A liquid chromatographic-mass spectrometric procedure for analysis of pentaerythrityl tetranitrate metabolites - Development, validation and application to ovine serum and human plasma samples.

Pentaerythrityl tetranitrate (PETN) is an established drug in the treatment of coronary heart disease and heart failure. It is assumed, that the vasodilative and vasoprotective effects of PETN also have a positive impact on pregnant patients with impaired placental perfusion and studies evaluating the effect of PETN in risk pregnancies have been carried out. In the context of these clinical trials, measuring of serum levels of PETN and its metabolites pentaerythrityl trinitrate (PETriN), pentaerythrityl dinitrate (PEDN), pentaerythrityl mononitrate (PEMN) and pentaerythritol (PE) were required. To evaluate the transfer of PETN and its metabolites (PEXN) from the mother to the fetus using samples from a human clinical trial and animal study, the present work aimed to develop a rapid and simple method to simultaneously analyze PEXN in human and ovine samples. A method employing a rapid and simple liquid-liquid extraction followed by reversed-phase (C18) liquid chromatography coupled to high-resolution mass spectrometry with negative electrospray ionization was developed and validated for the detection of PETN and PEXN in human and ovine samples. PE could only be qualitatively detected at higher concenrations. Method validation requirements, including accuracy, repeatability and intermediate precision were fulfilled in ovine and human samples for all other PEXN with exception PETriN in human samples. The recovery (RE) in ovine samples was 76.7 % ± 12 % for PEMN, 98 % ± 23 % for PEDN, 94 % ± 22 % for PETriN, in human samples RE was 59 % ± 16 % for PEMN, 67 % ± 19 % for PEDN, 71 % ± 17 %. The matrix effects (ME) in ovine samples were 90 % ± 11 % for PEMN, 70 % ± 30 % for PEDN, 107 % ± 17 % for PETriN, in human samples the ME were 93 % ± 13 % for PEMN, 84 % ± 17 % for PEDN, 98 % ± 16 % for PETriN. The limits of quantification (LOQ) in ovine samples were 1.0 ng/mL for PETriN and 0.1 ng/mL for PEMN and PEDN. The LOQs in human samples were 5.0 ng/mL for PETriN and 0.3 ng/mL for PEMN und PEDN. The newly developed method was used to analyze 184 ovine serum samples and 18 human plasma samples. In ovine maternal samples, the highest observed PEDN concentration was 3.5 ng/mL and the highest PEMN concentration was 10 ng/mL, the respective concentrations in fetal serum samples were 4.9 ng/mL for PEDN and 5.4 ng/mL for PEMN. PETriN was only detected in traces in maternal and fetal samples, whereas PETN could not be detected at all. In human maternal samples, the highest concentration for PEDN was 27 ng/mL and for PEMN 150 ng/mL. In umbilical cord plasma, concentrations of 2.3 ng/mL for PEDN and 73 ng/mL for PEMN were detected. Although the PEMN and PEDN concentrations in the human samples were several times higher than in ovine samples, neither PETN nor PETriN signals could be detected. These results demonstrated that the metabolites were transferred from mother to fetus with a slight time delay.

Transplacental migration of maternal natural killer and T cells assessed by ex vivo human placenta perfusion.

INTRODUCTION: The transplacental passage of cells between a mother and her fetus, known as microchimerism, is a less studied process during pregnancy. The frequency of maternal microchimeric cells in fetal tissues in physiological pregnancies and mechanisms responsible for transplacental cell trafficking are poorly understood. This study aimed to evaluate the placental trafficking of maternal peripheral blood mononuclear cells (PBMC) using human ex vivo placenta perfusion. METHODS: Ten placentas and maternal PBMC were obtained after healthy pregnancies. Flow cytometry was used to characterize PBMC subtypes. They showed a higher percentage of CD3+ T cells compared to CD56+ NK cells. The isolated PBMC were stained with a fluorescent dye and perfused through the maternal circuit of the placenta in an ex vivo perfusion system. Subsequent immunofluorescence staining for CD3+ T cells and CD56+ NK cells was performed on placental tissue sections, and the number of detectable PBMC in different tissue areas was counted using fluorescence microscopy. RESULTS: The applied method allowed discrimination of perfused autologous maternal cells from cells resident in the placenta before perfusion. Further, it allows additional immunohistochemical labelling and distinction of immune cell subsets. Perfused PBMC were detected in all analyzed placentas, mostly in contact to the syncytiotrophoblast. CD3+ T cells were identified more frequently than CD56+ NK cells and some CD3+ T cells were found inside fetoplacental tissues and vasculature. The results indicate that also other PBMCs than T or NK cells adhere to or enter villous tissue, but they have not been specified in this analysis. DISCUSSION: Previous studies have detected maternal cells in the fetal circulation which we could mimick in our ex vivo placenta perfusion experiments with fluorescence labelled autologous maternal PBMC. The applied experimental settings did not allow comparison of transmigration abilities of PBMC subsets, but slight modifications of the model will permit further studies of cell transfer processes and microchimerism in pregnancy.

Vaginal and neonatal microbiota in pregnant women with preterm premature rupture of membranes and consecutive early onset neonatal sepsis.

BACKGROUND: Preterm premature rupture of membranes (PPROM), which is associated with vaginal dysbiosis, is responsible for up to one-third of all preterm births. Consecutive ascending colonization, infection, and inflammation may lead to relevant neonatal morbidity including early-onset neonatal sepsis (EONS). The present study aims to assess the vaginal microbial composition of PPROM patients and its development under standard antibiotic therapy and to evaluate the usefulness of the vaginal microbiota for the prediction of EONS. It moreover aims to decipher neonatal microbiota at birth as possible mirror of the in utero microbiota. METHODS: As part of the PEONS prospective multicenter cohort study, 78 women with PPROM and their 89 neonates were recruited. Maternal vaginal and neonatal pharyngeal, rectal, umbilical cord blood, and meconium microbiota were analyzed by 16S rRNA gene sequencing. Significant differences between the sample groups were evaluated using permutational multivariate analysis of variance and differently distributed taxa by the Mann-Whitney test. Potential biomarkers for the prediction of EONS were analyzed using the MetaboAnalyst platform. RESULTS: Vaginal microbiota at admission after PPROM were dominated by Lactobacillus spp. Standard antibiotic treatment triggers significant changes in microbial community (relative depletion of Lactobacillus spp. and relative enrichment of Ureaplasma parvum) accompanied by an increase in bacterial diversity, evenness and richness. The neonatal microbiota showed a heterogeneous microbial composition where meconium samples were characterized by specific taxa enriched in this niche. The vaginal microbiota at birth was shown to have the potential to predict EONS with Escherichia/Shigella and Facklamia as risk taxa and Anaerococcus obesiensis and Campylobacter ureolyticus as protective taxa. EONS cases could also be predicted at a reasonable rate from neonatal meconium communities with the protective taxa Bifidobacterium longum, Agathobacter rectale, and S. epidermidis as features. CONCLUSIONS: Vaginal and neonatal microbiota analysis by 16S rRNA gene sequencing after PPROM may form the basis of individualized risk assessment for consecutive EONS. Further studies on extended cohorts are necessary to evaluate how far this technique may in future close a diagnostic gap to optimize and personalize the clinical management of PPROM patients. TRIAL REGISTRATION: NCT03819192, ClinicalTrials.gov. Registered on January 28, 2019.

Impact of tissue processing on microbiological colonization in the context of placentophagy.

A mother's postpartum ingestion of raw or processed placental tissue-referred to as human maternal placentophagy-is an emerging health trend observed in industrialized nations. Placenta is commonly consumed as small pieces of raw tissue, or as raw or steamed dehydrated pulverized and encapsulated tissue. To investigate the potential neonatal health risks of this behavior, the present study focused on microbial colonization of processed placenta preparations with potentially pathogenic bacteria Streptococcus agalactiae (Group-B-Streptococci; GBS) and Escherichia coli (E. coli). In the clinical approach placentas from 24 mothers were analyzed. Two placentas, from 13 mothers with confirmed positive maternal GBS status, showed GBS-growth on their surface (2/13; 15.4%) independent from delivery mode or antibiotic treatment. All processed samples (n = 24) were free from GBS. In the experimental approach, a standardized inoculation protocol was introduced to resemble ascending vaginal and hematogenous colonization. Six placentas from elective term C-sections of GBS negative mothers were collected and artificially inoculated with highly concentrated suspensions of GBS and E. coli. Heat processing significantly reduced the number of colony forming units (CFU) for GBS and E. coli. Our results suggest placentophagy of processed tissue is an unlikely source of clinical infection.

Evaluation of microbiome enrichment and host DNA depletion in human vaginal samples using Oxford Nanopore's adaptive sequencing.

Metagenomic sequencing is promising for clinical applications to study microbial composition concerning disease or patient outcomes. Alterations of the vaginal microbiome are associated with adverse pregnancy outcomes, like preterm premature rupture of membranes and preterm birth. Methodologically these samples often have to deal with low relative amounts of prokaryotic DNA and high amounts of host DNA (> 90%), decreasing the overall microbial resolution. Nanopore's adaptive sampling method offers selective DNA depletion or target enrichment to directly reject or accept DNA molecules during sequencing without specialized sample preparation. Here, we demonstrate how selective 'human host depletion' resulted in a 1.70 fold (± 0.27 fold) increase in total sequencing depth, providing higher taxonomic profiling sensitivity. At the same time, the microbial composition remains consistent with the control experiments. The complete removal of all human host sequences is not yet possible and should be considered as an ethical approval statement might still be necessary. Adaptive sampling increased microbial sequencing yield in all 15 sequenced clinical routine vaginal samples, making it a valuable tool for clinical surveillance and medical-based research, which can be used in addition to other host depletion methods before sequencing.

Molecular characteristics of established trophoblast-derived cell lines.

INTRODUCTION: Research on human placental development and function lacks a conclusive in vivo model. To investigate the intracellular molecular mechanisms in trophoblast cells, different cell lines have been established during the last decades. So far, none of these accomplishes all features of primary trophoblast, thus their suitability as well as the transferability of the results has been discussed. The aim of this study is to assess molecular markers and features matching different trophoblast subpopulations in trophoblastic cell lines to provide orientation on their suitability and relevance for distinct research questions. METHODS: The commonly used trophoblastic cell lines, BeWo, JEG-3, HTR-8/SVneo, AC1-M59, AC1-M32, ACH-3P and Swan71 were selected. qPCR and immunoblotting were used to determine expression of characteristic molecular markers. C14MC, C19MC and miR-371-3 miRNA expression were investigated by real time PCR. Proliferation, migration and network stabilization assays were performed. Hormone secretion was determined by chemiluminescent-immunoassays. DNA profiles were obtained by Short Tandem Repeat (STR)-genotyping. RESULTS: Immortalized cell lines differ from choriocarcinoma-derived ones in the expression of HLA-G, E-cadherin, N-cadherin, VE-cadherin, cadherin-11, cytokeratin 7, vimentin, ADAM12 and PRG2. Compared to choriocarcinoma-derived cell lines, expression of C19MC and hormone secretion were almost absent in immortalized cell lines. Conversely, they express C14MC and exhibit higher migration and network stabilization. DISCUSSION: The data presented will help justify the use of a cell line to evaluate distinct features of trophoblast biology and pathology. In general, characteristics and markers of choriocarcinoma derived cell lines seem to be more similar to in vivo trophoblast than immortalized cell lines and thus might be regarded as more suitable models.

Fetal exposure to environmental chemicals; insights from placental perfusion studies.

INTRODUCTION: The burden of environmental chemicals in the human population is ubiquitous and especially problematic in pregnancy due to potential exposure of the vulnerable fetus. According to the Developmental Origins of Health and Disease hypothesis, the fetal period is highly sensitive to exposure to environmental factors that will determine the development of diseases later in life. A range of environmental chemicals has been studied in the ex vivo placental perfusion model, which is a human model using the intact placenta directly after birth to study the placental transfer and metabolism of selected compounds. METHODS: Here, we reviewed the existing knowledge on human placental perfusion of environmental chemicals in order to identify potential correlations between placental transfer and properties of chemicals and areas of future research needs. RESULTS: We found 32 studies of the following groups of environmental chemicals: pesticides, persistent organic pollutants (POPs), plastics and byproducts, phyto/myco-estrogens and fungal toxins, byproducts from heating/curing food, combustion in traffic and industry, and metals. The studies showed highly distinct transfer rates from very fast transport to the fetal side to negligible transfer. DISCUSSION: In general, a low molecular weight favors placental translocation, but placental translocation is dependent on other physicochemical properties of the substances, claiming the need for more standardized studies and proper quantitative structure-activity relationship (QSAR) analysis. Overall, we recommend using placental perfusion studies in the risk assessment of environmental toxicants, to determine the transfer, metabolism and toxic effects of this diverse class of substances, on the human term placenta.

Lethal Neonatal Respiratory Failure by Perinatal Transmission of Ureaplasma Parvum after Maternal PPROM.

A primiparous pregnant woman was admitted due to preterm premature rupture of membranes (PPROM) at 27+0 week of gestational age (WGA). Conventional vaginal microbiological analysis had no pathological finding. Management decisions based on national guidelines included antenatal corticoids, tocolytics and antibiotics. Unstoppable efforts of preterm labor in 28+0 WGA and supposed amniotic infection syndrome necessitated emergency cesarean section. The preterm infant underwent NICU therapy, developed an early-onset neonatal sepsis and therapy-refractory pulmonary insufficiency with consecutive right heart failure, resulting in death on the 36th day of life. Microbiota analyses by 16Sr DNA sequencing was performed from maternal vaginal swabs and from neonatal pharyngeal swabs. Maternal antibiotic treatment resulted in depletion of physiological vaginal colonization with Lactobacillus crispatus. Ureaplasma parvum became the dominant vaginal microorganism at delivery and was detected in high relative abundance in the neonatal specimen. Progressive radiological air-space changes and interstitial pathologies associated with Ureaplasma infection (bronchopulmonary dysplasia type III) were seen early at the 3rd and distinctly from 14th day of life. This clearly demonstrates the need of vaginal colonization diagnostics in PPROM patients and awareness of the consecutive risks in the preterm. Vaginal microbiome analysis may allow individualized and targeted maternal and fetal diagnostic, prophylactic and therapeutic strategies to identify, protect and treat the high-risk neonates after PPROM.

Beyond Uterine Natural Killer Cell Numbers in Unexplained Recurrent Pregnancy Loss: Combined Analysis of CD45, CD56, CD16, CD57, and CD138.

Changes in the number and cytotoxic potential of uterine Natural Killer (uNK) cells have been associated with reduced fertility. To provide a better characterization of immunophenotypes in the endometrium of women with uRPL (unexplained recurrent pregnancy loss), we examined the applicability of a set of five immune cell markers. The concentration (cells/mm2) of CD45+ leukocytes, CD56+ uNK cells, and CD138+ plasma cells as well as of CD16+ and CD57+ cells, which indicate high cytotoxic uNK cells, were assessed by immunohistochemistry in endometrial biopsies from 61 uRPL patients and 10 controls. Control fertile endometria presented 90-300 CD56+ uNK cells/mm2. uRPL cases were classified in subgroups of low (uRPL-CD56low < 90 cells/mm2), normal (uRPL-CD56normal 90-300 cells/mm2), and high uNK cell counts (uRPL-CD56high > 300 cells/mm2). Some cases from the uRPL-CD56low and uRPL-CD56normal subgroups showed elevated proportions of cytotoxic CD16+ and CD57+ cells in relation to CD56+ cells. In the uRPL-CD56high subgroup, the CD57/CD56 ratio was reduced in most samples and the CD16/CD56 ratio was unaltered. Analysis of CD138 excluded the influence of chronic endometritis on these observations. Our results reinforce a link between uRPL and a dysfunctional endometrial environment associated with distinct immune cell profiles.

Placenta - Worth Trying? Human Maternal Placentophagia: Possible Benefit and Potential Risks.

The use of placenta preparations as an individual puerperal remedy can be traced back to historical, traditional practices in Western and Asian medicine. To evaluate the ingestion of processed placenta as a puerperal remedy, the potential risks (trace elements, microorganisms) and possible benefit (hormones in the placental tissue) of such a practice are discussed in this article based on a literature review.

Comparison of sample preparation techniques and data analysis for the LC-MS/MS-based identification of proteins in human follicular fluid.

The proteomic analysis of complex body fluids by liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis requires the selection of suitable sample preparation techniques and optimal parameter settings in data analysis software packages to obtain reliable results. Proteomic analysis of follicular fluid, as a representative of a complex body fluid similar to serum or plasma, is difficult as it contains a vast amount of high abundant proteins and a variety of proteins with different concentrations. However, the accessibility of this complex body fluid for LC-MS/MS analysis is an opportunity to gain insights into the status, the composition of fertility-relevant proteins including immunological factors or for the discovery of new diagnostic and prognostic markers for, for example, the treatment of infertility. In this study, we compared different sample preparation methods (FASP, eFASP and in-solution digestion) and three different data analysis software packages (Proteome Discoverer with SEQUEST, Mascot and MaxQuant with Andromeda) combined with semi- and full-tryptic databank search options to obtain a maximum coverage of the follicular fluid proteome. We found that the most comprehensive proteome coverage is achieved by the eFASP sample preparation method using SDS in the initial denaturing step and the SEQUEST-based semi-tryptic data analysis. In conclusion, we have developed a fractionation-free methodical workflow for in depth LC-MS/MS-based analysis for the standardized investigation of human follicle fluid as an important representative of a complex body fluid. Taken together, we were able to identify a total of 1392 proteins in follicular fluid.

Human placentophagy: Effects of dehydration and steaming on hormones, metals and bacteria in placental tissue.

INTRODUCTION: Human maternal placentophagy, the behavior of ingesting the own raw or processed placenta postpartum, is a growing trend by women of western societies. This study aims to identify the impact of dehydration and steaming on hormone and trace element concentration as well as microbial contamination of placental tissue. METHODS: A total of nine placentas have been processed: six were studied for hormone and trace element concentrations; eight were studied for microbial contamination. The concentrations of CRH, hPL, oxytocin and ACTH in samples of raw, steamed dehydrated and raw dehydrated placental tissue were detected using ELISA. A yeast bioassay was performed in order to detect estrogen equivalent (EEQ) and gestagen equivalent (PEQ) active substances. Elements (As, Cd, Fe, Pb, Se, Hg) were analyzed using ICP-MS. Isolated colonies from tissue and placenta swab samples were identified using Vitek MS. RESULTS: Following mean hormone concentrations were detected in raw placental tissue: CRH (177.88 ng/g), hPL (17.99 mg/g), oxytocin (85.10 pg/g), ACTH (2.07 ng/g), estrogen equivalent active substances (46.95 ng/g) and gestagen equivalent active substances (2.12 µg/g). All hormones were sensitive to processing with a significant concentration reduction through steaming and dehydration. Microorganisms mainly from the vaginal flora were detected on placenta swab samples and samples from raw, steamed, dehydrated and steamed dehydrated tissue and mostly disappeared after dehydration. According to regulations of the European Union the concentrations of potentially toxic elements (As, Cd, Hg, Pb) were below the toxicity threshold for foodstuffs. CONCLUSION: The commonly used protocols for preparation of placenta for its individual oral ingestion reduce hormone concentrations and bacterial contamination.

Human serum alters cell culture behavior and improves spheroid formation in comparison to fetal bovine serum.

BACKGROUND: The use of fetal bovine serum (FBS) as growth supplement for human cell and tissue culture is widely spread in basic research as well as in clinical approaches, although several limitations must be considered, such as unstable composition and availability, biosafety and ethical aspects. Regarding interspecies differences, xenogeneic growth factors may evoke incompatibilities and non-desired interactions with human cells resulting in imprecise outcome of human-relevant data. METHODS: In this study the functionality of human serum (HS) has been investigated in comparison to FBS by assessing proliferation, migration and invasion of the human cervical cancer cell lines SiHa and HeLa. The effects of both sera on spheroid formation were analyzed microscopically. RESULTS: Both, FBS and HS, stimulate cell proliferation and migration similarly, whereas HS significantly enhanced cell invasion. The spheroid formation assay revealed remarkable differences between both sera, especially for SiHa cells. While in FBS supplemented medium cells only formed loose aggregates, HS induced regularly shaped spheroids under all tested conditions. CONCLUSION: We were able to demonstrate that HS and FBS differently influence behavior of cells in culture which may have an impact on experimental results, especially in 3D cultures.

Improvement of a tissue maceration technique for the determination of placental involvement in schistosomiasis.

Schistosomiasis in pregnancy may cause low birth weight, prematurity and stillbirth of the offspring. The placenta of pregnant women might be involved when schistosome ova are trapped in placental tissue. Standard histopathological methods only allow the examination of a limited amount of placental tissue and are therefore not sufficiently sensitive. Thus, placental schistosomiasis remains underdiagnosed and its role in contributing to schistosomiasis-associated pregnancy outcomes remains unclear. Here we investigated an advanced maceration method in order to recover a maximum number of schistosome ova from the placenta. We examined the effect of different potassium hydroxide (KOH) concentrations and different tissue fixatives with respect to maceration success and egg morphology. Placental tissue was kept either in 0.9% saline, 5% formalin or 70% ethanol and was macerated together with Schistosoma mansoni infested mouse livers and KOH 4% or 10%, respectively. We found that placenta maceration using 4% KOH at 37°C for 24 h was the most effective method: placental tissue was completely digested, egg morphology was well preserved and alkaline concentration was the lowest. Ethanol proved to be the best fixative for this method. Here we propose an improved maceration technique in terms of sensitivity, safety and required skills, which may enable its wider use also in endemic areas. This technique may contribute to clarifying the role of placental involvement in pregnant women with schistosomiasis.

Generation of Multicellular Breast Cancer Tumor Spheroids: Comparison of Different Protocols.

Multicellular tumor spheroids are widely used models in tumor research. Because of their three dimensional organization they can simulate avascular tumor areas comprising proliferative and necrotic cells. Nonetheless, protocols for spheroid generation are still inconsistent. Therefore, in this study the breast cancer cell lines MCF-7, MDA-MB-231 and SK-BR-3 have been used to compare different spheroid generation models including hanging drop, liquid overlay and suspension culture techniques, each under several conditions. Experimental approaches differed in cell numbers (400-10,000), media and additives (25 % methocel, 25 % methocel plus 1 % Matrigel, 3.5 % Matrigel). In total, 42 different experimental setups have been tested. Generation of spheroids was evaluated by light microscopy and the structural composition was assessed immunohistochemically by means of Ki-67, cleaved poly (ADP-ribose) polymerase (cPARP) and mucin-1 (MUC-1) expression. Although the tested cell lines diverged widely in their capacity of forming spheroids we recommend hanging drops supplemented with 25 % methocel as the most reliable and efficient method with regard to success of generation of uniform spheroids, costs, experimental complexity and time expenditure in the different cell lines. MCF-7 cells formed spheroids under almost all analyzed conditions, and MDA-MB-231 cells under only one protocol (liquid overlay technique, 3.5 % Matrigel), while SK-BR-3 did not under neither condition. Therefore, we outline specific methods and recommend the use of adapted and standardized spheroid generation protocols for each cell line.

Oncostatin M and leukaemia inhibitory factor trigger signal transducer and activator of transcription 3 and extracellular signal-regulated kinase 1/2 pathways but result in heterogeneous cellular responses in trophoblast cells.

Leukaemia inhibitory factor (LIF) and oncostatin M (OSM) are pleiotropic cytokines present at the implantation site that are important for the normal development of human pregnancy. These cytokines share the cell membrane receptor subunit gp130, resulting in similar functions. The aim of this study was to compare the response to LIF and OSM in several trophoblast models with particular regard to intracellular mechanisms and invasion. Four trophoblast cell lines with different characteristics were used: HTR-8/SVneo, JEG-3, ACH-3P and AC1-M59 cells. Cells were incubated with LIF, OSM (both at 10ngmL(-1)) and the signal transducer and activator of transcription (STAT) 3 inhibitor S3I-201 (200µM). Expression and phosphorylation of STAT3 (tyr(705)) and extracellular regulated kinase (ERK) 1/2 (thr(202/204)) and the STAT3 DNA-binding capacity were analysed by Western blotting and DNA-binding assays, respectively. Cell viability and invasiveness were assessed by the methylthiazole tetrazolium salt (MTS) and Matrigel assays. Enzymatic activity of matrix metalloproteinase (MMP)-2 and MMP-9 was investigated by zymography. OSM and LIF triggered phosphorylation of STAT3 and ERK1/2, followed by a significant increase in STAT3 DNA-binding activity in all tested cell lines. Stimulation with LIF but not OSM significantly enhanced invasion of ACH-3P and JEG-3 cells, but not HTR-8/SVneo or AC1-M59 cells. Similarly, STAT3 inhibition significantly decreased the invasiveness of only ACH-3P and JEG-3 cells concomitant with decreases in secreted MMP-2 and MMP-9. OSM shares with LIF the capacity to activate ERK1/2 and STAT3 pathways in all cell lines tested, but their resulting effects are dependent on cell type. This suggests that LIF and OSM may partially substitute for each other in case of deficiencies or therapeutic interventions.

STAT5 is Activated by Epidermal Growth Factor and Induces Proliferation and Invasion in Trophoblastic Cells.

Epidermal growth factor (EGF) is expressed by decidual and trophoblast cells and influences manifold cellular functions during embryo implantation. Thus far, signaling of EGF via Signal Transducer and Activator of Transcription 5 (STAT5) has been only partially investigated. STAT5 stimulates proliferation and cell cycle progression in several cell types. Its dysregulation is associated with pregnancy. The aim of this study was to investigate STAT5 activation and function mediated by EGF in 2 trophoblastic cell lines, namely, HTR8/SVneo and JAR. Additionally, expression of STAT5B messenger RNA (mRNA) in trophoblast models has been compared to that of primary cells isolated from term placentas. Our results demonstrate the highest STAT5B mRNA expression in isolated trophoblast cells, lower expression in HTR8/SVneo cells, and the significantly lowest in JAR cells. Moreover, EGF-mediated STAT5 activation increases cell proliferation and viability in both cell lines. The STAT5 knockdown results in significant decrease in cell viability induced by EGF. Only in HTR8/SVneo cells, invasion decreases after STAT5 silencing and this effect cannot be rescued by further addition of EGF. These results demonstrate that STAT5 activated by EGF constitutes an important cascade for the regulation of cell proliferation and invasion in trophoblast cells.

Stimulation of the JAK/STAT pathway by LIF and OSM in the human granulosa cell line COV434.

The development of the follicle and competent oocyte is highly coordinated, requiring interplay among several systems. These implicate endocrine, immune, and metabolic signals, intrafollicular paracrine factors from theca, mural, and cumulus granulosa cells, and the oocyte itself. Granulosa cells play a key role in their interaction. COV434 is one of the few human granulosa cell lines that can be used as an in vitro model for ovarian research. We aimed to evaluate the possible activation of the Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway by IL-6-type cytokines leukemia inhibitory factor (LIF) and oncostatin M (OSM) in COV434 cells. Expression of GP130 (glycoprotein 130), STAT3 (signal transducer and activators of transcription 3), PIAS3 (protein inhibitor of activated STAT 3), and SOCS3 (suppressor of cytokine signaling 3) genes after stimulation with LIF or OSM was assessed using RT-qPCR (real-time PCR). GP130 transcripts were significantly upregulated after incubation with LIF or OSM for 24h. Expression of the STAT3 gene was stimulated only after incubation with LIF, but not OSM. SOCS3 showed significant upregulation for all time periods and the levels of PIAS3 were initially down- and after 24h upregulated. Furthermore, the major signaling components of the JAK/STAT pathway, GP130 and STAT3, and the kinase activation patterns of STAT3, were examined at protein level. We found constitutive protein expression for GP130, STAT3, pSTAT3(ser727) and upregulation of pSTAT3(tyr705) by LIF and OSM. Our results demonstrate the activation of the JAK/STAT pathway by LIF and OSM in human granulosa cells.

Only humans have human placentas: molecular differences between mice and humans.

The placenta is one of the organs with the highest evolutionary diversity among animal species. In consequence, an animal model that reflects human placentation exactly does not exist. However, the mouse is the most frequently used animal model for placenta and pregnancy research. It possesses a hemochorial placenta, which is similar, but also different from the human placenta. The question whether the similarities are sufficient for the achievement of useful results with regard to human pregnancy was debated recently at the 11th Congress of the European Society for Reproductive Immunology (Budapest, Hungary). Here, we discuss the molecular features of the human placenta that are restricted to primates or even to humans. Many of the primate-specific genetic novelties, e.g., the large microRNA cluster on chromosome 19, have been detected during the last 10-15 years and could not be referred to in earlier discussions. Now, in the light of recent findings and a better understanding of interspecies differences, we conclude that the mouse model is often overvalued. Owing to the increasing number of known human-specific factors in human placentation we consider that many aspects of human placentation can only be understood on the basis of experiments on human cells and tissues in combination with data collections from human subject studies.