Human CD56+CD39+ dNK cells support fetal survival through controlling trophoblastic cell fate: immune mechanisms of recurrent early pregnancy loss.

Decidual natural killer (dNK) cells are the most abundant immune cells at the maternal-fetal interface during early pregnancy in both mice and humans, and emerging single-cell transcriptomic studies have uncovered various human dNK subsets that are disrupted in patients experiencing recurrent early pregnancy loss (RPL) at early gestational stage, suggesting a connection between abnormal proportions or characteristics of dNK subsets and RPL pathogenesis. However, the functional mechanisms underlying this association remain unclear. Here, we established a mouse model by adoptively transferring human dNK cells into pregnant NOG (NOD/Shi-scid/IL-2Rγnull) mice, where human dNK cells predominantly homed into the uteri of recipients. Using this model, we observed a strong correlation between the properties of human dNK cells and pregnancy outcome. The transfer of dNK cells from RPL patients (dNK-RPL) remarkably worsened early pregnancy loss and impaired placental trophoblast cell differentiation in the recipients. These adverse effects were effectively reversed by transferring CD56+CD39+ dNK cells. Mechanistic studies revealed that CD56+CD39+ dNK subset facilitates early differentiation of mouse trophoblast stem cells (mTSCs) towards both invasive and syncytial pathways through secreting macrophage colony-stimulating factor (M-CSF). Administration of recombinant M-CSF to NOG mice transferred with dNK-RPL efficiently rescued the exacerbated pregnancy outcomes and fetal/placental development. Collectively, this study established a novel humanized mouse model featuring functional human dNK cells homing into the uteri of recipients and uncovered the pivotal role of M-CSF in fetal-supporting function of CD56+CD39+ dNK cells during early pregnancy, highlighting that M-CSF may be a previously unappreciated therapeutic target for intervening RPL.

Upregulation of MMPs in placentas of patients with gestational diabetes mellitus: Involvement of the PI3K/Akt pathway.

In recent years, there has been a notable rise in the incidence of pregnancies complicated by gestational diabetes mellitus (GDM), characterized by glucose intolerance first identified during pregnancy. Analysis of placental tissue has revealed that placentas from women with GDM tend to be larger and heavier compared to control placentas, indicating potential changes in trophoblast proliferation, differentiation, and apoptosis. In this study, transcriptome sequencing was conducted on placentas obtained from both normal pregnancies and pregnancies with GDM to investigate the molecular mechanisms underlying this condition. The original sequencing data were subjected to sequencing analysis, resulting in the identification of 935 upregulated genes and 256 downregulated genes. The KEGG and GO analysis techniques on differential genes uncovered evidence suggesting that the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway may contribute to the pathogenesis of GDM. Subsequent analysis indicated that the expression levels of matrix metalloproteinases (MMP) 11, MMP12, MMP14, and MMP15, which are regulated by the PI3K/Akt pathway, were upregulated in the placentas of patients with GDM when compared to those of individuals with normal placental function. Additionally, our investigation into alternative splicing patterns revealed an increase in exon skipping alternative splicing of CSF3R in the placenta of patients with GDM compared to that in the control group. The CSF3R-PI3K-MMP pathway is speculated to regulate the pathogenesis of GDM.

Cx40 Levels Regulate Hypoxia-Induced Changes in the Migration, Proliferation, and Formation of Gap Junction Plaques in an Extravillous Trophoblast Cell Model.

BACKGROUND: Extravillous trophoblasts (EVTs) form stratified columns at the placenta-uterus interface. In the closest part to fetal structures, EVTs have a proliferative phenotype, whereas in the closest part to maternal structures, they present a migratory phenotype. During the placentation process, Connexin 40 (Cx40) participates in both the proliferation and migration of EVTs, which occurs under hypoxia. However, a possible interaction between hypoxia and Cx40 has not yet been established. METHODS: We developed two cellular models, one with "low Cx40" (Jeg-3), which reflected the expression of this protein found in migratory EVTs, and one with "high Cx40" (Jeg-3/hCx40), which reflected the expression of this protein in proliferative cells. We analyzed the migration and proliferation of these cells under normoxic and hypoxic conditions for 24 h. Jeg-3 cells under hypoxia increased their migratory capacity over their proliferative capacity. However, in Jeg-3/hCx40, the opposite effect was induced. On the other hand, hypoxia promoted gap junction (GJ) plaque formation between neighboring Jeg-3 cells. Similarly, the activation of a nitro oxide (NO)/cGMP/PKG-dependent pathway induced an increase in GJ-plaque formation in Jeg-3 cells. CONCLUSIONS: The expression patterns of Cx40 play a crucial role in shaping the responses of EVTs to hypoxia, thereby influencing their migratory or proliferative phenotype. Simultaneously, hypoxia triggers an increase in Cx40 gap junction (GJ) plaque formation through a pathway dependent on NO.

A study on methods for preimplantation genetic testing (PGT) on in vivo- and in vitro-produced equine embryos, with emphasis on embryonic sex determination.

Two methods for preimplantation genetic testing (PGT) have been described for equine embryos: trophoblast cell biopsy (TCB) or blastocoele fluid aspiration (BFA). While TCB is widely applied for both in vivo- and in vitro-produced embryos, BFA has been mostly utilized for in vivo-produced embryos. Alternative methods for PGT, including analysis of cell-free DNA (CFD) in the medium where in vitro-produced embryos are cultured, have been reported in humans but not for equine embryos. In Experiment 1, in vivo- (n = 10) and in vitro-produced (n = 13) equine embryos were subjected to BFA, cultured for 24 h, then subjected to TCB, and cultured for additional 24 h. No detrimental effect on embryonic diameter or re-expansion rates was observed for either embryo group (P > 0.05). In Experiment 2, the concordance (i.e., agreement on detecting the same embryonic sex using two techniques) among BFA, TCB, and the whole embryo (Whole) was studied by detecting the sex-determining region Y (SRY) or testis-specific y-encoded protein 1 (TSPY) (Y-chromosome), and androgen receptor (AR; X-chromosome) genes using PCR. Overall, a higher concordance for detecting embryonic sex was observed among techniques for in vivo-produced embryos (67-100 %; n = 14 embryos) than for in vitro-produced embryos (31-92 %; n = 13 embryos). The concordance between sample types increased when utilizing TSPY (77-100 %) instead of SRY (31-100 %) as target gene. In Experiment 3, CFD analysis was performed on in vitro-produced embryos to determine embryonic sex via PCR (SRY [Y-chromosome] and amelogenin - AMEL [X- and Y-chromosomes]). Overall, CFD was detected in all medium samples, and the concordance between CFD sample and the whole embryo was 60 % when utilizing SRY and AMEL genes. In conclusion, equine embryos can be subjected to two biopsy procedures (24 h apart) without apparent detrimental effects on embryonic size. For in vivo-, but not for in vitro-produced equine embryos, BFA can be considered a potential alternative to TCB for PGT. Finally, CFD can be further explored as a non-invasive method for PGT in in vitro produced equine embryos.

NR2F1 overexpression alleviates trophoblast cell dysfunction by inhibiting GDF15/MAPK axis in preeclampsia.

Abnormal functions of trophoblast cells are associated with the pathogenesis of preeclampsia (PE). Nuclear receptor subfamily 2 group F member 1 (NR2F1) acts as a transcriptionally regulator in many diseases, but its role in PE remains unknown. Hypoxia/reoxygenation (H/R)-stimulated HTR-8/SVneo cells were used to mimic PE injury in vitro. NR2F1 overexpression alleviated trophoblast apoptosis, as evidenced by the decreased number of TUNEL-positive cells and the downregulation of caspase 3 and caspase 9 expression in cells. NR2F1 overexpression increased the invasion and migration ability of HTR-8/SVneo cells, accompanied by increased protein levels of matrix metalloproteinase (MMP)-2 and MMP-9. mRNA-seq was applied to explore the underlying mechanism of NR2F1, identifying growth differentiation factor 15 (GDF15) as the possible downstream effector. Dual-luciferase reporter, ChIP-qPCR, and DNA pull-down assays confirmed that NR2F1 bound to the promoter of GDF15 and transcriptionally inhibited its expression. GDF15 overexpression increased apoptosis and decreased the ability of invasion and migration in HTR-8/SVneo cells expressing NR2F1. MAPK pathway was involved in the regulation of PE. Administration of p38 inhibitor, ERK inhibitor, and JNK inhibitor reversed the effect of simultaneous overexpression NR2F1 and GDF15 on trophoblast apoptosis, invasion, and migration. Our findings demonstrated that NR2F1 overexpression inhibited trophoblast apoptosis and promoted trophoblast invasion and migration. NR2F1 might negatively regulate GDF15 expression by binding to its promoter region, which further inhibited MAPK signaling pathway in PE. Our study highlights that NR2F1 might sever as a potential target in PE.

Vitamin C inactivates c-Jun N-terminal kinase to stabilize heart and neural crest derivatives expressed 1 (Hand1) in regulating placentation and maintenance of pregnancy.

Vitamin C (VC) serves as a pivotal nutrient for anti-oxidation process, metabolic responses, and stem cell differentiation. However, its precise contribution to placenta development and gestation remains obscure. Here, we demonstrated that physiological levels of VC act to stabilize Hand1, a key bHLH transcription factor vital for the development trajectory of trophoblast giant cell (TGC) lineages, thereby promoting the differentiation of trophoblast stem cells into TGC. Specifically, VC administration inactivated c-Jun N-terminal kinase (JNK) signaling, which directly phosphorylates Hand1 at Ser48, triggering the proteasomal degradation of Hand1. Conversely, a loss-of-function mutation at Ser48 on Hand1 not only significantly diminished both intrinsic and VC-induced stabilization of Hand1 but also underscored the indispensability of this residue. Noteworthy, the insufficiency of VC led to severe defects in the differentiation of diverse TGC subtypes and the formation of labyrinth's vascular network in rodent placentas, resulting in failure of maintenance of pregnancy. Importantly, VC deficiency, lentiviral knockdown of JNK or overexpression of Hand1 mutants in trophectoderm substantially affected the differentiation of primary and secondary TGC in E8.5 mouse placentas. Thus, these findings uncover the significance of JNK inactivation and consequential stabilization of Hand1 as a hitherto uncharacterized mechanism controlling VC-mediated placentation and perhaps maintenance of pregnancy.

Alpha 1,3 N-Acetylgalactosaminyl Transferase (GTA) Impairs Invasion Potential of Trophoblast Cells in Preeclampsia.

Preeclampsia (PE) is a pregnancy-specific disorder associated with shallow invasion of the trophoblast cells and insufficient remodeling of the uterine spiral artery. Protein glycosylation plays an important role in trophoblast cell invasion. However, the glycobiological mechanism of PE has not been fully elucidated. In the current study, employing the Lectin array, we found that soybean agglutinin (SBA), which recognizes the terminal N-acetylgalactosamine α1,3-galactose (GalNAc α1,3 Gal) glycotype, was significantly increased in placental trophoblast cells from PE patients compared with third-trimester pregnant controls. Upregulating the expression of the key enzyme α1,3 N-acetylgalactosaminyl transferase (GTA) promoted the biosynthesis of terminal GalNAc α1,3 Gal and inhibited the migration/invasion of HTR8/SVneo trophoblast cells. Moreover, the methylation status of GTA promoter in placental tissues from PE patients was lower than that in the third trimester by methylation-specific PCR (MSP) and bisulfite sequencing PCR (BSP) analysis. Elevated GTA expression in combination with the DNA methylation inhibitor 5-azacytidine (5-AzaC) treatment increased the glycotype biosynthesis and impaired the invasion potential of trophoblast cells, leading to preeclampsia. This study suggests that elevated terminal GalNAc α1,3 Gal biosynthesis and GTA expression may be applied as the new markers for evaluating placental function and the auxiliary diagnosis of preeclampsia.

TFEB safeguards trophoblast syncytialization in humans and mice.

Nutrient sensing and adaptation in the placenta are essential for pregnancy viability and proper fetal growth. Our recent study demonstrated that the placenta adapts to nutrient insufficiency through mechanistic target of rapamycin (mTOR) inhibition-mediated trophoblast differentiation toward syncytiotrophoblasts (STBs), a highly specialized multinucleated trophoblast subtype mediating extensive maternal-fetal interactions. However, the underlying mechanism remains elusive. Here, we unravel the indispensable role of the mTORC1 downstream transcriptional factor TFEB in STB formation both in vitro and in vivo. TFEB deficiency significantly impaired STB differentiation in human trophoblasts and placenta organoids. Consistently, systemic or trophoblast-specific deletion of Tfeb compromised STB formation and placental vascular construction, leading to severe embryonic lethality. Mechanistically, TFEB conferred direct transcriptional activation of the fusogen ERVFRD-1 in human trophoblasts and thereby promoted STB formation, independent of its canonical function as a master regulator of the autophagy-lysosomal pathway. Moreover, we demonstrated that TFEB directed the trophoblast syncytialization response driven by mTOR complex 1 (mTORC1) signaling. TFEB expression positively correlated with the reinforced trophoblast syncytialization in human fetal growth-restricted placentas exhibiting suppressed mTORC1 activity. Our findings substantiate that the TFEB-fusogen axis ensures proper STB formation during placenta development and under nutrient stress, shedding light on TFEB as a mechanistic link between nutrient-sensing machinery and trophoblast differentiation.

Human trophoblast invasion and migration are mediated by the YAP1-CCN1 pathway: defective signaling in trophoblasts during early-onset severe preeclampsia.

The transcription coactivator YAP1 mediates the major effects of the Hippo signaling pathway. The CCN family is a small group of glycoproteins known to be downstream effectors of YAP1 in diverse tissues. However, whether CCN family members mediate the effects of YAP1 in human trophoblasts is unknown. In this study, placental expression of both YAP1 and CCN1 was found to be impaired in pregnancies complicated by early-onset severe preeclampsia (sPE). CCN1 was expressed not only in cytotrophoblasts, trophoblast columns and mesenchymal cells, similar to active YAP1, but also in syncytiotrophoblasts of normal first-trimester placental villi; moreover, decidual staining of active YAP1 and CCN1 was found in both interstitial and endovascular extravillous trophoblasts. In cultured immortalized human trophoblastic HTR-8/SVneo cells, knockdown of YAP1 decreased CCN1 mRNA and protein expression and led to impaired cell invasion and migration. Also, CCN1 knockdown negatively affected HTR-8/SVneo cell invasion and migration but not viability. YAP1 knockdown was further found to impair HTR-8/SVneo cell viability via G0/G1 cell cycle arrest and apoptosis, while CCN1 knockdown had minimal effect on cell cycle arrest and no effect on apoptosis. Accordingly, treatment with recombinant CCN1 partially reversed the YAP1 knockdown-induced impairment in trophoblast invasion and migration but not in viability. Thus, CCN1 mediates the effects of YAP1 on human trophoblast invasion and migration but not apoptosis, and decreased placental expression of YAP1 and CCN1 in pregnancies complicated by early-onset sPE might contribute to the pathogenesis of this disease.

CXC chemokine receptor type 5 may induce trophoblast dysfunction and participate in the processes of unexplained missed abortion, wherein p-ERK and interleukin-6 may be involved.

Chemokines regulate the trophoblast dysfunction involved in the occurrence and development of pathological pregnancy, including missed abortions. In particular, CXC chemokine receptor type 5 mediates cell proliferation, migration, and inflammation; nonetheless, its role in missed abortions remains unclear. This study aimed to examine the expression of CXC chemokine receptor type 5 in missed abortions and to investigate the effects of CXC chemokine receptor type 5 on the biological behaviour of trophoblasts, as well as the underlying mechanisms. Our results indicated that CXC chemokine receptor type 5 was upregulated in the villi of women who experienced unexplained missed abortions, as compared with those who had normal pregnancies. CXC chemokine receptor type 5 inhibited the proliferation and migration of human first-trimester trophoblast/simian virus cells but promoted cell apoptosis. With respect to its mechanisms, CXC chemokine receptor type 5 activated the extracellular signal-regulated protein kinase 1/2 signalling pathway and upregulated the secretion of interleukin-6; however, it had no effect on the secretion of tumour necrosis factor-α. In conclusion, our findings suggest that CXC chemokine receptor type 5 induces trophoblast dysfunction and participates in the processes of unexplained missed abortions, wherein p-ERK and interleukin-6 may be involved.

Combined analysis of the effects of hypoxia and oxidative stress on DNA methylation and the transcriptome in HTR-8/SVneo trophoblast cells.

The alterations in DNA methylation and transcriptome in trophoblast cells under conditions of low oxygen and oxidative stress have major implications for pregnancy-related disorders. However, the exact mechanism is still not fully understood. In this study, we established models of hypoxia (H group) and oxidative stress (HR group) using HTR-8/SVneo trophoblast cells and performed combined analysis of genome-wide DNA methylation changes using reduced representation bisulphite sequencing and transcriptome expression changes using RNA sequencing. Our findings revealed that the H group exhibited a higher number of differentially methylated genes and differentially expressed genes than the HR group. In the H group, only 0.90% of all differentially expressed genes displayed simultaneous changes in DNA methylation and transcriptome expression. After the threshold was expanded, this number increased to 6.29% in the HR group. Notably, both the H group and HR group exhibited concurrent alterations in DNA methylation and transcriptome expression within Axon guidance and MAPK signalling pathway. Among the top 25 differentially methylated KEGG pathways in the promoter region, 11 pathways were commonly enriched in H group and HR group, accounting for 44.00%. Among the top 25 KEGG pathways in transcriptome with significant differences between the H group and HR group, 10 pathways were consistent, accounting for 40.00%. By integrating our previous data on DNA methylation from preeclamptic placental tissues, we identified that the ANKRD37 and PFKFB3 genes may contribute to the pathogenesis of preeclampsia through DNA methylation-mediated transcriptome expression under hypoxic conditions.

Epigenetic signatures of trophoblast lineage and their biological functions.

Trophoblasts play a crucial role in embryo implantation and in interacting with the maternal uterus. The trophoblast lineage develops into a substantial part of the placenta, a temporary extra-embryonic organ, capable of undergoing distinctive epigenetic events during development. The critical role of trophoblast-specific epigenetic signatures in regulating placental development has become known, significantly advancing our understanding of trophoblast identity and lineage development. Scientific efforts are revealing how trophoblast-specific epigenetic signatures mediate stage-specific gene regulatory programming during the development of the trophoblast lineage. These epigenetic signatures have a significant impact on blastocyst formation, placental development, as well as the growth and survival of embryos and fetuses. In evolution, DNA hypomethylation in the trophoblast lineage is conserved, and there is a significant disparity in the control of epigenetic dynamics and the landscape of genomic imprinting. Scientists have used murine and human multipotent trophoblast cells as in vitro models to recapitulate the essential epigenetic processes of placental development. Here, we review the epigenetic signatures of the trophoblast lineage and their biological functions to enhance our understanding of placental evolution, development, and function.

The effects of leptin on human cytotrophoblast invasion are gestational age and dose-dependent.

Introduction: Leptin and its receptors are expressed by the human placenta throughout gestation, yet the role of leptin in early human placental development is not well characterized. Leptin is overexpressed in the placentas from preeclamptic (PE) pregnancies. PE can result from the impaired invasion of fetal placental cells, cytotrophoblasts (CTBs), into the maternal decidua. We hypothesized that elevated leptin levels would impair human CTB invasion. Methods: The effects of leptin on the invasion of human CTBs were evaluated in three cell models, HTR-8/SVneo cells, primary CTBs, and placental villous explants using invasion assays. Further, leptin receptor expression was characterized in all three cell models using RT-PCR. Further phosphokinase assays were performed in HTR-8/SVneo cells to determine signaling pathways involved in CTB invasion in response to differential leptin doses. Results: We found that, prior to 8 weeks gestation, leptin promoted CTB invasion in the explant model. After 11 weeks gestation in explants, primary CTBs and in HTR-8/SVneo cells, leptin promoted invasion at moderate but not at high concentrations. Further, leptin receptor characterization revealed that leptin receptor expression did not vary over gestation, however, STAT, PI3K and MAPK pathways showed different signaling in response to varied leptin doses. Discussion: These data suggest that the excess placental leptin observed in PE may cause impaired CTB invasion as a second-trimester defect. Leptin's differential effect on trophoblast invasion may explain the role of hyperleptinemia in preeclampsia pathogenesis.

Evaluating the role of DNA methyltransferases in trophoblast fusion and preeclampsia development: Insights from methylation-regulated genes.

The etiology of preeclampsia (PE), a complex and multifactorial condition, remains incompletely understood. DNA methylation, which is primarily regulated by three DNA methyltransferases (DNMTs), DNMT1, DNMT3A, and DNMT3B, plays a vital role in early embryonic development and trophectoderm differentiation. Yet, how DNMTs modulate trophoblast fusion and PE development remains unclear. In this study, we found that the DNMTs expression was downregulated during trophoblast cells fusion. Downregulation of DNMTs was observed during the reconstruction of the denuded syncytiotrophoblast (STB) layer of placental explants. Additionally, overexpression of DNMTs inhibited trophoblast fusion. Conversely, treatment with the DNA methylation inhibitor 5-aza-CdR decreased the expression of DNMTs and promoted trophoblast fusion. A combined analysis of DNA methylation data and gene transcriptome data obtained from the primary cytotrophoblasts (CTBs) fusion process identified 104 potential methylation-regulated differentially expressed genes (MeDEGs) with upregulated expression due to DNA demethylation, including CD59, TNFAIP3, SDC1, and CDK6. The transcription regulation region (TRR) of TNFAIP3 showed a hypomethylation with induction of 5-aza-CdR, which facilitated CREB recruitment and thereby participated in regulating trophoblast fusion. More importantly, clinical correlation analysis of PE showed that the abnormal increase in DNMTs may be involved in the development of PE. This study identified placental DNA methylation-regulated genes that may contribute to PE, offering a novel perspective on the role of epigenetics in trophoblast fusion and its implication in PE development.

In vitro generation of trophoblast like stem cells from goat pluripotent stem cells.

Since the first mouse induced pluripotent stem cells (iPSCs) was derived, the in vitro culture of domestic iPSCs functionally and molecularly comparable with mouse iPSCs has been a challenge. Here, we established dairy goat iPSCs (giPSCs) from goat ear fibroblast cells with mouse iPSCs morphology, the expression of pluripotent markers and differentiation ability in vitro delivered by piggyBac transposon with nine Dox-inducible exogenous reprogramming factors. These reprogramming factors were bOMSK (bovine OCT4, CMYC, SOX2, and KLF4), pNhL (porcine NANOG and human LIN28), hRL (human RARG and LRH1), and SV40 Large T. Notably, AF-giPSCs (induced in activin A and bFGF condition) were capable of differentiation in embryoid bodies in vitro and could contribute to interspecies chimerism in mouse E6.5 embryos in vitro, demonstrating that AF-giPSCs have the developmental capability to generate some embryonic cell lineages. Moreover, Wnt/ß-catenin signaling has an important role in driving goat induced trophoblast-like stem cells (giTLSCs) from Dox-independent giPSCs. This study will support further establishment of the stable giPSC lines without any integration of exogenous genes.

MIR193BHG inhibits the proliferation, migration and invasion of trophoblasts by upregulating p53.

Aberrant expression of long non-coding RNAs (lncRNAs) serves a crucial role in the biological function of trophoblasts and contributes to preeclampsia (PE). lncRNA MIR193BHG expression is increased in PE placental tissues. In the present study, the effects of MIR193BHG on the function of trophoblasts were assessed to elucidate its underlying molecular mechanisms. The subcellular localization of MIR193BHG in HTR-8/SVneo human first-trimester extravillous trophoblast cells was determined using a fluorescent in situ hybridization assay and by conducting nucleocytoplasmic separation. The effect of MIR193BHG knockdown or overexpression on proliferation, migration, invasion and apoptosis was evaluated in vitro using Cell Counting Kit-8, wound healing, Transwell and flow cytometry assays. RNA-sequencing, Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis and protein-protein interaction network construction were subsequently performed to screen the downstream molecules regulated by MIR193BHG. Finally, rescue experiments were conducted to ascertain whether MIR193BHG influenced the biological function of trophoblasts via p53. MIR193BHG was predominantly localized in the nucleus of HTR-8/SVneo cells and overexpression of MIR193BHG significantly inhibited proliferation, migration and invasion, while increasing the rate of apoptosis of HTR-8/SVneo cells. Knockdown of MIR193BHG had the opposite effect. Furthermore, overexpression of MIR193BHG led to increases in both mRNA and protein levels of p53 compared with the control group, and knockdown of p53 rescued the effects induced by overexpression of MIR193BHG on cell proliferation, migration and invasion, while partially counteracting its effects on apoptosis of HTR-8/SVneo cells. In conclusion, the findings of the present study suggested that MIR193BHG served a critical role in progression of PE by regulating the expression of p53, and may be a novel therapeutic target for PE.

JPT2 in subclinical hypothyroidism-related miscarriage as a transcription co-factor: involvement of LEPR/STAT3 activation.

BACKGROUND AND PURPOSE: Subclinical hypothyroidism (SCH) has been identified to be associated with implantation failure, in which the dysfunction of trophoblast cells is involved. In this study, the transcriptomics of aborted placenta from SCH rats were analyzed. Jupiter microtubule-associated homolog 2 (JPT2) was downregulated in the aborted placenta. This study aims to investigate its role in SCH-associated miscarriage. METHODS: Spontaneous abortion was observed in SCH rats generated by thyroidectomy combined with levothyroxine administration. The transcriptomics analysis was performed using aborted placenta. Afterward, the effects of JPT2 on trophoblast cells were explored using gain-and loss-of-function experiments. RESULTS: Transcriptomics analysis showed 1286 downregulated genes and 2300 upregulated genes in the aborted placenta, and JPT2 was significantly downregulated in the aborted placenta from SCH rats. Afterward, gain-and loss-of-function experiments exhibited that overexpression of JPT2 promoted the proliferation, migration, invasion, spheroid formation of HTR-8/SVneo trophoblast cells and their attachment to endometrial stromal cells, while these biological behaviors were suppressed by JPT2 knockdown. Furthermore, JPT2 accelerated the transcription of leptin receptor (LEPR), and activated signal transducer and activator of transcription 3 (STAT3) signal in a transcription factor AP-2γ-dependent manner. In addition, silencing of LEPR abolished the role of JPT2. CONCLUSION: Our results revealed that JPT2, which was downregulated in the aborted placenta from SCH rats, promoted proliferation, migration, invasion, spheroid formation, and attachment of trophoblast cells via regulating LEPR/STAT3 axis as a transcription co-factor. It is indicated that low expression of JPT2 may contribute to the abortion in individuals with SCH.

Immunohistochemical localization of HCA1 receptor in placenta in presence of fetal growth restriction.

INTRODUCTION: Glucose metabolism produces lactate and hydrogen ions in an anaerobic environment. Fetuses with intrauterine growth restriction are considered to become progressively lactacidemic as well as hypoxic. Roles of lactate in the placenta in the presence of fetal growth restriction (FGR) remain to be clarified. METHODS: Immunohistochemical localization of lactate-related substances, such as a receptor for lactate (hydroxy-carboxylic acid 1 receptor (HCA1 receptor/GPR81)), monocarboxylate transporters (MCTs) for lactate, lactate dehydrogenases (LDHs), and proteins expressed in syncytiotrophoblasts or cytotrophoblasts was examined in placentas of appropriate weight for gestational age (AGA) fetus and those showing FGR. RESULTS: Immunoreactivity for the HCA1 receptor was present in the cytoplasm of some trophoblasts, predominantly localized to their basal (fetus-facing) side, and was frequently colocalized with that for E-cadherin or serine peptidase inhibitor, Kunitz type 1 (SPINT1), a marker protein of cytotrophoblasts. Immunoreactivity for MCT1 and MCT4 was present on the basal and the microvillous (maternal-facing) membranes of trophoblasts in both groups, respectively. Clear immunoreactivity for LDHA and LDHB was also observed in the cytoplasm of trophoblasts, mainly localized to their basal side. However, there were no significant differences in immunohistochemically stained areas of lactate-related substances between AGA and late-onset FGR groups. On the other hand, there were correlations between coefficients of the presence of chorioamnionitis and the values of LDHB and E-cadherin. DISCUSSION: Immunohistochemical localization of the HCA1 receptor was predominantly observed in the cytoplasm located on the basal side of trophoblasts, suggesting a role of lactate in human placental development, including syncytialization.

CREG1 promotes bovine placental trophoblast cells exosome release by targeting IGF2R and participates in regulating organoid differentiation via exosomes transport.

BACKGROUND: Placental exosomes are a kind of intercellular communication media secreted by placental cells during pregnancy, exosomogenesis and release are regulated by many secretory glycoproteins. CREG1 is a kind of secreted glycoprotein widely expressed in various organs and tissues of the body, which inhibits cell proliferation and enhances cell differentiation. The aim of this study was to explore the role of CREG1 in regulating exosomogenesis during the proliferation and differentiation of placental trophoblast cells in early pregnant dairy cows by targeting IGF2R and participating in regulating organoid differentiation via exosomes transport. METHODS: Molecular biological methods were firstly used to investigate the expression patterns of CREG1, IGF2R and exosomal marker proteins in early placental development of pregnant dairy cows. Subsequently, the effects of CREG1 on the formation and release of bovine placental trophoblast (BTCs) derived exosomes by targeting IGF2R were investigated. Further, the effects of CREG1 on the change of gene expression patterns along with the transport of exosomes to recipient cells and participate in regulating the differentiation of organoids were explored. RESULTS: The expression of CREG1, IGF2R and exosomal marker proteins increased with the increase of pregnancy months during the early evolution of placental trophoblast cells in dairy cows. Overexpression of Creg1 enhanced the genesis and release of exosomes derived from BTCs, while knocking down the expression of Igf2r gene not only inhibited the genesis of exosomes, but also inhibited the genesis and release of exosomes induced by overexpression of CREG1 protein. Interestingly, IGF2R can regulate the expression of CREG1 through reverse secretion. What's more, the occurrence and release of trophoblast-derived exosomes are regulated by CREG1 binding to IGF2R, which subsequently binds to Rab11. CREG1 can not only promote the formation and release of exosomes in donor cells, but also regulate the change of gene expression patterns along with the transport of exosomes to recipient cells and participate in regulating the early development of placenta. CONCLUSIONS: Our study confirmed that CREG1 is involved in the exosomogenesis and release of exosomes during the proliferation and differentiation of placental trophoblast cells in early pregnant dairy cows by targeting IGF2R, and is involved in the regulation of organoid differentiation through exosome transport.

N6-methyladenosine dynamics in placental development and trophoblast functions, and its potential role in placental diseases.

N6-methyladenosine (m6A) is the most abundant modification controlling RNA metabolism and cellular functions, but its roles in placental development are still poorly understood. Here, we characterized the synchronization of m6A modifications and placental functions by mapping the m6A methylome in human placentas (n = 3, each trimester), revealing that the dynamic patterns of m6A were associated with gene expression homeostasis and different biological pathways in placental development. Then, we generated trophoblast-specific knockout mice of Wtap, a critical component of methyltransferase complex, and demonstrated that Wtap was essential for trophoblast proliferation, placentation and perinatal growth. Further in vitro experiments which includes cell viability assays and series molecular binding assays demonstrated that WTAP-m6A-IGF2BP3 axis regulated the RNA stability and translation of Anillin (ANLN) and VEGFA, promoting trophoblast proliferation and secretion. Dysregulation of this regulatory axis was observed in placentas from pregnancies with fetal growth restriction (FGR) or preeclampsia, revealing the pathogenic effects of imbalanced m6A modifications. Therefore, our findings provide novel insights into the functions and regulatory mechanisms of m6A modifications in placental development and placental-related gestational diseases.