Nontubal ectopic pregnancies: overview of diagnosis and treatment.

Nontubal ectopic pregnancies occur as a result of embryo implantation outside the uterine cavity and fallopian tubes. Sites include ovary, cervix, abdominal cavity, interstitial portion of fallopian tube, and cesarean scar. Nontubal pregnancies are uncommon. Nonspecific signs and symptoms of nontubal ectopic pregnancies make diagnosis challenging and, in many cases, significantly delayed, resulting in a high rate of morbidity. Although surgical management remains the mainstay of treatment, there is growing evidence that some of these can be managed medically or with the use of a combination of medical and surgical approaches with good outcome. This review summarizes the current diagnostic modalities, therapeutic options, and outcomes for nontubal ectopic pregnancies. Diagnostic and management options may be limited, especially in resource-restricted settings. Therefore, an understanding of the available options is critical. It needs to be emphasized that the rarity of cases and the difficulties in organizing ethically justified randomized trials result in the lack of well-established management guidelines for nontubal ectopic pregnancies.

Fertility Preservation for Transgender Males: Counseling and Timing of Treatment.

Fertility-preservation counseling in the transgender patient population is recommended by multiple organizations, including the American Society for Reproductive Medicine, the World Professional Association for Transgender Health, and the Endocrine Society. The optimal time to pursue fertility preservation has not been established, and data on potential effects of testosterone therapy on future reproductive potential are limited. This Current Commentary seeks to elucidate the most appropriate time to perform oocyte cryopreservation in relation to time on and off testosterone therapy, age of the individual, and emotional effect of treatment. Although there have been multiple studies that have demonstrated successful oocyte cryopreservation regardless of testosterone exposure, the data on live-birth rates after oocyte cryopreservation are limited. Moreover, the process of oocyte cryopreservation may have a significant negative emotional effect on the transgender male given the feminizing effects of gonadotropin stimulation, as well as the invasiveness of pelvic ultrasonograms and the oocyte-retrieval procedure. With our review, we demonstrate that a comprehensive, individualized approach to fertility-preservation counseling and timing to pursue treatment are essential. Postponing fertility-preservation procedures until patients have reached early adulthood might be considered to avoid the potential effect on mental health, without compromising outcomes.

Lipophilic statins inhibit growth and reduce invasiveness of human endometrial stromal cells.

PURPOSE: To compare effects of lipid-soluble statins (simvastatin, lovastatin, atorvastatin) and water-soluble statin (pravastatin) on growth and invasiveness of human endometrial stromal (HES) cells. METHODS: Endometrial biopsies were collected during the proliferative phase from five volunteers. HES cells were isolated and cultured in the absence or in the presence of simvastatin, lovastatin, atorvastatin, and pravastatin. Effects of statins on DNA synthesis, cell viability, activity of caspases 3/7 and invasiveness were evaluated. RESULTS: The proliferation of HES cells was significantly decreased by simvastatin (by 47-89%), lovastatin (by 46-78%), and atorvastatin (by 21-48%) in a concentration-dependent manner. Activity of executioner caspases 3/7 was significantly increased by simvastatin (by 10-25%), lovastatin (by 19%) and atorvastatin (by 7-10%) in a concentration-dependent manner. The greatest effects were observed in response to simvastatin. Accounting for the effects of statins on cell number, the invasiveness of HES cells was significantly decreased in cells treated with simvastatin (by 49%), lovastatin (by 54%), and atorvastatin (by 53%). Pravastatin had little or no effects on any of the tested endpoints. CONCLUSIONS: Present findings demonstrate that only lipid-soluble among tested statins were effective in inhibition of growth and invasiveness of HES cells. These findings may have clinical relevance in treatment of endometriosis.

Advances in Fertility Preservation for Young Women With Cancer.

Female patients of reproductive age with cancer often require treatment that can compromise their future fertility. Treatment-related infertility is an important cancer survivorship issue and is associated with depression and diminished quality of life. Recent advances in reproductive health care provide the opportunity to preserve fertility prior to the initiation of cancer therapy. Clinical guidelines recommend that oncology providers counsel patients about the risk of treatment-related infertility and fertility preservation options, and that they refer those who are interested in fertility preservation to fertility specialists. Guidelines endorse the use of assisted reproductive techniques (ART) provided by reproductive endocrinologists to preserve fertility in young female patients with cancer. In addition, ovarian suppression with gonadotropin-releasing hormone (GnRH) agonists may be considered for ovarian protection during chemotherapy. This article reviews currently available and emerging ART for fertility preservation in female patients of reproductive age with cancer and current data supporting the use of ovarian suppression for ovarian protection during chemotherapy in this population. We also review the uptake of fertility services and discuss barriers to fertility preservation in female patients of reproductive age with cancer.

Resveratrol potentiates effects of simvastatin on inhibition of rat ovarian theca-interstitial cells steroidogenesis.

BACKGROUND: Polycystic ovary syndrome (PCOS) is characterized by ovarian enlargement, hyperplastic theca compartment and increased androgen production due to, at least in part, excessive expression of several key genes involved in steroidogenesis. Previously, our group has demonstrated that simvastatin, competitive inhibitor of 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-CoA reductase), a rate-limiting step of the mevalonate pathway, reduces rat-theca interstitial cell steroidogenesis by inhibiting Cyp17a1 gene expression, the key enzyme of the androgen biosynthesis pathway. Recently, we demonstrated that resveratrol, a bioflavonoid abundant in red grapes, decreases rat theca-interstitial cell steroidogenesis and this suppressive effect is mediated through mechanisms independent of the mevalonate pathway. The present study evaluated the effect of combining simvastatin and resveratrol treatments on rat theca-interstitial cell steroidogenesis. METHODS: Rat theca-interstitial cells isolated from 30 day-old female rats were cultured for up to 48 h with or without simvastatin (1 µM) and/or resveratrol (3-10 µM). Steroidogenic enzymes gene expression was evaluated by quantitative real time PCR and steroid levels were measured by liquid chromatography-mass spectrometry. Comparisons between groups were performed using ANOVA and Tukey test. RESULTS: Resveratrol potentiated inhibitory effects of simvastatin on androstenedione and androsterone production in theca-interstitial cells. This suppressive effect correlated with profound inhibition in Cyp17a1 mRNA expression in the presence of a combination of resveratrol and simvastatin. CONCLUSIONS: The present findings indicate that resveratrol potentiates the simvastatin-induced inhibitory effect on theca-interstitial cell androgen production, raising the possibility of development of novel treatments of PCOS.

Comparison of effects of different statins on growth and steroidogenesis of rat ovarian theca-interstitial cells.

Statins are competitive inhibitors of 3-hydroxy-3-methyl-glutaryl-CoA reductase, the rate-limiting enzyme of the cellular production of cholesterol and other products of the mevalonate pathway. Statins exert hepatic and extrahepatic effects, modulating the function of various tissues and organs, including ovaries. Previously, we have demonstrated that simvastatin inhibited cellular proliferation and reduced androgen production by ovarian theca-interstitial cells. The above actions are of translational relevance to the most common endocrine disorder among women in reproductive age: polycystic ovary syndrome. However, different statins may have distinctly different profiles of effects on cholesterol and androgens. The present study was designed to compare the effects of several statins on growth and steroidogenesis of rat theca-interstitial cells. The cells were incubated in the absence (control) or in the presence of simvastatin, lovastatin, atorvastatin, or pravastatin. Assessment of effects of statins on cell growth was carried out by evaluation of DNA synthesis and by estimation of the number of viable cells. Effects on steroidogenesis were evaluated by quantification of steroid production and expression of mRNA for the key enzyme regulating androgen production: Cyp17a1. Among tested statins, simvastatin exerted the greatest inhibitory effects on all tested parameters. The rank order of the effects of the tested statins is as follows: simvastatin > lovastatin > atorvastatin ≥ pravastatin. While the lipophilicity is likely to play a major role in determining the ability of statins to act on nonhepatic cells, other factors unique to individual cell types are also likely to be relevant.

Resveratrol potentiates effect of simvastatin on inhibition of mevalonate pathway in human endometrial stromal cells.

CONTEXT: Growth of endometriotic lesions in rodent model of endometriosis is inhibited by resveratrol, a natural polyphenol with antiproliferative and antiinflammatory properties, and simvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) activity. OBJECTIVE: The objective of the investigation was to study the mechanism of action of resveratrol and its interactions with simvastatin, focusing on cholesterol biosynthesis and HMGCR gene expression and protein activity in primary cultures of human endometrial stromal (HES) cells. METHODS: HES cells were obtained from healthy volunteers. Biosynthesis of cholesterol was assessed by measuring the conversion of [(14)C]acetate to [(14)C]cholesterol. HMGCR mRNA transcripts were quantified by real-time PCR, protein expression by Western blot analysis, and enzyme activity by measuring the conversion of [3-(14)C]3-hydroxy-3-methyl-glutaryl-coenzyme A to [(14)C]mevalonic acid lactone in HES cell microsomes. RESULTS: Resveratrol inhibited cholesterol biosynthesis, HMGCR mRNA, and enzyme activity. Simvastatin inhibited cholesterol biosynthesis and enzyme activity but increased HMGCR mRNA and protein expression. Resveratrol potentiated the inhibitory effects of simvastatin on cholesterol biosynthesis and HMGCR enzyme activity and abrogated the stimulatory effects of simvastatin on HMGCR mRNA transcripts and protein expression. CONCLUSIONS: Resveratrol inhibits key steps of the mevalonate pathway by mechanisms that are partly complementary to and partly comparable with simvastatin via reducing both expression and activity of HMGCR. A combination of resveratrol and simvastatin may be of potential clinical relevance to development new treatments of human endometriosis.

Effects of simvastatin on retinoic acid system in primary human endometrial stromal cells and in a chimeric model of human endometriosis.

CONTEXT: Retinoic acid (RA) may promote survival or apoptosis of cells, depending on the levels of binding proteins: apoptosis-inducing cellular RA binding protein 2 (CRABP2), and cell survival-promoting fatty acid binding protein 5 (FABP5). Increased cellular uptake of retinol and altered actions of RA related to reduced expression of CRABP2 may contribute to the development of endometriosis. Recently statins have been shown to inhibit growth of human endometrial stromal (HES) cells and to reduce the number and size of endometriotic implants in experimental models of this disorder. OBJECTIVE: The objective of the study was to determine whether effects of simvastatin on HES cells and experimental endometriotic implants are related to the modulation of the RA system. METHODS: Effects of simvastatin and RA on proliferation and apoptosis of HES cells were evaluated. Expression of stimulated by RA 6 (STRA6), CRABP2, and FABP5 was determined by real-time PCR and Western blotting. Effects of simvastatin were also evaluated in a nude mouse model of human endometriosis. RESULTS: Simvastatin potentiated an inhibitory effect of RA on growth of HES cells. In HES cells, simvastatin induced expression of STRA6 and CRABP2 but not FABP5. Similarly, simvastatin treatment of nude mice bearing human endometrial xenografts led to an increased expression of CRABP2 and STRA6 proteins in ectopic lesions. CONCLUSIONS: Simvastatin interacts with the RA system, inducing the expression of the key protein regulating the uptake of retinol (STRA6) and the expression of apoptosis-promoting CRABP2. These effects may contribute to cooperative apoptosis-inducing effects of simvastatin and RA and support the examination of these compounds in the treatment of endometriosis.

Letrozole increases ovarian growth and Cyp17a1 gene expression in the rat ovary.

OBJECTIVE: To evaluate the effects of letrozole on ovarian size and steroidogenesis in vivo, as well as on proliferation and steroidogenesis of theca-interstitial cells alone and in coculture with granulosa cells using an in vitro model. DESIGN: In vivo and in vitro studies. SETTING: Research laboratory. ANIMAL(S): Immature Sprague-Dawley female rats. INTERVENTION(S): In vivo effects of letrozole were studied in intact rats receiving either letrozole (90-day continuous-release SC pellets, 400 µg/d) or placebo pellets (control group). In in vitro experiments, theca cells were cultured alone or in coculture with granulosa cells in the absence or presence of letrozole. MAIN OUTCOME MEASURE(S): Deoxyribonucleic acid synthesis was determined by thymidine incorporation assay; steroidogenesis by mass spectrometry; and steroidogenic enzyme messenger RNA (mRNA) expression by polymerase chain reaction. RESULT(S): In vivo, letrozole induced an increase in ovarian size compared with the control group and also induced a profound increase of androgen, LH levels, and Cyp17a1 mRNA expression. Conversely, a decrease in Star, Cyp11a1, and Hsd3b1 transcripts was observed in letrozole-exposed rats. In vitro, letrozole did not alter either theca cell proliferation or Cyp17a1 mRNA expression. Similarly, letrozole did not affect Cyp17a1 transcripts in granulosa-theca cocultures. CONCLUSION(S): These findings suggest that letrozole exerts potent, but indirect, effect on growth of rat ovary and dramatically increases androgen levels and Cyp17a1 mRNA expression, the key enzyme regulating the androgen biosynthesis pathway. The present findings reveal novel mechanisms of action of letrozole in the rat ovary.

Effects of resveratrol on growth and function of rat ovarian granulosa cells.

OBJECTIVE: To evaluate the effects of resveratrol on growth and function of granulosa cells. Previously, we demonstrated that resveratrol exerts profound proapoptotic effects on theca-interstitial cells. DESIGN: In vitro study. SETTING: Research laboratory. ANIMAL(S): Immature Sprague-Dawley female rats. INTERVENTION(S): Granulosa cells were cultured in the absence or presence of resveratrol. MAIN OUTCOME MEASURE(S): DNA synthesis was determined by thymidine incorporation assay, apoptosis by activity of caspases 3/7, cell morphology by immunocytochemistry, steroidogenesis by mass spectrometry, antimüllerian hormone (AMH), and vascular endothelial growth factor (VEGF) expression by polymerase chain reaction and Western blot. RESULT(S): Resveratrol induced a biphasic effect on DNA synthesis, whereby a lower concentration stimulated thymidine incorporation and higher concentrations inhibited it. Additionally, resveratrol slightly increased the cell number and modestly decreased the activity of caspases 3/7 with no effect on cell morphology or progesterone production. However, resveratrol decreased aromatization and VEGF expression, whereas AMH expression remained unaltered. CONCLUSION(S): Resveratrol, by exerting cytostatic but not cytotoxic effects, together with antiangiogenic actions mediated by decreased VEGF in granulosa cells, may alter the ratio of theca-to-granulosa cells and decrease vascular permeability, and therefore may be of potential therapeutic use in conditions associated with highly vascularized theca-interstitial hyperplasia and abnormal angiogenesis, such as those seen in women with polycystic ovary syndrome.

Resveratrol reduces steroidogenesis in rat ovarian theca-interstitial cells: the role of inhibition of Akt/PKB signaling pathway.

Polycystic ovary syndrome is characterized by theca-interstitial hyperplasia and increased expression of steroidogenic genes, leading to excessive androgen production. Resveratrol, a natural polyphenol, promotes apoptosis and reduces rat theca-interstitial cell growth, in part by inhibiting the mevalonate pathway and decreasing the availability of substrates of isoprenylation [farnesyl-pyrophosphate (FPP) and geranylgeranyl-pyrophosphate (GGPP)]. This study evaluated the effect of resveratrol on rat theca-interstitial cell steroidogenesis. Because resveratrol may activate sirtuins, this study also investigated whether steroidogenesis was affected by sirtuin inhibitors (nicotinamide, sirtinol). Theca-interstitial cells were cultured with or without resveratrol (1-10 µm), GGPP (30 µm), FPP (30 µm), nicotinamide (1 mm), and/or sirtinol (10 µm). Resveratrol did not affect progesterone levels but reduced androgen production in a concentration-dependent fashion (androstenedione by up to 78% and androsterone by up to 76%). This inhibitory effect correlated with a decrease in mRNA expression of genes regulating androgen production, especially Cyp17a1 (by up to 73%). GGPP and FPP had no effect on androgen levels and Cyp17a1 mRNA levels and did not alter the effects induced by resveratrol. Similarly, sirtuin inhibitors did not reverse resveratrol-induced inhibition of steroidogenesis. However, resveratrol decreased activity of serine-threonine kinase/protein kinase B pathway, a cell-signaling pathway involved in ovarian steroidogenesis. The present findings indicate that resveratrol reduces androgen production primarily by inhibiting Cyp17a1 mRNA expression, and this inhibition may be mediated, in part, by blocking the activity of the serine-threonine kinase/protein kinase B pathway. These findings may be of clinical relevance to conditions associated with excessive production of androgens by theca cells, such as polycystic ovary syndrome.

Simvastatin decreases invasiveness of human endometrial stromal cells.

Recently we reported that statins, the competitive inhibitors of the key enzyme regulating the mevalonate pathway, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), decrease proliferation of human endometrial stromal (HES) cells. Furthermore, we found that simvastatin treatment reduces the number and the size of endometrial implants in a nude mouse model of endometriosis. The present study was undertaken to investigate the effect of simvastatin on HES cell invasiveness and on expression of selected genes relevant to invasiveness: matrix metalloproteinase 2 (MMP2), MMP3, tissue inhibitor of matrix metalloproteinase 2 (TIMP2), and CD44. Because statin-induced inhibition of HMGCR reduces the production of substrates for isoprenylation-geranylgeranyl pyrophosphate (GGPP) and farnesyl pyrophosphate (FPP)-the effects of GGPP and FPP were also evaluated. Simvastatin induced a concentration-dependent reduction of invasiveness of HES cells. This effect of simvastatin was abrogated by GGPP but not by FPP. Simvastatin also reduced the mRNA levels of MMP2, MMP3, and CD44, but increased TIMP2 mRNA; all these effects of simvastatin were partly or entirely reversed in the presence of GGPP. The present findings provide a novel mechanism of action of simvastatin on endometrial stroma that may explain reduction of endometriosis in animal models of this disease. Furthermore, the presently described effects of simvastatin are likely mediated, at least in part, by inhibition of geranylgeranylation.

Simvastatin reduces steroidogenesis by inhibiting Cyp17a1 gene expression in rat ovarian theca-interstitial cells.

Polycystic ovary syndrome (PCOS) is characterized by ovarian enlargement, theca-interstitial hyperplasia, and increased androgen production by theca cells. Previously, our group has demonstrated that statins (competitive inhibitors of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase, a rate-limiting step of the mevalonate pathway) reduce proliferation of theca-interstitial cells in vitro and decrease serum androgen levels in women with PCOS. The present study evaluated the effect of simvastatin on rat ovarian theca-interstitial cell steroidogenesis. Because actions of statins may be due to reduced cholesterol availability and/or isoprenylation of proteins, the present study also investigated whether steroidogenesis was affected by cell- and mitochondrion-permeable 22-hydroxycholesterol, isoprenylation substrates (farnesyl-pyrophosphate [FPP] and geranylgeranyl-pyrophosphate [GGPP]), as well as selective inhibitors of farnesyltransferase (FTI) and geranylgeranyltransferase (GGTI). Theca-interstitial cells were cultured for 12, 24, and 48 h with or without simvastatin, GGPP, FPP, FTI, GGTI, and/or 22-hydroxycholesterol. Simvastatin decreased androgen levels in a time- and concentration-dependent fashion. This inhibitory effect correlated with a decrease in mRNA levels of Cyp17a1, the gene encoding the key enzyme regulating androgen biosynthesis. After 48 h, GGPP alone and FPP alone had no effect on Cyp17a1 mRNA expression; however, the inhibitory action of simvastatin was partly abrogated by both GGPP and FPP. The present findings indicate that statin-induced reduction of androgen levels is likely due, at least in part, to the inhibition of isoprenylation, resulting in decreased expression of CYP17A1.

Resveratrol inhibits the mevalonate pathway and potentiates the antiproliferative effects of simvastatin in rat theca-interstitial cells.

OBJECTIVE: To examine the mechanisms of action of resveratrol and its interaction with simvastatin on growth and the mevalonate pathway in rat theca-interstitial cells. DESIGN: In vitro study. SETTING: Research laboratory. ANIMAL(S): Immature Sprague-Dawley female rats. INTERVENTION(S): Theca-interstitial cells were cultured in the absence or presence of resveratrol, simvastatin, mevalonic acid, farnesyl pyrophosphate, and/or geranylgeranyl pyrophosphate. MAIN OUTCOME MEASURE(S): DNA synthesis was assessed by thymidine incorporation assay; 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) expression and activity were evaluated with the use of quantitative real-time polymerase chain reaction, Western blot analysis, and HMGCR activity assay. Cholesterol synthesis was determined by the conversion of [(14)C]-acetate to [(14)C]-cholesterol. RESULT(S): Resveratrol potentiated the simvastatin-induced inhibition on cell proliferation in a concentration-dependent manner. Inhibitory effects of resveratrol were partly abrogated by the addition of mevalonic acid, farnesyl pyrophosphate, and geranylgeranyl pyrophosphate. Resveratrol reduced HMGCR expression and activity, and decreased cholesterol synthesis. In contrast, simvastatin inhibited HMGCR activity with a compensatory increase in HMGCR expression. Resveratrol counteracted this effect of simvastatin on HMGCR expression but augmented the simvastatin-induced inhibition on HMGCR activity and cholesterol synthesis. CONCLUSION(S): Resveratrol inhibits the mevalonate pathway via distinctly different mechanisms than statins. These observations demonstrate a novel mechanism of action of resveratrol and underscore the potential translational/clinical relevance of resveratrol interactions with simvastatin.

Novel therapies targeting endometriosis.

Endometriosis is an often painful disorder in which the endometrial glands and stroma grow outside the uterus. The disease affects women's quality of life and is a common cause of infertility. In this review, we describe promising new developments in the field based on in vitro assays and rodent models, each of which has the potential to be beneficial in the treatment of this disease. We will specifically describe the role of anti-inflammatory drugs, selective estrogen, or progesterone modulators, statins, antiangiogenic agents, and the potential for targeting stem cells as likely methods to hone in and eliminate endometriosis. The most promising of these potential therapies are currently slated for further testing in both rodent and nonhuman primate trials.

Resveratrol inhibits development of experimental endometriosis in vivo and reduces endometrial stromal cell invasiveness in vitro.

Endometriosis is a common gynecologic disorder characterized by ectopic attachment and growth of endometrial tissues. Resveratrol is a natural polyphenol with antiproliferative and anti-inflammatory properties. Our objective was to study the effects of resveratrol on human endometriotic implants in a nude mouse model and to examine its impact on human endometrial stromal (HES) cell invasiveness in vitro. Human endometrial tissues were obtained from healthy donors. Endometriosis was established in oophorectomized nude mice by intraperitoneal injection of endometrial tissues. Mice were treated with 17ß-estradiol (8 mg, silastic capsule implants) alone (n = 16) or with resveratrol (6 mg/mouse; n = 20) for 10-12 and 18-20 days beginning 1 day after tissue injection. Mice were killed and endometrial implants were evaluated. A Matrigel invasion assay was used to examine the effects of resveratrol on HES cells. We assessed number and size of endometriotic implants in vivo and Matrigel invasion in vitro. Resveratrol decreased the number of endometrial implants per mouse by 60% (P < 0.001) and the total volume of lesions per mouse by 80% (P < 0.001). Resveratrol (10-30 µM) also induced a concentration-dependent reduction of invasiveness of HES by up to 78% (P < 0.0001). Resveratrol inhibits development of endometriosis in the nude mouse and reduces invasiveness of HES cells. These observations may aid in the development of novel treatments of endometriosis.

Statins inhibit growth of human theca-interstitial cells in PCOS and non-PCOS tissues independently of cholesterol availability.

CONTEXT: Polycystic ovary syndrome (PCOS) is associated with ovarian enlargement, prominent theca-interstitial hyperplasia, and excessive androgen production. Recent clinical trials have demonstrated that statins, 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors, decrease androgen levels in women with PCOS. OBJECTIVE: The present study evaluated the effect of statins on proliferation of human ovarian theca-interstitial cells. DESIGN AND SETTINGS: In vitro experiments were performed in the university research laboratory. PATIENTS: Human theca-interstitial cells were isolated from ovaries of PCOS (n=4) and non-PCOS (n=4) patients. MAIN OUTCOME MEASURES: The cells were incubated for 48 h without additives (control) or with simvastatin (3-30 µm), mevastatin (3-30 µm), and/or the cell- and mitochondrion-permeable form of cholesterol (22-hydroxycholesterol; 10 µm). To determine whether the effects of statins could be affected by leukocytes, the experiment was carried out on cells not purified of leukocytes and cells purified using anti-CD-45 immunomagnetic beads. The effect of statins on proliferation was evaluated by determination of DNA synthesis using radiolabeled thymidine-incorporation assay and by quantification of viable cells using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenil)-2H-tetrazolium assay. RESULTS: Statins induced an inhibition of DNA synthesis in both the absence and the presence of 22-hydroxycholesterol; furthermore, 22-hydroxycholesterol alone also inhibited DNA synthesis. These effects of statins and 22-hydroxycholesterol were confirmed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenil)-2H-tetrazolium assay. Comparable inhibition of proliferation was observed in cells obtained from women with and without PCOS and in cell preparations treated and not treated with anti-CD-45 immunomagnetic beads. CONCLUSIONS: Statins inhibit proliferation of human theca-interstitial cells irrespective of the availability of cholesterol and independently of leukocytes both in normal and PCOS ovaries.