BACKGROUND: Trophoblast homeostasis and differentiation require a proper endoplasmic reticulum (ER) function. The Krüppel-like factor-6 (KLF6) transcription factor modulates trophoblast migration, differentiation, and reactive oxygen species (ROS) production. Since ROS may impact on ER homeostasis, we assessed whether downregulation of KLF6 altered the unfolded protein response (UPR) and cellular process associated with ER homeostasis.
METHODS: Protein and RNA expression were analyzed by Western blot and qRT-PCR, respectively, in extravillous trophoblast HTR-8/SVneo cells silenced for KLF6. Apoptosis was detected by flow cell cytometry using Annexin V Apoptosis Detection Kit. Protein trafficking was assessed by confocal microscopy of a reporter fluorescent protein whose release from the ER was synchronized.
RESULTS: KLF6 downregulation reduced the expression of BiP, the master regulator of the UPR, at protein, mRNA, and pre-mRNA levels. Ire1α protein, XBP1 splicing, and DNAJB9 mRNA levels were also reduced in KLF6-silenced cells. Instead, PDI, Ero1α, and the p-eIF2α/eIF2α ratio as well as autophagy and proteasome dependent protein degradation remained unchanged while intracellular trafficking was increased. Under thapsigargin-induced stress, KLF6 silencing impaired BiP protein and mRNA expression increase, as well as the activation of the Ire1α pathway, but it raised the p-eIF2α/eIF2α ratio and CHOP protein levels. Nevertheless, apoptosis was not increased.
CONCLUSIONS: Results provide the first evidence of KLF6 as a modulator of the UPR components. The increase in protein trafficking and protection from apoptosis, observed in KLF6-silenced cells, are consistent with its role in extravillous trophoblast migration and differentiation.

Preeclampsia (PE) is a life-threatening pregnancy-specific complication with controversial mechanisms and no effective treatment except delivery is available. Currently, increasing researchers suggested that PE shares pathophysiologic features with protein misfolding/aggregation disorders, such as Alzheimer disease (AD). Evidences have proposed defective autophagy as a potential source of protein aggregation in PE. Endoplasmic reticulum-selective autophagy (ER-phagy) plays a critical role in clearing misfolded proteins and maintaining ER homeostasis. However, its roles in the molecular pathology of PE remain unclear. We found that lncRNA DUXAP8 was upregulated in preeclamptic placentae and significantly correlated with clinical indicators. DUXAP8 specifically binds to PCBP2 and inhibits its ubiquitination-mediated degradation, and decreased levels of PCBP2 reversed the activation effect of DUXAP8 overexpression on AKT/mTOR signaling pathway. Function experiments showed that DUXAP8 overexpression inhibited trophoblastic proliferation, migration, and invasion of HTR-8/SVneo and JAR cells. Moreover, pathological accumulation of swollen and lytic ER (endoplasmic reticulum) was observed in DUXAP8-overexpressed HTR8/SVneo cells and PE placental villus trophoblast cells, which suggesting that ER clearance ability is impaired. Further studies found that DUXAP8 overexpression impaired ER-phagy and caused protein aggregation medicated by reduced FAM134B and LC3II expression (key proteins involved in ER-phagy) via activating AKT/mTOR signaling pathway. The increased level of FAM134B significantly reversed the inhibitory effect of DUXAP8 overexpression on the proliferation, migration, and invasion of trophoblasts. In vivo, DUXAP8 overexpression through tail vein injection of adenovirus induced PE-like phenotypes in pregnant rats accompanied with activated AKT/mTOR signaling, decreased expression of FAM134B and LC3-II proteins and increased protein aggregation in placental tissues. Our study reveals the important role of lncRNA DUXAP8 in regulating trophoblast biological behaviors through FAM134B-mediated ER-phagy, providing a new theoretical basis for understanding the pathogenesis of PE.

Extracellular vesicles (EVs) serve as messengers for intercellular communication, yet the precise mechanisms by which recipient cells interpret EV messages remain incompletely understood. In this study, we explored how the origin of EVs, their protein cargo, and the recipient cell type influence the cellular response to EVs within an embryo implantation model. We treated two types of EVs to 6 different recipient cell types and expression of zinc finger protein 81 (ZNF81) gene expression in the recipient cells were quantified using quantitative polymerase chain reaction (qPCR). The proteomic contents of the EV cargos were also analyzed. The results showed that downregulation of the ZNF81 gene was a specific cellular response of receptive endometrial epithelial cells to trophoblast derived EVs. Protein cargo analysis revealed that the proteomic profile of EVs depends on their cell of origin and therefore may affect the recipient cell response to EVs. Furthermore, trophoblastic EVs were found to be specifically enriched with transcription factors such as CTNNB1 (catenin beta-1), HDAC2 (histone deacetylase 2), and NOTCH1 (neurogenic locus notch homolog protein 1), which are known regulators of ZNF81 gene expression. The current study provided compelling evidence supporting the existence of EV specificity, where the characteristics of both the EVs and the recipient cell type collectively contribute to regulating EV target specificity. Additionally, EV protein cargo analysis suggested a potential association between transcription factors and the specific functionality of trophoblastic EVs. This in vitro embryo implantation model and ZNF81 read-out provides a unique platform to study EV specific functionality in natural cell-cell communication.

The aberrant invasive capability of trophoblast cells is widely acknowledged as a primary mechanism underlying RSA. Recently, IGF2BP3 has been implicated in various cancers due to its influence on cellular invasion and migration. However, whether IGF2BP3 involve in the occurrence of RSA and the specific functions it assumes in the development of RSA remain elusive. In our study, we firstly collected villous tissues from RSA and those with normal pregnancies individuals to performed Protein sequencing and then detected the expression of IGF2BP3 through Western blot, qRT-PCR and immunohistochemistry. Secondly, we analyzed the single-cell data (GSE214607) to assess the expression of IGF2BP3 in invasive EVT trophoblasts. Thirdly, we utilized lentivirus technology to establish HTR-8/SVneo cell lines with stable IGF2BP3 knockdown and RNA-seq analysis was employed to investigate the GO functional pathway enrichment of IGF2BP3. Meanwhile, the effect of IGF2BP3 knockdown on trophoblast cells apoptosis, migration, and ferroptosis was evaluated through functional experiments. Additionally, LPS-induced abortion animal model was constructed to evaluate IGF2BP3 expression in placental tissues. A significant downregulation of IGF2BP3 was observed in the villous tissues of RSA patient, a finding corroborated by subsequent single cell sequencing results. Furthermore, it suggested that IGF2BP3 may be involved in the migration and apoptotic processes of trophoblast cells. Mechanistic research indicated that IGF2BP3 knockdown could compromise GPX4 mRNA stability, leading to the promotion of ferroptosis. Finally, our investigation observed the down-regulation of IGF2BP3 expression in placental villous tissues of an LPS-induced abortion animal model. Our findings revealed that IGF2BP3 was downregulated in the villous tissues of RSA patients. Mechanically, down-regulation of IGF2BP3 may induce RSA by promoting GPX4-mediated ferroptosis and inhibiting trophoblast invasion and migration. Our study may provide new targets and research directions for the pathogenesis of RSA.

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.

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.

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.

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.

This study aimed to investigate placental microblood flow perfusion in fetal growth restriction (FGR) both pre- and post-delivery, and explore the influence of LINC00473 and its downstream targets on FGR progression in trophoblast cells. Placental vascular distribution, placental vascular index (VIMV), CD34 expression, and histological changes were compared between control and FGR groups. FGR-related differentially expressed genes (DEGs) were analyzed and validated by quantitative real-time polymerase chain reaction (qPCR) and immunohistochemistry (IHC) in placentae. In vitro experiments examined the regulatory relationships among LINC00473, miR-5189-5p, and StAR, followed by investigations into their impacts on cell proliferation and apoptosis. FGR placentae exhibited irregular shapes, uneven parenchymal echo, stromal dysplasia, ischemic infarction, and variable degrees of thickening in some cases. FGR samples showed less prominent mother vessel lakes, significantly lower VIMV, and decreased CD34 expression. Hematoxylin & eosin (H&E) staining revealed placental fibrosis, fibrin adhesion, infarction, and interstitial dysplasia in FGR. LINC00473, miR-5189-5p, and StAR were identified as DEG, with qPCR demonstrating a significant increase in LINC00473 and a decrease in miR-5189-5p in FGR, while both qPCR and IHC indicated a significant increase in StAR expression. LINC00473 served as an endogenous sponge against miR-5189-5p in human HTR-8/SV neo cells, and StAR expression was regulated by both LINC00473 and miR-5189-5p. Dysregulation of these genes affected cell proliferation and apoptosis. Pathological changes in the placenta are significant contributors to FGR, with placental microblood flow potentially serving as an indicator for monitoring its progression. LINC00473 and its downstream targets may modulate trophoblasts proliferation and apoptosis, thus influencing the onset of FGR, suggesting novel avenues for diagnosis and treatment.

Copper pollution has attracted global environmental concern. Widespread Cu pollution results in excessive Cu accumulation in human. Epidemiological studies and animal experiments revealed that Cu exposure might have reproductive toxicity. Cuproptosis is a recently reported Cu-dependent and programmed cell death pattern. However, the mechanism by which copper exposure might cause cell cuproptosis is largely unknown. We chose trophoblast cells as cell model and found that copper exposure causes trophoblast cell cuproptosis. In mechanism, copper exposure up-regulates lnc-HZ11 expression levels, which increases intracellular Cu2+ levels and causes trophoblast cell cuproptosis. Knockdown of lnc-HZ11 efficiently reduces intracellular Cu2+ levels and alleviate trophoblast cell cuproptosis, which could be further alleviated by co-treatment with DC or TEPA. These results discover novel toxicological effects of copper exposure and also provide potential target for protection trophoblast cells from cuproptosis in the presence of excessive copper exposure.