Leptin protects placental cells from apoptosis induced by acidic stress.

Development of the human placenta is critical for a successful pregnancy. The placenta allows the exchange of oxygen and carbon dioxide and is crucial to manage acid-base balance within a narrow pH. It is known that low pH levels are a risk of apoptosis in several tissues. However, there has been little discussion about the effect of acidic stress in the placenta. Leptin is produced by the placenta with a trophic autocrine effect. Previous results of our group have demonstrated that leptin prevents apoptosis of trophoblast cells under different stress conditions such as serum deprivation and hyperthermia. The purpose of the present work is to evaluate acidic stress consequences in trophoblast explant survival and to determine leptin action in these conditions. For this objective, term human trophoblast explants were cultured at physiological pH (pH 7.4) and at acidic pH (pH 6.8) in the presence or absence of leptin. Western blot assays were performed to study the abundance of active caspase-3 and the p89 fragment of PARP-1. Pro-apoptotic and pro-survival members of Bcl-2 family, as Bax, t-Bid, and Bcl-2, were studied. Moreover, p53 pathway was also evaluated including Mdm-2, the main p53 regulator. Active caspase-3 and cleaved PARP-1 abundances were increased at low extracellular pH. Moreover, t-Bid levels were also augmented as well as p53 expression and phosphorylation on S46. Leptin treatment prevents the consequences of acidosis, decreasing p53 expression and increasing Mdm-2 expression. In summary, this work demonstrated for first time that low pH induces apoptosis of human trophoblast explants involving apoptotic intrinsic pathway, and leptin impairs this effect.

Effectiveness of a continuous-use ring-shaped vaginal pessary without support for advanced pelvic organ prolapse in postmenopausal women.

INTRODUCTION AND HYPOTHESIS: There is considerable variation in the clinical management of pessaries. This study was aimed at exploring the efficacy of the continuous use of ring pessaries without support for the treatment of advanced pelvic organ prolapse (POP) in nonhysterectomized postmenopausal women. METHODS: We conducted this prospective study of fitted pessaries between January 2013 and June 2015 in the Department of Obstetrics and Gynecology at Macarena Hospital, Seville University, Spain. A total of 171 nonhysterectomized postmenopausal patients with symptomatic POP (stages III and IV) were counseled for two treatment options: either surgery or vaginal pessary. A total of 94 patients who agreed to use the vaginal ring pessary were included. A successful fitting was defined as the continued use of the device until the end of the study (a median 27-month follow-up). The data were analyzed with continuity correction tests, Mann-Whitney U tests, and Fisher's exact test. RESULTS: Pessary use was continued by 80.8% of the patients. Most discontinuations (50.0%) occurred within the first week after device insertion. The adverse events rate was 31.6%, and all adverse events were Clavien-Dindo grade I. The complications were extrusion of the pessary (18.4%), bleeding or excoriation (10.5%), and pain or vaginal discharge (2.6%). No major complications occurred. CONCLUSIONS: The ring pessary without support was successfully fitted in patients with advanced POP, resulting in a high success rate. There were few side effects and complications associated with continuous use of this pessary without periodic removal or replacement.

Trends in contraception use in Spanish adolescents and young adults (15 to 24 years) between 2002 and 2008.

OBJECTIVE: To assess changes in the use of contraceptive methods, and induced abortion rates, in Spanish adolescents and young adults aged 15 to 24 years, between 2002 and 2008. STUDY DESIGN: Representative samples of Spanish men and women aged 15 to 24 years were surveyed in 2002 (N = 1826) and 2008 (N = 2000). RESULTS: The rate of use of contraceptive methods increased from 61% in men and 60% in women in 2002 to 80% and 75%, respectively, in 2008. The most commonly used method was the condom (51% in 2002 and 71% in 2008), followed by the contraceptive pill (18% in 2002 and 18% in 2008). None of the adolescents and young adults surveyed used natural methods or the diaphragm, or had undergone sterilisation. The induced abortion rate increased from 9.28 to 13.48 per 1000 women in the group aged between 15 and 19 years, and from 14.37 to 21.05 per 1000 women in the group aged 20 to 24 years. CONCLUSION: Despite an increase in the use of effective birth control methods, the rates of abortion rose during the study period, which may indicate that compliance with the use of condoms is inadequate. There is an urgent need to develop educational campaigns or to design specific policies addressing contraception-related issues for young people.

Contraceptive practices of women requesting induced abortion in Spain: a cross-sectional multicentre study.

OBJECTIVES: To collect information on (i) contraceptive methods used immediately before unwanted pregnancy, and (ii) planned contraception following induced abortion among Spanish residents. METHODS: Eight centres officially accredited to perform abortions participated in a cross-sectional study. The study population included 2475 women requesting induced abortion between 1 January and 31 March 2007. RESULTS: Contraceptive methods used before unwanted pregnancy were condoms in 40% of the cases, combined hormonal contraception in 14%, and other methods (mainly natural methods and withdrawal) in 10%. Thirty-six percent of women did not use any contraceptive method. Failure of the method due to incorrect use was reported by 77% of those using condoms and by 84% of those using hormonal contraception. Only 23% of women planned to use a contraceptive method after abortion. Almost half of the women were immigrants. CONCLUSION: Despite the use of effective contraceptive methods, the majority of women requesting induced abortion in Spain became pregnant during the use of these methods. These data reflect a lack of compliance. More education programmes are needed to improve compliance rates. The long-acting reversible contraceptive methods could constitute an appropriate approach.

Development and validation of the SEC-QOL questionnaire in women using contraceptive methods.

OBJECTIVES: Develop and validate a Spanish society of contraception quality-of-life (SEC-QOL) questionnaire to assess the impact of contraceptive methods on the health-related quality of life (HRQOL) of women. METHODS: SEC-QOL was developed following a standardized procedure including review of the literature, interviews with contraception users, and the administration of a pilot questionnaire to 187 women. SEC-QOL consists of 19 items and includes five dimensions. To validate the questionnaire, a multicenter, observational, prospective study was conducted in Spain. The following three study groups were defined: group A (n = 129) comprised women using effective contraceptive methods; group B (n = 251), comprised women about to start using an effective method; and group C (n = 73) comprised women using no or poorly effective contraception. All women attended baseline and final visits (4 ± 1 months). Participants completed the SEC-QOL, psychological well-being index, EuroQol five-dimensional questionnaire, and perceived health state questionnaires. RESULTS: At baseline, women from group A had a better HRQOL in all SEC-QOL dimensions, except for breast symptoms. Heavier menstrual bleeding, more androgenic and breast symptoms, menstrual pain, and not using hormonal contraceptive methods were associated with lower HRQOL. SEC-QOL scores showed moderate correlations to psychological well-being index and slightly lower correlation to EuroQol five-dimensional questionnaire scores. At follow-up, HRQOL had improved in all groups; most markedly in group B, which obtained an average effect size of 0.59. The minimum important difference was established as a 3.4-point change in the global SEC-QOL score. SEC-QOL obtained a Cronbach's α of 0.88 and an intraclass correlation coefficient of 0.82. CONCLUSIONS: SEC-QOL is a valid, reliable, and sensitive to change questionnaire for use in daily clinical practice and future research projects on contraception.

Human amniotic membrane conditioned medium inhibits proliferation and modulates related microRNAs expression in hepatocarcinoma cells.

The placental stem cells have called the focus of attention for their therapeutic potential to treat different diseases, including cancer. There is plenty evidence about the antiproliferative, antiangiogenic and proapoptotic properties of the amniotic membrane. Liver cancer is the fifth cause of cancer in the world, with a poor prognosis and survival. Alternative treatments to radio- or chemotherapy have been searched. In this work we aimed to study the antiproliferative properties of the human amniotic membrane conditioned medium (AM-CM) in hepatocarcinoma cells. In addition, we have analyzed the regulation of pro and antiOncomiRs expression involved in hepatocarcinoma physiology. We have determined by 3H-thymidine incorporation assay that AM-CM inhibits DNA synthesis in HepG2 cells after 72 h of treatment. AM-CM pure or diluted at 50% and 25% also diminished HepG2 and HuH-7 cells viability and cell number. Furthermore, AM-CM induced cell cycle arrest in G2/M. When proliferation mechanisms were analyzed we found that AM-CM reduced the expression of both Cyclin D1 mRNA and protein. Nuclear expression of Ki-67 was also reduced. We observed that this CM was able to promote the expression of p53 and p21 mRNA and proteins, leading to cell growth arrest. Moreover, AM-CM induced an increase in nuclear p21 localization, observed by immunofluorescence. As p53 levels were increased, Mdm-2 expression was downregulated. Interestingly, HepG2 and HuH-7 cells treatment with AM-CM during 24 and 72 h produced an upregulation of antiOncomiRs 15a and 210, and a downregulation of proOncomiRs 206 and 145. We provide new evidence about the promising novel applications of human amniotic membrane in liver cancer.

Proliferation and survival of human amniotic epithelial cells during their hepatic differentiation.

Stem cells derived from placental tissues are an attractive source of cells for regenerative medicine. Amniotic epithelial cells isolated from human amnion (hAECs) have desirable and competitive characteristics that make them stand out between other stem cells. They have the ability to differentiate toward all three germ layers, they are not tumorigenic and they have immunosuppressive properties. Although liver transplantation is the best way to treat acute and chronic hepatic failure patients, there are several obstacles. Recently, stem cells have been spotlighted as alternative source of hepatocytes because of their potential for hepatogenic differentiation. In this work, we aimed to study the proliferation and survival of the hAECs during their hepatic differentiation. We have also analyzed the changes in pluripotency and hepatic markers. We differentiated amniotic cells applying a specific hepatic differentiation (HD) protocol. We determined by qRT-PCR that hAECs express significant levels of SOX-2, OCT-4 and NANOG during at least 15 days in culture and these pluripotent markers diminish during HD. SSEA-4 expression was reduced during HD, measured by immunofluorescence. Morphological characteristics became more similar to hepatic ones in differentiated cells and representative hepatic markers significantly augmented their expression, measured by qRT-PCR and Western blot. Cells achieved a differentiation efficiency of 75%. We observed that HD induced proliferation and promoted survival of hAECs, during 30 days in culture, evaluated by 3H-thymidine incorporation and MTT assay. HD also promoted changes in hAECs cell cycle. Cyclin D1 expression increased, while p21 and p53 levels were reduced. Immunofluorescence analysis showed that Ki-67 expression was upregulated during HD. Finally, ERK 1/2 phosphorylation, which is intimately linked to proliferation and cell survival, augmented during all HD process and the inhibition of this signaling pathway affected not only proliferation but also differentiation. Our results suggest that HD promotes proliferation and survival of hAECs, providing important evidence about the mechanisms governing their hepatic differentiation. We bring new knowledge concerning some of the optimal transplantation conditions for these hepatic like cells.

Sp1 transcription factor is a modulator of estradiol leptin induction in placental cells.

INTRODUCTION: Pleiotropic effects of leptin have been identified in reproduction and pregnancy, particularly in the placenta, where it functions as an autocrine hormone. The synthesis of leptin in normal trophoblastic cells is regulated by different endogenous biochemical agents, but the regulation of placental leptin expression is still poorly understood. We have previously reported that 17ß-estradiol up-regulates placental leptin expression through genomic and nongenomic mechanisms. METHODS: To improve the understanding of estrogen receptor mechanisms in regulating leptin gene expression, we examined Sp1 transcription factor effect on estradiol leptin induction in human BeWo cell line. RESULTS: We demonstrated that Sp1 induces leptin expression determined by qRT-PCR, Western blot and transient transfection experiments. We also found that estradiol induction effect on leptin expression is enhanced by the over expression of Sp1 factor. Moreover, estradiol effect was not evidenced when Sp1 binding site on leptin promoter is mutated, suggesting that estradiol action is dependent on Sp1. On the other hand we showed data that demonstrate that Sp1 induction of leptin expression is insensitive to the antiestrogen ICI 182 780. By over expression experiments, we have also found that Sp1 effect on leptin expression could be mediated by estrogen receptor alpha. Supporting this idea, the downregulation of estrogen receptor alpha level through a specific siRNA, abolished Sp1 effect on leptin expression. DISCUSSION: Taken together all these evidences suggest a cooperative behavior between estrogen receptor alpha and Sp1 transcription factors to induce leptin transcription.

The alternative Epac/cAMP pathway and the MAPK pathway mediate hCG induction of leptin in placental cells.

Pleiotropic effects of leptin have been identified in reproduction and pregnancy, particularly in the placenta, where it works as an autocrine hormone. In this work, we demonstrated that human chorionic gonadotropin (hCG) added to JEG-3 cell line or to placental explants induces endogenous leptin expression. We also found that hCG increased cAMP intracellular levels in BeWo cells in a dose-dependent manner, stimulated cAMP response element (CRE) activity and the cotransfection with an expression plasmid of a dominant negative mutant of CREB caused a significant inhibition of hCG stimulation of leptin promoter activity. These results demonstrate that hCG indeed activates cAMP/PKA pathway, and that this pathway is involved in leptin expression. Nevertheless, we found leptin induction by hCG is dependent on cAMP levels. Treatment with (Bu)(2)cAMP in combination with low and non stimulatory hCG concentrations led to an increase in leptin expression, whereas stimulatory concentrations showed the opposite effect. We found that specific PKA inhibition by H89 caused a significant increase of hCG leptin induction, suggesting that probably high cAMP levels might inhibit hCG effect. It was found that hCG enhancement of leptin mRNA expression involved the MAPK pathway. In this work, we demonstrated that hCG leptin induction through the MAPK signaling pathway is inhibited by PKA. We observed that ERK1/2 phosphorylation increased when hCG treatment was combined with H89. In view of these results, the involvement of the alternative cAMP/Epac signaling pathway was studied. We observed that a cAMP analogue that specifically activates Epac (CPT-OMe) stimulated leptin expression by hCG. In addition, the overexpression of Epac and Rap1 proteins increased leptin promoter activity and enhanced hCG. In conclusion, we provide evidence suggesting that hCG induction of leptin gene expression in placenta is mediated not only by activation of the MAPK signaling pathway but also by the alternative cAMP/Epac signaling pathway.

Attitudes of Spanish women toward premenstrual symptoms, premenstrual syndrome and premenstrual dysphoric disorder: results of a nationwide survey.

OBJECTIVE: To assess the attitudes of women with premenstrual symptoms in relation to their perception of complaints and request for medical advice. STUDY DESIGN: Cross-sectional study of a representative cohort of 2018 Spanish women aged 15-49 years. Participants were personally interviewed at home and completed the premenstrual symptoms screening tool. RESULTS: A total of 1554 women (73.7%) complained of some of the premenstrual symptoms during the last 12 menstrual cycles. The prevalence of moderate or severe premenstrual syndrome (PMS) was 8.9% and the prevalence of premenstrual dysphoric disorder (PMDD) 1.1%. Only 291 (18.7%) women had sought medical advice. The main reason given by 90.6% of symptomatic women for not seeking medical consultation was to consider that symptoms were normal. A total of 175 (60.1%) women received pharmacological treatment (hormonal contraceptives in 95% followed by analgesics in 50% and anti-inflammatory agents in 44%), 20% were not treated because physicians considered that symptoms were not important and would disappear spontaneously, and 12% received only advice to change life style. CONCLUSION: Women suffering from PMS or PMDD do not usually seek medical advice and among those seeking medical care, in many cases, an adequate response to their demands is not obtained.

Prevalence of premenstrual syndrome and premenstrual dysphoric disorder in a representative cohort of Spanish women of fertile age.

OBJECTIVE: To assess the prevalence of premenstrual symptoms, premenstrual syndrome and premenstrual dysphoric disorder in a cohort of women of fertile age representative of the general Spanish population. STUDY DESIGN: During the period between November 27th and December 22nd, 2008, a cross-sectional nationwide survey was conducted among a cohort of Spanish women aged between 15 and 49 years. Participants were personally interviewed at home and completed the premenstrual symptoms screening tool. RESULTS: Of the 2108 participants, 1554 women (73.7%) complained of some of the premenstrual symptoms during the last 12 menstrual cycles. A total of 1415 (91%) women presented isolated symptoms and 139 (8.9%) a moderate/severe premenstrual syndrome. Twenty-four (1.1%) women fulfilled criteria for a diagnosis of premenstrual dysphoric disorder. CONCLUSION: The prevalence of premenstrual symptoms (73.7%) and premenstrual dysphoric disorder (1.1%) in Spanish women aged between 15 and 49 years is similar to that reported in other Western countries. Only women with moderate or severe premenstrual syndrome or premenstrual dysphoric disorder, however, had daily life activities significantly impaired by premenstrual symptoms.

Trends in the use of contraceptive methods and voluntary interruption of pregnancy in the Spanish population during 1997-2007.

BACKGROUND: This study was designed to acquire information about the use of contraceptive methods in order to reduce the number of elective abortions. STUDY DESIGN: Since 1997, representative samples of Spanish women of childbearing potential (15-49 years) have been surveyed by the Daphne Team every 2 years to gather data of contraceptive methods used. RESULTS: During the study period, 1997 to 2007, the overall use of contraceptive methods increased from 49.1% to 79.9%. The most commonly used method was the condom (an increase from 21% to 38.8%), followed by the pill (an increase from 14.2% to 20.3%). Female sterilization and IUDs decreased slightly and were used by less than 5% of women in 2007. The elective abortion rate increased from 5.52 to 11.49 per 1000 women. CONCLUSIONS: The factors responsible for the increased rate of elective abortion need further investigation.

Is the vagina an adequate route for the administration of hormonal contraceptives?

The vaginal route of drug administration provides women with a valid alternative to more conventional methods of contraception. Drugs absorbed in the upper part of the vagina can bypass the liver and, if metabolized, are subject to a reduced hepatic first-pass effect. Current vaginally-administered contraceptive formulations deliver similar doses of gestagens to those provided by oral methods but release lower amounts of oestrogens. This results in a systemic exposure to gestagens similar to that achieved via other routes, thereby maintaining contraceptive efficacy while limiting systemic, but not uterine, exposure to oestrogen. In this way, the probability of systemic oestrogen-related adverse effects are theoretically reduced without compromising cycle control. In addition, the fact that the effects of a contraceptive ring last a complete cycle makes it more user-friendly than other methods and results in better patient compliance. The present review will explain in detail the specificities of this route of delivery of hormonal contraception and will compare it to more classic forms of contraception received via the oral (pill), intramuscular (injected), transdermic (patch) and subcutaneous (implants) routes of administration.