Association between Doppler assessment and secondary cesarean delivery for intrapartum fetal compromise in small-for-gestational-age fetuses.

PURPOSE: To elucidate the association between arterial and venous Doppler ultrasound parameters and the risk of secondary cesarean delivery for intrapartum fetal compromise (IFC) and neonatal acidosis in small-for-gestational-age (SGA) fetuses. METHODS: This single-center, prospective, blinded, cohort study included singleton pregnancies with an estimated fetal weight (EFW) < 10th centile above 36 gestational weeks. Upon study inclusion, all women underwent Doppler ultrasound, including umbilical artery (UA) pulsatility index (PI), middle cerebral artery (MCA) PI, fetal aortic isthmus (AoI) PI, umbilical vein blood flow (UVBF), and modified myocardial performance index (mod-MPI). Primary outcome was defined as secondary cesarean section due to IFC. RESULTS: In total, 87 SGA pregnancies were included, 16% of which required a cesarean section for IFC. Those fetuses revealed lower UVBF corrected for abdominal circumference (AC) (5.2 (4.5-6.3) vs 7.2 (5.5-8.3), p = 0.001). There was no difference when comparing AoI PI, UA PI, ACM PI, or mod-MPI. No association was found for neonatal acidosis. After multivariate logistic regression, UVBF/AC remained independently associated with cesarean section due to IFC (aOR 0.61 [0.37; 0.91], p = 0.03) and yielded an area under the curve (AUC) of 0.78 (95% CI, 0.67-0.89). A cut-off value set at the 50th centile of UVBF/AC reached a sensitivity of 86% and specificity of 58% for the occurrence of cesarean section due to IFC (OR 8.1; 95% CI, 1.7-37.8, p = 0.003). CONCLUSION: Low levels of umbilical vein blood flow (UVBF/AC) were associated with an increased risk among SGA fetuses to be delivered by cesarean section for IFC.

Fibrinolytic potential as a risk factor for postpartum hemorrhage.

Background: Postpartum hemorrhage (PPH) is still the leading cause of maternal morbidity and mortality worldwide. While impaired fibrin polymerization plays a crucial role in the development and progress of PPH, recent approaches using viscoelastic measurements have failed to sensitively detect early changes in fibrinolysis in PPH. This study aimed to evaluate whether women experiencing PPH show alterations in POC-VET fibrinolytic potential during childbirth and whether fibrinolytic potential offers benefits in the prediction and treatment of PPH. Methods: Blood samples were collected at three different timepoints: T0 = hospital admission (19 h ± 18 h prepartum), T1 = 30-60 min after placental separation, and T2 = first day postpartum (19 h ± 6 h postpartum). In addition to standard laboratory tests, whole-blood impedance aggregometry (Multiplate) and viscoelastic testing (VET) were performed using the ClotPro system, which included the TPA-test lysis time, to assess the POC-VET fibrinolytic potential, and selected coagulation factors were measured. The results were correlated with blood loss and clinical outcome markers. Severe PPH was defined as a hemoglobin drop > 4g/dl and/or the occurrence of shock or the need for red blood cell transfusion. Results: Blood samples of 217 parturient women were analyzed between June 2020 and December 2020 at Heidelberg University Women's Hospital, and 206 measurements were eligible for the final analysis. Women experiencing severe PPH showed increased fibrinolytic potential already at the time of hospital admission. When compared to non-PPH, the difference persisted 30-60 min after placental separation. A higher fibrinolytic potential was accompanied by a greater drop in fibrinogen and higher d-dimer values after placental separation. While 70% of women experiencing severe PPH showed fibrinolytic potential, 54% of those without PPH showed increased fibrinolytic potential as well. Conclusion: We were able to show that antepartal and peripartal fibrinolytic potential was elevated in women experiencing severe PPH. However, several women showed high fibrinolytic potential but lacked clinical signs of PPH. The findings indicate that high fibrinolytic potential is a risk factor for the development of coagulopathy, but further conditions are required to cause PPH.

CDK7/12/13 inhibition targets an oscillating leukemia stem cell network and synergizes with venetoclax in acute myeloid leukemia.

The heterogeneous response of acute myeloid leukemia (AML) to current anti-leukemic therapies is only partially explained by mutational heterogeneity. We previously identified GPR56 as a surface marker associated with poor outcome across genetic groups, which characterizes two leukemia stem cell (LSC)-enriched compartments with different self-renewal capacities. How these compartments self-renew remained unclear. Here, we show that GPR56+ LSC compartments are promoted in a complex network involving epithelial-to-mesenchymal transition (EMT) regulators besides Rho, Wnt, and Hedgehog (Hh) signaling. Unexpectedly, Wnt pathway inhibition increased the more immature, slowly cycling GPR56+ CD34+ fraction and Hh/EMT gene expression, while Wnt activation caused opposite effects. Our data suggest that the crucial role of GPR56 lies in its ability to co-activate these opposing signals, thus ensuring the constant supply of both LSC subsets. We show that CDK7 inhibitors suppress both LSC-enriched subsets in vivo and synergize with the Bcl-2 inhibitor venetoclax. Our data establish reciprocal transition between LSC compartments as a novel concept underlying the poor outcome in GPR56high AML and propose combined CDK7 and Bcl-2 inhibition as LSC-directed therapy in this disease.

A New Practical Decision Rule to Better Differentiate BI-RADS 3 or 4 Breast Masses on Breast Ultrasound.

OBJECTIVES: The BI-RADS classification provides a standardized way to describe ultrasound findings in breast cancer diagnostics. However, there is little information regarding which BI-RADS descriptors are most strongly associated with malignancy, to better distinguish BI-RADS 3 (follow-up imaging) and 4 (diagnostic biopsy) breast masses. METHODS: Patients were recruited as part of an international, multicenter trial (NCT02638935). The trial enrolled 1294 women (6 excluded) categorized as BI-RADS 3 or 4 upon routine B-mode ultrasound examination. Ultrasound images were evaluated by three expert physicians according to BI-RADS. All patients underwent histopathological confirmation (reference standard). We performed univariate and multivariate analyses (chi-square test, logistic regression, and Krippendorff's alpha). RESULTS: Histopathologic evaluation showed malignancy in 368 of 1288 masses (28.6%). Upon performing multivariate analysis, the following descriptors were significantly associated with malignancy (P < .05): age ≥50 years (OR 8.99), non-circumscribed indistinct (OR 4.05) and microlobulated margin (OR 2.95), nonparallel orientation (OR 2.69), and calcification (OR 2.64). A clinical decision rule informed by these results demonstrated a 97% sensitivity and missed fewer cancers compared to three physician experts (range of sensitivity 79-95%) and a previous decision rule (sensitivity 59%). Specificity was 44% versus 22-83%, respectively. The inter-reader reliability of the BI-RADS descriptors and of the final BI-RADS score was fair-moderate. CONCLUSIONS: A patient should undergo a diagnostic biopsy (BI-RADS 4) instead of follow-up imaging (BI-RADS 3) if the patient is 50 years or older or exhibits at least one of the following features: calcification, nonparallel orientation of mass, non-circumscribed margin, or posterior shadowing.

A Web-Based Survey Assessing the Attitudes of Health Care Professionals in Germany Toward the Use of Telemedicine in Pregnancy Monitoring: Cross-Sectional Study.

BACKGROUND: The demand for fetal monitoring and constant reassurance is high in pregnant women. Consequently, pregnant women use various health apps and are more likely to visit emergency departments due to subjective but nonurgent complaints. However, electronic health (eHealth) and mobile health (mHealth) solutions are rarely used to prevent nonurgent emergency consultations. To implement modern care solutions, a better understanding of the attitudes, fears, and hopes of health care professionals toward eHealth and mHealth is needed. OBJECTIVE: The aim of this study was to investigate the attitudes of health care professionals in obstetrics toward telemedicine. METHODS: A quantitative Web-based survey on health care professionals in obstetrics in Germany was conducted. The participants included nurses, midwives, and physicians of all age groups and job positions working in hospitals that provide various levels of health care. The questionnaire comprised 24 questions about the characteristics of the study population, views about emergency consultations in obstetrics, attitude toward telemedicine, job satisfaction, and sleeping behavior. RESULTS: In total, 244 health care professionals participated in the Web-based survey. In general, health care professionals were skeptical (170/233, 72.9%) about the use of telemedicine in obstetrics; however, 55.8% (130/233) recognized its potential. Moreover, 72% (62/86) of physicians were optimistic in using apps for pregnancy monitoring, whereas 36.1% (47/130) of nonphysicians (P<.001) were not. Significantly, more nonphysicians rejected such developments (75/130, 57.7% rejected) compared with physicians (24/86, 28%; P<.001). We also found that obstetricians with more than 10 years of work-experience are more skeptical; however, approximately 49% (18/37) of them believed that telemedicine could reduce nonurgent emergency consultations, whereas 73.2% (106/145) of obstetricians with less than 5 years of experience (P=.01) thought otherwise. Our survey revealed a high job satisfaction and a prevalence of regular sleeping problems of 45.9% (91/198) among health care professionals in obstetrics. Surprisingly, both job satisfaction and sleeping problems were independent from the number of night shifts per month (P=.77 and P=.99, respectively). Yet, 56.6% (112/198) of the survey participants thought they would be happier with their job if they had to work fewer night shifts per month. CONCLUSIONS: Our study reveals an ambivalent attitude toward the use of telemedicine among health care professionals in obstetrics in Germany at the moment. Efforts to promote the use of telemedicine should focus on nurses and midwives because these groups are the most skeptical. By contrast, particularly young physicians recognize the potential of apps in patient care and would like to use such technology in pregnancy monitoring.

Initial results of the FUSION-X-US prototype combining 3D automated breast ultrasound and digital breast tomosynthesis.

PURPOSE: To determine the feasibility of a prototype device combining 3D-automated breast ultrasound (ABVS) and digital breast tomosynthesis in a single device to detect and characterize breast lesions. METHODS: In this prospective feasibility study, the FUSION-X-US prototype was used to perform digital breast tomosynthesis and ABVS in 23 patients with an indication for tomosynthesis based on current guidelines after clinical examination and standard imaging. The ABVS and tomosynthesis images of the prototype were interpreted separately by two blinded experts. The study compares the detection and BI-RADS® scores of breast lesions using only the tomosynthesis and ABVS data from the FUSION-X-US prototype to the results of the complete diagnostic workup. RESULTS: Image acquisition and processing by the prototype was fast and accurate, with some limitations in ultrasound coverage and image quality. In the diagnostic workup, 29 solid lesions (23 benign, including three cases with microcalcifications, and six malignant lesions) were identified. Using the prototype, all malignant lesions were detected and classified as malignant or suspicious by both investigators. CONCLUSION: Solid breast lesions can be localized accurately and fast by the Fusion-X-US system. Technical improvements of the ultrasound image quality and ultrasound coverage are needed to further study this new device. KEY POINTS: The prototype combines tomosynthesis and automated 3D-ultrasound (ABVS) in one device. It allows accurate detection of malignant lesions, directly correlating tomosynthesis and ABVS data. The diagnostic evaluation of the prototype-acquired data was interpreter-independent. The prototype provides a time-efficient and technically reliable diagnostic procedure. The combination of tomosynthesis and ABVS is a promising diagnostic approach.

Stimulation of chemokines in human endometrial stromal cells by tumor necrosis factor-α and interferon-γ is similar under apoptotic and non-apoptotic conditions.

PURPOSE: Tumor necrosis factor (TNF)-α and interferon (IFN)-γ are pro-inflammatory cytokines which have been shown to be involved in the pathophysiology of implantation disorders. Both cytokines in combination are able to sensitize primarily resistant human endometrial stromal cells (ESCs) to Fas-induced apoptosis. Since CCL (CC-chemokine ligand) 5 and CCL2 are important regulators of the endometrial immune cell population, we examined the impact of TNF-α and IFN-γ on these two chemokines under non-apoptotic and apoptotic conditions. METHODS: ESCs were isolated from hysterectomy specimens, decidualized in vitro and incubated with TNF-α, IFN-γ, an activating anti-Fas antibody and a caspase-inhibitor. CCL5 and CCL2 were measured using ELISA and real-time RT-PCR. Apoptosis was determined by flow cytometry, and cellular viability and membrane integrity were measured by fluorescent assays. RESULTS: The secretion of CCL5 and CCL2 was stimulated in undifferentiated and decidualized ESCs by the combination of TNF-α and IFN-γ under non-apoptotic as well as apoptotic (with Fas-stimulation in parallel) conditions. TNF-α or IFN-γ alone did not have this effect. The stimulatory influence of TNF-α plus IFN-γ on CCL5 and CCL2 in ESCs was also seen on the transcriptional level. Inhibition of cell death by a caspase-inhibitor had no influence on the secretion of CCL5 and CCL2 in ESCs under apoptotic stimulation. CONCLUSION: TNF-α and IFN-γ modulate the secretion of chemokines in ESCs independently of Fas-induced apoptosis. These results suggest a constant response pattern on pro-inflammatory cytokines within the population of human ESCs.

Inter-rater reliability and double reading analysis of an automated three-dimensional breast ultrasound system: comparison of two independent examiners.

PURPOSE: Breast ultrasound could be a valuable tool complementary to mammography in breast cancer screening. Automated 3D breast ultrasound (ABUS) addresses challenges of hand-held ultrasound and could allow double reading analysis of ultrasound images. This trial assesses the inter-rater reliability and double reading analysis of an ABUS system. METHODS: To assess the reproducibility and diagnostic validity of the ABUS system, SomoV™, a blinded double reading analysis, was performed in 1019 patients (2038 breasts) by two examiners (examiner A/B) and compared to single reading results, as well as to the reference standard regarding its diagnostic validity. Cohen's kappa coefficients were calculated to measure the inter-rater reliability and agreement of the different diagnostic modalities. Patient comfort and time consumption for image acquisition and reading were analyzed descriptively as secondary objectives. RESULTS: Analysis of inter-rater reliability yielded agreement in 81.6% (κ = 0.37; p < 0.0001) showing fair agreement. Single reading analysis of SomoV™ exams (examiner A/examiner B) compared to reference standard showed good specificity (examiner A: 88.3%/examiner B: 84.5%), fair inter-rater agreement (examiner A: κ = 0.31/examiner B: κ = 0.31), and adequate sensitivity (examiner A: 53.1%/examiner B: 64.2%). Double reading analysis yielded good sensitivity and specificity (73.7 and 77.7%). Mammography (n = 1911) alone detected 160 of 176 carcinomas (sensitivity 90.1%). Adding SomoV™ to mammography would have detected 12 additional carcinomas, resulting in a higher sensitivity of 97.7%. CONCLUSION: SomoV™ is a promising technique with good sensitivity, high patient comfort, and fair inter-examiner reliability. It allows double reading analysis that, in combination with mammography, could increase detection rates in breast cancer screening.

Aberrant ICOS+ -T cell differentiation in women with spontaneous preterm labor.

PROBLEM: Recent studies revealed appropriate differentiation of recent thymic emigrant (RTE)-regulatory T cells (Tregs) to be crucial for maintaining healthy pregnancy. Currently, the role of responder T cells (Tresps) is not known. METHOD OF STUDY: Six-color flow cytometric analysis was used to detect differences in the differentiation of highly proliferative inducible co-stimulatory (ICOS)+ -RTE-Tregs/Tresps and apoptosis-sensitive ICOS- -RTE-Tregs/Tresps into mature naïve (MN)-, CD31+ -memory and CD31- -memory Tregs/Tresps in women with spontaneous preterm labor (sPL) compared to healthy pregnancy. RESULTS: Healthy pregnancy was characterized by an increased differentiation of ICOS+ - and ICOS- -RTE-Tregs, as well as ICOS+ -RTE-Tresps via CD31+ -memory Tregs/Tresps into CD31- -memory Tregs/Tresps. Women with sPL showed an early interruption of RTE-Treg/Tresp differentiation. Instead, ICOS+ -MN-Tresps and partly ICOS- -MN-Tregs differentiated into CD31- -memory Tregs/Tresps, causing a significant reduction of both ICOS+ -Tregs and ICOS+ -Tresps, but an increase of ICOS- -Tresps within total CD4+ -T-helper cells. CONCLUSION: Aberrant differentiation of ICOS+ -T cells is associated with sPL.

The presence of heparins during decidualization modulates the response of human endometrial stromal cells to IL-1ß in vitro.

PURPOSE: The aim of this paper is to study the impact of heparin on the response of human endometrial stromal cells (ESCs) to interleukin (IL)-1ß during decidualization in vitro. METHODS: ESCs were isolated from hysterectomy specimens of premenopausal women undergoing hysterectomy for benign reasons; decidualized in vitro and incubated in parallel with unfractionated heparin or tinzaparin; and stimulated with IL-1ß at days 0, 3, 6, and 9 during decidualization. IL-6, IL-11, and leukemia inhibitory factor (LIF) were analyzed using ELISAs and real-time RT-PCR. Cell viability was determined by a fluorometric assay. RESULTS: IL-1ß dose-dependently stimulated IL-6, IL-11, and LIF in distinct patterns in ESCs during decidualization. Unfractionated heparin as well as tinzaparin attenuated the IL-1ß-mediated induction of IL-6, IL-11, and LIF on protein and messenger RNA (mRNA) levels. The relative effects of heparin and tinzaparin were getting more pronounced during the time course of decidualization. CONCLUSIONS: Unfractionated heparin and the low molecular weight heparin tinzaparin have modulating effects on IL-1ß-induced endometrial cytokines of the IL-6 family during decidualization. These effects of heparins beyond their classical anti-coagulatory properties might have implications on the regulation of endometrial receptivity and early implantation.

Interferon-γ differentially modulates the impact of tumor necrosis factor-α on human endometrial stromal cells.

The pro-inflammatory T helper (Th)-1 cytokines, tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), are immunological factors relevant at the feto-maternal interface and involved in the pathophysiology of implantation disorders. The synergistic action of the two cytokines has been described with regard to apoptotic cell death and inflammatory responses in different cell types, but little is known regarding the human endometrium. Therefore, we examined the interaction of TNF-α and IFN-γ in human endometrial stromal cells (ESCs). ESCs were isolated from specimens obtained during hysterectomy and decidualized in vitro. Cells were incubated with TNF-α, IFN-γ or signaling-inhibitor. Insulin-like growth factor binding protein (IGFBP)-1, prolactin (PRL), leukemia inhibitory factor (LIF), interleukin (IL)-6, IL-8, regulated on activation normal T-cell expressed and secreted protein (RANTES) and monocyte chemotactic protein (MCP)-1 were measured using ELISA and real-time RT-PCR. Nuclear factor of transcription (NF)-κB and its inhibitor (IκBα) were analyzed by in-cell western assay and transcription factor assay. TNF-α inhibited and IFN-γ did not affect the decidualization of ESCs. In contrast, IFN-gamma differentially modulated the stimulating effect of TNF-alpha on cytokines by enhancing IL-6, RANTES and MCP-1 and attenuating LIF mRNA expression. These effects were time- and dose-dependent. IFN-γ had no impact on the initial activation of NF-κB signaling. Histone-deacetylase activity was involved in the modulating effect of IFN-γ on RANTES secretion. These observations showed a distinct pattern of interaction of the Th-1 cytokines, TNF-α and IFN-γ in the human endometrium, which could play an important role in the pathophysiology of implantation disorders.

The role of recent thymic emigrant-regulatory T-cell (RTE-Treg) differentiation during pregnancy.

During pregnancy, regulatory T cells (Tregs) have a key role in maternal immune tolerance to the semi-allogeneic fetus. Our previous results showed that the naive CD45RA(+)-Treg pool is functionally improved in pregnant women compared with non-pregnant women. Therefore, we examined the thymic output and differentiation of CD45RA(+)CD31(+) recent thymic emigrant (RTE)-Tregs during normal pregnancy and in the presence of preeclampsia. With the onset of pregnancy, the composition of the total CD4(+)CD127(low+/-)FoxP3(+)-Treg pool changed in the way that its percentage of RTE- and CD45RA(-)CD31(+)-memory Tregs decreased strongly, whereas that of the CD45RA(+)CD31(-)-mature naive (MN)-Tregs did not change and that of the CD45RA(-)CD31(-)-memory Tregs increased complementary. Thereby, the ratio of RTE-/MN-Tregs decreased from 1.0 to 0.7 leading to a significant increase in the suppressive activity of the naive CD45RA(+)-Treg pool. This effect was confirmed by re-assembling separated RTE- and MN-Tregs from non-pregnant women in the ratio of pregnant women. The suppressive activity of both separated naive Treg subsets was equally high in non-pregnant and pregnant women, but considerably reduced in preeclampsia patients, who showed significantly increased percentages of CD45RA(-)CD31(+)-memory Tregs, but decreased percentages of RTE- and MN-Tregs. Our results suggest a reduced thymic output of RTE-Tregs during pregnancy, which causes a decrease in the ratio of RTE-/MN-Tregs and thus an increase in the differentiation of RTE-Tregs towards CD45RA(-)CD31(-)-memory Tregs. Presumably, this differentiation of RTE-Tregs, which was impaired in preeclampsia patients, ensures the improved suppressive activity of the CD45RA(+)-naive Treg pool and thus retains the maintenance of pregnancy.

Heparin modulates chemokines in human endometrial stromal cells by interaction with tumor necrosis factor α and thrombin.

OBJECTIVE: To study the impact of heparins on chemokines in decidualized human endometrial stromal cells (ESCs) in vitro. DESIGN: In vitro experiment. SETTING: Research laboratory. PATIENT(S): Premenopausal women undergoing hysterectomy for benign reasons. INTERVENTION(S): ESCs were isolated from hysterectomy specimens, decidualized in vitro and incubated with unfractionated heparin and low-molecular-weight heparins (LMWHs) as well as tumor necrosis factor (TNF) α or thrombin with or without heparins. MAIN OUTCOME MEASURE(S): Chemokines CXCL1, CXCL5, CXCL8, CCL2, and CCL5 were measured with the use of ELISA, and CXCL5, CXCL8, CCL2, and CCL5 were detected with the use of real-time reverse-transcription polymerase chain reaction. Cell viability was determined with the use of a fluorometric assay. RESULT(S): TNF-α and thrombin stimulated distinct patterns of chemokines in ESCs. Unfractionated heparin and LMWHs attenuated the TNF-α-mediated induction of CXCL8 and enhanced CXCL5, CCL2, and CCL5. The stimulating effect of thrombin on CXCL8 could be inhibited by heparin, whereas heparin had no impact on thrombin-induced CXCL1 and CCL2. Nuclear factor of transcription κB signaling mediated the effects of TNF-α. The effects of thrombin were mediated via extracellular signal-regulated protein kinases 1/2. CONCLUSION(S): Heparins have modulating effects on TNF-α- and thrombin-induced endometrial chemokines, which might have implications in the regulation of endometrial receptivity and early implantation.

Constitutive activity of Erk1/2 and NF-κB protects human endometrial stromal cells from death receptor-mediated apoptosis.

Apoptosis in the human endometrium plays an essential role for endometrial receptivity and early implantation. A dysbalance of pro- and anti-apoptotic events in the secretory endometrium seems to be involved in implantation disorders and consecutive pregnancy complications. However, little is known about the mechanisms regulating apoptosis-sensitivity in the human endometrium. Therefore this study was performed to identify molecular mechanisms underlying the resistance toward apoptosis in human endometrial stromal cells (ESCs). Human ESCs were isolated from hysterectomy specimens and used as undifferentiated cells or after decidualization in vitro. Cells were incubated with an activating anti-Fas antibody, tumor-necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), TNF-α and inhibitors of protein- and RNA-syntheses, a caspase-inhibitor and inhibitors of extracellular signal regulated kinase (Erk)1/2, nuclear factor (NF)-κB and Akt. Apoptosis was measured by flow cytometric detection of hypodiploid nuclei. Caspase-activity was detected by luminescencent assays. Several pro- and anti-apoptotic molecules and the activation of Erk1/2, NF-κB and Akt were analyzed by in-cell Western assays or flow cytometry. Inhibition of protein- and RNA-syntheses differentially sensitized human ESCs for death receptor-mediated apoptosis in a caspase-dependent manner, based on the up-regulation of the death receptors Fas and TRAIL-R2. The constitutive activity of Erk1/2 and NF-κB could be identified as a reason for the apoptosis-resistance of human ESCs. These results suggest the pro-survival signaling pathways Erk1/2 and NF-κB as key regulators of the sensitivity of human ESCs for death receptor-mediated apoptosis. The modulation of these pathways might play an important role in the physiology of implantation.

Heparin inhibits TNF-α signaling in human endometrial stromal cells by interaction with NF-κB.

Heparins have been shown to be beneficial for the prevention of habitual miscarriages and repeated implantation failure, independently of their function as anticoagulants. The pro-inflammatory cytokine tumor necrosis factor (TNF)-α plays a role in the pathogenesis of these early pregnancy complications. Therefore, we examined the impact of heparin on TNF-α signaling in human endometrial stromal cells (ESCs) in vitro. Human ESCs were isolated from hysterectomy specimens and either used as undifferentiated cells or after decidualization in vitro. Cells were incubated with TNF-α, unfractionated heparin and signaling- and transporter-inhibitors. Interleukin (IL)-8 and -6 were measured using ELISAs and real-time RT-PCR. Nuclear factor of transcription (NF)-κB and its inhibitor IκBα were analyzed by in-cell western assays and confocal microscopy. Activation of NF-κB was determined in nuclear extracts using a specific transcription factor assay. Cellular internalization of heparin was detected by a heparin-uptake assay. Unfractionated heparin significantly suppressed the TNF-α-induced and NF-κB-mediated secretion and expression of IL-8 and -6 as well as other molecules in decidualized and undifferentiated human ESCs. Thereby heparin had no influence on the degradation of IκBα and the phosphorylation of NF-κB, but it inhibited the activity of NF-κB in the nucleus. An active heparin-uptake into the cells was the prerequisite for these heparin-effects. Unfractionated heparin is able to inhibit TNF-α/NF-κB-mediated inflammatory effects in the human endometrium independently of its classical function as an anticoagulant. These observations further underline on a molecular level the beneficial anti-inflammatory effects of heparin in women suffering from implantation disorders even in the absence of a thrombophilia.

The molecular charge and size of heparins determine their impact on the decidualization of human endometrial stromal cells.

Heparin modulates the decidualization of human endometrial stromal cells (ESCs), but the molecular mechanisms behind these effects are still unknown. In the present study, we further specified this biological effect of heparin in human ESCs in vitro. ESCs were isolated from hysterectomy specimens, decidualized over 12 days using progesterone and 17ß-estradiol and incubated with thrombin, factor Xa (FXa), unfractionated heparin, dextran sulfate, danaparoid or different low-molecular-weight heparins (LMWHs). Production of insulin-like growth factor (IGF)-I, prolactin (PRL) and IGF-binding protein (IGFBP)-1 by ESCs was measured using ELISAs. Like heparin, thrombin and FXa cause an increase in IGF-I in ESCs, suggesting an action of heparin independent from its anticoagulatory effects. This was supported by demonstrating the induction of the same effects on IGF-I, PRL and IGFBP-1 as heparin by dextran sulfate, a polysaccharide of similar size and charge as heparin, but without anticoagulatory properties. LMWHs with the same anti-FXa activity as heparin showed less pronounced effects on ESCs than heparin, whereas the very short pentasaccharide fondaparinux (17 kDa) had barely any effect, further supporting the primary role of molecular size and charge mediating these biological effects of heparin on ESCs. In conclusion, the effects of heparin on the decidualization of human ESCs seem to be independent of its anticoagulatory function, but rather depend on the charge and the size of this polysulfated glycosaminoglycan. Therefore, highly sulfated polysaccharides with a molecular weight >17 kDa might be an interesting pharmacological approach for the therapy of endometrial pathologies, e.g. the treatment of women suffering from recurrent miscarriage or repeated implantation failure.

Non-apoptotic Fas-induced regulation of cytokines in undifferentiated and decidualized human endometrial stromal cells depends on caspase-activity.

Fas has originally been described as a member of the death-receptor family, mediating apoptosis upon stimulation by Fas-ligand (FasL). However, Fas expressing human endometrial stromal cells (ESCs) are resistant to Fas-mediated apoptosis. Since the implanting embryo secretes FasL, we examined whether Fas mediates non-apoptotic effects in human ESCs in vitro. ESCs were isolated from hysterectomy specimens, decidualized using progesterone and 17ß-estradiol and incubated with an activating anti-Fas antibody, recombinant FasL and a caspase-inhibitor. Leukemia inhibitory factor (LIF), interleukin (IL)-11, -6, -8, monocyte chemoattractant protein (MCP)-1 and RANTES (Regulated on Activation Normal T cell Expressed and Secreted) were measured using ELISA and real-time RT-PCR. Viability of ESCs was determined using an MTT assay. Caspase-activity was measured by luminescent assays. Activation of nuclear factor (NF)-κB was detected by in-cell western and transcription factor assays. LIF and IL-11 in undifferentiated and IL-8 in decidualized ESCs were up-regulated by non-apoptotic Fas-signaling. In contrast, IL-6, MCP-1 and RANTES were not regulated by Fas. Caspases were activated upon Fas-stimulation and the Fas-mediated effects on LIF, IL-11 and -8 were reversed by caspase-inhibition. The transcription factor NF-κB was not activated in ESCs after stimulation of Fas. These results suggest a differential regulatory role of caspase-dependent Fas-signaling at the feto-maternal interface during early implantation. Remarkably, this typical death machinery mediates non-apoptotic effects in the human endometrium rather than inducing apoptosis.

Heparin inhibits interferon-γ signaling in human endometrial stromal cells by interference with the cellular binding of interferon-γ.

OBJECTIVE: To examine the impact of heparins on interferon-γ (IFN-γ) signaling in human endometrial stromal cells (ESCs) in vitro. DESIGN: In vitro experiment. SETTING: Research laboratory at a medical university center. PATIENT(S): Premenopausal women undergoing hysterectomy for benign reasons. INTERVENTION(S): The ESCs were isolated from hysterectomy specimens, decidualized in vitro using P and 17ß-E(2), and incubated with recombinant IFN-γ, unfractionated heparin, and low molecular weight heparins (LMWHs). MAIN OUTCOME MEASURE(S): Interferon response factor 1 (IRF-1) and N-myc interactor (Nmi) messenger RNA (mRNA) were measured using real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Phosphorylation of signal transducer and activator of transcription 1 (STAT-1) was detected by an in-cell Western assay, expression of the IFN-γ receptor by flow cytometry. Cell-bound IFN-γ was determined in lysates by an ELISA. RESULT(S): Heparin and LMWHs inhibit the IFN-γ-mediated induction of IRF-1, but not Nmi in undifferentiated and decidualized ESCs. The phosphorylation of signal transducer and activator of transcription 1 STAT-1 upon IFN-γ stimulation is inhibited as well. Heparin has no effect on the IFN-γ receptor in ESCs, but inhibits the binding of IFN-γ to the cells. CONCLUSION(S): Unfractionated heparin, as well as LMWHs, are able to inhibit IFN-γ signaling in human ESCs and therefore might be clinically interesting agents to modulate the actions of this proinflammatory cytokine at the implantation site.

Heparin and low-molecular-weight heparins modulate the decidualization of human endometrial stromal cells.

OBJECTIVE: To examine the impact of unfractionated heparin and low-molecular-weight heparins (LMWHs) on the decidualization of human endometrial stromal cells (ESCs) in vitro. DESIGN: In vitro experiment. SETTING: Research laboratory at a medical university center. PATIENT(S): Premenopausal women undergoing hysterectomy for benign reasons. INTERVENTION(S): The ESCs were isolated from hysterectomy specimens, decidualized in vitro using progesterone and 17beta-estradiol, and incubated with unfractionated heparin and three different LMWHs. MAIN OUTCOME MEASURE(S): Insulin-like growth factor-binding protein (IGFBP) 1, PRL, and insulin-like growth factor (IGF) I were measured using ELISA and real-time reverse-transcription polymerase chain reaction. Cell viability was determined by a fluorometric assay. Intracellular cyclic adenosine 3',5'-monophosphate (cAMP) was measured using a luminescent assay. RESULT(S): Heparin dose- and time-dependently delayed the production of IGFBP-1 and amplified the levels of PRL and IGF-I in ESCs during decidualization in vitro. Similar effects were seen under the influence of the three different LMWHs. Intracellular cAMP was increased in decidualizing ESCs under the influence of heparin and LMWHs. CONCLUSION(S): Unfractionated heparin as well as LMWHs are able to modulate the decidualization of human ESCs in vitro and therefore might be useful to control endometrial differentiation and receptivity in assisted reproduction.