FGFR1 regulates trophectoderm development and facilitates blastocyst implantation.
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ABSTRACT:
SUBMITTER: Kurowski A
PROVIDER: S-EPMC6370302 | biostudies-literature | 2019 Feb
REPOSITORIES: biostudies-literature
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FGFR1 regulates trophectoderm development and facilitates blastocyst implantation.
Kurowski Agata A Molotkov Andrei A Soriano Philippe P
Developmental biology 20181212 1
FGF signaling plays important roles in many aspects of mammalian development. Fgfr1<sup>-/-</sup> and Fgfr1<sup>-/-</sup>Fgfr2<sup>-/-</sup> mouse embryos on a 129S4 co-isogenic background fail to survive past the peri-implantation stage, whereas Fgfr2<sup>-/-</sup> embryos die at midgestation and show defects in limb and placental development. To investigate the basis for the Fgfr1<sup>-/-</sup> and Fgfr1<sup>-/-</sup>Fgfr2<sup>-/-</sup> peri-implantation lethality, we examined the role of FGFR ...[more]
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Project description:BackgroundA key step in embryo implantation is the adhesion to and invasion of the endometrium by the blastocyst trophectoderm. The envelope proteins of HERV-W and -FRD (human endogenous retrovirus-W and -FRD), syncytin-1 and syncytin-2, are mainly distributed in the placenta, and play important roles in the development of the placenta. The placenta originates from the trophectoderm of the blastocyst. It is unclear whether the envelope proteins of HERV-W and -FRD have an effect on the development of the trophectoderm and whether they have any association with the implantation of the blastocyst.MethodsThe whole-genome amplification products of the human blastocyst trophectoderm were used to measure the copy number of syncytin-1 and syncytin-2 using real time qPCR. In addition, clinical data associated with the outcome of pregnancies was collected, and included age, body mass index (BMI), basic follicle stimulating hormone(bFSH), rate of primary infertility and oligo-astheno-teratospermia, the thickness of the endometrium on the day of endometrial transformation, the levels of estrogen and progestin on the transfer day, the days and the morphological scores of the blastocysts. The expression of mRNA and the copy numbers of syncytin-1 and syncytin-2 in H1 stem cells, and in differentiated H1 cells, induced by BMP4, were measured using real time qPCR.ResultsThe relative copy number of syncytin-1 in the pregnant group (median: 424%, quartile: 232%-463%, p < 0.05) was significantly higher than in the non-pregnant group (median: 100%, quartile: 81%-163%). There was a correlation (r s = 0.681, p < 0.001) between the copy number of syncytin-1 and blastocyst implantation after embryo transfer. As the stem cells differentiated, the expression of NANOG mRNA decreased, and the expression of caudal type homeobox 2(CDX2) and β-human chorionic gonadotropin (β-hCG) mRNAs increased. Compared to the undifferentiated cells, the relative expression of the syncytin-1 mRNA was 1.63 (quartile: 0.59-6.37, p > 0.05), 3.36 (quartile: 0.85-14.80, p > 0.05), 10.85 (quartile: 3.39-24.46, p < 0.05) and 67.81 (quartile: 54.07-85.48, p < 0.05) on day 1, 3, 5 and 7, respectively, after the differentiation. The relative expression of syncytin-2 was 5.34 (quartile: 4.50-10.30), 7.90 (quartile: 2.46-14.01), 57.44 (quartile: 38.35-103.87) and 344.76 (quartile: 267.72-440.10) on day 1, 3, 5 and 7, respectively, after the differentiation (p < 0.05). The copy number of syncytin-1 increased significantly during differentiation.ConclusionPreceding the transfer of frozen embryos, the increased copy number of syncytin-1 in the blastocyst trophectoderm was associated with good outcomes of pregnancies.
| S-EPMC7678462 | biostudies-literature
Project description:A systematic review of the literature showed that trophectoderm biopsy could assist in the selection of healthy embryos for uterine transfer without affecting implantation rates. However, previous studies attempting to establish the relationship between trophectoderm gene expression profiles and implantation competency using either microarrays or RNA sequencing strategies, were not sufficiently optimized to handle the exceptionally low RNA inputs available from biopsied material. In this pilot study, we report that differential gene expression in human trophectoderm biopsies assayed by an ultra-sensitive next generation RNA sequencing strategy could predict blastocyst implantation competence. RNA expression profiles from isolated human trophectoderm cells were analysed with established clinical pregnancy being the primary endpoint. Following RNA sequencing, a total of 47 transcripts were found to be significantly differentially expressed between the trophectoderm cells from successfully implanted (competent) versus unsuccessful (incompetent) blastocysts. Of these, 36 transcripts were significantly down-regulated in the incompetent blastocysts, including Hydroxysteroid 17-Beta Dehydrogenase 1 (HSD17B1) and Cytochrome P450 Family 11 Subfamily A Member 1 (CYP11A1), while the remaining 11 transcripts were significantly up-regulated, including BCL2 Antagonist/Killer 1 (BAK1) and KH Domain Containing 1 Pseudogene 1 (KHDC1P1) of which the latter was always detected in the incompetent and absent in all competent blastocysts. Ontological analysis of differentially expressed RNAs revealed pathways involved in steroidogenic processes with high confidence. Novel differentially expressed transcripts were also noted by reference to a de novo sequence assembly. The selection of the blastocyst with the best potential to support full-term pregnancy following single embryo transfer could reduce the need for multiple treatment cycles and embryo transfers. The main limitation was the low sample size (N = 8). Despite this shortcoming, the pilot suggests that trophectoderm biopsy could assist with the selection of healthy embryos for embryo transfer. A larger cohort of samples is needed to confirm these findings. Abbreviations: AMA: advanced maternal age; ART: assisted reproductive technology; CP: clinical pregnancy; DE: differential expression; FDR: false discovery rate; IVF: in vitro fertilization; LD PCR: long distance PCR; qRT-PCR: quantitative real-time PCR; SET: single embryo transfer; TE: trophectoderm.
| S-EPMC6816490 | biostudies-literature
Project description:Mechanisms of implantation such as determination of the attachment pole, fetal-maternal communication, as well as underlying causes of implantation failure are largely unexplored. Here we performed single-cell RNA sequencing (scRNA-seq) on peri-implantation embryos from both humans and mice to explore trophectoderm development and embryo–endometrium crosstalk. We found that the transcriptomes of polar and mural trophectoderm (TE) diverged after embryos hatched from the zona pellucida in both species, with polar TE being more mature than mural TE. The implantation poles show similarities in cell cycle activities, as well as in expression of genes critical for implantation and placentation. Embryos that either fail to attach in vitro or fail to implant in vivo, show abnormalities in pathways related to energy production, protein metabolism and 18S rRNA m6A methylation. These findings uncover the gene expression characteristics of humans and mice TE differentiation during the peri-implantation period and provide new insights into embryo implantation.
2022-08-01 | GSE133200 | GEO
Project description:STUDY QUESTION:What is the impact of adenosine monophosphate-activated protein kinase (AMPK) activation on blastocyst formation, gene expression, and tight junction formation and function? SUMMARY ANSWER:AMPK activity must be tightly controlled for normal preimplantation development and blastocyst formation to occur. WHAT IS KNOWN ALREADY:AMPK isoforms are detectable in oocytes, cumulus cells and preimplantation embryos. Cultured embryos are subject to many stresses that can activate AMPK. STUDY DESIGN, SIZE, DURATION:Two primary experiments were carried out to determine the effect of AICAR treatment on embryo development and maintenance of the blastocoel cavity. Embryos were recovered from superovulated mice. First, 2-cell embryos were treated with a concentration series (0-2000 ?M) of AICAR for 48 h until blastocyst formation would normally occur. In the second experiment, expanded mouse blastocysts were treated for 9 h with 1000 ?M AICAR. PARTICIPANTS/MATERIALS, SETTING, METHODS:Outcomes measured included development to the blastocyst stage, cell number, blastocyst volume, AMPK phosphorylation, Cdx2 and blastocyst formation gene family expression (mRNAs and protein measured using quantitative RT-PCR, immunoblotting, immunofluorescence), tight junction function (FITC dextran dye uptake assay), and blastocyst ATP levels. The reversibility of AICAR treatment was assessed using Compound C (CC), a well-known inhibitor of AMPK, alone or in combination with AICAR. MAIN RESULTS AND THE ROLE OF CHANCE:Prolonged treatment with AICAR from the 2-cell stage onward decreases blastocyst formation, reduces total cell number, embryo diameter, leads to loss of trophectoderm cell contacts and membrane zona occludens-1 staining, and increased nuclear condensation. Treatment with CC alone inhibited blastocyst development only at concentrations that are higher than normally used. AICAR treated embryos displayed altered mRNA and protein levels of blastocyst formation genes. Treatment of blastocysts with AICAR for 9 h induced blastocyst collapse, altered blastocyst formation gene expression, increased tight junction permeability and decreased CDX2. Treated blastocysts displayed three phenotypes: those that were unaffected by treatment, those in which treatment was reversible, and those in which effects were irreversible. LARGE SCALE DATA:Not applicable. LIMITATIONS, REASONS FOR CAUTION:Our study investigates the effects of AICAR treatment on early development. While AICAR does increase AMPK activity and this is demonstrated in our study, AICAR is not a natural regulator of AMPK activity and some outcomes may result from off target non-AMPK AICAR regulated events. To support our results, blastocyst developmental outcomes were confirmed with two other well-known small molecule activators of AMPK, metformin and phenformin. WIDER IMPLICATIONS OF THE FINDINGS:Metformin, an AMPK activator, is widely used to treat type II diabetes and polycystic ovarian disorder (PCOS). Our results indicate that early embryonic AMPK levels must be tightly regulated to ensure normal preimplantation development. Thus, use of metformin should be carefully considered during preimplantation and early post-embryo transfer phases of fertility treatment cycles. STUDY FUNDING AND COMPETING INTEREST(S):Canadian Institutes of Health Research (CIHR) operating funds. There are no competing interests.
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Project description:The establishment of a functional trophectoderm (TE) epithelium is an essential prerequisite for blastocyst formation and placentation. Transcription coactivator yes-associated protein (YAP), a downstream effector of the hippo signaling pathway, is required for specification of both the TE and epiblast lineages in mice. However, the biological role of YAP in porcine blastocyst development is not known. Here, we report that maternally derived YAP protein is localized to both the cytoplasm and nuclei prior to the morula stage and is then predominantly localized to the TE nuclei in blastocysts. Functionally, maternal YAP knockdown severely impeded blastocyst formation and perturbed the allocation of the first two lineages. The treatment of embryos with verteporfin, a pharmacological inhibitor of YAP, faithfully recapitulated the phenotype observed in YAP deleted embryos. Mechanistically, we found that maternal YAP regulates multiple genes which are important for lineage commitment, tight junction assembly, and fluid accumulation. Consistent with the effects on tight junction gene expression, a permeability assay revealed that paracellular sealing was defective in the trophectoderm epithelium. Lastly, YAP knockdown in a single blastomere at the 2-cell stage revealed that the cellular progeny of the YAP+ blastomere were sufficient to sustain blastocyst formation via direct complementation of the defective trophectoderm epithelium. In summary, these findings demonstrate that maternal YAP facilitates porcine blastocyst development through transcriptional regulation of key genes that are essential for lineage commitment, tight junction assembly, and fluid accumulation.
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2021-09-15 | GSE177689 | GEO
Project description:Zika virus (ZIKV) infection results in an increased risk of spontaneous abortion and poor intrauterine growth although the underlying mechanisms remain undetermined. Little is known about the impact of ZIKV infection during the earliest stages of pregnancy, at pre- and peri-implantation, because most current ZIKV pregnancy studies have focused on post-implantation stages. Here, we demonstrate that trophectoderm cells of pre-implantation human and mouse embryos can be infected with ZIKV, and propagate virus causing neural progenitor cell death. These findings are corroborated by the dose-dependent nature of ZIKV susceptibility of hESC-derived trophectoderm cells. Single blastocyst RNA-seq reveals key transcriptional changes upon ZIKV infection, including nervous system development, prior to commitment to the neural lineage. The pregnancy rate of mice is >50% lower in pre-implantation infection than infection at E4.5, demonstrating that pre-implantation ZIKV infection leads to miscarriage. Cumulatively, these data elucidate a previously unappreciated association of pre- and peri-implantation ZIKV infection and microcephaly.
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Project description:Implantation is a hallmark of mammalian embryogenesis during which embryos establish their contacts with the maternal endometrium, remodel, and undertake growth and differentiation. The mechanisms and sequence of events through which embryos change their shape during this transition are largely unexplored. Here, we show that the first extraembryonic lineage, the polar trophectoderm, is the key regulator for remodeling the embryonic epiblast. Loss of its function after immuno-surgery or inhibitor treatments prevents the epiblast shape transitions. In the mouse, the polar trophectoderm exerts physical force upon the epiblast, causing it to transform from an oval into a cup shape. In human embryos, the polar trophectoderm behaves in the opposite manner, exerting a stretching force. By mimicking this stretching behavior in mouse embryogenesis, we could direct the epiblast to adopt the disc-like shape characteristic of human embryos at this stage. Thus, the polar trophectoderm acts as a conserved regulator of epiblast shape.
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Project description:Shortly after fertilization, human embryos implant into the uterus. This requires the formation of a blastocyst consisting of a sphere encircling a cavity lodging the embryo proper. Stem cells can form blastocyst models, which we termed blastoid. Here we show that naïve human pluripotent stem cells (hPSCs) triply inhibited for the Hippo, TGFb, and ERK pathways consistently and efficiently (>70%) form blastoids that generate transcriptional pre-implantation (>95%) analogs of the three founding lineages (trophoblast, epiblast, hypoblast), and in the sequential and timely manner of blastocysts. Blastoids spontaneously form an axis marked by maturation of the polar region, which acquires the potential to specifically attach to hormonally-stimulated endometrial cells, as during in utero implantation. Such human blastoids are scalable, versatile, and ethical models to explore human implantation and development.
2021-09-15 | GSE177616 | GEO