Ariel Fuentes

Augmented cell survival in eutopic endometrium from women with endometriosis: Expression of c-myc, TGF-beta1 and bax genes

BackgroundEndometriosis is a common gynaecological disorder characterized by the presence of endometrial tissue outside of the uterus. The fragments in normal menstruation are composed of necrotic and living cells, which do not survive in ectopic locations because of programmed cell death. The aim of this study was to evaluate if the balance between cell proliferation and apoptosis is changed in eutopic endometrium from women with endometriosis throughout the menstrual cycle by studying bax (pro-apoptotic), c-myc (regulator of cell cycle) and TGF-beta1 (involved in cell differentiation) genes.MethodsEutopic endometrium was obtained from: 30 women with endometriosis (32.8 +/- 5 years) and 34 fertile eumenorrheic women (36 +/- 5.3 years). We analyzed apoptosis (TUNEL: DNA fragmentation); cell proliferation (immunohistochemistry (IHC) for Ki67); c-myc, bax and TGF-beta1 mRNA abundance (RT-PCR) and TGF-beta1 protein (IHC) in endometrial explants.ResultsCell proliferation strongly decreased from proliferative to late secretory phases in glands, but not in stroma, in both endometria. Positive staining in glands and stroma from proliferative endometrium with endometriosis was 1.9- and 2.2-fold higher than control endometrium, respectively (p < 0.05). Abundance of c-myc mRNA was 65% higher in proliferative endometrium from endometriosis than normal tissue (p < 0.05). TGF-beta1 (mRNA and protein) augmented during mid secretory phase in normal endometrium, effect not observed in endometrium with endometriosis. In normal endometrium, the percentage of apoptotic epithelial and stromal cells increased more than 30-fold during late secretory phase. In contrast, in endometrium from endometriosis, not only this increase was not observed, besides bax mRNA decreased 63% versus normal endometrium (p < 0.05). At once, in early secretory phase, apoptotic stromal cells increased 10-fold with a concomitant augment of bax mRNA abundance (42%) in endometria from endometriosis (p < 0.05).ConclusionAn altered expression of c-myc, TGF-beta1 and bax was observed in eutopic endometrium from endometriosis, suggesting its participation in the regulation of cell survival in this disease. The augmented cell viability in eutopic endometrium from these patients as a consequence of a reduction in cell death by apoptosis, and also an increase in cell proliferation indicates that this condition may facilitate the invasive feature of the endometrium.

The human corpus luteum: life cycle and function in natural cycles.

OBJECTIVE To summarize recent advances in the understanding of the endocrine signaling pathways between the hypothalamus, pituitary, and human corpus luteum (CL); to examine the major paracrine and autocrine mechanisms and the key genes and proteins involved in CL development, function, and regression in natural cycles; to review the endocrine and molecular response of the midluteal phase CL to in vivo administration of human chorionic gonadotropin (hCG); and to describe the ultrasonographic and Doppler evaluation of the ovary and endometrium throughout the luteal phase. DESIGN Published data in the literature, including the basic and clinical research studies of the authors. SETTING University-affiliated hospital and research centers. PATIENT(S) None. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Clinical and molecular analysis of human CL function. RESULT(S) The endocrine function of the subpopulations of luteal cells is critical for the maintenance of CL function, including neovacularization and steroid hormones production. We consider the key genes and proteins that favor development of luteal structure and function throughout the menstrual cycle and in our model of hCG treatment resembling early pregnancy. CONCLUSION(S) These data indicate that the functional lifespan of the CL depends on paracrine and autocrine mechanisms. Therefore, the significance of the key genes and proteins that we analyze in lutein cells during CL development, function, demise, and rescue by hCG is likely to bring new therapeutic applications for the management of fertility defects and the control of fertility.

Recent cigarette smoking and assisted reproductive technologies outcome

OBJECTIVE To assess the association between recent cigarette smoking (CS) in female and male partners and assisted reproduction technology (ART) outcomes. DESIGN Cohort prospective study. SETTING University ART program in Chile. PATIENT(S) One hundred sixty-six couples seeking pregnancy through ART. INTERVENTION(S) Follicular fluid (FF) and serum cotinine concentrations were measured in female partners. Self-reported CS data were collected through personal interviews. MAIN OUTCOME MEASURE(S) The association between female recent smoking, assessed by FF and serum cotinine concentrations, and ART outcomes, such as number of ova retrieved and implantation rates, and the association between self-reported male recent smoking and live birth rates. RESULT(S) A significant age-adjusted association between increased FF cotinine level and decreased number of ova retrieved was found. The male partners smoking habit significantly decreased the live birth rate from 21.1% to 7.8%. Serum cotinine concentrations paralleled those of FF. CONCLUSION(S) The hypothesis of a detrimental effect of recent female smoking over implantation rates is rejected. However, recent male smoking is associated with significantly decreased live birth rates even after adjusting for confounders. Female recent smoking was significantly associated with decreased number of retrieved ova.

Nuclear factor κB pathway and interleukin-6 are affected in eutopic endometrium of women with endometriosis

In order to investigate the role of the nuclear factor kappaB (NFKB) pathway on gene expression in the eutopic endometrium in endometriosis, and in particular of interleukin-6 (IL6), we evaluated RELA, IkappaB kinase (CHUK), NFKBIA and IL6 expressions and NFKB DNA binding in eutopic endometrium from women with endometriosis. Eutopic endometrium was obtained from 37 women with endometriosis and 42 fertile women during laparoscopy. We analysed RELA, CHUK, NFKBIA and IL6 mRNA levels (RT-PCR); RELA, CHUK and NFKBIA proteins and p-NFKBIA/NFKBIA ratio (western blot); and NFKB binding (DNA shift assay) and IL6 concentration (ELISA) in endometrial explants. Our results indicate that mRNA and cytoplasmic proteins of RELA and CHUK exhibit constant levels in normal endometrium during the menstrual cycle. A dramatic increase (P<0.05) in NFKBIA mRNA expression, RELA nuclear presence and the mRNA and the protein of IL6 during late secretory phase was also observed in this tissue. By contrast, in eutopic endometrium from endometriosis patients, a decrease (P<0.05) in IL6 mRNA and protein (61%), NFKBIA mRNA (46%), p-NFKBIA/NFKBIA ratio (42%), RELA nuclear stromal (68%) and CHUK (48%) proteins were found exclusively during the late secretory phase compared with normal endometrium. In conclusion, the canonical activation of NFKB pathway is deregulated and may have reduced transcriptional function affecting NFKBIA and IL6 expression, genes related local proinflammatory processes. These molecular alterations observed during the late secretory phase in eutopic endometrium from endometriosis patients constitute a NFKB system dysfunction, suggesting that NFKB could be an important factor in endometriosis aetiology.

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.

Association between MMP1 and MMP9 activities and ICAM1 cleavage induced by tumor necrosis factor in stromal cell cultures from eutopic endometria of women with endometriosis

Endometriosis is a benign gynecological pathology in which immune system deregulation may play a role in its initiation and progression. In endometriotic lesions, intercellular adhesion molecule-1 (ICAM1) is released from the cell membrane by proteolytic cleavage of its extracellular domain, a process that coincides with increased expression and proteolytic activity of metalloproteinases such as MMP1 and MMP9. The objective of our study was to investigate the association between MMP1 and MMP9 activities and ICAM1 cleavage mediated by tumor necrosis factor (TNF) in eutopic endometrial stromal cells from women with and without (control) endometriosis during culture. The RNA was evaluated by RT-PCR, and the protein was determined by western blot (ICAM1, MMP1), casein or gelatin zymographies (secreted active MMP1 or MMP9 respectively), ELISA (soluble ICAM1 (sICAM1)), and fluorescence assay (secreted active MMP1). Under basal conditions, proMMP9 dimer and MMP9 were higher in endometriosis cell cultures. In stromal cultures derived from control women and those with endometriosis, TNF augmented the intracellular proMMP1 (1.2-fold in control stromal cells) and ICAM1 (1.4- and 1.9-fold), greatly increased MMP1 and proMMP9 levels, and the sICAM1 concentration (2.3- and 4.3-fold) in their media compared with basal levels. The combination of TNF and MMP9 increased the sICAM1 concentration 14-fold in the endometriosis cell media, whereas GM6001 inhibited the stimulatory effect of TNF in both cell cultures. The deregulation of MMP9, and the TNF participation in the MMP1 and proMMP9 secretions, in the MMP9 expression and in the expression and cleavage of ICAM1 may contribute to the pathophysiology of this disease.

Prospective Identification of Pregnant Women Drinking Four or More Standard Drinks (≥ 48 g) of Alcohol Per Day

We aimed to identify drinking rates in a prospectively identified cohort of pregnant women, and subsequently, to identify the drinkers of 48 g or more alcohol/day among them, by using complementary methods for verifying self-reported drinking habits. A research team of social workers and health professionals at the Maipú Clinic, located in a lower middle class neighborhood of Santiago, Chile, conducted interviews of women attending a prenatal clinic between August 1995 and July 2000. Women whose interview responses met predefined criteria (identified in the text) were further evaluated by home visits. We interviewed 9,628 of 10,917 (88%) women receiving prenatal care. By initial interview, 42.6% of women reported no drinking, 57.4% some alcohol consumption, and 3.7% consuming at least one standard drink (15 mL of absolute alcohol) per day. Of the 887 women who had home visits, 101 were identified as consuming on average at least 4 drinks/day (48 g). To determine the best home visit questionnaire items for identifying those drinking at least 4 drinks per day, 48 women who openly admitted drinking this amount were compared with 786 women who were not considered drinkers after the home visit. The 48 self-reported 48 g/day drinkers were significantly more likely to get tipsy when drinking before (p = 0.01) or during (p < 0.0001) pregnancy, to have started drinking at a younger age (p = 0.007), or to exhibit signs of low self-esteem (p < 0.0001), sleep or appetite problems (p < 0.0001), bad interpersonal relationships (p < 0.0001) or having family members with fetal alcohol syndrome features (p < 0.009). In conclusion, using complementary methods of alcohol misuse ascertainment during pregnancy, we found that at least 1% of pregnant women in a Santiago, Chile, clinic population were drinking at levels that are clearly dangerous to the fetus (48 g/day or more). We identified specific interview questions that may help screen for alcohol use of 48 g/day or more in pregnant women.

Decreased anti-Müllerian hormone concentration in follicular fluid of female smokers undergoing artificial reproductive techniques.

BACKGROUND Several reports indicate that women who smoke have an increased risk of failure to conceive compared with their non-smoker counterparts. Here, we assessed the effect of smoking during the Assisted Reproduction Therapy (ART) on a potential marker of ovarian reserve, anti-müllerian hormone (AMH) in the follicular fluid (FF). MATERIALS AND METHODS This was a cohort prospective study to assess the association between cigarette smoking and AMH concentrations in FF in fifty-six women undergoing their first ART cycle. Self-reported smoking status over time was also collected through personal interview. The main outcome measured was the association between current smoking and AMH concentrations in FF. Smoking status was assessed by FF cotinine concentrations. Analysis of covariance was performed to test statistical interaction between the main outcome and confounders. RESULTS The mean concentration of AMH in follicular fluid was significantly decreased among smokers (1.02±0.14 vs. 1.74±0.15, P<0.05). No statistical interaction was found between this difference in AMH concentrations and confounders like age and BMI. Thus, our data support the idea that AMH is decreased in active smokers across the fertile age. CONCLUSIONS The hypothesis of decreased AMH concentration in follicular fluid in female smokers was confirmed. The mechanisms through which cigarette smoking induces this fall in AMH are unknown and additional research is needed to improve our comprehension of the negative impact of smoking on ART outcomes.

P450Arom induction in isolated control endometrial cells by peritoneal fluid from women with endometriosis

OBJECTIVE To study the effect of peritoneal fluid from women with (PF-E) and without (PF-C) endometriosis on P(450)Arom expression in endometrial cells. DESIGN Experimental study. SETTING University research unit. PATIENT(S) Forty women of reproductive age with (n = 22) or without (control; n = 18) endometriosis. INTERVENTION(S) Peritoneal fluid and eutopic endometrial samples were obtained during surgery from women with (n = 13 and 9, respectively) and without (n = 4 and 14, respectively) endometriosis. MAIN OUTCOME MEASURE(S) Expression study for P(450)Arom, steroid factor 1 (SF-1), chicken ovalbumin upstream transcription factor I (COUP-TFI), and COUP-TFII messenger RNA (reverse transcriptase-polymerase chain reacion) and/or protein (immunoblot) in isolated endometrial epithelial cells transfected or not with expression vector containing SF-1, COUP-TFI, or COUP-TFII complementary DNAs. RESULT(S) Basal messenger RNA and/or protein expression of P(450)Arom and SF-1 were augmented in endometriosis, and that of COUP-TF was diminished. In control cells, (Bu)(2)cAMP and PF-E increased P(450)Arom and SF-1 expression (but not COUP-TF expression) in a dose-dependent way, an effect not observed with PF-C, adsorbed PF-E, or 10(-5) M indomethacin. Transfected cells confirmed these results. Any treatments modified the studied molecules in endometriosis cells. CONCLUSION(S) These data indicate that molecules contained in PF-E favor an estrogenic microenvironment, suggesting a role in the etiopathogenesis of endometriosis enabling the survival, maintenance, and growth of endometrial implants in the ectopic locations.

Assessment of diagnostic competence of plasmatic androgens on polycystic ovary syndrome based on receiver operator characteristic curves

Objective. This study was designed to assess the diagnostic potency of different androgens in hyperandrogenaemia criterion on polycystic ovary syndrome (PCOS) based on receiver operator characteristic (ROC) curves analysis. Methods. We evaluated 55 PCOS patients and 27 healthy fertile women (control). Androgen evaluation included bio-available testosterone (BAT) by ammonium sulphate precipitation, Free Testosterone Index (FTI), androstenedione (A), total testosterone and dehydroepiandrosterone sulphate (DHEA-S). Results. The androgen tests with the best diagnostic capacities were FTI and BAT. Although T and A had similar diagnostic potencies, A detected 5% of PCOS patients that could not be recognised by FTI, BAT (%), or T. The association of FTI, BAT (%) and A identified 96.36% of the hyperandrogenaemic patients. DHEA-S showed a wide dispersion of values and therefore poor discriminatory competence. Discussion. This study suggests that routine androgen evaluation in PCOS should include FTI, BAT and A to avoid misdiagnosis. ROC curve analysis of these tests on patients with the complete spectrum of PCOS phenotypes is needed to confirm these results.