Blastocyst activation engenders transcriptome reprogram affecting X-chromosome reactivation and inflammatory trigger of implantation.

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.

SIRT1 facilitates primordial follicle recruitment independent of deacetylase activity through directly modulating Akt1 and mTOR transcription.

In female mammals, the majority of primordial follicles (PFs) are physiologically quiescent, and only a few of them are activated and enter the growing follicle pool. Specific molecules, such as mammalian target of rapamycin (mTOR) and the serine/threonine kinase Akt (AKT), have been proven to be important for PF activation. However, how the transcription of these genes is regulated is not clear. Although activators of mTOR or AKT have been successfully used to rescue the fertility of patients with premature ovarian insufficiency, the low efficacy and unclear safety profile of these drugs hinder their clinical use in the in vitro activation (IVA) of PFs. Here, sirtuin 1 (SIRT1), an NAD-dependent deacetylase, was demonstrated to activate mouse PFs independent of its deacetylase activity. SIRT1 was prominently expressed in pregranulosa cells (pGCs) and oocytes, and its expression was increased during PF activation. PF activation was achieved by either up-regulating SIRT1 with a specific activator or overexpressing SIRT1. Moreover, SIRT1 knockdown in oocytes or pGCs could significantly suppress PF activation. Further studies demonstrated that SIRT1 enhanced both Akt1 and mTOR expression by acting more as a transcription cofactor, directly binding to the respective gene promoters, than as a deacetylase. Importantly, we explored the potential clinical applications of targeting SIRT1 in IVA via short-term treatment of cultured ovaries from mice and human ovarian tissues to activate PFs by applying the SIRT1 activator resveratrol. RSV-induced IVA could be a candidate strategy to develop more efficient procedures for future clinical treatment of infertility.-Zhang, T., Du, X., Zhao, L., He, M., Lin, L., Guo, C., Zhang, X., Han, J., Yan, H., Huang, K., Sun, G., Yan, L., Zhou, B., Xia, G., Qin, Y., Wang, C. SIRT1 facilitates primordial follicle recruitment independent of deacetylase activity through directly modulating Akt1 and mTOR transcription.

Runx2 acts downstream of C/EBPß to regulate the differentiation of uterine stromal cells in mice.

Although Runx2 is involved in the regulation of cellular differentiation, its physiological roles in the differentiation of uterine stromal cells during decidualization still remain unknown. The aim of this study was to examine the expression, regulation and function of Runx2 in mouse uterus during decidualization. The results showed that Runx2 was highly expressed in the decidua and oil-induced decidualized cells. In the uterine stromal cells, recombinant human Runx2 (rRunx2) could induce the expression of Prl8a2 and Prl3c1 which are two well-known differentiation markers for decidualization, while inhibition of Runx2 with specific siRNA reduced their expression. Further study found that rRunx2 could improve the expression of Prl8a2 and Prl3c1 in the C/EBPß siRNA-transfected stromal cells. In the stromal cells, cAMP analogue 8-Br-cAMP could induce the expression of Runx2. Moreover, the induction was blocked by PKA inhibitor H89. Simultaneously, attenuation of C/EBPß with siRNA could also reduce the cAMP-induced Runx2 expression. Furthermore, siRNA-mediated silencing of Runx2 expression alleviated the effects of cAMP on the differentiation of stromal cells. Runx2 might act downstream of C/EBPß to regulate the expression of Cox-2, Vegf and Mmp9 in the uterine stromal cells. Collectively, Runx2 may play an important role during mouse decidualization.

Differential expression and regulation of Runx1 in mouse uterus during the peri-implantation period.

Runx1 transcription factor is a key developmental regulator. However, little is known about the effects of Runx1 on embryo implantation and decidualization. The aim of this study is to examine the expression and regulation of Runx1 in mouse uterus during the peri-implantation period. There was no evident Runx1 mRNA signal on days 1-4 of pregnancy. On day 5 of pregnancy, Runx1 mRNA was mainly localized in the subluminal stroma surrounding the implanting blastocyst. A similar result was observed in the estrogen-activated implantation uterus. Simultaneously, a high level of Runx1 mRNA expression was detected on days 6-8 of pregnancy and under artificial decidualization. 8-Br-cAMP could induce the expression of Runx1 mRNA in the uterine stromal cells. Moreover, the induction was obviously blocked by PKA inhibitor H89. Inhibition of Runx1 with specific siRNA could decrease the proliferation of stromal cells and expression of decidual markers Prl8a2 and Prl3c1 in the uterine stromal cells. Further study found that inhibition of Runx1 could also suppress the expression of Cox-2, mPGES-1 and Mmp2 genes in uterine stromal cells. Estrogen and progesterone could induce the expression of Runx1 mRNA in ovariectomized mouse uterus and uterine stromal cells. Taken together, these data suggest that Runx1 may play an important role during mouse decidualization.

The causal role of circulating immunity-inflammation in preeclampsia: A Mendelian randomization.

Patients with systemic autoimmune diseases, such as systemic lupus erythematosus, were at a higher risk for preeclampsia. The causal relationship between immunological inflammation and preeclampsia (PE) remains uncertain. We aimed to investigate the causal relationship between circulating immune inflammation and PE. Genetically predicted blood immune cells and circulating inflammatory proteins were identified using two genome-wide association studies (GWAS). We used a two-sample Mendelian randomization (MR) method to determine whether circulating immunological inflammation causes PE. Our findings indicated that ten immunophenotypes were identified to be significantly associated with PE risk: CD62L- Dendritic Cell Absolute Count, CD86+ myeloid Dendritic Cell %Dendritic Cell, CD62L- myeloid Dendritic Cell Absolute Count, CD86+ myeloid Dendritic Cell Absolute Count, CD62L- myeloid Dendritic Cell %Dendritic Cell, CD62L- CD86+ myeloid Dendritic Cell %Dendritic Cell, CD62L- CD86+ myeloid Dendritic Cell Absolute Count, CD16 on CD14+ CD16+ monocyte, HLA DR+ Natural Killer Absolute Count, and T cell Absolute Count. Ninety-one inflammation-related proteins had no statistically significant effect on PE following false discovery rate (FDR) correction. Certain proteins exhibited unadjusted low p-values that merited mention. These proteins include interleukin-10 (OR = 0.76, 95%CI = 0.63-0.93, p = .006), fibroblast growth factor 21 (OR = 1.23, 95%CI = 1.01-1.47, p = .035), and Caspase 8 (OR = 0.65, 95%CI = 0.50-0.85, p = .001). The ELISA analysis demonstrated elevated levels of FGF-21 and decreased levels of IL-10 and Caspase-8 in the plasma of patients with PE. These findings reveal that immunophenotypes and circulating inflammatory proteins may induce PE, confirming the importance of peripheral Immunity-Inflammation in PE. The discovery has the potential to lead to earlier detection and more effective treatment techniques.

The presence of blastocyst within the uteri facilitates lumenal epithelium transformation for implantation via upregulating lysosome proteostasis activity.

ABBREVIATIONS: ACTB: actin beta; AREG: amphiregulin; ATP6V0A4: ATPase, H+ transporting, lysosomal V0 subunit A4; Baf A1: bafilomycin A1; BSA: bovine serum albumin; CLDN1: claudin 1; CTSB: cathepsin B; DEGs: differentially expressed genes; E2: 17ß-estradiol; ESR: estrogen receptor; GATA2: GATA binding protein 2; GLA: galactosidase, alpha; GO: gene ontology; HBEGF: heparin-binding EGF-like growth factor; IGF1R: insulin-like growth factor 1 receptor; Ihh: Indian hedgehog; ISH: in situ hybridization; LAMP1: lysosomal-associated membrane protein 1; LCM: laser capture microdissection; Le: lumenal epithelium; LGMN: legumain; LIF: leukemia inhibitory factor; LIFR: LIF receptor alpha; MSX1: msh homeobox 1; MUC1: mucin 1, transmembrane; P4: progesterone; PBS: phosphate-buffered saline; PCA: principal component analysis; PPT1: palmitoyl-protein thioesterase 1; PGR: progesterone receptor; PSP: pseudopregnancy; PTGS2/COX2: prostaglandin-endoperoxide synthase 2; qPCR: quantitative real-time polymerase chain reaction; SP: pregnancy; TFEB: transcription factor EB.

Menin directs regionalized decidual transformation through epigenetically setting PTX3 to balance FGF and BMP signaling.

During decidualization in rodents, uterine stroma undergoes extensive reprograming into distinct cells, forming the discrete regions defined as the primary decidual zone (PDZ), the secondary decidual zone (SDZ) and the layer of undifferentiated stromal cells respectively. Here we show that uterine deletion of Men1, a member of the histone H3K4 methyltransferase complex, disrupts the terminal differentiation of stroma, resulting in chaotic decidualization and pregnancy failure. Genome-wide epigenetic profile reveals that Men1 binding in chromatin recapitulates H3K4me3 distribution. Further transcriptomic investigation demonstrates that Men1 directly regulates the expression of PTX3, an extra-cellular trap for FGF2 in decidual cells. Decreased Ptx3 upon Men1 ablation leads to aberrant activation of ERK1/2 in the SDZ due to the unrestrained FGF2 signal emanated from undifferentiated stromal cells, which blunt BMP2 induction and decidualization. In brief, our study provides genetic and molecular mechanisms for epigenetic rewiring mediated decidual regionalization by Men1 and sheds new light on pregnancy maintenance.

Maternal anxiety affects embryo implantation via impairing adrenergic receptor signaling in decidual cells.

Recurrent implantation failure (RIF) is defined as the failed pregnancy after good embryo transfer over 3 cycles during in vitro fertilization (IVF).The human endometrium plays a vital role in providing the site for embryo implantation, with several factors implicated in unsatisfactory endometrial receptivity in RIF. Our present results revealed that women with pregnancy loss or infertility have a higher serum epinephrine level, indicating a potential correlation between psychological stress and pregnancy failure. RNA-sequencing of the tissues collected at the endometrial receptive phase in normal and RIF women showed that stress hormones could affect the functional status of endometrial receptivity. Subsequent analysis revealed that the epinephrine signaling acts as an important regulator of endometrial receptivity through the PI3K-AKT and FOXO1 signaling pathways. We also found that patients with RIF show attenuated expression of the alpha-2C-adrenergic receptor (ADRA2C) and that its down regulation induced by high level epinephrine could inhibit the decidualization. Early pregnant mice treated with stress showed high serum epinephrine levels, defective uterine adrenergic receptor expression, and low pregnancy rates. Altogether, our findings indicate that mental stress during early pregnancy can alter the functional status of endometrial receptivity.

Sequential activation of uterine epithelial IGF1R by stromal IGF1 and embryonic IGF2 directs normal uterine preparation for embryo implantation.

Embryo implantation in both humans and rodents is initiated by the attachment of a blastocyst to the uterine epithelium. For blastocyst attachment, the uterine epithelium needs to transform at both the structural and molecular levels first, and then initiate the interaction with trophectoderm. Any perturbation during this process will result in implantation failure or long-term adverse pregnancy outcomes. Endocrine steroid hormones, which function through nuclear receptors, combine with the local molecules produced by the uteri or embryo to facilitate implantation. The insulin-like growth factor (IGF) signaling has been reported to play a vital role during pregnancy. However, its physiological function during implantation remains elusive. This study revealed that mice with conditional deletion of Igf1r gene in uteri suffered from subfertility, mainly due to the disturbed uterine receptivity and abnormal embryo implantation. Mechanistically, we uncovered that in response to the nidatory estrogen on D4 of pregnancy, the epithelial IGF1R, stimulated by the stromal cell-produced IGF1, facilitated epithelial STAT3 activation to modulate the epithelial depolarity. Furthermore, embryonic derived IGF2 could activate both the epithelial ERK1/2 and STAT3 signaling through IGF1R, which was critical for the transcription of Cox2 and normal attachment reaction. In brief, our data revealed that epithelial IGF1R was sequentially activated by the uterine stromal IGF1 and embryonic IGF2 to guarantee normal epithelium differentiation during the implantation process.

HOXA10 co-factor MEIS1 is required for the decidualization in human endometrial stromal cell.

Decidualization is a critical process for embryo implantation and pregnancy maintenance in humans. The homeobox gene HOXA10 has been widely studied in endometrial receptivity establishment and decidualization. MEIS1, a three-amino-acid loop extension (TALE) family homeobox gene, has been proven to be a co-factor for HOXA10 in mouse uterus. However, the interaction between MEIS1 and HOXA10 in the human decidual cells remains to be elucidated. siRNA and CRISPR-Cas9 were employed to knockdown and knockout MEIS1 in the cultured human endometrial stromal cells, and it was found that MEIS1 deficiency leads to impaired decidualization. The physical interaction between the MEIS1 and HOXA10 in human endometrial stromal cell was confirmed by immunoprecipitation. Moreover, KAT2B and ETA were proved to be downregulated in the absence of MEIS1, and luciferase reporter and ChIP assays demonstrated that MEIS1-HOXA10 complex binds to the promoters of KAT2B and ETA and regulates their activity. Overexpression of KAT2B and ETA can partially rescue the decidualization defects in MEIS1-knockout HESCs. Taken together, these data suggest that MEIS1 plays an indispensable role in decidualization in human endometrial stromal cells, and MEIS1 interacts with HOXA10 to regulate the downstream genes, such as KAT2B and ETA. These findings will contribute to our understanding about the regulatory network in the process of decidualization in humans.

An exaggerated epinephrine-adrenergic receptor signaling impairs uterine decidualization in mice.

Our understanding of the relationship between stress-derived epinephrine and early pregnancy failure remains incomplete. Here, we explored the effect of epinephrine exposure on early pregnancy and pseudopregnancy in mice. Increased expression of adrenergic receptors Adra1b, Adra2b and Adrb2 was observed during decidualization and post-implantation embryogenesis was delayed or survival impaired. Epinephrine treatment also impaired decidualization in both the gravid and pseudopregnant uterus, suggesting the effect on decidualization was independent of the conceptus. This included a suppression of endometrial stroma cell proliferation and of key decidualization regulators, including COX2, BMP2 and WNT4. Collectively, these data demonstrate that maternal epinephrine exposure during early pregnancy impairs uterine decidualization and embryo development, underlying early pregnancy failure.

High FOXK1 expression correlates with poor outcomes in hepatocellular carcinoma and regulates stemness of hepatocellular carcinoma cells.

AIMS: Forkhead box (FOX) proteins constitute a huge family of transcriptional regulators, which are involved in a wide range of cancers. FOXK1 is a little studied member of FOXK subfamily. This study aimed to investigate the potential prognostic value of FOXK1 in human hepatocellular carcinoma (HCC) and explore potential underlying mechanisms. MAIN METHODS: We performed bioinformatic analyses to evaluate the prognostic value of FOXK1 expression in human HCC and to reveal the underlying mechanism by which FOXK1 regulates HCC. RT-PCR, FACS analysis and sphere formation assay were carried out to investigate the role of FOXK1 in regulating liver cancer stem cells. KEY FINDINGS: Our results demonstrated that FOXK1 was overexpressed in human HCC and positively correlated with cancer progression. DNA hypomethylation and gene copy number variation contributed to the overexpression of FOXK1. Importantly, high FOXK1 expression was associated with both low overall survival probability (OS) and low relapse free survival probability (RFS) of HCC patients. Intriguingly, we found that high FOXK1 expression was correlated with activation of stem cell-regulating pathways in human HCC. Knockdown of FOXK1 resulted in downregulation of the cancer stem cell marker EpCAM and ALDH1 and decreased sphere-forming ability of hepatocellular carcinoma cells. SIGNIFICANCE: Overall, our study identified FOXK1 as a new biomarker for prognosis of HCC patients and revealed its role in regulating stemness of hepatocellular carcinoma cells.

Hmgn5 functions downstream of Hoxa10 to regulate uterine decidualization in mice.

Although Hmgn5 is involved in the regulation of cellular proliferation and differentiation, its physiological function during decidualization is still unknown. Here we showed that Hmgn5 was highly expressed in the decidual cells. Silencing of Hmgn5 expression by specific siRNA reduced the proliferation of uterine stromal cells and expression of Ccnd3 and Cdk4 in the absence or presence of estrogen and progesterone, whereas overexpression of Hmgn5 exhibited the opposite effects. Simultaneously, Hmgn5 might induce the expression of Prl8a2 and Prl3c1 which were 2 well-known differentiation markers for decidualization. In the uterine stromal cells, cAMP analog 8-Br-cAMP and progesterone could up-regulate the expression of Hmgn5, but the up-regulation was impeded by H89 and RU486, respectively. Attenuation of Hmgn5 expression could block the differentiation of uterine stromal cells in response to cAMP and progesterone. Further studies found that regulation of cAMP and progesterone on Hmgn5 expression was mediated by Hoxa10. During in vitro decidualization, knockdown of Hmgn5 could abrogate Hoxa10-induced upregulation of Prl8a2 and Prl3c1, while overexpression of Hmgn5 reversed the inhibitory effects of Hoxa10 siRNA on the expression of Prl8a2 and Prl3c1. In the stromal cells undergoing decidualization, Hmgn5 might act downstream of Hoxa10 to regulate the expression of Cox-2, Vegf and Mmp2. Collectively, Hmgn5 may play an important role during mouse decidualization.

Hmgn1 acts downstream of C/EBPß to regulate the decidualization of uterine stromal cells in mice.

Although Hmgn1 is involved in the regulation of gene expression and cellular differentiation, its physiological roles on the differentiation of uterine stromal cells during decidualization still remain unknown. Here we showed that Hmgn1 mRNA was highly expressed in the decidua on days 6-8 of pregnancy. Simultaneously, increased expression of Hmgn1 was also observed in the artificial and in vitro induced decidualization models. Hmgn1 induced the proliferation of uterine stromal cells and expression of Ccna1, Ccnb1, Ccnb2 and Cdk1 in the absence of estrogen and progesterone. Overexpression of Hmgn1 could enhance the expression of Prl8a2 and Prl3c1 which were 2 well-known differentiation markers for decidualization, whereas inhibition of Hmgn1 with specific siRNA could reduce their expression. Further studies found that Hmgn1 could mediate the effects of C/EBPß on the expression of Prl8a2 and Prl3c1 during in vitro decidualization. In the uterine stromal cells, cAMP analog 8-Br-cAMP could stimulate the expression of Hmgn1 via C/EBPß. Moreover, siRNA-mediated down-regulation of Hmgn1 could attenuate the effects of cAMP on the differentiation of uterine stromal cells. During in vitro decidualization, Hmgn1 might act downstream of C/EBPß to regulate the expression of Cox-2, mPGES-1 and Vegf. Progesterone could up-regulate the expression of Hmgn1 in the ovariectomized mouse uterus, uterine epithelial cells and stromal cells. Knockdown of C/EBPß with siRNA alleviated the up-regulation of progesterone on Hmgn1 expression. Collectively, Hmgn1 may play an important role during mouse decidualization.

Differential expression and regulation of Ido2 in the mouse uterus during peri-implantation period.

Ido2 is involved in tryptophan catabolism and immunity, but its physiological functions remain poorly understood. This study was undertaken to examine the expression and regulation of Ido2 gene in mouse uterus during the peri-implantation period. The results showed that Ido2 mRNA was highly expressed on day 4 of pregnancy and in the delayed implantation uterus. On days 5-8 of pregnancy, a low level of Ido2 expression was observed in the uteri. Simultaneously, Ido2 mRNA was also lowly expressed in the decidualized uterus. In the uterine stromal cells, 8-Br-cAMP could inhibit the expression of Ido2 mRNA. Moreover, Ido2 mRNA expression was gradually decreased after the stromal cells were treated with estrogen and progesterone and reached a nadir at 96 h. Further study found that overexpression of Ido2 could downregulate the expression of decidualization marker genes PRL, IGFBP1, and Dtprp under in vitro decidualization, while inhibition of Ido2 with devo-1-methyl-tryptophan (D-1-MT) could upregulate the expression of these marker genes. Under in vitro decidualization, overexpression of Ido2 could suppress the proliferation of uterine stromal cells and elevate the expression of Bax and MMP2 genes. On the contrary, Ido2 inhibitor D-1-MT could enhance the proliferation of stromal cells and expression of Bcl2 gene but decline the Bax/Bcl2 ratio. In the uterine stromal cells, estrogen and progesterone could induce the expression of Ido2 mRNA. These data indicate that Ido2 may be important for mouse embryo implantation and decidualization.

Expression, regulation and function of Hmgn3 during decidualization in mice.

Although Hmgn3 is involved in the regulation of development and cellular differentiation, its physiological roles on decidualization are still unknown. Here we showed that Hmgn3 was highly expressed in the decidua and decidualizing stromal cells. Overexpression of Hmgn3 variants, Hmgn3a or Hmgn3b, enhanced the expression of decidualization markers Prl8a2 and Prl3c1, whereas inhibition of Hmgn3 reduced their expression. Hmgn3 could mediate the effects of Hoxa10 and cAMP on the expression of Prl8a2 and Prl3c1. Further study found that Hmgn3 directed the process of decidualization through influencing the expression of Hand2. Progesterone could induce the expression of Hmgn3 in the ovariectomized mouse uterus, uterine epithelial cells and stromal cells. Knockdown of Hoxa10 with siRNA alleviated the induction of progesterone and cAMP on Hmgn3 expression. Simultaneously, siRNA-mediated down-regulation of Hmgn3 in the uterine stromal cells could attenuate the effects of progesterone, cAMP and Hoxa10 on the expression of Hand2. Collectively, Hmgn3 may play an important role during mouse decidualization.