Abstract
The present study investigated the role of the N, N{′ }-dimethylbiguanide metformin (50 mg/100 g body weight in 0.05 ml water, given orally with a canulla) in the prevention of endocrine and immune disorders provoked by the hyperandrogenization with dehydroepiandrosterone (DHEA) in prepuberal BALB/c mice. The treatment with DHEA (6 mg/100 g body weight in 0.1 ml oil) for 20 consecutive days, recreates a mouse model that resembles some aspects of the human polycystic ovary syndrome (PCOS). The treatment with DHEA did not modify either body mass index (BMI) or blood glucose levels, but did increase fasting insulin levels when compared with controls. Markers of ovarian function – serum estradiol (E), progesterone (P) and ovarian prostaglandin E (PGE) – were evaluated. The treatment with DHEA increased serum E and P levels while ovarian PGE diminished. When metformin was administered together with DHEA, serum insulin, E and P levels, and ovarian PGE values did not differ when compared with controls. Using flow cytometry assays we found that the treatment with DHEA diminished the percentage of the CD4 + T lymphocyte population and increased the percentage of the CD8 + T lymphocyte population from both ovarian tissue and retroperitoneal lymph nodes. However, when metformin was administered together with DHEA, the percentages of CD4 + and CD8 + T lymphocyte populations from both ovarian tissue and retroperitoneal lymph nodes were similar to those observed in controls. Finally, when DHEA was administered alone it increased the serum tumor necrosis factor-alpha (TNF-α ) levels when compared with controls; however, when metformin was administered together with DHEA, serum TNF-α levels were similar to controls. These results indicate that metformin is able, directly or indirectly, to avoid the endocrine and immune alterations produced when mice are hyperandrogenized with DHEA.
Introduction
Polycystic ovary syndrome (PCOS) – which is characterized by hyperandrogenemia, hirsutism, oligo- or amenorrhea and anovulation – is one of the most common endocrinological diseases encountered in premenopausal women (Franks 1995, Asuncion et al. 2000). It has been reported that hyperinsulinemia, frequently associated with PCOS, increases both the risk of cardiovascular diseases and the development of diabetes mellitus (Abbott et al. 2002). Although during the last decade several clues have emerged from human and animal studies, little is known about the etiology and pathophysiology of PCOS. The battery of animal models used for the study of polycystic ovaries has allowed researchers to investigate different aspects of the pathology (Billiar et al. 1985, Szkiewicz & Uilenbroek 1998, Weil et al. 1999, West et al. 2001, Abbott et al. 2002). After it was found that dehydroepiandrosterone (DHEA) levels were increased in women with PCOS (Malesh & Greenblatt 1962), Roy et al.(1962) produced an animal model using DHEA for induction of PCOS. Subsequent studies established that the DHEA–PCOS murine model exhibits some of the salient features of human PCOS, such as hyperandrogenism, abnormal maturation of ovarian follicules and anovulation (Lee et al. 1991, 1998, Anderson et al. 1992, Henmi et al. 2001). These findings, together with the fact that increasing evidence indicates that DHEA has, in addition, potent immunoregulatory functions (Meikle et al. 1992, Okabe et al. 1995, Hernandez Pardo et al. 1998, Zhang et al. 1999) led us to use the DHEA–mice model to study some aspects related to the endocrine and immune responses involved.
In a previous study, we have demonstrated that treatment of prepuberal BALB/c mice with DHEA alters ovarian morphology, induces ovarian cysts, increases serum 17 beta estradiol (E) and progesterone (P) levels, and modifies ovarian prostaglandin E (PGE) concentration. Therefore, the treatment with DHEA led to a selective ovarian CD4 + and CD8 + T lymphocyte expression characterized by both a diminution of the percentage of CD4 + lymphocytes and an increase in the percentage of CD8 + T lymphocytes (Luchetti et al. 2004). In view of our previous studies and those reported by Lu et al.(2002), who demonstrated that production of cytokines by B cells is controlled by an enriched CD8 + T lymphocyte population and steroid hormones, experiments were designed to evaluate whether DHEA was able to lead to a selective T lymphocyte population that could, in turn, regulate cytokine production. Considering the fact that tumor necrosis factor-alpha (TNF-α ) regulates androgen production by theca cells (Sayin et al. 2003) and that it has been reported to be associated with PCOS (Deshpande et al. 2000, Araya et al. 2002, Korhonen et al. 2002, Peral et al. 2002), the aim of this study was to investigate the concentration of serum TNF-α after treatment with DHEA.
Multiple concomitant therapies have been applied in PCOS to address the variety of symptoms and to achieve better results. Recent studies have investigated the role of a family of insulin-sensitizing agents: the biguanides. Thus, the use of metformin (N, N{′ }-dimethylbiguanide) is becoming increasingly accepted and widespread. However, this drug is being clinically used without a complete understanding of the mechanisms involved. Metformin has been shown to be useful in the reduction of insulin resistance by restoring insulin sensitivity (Fedorcsak et al. 2003, Harbone et al. 2003, Lord et al. 2003). In addition, it has been demonstrated that metformin is able, either directly or indirectly, to regulate ovarian steroidogenesis (La Marca et al. 2002, Mansfield et al. 2003).
Controversial results have been reported with regards to metformin and its relationship with the immune system. In patients with type-2 diabetes, the biguanide enhances the tyrosine kinase activity of the insulin receptor by modulating the plasma cell differentiation antigen (PC-1) (Stefanovic et al. 1999). Nevertheless, Ruat et al.(2003) have failed to demonstrate any relationship between metformin and proliferation assays of T cells of lymph nodes.
The aim of the present work was to assess the efficacy of metformin on some immune and endocrine parameters altered after hyperandrogenization with DHEA. We therefore evaluated body mass index (BMI), the glucose:insulin ratio (in order to determine the homeostasis model assessment (HOMA) index), and serum E and P levels (as indicators of ovarian function). We also quantified ovarian PGE production, because PGE is not only involved in inflammatory processes and displays immunomodulatory properties, but is also altered in PCOS (Navarra et al. 1996, Wojtowicz-Praga 1997). With regards to the immune system we also designed experiments to investigate the role of metformin in modulating both the CD4 + and CD8 + T cell population of ovarian tissue and secondary immune tissues (axillar and retroperitoneal lymph nodes). In addition, the production of the proinflammatory cytokine TNF-α was evaluated.
Materials and Methods
Animals and experimental protocol
The hyperandrogenized environment of PCOS was reproduced in mice by injection of DHEA (Luchetti et al. 2004). Briefly, female prepuberal (25 days old) mice of the BALB/c strain were injected daily with DHEA (6 mg/100 g body weight, dissolved in 0.10 ml sesame oil) for 20 consecutive days (DHEA group). The animals of the DHEA + metformin group were injected with DHEA and given metformin orally (50 mg/100 g body weight in 0.05 ml water, given orally with a canulla) for 20 days. The dose of metformin administered was equivalent to that used in the treatment of women with PCOS. The controls consisted of three groups: (a) animals injected with oil (0.1 ml) and given water orally (0.05 ml) for 20 consecutive days (control vehicle); (b) the metforminalone group which consisted of mice treated orally with 50 mg metformin/kg body weight in 0.05 ml water for 20 days; (c) the untreated group formed by animals that did not receive treatment. Mice were housed under controlled temperature (22 ° C) and illumination (14 h light:10 h darkness; lights on at 0500 h) and were allowed free access to Purina rat chow and water. All procedures involving animals were conducted in accordance with the Animal Care and Use Committee of Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) 1996.
Throughout the whole treatment, the animals were weighed daily and, in addition, the stage of the sexual cycle was determined. Vaginal smears were taken daily up to the day of autopsy. As previously found (Luchetti et al. 2004), mice treated with DHEA remained in constant estrus. In contrast, mice administered with DHEA together with metformin showed irregular sexual cycles. None of the animals in the experimental groups displayed a complete sexual cycle at the time of killing. These findings led us to use, for the control and metforminalone groups, only those animals that at the time of killing were in the estrus stage of the sexual cycle. In other words, in order to compare the different groups, we have treated sufficient animals from groups other than the DHEA-alone group to ensure that there were ten animals per group at estrus stage on the day of killing.
After 20 days of treatment, ten mice (45 days old) per group were anesthetized with ether and killed by decapitation. Blood was collected and fasting glucose levels were immediately determined. Then, serum was isolated and stored at −70 ° C until assayed for E, P, insulin and TNF-α . Twenty freshly dissected ovaries per group were divided as follows: ten ovaries of each group were prepared to evaluate CD4 + and CD8 + T lymphocyte expression by flow cytometry assay; ten ovarian tissues were immediately frozen at −70 ° C until PGE radio-immunoassay. Lymphoid tissues (axillar and retroperitoneal nodes) from the ten animals of each group were immediately collected and processed to determine the CD4 + and CD8 + T cell expression by flow cytometry assay. All experiments were repeated three times.
Assays for glucose and insulin quantification
Fasting blood glucose was determined by using the Haemoglukotest (Roche) test strips for visual determination in the range of 20–800 mg/100 ml (1–44 mmol/l). The test principle uses the glucoseoxidase/peroxidase reaction. Results are expressed in millimoles of glucose per liter.
Serum insulin levels were assayed by the Coat-A-Count insulin method (Diagnostic Products Corporation, LA, USA) following the manufacturer’s instructions. Briefly, the determination consists of a solid-phase 125I radioimmunoassay designed for the quantitative measurement of insulin in serum. The antibody is immobilized to the wall of a polypropylene tube and simply decanting the supernatant suffices to terminate the competition and to isolate the antibody- bound fraction of the radiolabeled insulin. The samples were counted in a gamma counter. Analytical sensitivity was 1.2 μ IU insulin/serum ml.
E and P determination
Serum E levels were evaluated as described previously (Luchetti et al. 2004). Briefly, the blood was allowed to clot and the serum removed and frozen until E and P concentrations were determined by radioimmunoassay. Antisera were provided by Dr G D Niswender (Colorado State University, Fort Collins, CO, USA). The E antiserum showed low cross-reactivity: < 1% for P and testosterone, < 5% for estriol and < 10% for estrone. The P antiserum was highly specific and showed low cross-reactivity: < 2% for 20-α-dihydroprogesterone and deoxycorticosterone, and 1% for other steroids normally present in serum. Results are expressed in nanograms per serum milliliter.
Prostaglandin radioimmunoassay
The measurement of PGE was carried out in the incubation media of ovarian tissues as previously reported (Luchetti et al. 2004). Briefly, the tissue (each ovary) was weighed and incubated in Krebs–Ringer–bicarbonate (KRB) with glucose (11.0 mmol/l) as external substrate (pH 7.0) for 1 h in a Dubnoff metabolic shaker under an atmosphere of 5% CO2 in 95% O2 at 37 ° C. At the end of the incubation period, the tissue was removed and the solution acidified to pH 3.0 with 1 M HCl and extracted for prostaglandin determination three times with 1 volume of ethyl acetate. Pooled ethyl acetate extracts were dried under an atmosphere of N2 and stored at −20 ° C until prostaglandin radioimmunoassay was perfomed. PGE was quantified using a rabbit antiserum from Sigma. Sensitivity was 10 pg/tube and cross-reactivity was 100% with PGE and < 0.1% with other prostaglandins. Results are expressed in picograms PGE per milligram of protein. Ovarian protein content was determined using the Bradford method (1976).
Flow cytometry
To carry out the flow cytometry assays, ovarian tissue and lymph nodes (axillar and retroperitoneal) must be dispersed (Luchetti et al. 2004). Briefly, tissues were enzymatically dissociated in culture medium (medium 199, 25 mM Hepes, 26 mM NaHCO3 and 50 UI/ml penicillin) with collagenase (trypsinfree, 740 IU/100 mg tissue) and DNAse (14 IU/100 mg tissue). After 90 min, cells were washed twice with culture medium, twice with Dulbecco—phosphate-buffered saline free of Ca2+ and Mg2+ (PBS) and twice with culture medium containing EDTA (1 mM). To remove blood cells, suspensions were applied to Ficollhystopaque gradient 1.077 (Sigma), centrifuged at 400 g for 45 min and washed with PBS/0.1% BSA. Cells were counted in a hemocytometer (viability was >80% as assessed by the trypan blue exclusion method) and then processed for direct immunofluorescence. Thus, 100 μ l of each cellular suspension, at a concentration of 106 cells/ml, were incubated for 30 min at 4 ° C with: (a) 30 μ l phycoerythrin (PE) Rat IgG2a K Isotype Control plus 30 μ l fluorescein isothiocyanate (FITC) Rat IgG2a K Iso-type Control (eBioscience, San Diego, CA, USA) corresponding to the isotype control sample; or (b) 4 μ l PE anti-mouse CD4 plus 4 μ l FITC anti-mouse CD8 (eBioscience, USA), corresponding to control, DHEA and DHEA + metformin assay according to the cellular suspension. Antibodies were used at saturating concentrations, as established after titration by flow cytometry. Samples were then washed with PBS and PBS-EDTA, fixed with 4% paraformaldehyde and stored at 4 ° C in darkness until the analysis was performed within 6 days of labeling. Fluorescence analysis was evaluated with FACScan and Winmdi 2.8 software (Scripps Research Institute). Both ovarian and lymph suspensions were analyzed using different physical characteristics (i.e. size and complexity), using both forward (FSC: cell size) and side scatter (SSC: cell complexity) parameters. Thus, the settings correlated to cellular size and granularity of mouse T lymphocytes. The resulting gate and quadrant were maintained throughout the analysis. Flow cytometric determination was performed using standard fluorescence 1 (FL1; FITC anti-mouse CD8 + T lymphocyte) and fluorescence 2 (FL2; PE-anti-mouse CD4 + T lymphocyte). The analysis was based on quantification of 50 000 cells for each assay for ovarian tissue and 10 000 cells for lymph nodes.
Serum TNF-α determination
A TNF-α enzyme immunometric assay (EIA) kit (Assay Design’s mouse, MI, USA) was employed following the manufacturer’s instructions to quantify serum TNF-α from samples of the three experimental groups. Results are expressed in picograms per serum milliliter.
Statistical analysis
Statistical analyses were carried out using the Instat program (GraphPAD software, San Diego, CA, USA). ANOVA was performed using Newman–Keuls test to compare all pairs of columns and P < 0.05 was considered significant. All results are presented as the means± s.e.m.
Results
Effect of metformin on BMI, homeostasis model assessment, fasting glucose and insulin levels
These experiments were performed to determine whether the treatment with metformin was able to modify some parameters of the homeostasis and to study the role, if any, of metformin when administered together with DHEA. The weight of the animals was not modified by any of the treatments (Table 1), and thus the BMI (defined as weight (kg)/height (m2)) was similar in all the groups analyzed (Table 1). Glucose levels did not vary in any of the groups studied (Fig. 1A), while fasting insulin levels increased with DHEA treatment when compared with controls (untreated, control vehicle and metformin-alone groups). HOMA index (defined as: HOMA = insulin (μ UI/ml) × glucose (mmol/l):BMI (kg/m2)) increased after the treatment with DHEA when compared with controls. When metformin was administered together with DHEA, the effects of DHEA on both insulin levels (Fig. 1B) and the HOMA index were attenuated (Fig. 1c).
Effect of metformin on ovarian function: serum E and P levels and ovarian PGE production
To evaluate both ovarian function after treatment with DHEA and the possible role of metformin as a treatment for the ovarian anomalies produced by DHEA, we designed experiments to quantify serum E and P levels, and ovarian PGE production. Treatment with DHEA for 20 consecutive days increased both serum E and P levels (Fig. 2A and B) when compared with controls (untreated, control vehicle and metformin alone). When metformin was administered together with DHEA, serum E and P levels showed similar patterns to those of control values (Fig. 2A; DHEA + metformin).
On the other hand, the production of PGE by ovarian tissue was diminished after treatment with DHEA (Fig. 2C), but did not significantly differ from the control values when metformin was administered together with DHEA (Fig. 2C).
Role of metformin in ovarian CD4 + and CD8 + T lymphocyte expression
Flow cytometry analysis was employed to determine both the effect of DHEA treatment on the expression of ovarian CD4 + (or helpers) and CD8 + (or cytotoxic/suppressors) T lymphocytes and the role of metformin when administered together with DHEA. For the 50 000 cells analyzed, we found that the control groups showed equivalent percentages of ovarian CD4 + and CD8 + T lymphocytes (Fig. 3). However, treatment with DHEA diminished the percentage of CD4 + T cells (17 ± 5%) and increased the percentage of CD8 + T cells (83 ± 4%) when compared with controls (Fig. 3). In contrast, when metformin was administered together with DHEA, we observed the same percentages of CD4 + and CD8 + T cells as in the controls (Fig. 3).
Role of metformin in T lymphocyte expression in lymph nodes
In order to establish whether the effects of DHEA treatment and the administration of metformin together with DHEA also involved secondary lymphoid tissues, the percentages of CD4 + and CD8 + T cells were quantified in both axillar and retroperitoneal lymph nodes. Figure 4A illustrates the flow cytometry analysis of axillar nodes. It can be seen that all the control groups yielded equivalent percentages of CD4 + and CD8 + T lymphocytes, and that neither the treatment with DHEA nor treatment with DHEA and metformin together were able to modify the percentages of CD4 + and CD8 + T lymphocytes from axillar nodes.
The flow cytometry analysis of retroperitoneal lymph nodes (Fig. 4B) showed equivalent percentages of CD4 + and CD8 + T lymphocytes in all the control groups. However, treatment with DHEA diminished the percentage of CD4 + T cells, and increased the CD8 + T cells from retroperitoneal lymph nodes (Fig. 4B; CD4 + , 24 ± 6%; CD 8 + , 64 ± 4%). In contrast, when metformin was administered together with DHEA, the percentages of CD4 + and CD8 + T cells from retroperitoneal lymph nodes were similar to those of controls (Fig. 4B).
Discussion
In this work, we studied some aspects related to both the endocrine and the immune responses in a polycystic ovarian condition induced by hyperandrogenization with DHEA in BALB/c mice. The aim of the present study was also to investigate the ability of metformin to modulate these aspects.
The mechanism by which administration of DHEA brings about the cascade of hormonal events that produces ovarian failure remains unknown. However, it is known that the experimental model used in this work reproduces the main aspects of human PCOS (Lee et al. 1991, 1998, Anderson et al. 1992, Henmi et al. 2001). Our data show that the hyperandrogenic environment recreated by the treatment with DHEA did not modify the weight of the animals or, consequently, the BMI. We could then infer that neither hyperlipidemia nor obesity, which can sometimes be associated with PCOS (Franks et al. 1997, Abbott et al. 2002), were induced in this animal model.
Both hyperinsulinemia and hyperandrogenism play a pathogenic role in PCOS since they contribute to anovulation (Shoupe et al. 1983, Dunaif et al. 1989, Franks et al. 2000), impair folliculogenesis and affect follicular development (Dunaif et al. 1989, Gougeon 1996, Musso et al. 2005). For these reasons, insulin-sensitizing drugs such as metformin can improve the rate of spontaneous ovulation (De Leo et al. 1999, Glueck et al. 1999, Vandermolen et al. 2001). Although we did not directly assess insulin sensitivity, we measured surrogate markers of insulin sensitivity such as fasting serum insulin, fasting blood glucose and the HOMA index (which reflects the glucose–insulin relationship). Our results showed that treatment with DHEA increased the serum insulin levels without affecting fasting glucose, while treatment with metformin and DHEA together led to a HOMA index similar to that of controls. Although metformin is usually used for the treatment of chronic obese, insulin-resistant type-2 diabetic, and PCOS patients (Fedorcsak et al. 2003, Harbone et al. 2003, Lord et al. 2003), the role of this drug during conditions of normal glucose concentration remains unknown. Our findings are in agreement with previous studies that have demonstrated that metformin increases peripheral insulin sensitivity in non-diabetic women with PCOS (Diamanti-Kandarakis et al. 1998, Moghetti et al. 2000, Vandermolen et al. 2001).
Since metformin modulates insulin concentration and, in turn, insulin controls ovarian steroidogenesis, it can be said that metformin acts indirectly on the steroidogenic activity of theca and granulosa cells (La Marca et al. 2002). However, recently, Mansfield et al.(2003) have demonstrated that metformin exerts a direct effect on cultured ovarian cells. Therefore, we could speculate that metformin would regulate ovarian steroidogenesis both by modulating insulin levels and by acting directly on ovarian cells. These two pathways would contribute to ensure the accuracy of ovarian function. However, we are designing further experiments to clarify both this point and the molecular mechanisms involved in the action of metformin.
The data presented here show that mice from the DHEA group exhibited increased levels of both serum E and P and were in constant estrus. In view of the fact that only those follicles that show significant amounts of aromatizable androgens and low production of E are classified as selectable follicles (i.e. appropriate to ovulate) (Gougeon 1996), we can assume that in addition to the hyperandrogenized environment created by the daily injection of DHEA, the enhanced concentration of serum E would result in unfavorable conditions for producing follicles destined for ovulation. Lee et al.(1992), who worked with the same animal model, have reported a similar hormonal profile. This profile suggests an increased steroidogenic activity, which is widely described in PCOS (Franks et al. 2000, Abbott et al. 2002). Evidence shows that follicles from anovulatory women with PCOS hypersecrete E when compared with size-matched follicles from normal ovaries or polycystic ovaries from ovulatory women (Franks et al. 2000, Mendonca et al. 2004, Doi et al. 2005). These findings also support those of the current study.
In the present work, also in accordance with previous findings (Vandermolen et al. 2001, Harbone et al. 2003, Kazerooni & Dehghan-Kooshkghazi 2003, Weerakiet et al. 2004), we demonstrate that the administration of metformin together with DHEA prevented the effect of hyperandrogenization, i.e. the increase of serum E and P levels. As discussed above, these data would represent the result of both indirect and direct actions of metformin in modulating ovarian steroidogenesis.
Since animals from the DHEA group remained at constant estrus throughout the treatment, we suggest that the hyperandrogenization induced with DHEA resulted in anovulation. Conversely, neither the animals from the control groups nor those from the DHEA + metformin group showed a complete sexual cycle. However, they did show irregular cycles. Therefore, we suggest that the administration of metformin together with DHEA creates an endocrine condition that allows the animals to start to cycle, and that the irregularity of sexual cycles could be attributed to the immature condition of the animals (45 days old at the time of killing) rather than to inefficiency of the treatment with metformin.
Considering that prostaglandins are involved in the paracrine regulation of the rupture of ovarian follicles associated with ovulation (Priddy & Killick 1993) and that PGE has been reported to have immunomodulatory properties by modulating cytokine production (Kuroda & Yamashita 2003, Lakier Smith 2003, Yang et al. 2003), we also evaluated whether the ovarian concentration of PGE was modified by DHEA-induced hyperandrogenization. The fact that the treatment with DHEA reduced ovarian PGE production was an expected result since DHEA-hyperandrogenized animals not only did not start to cycle but also showed increased levels of TNF-α when compared with controls. Although we have previously reported that the treatment with DHEA increased ovarian PGE (Luchetti et al. 2004), this apparently controversial result could be explained by the fact that, in that report, the dose of DHEA administered to prepuberal mice was lower than that used here. The dose of DHEA used in the present report correlates better with the concentration of DHEA described in women with PCOS (Malesh & Greenblatt 1962, Roy et al. 1962, Lee et al. 1991, 1998, Anderson et al. 1992, Henmi et al. 2001). We also found an inverse relationship between the concentration of DHEA and both the ovarian PGE production and the expression of cyclooxygenase (COX) (the enzyme that synthesizes PGE) (data not shown). The last observation is due to the fact that it has been demonstrated that prostaglandins down-regulate their own synthesis (Ivanov & Romanovsky 2004). We therefore assume that the higher dose of DHEA injected in the present report led to an accumulation of ovarian PGE which in turn could inhibit the expression of COX and consequently the production of ovarian PGE, as observed here.
On the other hand, we found that animals from the DHEA + metformin group showed a pattern of ovarian PGE synthesis similar to that of the control groups. Although the action of this biguanide on improving lipid metabolism has been widely reported (Caballero 2004, Dominguez & Sowers 2005, Rautio et al. 2005), our data represent the first evidence that metformin modulates the production of ovarian prostaglandins.
TNF has been found to be increased in patients with PCOS (Sayin et al. 2003). In addition, a mutation of the TNF receptor has been associated with hyperandrogenism (Peral et al. 2002). In agreement with these findings, we found that animals treated with DHEA had higher serum TNF-α levels when compared with controls, a fact that correlates both with increased levels of PGE and with anovulatory cycles. It has been reported that TNF-α modulates steroidogenesis of both granulosa and theca-interstitial cells by a mechanism independent of those induced by insulin and insulin-like growth factor-I (IGF-I) (Spaczynski et al. 1999). For this reason we could infer that the increase of serum TNF-α observed in animals from the DHEA group would be an additional mechanism to those involved in hyperinsulinemia, which impair ovarian steroidogenesis. In the present work, we demonstrated that the administration of metformin together with DHEA led to serum TNF-α levels similar to control values. In the literature, conflicting results have been reported with regards to the relationship between metformin and the regulation of TNF-α (Cacicedo et al. 2004, Bruun et al. 2005, Di Gregorio et al. 2005, Kiortsis et al. 2005).
Finally, the data presented here show that DHEA-induced hyperandrogenization increased the percentage of the CD8 + T population and diminished the percentage of CD4 + T lymphocytes when compared with controls. Although the receptor for DHEA has not been identified yet, a specific DHEA binding activity has been detected in T cells (Meikle et al. 1992, Okabe et al. 1995) and for this reason we hypothesized that endocrine disturbances could be directly related to T lymphocyte differentiation. It has been documented, for example, that expression of the most mature thymocytes is regulated by P and E levels, and is also related to the high propensity of autoimmune diseases in females (Leposavic et al. 2001, Obradovic et al. 2001). Moreover, Yan et al.(2000) have reported that both autoimmune premature ovarian failure (POF) and insulin-dependent diabetes mellitus (IDDM) patients present increased numbers of CD8 + T cells. In addition, it has been demonstrated that production of cytokines by B cells is controlled by an enriched CD8 + T population (Lu et al. 2002).
Data presented here show that metformin treatment resulted in similar percentages of CD4 + and CD8 + T lymphocytes to those seen in the controls. As metformin modulates P and E levels and since, in turn, P and E control T lymphocyte differentiation (Yan et al. 2000, Leposavic et al. 2001, Obradovic et al. 2001, Lu et al. 2002), we can suggest that metformin would act indirectly in modulating the percentages of ovarian CD4 + and CD8 + T lymphocytes. However, other pathways (such as regulation of reactive oxygen species and the induction of AMP-activated protein kinase (AMPK) of T lymphocytes) described in other tissues (Bonnefont-Rousselot et al. 2003, Cacicedo et al. 2004, Huypens et al. 2005, McCarthy 2005) must not be disregarded and are currently being studied at our laboratory. Both the fact that the T cell population from axillar nodes was not affected during DHEA- induced hyperandrogenization and that the T cell population from retroperitoneal lymph nodes was affected – and also the fact that hyperandrogenization induced a similar T cell population when samples from retroperitoneal lymph tissue were compared with those obtained from ovarian tissue – lead us to suggest that the local inflammatory status would be contributing to a selective differentiation of T cells. According to this hypothesis, lymph nodes and sex steroids are related to different systems, thus suggesting a coordinated organ-specific and steroid hormone relationship (Chantakru et al. 2003). Moreover, the E receptor expressed by follicular dendritic cells in lymph nodes has recently been proposed as a novel pathological marker (Sapino et al. 2003) and the deficiency of E caused by ovariectomy or menopause is involved in the T lymphocyte status (Safadi et al. 2000). In summary, the present study describes the role of metformin in the regulation of some aspects of the intricate network that relates the endocrine and the immune pathway in a hyperandrogenized environment. As the development of immune cell surface markers is beginning to be used in diagnosis prior to the development of complete ovarian failure, we believe that understanding the role of the immune processes involved in PCOS could also be important in the manipulation of this pathology.
Funding
These studies were supported by PIP CONICET (reference 6051) and Agencia Nacional de Promoción Científica y Tecnológica (PICT) (reference 05-10901).
Effect of DHEA on BMI.
| Untreated | Control vehicle | Metformin-alone | DHEA | DHEA + metformin | |
|---|---|---|---|---|---|
| Age (days) | 45 | 45 | 45 | 45 | 45 |
| Weight (g) | 14.0 ± 1.2 | 15.0 ± 3.0 | 14.0 ± 2.5 | 14.8 ± 1.8 | 15.2 ± 2.4 |
| BMI (kg/m2) | 5.6 ± 0.2 | 5.7 ± 0.5 | 5.7 ± 0.4 | 5.4 ± 0.6 | 5.3 ± 0.8 |
Role of DHEA in (A) fasting glucose levels, (B) fasting insulin levels and (C) HOMA index and effect of metformin treatment. P < 0.001, b vs a; P < 0.05, c vs a. Each group represents the mean± s.e.m. of ten different animals.
Citation: Reproduction 131, 3; 10.1530/rep.1.00941
Role of DHEA in (A) fasting glucose levels, (B) fasting insulin levels and (C) HOMA index and effect of metformin treatment. P < 0.001, b vs a; P < 0.05, c vs a. Each group represents the mean± s.e.m. of ten different animals.
Citation: Reproduction 131, 3; 10.1530/rep.1.00941
Role of DHEA in (A) fasting glucose levels, (B) fasting insulin levels and (C) HOMA index and effect of metformin treatment. P < 0.001, b vs a; P < 0.05, c vs a. Each group represents the mean± s.e.m. of ten different animals.
Citation: Reproduction 131, 3; 10.1530/rep.1.00941
(A) Serum 17 beta estradiol levels from mice from control groups, and the DHEA and DHEA + metformin groups. (B) Serum progesterone levels from mice from control groups, and the DHEA and DHEA + metformin groups. (C) Ovarian PGE concentration from mice from control groups, and the DHEA and DHEA + metformin groups. Each column represents the mean± s.e.m. of ten measurements from different animals. ***P < 0.001.
Citation: Reproduction 131, 3; 10.1530/rep.1.00941
(A) Serum 17 beta estradiol levels from mice from control groups, and the DHEA and DHEA + metformin groups. (B) Serum progesterone levels from mice from control groups, and the DHEA and DHEA + metformin groups. (C) Ovarian PGE concentration from mice from control groups, and the DHEA and DHEA + metformin groups. Each column represents the mean± s.e.m. of ten measurements from different animals. ***P < 0.001.
Citation: Reproduction 131, 3; 10.1530/rep.1.00941
(A) Serum 17 beta estradiol levels from mice from control groups, and the DHEA and DHEA + metformin groups. (B) Serum progesterone levels from mice from control groups, and the DHEA and DHEA + metformin groups. (C) Ovarian PGE concentration from mice from control groups, and the DHEA and DHEA + metformin groups. Each column represents the mean± s.e.m. of ten measurements from different animals. ***P < 0.001.
Citation: Reproduction 131, 3; 10.1530/rep.1.00941
Percentage of ovarian T cell phenotypes from mice from control groups (untreated, control vehicle and metformin alone), and from the DHEA and DHEA + metformin groups, determined by flow cytometry assay. Each column represents the mean+s.e.m. of ten measurements from different animals. ***P < 0.001. Upper panels show a representative flow cytometry analysis using forward (FSC; cell size) and side scatter (SSC; cell complexity) parameters, and dotplot analysis using both standard fluorescence 1 (FL1; FITC anti-mouse CD8+T lymphocyte) and 2 (FL2; PE anti-mouse CD4+T lymphocyte).
Citation: Reproduction 131, 3; 10.1530/rep.1.00941
Percentage of ovarian T cell phenotypes from mice from control groups (untreated, control vehicle and metformin alone), and from the DHEA and DHEA + metformin groups, determined by flow cytometry assay. Each column represents the mean+s.e.m. of ten measurements from different animals. ***P < 0.001. Upper panels show a representative flow cytometry analysis using forward (FSC; cell size) and side scatter (SSC; cell complexity) parameters, and dotplot analysis using both standard fluorescence 1 (FL1; FITC anti-mouse CD8+T lymphocyte) and 2 (FL2; PE anti-mouse CD4+T lymphocyte).
Citation: Reproduction 131, 3; 10.1530/rep.1.00941
Percentage of ovarian T cell phenotypes from mice from control groups (untreated, control vehicle and metformin alone), and from the DHEA and DHEA + metformin groups, determined by flow cytometry assay. Each column represents the mean+s.e.m. of ten measurements from different animals. ***P < 0.001. Upper panels show a representative flow cytometry analysis using forward (FSC; cell size) and side scatter (SSC; cell complexity) parameters, and dotplot analysis using both standard fluorescence 1 (FL1; FITC anti-mouse CD8+T lymphocyte) and 2 (FL2; PE anti-mouse CD4+T lymphocyte).
Citation: Reproduction 131, 3; 10.1530/rep.1.00941
Percentage of (A) T cell phenotypes from axillar lymph nodes and (B) T cell phenotypes from retroperitoneal lymph nodes determined by flow cytometry assay. Each column represents the mean± s.e.m. of ten measurements from different animals. ***P < 0.001. Upper panels show a representative flow cytometry analysis using forward (FSC; cell size) and side scatter (SSC; cell complexity) parameters, and dot-plot analysis using both standard fluorescence 1 (FL1; FITC anti-mouse CD8+T lymphocyte) and 2 (FL2; PE anti-mouse CD4+T lymphocyte).
Citation: Reproduction 131, 3; 10.1530/rep.1.00941
Percentage of (A) T cell phenotypes from axillar lymph nodes and (B) T cell phenotypes from retroperitoneal lymph nodes determined by flow cytometry assay. Each column represents the mean± s.e.m. of ten measurements from different animals. ***P < 0.001. Upper panels show a representative flow cytometry analysis using forward (FSC; cell size) and side scatter (SSC; cell complexity) parameters, and dot-plot analysis using both standard fluorescence 1 (FL1; FITC anti-mouse CD8+T lymphocyte) and 2 (FL2; PE anti-mouse CD4+T lymphocyte).
Citation: Reproduction 131, 3; 10.1530/rep.1.00941
Percentage of (A) T cell phenotypes from axillar lymph nodes and (B) T cell phenotypes from retroperitoneal lymph nodes determined by flow cytometry assay. Each column represents the mean± s.e.m. of ten measurements from different animals. ***P < 0.001. Upper panels show a representative flow cytometry analysis using forward (FSC; cell size) and side scatter (SSC; cell complexity) parameters, and dot-plot analysis using both standard fluorescence 1 (FL1; FITC anti-mouse CD8+T lymphocyte) and 2 (FL2; PE anti-mouse CD4+T lymphocyte).
Citation: Reproduction 131, 3; 10.1530/rep.1.00941
Serum TNF from mice from control groups (untreated, control vehicle and metformin alone), the DHEA group and the DHEA + metformin group. Each column represents the mean+s.e.m. of ten measurements from different animals. ***P < 0.001.
Citation: Reproduction 131, 3; 10.1530/rep.1.00941
Serum TNF from mice from control groups (untreated, control vehicle and metformin alone), the DHEA group and the DHEA + metformin group. Each column represents the mean+s.e.m. of ten measurements from different animals. ***P < 0.001.
Citation: Reproduction 131, 3; 10.1530/rep.1.00941
Serum TNF from mice from control groups (untreated, control vehicle and metformin alone), the DHEA group and the DHEA + metformin group. Each column represents the mean+s.e.m. of ten measurements from different animals. ***P < 0.001.
Citation: Reproduction 131, 3; 10.1530/rep.1.00941
References
Abbott DH, Dumesic DA & Franks S2002 Developmental origin of polycystic ovary syndrome – a hypothesis. Journal of Endocrinology 174 1–5.
Anderson E, Lee MT & Lee G1992 Cystogenesis of the ovarian antral follicle of the rat: ultrastructural changes and hormonal profile following the administration of dehydroepiandrosterone. Anatomical Record 234 359–382.
Araya AV, Aguirre A, Romero C, Miranda C, Molina MC & Ferreira A2002 Evaluation of tumor necrosis factor alpha production in ex vivo short term culture whole blood from women with polycystic ovary syndrome. European Cytokine Network 13 419–424.
Asuncion M, Calvo RM, San Millan JL, Sancho J, Avila S & Escobar-Morreale HF2000 A prospective study of the prevalence of the polycystic ovary syndrome in unselected Caucasian women from Spain. Journal of Clinical Endocrinology and Metabolism 85 2434–2438.
Billiar RB, Richardson D, Anderson E, Mahajan D & Little B1985 The effect of chronic and acyclic elevation of circulating androstenedione or estrone concentrations on ovarian function in the rhesus monkey. Endocrinology 116 2209–2220.
Bonnefont-Rousselot D, Raji B, Walrand M, Gardes-Albert M, Jore D, Legrand A, Peynet J & Vasson MP2003 An intracellular modulation of free radical production could contribute to the beneficial effects of metformin towards oxidative stress. Metabolism 52 586–589.
Bradford M1976 A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72 248–254.
Bruun JM, Lihn AS, Pedersen SB & Richelsen B2005 Monocyte chemoattractant protein-1 release is higher in visceral than subcutaneous human adipose tissue (AT): implication of macrophages resident in the AT. Journal of Clinical Endocrinology and Metabolism 90 2282–2289.
Caballero AE2004 Endothelial dysfunction, inflammation, and insulin resistance: a focus on subjects at risk for type 2 diabetes. Current Diabetic Report 4 237–246.
Cacicedo JM, Yagihashi N, Keaney JF Jr, Ruderman NB & Ido Y2004 AMPK inhibits fatty acid-induced increases in NF-kappaB transactivation in cultured human umbilical vein endothelial cells. Biochemical and Biophysical Research Communications 26 1204–1209.
Chantakru S, Wang WC, Van den Heuvel M, Bashar S, Simpson A, Chen Q, Croy BA & Evans SS2003 Coordinate regulation of lymphocyte-endothelial interactions by pregnancy-associated hormones. Journal of Immunology 171 4011–4019.
De Leo V, La Marca A, Ditto A, Morgante G & Cianci A1999 Effects of metformin on gonadotropin-induced ovulation in women with polycystic ovary syndrome. Fertility and Sterility 72 282–285.
Deshpande RR, Chang MY, Chapman JC & Michael SD2000 Alteration of cytokine production in follicular cystic ovaries induced in mice by neonatal estradiol injection. American Journal of Reproduction and Immunology 44 80–88.
Diamanti-Kandarakis E, Kouli C, Tsianateli T & Bergiele A1998 Therapeutic effects of metformin on insulin resistance and hyper-androgenism in polycystic ovary syndrome. European Journal of Endocrinology 138 269–274.
Di Gregorio GB, Yao-Borengasser A, Rasouli N, Yarma V, Lu T, Miles LM, Ranganathan G, Peterson CA, McGehee RE & Kern PA2005 Expression of CD68 and macrophage chemoattractant protein-1 genes in human adipose and muscle tissues: association with cytokine expression, insulin resistance, and reduction by pioglitazone. Diabetes 54 2305–2313.
Doi SA, Al-Zaid M, Towers PA, Scott CJ & Al-Shoumer KA2005 Irregular cycles and steroid hormones in polycystic ovary syndrome. Human Reproduction 20 2402–2408.
Dominguez LJ & Sowers JR2005 Metabolic syndrome therapy: prevention of vascular injury by antidiabetic agents. Current Hypertension Reports 7 110–116.
Dunaif A, Segal KR, Futterweit W & Dobriansky A1989 Profound peripheral insulin resistance, independent of obesity in polycystic ovary syndrome. Diabetes 38 1165–1174.
Fedorcsak P, Dale PO, Storeng R, Abyholm T & Tanbo T2003 The effect of metformin on ovarian stimulation and in vitro fertilization in insulin-resistant women with polycystic ovary syndrome: an open-label randomized cross-over trial. Gynecological Endocrinology 17 207–214.
Franks S1995 Polycystic ovary syndrome. New England Journal of Medicine 333 853–861.
Franks S, Gharani N, Waterworth D, Batty S, White D, Williamson R & McCarthy M1997 The genetic basis of polycystic ovary syndrome. Human Reproduction 12 2641–2648.
Franks S, Mason H & Willis D2000 Follicular dynamics in the poly-cystic ovary syndrome. Molecular Cellular Endocrinology 25 49–52.
Glueck CJ, Wang P, Fontaine R, Tracy T & Sieve-Smith L1999 Metformin-induced resumption of normal menses in 39 of 43 (91%) previously amenorrheic women with the polycystic ovary syndrome. Metabolism 48 511–519.
Gougeon A1996 Regulation of ovarian follicular development in primates: facts and hypotheses. Endocrine Reviews 17 121–155.
Harbone L, Fleming R, Lyall H, Sattar N & Norman J2003 Metformin or antiandrogen in the treatment of hirsutism in polycystic ovary syndrome. Journal of Clinical Endocrinology and Metabolism 88 4116–4123.
Henmi H, Endo T, Nagasawa K, Hayashi T, Chida M, Akutagawa N, Iwasaki M, Kitajima Y, Kiya T, Nishikawa A, Manase K & Kudo R2001 Lysyl oxidase and MMP-2 expression in dehydroepiandrosterone-induced polycystic ovary in rats. Biology of Reproduction 64 157–162.
Hernandez Pardo R, Streber MDL, Orozco H, Arriaga K, Pavon L, Al-Nakhli SA & Rook GA1998 The effects of androstenedione and dehydroepiandrosterone on the course and cytokine profile of tuberculosis in BALB/c mice. Immunology 95 234–241.
Huypens P, Quartier E, Pipeleers D & Van de Casteele M2005 Metformin reduces adiponectin protein expression and release in 3T3-L1 adipocytes involving activation of AMP activated protein kinase. European Journal of Pharmacology 518 90–95.
Ivanov AI & Romanovsky AA2004 Prostaglandin E2 as a mediator of fever: synthesis and catabolism. Frontiers in Bioscience 1 1977–1993.
Kazerooni T & Dehghan-Kooshkghazi M2003 Effects of metformin therapy on hyperandrogenism in women with polycystic ovarian syndrome. Gynecological Endocrinology 17 51–56.
Kiortsis DN, Filippatos TD & Elisaf MS2005 The effects of orlistat on metabolic parameters and other cardiovascular risk factors. Diabetes and Metabolism 31 15–22.
Korhonen S, Romppanen EL, Hiltunen M, Mannernaa A, Punnonen K, Hippelainen M & Heinoneu D2002 Lack of association between C-850T polymorphism of the gene encoding tumor necrosis factor-alpha and polycystic ovary syndrome. Gynecological Endocrinology 16 271–274.
Kuroda E & Yamashita U2003 Mechanisms of enhanced macrophage-mediated prostaglandin E2 production and its suppressive role in Th1 activation in Th2-dominant BALB/c mice. Journal of Immunology 15 757–764.
Lakier Smith L2003 Overtraining, excessive exercise, and altered immunity: is this a T helper-1 versus T helper-2 lymphocyte response? Sports and Medicine 33 347–364.
La Marca A, Morgante G, Palumbo M, Cianci A, Petraglia F & De Leo V2002 Insulin-lowering treatment reduces aromatase activity in response to follicle-stimulating hormone in women with polycystic ovary syndrome. Fertility and Sterility 78 1234–1239.
Lee GY, Croop JM & Anderson E1998 Multidrug resistance gene expression correlates with progesterone production in dehydroepiandrosterone-induced polycystic equine chorionic gonadotropin-stimulated ovaries of prepuberal rats. Biology of Reproduction 58 330–337.
Lee MT, Anderson E & Lee GY1991 Changes in ovarian morphology and serum hormones in the rat after treatment with dehydroepiandrosterone. Anatomical Record 231 185–192.
Leposavic G, Obradovic S, Kosec D, Pejcic-Karapetrovic B & Vidic-Dankovic B2001 In vivo modulation of the distribution of thymocyte subsets by female sex steroid hormones. International Immunopharmacology 1 1–12.
Lord JM, Flight IH & Norman RJ2003 Insulin-sensitising drugs (metformin, troglitazone, rosiglitazone, pioglitazone, D-chiroinositol) for polycystic ovary syndrome. Cochrane Database System Reviews 3 CD0033053.
Lu FX, Abel K, Ma Z, Rourke T, Lu D, Torten J, McChesney M & Miller CJ2002 The strength of B cell immunity in female rhesus macaques is controlled by CD8 + T cells under the influence of ovarian steroid hormones. Clinical Experimental Immunology 128 10–20.
Luchetti CG, Solano ME, Sander V, Barreiro-Arcos ML, Gonzalez C, Di Girolamo G, Chiocchio S, Cremaschi G & Motta AB2004 Effects of dehydroepiandrosterone on ovarian cystogenesis and immune function. Journal of Reproductive Immunology 64 59–74.
McCarthy MF2005 AMPK activation as a strategy for reversing the endothelial lipotoxicity underlying the increased vascular risk associated with insulin resistance syndrome. Medical Hypotheses 64 1211–1215.
Malesh VB & Greenblatt RB1962 Isolation of dehydroepiandrosterone and 17α - hydroxy- pregnenolone from polycystic ovaries of the Stein–Leventhal syndrome. Journal of Clinical Endocrinology and Metabolism 22 441–448.
Mansfield R, Galea R, Brincat M, Hole D & Mason H2003 Metformin has direct effects on human ovarian steroidogenesis. Fertility and Sterility 79 956–962.
Meikle AW, Dorchuck RW, Araneo BA, Stringham JD, Evans TG, Spruance SL & Daynes RA1992 The presence of dehydroepiandrosterone-specific receptor binding complex in murine T cells. Journal of Steroid Biochemisry and Molecular Biology 42 293–304.
Mendonca HC, Montenegro RM, Foss MC, Silva de Sa MF & Ferriani RA2004 Positive correlation of serum leptin with estradiol levels in patients with polycystic ovary syndrome. Brazilian Journal of Medical and Biological Research 37 729–736.
Moghetti P, Castello R, Negri C, Tosi F, Perrone F, Caputo M, Zanolin E & Muggeo M2000 Metformin effects on clinical features, endocrine and metabolic profiles, and insulin sensitivity in polycystic ovary syndrome: a randomized, double-blind, placebo-controlled 6 month trial, followed by open, long-term clinical evaluation. Journal of Clinical Endocrinology and Metabolism 85 139–146.
Musso C, Shawker T, Cochran E, Javor ED, Young J & Gorden P2005 Clinical evidence that hyperinsulinaemia independent of gonadotropins stimulates ovarian growth. Clinical Endocrinology (Oxford) 63 73–78.
Navarra P, Andreani CL, Lazzarin N, Pierro E, Mirtella A, Lanzone A & Mancuso S1996 Increased production and release of prostaglandin E2 by human granulosa cells from polycystic ovaries. Prostaglandins 52 187–197.
Obradovic S, Vidic-Dankovic B, Pejcic-Karapetrovic B, Kosec D & Leposavic G2001 In vivo modulation of the splenocyte yield and composition by female sex steroid hormones. Pharmazie 56 235–238.
Okabe T, Haji M, Takayanagi R, Adachi M, Imasaki K, Kurimoto F, Watanabe T & Nawata H1995 Up-regulation of high-affinity dehydroepiandrosterone binding activity by dehydroepiandrosterone in activated human T lymphocytes. Journal of Clinical Endocrinology and Metabolism 80 2993–2996.
Peral B, San Millan JL, Castello R, Morghetti P & Escobar-Morreale HF2002 Comment: the methionine 196 arginine polymorphism in exon 6 of the TNF receptor 2 gene (TNFRSFIB) is associated with the polycystic ovary syndrome and hyperandrogenism. Journal of Clinical Endocrinology and Metabolism 87 3977–3983.
Priddy AR & Killick SR1993 Eicosanoids and ovulation. Prostaglandins Leukotrienes and Essential Fatty Acids 49 827–1993.
Rautio K, Tapanainen JS, Ruokonen A & Mouri-Papunen LC2005 Effects of metformin and ethinyl estradiolcyproterone acetate on lipid levels in obese and non-obese women with polycystic ovary syndrome. European Journal of Endocrinology 152 269–275.
Roy S, Mahesh VB & Greenblatt RB1962 Effect of dehydroepiandrosterone and δ 4-androstenedione on the reproductive organs of female rats: production of cystic changes in the ovary. Nature 196 2–6.
Ruat C, Faure L, Choquet-Kastylevsky G, Ravel G & Descotes J2003 Tritiated thymidine incorporation does not enhance sensitivity of the politeal lymph node assay. Toxicology 188 29–33.
Safadi FF, Dissanayake IR, Goodman GG, Jago RA, Baker AE, Bowman AR, Sass DA, Popoff SN & Epstein S2000 Influence of estrogen deficiency and replacement on T-cell populations in rat lymphoid tissues and organs. Endocrine 12 81–88.
Sapino A, Cassoni P, Ferrero E, Bongiovanni M, Righi L, Fortunati N, Crafa P, Chiarle R & Bussolati G2003 Estrogen receptor alpha is a novel marker expressed by follicular dendritic cells in lymph nodes and tumor-associated lymphoid infiltrates. American Journal of Pathology 163 1313–1320.
Sayin NC, Gucer F, Balkanli-Kaplan P, Yuce MA, Ciftci S, Kucuk M & Yardim T2003 Elevated serum TNF-alpha levels in normal-weight women with polycystic ovaries syndrome. Journal of Reproductive Medicine 48 165–170.
Shoupe D, Kumar DD & Lobo RA1983 Insulin resistance in poly-cystic ovary syndrome. American Journal of Obstetrics and Gynecology 147 588–592.
Spaczynski RZ, Arici A & Duleba AJ1999 Tumor necrosis factor-alpha stimulates proliferation of rat ovarian theca-interstitial cells. Biology of Reproduction 61 993–998.
Stefanovic V, Antic S, Mitic-Zlatkovic M & Vlahovic P1999 Reversal of increased lymphocyte PC-1 activity in patients with type 2 diabetes treated with metformin. Diabetes and Metabolism Research Reviews 15 400–404.
Szkiewicz D & Uilenbroek JT1998 Polycystic ovary syndrome –searching for an animal model. Journal of Medicine 29 259–275.
Vandermolen DT, Ratts VS, Evans WS, Stovall DW, Kauma SW & Nestler JE2001 Metformin increases the ovulation rate and pregnancy rate from clomiphene citrate in patients with polycystic ovary syndrome who are resistant to clomiphene citrate alone. Fertility and Sterility 75 310–315.
Weerakiet S, Srisombut C, Rojanasakul A, Panburana P, Thakkinstian A & Herabutya Y2004 Prevalence of gestational diabetes mellitus and pregnancy outcomes in Asian women with polycystic ovary syndrome. Gynecological Endocrinology 19 134–140.
Weil S, Vendola K, Zhou J & Bondy CA1999 Androgen and follicle-stimulating hormone interactions in primate ovarian follicle development. Journal of Clinical Endocrinology and Metabolism 84 2951–2956.
West C, Foster DL, Evans NP, Robinson J & Padmanabhan V2001 Intra-follicular activin availability is altered in prenatally-androgenized lambs. Molecular and Cellular Endocrinology 185 51–59.
Wojtowicz-Praga S1997 Reversal of tumor-induced immunosuppression: a new approach to cancer therapy. Journal of Immunotherapy 20 165–177.
Yan G, Schoenfeld D, Penney C, Hurxthal K, Taylor AE & Faustman D2000 Identification of premature ovarian failure patients with underlying autoimmunity. Journal of Women’s Health and Gender Based Medicine 9 275–287.
Yang L, Yamagata N, Yadav R, Brandon S, Courtney RL, Morrow JD, Shyr Y, Boothby M, Joyce S, Carbone DP & Breyer RM2003 Cancer-associated immunodeficiency and dendritic cell abnormalities mediated by the prostaglandin EP2 receptor. Journal of Clinical Investigation 111 727–735.
Zhang Z, Araghi-Niknam M, Liang B, Inserra P, Ardestani SK, Jiang S, Chow S & Watson RR1999 Prevention of immune dysfunction and vitamin E loss by dehydroepiandrosterone and melatonin supplementation during murine retrovirus infection. Immunology 96 291–297.
