Upregulation of Toll-like receptor 4 through anti-miR-Let-7a enhances blastocyst attachment to endometrial cells in mice.

Despite encouraging advances in fertility technology, the success rate of an ongoing pregnancy is relatively low and predominantly associated with implantation failure. Inflammatory responses are beneficial in the fetomaternal interface and supposedly accelerate the chances for successful implantation. The current study aims to determine the effect of Toll-like receptor 4 (TLR4) overexpression in mouse blastocysts via Let-7a downregulation using intracytoplasmic sperm injection-sperm-mediated gene transfer on embryo attachment rate. The pLenti-III-GFP-miR-Off-Let-7a vector was transmitted to oocytes derived via in vitro maturation (IVM) and in vivo oocytes by using NaOH-treated spermatozoa. Let-7a and TLR4 expression levels were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR), immunocytochemistry, and western blot analysis in both oocytes and embryos. Blastocyst adhesion on the endometrial cells was monitored by microscopic analysis. qRT-PCR results showed that Let-7a expression decreased in the IVM (GV-MII) oocytes compared to the in vivo oocyte (MII) group (p < .05). TLR4 showed a higher expression in GV-MII oocytes at both the gene and protein levels (p < .05). Following anti-miR-Let-7a transmission, the TLR4 expression level was significantly upregulated in embryos compared with the control groups (p < .05). Attachment and migration of trophoblasts cells towards endometrial cells dramatically increased compared to the control group (p < .05). Based on our results, we concluded that Let-7a might mediate embryo attachment through regulation of TLR4 expression levels.

Does blastomere removal alter the expression level of miR-Let7a and its target genes following mouse embryo biopsy?

Embryo manipulations may cause the misexpression of various genes, most of which play critical roles in the regulation of implantation. This study aimed to evaluate the effects of embryo biopsy on the expression of miR-Let-7a and its gene targets including ErbB4, Tgf-α, Itg-αv, Itg ß3 on the implantation of mouse embryo. Embryos were produced by in vitro fertilization followed by blastomere biopsy at the eight-cell stage. The effects of blastomere removal on the expression of genes ErbB4, Tgf-α, Itg αv, Itg ß3, and miR-Let-7a as well as the alteration of the blastocyst cell number were compared in both biopsied and non-biopsied groups. Finally, blastocyst attachment was assessed on culture dishes precoated with Fibronectin. The results revealed that there were no significant differences between the biopsied and non-biopsied embryos with reference to the blastocyst formation rates, the average inner cell mass, trophectoderm cell number, and percentage of attachment of blastocysts (P > 0.05). The expression of ErbB4, Itg-ß3, Itg-αv, TGF-α transcripts, and miR-Let-7a in blastocysts biopsied embryos did not differ from the non-biopsied blastocysts (P > 0.05). The results demonstrated that the preimplantation embryo development and attachment of biopsied embryos in vitro is not adversely affected by one blastomere biopsy at the eight-cell stage embryo.

Characterization of multitype colonies originating from porcine blastocysts produced in vitro.

Many types of embryonic stem cells have been induced from pre-implantation blastocysts to study the specification of early lineages. Various cell lines have been established using chemicals, including excessive inhibitory molecules. Previous studies have also aimed to purify cell populations representing a single embryonic lineage from a protocol. In this study, we used a novel culture condition to induce cells from blastocyst seeding and analyzed their characteristics. Next, signaling inhibitors were introduced during the cell culture period. Furthermore, we investigated the cell types using RNA sequencing. Each type of cell population showed a distinct morphology and reactivity with alkaline phosphatase. Marker proteins enabled each cell type to be distinguished by immunocytochemistry, and genes such as Sox17, Gata4, Gata6, T, and Cdx2 showed applicability for the discrimination of cell types. Signaling inhibitors suppressed the production of some cell types, and gene expression and marker protein patterns were collapsed. RNA-sequencing suggested cell-type-specific marker genes and the correlation among samples. In conclusion, four types of cells could be induced from porcine embryos using a single protocol, and they could be isolated manually. Our data will help promote the study of lineage segregation based on embryonic cells.

Infertile human endometrial organoid apical protein secretions are dysregulated and impair trophoblast progenitor cell adhesion.

Introduction: Embryo implantation failure leads to infertility. As an important approach to regulate implantation, endometrial epithelial cells produce and secrete factors apically into the uterine cavity in the receptive phase to prepare the initial blastocyst adhesion and implantation. Organoids were recently developed from human endometrial epithelium with similar apical-basal polarity compared to endometrial gland making it an ideal model to study endometrial epithelial secretions. Methods: Endometrial organoids were established using endometrial biopsies from women with primary infertility and normal fertility. Fertile and infertile organoids were treated with hormones to model receptive phase of the endometrial epithelium and intra-organoid fluid (IOF) was collected to compare the apical protein secretion profile and function on trophoblast cell adhesion. Results: Our data show that infertile organoids were dysregulated in their response to estrogen and progesterone treatment. Proteomic analysis of organoid apical secretions identified 150 dysregulated proteins between fertile and infertile groups (>1.5-fold change). Trophoblast progenitor spheroids (blastocyst surrogates) treated with infertile organoid apical secretions significantly compromised their adhesion to organoid epithelial cell monolayers compared to fertile group (P < 0.0001). Discussion: This study revealed that endometrial organoid apical secretions alter trophoblast cell adhesiveness relative to fertility status of women. It paves the way to determine the molecular mechanisms by which endometrial epithelial apical released factors regulate blastocyst initial attachment and implantation.

Uterine Epithelial LIF Receptors Contribute to Implantation Chamber Formation in Blastocyst Attachment.

Recent studies have demonstrated that the formation of an implantation chamber composed of a uterine crypt, an implantation-competent blastocyst, and uterine glands is a critical step in blastocyst implantation in mice. Leukemia inhibitory factor (LIF) activates signal transducer and activator of transcription 3 (STAT3) precursors via uterine LIF receptors (LIFRs), allowing successful blastocyst implantation. Our recent study revealed that the role of epithelial STAT3 is different from that of stromal STAT3. However, both are essential for blastocyst attachment, suggesting the different roles of epithelial and stromal LIFR in blastocyst implantation. However, how epithelial and stromal LIFR regulate the blastocyst implantation process remains unclear. To investigate the roles of LIFR in the uterine epithelium and stroma, we generated Lifr-floxed/lactoferrin (Ltf)-iCre (Lifr eKO) and Lifr-floxed/antimüllerian hormone receptor type 2 (Amhr2)-Cre (Lifr sKO) mice with deleted epithelial and stromal LIFR, respectively. Surprisingly, fertility and blastocyst implantation in the Lifr sKO mice were normal despite stromal STAT3 inactivation. In contrast, blastocyst attachment failed, and no implantation chambers were formed in the Lifr eKO mice with epithelial inactivation of STAT3. In addition, normal responsiveness to ovarian hormones was observed in the peri-implantation uteri of the Lifr eKO mice. These results indicate that the epithelial LIFR-STAT3 pathway initiates the formation of implantation chambers, leading to complete blastocyst attachment, and that stromal STAT3 regulates blastocyst attachment without stromal LIFR control. Thus, uterine epithelial LIFR is critical to implantation chamber formation and blastocyst attachment.

Endoglin (CD105) coordinates the process of endometrial receptivity for embryo implantation.

Endoglin is a TGF-ß receptor that is expressed in uterine endothelial and stromal cells in addition to trophoblast expression. However, the functional importance of endoglin in the embryo implantation process is not clear. We observed endoglin expression in the endometrium throughout the stages of its receptivity; however, its expression was enhanced during the receptive stage. Endoglin expression was predominant in epithelial cells of the lumen and glands, but showed a milder expression in stromal cells. Endoglin expression was initially observed in the primary decidual zone and later extended to the secondary decidua zone. Knockdown of endoglin via siRNA reduced the implantation sites along with the blastocyst numbers. Mouse blastocyst with endoglin-silenced endometrial epithelial cells (human and mouse origin) showed poor trophoblast outgrowth, which suggests an essential role for endoglin during endometrial receptivity. In conclusion, our findings reveal the association of endoglin with endometrial receptivity, which is important for embryo attachment.

Physiological and molecular determinants of embryo implantation.

Embryo implantation involves the intimate interaction between an implantation-competent blastocyst and a receptive uterus, which occurs in a limited time period known as the window of implantation. Emerging evidence shows that defects originating during embryo implantation induce ripple effects with adverse consequences on later gestation events, highlighting the significance of this event for pregnancy success. Although a multitude of cellular events and molecular pathways involved in embryo-uterine crosstalk during implantation have been identified through gene expression studies and genetically engineered mouse models, a comprehensive understanding of the nature of embryo implantation is still missing. This review focuses on recent progress with particular attention to physiological and molecular determinants of blastocyst activation, uterine receptivity, blastocyst attachment and uterine decidualization. A better understanding of underlying mechanisms governing embryo implantation should generate new strategies to rectify implantation failure and improve pregnancy rates in women.