Expression, localization and control of activin A release from human umbilical vein endothelial cells.

Activin-A is a member of the TGFß superfamily found in maternal and umbilical cord blood throughout gestation. We investigated whether human umbilical vein endothelial cells (HUVEC) express activin-A in vivo and tested the effects of vasoactive (endothelin-1), pro-inflammatory (interferon-γ, interleukin-8) and anti-inflammatory (dexamethasone, urocortin) factors on activin-A release by isolated HUVEC in vitro. Activin ßA subunit protein and mRNA were strongly localized in the endothelial cells of umbilical veins and were also detectable in scattered cells of the cord connective tissue. Dimeric activin-A was detected in the HUVEC culture medium at picomolar concentrations. Activin-A release by HUVEC decreased after cell incubation with urocortin (p < 0.01), whereas no effect was observed with interleukin-8, interferon-γ, endothelin-1 or dexamethasone. In summary, activin-A is present in the human umbilical vein endothelium in vivo and is produced and released by isolated HUVEC. Activin-A secretion is inhibited in vitro by urocortin, a neuropeptide with predominantly anti-inflammatory action.

Serum antimüllerian hormone measurements with second generation assay at two distinct menstrual cycle phases for prediction of cycle cancellation, pregnancy and live birth after in vitro fertilization.

PURPOSE: This study investigated the usefulness of serum antimüllerian hormone (AMH) measurements at two distinct menstrual cycle phases to predict in vitro fertilization (IVF) outcomes. METHODS: This was a prospective observational study enrolling 135 consecutive patients referred for conventional IVF or ICSI in a university hospital. Blood samples were obtained for serum AMH measurements on days 3 and 18-20, while transvaginal ultrasound was performed for antral follicle count (AFC) at day 3 of the menstrual cycle immediately before treatment. AMH was measured with the new Beckman Coulter Generation II (GenII) assay. The main outcome measures were cycle cancellation due to poor ovarian response, clinical pregnancy, and live birth. RESULTS: There was a strong correlation between AMH levels measured at day 3 and day 18-20 of the menstrual cycle (r = 0.837; P < 0.0001). Day 18-20 serum AMH was comparable to day 3 serum AMH and AFC for the prediction of cycle cancellation (areas under the ROC curve were 0.84 for day 3 AMH, 0.89 for day 18-20 AMH, and 0.80 for AFC). Day 18-20 AMH had a modest predictive value for pregnancy or live birth (area under ROC curve 0.71 for both), which was comparable to that of day 3 AMH; however, AFC had no predictive value for these outcomes. CONCLUSIONS: Day 18-20 AMH was comparable to day 3 AMH for the prediction of cycle cancellation, clinical pregnancy, and live birth after IVF. Both AMH measurements were accurate for the prediction of cancellation but were significantly less useful for the prediction of pregnancy or live birth.

Urocortin 2 and urocortin 3 in endometriosis: evidence for a possible role in inflammatory response.

Urocortin 2 (Ucn 2) and urocortin 3 (Ucn 3) are neuropeptides expressed by human endometrium. This study evaluated (i) the expression of Ucn 2 and Ucn 3 mRNA in endometriotic lesions and in endometrium of women with endometriosis; (ii) the effect of Ucn 2 and Ucn 3 on cytokines secretion from cultured endometrial stromal cells. Endometriotic tissue was collected from endometrioma (n=39); endometrial specimens were obtained from women with (n=39) and without (n=41) endometriosis throughout menstrual cycle. Tissue specimens were analysed for Ucn 2 and Ucn 3 mRNA expression and peptide localization; the effects of Ucn 2 or Ucn 3 on tumour necrosis factor (TNF-α) and interleukin (IL-4) secretion from cultured endometrial stromal cells was studied. Ucn 2 and Ucn 3 mRNA expression and localization were assessed by RT-PCR and by immuohistochemistry, respectively; cytokines secretion were measured by ELISA. Results showed that endometriotic tissue expressed both Ucn 2 and Ucn 3, with Ucn 3 expression higher in ectopic than in eutopic endometrium. Endometrial Ucn 2 mRNA expression in controls showed peak values at early proliferative phase, while in endometriotic patients low expression and no significant changes throughout menstrual cycle were observed. Endometrial Ucn 3 mRNA expression was highest in late secretory phase in controls, while in endometriotic patients low levels and no menstrual-cycle-related changes were found. When added to cultured endometrial cell cultures, Ucn 2 significantly increased TNF-α (P<0.01) and IL-4 (P<0.001), while Ucn 3 induced an increase of IL-4 secretion (P<0.01). In conclusion, endometriotic tissue expressed and localized Ucn 2 and Ucn 3; patients with endometriosis showed Ucn 2 and Ucn 3 mRNA expression in eutopic endometrium lower than in control group, with no endometrial cycle-related changes. Ucn 2 and Ucn 3-modulated TNF-α and IL-4 secretion from culture endometrial cells. These data suggest a possible involvement of Ucn 2 and Ucn 3 in the mechanisms of endometriosis.

Differential expression of follistatin and FLRG in human breast proliferative disorders.

BACKGROUND: Activins are growth factors acting on cell growth and differentiation. Activins are expressed in high grade breast tumors and they display an antiproliferative effect inducing G0/G1 cell cycle arrest in breast cancer cell lines. Follistatin and follistatin- related gene (FLRG) bind and neutralize activins. In order to establish if these activin binding proteins are involved in breast tumor progression, the present study evaluated follistatin and FLRG pattern of mRNA and protein expression in normal human breast tissue and in different breast proliferative diseases. METHODS: Paraffin embedded specimens of normal breast (NB - n = 8); florid hyperplasia without atypia (FH - n = 17); fibroadenoma (FIB - n = 17); ductal carcinoma in situ (DCIS - n = 10) and infiltrating ductal carcinoma (IDC - n = 15) were processed for follistatin and FLRG immunohistochemistry and in situ hybridization. The area and intensity of chromogen epithelial and stromal staining were analyzed semi-quantitatively. RESULTS: Follistatin and FLRG were expressed both in normal tissue and in all the breast diseases investigated. Follistatin staining was detected in the epithelial cytoplasm and nucleus in normal, benign and malignant breast tissue, with a stronger staining intensity in the peri-alveolar stromal cells of FIB at both mRNA and protein levels. Conversely, FLRG area and intensity of mRNA and protein staining were higher both in the cytoplasm and in the nucleus of IDC epithelial cells when compared to NB, while no significant changes in the stromal intensity were observed in all the proliferative diseases analyzed. CONCLUSION: The present findings suggest a role for follistatin in breast benign disease, particularly in FIB, where its expression was increased in stromal cells. The up regulation of FLRG in IDC suggests a role for this protein in the progression of breast malignancy. As activin displays an anti-proliferative effect in human mammary cells, the present findings indicate that an increased FST and FLRG expression in breast proliferative diseases might counteract the anti-proliferative effects of activin in human breast cancer.

Effect of a combination of ethinylestradiol 30 microg and drospirenone 3 mg on tolerance, cycle control, general well-being and fluid-related symptoms in women with premenstrual disorders requesting contraception.

PURPOSE: Positive effects on premenstrual symptoms have been observed with low-dose oral contraceptives. Drospirenone is a synthetic progestogen with antiandrogenic and antimineralocorticoid effects. This open-label, multicenter study evaluated the effects of a combination of ethinylestradiol 30 microg and drospirenone 3 mg on safety, cycle control, general well-being and fluid-related symptoms in women with premenstrual disorders requesting contraception. MATERIALS AND METHODS: A total of 241 healthy volunteers with symptoms of premenstrual disorder was enrolled in the study. Of the final sample, 203 completed the six-cycle treatment and were included in the efficacy analysis whereas 236 were included in the tolerability analysis. The subjects recruited to the study were required to fill up the Psychological General Well-Being Index (PGWBI). RESULTS: There was no significant change in body weight or blood pressure throughout the treatment. Adverse events reported by patients during treatment consisted of those already known to be associated with oral contraceptive use. PGWBI scores were significantly higher after six cycles of treatment compared with baseline values (p<.0001). A total of 198 (84.2%) subjects reported a great improvement in premenstrual symptoms. CONCLUSIONS: The results of this study confirm that oral use of a combination of ethinylestradiol 30 microg and drospirenone 3 mg provides good cycle control, is well tolerated and has a positive impact on symptoms of premenstrual disorder.

Urocortin 1 expression and secretion by human umbilical vein endothelial cells: In vitro effects of interleukin 8, interferon γ, lipopolysaccharide, endothelin 1, prostaglandin F-2α, estradiol, progesterone and dexamethasone.

Urocortin 1 (Ucn1) is a 40-amino-acid peptide that has vasodilatory activity and displays immunomodulatory and antioxidant properties. Maternal and cord plasma Ucn1 levels are increased in preeclampsia and preterm labor, but the mechanisms of such increase are poorly known. Thus, we investigated Ucn1 localization in human umbilical cord and assessed some potential stimuli to Ucn1 release by human umbilical vein endothelial cells (HUVEC). Human umbilical cords were obtained at uncomplicated term pregnancy (n=11). Ucn1 localization was assessed by immunohistochemistry and quantified. HUVEC were grown in vitro to confluence, then incubated with serial concentrations of interleukin (IL)-8, interferon (INF)-γ, lipopolysaccharide (LPS), endothelin (ET)-1, prostaglandin (PG)F-2α, estradiol, progesterone and dexamethasone and Ucn1 concentrations were measured in the supernatants. Ucn1 was immunolocalized with similar intensity in umbilical cord arteries, vein and Wharton's jelly. Ucn1 mRNA was detected in all HUVEC cultures and Ucn1 peptide was detectable in culture medium from untreated cells at different time points. Incubation with IFN-γ increased Ucn1 secretion in a dose-dependent manner. Treatments with IL-8, LPS, ET-1 and dexamethasone were able to increase three to fourfold Ucn1 release from cultured endothelial cells. In conclusion, umbilical vessels express Ucn1 and may be a contributive source of Ucn1 release into fetal-placental circulation. IL-8, IFN-γ, LPS, ET-1 and dexamethasone promote Ucn1 secretion from cultured HUVEC.

Evaluation of endometrial activin A secretion for prediction of pregnancy after intrauterine insemination.

OBJECTIVE: To measure activin A concentrations in uterine washing fluid collected from women with couple infertility undergoing intrauterine insemination (IUI). DESIGN: Retrospective case-control study. SETTING: Tertiary university center for women's care. PATIENT(S): Fifty women, of whom 25 became pregnant after up to three IUI attempts and 25 did not. INTERVENTION(S): Endocrine and clinical evaluation, semen analyses and hypo-osmotic swelling test, ovarian stimulation, endometrial thickness measurement by ultrasound, uterine washing fluid collection by sonohysterography, and IUI. MAIN OUTCOME MEASURE(S): Activin A measurement by enzyme-linked immunosorbent assay, receiver operating characteristics curve analysis, and pregnancy rates after IUI; sensitivity, specificity, positive and negative likelihood ratios of activin A, and endometrial thickness for pregnancy prediction. RESULT(S): Activin A was measurable in all samples evaluated, and the levels (mean +/- SEM) were statistically significantly higher in the pregnant (0.08 +/- 0.01 ng/mL) than in the nonpregnant (0.022 +/- 0.001 ng/mL) group. Activin A at the cut off of 0.04 ng/mL achieved a sensitivity of 76.0 % (95% CI, 54.9%-90.6%) and a specificity of 100% (95% CI, 86.2%-100%) as a single marker for prediction of pregnancy. CONCLUSION(S): Activin A is secreted into uterine lumen; the levels in endometrial washing fluids were higher in women who subsequently became pregnant after IUI, so its measurement may be useful in predicting successful implantation.

Activins and related proteins in the establishment of pregnancy.

Activin A and related proteins (inhibins, follistatin [FS], follistatin-related gene [FLRG], endometrial bleeding associated factors [ebaf]) are involved in the complex mechanisms allowing the establishment and the maintenance of pregnancy. As a consequence of ovarian progesterone stimuli, activin A is expressed and secreted by the stromal endometrial cells, which locally induces the decidualization process, a prerequisite for implantation. Moreover, activin A does influence the implantation phase, also enhancing cytotrophoblast differentiation, indirectly, by increasing the expression of other molecules involved in embryo implantation, such as matrix metalloproteinases (MMPs) and leukemia inhibitory factor (LIF). The local derangement of activin A pathway in some pregnancy disorders (incomplete and complete miscarriages, recurrent abortion, and ectopic pregnancy [EP]) further sustains the hypothesis that activin A and its related proteins play a relevant role in the establishment of pregnancy.

Activin A, activin receptor type II, nodal, and cripto mRNA are expressed by eutopic and ectopic endometrium in women with ovarian endometriosis.

Activin A is a dimeric protein that regulates endometrial functions by signaling at its receptors, namely type I (ActRI) and type II (ActRII). Nodal is an activin competitor that requires the coreceptor cripto to assemble its signaling pathway through ActRI and ActRII. In the current study, we evaluated the expression of activin A, ActRII, nodal, and cripto in eutopic and ectopic endometrium collected from women with ovarian endometrioma (n = 15) and in eutopic endometrium of healthy participants (n = 15). Eutopic endometrial samples were evaluated according to the stage of menstrual cycle. Total RNA was extracted from tissue homogenates and analyzed by real-time polymerase chain reaction (PCR). Activin A messenger RNA (mRNA) expression in eutopic endometrium of patients with endometriosis was significantly higher than in controls (P < .001) with a 10.2-fold and 7.3-fold increase in the proliferative and secretory phases, respectively. ActRII and nodal mRNA expression were found to be similar in patients with and without endometriosis, while cripto mRNA was markedly lower in eutopic (fold change = 0.03 at proliferative phase, P < .001) and ectopic endometrium (fold change = 0.14, P < .001) of women with endometriosis compared with eutopic endometrium from healthy controls. In conclusion, the altered endometrial expression of activin A and cripto during the menstrual cycle and the differences observed in the endometriotic tissue support the involvement of the activin system in endometrial changes of women with endometriosis.