Fernando Gabler

Expression of steroid receptors and proteins related to apoptosis in endometria of women with polycystic ovary syndrome

OBJECTIVE To evaluate the expression of steroid hormone receptors, proteins related to apoptosis, and cell proliferation in endometria from women with polycystic ovary syndrome (PCOS). DESIGN Case-control study. SETTING Hospital research unit. PATIENT(S) Eight women with PCOS and 12 fertile healthy women of similar age to those with PCOS. INTERVENTION(S) Endometrial samples were obtained from women with PCOS (PCOSE) and normal (NE) women during the proliferative phase of the menstrual cycle. MAIN OUTCOME MEASURE(S) Expression studies (immunohistochemistry and reverse transcription-polymerase chain reaction) and DNA fragmentation [TdT-mediated dUTP nick end labeling (TUNEL)]. RESULT(S) In stroma, protein expression of estrogen receptor alpha, Bcl-2, and Bax was higher in PCOSE than in NE; epithelial cells had a greater expression of androgen receptor in the nucleus and a lower expression in the cytoplasm of PCOSE. Cell proliferation was higher in the epithelia of NE, while the expression of caspase-3 and DNA fragmentation was similar in both groups. The bax mRNA expression was higher in PCOSE, and bcl-2 mRNA expression was similar between groups. A higher bcl-2/bax relative ratio in PCOSE was observed. CONCLUSION(S) An alternating expression of proteins related to cell survival in endometria from PCOSE may potentially be associated with the disruption of their endometrial cell cycle.

Role of nitric oxide and bcl-2 family genes in the regulation of human endometrial apoptosis☆

OBJECTIVE To investigate the role of nitric oxide (NO) and death regulatory genes, bcl-2 and bax, in human endometria apoptosis. DESIGN Expression of bcl-2, bax, NO synthases (NOS), and the apoptotic effect of L-arginine on endometrial explants in vitro. SETTING Prospective study. PATIENT(S) Thirty-seven eumenorrheic women. INTERVENTION(S) Endometrial samples were obtained with Pipelle suction curette after women signed institutional informed consent forms. MAIN OUTCOME MEASURE(S) DNA fragmentation (TUNEL), immunohistochemistry, and reverse transcription polymerase chain reaction. RESULT(S) Apoptosis was detected in mid and late secretory endometria. L-arginine induced an increase in apoptosis in stroma (threefold), glands (eightfold), and surface epithelia (fourfold) in proliferative but not secretory endometria explants. Immunostaining of Bcl-2 was almost absent in the secretory endometria, whereas Bax increased in the stroma at the end of the menstrual cycle, coincident to the decrease in the bcl-2/bax mRNA relative ratio (P<.05) observed in secretory endometria. CONCLUSION(S) The induction of DNA fragmentation by L-arginine on proliferative endometria suggests that NO may be involved in the endometrial apoptotic process, whose control may be related predominantly to the changes of Bcl-2 expression.

Metformin augments the levels of molecules that regulate the expression of the insulin-dependent glucose transporter GLUT4 in the endometria of hyperinsulinemic PCOS patients

STUDY QUESTION Does treatment with the insulin sensitizer metformin modify the levels and activation of proteins related to the expression of the insulin-dependent glucose transporter (GLUT4), such as adenosine monophosphate-activated protein kinase (AMPK) and myocyte enhancer factor 2A (MEF2A), in endometria from hyperinsulinemic hyperandrogenemic polycystic ovary syndrome (PCOS h-Ins) patients? SUMMARY ANSWER In PCOS h-Ins patients, metformin increases endometrial levels of GLUT4 mRNA and protein levels by normalizing the quantity and activation of molecules that regulate GLUT4 expression to healthy values. These changes could improve endometrial metabolic function. WHAT IS ALREADY KNOWN PCOS is an endocrine-metabolic disorders closely associated with insulin resistance. In particular, the insulin signaling pathway is impaired in endometria from these patients and the concentration of GLUT4, as well as the molecules involved in its translocation to the cell surface, is decreased. However, there are limited data about the mechanisms that regulate the GLUT4 expression in the endometria and the effect of metformin on them. STUDY DESIGN, SIZE AND DURATION This is a case-control study in the setting of a research unit, approved by the Ethical Committees of our institution. The groups whose endometria were studied were PCOS h-Ins (n = 8); PCOS patients with hyperandrogenemia hyperinsulinemia taking only metformin for at least 3 months (PCOS-MTF, n = 8) and healthy fertile women at the time of hysterectomy because of benign pathology as controls (CE, n = 8). PARTICIPANTS/MATERIALS, SETTING, METHODS Steroids and sex hormone-binding globulin were measured and glucose and insulin levels were evaluated during an oral glucose tolerance test. Protein levels for αAMPK (catalytic subunit of AMPK), phosphorylated (p)-AMPKαThr(172) (activating phosphorylation site), MEF2A, p-MEF2AThr312 (activating phosphorylation site) and GLUT4 were assessed by western blot and immunohistochemistry. In addition, GLUT4 gene expression was evaluated by RT-PCR. MAIN RESULTS AND THE ROLE OF CHANCE We found significantly lower levels of MEF2A and p-MEF2AThr312 in PCOS h-Ins compared with CE endometria (P < 0.05). Also, we detected lower levels of p-AMPKαThr(172) in PCOS h-Ins endometria compared with the PCOS-MTF group (P < 0.05). The ratios of phospho-AMPK/total AMPK and phospho-MEF2A/total MEF2A were significantly increased in the PCOS-MTF compared with the PCOS h-Ins group (P < 0.05). The RT-PCR experiments showed lower levels of GLUT4 mRNA transcripts in PCOS h-Ins compared with PCOS-MTF-treated group (P < 0.05), the protein levels of GLUT4 were decreased in a similar way. LIMITATIONS, REASONS FOR CAUTION The limited number of patients included in this study who presented large clinical variability. Therefore, it would be necessary to recruit a greater number of patients to minimize our data dispersion in order to prove the clinical benefits of metformin described by others. WIDER IMPLICATIONS OF THE FINDINGS Since the insulin sensitizer metformin increases the expression of the GLUT4, it may improve endometrial physiology in PCOS patients and, therefore, promote better reproductive outcomes. These results suggest that in PCOS patients, metformin may act directly at the endometrial level and decrease insulin resistance condition by increasing the expression of GLUT4 and, in this way, indirectly restore endometrial function. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by Fondo Nacional de Desarrollo Científico y Tecnológico (grant number 1095127 to M.V.). None of the authors has any conflict of interest to declare.

Role of the transcriptional factors FOXO1 and PPARG on gene expression of SLC2A4 in endometrial tissue from women with polycystic ovary syndrome

Fifty to seventy percent of patients with polycystic ovary syndrome (PCOS) present hyperinsulinemia. On the other hand, reports indicate that forkhead box class O 1 (FOXO1) and peroxisome proliferator-activated receptor-gamma (PPARG) are involved in the insulin signaling pathway, regulating the gene expression of SLC2A4 (GLUT4). The negative effect of FOXO1 over PPARG transcription disappears when FOXO1 is phosphorylated (p-FOXO1) and excluded from the nucleus, whereas PPARG can suppress gene expression of SLC2A4. Scarce knowledge is available in endometrium of women with PCOS and hyperinsulinemia (PCOSE h-Ins) about the role of these factors. We aimed to evaluate whether the endocrine and metabolic status of PCOS modify the levels of gene and protein expression of FOXO1, PPARG, and SLC2A4 in the endometria from hyperinsulinemic PCOS women compared with controls. In endometria from control (CE, n=7) or PCOSE h-Ins (n=7), we determined the subcellular location and protein levels of p-FOXO1Ser319 and FOXO1/FOXO4 by immunohistochemistry and western blot respectively; gene and/or protein levels of PPARG and SLC2A4 were evaluated by RT-PCR and/or western blot. Cytoplasm location for FOXO1 and p-FOXO1Ser319 was immunodetected in both groups of endometria, showing significantly higher staining in PCOSE h-Ins for these proteins (P<0.05). In PCOSE h-Ins, gene and protein levels of PPARG were significantly higher than in CE, whereas SLC2A4 mRNA was decreased (P<0.05). In conclusion, the derepression of PPARG transcription by the high levels of p-FOXO1Ser319 could partially account for the lower levels of SLC2A4 found in PCOSE h-Ins, suggesting an alteration of the endometrial function in these patients.

Activities of steroid metabolic enzymes in secretory endometria from untreated women with Polycystic Ovary Syndrome.

Polycystic Ovary Syndrome (PCOS) is an endocrine-metabolic pathology related with infertility and recurrent miscarriage. We have previously shown that the endometrium of these patients can exhibit a potentially higher sensitivity to estrogen action, being estrogens important regulators of the cell cycle and tissue homeostasis. The effect of estrogens on tissues depends on their in situ availability, which is in part regulated by the activity of steroid metabolic enzymes within the tissues. Therefore, the objective of the present study was to analyze if the activity and/or expression of steroid metabolic enzymes in endometria from women with PCOS differ from controls. For this purpose, the activity of the enzymes was determined by using radiometric assays and the mRNA levels measured by semi-quantitative RT-PCR. Both assays were assessed in endometria obtained during mid secretory phase from control (CE, n=12) and PCOS women (PCOSE, n=11). For the statistical analyses, Mann-Whitney and Students t-tests were used to compare CE and PCOSE, considering a p value <0.05 significantly different. The results showed an increase in the sulfatase activity in PCOS respect to control endometria (200+/-28pmol/mg vs. 115+/-13pmol/mgproth; p<0.05), in agreement with the higher mRNA levels found for the enzyme in PCOSE. In addition, a PCOSE exhibited lower activity of sulfotransferase respect to the control group (50+/-21pmol/mg vs. 124+/-10pmol/mgproth; p<0.05), whereas a higher level of 17beta-hydroxysteroid dehydrogenase type 1mRNA was found in PCOSE compared with the control tissues (p<0.05). The activity of 17beta-hydroxysteroid dehydrogenase type 2 and the mRNA levels of sulfotransferase were similar in both groups; meanwhile, the expression of aromatase was undetectable. These data indicate that the sulfatase pathway could play an important role in the local production of estrogens in PCOSE from secretory phase. This potentially higher bioavailability of estrogens in endometria from PCOS women could influence the deregulation of tissue homeostasis that we have previously reported, and could partially explain the poor reproductive performance observed in this group of patients.

Tyrosine kinase A receptor (trkA): a potential marker in epithelial ovarian cancer.

OBJECTIVES To evaluate the role of trkA receptor as a potential tumor marker in serous epithelial ovarian cancer and its relationship with the angiogenic factors expression as vascular endothelial growth factor (VEGF) and nerve growth factor (NGF). Additionally, to examine whether NGF and VEGF secreted by epithelial ovarian cancer (EOC) explants and from epithelial ovarian cancer cell line (A2780) are involved in the process of angiogenesis, such as cellular proliferation, migration and differentiation of the human endothelial cell line (EA.hy926). METHODS The mRNA levels of VEGF, NGF and trkA receptors were measured using PCR in 60 ovarian samples. Cellular localization and semi-quantitative estimation of VEGF, NGF, total trkA and p-trkA was performed using IHC in epithelial cells. NGF, total trkA and p-trkA protein were also evaluated in endothelial cells from the same tissues. Human endothelial cell line EA.hy926 was cultured with conditioned media obtained from both EOC explants and from the A2780 cell line, with or without NGF stimulus. RESULTS Significantly higher levels of NGF, total trkA and p-trkA protein expressions were observed in epithelial and endothelial cells in poorly differentiated EOC versus normal ovary. Interestingly, the p-trkA receptor expression level showed the most significant difference and its presence was only found in borderline tumor and EOC samples indicating the importance of trkA receptor in EOC as a potential tumor marker. A significant increase in proliferation, migration and differentiation of EA.hy926 cells was observed with NGF, and this effect was significantly reverted when NGF was immuno-blocked and when a trkA inhibitor was used, showing that NGF is an important angiogenic factor in EOC by activating its trkA receptor. CONCLUSION These results indicate that p-trkA may be considered as a new potential tumor marker in EOC, and that NGF may also act as a direct angiogenic factor in EOC.

The effect of overweight and obesity on proliferation and activation of AKT and ERK in human endometria.

OBJECTIVE To examine whether overweight and obesity could lead to increased endometrial proliferation and activation of AKT and ERK1,2 in cycling premenopausal women. METHODS Endometrial and blood samples were obtained from women with normal endometrial histology, and allocated into three groups-normal-weight, overweight and obese-according to the subjects body mass index (BMI). Samples from obese patients with type-I endometrial cancer (EC) were included as a control. Cell proliferation was measured by immunohistochemical detection of Ki67 and phosphorylated histone H3 (p-H3). AKT and ERK1,2 activation was assessed by Western blot. Circulating steroids, leptin and insulin were measured by immunoassays. RESULTS In endometrial samples with normal histology, epithelial cell proliferation was higher in the overweight and obese groups versus the normal-weight set (P<0.05). Proliferation indexes were positively correlated with the subjects BMI and serum levels of estrogen, leptin and insulin (P<0.05). Increased phosphorylated AKT (pAKT) (1.6-fold) and ERK1,2 (pERK1,2) (8.7-fold) were observed in endometria from obese with respect to normal-weight subjects (P<0.05). Similarly, increased phosphorylation of AKT (0.7-fold) and ERK1,2 (2.3-fold) was detected in endometria from overweight as compared with the normal-weight group (P<0.05). In women with EC, we found a significant increase in endometrial proliferation, and in pAKT and pERK1,2 expression levels when compared to patients with normal endometrial histology. CONCLUSION These results show correlation between obesity (and overweight) and increased endometrial cell proliferation, and the activation of AKT and ERK1,2. These features could be related with the higher risk to develop type-I EC in overweight and obese women.

Involvement of Akt, Ras and cell cycle regulators in the potential development of endometrial hyperplasia in women with polycystic ovarian syndrome

OBJECTIVE To examine whether the abundance, localization, and/or activity of cell cycle regulators CDK2, Cyclin E, p27, and survival proteins AKT and Ras in PCOS-associated endometria (with and without hyperplasia) differ from non-PCOS endometria. METHODS The expression of CDK2, Cyclin E, p27, AKT and Ras was measured by immunohistochemistry and/or Western blot in 9 normal endometria (NE), 12 endometria from PCOS patients without endometrial hyperplasia (PCOSE), 7 endometria from PCOS women with endometrial hyperplasia (HPCOSE), and 9 endometria from patients with endometrial hyperplasia (HE). The activity of CDK2 was assessed by an in vitro kinase assay. RESULTS CDK2, Cyclin E and p27 proteins were expressed mainly in the endometrial epithelial cells of the studied groups. No change in the activity of CDK2 was observed in total extracts obtained from the tissue samples. However, the nuclear expression of CDK2 in epithelial cells was slightly elevated in PCOSE and significantly increased in HPCOSE when compared to NE. Higher expression of p27 was detected in the cytoplasm of epithelial cells of PCOSE and HPCOSE when compared to NE. Also, we found an increment in Ser473-AKT phosphorylation and an over-expression of the Ras oncogene in endometria of patients with PCOS. CONCLUSION The PCOS condition is associated with increased Ser473-AKT phosphorylation, elevated expression of Ras, increased cytoplasmic abundance of p27, and increased nuclear abundance of CDK2 in the endometrial epithelial cells. These biological events could potentially provide a chance for endometrial cells from PCOS patients to exit the controlled cell cycle and become hyperplastic at a later stage.

In situ estrogen metabolism in proliferative endometria from untreated women with polycystic ovarian syndrome with and without endometrial hyperplasia.

The aim of the present investigation was to study whether the endocrinological status of women bearing polycystic ovarian syndrome (PCOS) affects the endometrial in situ steroid metabolism. For this purpose, we evaluated the mRNA levels (RT-PCR), and the activity of steroid metabolic enzymes: P450 aromatase, steroid sulfatase (STS), estrogen sulfotransferase (EST) and 17beta-hydroxysteroid dehydrogenase (17beta-HSD) in 23 samples of normal endometria (CE), 18 PCOS endometria without treatment (PCOSE), 10 specimens from PCOS women with endometrial hyperplasia (HPCOSE), and 7 endometria from patients with endometrial hyperplasia not associated to PCOS (EH). The data showed lower levels of STS mRNA for PCOSE and HPCOSE (p<0.05, p<0.01, respectively) and of EST for HPCOSE and EH compared to control (p<0.05). However, higher levels for EST mRNA were obtained in PCOSE (p<0.05) versus CE. The mRNA and protein levels for P450 aromatase were undetectable in all analyzed endometria. The relationship between the activities of STS and EST was lower in PCOSE and HPCOSE (p<0.05) versus CE. The ratio between the mRNA from 17beta-HSD type 1/type 2 was higher in PCOSE (p<0.05), whereas, a diminution in the 17beta-HSD type 2 activity was observed in PCOSE (p<0.05). These results indicate that the activity of enzymes related to the steroid metabolism in analyzed PCOSE differ from those found in the CE. Consequently, PCOSE may present an in situ deregulation of the steroid metabolism.

Levels of Rabs and WAVE family proteins associated with translocation of GLUT4 to the cell surface in endometria from hyperinsulinemic PCOS women

BACKGROUND Polycystic ovary syndrome (PCOS) is an endocrine-metabolic disorder highly associated with insulin resistance and compensatory hyperinsulinemia. It is known that the insulin signaling pathway is impaired in endometria from PCOS hyperinsulinemic women, but no information is available about molecules associated with cell surface GLUT4 translocation. We therefore evaluated the protein levels of AS160 target molecules, Rab8A and Rab10, and the WAVE family proteins involved in the cortical-actin remodeling, Neural Wiskott-Aldrich Syndrome Protein (N-WASP) and WASP, in endometria from hyperinsulinemic PCOS women and controls. METHODS Protein levels were assessed by western blot, immunohistochemistry and immunofluorescence in proliferative (PE = 7) and secretory (SE = 7) phase endometria from control women and in endometria from hyperinsulinemic PCOS women (PCOS h-INS = 7). RESULTS Similar levels were detected for Rab10 in the three studied groups; however, Rab8A levels decreased in SE (P < 0.05) while higher levels were obtained in PCOSE h-INS compared with PE (P < 0.05). In the normal menstrual cycle, Neural Wiskott-Aldrich syndrome protein (N-WASP) and WASP levels were increased in SE versus PE (P < 0.05), but in PCOSE h-INS, the levels were diminished compared with PE (P < 0.05). CONCLUSIONS SE is characterized by protein expression changes associated with glucose uptake. In endometria from PCOS women with hyperinsulinemia, reduced levels of WAVE family proteins could compromise the cell surface GLUT4 exposure and the consequent glucose uptake in this tissue.