l'âge adulte, causant plusieurs effets nocifs aux niveaux reproductif et comportemental. Puisque l'aromatase est. une enzyme qui catalyse la conversion des androgènes en œstrogènes, et qu'elle est. responsable d'un équilibre adéquat des hormones sexuelles chez les hommes et les femmes, l'administration de molécules agissant comme freinateurs peut contribuer à restaurer une activité enzymatique anormale. Une étude prospective pilote a été menée pour étudier l'effet du D-chiro-inositol, un potentiel freinateur de l'aromatase, sur les taux sériques de testostérone, estradiol, estrone, déhydroépiandrostérone et d'épiandrostérone d'un groupe d'hommes adultes volontaires. Le glucose, l'insuline, l'hormone folliculostimulante, l'hormone lutéinisante, l'inhibine B, le D-chiro-inositol et les taux sériques de myo-inositol ont également été mesurés. RéSULTATS: Les hommes volontaires ont été sélectionnés selon l'âge et l'indice de masse corporelle. Les hommes qui présentaient une glycémie et/ou un statut hormonal altérés en raison d'un âge avancé ou d'un poids anormal, ont été inclus dans l'étude. Chacun des 10 volontaires enrôlés a pris du D-chiro-inositol (1 g/jour) par voie orale pendant un mois. Des dosages sériques des marqueurs sélectionnés ont été réalisés avant (témoin) et après le traitement. L'administration de D-chiro-inositol a été associée à une réduction des taux sériques de l'estrone (− 85.0%) et de l'estradiol (− 14,4%), et a une augmentation des taux sériques de testostérone (+ 23,4%) et de déhydroépiandrostérone (+ 13,8%). En outre, les taux d'épiandrostérone étaient plus élevés (39%) après le traitement. D'autre part, les taux d'hormone folliculostimulante, d'hormone lutéinisante et d'inhibine B n'ont pas été modifiés. Une tendance à la diminution de la glycémie, de l'insulinémie et de l'indice d'évaluation du modèle homéostatique a été observée après traitement par D-chiro-inositol, bien que les différences n'aient pas atteint une signification statistique. Le traitement par D-chiro-inositol n'a causé aucun effet indésirable notable. CONCLUSIONS: L'augmentation des androgènes et la diminution des œstrogènes semblent confirmer que le D-chiro-inositol agit comme un freinateur de l'aromatase, mais avec un mécanisme d'action encore inconnu. Cette étude pilote ouvre de nouvelles perspectives de recherche et d'applications thérapeutiques pour le D-chiro-inositol à différents dosages et durées de traitement.

High Doses of D-Chiro-Inositol Alone Induce a PCO-Like Syndrome and Other Alterations in Mouse Ovaries.

Administration of 1000-1500 mg/day D-Chiro-Inositol (DCIns) or a combination of Myo-Inositol (MyoIns) and DCIns in their plasma molar ratio (40:1) for three or more months are among recommended treatments for metabolic syndrome and/or Polycystic Ovary Syndrome (PCOS). We previously confirmed the efficacy of this formulation (8.2 mg/day MyoIns and 0.2 mg/day DCIns for 10 days) in a mouse PCOS model, but also observed negative effects on ovarian histology and function of formulations containing 0.4-1.6 mg/day DCIns. We therefore analyzed effects of higher doses of DCIns, 5, 10 and 20 mg/day, administered to young adult female mice for 21 days, on ovarian histology, serum testosterone levels and expression of the ovarian enzyme aromatase. Five mg/day DCIns (human correspondence: 1200 mg/day) altered ovarian histology, increased serum testosterone levels and reduced the amount of aromatase of negative controls, suggesting the induction of an androgenic PCOS model. In contrast, 10-20 mg/day DCIns (human correspondence: 2400-4800 mg/day) produced ovarian lesions resembling those typical of aged mice, and reduced serum testosterone levels without affecting aromatase amounts, suggesting a failure in steroidogenic gonadal activity. Notwithstanding physiological/biochemical differences between mice and humans, the observed pictures of toxicity for ovarian histology and function recommend caution when administering DCIns to PCOS patients at high doses and/or for periods spanning several ovulatory cycles.

Experts' opinion on inositols in treating polycystic ovary syndrome and non-insulin dependent diabetes mellitus: a further help for human reproduction and beyond.

Introduction: This Experts' opinion provides an updated scientific support to gynecologists, obstetricians, endocrinologists, nutritionists, neurologists and general practitioners on the use of Inositols in the therapy of Polycystic Ovary Syndrome (PCOS) and non-insulin dependent (type 2) diabetes mellitus (NIDDM).Areas covered: This paper summarizes the physiology of Myo-Inositol (MI) and D-Chiro-Inositol (DCI), two important molecules present in human organisms, and their therapeutic role, also for treating infertility. Some deep differences between the physiological functions of MI and DCI, as well as their safety and intestinal absorption are discussed. Updates include new evidence on the efficacy exerted in PCOS by the 40:1 MI/DCI ratio, and the innovative approach based on alpha-lactalbumin to overcome the decreased therapeutic efficacy of Inositols in some patients.Expert opinion: The evidence suggests that MI, alone or with DCI in the 40:1 ratio, offers a promising treatment for PCOS and NIDDM. However, additional studies need to evaluate some still unresolved issues, such as the best MI/DCI ratio for treating NIDDM, the potential cost-effectiveness of reduced gonadotropins administration in IVF due to MI treatment, or the benefit of MI supplementation in ovulation induction with clomiphene citrate in PCOS patients.

Risk of reduced intestinal absorption of myo-inositol caused by D-chiro-inositol or by glucose transporter inhibitors.

Background: D-chiro-inositol (DCI) and glucose transporter inhibitors may inhibit myo-inositol (MI) transporters, and the aim is to clinically evaluate their effect on MI absorption. Research design and methods: Fasting 18 healthy volunteers received orally 6000 mg MI, 6000 mg MI with 1000 mg DCI, and 6000 mg MI with SelectSIEVE® Apple PCQ and Sorbitol, Maltodextrin and Sucralose (PCQ-SMS), in three different phases with a washout period of 7 days. At each phase, blood samples were collected before administration, and every 60 minutes until 540 minutes after administration. MI plasma levels (µmol/L) were quantified by gas chromatography-mass spectrometry; maximum plasma concentration (Cmax), time to reach it (Tmax), and the area under the time-concentration curve of MI (AUC 0-540) were evaluated. Results: The Cmax of MI alone (Tmax = 180min) was 1.29-fold higher than those of MI with DCI (Tmax = 180min) (p < 0.001) and 1.69-fold higher than those of MI with PCQ-SMS (Tmax = 240min) (p < 0.001). The AUC 0-540 was reduced by 19.09% in MI plus DCI (p = 0.0118) and by 31.8% in MI plus PCQ-SMS (p < 0.001) as compared to MI alone. Conclusions: DCI, glucose transporter inhibitors and sugars, such as sorbitol and maltodextrin, seem to inhibit MI absorption, decreasing MI plasma concentration as compared to MI alone.

Alpha-lactalbumin Effect on Myo-inositol Intestinal Absorption: In vivo and In vitro.

BACKGROUND: Myo-inositol is a natural molecule with important therapeutic applications and an impaired oral absorption may result in a reduced clinical effect. Aim of this study was to determine if the combined oral administration of α-lactalbumin and myo-inositol in healthy subjects, could increase the plasma level of myo-inositol administered alone. In vitro studies on human differentiated intestinal Caco-2 cells were also conducted to identify the mechanisms involved in myo-inositol absorption. OBJECTIVE: The in vivo study was conducted on healthy volunteers in two phases. Subjects received a single oral myo-inositol dose. After 7 days washout, the same subjects were administered a single dose of myo-inositol and α-lactalbumin. Cmax, Tmax and AUC for myo-inositol in plasma were calculated from samples collected at different times. Transepithelial myo-inositol passage, with or without addition of digested α-lactalbumin, was measured in vitro in differentiated Caco-2 cells and compared to transepithelial electrical resistance and phenol red passage. RESULTS: The bioavailability of myo-inositol was modified by the concomitant administration of α- lactalbumin. Although peak concentration of myo-inositol at 180 min (Tmax) was similar for both treatments, administration of α-lactalbumin with myo-inositol in a single dose, significantly increased the plasma concentrations of myo-inositol compared to when administered alone. In vitro, myo-inositol absorption in Caco-2 cells was improved in the presence of digested α-lactalbumin, and this change was associated with an increase in tight junction permeability. CONCLUSION: Better myo-inositol absorption when orally administered with α-lactalbumin can be beneficial in non-responder patients. Preliminary in vitro findings suggest that peptides deriving from α- lactalbumin digestion may modulate tight junction permeability allowing increased absorption of myoinositol.

Combining treatment with myo-inositol and D-chiro-inositol (40:1) is effective in restoring ovary function and metabolic balance in PCOS patients.

Polycystic ovary syndrome (PCOS), a relevant cause of infertility, is a heterogeneous, endocrine disorder affecting up to 10-15% of women in reproductive age. Besides hyperandrogenism, insulin resistance (IR) plays a key role in such syndrome. Insulin-sensitizing drugs, such as Metformin, are effective in treating hyper-insulinemic PCOS patients. Recently, inositols - myo-inositol (MI) and D-chiro-inositol (DCI) - have shown to be an efficient and safe alternative in PCOS management, as both inositol isoforms are able to counteract downstream consequences of insulin resistance. Yet, whereas DCI contributes in mediating insulin activity mainly on non-ovarian tissues, MI displays specific effects on ovary, chiefly by modulating glucose metabolism and FSH-signaling. Moreover, MI may also improve ovarian functions by modulating steroid metabolism through non-insulin-dependent pathways. As DCI and MI activity likely involves different biological mechanisms, both inositol isoforms can be synergistically integrated according to a multitargeted design, by combining MI and DCI in a ratio corresponding to their physiological plasma relative amount (40:1). New experimental and clinical evidence with MI plus DCI evidenced the suitability of such integrated approach, and provided promising results. Further studies need to investigate thoroughly the molecular mechanism and confirm such preliminary data.

Immunomodulatory activities of alpha lipoic acid with a special focus on its efficacy in preventing miscarriage.

INTRODUCTION: Alpha lipoic acid (ALA) is an essential mitochondrial co-factor and, as a free molecule, it can exert multi-level immunomodulatory functions. Both ALA and its reduced form, dihydrolipoic acid (DHLA), are believed to be able to chelate heavy metals, to regenerate essential antioxidants and to repair important molecules damaged by oxidation. The largest part of the effects of ALA/DHLA couple can be explained by a specific stimulatory activity on Nrf2-dependent gene transcription and by the inhibition of NF-kB activity. These features have prompted its use as a drug for several diseases. Areas covered: This article surveys the main features of ALA/DHLA and its therapeutic effects. Its complex and differentiated function cannot simply be reduced to anti-inflammatory, antioxidant and detoxifying action. We highlight its capability to finely modulate several physiological pathways when unbalanced. In particular, we focus our attention on pregnancy, in relation to ALA administration by oral route and by a new formulation for vaginal delivery, in patients with threatened miscarriage. Expert opinion: Future efforts should be devoted to explaining carefully ALA/DHLA mechanism of action to reactivate the physiological balance when modified during pregnancy. On the other hand, ALA safety in pregnant women and its pharmacokinetics by vaginal route, have to be studied in depth. Moreover, ALA efficacy has to be confirmed in a much larger sample of patients.

Effect of beta-carotene-rich tomato lycopene beta-cyclase ( tlcy-b) on cell growth inhibition in HT-29 colon adenocarcinoma cells.

Lycopene beta-cyclase (tlcy-b) tomatoes, obtained by modulating carotenogenesis via genetic engineering, contain a large amount of beta-carotene, as clearly visible by their intense orange colour. In the present study we have subjected tlcy-b tomatoes to an in vitro simulated digestion and analysed the effects of digestate on cell proliferation. To this aim we used HT-29 human colon adenocarcinoma cells, grown in monolayers, as a model. Digested tomatoes were diluted (20 ml, 50 ml and 100 ml/l) in culture medium and added to the cells for different incubation times (24 h, 48 h and 72 h). Inhibition of cell growth by tomato digestate was dose-dependent and resulted from an arrest of cell cycle progression at the G0/G1 and G2/M phase and by apoptosis induction. A down-regulation of cyclin D1, Bcl-2 and Bcl-xl expression was observed. We also found that heat treatment of samples before digestion enhanced beta-carotene release and therefore cell growth inhibition. To induce with purified beta-carotene solubilised in tetrahydrofuran the same cell growth inhibition obtained with the tomato digestate, a higher amount of the carotenoid was necessary, suggesting that beta-carotene micellarised during digestion is utilised more efficiently by the cells, but also that other tomato molecules, reasonably made available during digestion, may be present and cooperate with beta-carotene in promoting cell growth arrest.

Intestinal and peripheral immune response to MON810 maize ingestion in weaning and old mice.

This study evaluated the gut and peripheral immune response to genetically modified (GM) maize in mice in vulnerable conditions. Weaning and old mice were fed a diet containing MON810 or its parental control maize or a pellet diet containing a GM-free maize for 30 and 90 days. The immunophenotype of intestinal intraepithelial, spleen, and blood lymphocytes of control maize fed mice was similar to that of pellet fed mice. As compared to control maize, MON810 maize induced alterations in the percentage of T and B cells and of CD4(+), CD8(+), gammadeltaT, and alphabetaT subpopulations of weaning and old mice fed for 30 or 90 days, respectively, at the gut and peripheral sites. An increase of serum IL-6, IL-13, IL-12p70, and MIP-1beta after MON810 feeding was also found. These results suggest the importance of the gut and peripheral immune response to GM crop ingestion as well as the age of the consumer in the GMO safety evaluation.

Assessment of the nutritional values of genetically modified wheat, corn, and tomato crops.

The genetic modification in fruit and vegetables could lead to changes in metabolic pathways and, therefore, to the variation of the molecular pattern, with particular attention to antioxidant compounds not well-described in the literature. The aim of the present study was to compare the quality composition of transgenic wheat ( Triticum durum L.), corn ( Zea mays L.), and tomato ( Lycopersicum esculentum Mill.) to the nontransgenic control with a similar genetic background. In the first experiment, Ofanto wheat cultivar containing the tobacco rab1 gene and nontransgenic Ofanto were used. The second experiment compared two transgenic lines of corn containing Bacillus thuringiensis "Cry toxin" gene (PR33P67 and Pegaso Bt) to their nontransgenic forms. The third experiment was conducted on transgenic tomato ( Lycopersicum esculentum Mill.) containing the Agrobacterium rhizogenes rolD gene and its nontransgenic control (cv. Tondino). Conventional and genetically modified crops were compared in terms of fatty acids content, unsaponifiable fraction of antioxidants, total phenols, polyphenols, carotenoids, vitamin C, total antioxidant activity, and mineral composition. No significant differences were observed for qualitative traits analyzed in wheat and corn samples. In tomato samples, the total antioxidant activity (TAA), measured by FRAP assay, and the naringenin content showed a lower value in genetically modified organism (GMO) samples (0.35 mmol of Fe (2+) 100 g (-1) and 2.82 mg 100 g (-1), respectively), in comparison to its nontransgenic control (0.41 mmol of Fe (2+) 100 g (-1) and 4.17 mg 100 g (-1), respectively). On the basis of the principle of substantial equivalence, as articulated by the World Health Organization, the Organization for Economic Cooperation and Development, and the United Nations Food and Agriculture Organization, these data support the conclusion that GM events are nutritionally similar to conventional varieties of wheat, corn, and tomato on the market today.

The growth-inhibitory effects of tomatoes digested in vitro in colon adenocarcinoma cells occur through down regulation of cyclin D1, Bcl-2 and Bcl-xL.

In the present study, we utilised an in vitro digestion procedure to deliver molecules contained in tomatoes to cultured cells and to analyse potential mechanisms underlying the antitumoural effects of tomatoes reported in the literature. Ripe tomatoes underwent in vitro simulated digestion and the aqueous fraction obtained was delivered to HT-29 and HCT-116 colon adenocarcinoma cells. The amount of lycopene released during digestion and transferred to the aqueous fraction during digestion was 10-fold lower than that present in tomato homogenate before digestion. The carotenoid was accumulated by colon adenocarcinoma cells in a dose-dependent manner after the addition of tomato digestate (20-100 ml/l) for 24 h. Tomato digestate inhibited the growth of HT-29 and HCT-116 cells in a dose-dependent manner. Growth inhibition resulted from an arrest of cell cycle progression at the G0/G1 phase and by apoptosis induction. A down regulation of cyclin D1, Bcl-2 and Bcl-xL expression was also observed, without apparent changes in p53, p21, p27 and Bax. In conclusion, the present data demonstrate that the in vitro digestion procedure represents a useful approach to supply tomato to colon cultured cells. Moreover, we have shown that tomato digestate is able to inhibit the growth of colon cancer cells by modulating the expression of regulators of the cell cycle and apoptosis.

Use of hormone replacement therapy in Italian women aged 50-70 years.

OBJECTIVES: The aim of the present cross-sectional analysis was to describe patterns and determinants of use of hormone replacement therapy (HRT) in a large sample of women attending mammographic screening. METHODS: Between 1999 and 2001, 8533 women aged 50-70 years were recruited by 11 screening centres, and structured interviews were made collecting information on socio-demographic characteristics, lifestyle habits, medical and reproductive history (overall response rate 87%). RESULTS: Current HRT use was reported by 6.9% of women (n=585), the average duration of use being 3.5 years; 13.2% were ever HRT users. Binomial and multinomial logistic regression (MLR) analyses showed that younger age, higher educational level, past mammographic examination and history of bilateral oophorectomy were the major predictors of current and ever HRT use. Current use was also more frequently reported by women who were thinner, nulliparae, had had induced menopause, had a later onset of menopause, with history of oral contraceptive use and hysterectomy without bilateral oophorectomy. By contrast, those who were diabetics or had positive history of cardiovascular disease were less likely to be current HRT users. No differences were found in HRT use according to marital status, type of occupation, menopausal status, smoking, history of breast cancer, hypercholesterolemia, hypertension and phlebitis. CONCLUSIONS: Our results support previous findings indicating that HRT use in Italian women is uncommon and of short duration. Current HRT users were less likely than non-users to report several characteristics associated with higher mortality and morbidity, in accordance with the healthy-user phenomenon described in other countries.