ResearchPad - blastocysts https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[The transition from local to global patterns governs the differentiation of mouse blastocysts]]> https://www.researchpad.co/article/elastic_article_14745 During mammalian blastocyst development, inner cell mass (ICM) cells differentiate into epiblast (Epi) or primitive endoderm (PrE). These two fates are characterized by the expression of the transcription factors NANOG and GATA6, respectively. Here, we investigate the spatio-temporal distribution of NANOG and GATA6 expressing cells in the ICM of the mouse blastocysts with quantitative three-dimensional single cell-based neighbourhood analyses. We define the cell neighbourhood by local features, which include the expression levels of both fate markers expressed in each cell and its neighbours, and the number of neighbouring cells. We further include the position of a cell relative to the centre of the ICM as a global positional feature. Our analyses reveal a local three-dimensional pattern that is already present in early blastocysts: 1) Cells expressing the highest NANOG levels are surrounded by approximately nine neighbours, while 2) cells expressing GATA6 cluster according to their GATA6 levels. This local pattern evolves into a global pattern in the ICM that starts to emerge in mid blastocysts. We show that FGF/MAPK signalling is involved in the three-dimensional distribution of the cells and, using a mutant background, we further show that the GATA6 neighbourhood is regulated by NANOG. Our quantitative study suggests that the three-dimensional cell neighbourhood plays a role in Epi and PrE precursor specification. Our results highlight the importance of analysing the three-dimensional cell neighbourhood while investigating cell fate decisions during early mouse embryonic development.

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<![CDATA[Comparison of two bovine serum pregnancy tests in detection of artificial insemination pregnancies and pregnancy loss in beef cattle]]> https://www.researchpad.co/article/5c5217cbd5eed0c4847945d9

Blood tests for early detection of pregnancy in cattle based on pregnancy-associated glycoproteins (PAGs) are commercially available. The objective of these studies were to compare the accuracy of blood tests to transrectal ultrasonography in detecting AI pregnancies, and to compare the accuracy of blood tests in predicting pregnancy loss. Beef cattle from 6 herds were synchronized using a recommended CIDR based protocol (Study 1: n = 460; Study 2: n = 472). Pregnancy status was determined by transrectal ultrasonography between days 28–40 following AI, blood samples were collected at this time. In study 2 a final pregnancy determination was performed at the end of the breeding season to determine pregnancy loss. Each serum sample was examined for PAG concentrations using a microtiter plate reader and/or scored by two technicians blind to pregnancy status and pregnancy loss. For study 1 Cohen’s kappa statistics were calculated to assess the agreement between each test and transrectal ultrasonography. For study 2 data was analyzed using the GLIMMIX procedure of SAS with herd as a random effect, and loss, age, and their interaction included in the model. Agreement was good to very good for each test. There was no difference (P = 0.79) in sensitivity, but a difference (P<0.01) in specificity of the assays (88%, 64%, 87%, 90%) and in the overall percent correct (93%, 84%, 93%, 93%). There was an effect of pregnancy loss (P = 0.04), age (P = 0.0002), and their interaction (P = 0.06) on PAG concentrations. In conclusion both pregnancy tests were accurate at detecting AI pregnancies, and were in very good agreement with transrectal ultrasonography. Both tests detected differences in PAGs among females that maintained and lost pregnancy; however, prediction proved to be difficult as most females were above the threshold and would have been considered pregnant on the day of testing.

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<![CDATA[Melatonin: The smart molecule that differentially modulates autophagy in tumor and normal placental cells]]> https://www.researchpad.co/article/5c40f7abd5eed0c484386589

Melatonin has protective roles in normal cells and cytotoxic actions in cancer cells, with effects involving autophagy and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) transcription factor pathways. Hypoxia/reoxygenation (H/R) induces oxidative damage and apoptosis. These consequences activate autophagy, which degrades damaged cellular content, as well as activates Nrf2 the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) transcription factor, and thereby the expression of protective genes. Melatonin has protective roles in normal cells and cytotoxic actions in cancer cells, with effects involving autophagy and Nrf2 pathways. The current study shows melatonin to differentially modulate autophagy and Nrf2 pathways in tumor and normal placental cells exposed to H/R. BeWo, a human placental choriocarcinoma cell line, and primary villous cytotrophoblasts isolated from normal term placenta, were maintained in normoxia (8% O2) for 24 h or exposed to hypoxia (0.5% of O2 for 4 h) followed by 20 h of normoxia, creating a situation of H/R, in the presence or absence of 1 mM melatonin. Melatonin induced a 7-fold increase in the activation of 5' adenosine monophosphate-activated protein kinase (AMPK)α, an upstream modulator of autophagy, rising to a 16-fold increase in BeWo cells co-exposed to H/R and melatonin, compared to controls. H/R induced autophagosome formation via the increased expression of Beclin-1 (by 94%) and ATG7 (by 97%) in BeWo cells. Moreover, H/R also induced autophagic activity, indicated by the by the 630% increase in P62, and increased Nrf2 by 314% in BeWo cells. In H/R conditions, melatonin reduced autophagic activity by 74% and Nrf2 expression activation by 300%, leading to BeWo cell apoptosis. In contrast, In human primary villous cytotrophoblasts, H/R induced autophagy and Nrf2, which melatonin further potentiated, thereby affording protection against H/R. This study demonstrates that melatonin differentially modulates autophagy and the Nrf2 pathway in normal vs. tumor trophoblast cells, being cytoprotective in normal cells whilst increasing apoptosis in tumoral trophoblast cells.

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<![CDATA[Bovine epididymal spermatozoa treatment for in vitro fertilization: Heparin accelerates fertilization and enables a reduction in coincubation time]]> https://www.researchpad.co/article/5c3d0184d5eed0c48403c11d

This study aimed to establish a protocol for in vitro embryo production using epididymal sperm (EP). Samples were obtained from ejaculated sperm (EJ) and the epididymis of 7 Gir bulls. First, the effect of heparin (+) on the viability, longevity (Experiment 1) and fertilization rates (Experiment 2) of the EP was evaluated. In experiment 2, a pool of EP and EJ sperm (n = 7) was coincubated with cumulus-oocyte complexes (COCs) for 0, 3, 6, 12 and 18 h, and the fertilization rate (FR) was evaluated. A third experiment was performed to test sperm treatments for IVP using the Percoll (P) or PureSperm (PS) gradients or a spTALP wash for sperm selection. Cleavage, blastocyst rate (BR) and embryo sex were evaluated. In experiment 4, embryos were produced using 6, 12, and 18 h of sperm-oocyte coincubation. The cleavage, BR, and total number and percentage of apoptotic cells were determined. Heparin affected EP viability, longevity and FR. After 6 h, 82% of the oocytes were fertilized in the EP+ group, a higher value (P<0.05) than that in the EJ (19%) and EP- (42%) groups. At 12 and 18 h, FR remained higher in the EP+ group, and a gradual increase in polyspermy was observed. The use of a P or PS gradient yielded a similar BR on D7 (54% and 52%), which was higher than the rate obtained using the washing method (37%). The embryos produced by EP and selected in a P or PS gradient resulted in a sex deviation in favor of male embryos (P>0.05). No differences (P>0.05) were observed among the groups that were coincubated for 6, 12 and 18 h with respect to embryo production, kinetics of development, total cell number and percentage of apoptotic cells. In conclusion, IVF time can be reduced to 6 h without affecting embryo production and quality. In addition, EP sperm selection can be performed by either a PS or P gradient.

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<![CDATA[FOXO1 regulates uterine epithelial integrity and progesterone receptor expression critical for embryo implantation]]> https://www.researchpad.co/article/5bfc623ad5eed0c484ec77fd

Successful embryo implantation requires a receptive endometrium. Poor uterine receptivity can account for implantation failure in women who experience recurrent pregnancy loss or multiple rounds of unsuccessful in vitro fertilization cycles. Here, we demonstrate that the transcription factor Forkhead Box O1 (FOXO1) is a critical regulator of endometrial receptivity in vivo. Uterine ablation of Foxo1 using the progesterone receptor Cre (PgrCre) mouse model resulted in infertility due to altered epithelial cell polarity and apoptosis, preventing the embryo from penetrating the luminal epithelium. Analysis of the uterine transcriptome after Foxo1 ablation identified alterations in gene expression for transcripts involved in the activation of cell invasion, molecular transport, apoptosis, β-catenin (CTNNB1) signaling pathway, and an increase in PGR signaling. The increase of PGR signaling was due to PGR expression being retained in the uterine epithelium during the window of receptivity. Constitutive expression of epithelial PGR during this receptive period inhibited expression of FOXO1 in the nucleus of the uterine epithelium. The reciprocal expression of PGR and FOXO1 was conserved in human endometrial samples during the proliferative and secretory phase. This demonstrates that expression of FOXO1 and the loss of PGR during the window of receptivity are interrelated and critical for embryo implantation.

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<![CDATA[Characteristics of candidate genes associated with embryonic development in the cow: Evidence for a role for WBP1 in development to the blastocyst stage]]> https://www.researchpad.co/article/5989db5cab0ee8fa60bdfe7a

The goal was to gain understanding of how 12 genes containing SNP previously related to embryo competence to become a blastocyst (BRINP3, C1QB, HSPA1L, IRF9, MON1B, PARM1, PCCB, PMM2, SLC18A2, TBC1D24, TTLL3 and WBP1) participate in embryonic development. Gene expression was evaluated in matured oocytes and embryos. BRINP3 and C1QB were not detected at any stage. For most other genes, transcript abundance declined as the embryo developed to the blastocyst stage. Exceptions were for PARM1 and WBP1, where steady-state mRNA increased at the 9–16 cell stage. The SNP in WBP1 caused large differences in the predicted three-dimensional structure of the protein while the SNP in PARM1 caused smaller changes. The mutation in WBP1 causes an amino acid substitution located close to a P-P-X-Y motif involved in protein-protein interactions. Moreover, the observation that the reference allele varies between mammalian species indicates that the locus has not been conserved during mammalian evolution. Knockdown of mRNA for WBP1 decreased the percent of putative zygotes becoming blastocysts and reduced the number of trophectoderm cells and immunoreactive CDX2 in the resulting blastocysts. WBP1 is an important gene for embryonic development in the cow. Further research to identify how the SNP in WBP1 affects processes leading to differentiation of the embryo into TE and ICM lineages is warranted.

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<![CDATA[Promoter Methylation Pattern Controls Corticotropin Releasing Hormone Gene Activity in Human Trophoblasts]]> https://www.researchpad.co/article/5989db51ab0ee8fa60bdc433

Placental CRH production increases with advancing pregnancy in women and its course predicts gestational length. We hypothesized that CRH gene expression in the placenta is epigenetically controlled setting gestational trajectories characteristic of normal and pathological pregnancies. Here we determined histone modification and DNA methylation levels and DNA methylation patterns at the CRH promoter in primary trophoblast cultures by chromatin immunoprecipitation combined with clonal bisulfite sequencing and identified the transcriptionally active epialleles that associate with particular histone modifications and transcription factors during syncytialisation and cAMP-stimulation. CRH gene expression increased during syncytial differentiation and cAMP stimulation, which was associated with increased activating and decreased repressive histone modification levels at the promoter. DNA methylation levels remained unchanged. The nine CpGs of the CRH proximal promoter were partially and allele-independently methylated displaying many (>100) epialleles. RNA-polymerase-II (Pol-II) bound only to three particular epialleles in cAMP-stimulated cells, while phospho-cAMP response element-binding protein (pCREB) bound to only one epiallele, which was different from those selected by Pol-II. Binding of TATA-binding protein increased during syncytial differentiation preferentially at epialleles compatible with Pol-II and pCREB binding. Histone-3 acetylation was detected only at epialleles targeted by Pol-II and pCREB, while gene activating histone-4 acetylation and histone-3-lysine-4 trimethylation occurred at CRH epialleles not associated with Pol-II or pCREB. The suppressive histone-3-lysine-27 trimethyl and–lysine-9 trimethyl modifications showed little or no epiallele preference. The epiallele selectivity of activating histone modifications and transcription factor binding demonstrates the epigenetic and functional diversity of the CRH gene in trophoblasts, which is controlled predominantly by the patterns, not the overall extent, of promoter methylation. We propose that conditions impacting on epiallele distribution influence the number of transcriptionally active CRH gene copies in the trophoblast cell population determining the gestational trajectory of placental CRH production in normal and pathological pregnancies.

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<![CDATA[Human Oocyte-Derived Methylation Differences Persist in the Placenta Revealing Widespread Transient Imprinting]]> https://www.researchpad.co/article/5989d9eeab0ee8fa60b6d6b4

Thousands of regions in gametes have opposing methylation profiles that are largely resolved during the post-fertilization epigenetic reprogramming. However some specific sequences associated with imprinted loci survive this demethylation process. Here we present the data describing the fate of germline-derived methylation in humans. With the exception of a few known paternally methylated germline differentially methylated regions (DMRs) associated with known imprinted domains, we demonstrate that sperm-derived methylation is reprogrammed by the blastocyst stage of development. In contrast a large number of oocyte-derived methylation differences survive to the blastocyst stage and uniquely persist as transiently methylated DMRs only in the placenta. Furthermore, we demonstrate that this phenomenon is exclusive to primates, since no placenta-specific maternal methylation was observed in mouse. Utilizing single cell RNA-seq datasets from human preimplantation embryos we show that following embryonic genome activation the maternally methylated transient DMRs can orchestrate imprinted expression. However despite showing widespread imprinted expression of genes in placenta, allele-specific transcriptional profiling revealed that not all placenta-specific DMRs coordinate imprinted expression and that this maternal methylation may be absent in a minority of samples, suggestive of polymorphic imprinted methylation.

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<![CDATA[MSX2 Induces Trophoblast Invasion in Human Placenta]]> https://www.researchpad.co/article/5989dab9ab0ee8fa60bade75

Normal implantation depends on appropriate trophoblast growth and invasion. Inadequate trophoblast invasion results in pregnancy-related disorders, such as early miscarriage and pre-eclampsia, which are dangerous to both the mother and fetus. Msh Homeobox 2 (MSX2), a member of the MSX family of homeobox proteins, plays a significant role in the proliferation and differentiation of various cells and tissues, including ectodermal organs, teeth, and chondrocytes. Recently, MSX2 was found to play important roles in the invasion of cancer cells into adjacent tissues via the epithelial-mesenchymal transition (EMT). However, the role of MSX2 in trophoblastic invasion during placental development has yet to be explored. In the present study, we detected MSX2 expression in cytotrophoblast, syncytiotrophoblast, and extravillous cytotrophoblast cells of first or third trimester human placentas via immunohistochemistry analysis. Furthermore, we found that the in vitro invasive ability of HTR8/SVneo cells was enhanced by exogenous overexpression of MSX2, and that this effect was accompanied by increased protein expression of matrix metalloproteinase-2 (MMP-2), vimentin, and β-catenin. Conversely, treatment of HTR8/SVneo cells with MSX2-specific siRNAs resulted in decreased protein expression of MMP-2, vimentin, and β-catenin, and reduced invasion levels in a Matrigel invasion test. Notably, however, treatment with the MSX2 overexpression plasmid and the MSX2 siRNAs had no effect on the mRNA expression levels of β-catenin. Meanwhile, overexpression of MSX2 and treatment with the MSX2-specific siRNA resulted in decreased and increased E-cadherin expression, respectively, in JEG-3 cells. Lastly, the protein expression levels of MSX2 were significantly lower in human pre-eclamptic placental villi than in the matched control placentas. Collectively, our results suggest that MSX2 may induce human trophoblast cell invasion, and dysregulation of MSX2 expression may be associated with pre-eclampsia.

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<![CDATA[The Principal Forces of Oocyte Polarity Are Evolutionary Conserved but May Not Affect the Contribution of the First Two Blastomeres to the Blastocyst Development in Mammals]]> https://www.researchpad.co/article/5989dad4ab0ee8fa60bb748d

Oocyte polarity and embryonic patterning are well-established features of development in lower species. Whether a similar form of pre-patterning exists in mammals is currently under hot debate in mice. This study investigated this issue for the first time in ovine as a large mammal model. Microsurgical trisection of unfertilized MII-oocytes revealed that cortical cytoplasm around spindle (S) contained significant amounts of total maternal mRNAs and proteins compared to matched cytoplast hemispheres that were located either near (NS) or far (FS) -to-spindle. RT-qPCR provided striking examples of maternal mRNA localized to subcellular substructures S (NPM2, GMNN, H19, PCAF, DNMT3A, DNMT1, and STELLA), NS (SOX2, NANOG, POU5F1, and TET1), and FS (GCN) of MII oocyte. Immunoblotting revealed that specific maternal proteins DNMT3A and NANOG were asymmetrically enriched in MII-spindle-half of the oocytes. Topological analysis of sperm entry point (SEP) revealed that sperm preferentially entered via the MII-spindle-half of the oocytes. Even though, the topological position of first cleavage plane with regard to SEP was quite stochastic. Spatial comparison of lipid content revealed symmetrical distribution of lipids between 2-cell blastomeres. Lineage tracing using Dil, a fluorescent dye, revealed that while the progeny of leading blastomere of 2-cell embryos contributed to more cells in the developed blastocysts compared to lagging counterpart, the contributions of leading and lagging blastomeres to the embryonic-abembryonic parts of the developed blastocysts were almost unbiased. And finally, separated sister blastomeres of 2-cell embryos had an overall similar probability to arrest at any stage before the blastocyst (2-cell, 4-cell, 8-cell, and morula) or to achieve the blastocyst stage. It was concluded that the localization of maternal mRNAs and proteins at the spindle are evolutionarily conserved between mammals unfertilized ovine oocyte could be considered polar with respect to the spatial regionalization of maternal transcripts and proteins. Even though, the principal forces of this definitive oocyte polarity may not persist during embryonic cleavages.

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<![CDATA[Systematic Review of Micro-RNA Expression in Pre-Eclampsia Identifies a Number of Common Pathways Associated with the Disease]]> https://www.researchpad.co/article/5989da52ab0ee8fa60b8e20d

Background

Pre-eclampsia (PE) is a complex, multi-systemic condition of pregnancy which greatly impacts maternal and perinatal morbidity and mortality. MicroRNAs (miRs) are differentially expressed in PE and may be important in helping to understand the condition and its pathogenesis.

Methods

Case-control studies investigating expression of miRs in PE were collected through a systematic literature search. Data was extracted and compared from 58 studies to identify the most promising miRs associated with PE pathogenesis and identify areas of methodology which could account for often conflicting results.

Results

Some of the most frequently differentially expressed miRs in PE include miR-210, miR-223 and miR-126/126* which associate strongly with the etiological domains of hypoxia, immunology and angiogenesis. Members of the miR-515 family belonging to the imprinted chromosome 19 miR cluster with putative roles in trophoblast invasion were also found to be differentially expressed. Certain miRs appear to associate with more severe forms of PE such as miR-210 and the immune-related miR-181a and miR-15 families. Patterns of miR expression may help pinpoint key pathways (e.g. IL-6/miR-223/STAT3) and aid in untangling the heterogeneous nature of PE. The detectable presence of many PE-associated miRs in antenatal circulatory samples suggests their usefulness as predictive biomarkers. Further progress in ascertaining the clinical value of miRs and in understanding how they might contribute to pathogenesis is predicated upon resolving current methodological challenges in studies. These include differences in diagnostic criteria, cohort characteristics, sampling technique, RNA isolation and platform-dependent variation in miR profiling.

Conclusion

Reviewing studies of PE-associated miRs has revealed their potential as informants of underlying target genes and pathways relating to PE pathogenesis. However, the incongruity in results across current studies hampers their capacity to be useful biomarkers of the condition.

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<![CDATA[Plasma Level of Placenta-Derived Macrophage-Stimulating Protein -Chain in Preeclampsia before 20 Weeks of Pregnancy]]> https://www.researchpad.co/article/5989db13ab0ee8fa60bccb03

Object

This study aimed to investigate the diagnostic value of placenta-derived macrophage-stimulating protein α-chain (MSP-α) before the 20th week of gestation for the early diagnosis of preeclampsia (PE).

Methods and Materials

Two parts of this nested case-control study were simultaneously executed, and 1500 pregnant women were recruited. A total of 124 pregnant women were included in the plasma analysis part of this study. The MSP-α plasma level was measured before the 20th week of gestation, and the participants were followed until delivery. A case group of 62 women with PE and a control group of 62 women matched by gestational age, maternal age, and pre-pregnancy BMI (with normotensive pregnancies) were evaluated. In the placenta analysis part of this nested case-control study, the placentas of 34 pregnant women were randomly obtained. The placental levels of MSP were measured in 17 individuals with PE (case group) and in 17 women with a normotensive pregnancy matched by gestational age and maternal age (control group).

Results

The plasma level of MSP-α was higher in the PE group than in the control group before the 20th week of gestation (p < 0.001). In addition, compared to the women with severe features in the PE group, those without severe features had a significantly higher plasma MSP-α level before the 20th week of gestation (p < 0.001). The area under the receiver operating characteristic curve (AUC) of MSP-α before the 20th week of gestation was 0.905 (95% CI, 0.811–0.962) for the women with early-onset PE without severe features. With regard to the placenta, the PE group (accumulated optical density, IOD [SUM] = 8862.37 ± 2064.42) exhibited increased MSP staining (more intense MSP staining or more extensive staining) compared with the control group (normal pregnancies (IOD [SUM] = 447.92 ± 114.72, P < 0.001). Furthermore, increased MSP staining was detected among the women without severe features compared with those with severe features in the PE group (IOD [SUM]: 12192.65 ± 5325.56 vs. 4104.83 ± 2383.06, P = 0.021).

Conclusion

According to the findings of this study, the plasma level of MSP-α may be associated with PE, and MSP-α may be considered a candidate protein for further analysis in studies of PE. Multicenter studies with larger sample sizes must be performed in the future to obtain accurate results regarding the predictive value of MSP-α in combination with other protein factors for the early diagnosis of PE.

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<![CDATA[Real-Time Tracking of BODIPY-C12 Long-Chain Fatty Acid in Human Term Placenta Reveals Unique Lipid Dynamics in Cytotrophoblast Cells]]> https://www.researchpad.co/article/5989db4bab0ee8fa60bda67e

While the human placenta must provide selected long-chain fatty acids to support the developing fetal brain, little is known about the mechanisms underlying the transport process. We tracked the movement of the fluorescently labeled long-chain fatty acid analogue, BODIPY-C12, across the cell layers of living explants of human term placenta. Although all layers took up the fatty acid, rapid esterification of long-chain fatty acids and incorporation into lipid droplets was exclusive to the inner layer cytotrophoblast cells rather than the expected outer syncytiotrophoblast layer. Cytotrophoblast is a progenitor cell layer previously relegated to a repair role. As isolated cytotrophoblasts differentiated into syncytialized cells in culture, they weakened their lipid processing capacity. Syncytializing cells suppress previously active genes that regulate fatty-acid uptake (SLC27A2/FATP2, FABP4, ACSL5) and lipid metabolism (GPAT3, LPCAT3). We speculate that cytotrophoblast performs a previously unrecognized role in regulating placental fatty acid uptake and metabolism.

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<![CDATA[Changes in Parthenogenetic Imprinting Patterns during Reprogramming by Cell Fusion]]> https://www.researchpad.co/article/5989da52ab0ee8fa60b8e381

Differentiated somatic cells can be reprogrammed into the pluripotent state by cell-cell fusion. In the pluripotent state, reprogrammed cells may then self-renew and differentiate into all three germ layers. Fusion-induced reprogramming also epigenetically modifies the somatic cell genome through DNA demethylation, X chromosome reactivation, and histone modification. In this study, we investigated whether fusion with embryonic stem cells (ESCs) also reprograms genomic imprinting patterns in somatic cells. In particular, we examined imprinting changes in parthenogenetic neural stem cells fused with biparental ESCs, as well as in biparental neural stem cells fused with parthenogenetic ESCs. The resulting hybrid cells expressed the pluripotency markers Oct4 and Nanog. In addition, methylation of several imprinted genes except Peg3 was comparable between hybrid cells and ESCs. This finding indicates that reprogramming by cell fusion does not necessarily reverse the status of all imprinted genes to the state of pluripotent fusion partner.

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<![CDATA[Interdependence of JAK-STAT and MAPK signaling pathways during EGF-mediated HTR-8/SVneo cell invasion]]> https://www.researchpad.co/article/5989db5cab0ee8fa60be002c

Invasion of trophoblast cells is spatio-temporally regulated by various cytokines and growth factors. In pregnancy, complications like preeclampsia, shallow invasion of trophoblast cells and low amounts of epidermal growth factor (EGF) have been reported. In the present study, regulatory mechanisms associated with EGF-mediated invasion in HTR-8/SVneo trophoblastic cells have been delineated. Treatment of HTR-8/SVneo cells with EGF (10 ng/ml) led to eight fold increase (p < 0.05) in invasion. Increased invasion of HTR-8/SVneo cells by EGF was associated with an increase in phosphorylation of ERK½. In addition, significant phosphorylation of STAT1 (ser 727) and STAT3 (both tyr 705 and ser 727 residues) was also observed, accompanied by a decrease in total STAT1. Inhibition of ERK½ phosphorylation by U0126 (10 μM) led to a significant decrease in EGF-mediated invasion with simultaneous decrease in the phosphorylated forms of STAT3 and STAT1. Decrease in total STAT1 was also reversed on inhibition of ERK½. Interestingly, inhibition of STAT3 by siRNA led to a significant decrease in EGF-mediated invasion of HTR-8/SVneo cells and phosphorylation of STAT1, but it did not have any effect on the activation of ERK½. On the other hand, inhibition of STAT1 by siRNA, also led to a significant decrease in the EGF-mediated invasion of HTR-8/SVneo cells, showed concomitant decrease in ERK½ phosphorylation and STAT3 phosphorylation at ser 727 residue. These results suggest cross-communication between ERK½ and JAK-STAT pathways during EGF-mediated increase in invasion of trophoblast cells; phosphorylation at ser 727 residue of both STAT3 and STAT1 appears to be critical.

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<![CDATA[No evidence for genome editing in mouse zygotes and HEK293T human cell line using the DNA-guided Natronobacterium gregoryi Argonaute (NgAgo)]]> https://www.researchpad.co/article/5989db5eab0ee8fa60be0b28

A recently published research article reported that the extreme halophile archaebacterium Natronobacterium gregoryi Argonaute enzyme (NgAgo) could cleave the cellular DNA under physiological temperature conditions in cell line and be implemented as an alternative to CRISPR/Cas9 genome editing technology. We assessed this claim in mouse zygotes for four loci (Sptb, Tet-1, Tet-2 and Tet-3) and in the human HEK293T cell line for the EMX1 locus. Over 100 zygotes were microinjected with nls-NgAgo-GK plasmid provided from Addgene and various concentrations of 5’-phosphorylated guide DNA (gDNA) from 2.5 ng/μl to 50 ng/μl and cultured to blastocyst stage of development. The presence of indels was verified using T7 endonuclease 1 assay (T7E1) and Sanger sequencing. We reported no evidence of successful editing of the mouse genome. We then assessed the lack of editing efficiency in HEK293T cell line for the EMX1 endogenous locus by monitoring the NgAgo protein expression level and the editing efficiency by T7E1 assay and Sanger sequencing. We reported that the NgAgo protein was expressed from 8 hours to a maximum expression at 48 hours post-transfection, confirming the efficient delivery of the plasmid and the gDNA but no evidence of successful editing of EMX1 target in all transfected samples. Together our findings indicate that we failed to edit using NgAgo.

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<![CDATA[miR-125b Enhances IL-8 Production in Early-Onset Severe Preeclampsia by Targeting Sphingosine-1-Phosphate Lyase 1]]> https://www.researchpad.co/article/5989dad5ab0ee8fa60bb7abf

Preeclampsia (PE) is one of the leading causes of maternal and perinatal mortality and morbidity. One of the main hallmarks observed in PE is impaired inflammation state. In the current study, we found that miR-125b was deregulated in placental tissues and plasma derived from PE patients, which suggest a potential association between this miRNA and the pathogenesis of PE. Overexpression of miR-125b significantly reduced SGPL1 expression, and luciferase assays confirmed that SGPL1 is a direct target of miR-125b. We also found that miR-125b enhanced IL-8 production by directly targeting sphingosine-1-phosphate lyase 1 (SGPL1), and this effect could be reversed by SGPL1 overexpression. In placentas derived from PE patients, a negative correlation of miR-125b and SGPL1 was observed, and IL-8 was validated to be increased in the circulation of PE patients. Our data demonstrated a critical role of miR-125b in IL-8 production and the development of PE.

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<![CDATA[A Patient Friendly Corifollitropin Alfa Protocol without Routine Pituitary Suppression in Normal Responders]]> https://www.researchpad.co/article/5989da0bab0ee8fa60b77ab2

The release of corifollitropin alfa simplifies daily injections of short-acting recombinant follicular stimulating hormone (rFSH), and its widely-used protocol involves short-acting gonadotropins supplements and a fixed GnRH antagonist regimen, largely based on follicle size. In this study, the feasibility of corifollitropin alfa without routine pituitary suppression was evaluated. A total of 288 patients were stimulated by corifollitropin alfa on cycle day 3 following with routine serum hormone monitoring and follicle scanning every other day after 5 days of initial stimulation, and a GnRH antagonist (0.25 mg) was only used prophylactically when the luteinizing hormone (LH) was ≧ 6 IU/L (over half of the definitive LH surge). The incidence of premature LH surge (≧ 10 IU/L) was 2.4% (7/288) before the timely injection of a single GnRH antagonist, and the elevated LH level was dropped down from 11.9 IU/L to 2.2 IU/L after the suppression. Two hundred fifty-one patients did not need any antagonist (87.2% [251/288]) throughout the whole stimulation. No adverse effects were observed regarding oocyte competency (fertilization rate: 78%; blastocyst formation rate: 64%). The live birth rate per OPU cycle after the first cryotransfer was 56.3% (161/286), and the cumulative live birth rate per OPU cycle after cyrotransfers was 69.6% (199/286). Of patients who did and did not receive GnRH antagonist during stimulation, no significant difference existed in the cumulative live birth rates (78.4% vs. 68.3%, p = 0.25). The results demonstrated that the routine GnRH antagonist administration is not required in the corifollitropin-alfa cycles using a flexible and hormone-depended antagonist regimen, while the clinical outcome is not compromised. This finding reveals that the use of a GnRH antagonist only occasionally may be needed.

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<![CDATA[Requirement of Leukemia Inhibitory Factor or Epidermal Growth Factor for Pre-Implantation Embryogenesis via JAK/STAT3 Signaling Pathways]]> https://www.researchpad.co/article/5989db06ab0ee8fa60bc846a

Leukemia inhibitory factor (LIF) plays a key role in the survivability of mouse embryos during pre-implantation. In this study, we verified the role of LIF by detecting gene expression in morula stage embryos through DNA microarray. Our results showed that LIF knockdown affected expression of 369 genes. After LIF supplementation, the epidermal growth factor (EGF) is most affected by LIF expression. To observe the correlation between LIF and EGF, the LIF knockdown embryos were supplemented with various growth factors, including LIF, EGF, GM-CSF, TGF, and IGF II. Only LIF and EGF caused the rate of blastocyst development to recover significantly from 52% of control to 83% and 93%, respectively. All of the variables, including the diameter of blastocysts, the number of blastomeres, and cells in ICM and TE, were almost restored. Moreover, EGF knockdown also impaired blastocyst development, which was reversed by LIF or EGF supplementation. The treatment with various signaling suppressors revealed that both EGF and LIF promoted embryonic development through the JAK/STAT3 signaling pathway. These data suggest that the EGF and LIF can be compensatory to each other during early embryonic development, and at least one of them is necessary for sustaining the normal development of pre-implantation embryos.

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<![CDATA[Top quality blastocyst formation rates in relation to progesterone levels on the day of oocyte maturation in GnRH antagonist IVF/ICSI cycles]]> https://www.researchpad.co/article/5989db5cab0ee8fa60bdfe0e

Cycles with progesterone elevation during controlled ovarian stimulation (COS) for IVF/ICSI are commonly managed with a “freeze-all” strategy, due to a well-recognized detrimental effect of high progesterone levels on endometrial receptivity. However, also a detrimental effect of elevated progesterone on day-3 embryo quality has recently been found with regards to top quality embryo formation rate. Because blastocyst culture and cryopreservation are largely adopted, we deemed relevant to determine whether this detrimental effect is also seen on blastocyst quality on day 5–6. This issue was investigated through a large two-center retrospective study including 986 GnRH antagonist IVF/ICSI cycles and using top quality blastocyst formation rate as the main outcome. Results showed that on multivariate analysis sperm motility (p<0.01) and progesterone levels at ovulation triggering (p = 0.01) were the only two variables that significantly predicted top quality blastocyst formation rate after adjusting for relevant factors including female age, BMI, basal AMH and total dose of FSH used for COS. More specifically, progesterone levels at induction showed an inverse relation with top quality blastocyst formation (correlation coefficient B = -1.08, 95% CI -1.9 to -0.02) and ROC curve analysis identified P level >1.49 ng/ml as the best cut-off for identification of patients at risk for the absence of top quality blastocysts (AUC 0.55, p<0.01). Our study is the first to investigate the top quality blastocyst formation rate in relation to progesterone levels in IVF/ICSI cycles, showing that increasing progesterone is associated with lower rates of top quality blastocyst. Hence, the advantages of prolonging COS to maximize the number of collected oocytes might eventually be hindered by a decrease in top quality blastocysts available for transfer, if increasing progesterone levels are observed. This observation extends the results of two recent studies focused on day-3 embryos and deserves further research.

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