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.

Spatially ordered embryo-like structures self-assembled from blastocyst-derived stem cells can be generated to mimic embryogenesis in vitro. However, the assembly system and developmental potential of such structures needs to be further studied. Here, we devise a nonadherent-suspension-shaking system to generate self-assembled embryo-like structures (ETX-embryoids) using mouse embryonic, trophoblast and extra-embryonic endoderm stem cells. When cultured together, the three cell types aggregate and sort into lineage-specific compartments. Signaling among these compartments results in molecular and morphogenic events that closely mimic those observed in wild-type embryos. These ETX-embryoids exhibit lumenogenesis, asymmetric patterns of gene expression for markers of mesoderm and primordial germ cell precursors, and formation of anterior visceral endoderm-like tissues. After transplantation into the pseudopregnant mouse uterus, ETX-embryoids efficiently initiate implantation and trigger the formation of decidual tissues. The ability of the three cell types to self-assemble into an embryo-like structure in vitro provides a powerful model system for studying embryogenesis.

Coordinated uterine-embryonic axis formation and decidual remodeling are hallmarks of mammalian post-implantation embryo development. Embryonic-uterine orientation is determined at initial implantation and synchronized with decidual development. However, the molecular mechanisms controlling these events remain elusive despite its discovery a long time ago. In the present study, we found that uterine-specific deletion of Rbpj, the nuclear transducer of Notch signaling, resulted in abnormal embryonic-uterine orientation and decidual patterning at post-implantation stages, leading to substantial embryo loss. We further revealed that prior to embryo attachment, Rbpj confers on-time uterine lumen shape transformation via physically interacting with uterine estrogen receptor (ERα) in a Notch pathway-independent manner, which is essential for the initial establishment of embryo orientation in alignment with uterine axis. While at post-implantation stages, Rbpj directly regulates the expression of uterine matrix metalloproteinase in a Notch pathway-dependent manner, which is required for normal post-implantation decidual remodeling. These results demonstrate that uterine Rbpj is essential for normal embryo development via instructing the initial embryonic-uterine orientation and ensuring normal decidual patterning in a stage-specific manner. Our data also substantiate the concept that normal mammalian embryonic-uterine orientation requires proper guidance from developmentally controlled uterine signaling.

Placenta formation during pregnancy requires chorioallantoic branching morphogenesis that involves establishing an amplifying feedback loop between Frizzled5 and Gcm1 to regulate branching initiation and trophoblast differentiation.

Among nearly 100 mammalian species, implantation can be suspended at blastocyst stage for a certain time and reactivated under favorable conditions, a phenomenon known as embryonic diapause. Until now, the underlying molecular mechanism governing embryonic diapause and reactivation for implantation remained largely unknown. Here we conducted the first integral proteomic analysis of blastocysts from diapause to reactivation by using a physiologically relevant mouse delayed implantation model. More than 6000 dormant and reactivated blastocysts were used for the proteomic analysis. A total of 2255 proteins were detected. Various cellular and molecular processes, including protein translation, aerobic glycolysis, pentose phosphate pathway, purine nucleotide biosynthesis, glutathione metabolism, and chromatin organization were identified as differentially regulated. In particular, we demonstrated a remarkable activation of mitochondria in blastocysts upon reactivation from dormancy, highlighting their essential physiological significance. Moreover, the activities of the endosome-lysosome system were prominently enhanced in the mural trophectoderm of reactivated blastocysts, accompanied by active phagocytosis at the fetal-maternal interface, suggesting a critical role in promoting trophoblast invasion. Collectively, we provided an integral proteomic view upon the regulatory network of blastocyst reactivation from diapause, which will help to better interpret the nature of embryonic diapause and reactivation in wild animals and to identify molecular indicators for selecting blastocysts with high implantation competency.

Multiple morphological abnormalities of flagella (MMAF) is one kind of severe teratozoospermia. Gene mutations reported in previous works only revealed the pathogenesis of approximately half of the MMAF cases, and more genetic defects in MMAF need to be explored. In the present study, we performed a genetic analysis on Han Chinese men with MMAF using whole‐exome sequencing. After filtering out the cases with known gene mutations, we identified five novel mutation sites in the DNAH2 gene in three cases from three families. These mutations were validated through Sanger sequencing and absent in all control individuals. In silico analysis revealed that these DNAH2 variations are deleterious. The spermatozoa with DNAH2 mutations showed severely disarranged axonemal structures with mitochondrial sheath defection. The DNAH2 protein level was significantly decreased and inner dynein arms were absent in the spermatozoa of patients. ICSI treatment was performed for two MMAF patients with DNAH2 mutations and the associated couples successfully achieved pregnancy, indicating good nuclear quality of the sperm from the DNAH2 mutant patients. Together, these data suggest that the DNAH2 mutation can cause severe sperm flagella defects that damage sperm motility. These results provide a novel genetic pathogeny for the human MMAF phenotype.

Estrogen and progesterone coupled with locally produced signaling molecules are essential for embryo implantation. However, the hierarchical landscape of the molecular pathways that governs this process remains largely unexplored. Here we show that the protein tyrosine phosphatase Shp2, a positive transducer of RTK signaling, is predominately localized in the nuclei in the periimplantation mouse uterus. Uterine-specific deletion of Shp2 exhibits reduced progesterone receptor (PR) expression and progesterone resistance, which derails normal uterine receptivity, leading to complete implantation failure in mice. Notably, the PR expression defects are attributed to the limited estrogen receptor α (ERα) activation in uterine stroma. Further analysis reveals that nuclear Shp2, rather than cytosolic Shp2, promotes the ERα transcription activity. This function is achieved by enhancing the Src kinasemediated ERα tyrosine phosphorylation, which facilitates ERα binding to Pgr promoter in an ERK-independent manner in periimplantation uteri. Besides uncovering a regulatory mechanism, this study could be clinically relevant to dysfunctional ERα-caused endometrial disorders in women.Shp2 | ERK signaling | Src kinase | estrogen receptor | uterine receptivity S uccessful implantation requires synchronization between an implantation-competent blastocyst and a receptive uterus. In humans, natural conception per cycle is poor (∼30%), and ∼75% of failed pregnancies are considered to be due to implantation failure (1). One-third of implantation failure is attributed to the embryo itself, whereas the remaining two-thirds appear to result from inadequate uterine receptivity (2). The endometrium enters into a receptive stage for blastocyst implantation only in a restricted time period termed "implantation window," which is dominated by precisely regulated proliferation and differentiation of endometrial epithelium and stroma under the influence of progesterone and estrogen (3).Estrogen and progesterone bind to the estrogen receptor (ER) and progesterone receptor (PR), respectively, coupled with specific cofactors to endure the optimal functions. The activity of these nuclear receptors is also regulated at the posttranslational level by various modifications, such as phosphorylation, which can influence the protein stability, interaction with the cofactors and DNA binding affinity. So far, a wide range of nuclear receptor cofactors have been identified to ensure the normal ER transcriptional activation, such as the well-known steroid receptor coactivator (SRC) family members, which bind with ERα in the chromatin to recruit the histone acetyltransferase p300 (P300) for transcriptional activation (4-6). It is conceivable that ordered and combinatorial recruitment of cofactors at a specific target promoter after ERα binding to DNA sequence was essential to ensure target gene transcription. Recent evidence shows that nuclear receptor coactivator 6 is essential for embryo implantation by maintaining the appropriate level and activity of uteri...

Implantation of the blastocyst into the uterus is the gateway for further embryonic development in mammals. Programming of blastocyst to an implantation-competent state known as blastocyst activation is the determining factor for implantation into the receptive uterus. However, it remains largely unclear how the blastocyst is globally programmed for implantation. Employing a delayed implantation mouse model, we show here that the blastocyst undergoes extensive programming essential for implantation. By analyzing the transcriptional profile of blastocysts with different implantation competency, we reveal the dynamic change in the biosynthesis, metabolism, and proliferation during blastocyst reactivation from diapause. We also demonstrate that reactivation of the X chromosome, one of the most important events during periimplantation of female embryonic development, is not completed even in blastocysts under conditions of dormancy, despite long term suspension in the uterus. Moreover, the mural trophectoderm (TE), but not the polar TE, differentiates to be more invasive through the weakened cell-cell tight junctions and extracellular matrices (ECMs). By analyzing the differentially expressed profile of secretory proteins, we further demonstrate that the blastocyst functions as a proinflammatory body to secrete proinflammatory signals, such as TNFα and S100A9, thereby triggering embryo-uterine attachment reaction during implantation. Collectively, our data systematically and comprehensively disclose the programming of blastocyst reactivation from diapause for implantation and uncover previously undefined roles of blastocyst during implantation.